Liposome Technology, Volume II Entrapment of Drugs and Other Materials Into Liposomes, Third Edition

Liposome TechnologyThird Edition

Volume IIEntrapment of Drugs and

Other Materials into Liposomes

Edited by

Gregory GregoriadisThe School of Pharmacy

University of Londonand

Lipoxen PLCLondon, U.K.

New York London

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Dedicated to the memory of my parents,Christos and Athena

Preface

Preface

, ,

The science and technology of liposomes as a delivery system for drugs andvaccines have evolved through a variety of phases that I have been privilegedto witness from the very beginning. The initial observation (1) that exposureof phospholipids to excess water gives rise to lamellar structures that are ableto sequester solutes led to the adoption of these structures (later to becomeknown as liposomes) as a model for the study of cell membrane biophysics.Solute sequestration into liposomes prompted a few years later the develop-ment of the drug delivery concept (2,3) and, in 1970, animals were for the firsttime injected with active-containing liposomes (3,4). Subsequent work inthe author’s laboratory and elsewhere worldwide on drug- and vaccine-containing liposomes and their interaction with the biological milieuin vivo culminated in the licensing of a number of injectable liposome-basedtherapeutics and vaccines. The history of the evolution of liposomes froma structural curiosity in the 1960s to a multifaceted, powerful tool fortransforming toxic or ineffective drugs into entities with improved pharma-cological profiles today has been summarized elsewhere (5,6).

The great strides made toward the application of liposomes in thetreatment and prevention of disease over nearly four decades are largelydue to developments in liposome technology; earlier achievements wereincluded in the previous two editions of this book (7,8). The avalanche ofnew techniques that came with further expansion of liposomology since

v

the second edition in 1992 has necessitated their inclusion into a radicallyupdated third edition. Indeed, so great is the plethora of the new materialthat very little from the second edition has been retained. As before, con-tributors were asked to emphasize methodology employed in their ownlaboratories since reviews on technology with which contributors have nopersonal experience were likely to be superficial for the purpose of thepresent book. In some cases, however, overviews were invited when it wasdeemed useful to reconnoiter distinct areas of technology. A typical chapterincorporates an introductory section directly relevant to the author’s subjectwith concise coverage of related literature. This is followed by a detailedmethodology section describing experiences from the author’s laboratoryand examples of actual applications of the methods presented, and, finally,by a critical discussion of the advantages or disadvantages of the method-ology presented vis-a-vis other related methodologies. The 55 chapterscontributed have been distributed logically into three volumes. Volume Ideals with a variety of methods for the preparation of liposomes and anarray of auxiliary techniques required for liposome characterization anddevelopment. Volume II describes procedures for the incorporation intoliposomes of a number of drugs selected for their relevance to current trendsin liposomology. Volume III is devoted to technologies generatingliposomes that can function in a ‘‘targeted’’ fashion and to approaches ofstudying the interaction of liposomes with the biological milieu.

It has been again a pleasure for me to undertake this task of bringingtogether so much knowledge, experience, and wisdom that has been sogenerously provided by liposomologist friends and colleagues. It is to behoped that the book will prove useful to anyone involved in drug delivery,especially those who have entered the field recently and need guidancethrough the vastness of related literature and the complexity and diversityof aspects of liposome use. I take this opportunity to thank Mrs. ConchaPerring for her many hours of help with the manuscripts and InformaHealthcare personnel for their truly professional cooperation.

Gregory Gregoriadis

REFERENCES

1. Bangham AD, Standish MM, Watkins JC. Diffusion of univalent ions across thelamellae of swollen phospholipids. J Mol Biol 1965; 13:238.

2. Gregoriadis G, Leathwood PD, Ryman BE. Enzyme entrapment in liposomes.FEBS Lett 1971; 14:95.

3. Gregoriadis G, Ryman BB. Fate of protein-containing liposomes injected intorats. An approach to the treatment of storage diseases. Eur J Biochem 1972;24:485.

vi Preface

4. Gregoriadis G. The carrier potential of liposomes in biology and medicine. NewEngl J Med 1976; 295:704–765.

5. Gregoriadis G. ‘‘Twinkling guide stars to throngs of acolytes desirous of yourmembrane semi-barriers. Precursors of bion, potential drug carriers...’’. J Lipo-some Res 1995; 5:329.

6. Lasic DD, Papahadjopoulos D (Eds), Medical Applications of Liposomes,Elsevier. Amsterdam 1998.

7. Gregoriadis G. Liposome Technology. CRC Press, Boca Raton, Volumes I, IIand III, 1984.

8. Gregoriadis G. Liposome Technology 2nd Edition. CRC Press, Boca Raton,Volumes I, II and III, 1992.

Preface vii

Acknowledgments

The individuals listed below in chronological order (1972–2006) worked inmy laboratory as postgraduate students, senior scientists, research assis-tants, post-doctoral fellows, technicians, visiting scholars, and Erasmus orSandwich students. I take this opportunity to express my gratitude for theircontributions to the science and technology of liposomes and other deliverysystems, as well as their support and friendship. I am most grateful tomy secretary of 14 years, Concha Perring, for her hard work, perseverance,and loyalty.

Rosemary A. Buckland (UK), Diane Neerunjun (UK), ChristopherD.V. Black (UK), Anthony W. Segal (UK), Gerry Dapergolas (Greece),Pamela J. Davisson (UK), Susan Scott (UK), George Deliconstantinos(Greece), Peter Bonventre (USA), Isobel Braidman (UK), Daniel Wreschner(Israel), Emanuel Manesis (Greece), Christine Davis (UK), Roger Moore(UK), Chris Kirby (UK), Jackie Clarke (UK), Pamela Large (UK), JudithSenior (UK), Ann Meehan (UK), Mon-Moy Mah (Malaysia), CatherineLemonias (Greece), Hishani Weereratne (Sri Lanka), Jim Mixson (USA),Askin Tumer (Turkey), Barbara Wolff (Germany), Natalie Garcon (France),Volkmar Weissig (USA), David Davis (UK), Alun Davies (UK), Jay R.Behari (India), Steven Seltzer (USA), Yash Pathak (India), Lloyd Tan (Sin-gapore), Qifu Xiao (China), Christine Panagiotidi (Greece), K.L. Kahl(New Zealand), Zhen Wang (China), Helena da Silva (Portugal), BrendaMcCormack (UK), M. Yasar Ozden (Turkey), Natasa Skalko (Croatia),John Giannios (Greece), Dmitry Genkin (Russia), Maria Georgiou (Cyprus),Sophia Antimisiaris (Greece), Becky J. Ficek (USA), Victor Kyrylenko(Ukraine), Suresh Vyas (India), Martin Brandl (Germany), Dieter Bachmann(Germany), Mayda Gursel (Turkey), Sabina Ganter (Germany), IshanGursel (Turkey), Maria Velinova (Bulgaria), Cecilia D’Antuono (Argentina),

ix

Ana Fernandes (Portugal), Cristina Lopez Pascual (Spain), Susana Morais(Portugal), Ann Young (UK), Yannis Loukas (Greece), Vassilia Vraka (Gre-ece), Voula Kallinteri (Greece), Fatima Era€��s (France), Jean Marie Verdier(France), Dimitri Fatouros (Greece), Veronika Muller (Germany), Jean-Christophe Olivier (France), Janny Zhang (China), Roghieh Saffie (Iran),Irene Naldoska (Polland), Sudaxina Murdan (Mauritius), Sussi Juul Hansen(Denmark), Anette Hollensen (Denmark), Yvonne Perrie (UK), Maria JoseSaez Alonso (Spain), Mercedes Valdes (Spain), Laura Nasarre (Spain), EveCrane (USA), Brahim Zadi (Algeria), Maria E. Lanio (Cuba), GernotWarnke (Germany), Elizabetta Casali (Italy), Sevtap Velipasaoglu (Turkey),Sara Lauria (Italy), Oulaya Belguenani (France), Isabelle Gyselinck (Bel-gium), Sigrun Lubke (Germany), Kent Lau (Hong Kong), Alejandro Soto(Cuba), Yanin Bebelagua (Cuba), Steve Yang (Taiwan), Filipe Rocha daTorre Assoreira (Portugal), Paola Genitrini (Italy), Guoping Sun (China),Malini Mital (UK), Michael Schupp (Germany), Karin Gaimann (Germany),Mia Obrenovic (Serbia), Sherry Kittivoravitkul (Thailand), Yoshie Maitani(Japan), Irene Papanicolaou (Greece), Zulaykho Shamansurova (Uzbeki-stan), Miriam Steur (Germany), Sanjay Jain (India), Ioannis Papaioannou(Greece), Maria Verissimo (Italy), Bruno da Costa (Portugal), Letizia FloresPrieto (Spain), Andrew Bacon (UK).

x Acknowledgments

Contents

Preface . . . . vAcknowledgments . . . . ixContributors . . . . xvii

1. Amphipathic Weak Base Loading into Preformed LiposomesHaving a Transmembrane Ammonium Ion Gradient:From the Bench to Approved Doxil . . . . . . . . . . . . . . . . . . 1Yechezkel BarenholzIntroduction . . . . 1Mechanism of Remote Loading by AS Gradient . . . . 2The Doxil Example for Remote Loading of

Amphipathic Weak Base into Liposomes . . . . 8‘‘Remote’’ Release . . . . 11Experimental Demonstration of DOX Remote

Loading to Form Doxil . . . . 13Summary of the Characterization of 100 nm DOX–SSL

Remote Loaded with DOX via TransmembraneAS Gradient . . . . 21

References . . . . 22

2. Encapsulation of Drugs Within Liposomes bypH-Gradient Techniques . . . . . . . . . . . . . . . . . . . . . . . . . 27David B. Fenske and Pieter R. CullisIntroduction . . . . 27

xi

The Formation of Large Unilamellar Vesicles byExtrusion Methods . . . . 30

Generation of pH Gradients via InternalCitrate Buffer . . . . 32

Generation of pH Gradients via TransmembraneAmmonia Gradients . . . . 38

Ionophore-Mediated Generation of pH Gradients viaTransmembrane Ion Gradients . . . . 40

Comparison of Loading Methods . . . . 44Conclusions . . . . 45References . . . . 45

3. Incorporation of Lipophilic Antitumor and AntiviralDrugs into the Lipid Bilayer of SmallUnilamellar Liposomes . . . . . . . . . . . . . . . . . . . . . . . . . . 51Reto Schwendener and Herbert SchottIntroduction . . . . 51Materials and Methods . . . . 54Results . . . . 57Conclusions and Prospects . . . . 58References . . . . 59

4. Liposome-Encapsulated Hemoglobin as an ArtificialOxygen Carrier . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63Vibhudutta Awasthi, Beth A. Goins, andWilliam T. PhillipsIntroduction . . . . 63Formulation Factors Influencing the Composition

of LEH . . . . 65Current Manufacturing Technology . . . . 73Freeze-Drying LEH . . . . 75Storage Stability . . . . 76Evaluation Techniques . . . . 77Summary . . . . 81References . . . . 82

5. An Original Lipid Complex System for Amphotericin B . . . 93Malika Larabi, Philippe Legrand, and Gillian BarrattIntroduction . . . . 93Preparation of Lipid Complex of AmB . . . . 96Physical Characterization . . . . 97Evaluation of Toxicity . . . . 102

xii Contents

Evaluation of Activity . . . . 105Conclusion . . . . 107References . . . . 108

6. Coupling of Peptides to the Surface of Liposomes—Application to Liposome-Based Synthetic Vaccines . . . . . 111Francis Schuber, Fatouma Said Hassane, and Benoıt FrischIntroduction . . . . 111Techniques for Coupling Peptides to the Surface

of Liposomes . . . . 112Targeted Liposome-Peptide Constructs . . . . 117Application of Liposome-Peptide Constructs

to Vaccination . . . . 118Conclusions . . . . 123References . . . . 125

7. Encapsulation of Nucleic Acid–Based Therapeutics . . . . . 131Norbert Maurer, Igor Zhigaltsev, and Pieter R. CullisIntroduction . . . . 131Methodology . . . . 132Results . . . . 135Conclusions . . . . 143References . . . . 146

8. Intraliposomal Trapping Agents for Improving In VivoLiposomal Drug Formulation Stability . . . . . . . . . . . . . . 149Daryl C. Drummond, Mark E. Hayes, Charles O.Noble IV, John W. Park, Dmitri B. Kirpotin,and Zexiong GuoandIntroduction . . . . 149Methods . . . . 151Factors Influencing In Vivo Drug Retention . . . . 159Colloidal and Chemical Stability

Considerations . . . . 164Conclusions . . . . 164References . . . . 166

9. Radiolabeling of Liposomes for Scintigraphic Imaging . . . 169Peter Laverman, Gert Storm, William T. Phillips,Ande Bao, and Beth A. GoinsIntroduction . . . . 169Scintigraphic Imaging . . . . 170

Contents xiii

The Choice of the Radionuclide . . . . 171Labeling Methods . . . . 172Concluding Remarks . . . . 181References . . . . 183

10. Liposomal Bisphosphonates for the Treatmentof Restenosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 187Hila Epstein, Eyal Afergan, Nickolay Koroukhov, GalitEisenberg, Dikla Gutman, and Gershon GolombIntroduction . . . . 187Inflammation and Restenosis . . . . 189Macrophage/Monocyte Inhibition by Liposomal Delivery

System of BPs . . . . 191Inhibition of Restenosis . . . . 197Conclusion . . . . 199References . . . . 200

11. Development of a Liposomal Vaccination Systemfor Immunity-Modulating Antitumor Therapy . . . . . . . . . 207Andreas Graser, Abdo Konur, and Alfred FahrIntroduction . . . . 207Methodology . . . . 208Results and Discussion . . . . 212Summary . . . . 218References . . . . 219

12. Influenza Virosomes as Adjuvants in Cancer Immunotherapy 221Reto Schumacher, Giulio C. Spagnoli, and Michel AdaminaIntroduction . . . . 221Production of IRIV . . . . 222In Vitro Characterization of IRIV . . . . 222In Vitro Evaluation of IRIV Cytotoxic

T-Cell Adjuvance . . . . 226Discussion . . . . 229References . . . . 231

13. Liposome-Based DNA/Protein Vaccines: Procedures forEntrapment and Immunization Studies . . . . . . . . . . . . . . 233Gregory Gregoriadis, Andrew Bacon, Brenda McCormack,Peter Laing, Benoıt Frisch, and Francis SchuberIntroduction . . . . 233Materials . . . . 235

xiv Contents

Entrapment of Plasmid DNA and Protein Vaccines intoLiposomes by the Dehydration–RehydrationProcedure . . . . 235

Immunization Studies . . . . 241References . . . . 243

14. Liposome-Polycation-DNA: A Nonviral Gene VectorTurned into a Potent Vaccine Carrier . . . . . . . . . . . . . . 245Lisa M. Shollenberger and Leaf HuangLiposome-Polycation-DNA Complexes . . . . 245LPDI and the Immune System . . . . 247Summary . . . . 250References . . . . 251

15. Automated Screening of Cationic Lipid Formulationsfor Transfection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 253Ulrich Massing and Peter JantscheffIntroduction . . . . 253Screening for Improved Cationic Lipids . . . . 259Combination of the Screening Approach with

Combinatorial Solid Phase Synthesis ofCationic Lipids . . . . 263

Conclusion and Future Directions . . . . 269References . . . . 269

16. Incorporation of Poly(Ethylene Glycol) Lipid intoLipoplexes: On-Line Incorporation Assessment andPharmacokinetics Advantages . . . . . . . . . . . . . . . . . . . . 273Nathalie Mignet, Mamonjy Cadet, Michel Bessodes, andDaniel SchermanIntroduction . . . . 273Why Lipoplex PEGylation Is Needed . . . . 274Examples of PEG-Lipids Suitable for Lipoplex

Incorporation . . . . 276PEG-Lipid Incorporation into Lipoplexes: Protocols and

Monitoring . . . . 283Pharmacokinetic Properties of

PEG-Lipoplexes . . . . 285PEG-Lipoplexes: What More Is Needed? . . . . 286References . . . . 289

Contents xv

17. Efficient Gene Transfer by Lipid/PeptideTransfection Complexes . . . . . . . . . . . . . . . . . . . . . . . . 293Scott A. Irvine, Stephen L. Hart, Jean R. McEwan, andFaiza AfzalIntroduction . . . . 293Therapeutic Gene Transfer . . . . 294Liposome and Peptides . . . . 294Complex Formation . . . . 295Targeting . . . . 297Nuclear Localization Sequence . . . . 305Summary . . . . 307References . . . . 308

18. Phospholipid- and Nonphospholipid-Based Vesiclesfor Drug and DNA Delivery to Mitochondria inLiving Mammalian Cells . . . . . . . . . . . . . . . . . . . . . . . 317Volkmar Weissig, Sarathi V. Boddapati, Shing-Ming Cheng,Gerard G. M. D’Souza, and Vladimir P. TorchilinIntroduction . . . . 317Mitochondriotropic Liposomes . . . . 322Bola-Lipid–Based Mitochondria-Specific

Delivery Systems . . . . 325Summary and Conclusion . . . . 335References . . . . 336

19. Spectral Imaging for the Investigation of the IntracellularFate of Liposomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . 341Ulrich S. Huth, Rolf Schubert, and Regine Peschka-SussIntroduction . . . . 341Initial Mode of Internalization . . . . 345Intracellular Trafficking . . . . 359Metabolic Activity . . . . 365Transcytosis . . . . 366General Considerations . . . . 368Conclusion . . . . 371References . . . . 372

Index . . . . 383

xvi Contents

Contributors

Michel Adamina Department of Surgery, Institute for Surgical Researchand Hospital Management, University of Basel, Basel, Switzerland

Eyal Afergan Department of Pharmaceutics, School of Pharmacy,Faculty of Medicine, The Hebrew University of Jerusalem,Jerusalem, Israel

Faiza Afzal Centre for Cardiovascular Genetics, University CollegeLondon, Rayne Institute, London, U.K.

Vibhudutta Awasthi Department of Radiology, University of TexasHealth Science Center at San Antonio, San Antonio, Texas, U.S.A.

Andrew Bacon Lipoxen PLC, London, U.K.

Ande Bao Department of Radiology, University of Texas Health ScienceCenter at San Antonio, San Antonio, Texas, U.S.A.

Yechezkel Barenholz Laboratory of Membrane and Liposome Research,The Hebrew University–Hadassah Medical School, Jerusalem, Israel

Gillian Barratt Universite Paris-Sud, Chatenay-Malabry, France

Michel Bessodes Unite Pharmacol. Chim. Genet., Universite ReneDescartes Paris, Paris, France

xvii

Sarathi V. Boddapati Department of Pharmaceutical Sciences, School of

Pharmacy, Bouve College of Health Sciences, Northeastern University,

Boston, Massachusetts, U.S.A.

Mamonjy Cadet Unite Pharmacol. Chim. Genet., Universite Rene

Descartes Paris, Paris, France

Shing-Ming Cheng Department of Pharmaceutical Sciences, School of



Pieter R. Cullis Department of Biochemistry and Molecular Biology,

University of British Columbia, Vancouver, British Columbia, Canada

Daryl C. Drummond Hermes Biosciences, Inc., South San Francisco,

California, U.S.A.

Gerard G. M. D’Souza Department of Pharmaceutical Sciences, School of



Galit Eisenberg Department of Pharmaceutics, School of Pharmacy,

Faculty of Medicine, The Hebrew University of Jerusalem, Jerusalem, Israel

Hila Epstein Department of Pharmaceutics, School of Pharmacy, Faculty

of Medicine, The Hebrew University of Jerusalem, Jerusalem, Israel

Alfred Fahr Lehrstuhl fur Pharmazeutische Technologie, Friedrich-

Schiller-Universitat Jena, Jena, Germany

David B. Fenske Department of Chemistry, University College of the

Fraser Valley, Abbotsford, British Columbia, Canada

Benoıt Frisch Laboratoire de Chimie Bioorganique, Faculte de

Pharmacie, Universite Louis Pasteur, Strasbourg-Illkirch, and

Chimie Enzymatique, Illkirch, France

Beth A. Goins Department of Radiology, University of Texas Health

Science Center at San Antonio, San Antonio, Texas, U.S.A.

Gershon Golomb Department of Pharmaceutics, School of Pharmacy,


xviii Contributors

Andreas Graser Pharmaceutical Technology Development Formulation

Liquids, F. Hoffmann-La Roche Ltd., Basel, Switzerland

Gregory Gregoriadis The School of Pharmacy, University of London, and

Lipoxen PLC, London, U.K.

Zexiong Guo First Affiliated Hospital of Jinan University, Guangzhou,

P.R. China

Dikla Gutman Department of Pharmaceutics, School of Pharmacy,


Stephen L. Hart Molecular Immunology Unit, Institute of Child Health,

London, U.K.

Mark E. Hayes Hermes Biosciences, Inc., South San Francisco,

California, U.S.A.

Leaf Huang University of Pittsburgh School of Pharmacy, Pittsburgh,

Pennsylvania, U.S.A.

Ulrich S. Huth Department of Pharmaceutical Technology and

Biopharmacy, Albert-Ludwigs University, Freiburg im Breisgau, Germany

Scott A. Irvine Molecular Immunology Unit, Institute of Child Health,

London, U.K.

Peter Jantscheff Department of Clinical Research, Tumor Biology

Center, Freiburg, Germany

Dmitri B. Kirpotin Hermes Biosciences, Inc., South San Francisco,

California, U.S.A.

Abdo Konur Klinikum Geb. 302T/TVZ Johannes Gutenberg-Universitat

Mainz, Mainz, Germany

Nickolay Koroukhov Department of Pharmaceutics, School of Pharmacy,


Peter Laing Lipoxen PLC, London, U.K.

Malika Larabi Department of Radiology/Nuclear Medicine, Lucas MRS

Imaging Center, Stanford, California, U.S.A.

Contributors xix

Peter Laverman Department of Nuclear Medicine, Radboud University

Nijmegen Medical Centre, Nijmegen, The Netherlands

Philippe Legrand Universite Paris-Sud, Chatenay-Malabry, France

Ulrich Massing Department of Clinical Research, Tumor Biology Center,

Freiburg, Germany

Norbert Maurer Department of Biochemistry and Molecular Biology,

University of British Columbia, Vancouver, British Columbia, Canada

Brenda McCormack Lipoxen PLC, London, U.K.

Jean R. McEwan Centre for Cardiovascular Genetics, University College

London, Rayne Institute, London, U.K.

Nathalie Mignet Unite Pharmacol. Chim. Genet., Universite Rene


Charles O. Noble IV Hermes Biosciences, Inc., South San Francisco,

California, U.S.A.

John W. Park University of California at San Francisco Comprehensive

Cancer Center, San Francisco, California, U.S.A.

Regine Peschka-Suss Department of Pharmaceutical Technology and

Biopharmacy, Albert-Ludwigs University, Freiburg im Breisgau, Germany

William T. Phillips Department of Radiology, University of Texas Health

Science Center at San Antonio, San Antonio, Texas, U.S.A.

Fatouma Said Hassane Laboratoire de Chimie Bioorganique, Faculte de

Pharmacie, Universite Louis Pasteur, Strasbourg-Illkirch, France

Daniel Scherman Unite Pharmacol. Chim. Genet., Universite Rene


Herbert Schott Institute of Organic Chemistry, Eberhard-Karls

University, Tuebingen, Germany

Francis Schuber Laboratoire de Chimie Bioorganique, Faculte de

Pharmacie, Universite Louis Pasteur, Strasbourg-Illkirch, and

Chimie Enzymatique, Illkirch, France

xx Contributors

Rolf Schubert Department of Pharmaceutical Technology andBiopharmacy, Albert-Ludwigs University, Freiburg im Breisgau, Germany

Reto Schumacher Department of Surgery, Institute for Surgical Researchand Hospital Management, University of Basel, Basel, Switzerland

Reto Schwendener Institute of Molecular Cancer Research, University ofZurich, Zurich, Switzerland

Lisa M. Shollenberger University of Pittsburgh School of Medicine,Pittsburgh, Pennsylvania, U.S.A.

Giulio C. Spagnoli Department of Surgery, Institute for Surgical Researchand Hospital Management, University of Basel, Basel, Switzerland

Gert Storm Department of Pharmaceutics, Utrecht Institute forPharmaceutical Sciences, Utrecht University, Utrecht, The Netherlands

Vladimir P. Torchilin Department of Pharmaceutical Sciences, School ofPharmacy, Bouve College of Health Sciences, Northeastern University,Boston, Massachusetts, U.S.A.

Volkmar Weissig Department of Pharmaceutical Sciences, School ofPharmacy, Bouve College of Health Sciences, Northeastern University,Boston, Massachusetts, U.S.A.

Igor Zhigaltsev Department of Biochemistry and Molecular Biology,University of British Columbia, Vancouver, British Columbia, Canada

Contributors xxi

1

Amphipathic Weak Base Loadinginto Preformed Liposomes Having

a Transmembrane AmmoniumIon Gradient: From the Bench

to Approved Doxil

Yechezkel Barenholz

Laboratory of Membrane and Liposome Research,The Hebrew University–Hadassah Medical School,

Jerusalem, Israel

INTRODUCTION

The main objective of using liposomes as drug carriers is to achieve selective,and sufficiently high, localization of ‘‘active’’ drug at disease sites such astumors and inflamed tissues. In addition, in order to achieve therapeutic effi-cacy, the liposomal encapsulated/associated drug should become availableto the target cells. In this respect, the liposome differs from other controlledrelease systems, in which drug release occurs either in plasma or at the site ofadministration. Selective localization can be obtained using either passive oractive targeting. Passive targeting is a process by which the physical proper-ties of the liposomes combined with the microanatomy of the vasculatureat the target tissue determine drug selective localization. Active targetingrequires, in addition to the ability to reach the disease site by passive target-ing, a homing device (antibody, receptor ligand, etc.) as part of the liposomesurface so that the liposomes can recognize the ‘‘sick’’ cells, bind to them

1

selectively, and either be internalized by these cells or be broken down byeither enzymatic hydrolysis or processes such as ultrasonic irradiation torelease the drug near the cell surface so it will be taken up by the target cells (1).

Doxil1, of which I had the pleasure of being one of the inventors(1–12), serves as an example of successful passive targeting to tumors inanimals and humans (1,3,4,13). The ammonium sulfate (AS) transmembraneinside high/outside low gradient-driven loading is also the basis for doxoru-bicin (DOX) loading for Targeted Doxil (14), which soon will be tested inclinical trials.

Both passive and active targeting share four common requirementsthat have to be met in order for the liposomes to become therapeutically effi-cacious (1,3,7). These are described in Table 1. Here we focus mainly on thesecond item of Table 1: how to achieve sufficient stable drug loading inthe liposomes. Achievement of this goal involves a paradox because reduc-ing the liposome to the necessary size results in reduction of its volume verygreatly (halving the radius results in 1⁄23 or 1⁄8 the volume). This paradox canbe overcome by remote loading driven by gradients such as that of AS, asused in the case of Doxil (1,2,6,7,10–12), or protons (16,17), as in the case ofMyocet (29,30), where DOX citrate is accumulated in the aqueous phaseof conventional liposomes. However, for drugs whose loading cannot be dri-ven by such gradients and can only be passively loaded, this goal is moredifficult to reach, especially when high drug doses are required. A good exampleof improving the passive loading process is sterically stabilized liposomes (SSL)loaded with cisplatin to form Stealth cisplatin (1,9,31). However, in the case ofStealth cisplatin, improved drug loading, is based on performing the lipid hydra-tion and extrusion at 65�C when the solubility is fourfold higher than at roomtemperature. Once 100-nm liposomes are formed, their nanovolume, due toenergetic considerations, prevents crystallization of the drug in the intralipo-some aqueous phase (31). This procedure is less favorable than the remoteloading, as drug-to-lipid ratio is still low, and a much higher (than in Doxil) doseof liposomes will be needed to attain a therapeutic dose. Also, such a mechanismmay prevent drug release and therefore will result in nonactive liposomes(1,31,32). Passive loading and means to improve it are outside the scope of thispaper, which will focus on the use of transmembrane intraliposome high/extra-liposome low ammonium ion gradients to load liposomes with amphipathicweak bases, and especially on DOX remote loading into SSL to form Doxil.

MECHANISM OF REMOTE LOADING BY AS GRADIENT

Background

Doxil, the first liposomal drug that was approved by the Food and DrugAdministration, in 1995, is a good example of the successful applicationof a transmembrane inner liposome high/outer liposome low ammoniumion gradient for remote loading of an amphipathic weak base, the anticancer

2 Barenholz

anthracycline, DOX. In the case of Doxil, sulfate is used as the anion of theammonium ion. The huge difference in the permeability coefficients (Pd)between the neutral ammonia (Pd¼ 0.12 cm/s) and the sulfate anion(Pd< 10�12 cm/s) combined with the efficient precipitation (gelation) ofDOX sulfate in the intraliposome aqueous phase and the low octanol/intra-liposome aqueous phase partition coefficient both play a major role in thesuccess of Doxil. The type (low molecular weight inorganic or organic, orpolymeric) and valency of anion that forms the ammonium salt can be used

Table 1 Requirements to Achieve Therapeutically Efficacious Passive Targeting ofLiposomes Loaded with Drugs and Their Solution

Main requirements to achievetherapeutically efficaciouspassive targeting of liposomes

Physicochemical and biophysical solutionsused to meet the requirements

Extended circulation time in intactform in the plasma

Development of sterically stabilizedliposomes (SSL) composed of high Tmlipids, cholesterol, and a lipopolymer, suchas 2000poly-(ethylene glycol methyl ether)-1,2-distearoyl-sn-glycero-3-phospho-ethanolamine triethyl ammoniumsalt (1,3–5,8,9,14,15)

Sufficient stable loading of drug inorder to reach disease site withliposomes loaded with drug at alevel needed to achievetherapeutic efficacy

Use of pH (16,17) or ammonium iongradients for remote (active) loading ofamphipathic weak bases (1–3,6–8,10–12,18–21) or acids (22,23)

Extravasation into diseased tissue(tumor or inflamed sites)

Having the liposomes small enough(<120 nm) to extravasate through the gapsin the tumor vascularate (24)

Getting active drug into target cells Releasing drug from liposomes throughselective drug leakage at site due to diseasedtissue properties, or using: collapsible iongradient (1,25), and/or liposomes sensitiveto secretory phospholipases (26,27) or byapplying physical means such as heat[thermosensitive SSL (45,46)], orultrasound (28) (Schroeder A, Avnir Y,Weisman S, et al. Nanoscale drug deliveryfrom liposomes using low-frequencyultrasound: mechanism and feasibility.Submitted for publication.) or byinternalization after activetargeting (14)

Amphipathic Weak Base Loading 3

to control the release rate of the liposome remote-loaded amphipathic weakbase (1,2,6,7,10–12,18,33,34).

The Role of the Ammonium Ion

Our studies in which gradients of various ammonium salts were used forloading amphipathic weak bases into the intraliposome aqueous phase dem-onstrate that the actual driving force for the loading itself is the ammoniumion gradient (33,35) and [Wasserman V, Kizelsztein P, Garbuzenko O, et al.The antioxidant tempamine (TMN): in vitro proapoptotic and neuroprotectiveeffects and optimization of liposomal encapsulation. Submitted for publica-tion. Here after cited as ‘‘Wasserman et al.’’]. The loading is related to theunique property of the ammonium ion to dissociate to a proton and neutralammonia (NH3) gas, thus having a very high permeability coefficient.

NHþ4Ðhigh pH

low pHNH3 þHþ ð1Þ

This dissociation is pH dependent. The higher the pH, the higher is the dis-sociation to form the neutral NH3 gas, whereas at lower there is moreammonium ion. pH there is more ammonium ion. The loading of amphi-pathic weak bases into the liposome requires the presence of NH3 in theintraliposome aqueous phase as, due to its very high permeability coeffi-cient, it diffuses across the membrane to the extraliposome aqueous phase(Fig. 1), leaving behind excess of protons and therefore creating (in additionto the ammonium gradient) a proton gradient:

½Hþ�liposome � ½Hþ�medium ð2Þ

Namely, the intraliposome aqueous phase becomes acidic.This acidification stops further dissociation of the NH4

þ and there-fore the leakage of NH3, thereby stabilizing the system. The amphipathicweak base when added to this liposome dispersion will diffuse throughthe liposome membrane and reach the intraliposome aqueous phase in itsuncharged (unprotonated) form. There it is protonated and uses the excessprotons, thereby elevating the pH, renewing the dissociation of NH4

þ toNH3 and Hþ, thus enabling the continuation of the loading cycle. This pro-cess can continue until all the ammonium ion is exchanged with the loadedamphipathic weak base.

Figure 1 (Facing page) (A) Mechanism of remote loading of doxorubicin by trans-membrane ammonium sulfate gradient. (B) Collapse of transmembrane ammoniumion gradient in SSL by nonactine induces doxorubicin release. (C) Collapse of trans-membrane proton gradient in SSL by nigericin induces collapse of transmembraneammonium ion gradient followed by release of DOX. Abbreviations: DOX, doxoru-bicin; SSL, sterically stabilized liposome.

4 Barenholz

Figure 1 (Caption on facing page)


The best way to prove the cardinal and obligatory role of the ammo-nium ion in the loading of amphipathic weak bases is to use the ionophorenonactine [Wasserman V, Kizelsztein P, Garbuzenko O, et al. The antioxi-dant tempamine (TMN): in vitro proapoptotic and neuroprotective effectsand optimization of liposomal encapsulation. Submitted for publication.],which exchanges ammonium ions with potassium ions ðNHþ4 !KþÞ. Non-actine is without effect on a proton gradient that is not derived from anammonium ion gradient and will not affect drug loading due to the latterproton gradient. In the presence of nonactine and potassium ions, there willbe exchange of NH4

þ by Kþ in the intraliposome aqueous phase and there-fore the ammonium ion gradient will collapse and the loading of amphipathicweak bases will be prevented irrespective of the anion that forms theammonium salt being either inorganic anions (i.e., chloride, sulfate, phos-phate) or organic, low molecular weight (such as citrate or glucuronate) (34) and(Wasserman et al.) or polymeric anions [such as dextran sulfate (34), heparinsulfate sucralfate]. Nonactine will induce release of amphipathic weak basesfrom the liposomes, but only if the loading was driven by a transmembraneammonium ion gradient (Fig. 1B). If the remote loading is driven by a pH gra-dient that is not ammonium-ion dependent, nonactine will not cause releaseof the amphipathic weak base (Table 2). Nonactine therefore acts differentlyfrom the ionophore nigericin, which exchanges between Hþ and Kþ. Nigericinprevents amphipathic weak base loading into liposomes for both transmem-brane proton and ammonium ion gradients (Fig. 1C and Table 2).

The Role of the Ammonium Salt Anion

The role of the ammonium salt anion is not the loading of the amphipathicweak base per se, but rather to control the stability of loading and the profileand rate of release of the amphipathic weak base from the liposome to theexternal aqueous phase. Two major factors that differentiate the differentanions are, firstly, their ability to induce precipitation/crystallization/gelation in the intraliposome aqueous phase (1,12), and secondly, their effecton the membrane/buffer and octanol/buffer partition coefficient of the am-phipathic weak base (1). Regarding the precipitation, the higher the amountof precipitated amphipathic weak base, the more stable is the loading andthe slower is its release rate (10–12,18,33,35) and (Wasserman et al.). Thereare also some risks involved in the precipitation which in some cases reducethe mechanical stability of the liposomes and change liposome shape (36).

Among low molecular weight anions (inorganic and organic) used toachieve ammonium ion-driven loading, the order of loading stability formost amphipathic weak bases studied is sulfate> citrate> phosphate>chloride> glucuronate (33,35) and (Wasserman et al.). Regarding polymericanions such as dextran sulfate (for dextran sulfate ammonium salt¼DSAS);results varied, in some cases, such as ciprofloxacin (34) and acridine orange

6 Barenholz

Tab

le2

Ch

ara

cter

izati

on

of�

10

0n

mS

SL

Rem

ote

Lo

ad

edw

ith

DO

Xv

iaT

ran

smem

bra

ne

Am

mo

niu

mS

ulf

ate

Gra

die

nta

Pro

per

tyM

ag

nit

ud

e

Tra

nsm

emb

ran

ep

roto

ng

rad

ien

t(D

pH

)

Tra

nsm

emb

ran

ea

mm

on

ium

ion

gra

die

nt

det

erm

ined

by

am

mo

niu

mel

ectr

od

eN

H4

ðÞ 2

SO

4

�� li

po

som

e=

NH

4ð

Þ 2S

O4

�� m

ediu

m�

10

00

Intr

ali

po

som

ea

qu

eou

sp

Hd

eter

min

edb

efo

reD

OX

loa

din

gu

sin

gp

yra

nin

ep

relo

ad

edin

lip

oso

mes

<5

.25

,b

ein

go

ut

of

the

ran

ge

of

the

mea

sure

men

to

fp

Hra

ng

efo

rp

Hd

eter

min

ati

on

by

py

ran

ine

(pH

5.2

–8

.0)

Det

erm

ina

tio

no

ftr

an

smem

bra

ne

pH

gra

die

nt

(in

ner

low

/o

ute

rh

igh

)as

Dp

HB

efo

reD

OX

loa

din

gB

yA

Od

istr

ibu

tio

n9

6.4

%b

yA

Od

istr

ibu

tio

nin

toli

po

som

e�

3.0

pH

un

its

By

14C

MA

dis

trib

uti

on

87

.5%

by

14C

MA

dis

trib

uti

on

into

lip

oso

mes

�3

.0p

Hu

nit

sþ

Nig

eric

in2

.0%

by

AO

dis

trib

uti

on

into

lip

oso

mes

��

03

.0%

by

14C

MA

dis

trib

uti

on

into

lip

oso

mes¼

3.0

%��

0þ

No

na

ctin

e4

.0%

by

AO

dis

trib

uti

on

into

lip

oso

mes

��

03

.0%

by

14C

MA

dis

trib

uti

on

into

lip

oso

mes

�0

DO

Xlo

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ing

%D

OX

loa

din

g�

90

.0%

Dp

Ha

fter

DO

Xlo

ad

ing

30

–3

5%

by

14C

MA

dis

trib

uti

on

into

lip

oso

mes

�1

.0p

Hu

nit

s

Dp

Ha

fter

DO

Xlo

ad

ing

by

14C

MA

dis

trib

uti

on

:þ

No

na

ctin

e2

%b

y1

4C

MA

dis

trib

uti

on

�0

þN

iger

icin

2%

by

14C

MA

dis

trib

uti

on

�0

aS

SL

sta

bil

ity

:si

zed

istr

ibu

tio

n,

lev

elo

ffr

eed

rug

,a

nd

Dp

Hre

ma

inu

na

lter

edfo

rm

ore

tha

nsi

xm

on

ths

sto

rag

ea

t4� C

;D

pH

for

bo

th%

14C

MA

an

d%

AO

are

base

do

nca

lib

rati

on

curv

es.

Ab

bre

via

tio

ns:

DO

X;

do

xo

rub

icin

;A

O,

acr

idin

eo

ran

ge;

MA

,m

eth

yla

min

e;S

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,st

eric

all

yst

ab

iliz

edli

po

som

e.


(AO), dextran sulfate anion is superior to sulfate anion in gradient stabiliza-tion. Another advantage of some polymeric anions such as dextran sulfate isthe high concentration of NH4 they carry per mOsmol (i.e., 100 mg/mL ofdextran sulfate average molecular weight of 8000, carries 0.6 mmol/mLof NH4

þ ions). The mode of precipitation/crystallization and especially theshape of the crystals (10–12,36) may affect shape of the liposomes and, there-fore, their performance. The implications of the effect of the anions that formthe ammonium salts on the pharmacokinetic and therapeutic performance ofliposomes (mainly sterically stabilized liposomes) as drug carriers are describedelsewhere (1,33,34).

THE DOXIL EXAMPLE FOR REMOTE LOADING OFAMPHIPATHIC WEAK BASE INTO LIPOSOMES

For Doxil-like DOX-loaded �100 SSL, the DOX remote loading via higherinside/lower outside transmembrane AS gradient showed distinct dif-ferences from loading via ammonium glucuronate (AG) gradient. In thiscomparison, that the only variable that differs between the two formulationsis the anion sulfate versus glucuronate. The liposomes themselves, externalmedium, and the drug (DOX) are identical in the two formulations. Table 3summarizes the comparison.

Therapeutic efficacy was compared in four different animal models.In all of them, both liposomal formulations show similar therapeutic effi-cacy and were much superior to free DOX, which resembles the controlof untreated mice.

To sum up, the comparison between AS–SSL and AG–SSL shows thatin both liposomes the DOX behaves like it is delivered via SSL and both dif-fer to a large extent from the free DOX. However, AG–SSL have somewhatfaster release rate and shorter t1/2

of the entrapped DOX. These differenceshave no significant effect on the antitumor therapeutic efficacy in animalmodels. Therefore, it is possible that such differences in release profile willenable reducing the incidence and severity of the skin toxicity syndromepalmar–plantar erythrodysesthesia (PPE), one of the major side effects ofDoxil (4). Studies with another amphipathic weak base, the antioxidanttempamine (TMN), reveal a similar behavior with respect to the effect ofthe anion of the ammonium salt on drug release (35) and Wasserman et al.For both drugs, DOX and TMN, the differences in rates of drug release canbe explained by the fact that most drug-sulfate salts are present in theliposomes as a precipitate, whereas drug-glucuronate salts are not. The dif-ferences between the permeability coefficient of sulfate and glucuronate aretoo small to explain differences in the release rate (Table 3).

Figure 1A describes the overall mechanism of loading DOX intoSSL under conditions that NH4ð Þ2SO4

� �liposome

� NH4ð Þ2SO4

� �medium

8 Barenholz

(1,2,7,10–12,18). The efficiency of DOX loading by this method and loadingstability are dependent on:

1. The large (�1012) difference in permeability coefficient of the neu-tral ammonia gas (10�1 cm/s) and the SO4

2� anion (>10�12 cm/s)(1,18);

2. The initial pH gradient, as result of the ammonium gradient, hav-ing the [Hþ]liposome >> [Hþ]medium transmembrane pH� 3.0 pHunits. There is a threshold of medium pH of �3.5; below thispH no quantitative level of DOX loading occurs, even above the

Table 3 Comparison Between 100-nm SSL Remote-Loaded with DOX via HigherInside/Lower Outside Transmembrane Gradients of Either 250 mM AmmoniumSulfate or of Ammonium Glucuronate

PropertyAS gradient

% releaseAG gradient

% release Free DOX

Plasma release at 37�C4 hr < 3.0� 2.0 < 3.0� 2.0 NR24 hr 6.5 20 NR96 hr 38 78 NR

Biological activity mM DOX mM DOX mM DOXIC50 M109–sensitive 9.8 1.4 0.56IC50 M109–resistant > 300 28.0 2.00IC50 C26 > 200 64.0 0.96

t1/2(hr) t1/2

(hr) t1/2(hr)

Pharmacokinetics intumor-bearing mice t1/2

24 16 <0.5

Blood concentrationfor equal dose at:

mg/mL plasma mg/mL plasma mg/mL plasma

4 hr 248 180 �24 hr 160 110 BD48 hr 85 50 BD

250 mM AS 250 mM AG �DOX visual precipitation

occurs at<2 mM DOX >150 mM DOX

Permeability coefficientPd (cm/s)

Sulfate anion Glucuronate anion NH3

<10�12 �10�12 1.2� 10�1

Note: NR—not relevant; BD—below detection.

Abbreviations: DOX, doxorubicin, SSL, sterically stabilized liposome; AS, ammonium sulfate;

AG, ammonium glucuronate.

Source: Ref. 33.


liposome forming lipid Tm and after a reasonable loadingtime (a few hours) (unpublished data);

3. The rate of loading is dependent on the cross talk between lipo-some lipid composition and temperature of loading. Efficientloading occurs only above the liposome-forming lipid Tm. Forliposomes made of liposome-forming lipids having Tm> 37�C,and there is a special benefit regarding stability if loading is per-formed above, while storage is below Tm, and therefore loadingstability is improved (1,10,35) and Wasserman et al.

4. The low solubility of (DOX)2–SO4 (>2mM), which also minimizesintraliposomal osmotic pressure and therefore helps keep liposomeintegrity (1);

5. The asymmetry in DOX partition coefficient (Kp):(Kp lip/external med>Kp lip/intralip med); (Kp oct/externalmed>Kp oct/intralip med) (1,7,37,38).

Kp is a partition coefficient between two phases, a usually less polarphase (either the liposome membrane or solvents such as octanol, oil, etc.)and a polar aqueous buffer or water as defined in the last entry of theabove-mentioned list (1,7,37,39–43).

The octanol/buffer Kp represents a partition coefficient between twobulk phases; it is less affected by the structure of the analyte and thereforeit cannot be used to predict the exact value of liposome membrane-to-bufferKp, which is also affected by the geometry of the analyte (41–44). However,it is accepted and established that the octanol-to-buffer Kp can help to pre-dict transmembrane passive diffusion (40). In the case of liposomes suchas Doxil, in which the internal aqueous phase (intraliposome aqueous phase)is different from the external liposome aqueous medium due to large differ-ences in the composition and pH of these two aqueous phases, there aretwo different liposome membrane-to-aqueous phase partition coefficients;this is referred to as asymmetry in the membrane-to-aqueous media partitioncoefficient.

This asymmetry means that the Kp of DOX in the extraliposomal med-ium supports influx in a direction opposite to the AS gradient (namely, intothe liposomes), while the Kp of DOX in the intraliposomal aqueous phaseacts to reduce partition into the membrane, thereby reducing the desorptionrate (koff) (1,7,35,37) and Wasserman et al. The reduction in DOX Kp

in the intraliposomal aqueous phase is driven by the still high concentration(>100 mM) of the ammonium ions remaining inside the intraliposomal aqu-eous phase after DOX remote loading. Therefore, it seems that AS plays amultifactorial role in the remote loading and retention of the loaded DOX inthe liposomes. For Doxil, the interplay between the above five aspects, whencombined with Doxil membrane composition and liposome size, determinesthe liposome performance.

10 Barenholz

Another issue, so far neglected, but which is especially relevant todrugs such as DOX, is their tendency to self-aggregate (1,10–12,18,38), form-ing oligomers of various mer number. This phenomenon results from thestacking of the planar aromatic and hydrophobic rings of the anthracyclinesdue to interaction between the p-electrons of the rings, reducing exposure ofhydrophobic surface area to water. This self-aggregation is facilitated byincreasing ionic strength. DOX dimers appear already at 1 mM and largeraggregates appear at higher DOX concentrations (38). The effect of suchassociation on therapeutic efficacy is not yet clear. However, based on sim-ple geometric considerations, it is obvious that nonmonomeric DOX cannotinteract with DNA in the same way as monomeric DOX, and the exact loca-tion between the two DNA strands should differ (38). Therefore, the way bywhich the drug is internalized (monomers vs. aggregated form) by the tumorcell may be an important factor in drug efficacy.

This important aspect was never seriously studied. Tumor treatmentbased on nontargeted Doxil does not face such a problem, as in most casesthe drug reaches the cells when released from the liposomes, after the lipo-somes get into the tumor interstitial fluid. That is, DOX in the interstitialfluid and in the cells is mainly in the form of monomers of DOX chlorideand, to a lesser degree, dimers. However, when the intact targeted Doxil[such as folate–Doxil (44)] is internalized via a receptor-mediated process,the drug reaches the acidic compartment of cells as DOX sulfate salt (44),and the apparent drug concentration in the intraliposomal aqueous phase(>200 mM) is much above the drug solubility product (1,11,12,38). A visibleprecipitation of DOX sulfate occurs already at a concentration of < 2 mM(Table 3), which is more than 100-fold lower than the intraliposomal DOX sul-fate concentration. Smaller aggregates (not visible to the naked eye) occur evenat lower concentrations. That is, a major (>100-fold) dilution is required beforeall of the drug will become monomeric. The internalization via a receptor-mediated endocytosis keeps the liposomes under an acidic condition that isnot supportive of DOX dissolution and/or fast release of the liposomes (asexplained by Fig. 1).

‘‘REMOTE’’ RELEASE

It seems that there is ‘‘no free lunch’’ and the ‘‘cost’’ of stable loading maybe a too-slow or no drug release at the target site. Using liposomes that are‘‘leaky’’ may result in release of a major fraction of the drug while the lipo-somes are still in circulation, thereby reaching the extravascular disease sitewith drug-poor liposomes. To overcome this limitation, one has to design aliposomal system that is stable upon storage and while circulating in vivoin the plasma, but loses at least part of this stability once the liposomesreach the disease target site. This is the case for Doxil, where the conditionsin the tumor interstitial fluid differ to a large extent from the conditions in


the plasma. Factors leading to Doxil release may include collapse or partialcollapse of the ammonium ion gradient and/or the activity of phos-pholipases that hydrolyze the liposome phospholipids (1,26,27), therebydestabilizing the liposome membrane.

An excellent demonstration for the role of rate of drug release ofthe liposome is the therapeutic efficacy comparison between the fate andperformance of Doxil SSL remote loaded with DOX and Stealth cisplatin(SSL) passively loaded with cisplatin (9). Both formulations are identicalin their size and lipid composition. Although Doxil demonstrates release ofDOX, Stealth cisplatin neither releases cisplatin in vitro nor in vivo (31,32).This suggests that the main factor in inducing drug release between the twoformulations is related to differences in drug transmembrane permeabilitycoefficients and/or the effect of the collapse of the ammonium ion gradient,which exists only in the case of remote-loaded Doxil. This comparison alsosuggests that, at least for these two formulations, phospholipases, includingsecretory phospholipases, are not playing a major role in drug release, whichmay be explained by the high mole percentage of cholesterol in these SSLmembranes. This explains why DOX is released from Doxil in vivo intumor-bearing mice and in humans (1,4,5), as proven directly from the findingof DOX metabolites in the tumor tissue (4), whereas Stealth cisplatin, whichlacks an ion or proton gradient, reaches the tumor at the same efficiency asDoxil but does not release cisplatin in the tumor site and therefore lacks effi-cacy (32). It remains to be studied if the performance of Doxil can be furtherimproved by changing the rate of release of DOX.

One approach tested is to destabilize the liposome membrane byremoval of one of its components through hyperthermia. This effect occurswhen the liposome lipid bilayer undergoes solid ordered (SO) to liquid dis-ordered (LD) phase transition and, therefore, requires lack of cholesterol inthe lipid bilayer (1,27,37,45). The idea is to use SSL, which will accumulatein the disease site. Once accumulation is achieved, mild hyperthermic expo-sure of the animal to temperatures in the range of 39�C to 40�C will induceSO to LD phase transition followed by release of one membrane compo-nent, leading to very fast (tens of seconds) release of DOX, and improvingtherapeutic efficacy (45,46). Thus, in order to benefit from this approach, theoptimal release rate of the drug has to be known. However, because the SSLused in this case lacks cholesterol, they may not retain drug during SSL pro-longed circulation. Also, our recent studies demonstrate that SSL lackingcholesterol have a lower capability to retain a pH gradient introducedthrough use of buffers such as citrate buffer upon storage at 4�C and uponincubation at 37�C (Garbuzenko et al. unpublished). The pH gradient sta-bility can be much improved if it is based on an AS gradient (Garbunzenkoet al. unpublished). Another approach to achieve remote release is the con-trolled remote collapse of the AS gradient by an ionophore or ionophores(1,10,25), as demonstrated in Figs. 1B and C.

12 Barenholz

A very different approach to induce remote destabilization of SSLand sterically stabilized targeted liposomes is the use of a detachable stericstabilizer, first demonstrated by Zalipsky and coworkers (47). Accordingly,removal of polyethylene glycol (PEG) headgroups from the liposomeswill increase their permeability or induce liposome collapse, which is thecase for dialeoyl phosphatidylethanolamine (DOPE)- enriched SSL (47).In the first detachable lipopolymer, the PEG attachment was based on a dis-ulfide bond and required strong thiolysis [by dithiothreitol (DTT)] to releasethe PEG moiety, leaving behind in the lipid bilayer a non-natural lipid.Recently Zalipsky et al. (48) improved the strategy for the reversible attach-ment of methoxyPEG by using an amino-containing anchor. The attachmentis based on a dithiobenzylurethane linkage. The PEG moiety is detached bymild thiolysis with cysteine at physiologically relevant concentration. Thefinal product of this thiolysis is phosphatidylethanolamine.

EXPERIMENTAL DEMONSTRATION OF DOX REMOTELOADING TO FORM DOXIL

Principles of Preparation of Liposomes Having TransmembraneAmmonium Ion Gradient

Many types of liposomes of different lipid composition and different sizeshaving a transmembrane AS gradient were prepared (10). These liposomesvaried: (i) in their liposome-forming phosphatidylcholine (PC), being withand without cholesterol and/or lipopolymer; (ii) in their size; and (iii) in theirmethod of preparation. The approaches for preparing these different liposomeformulations varies in their lipid hydration and downsizing. Table 1 in Haranet al. (10) gives a partial list of such liposome preparations. In all cases thescheme of liposome preparation can be summarized as described in Table 4.

Table 4 Steps in Preparation of Liposomes Having a Transmembrane AmmoniumSulfate Gradient

Step number Step description

1 Lipid hydration in ammonium sulfate solution to formmultilamellar vesicles

2 Liposome downsizing to desired size (this step is omitted if nodefined size is needed)

3 Formation of ammonium sulfate gradient by its removal from theextraliposome medium

4 Liposome loading with doxorubicin5 Removal of nonentrapped doxorubicin (in the case of Doxil this

step was omitted as loading is approaching 100%)


Preparation of �100 nm SSL Loaded with DOX ViaTransmembrane AS Gradient

Materials

Hydrogenated soybean PC (HSPC) was obtained from Lipoid (Ludwigshafen,Germany). N-carbamoyl-poly-(ethylene glycol methyl ether)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine triethyl ammonium salt (PEG-DSPE) (thePEG moiety of this phospholipid having a molecular mass of 2000 Da) wasa gift from Liposome Technology Inc. (Menlo Park, California, now ALZACorporation, Mountain View, California, U.S.A.) or obtained from Genzyme,(Liestal, Switzerland). Cholesterol was obtained from Sigma (St. Louis,Missouri, U.S.A.).

Drugs: DOX-HCL was obtained from Farmitalia Carlo Erba (Milan,Italy).

The purity of all lipids and anthracyclines exceeded 98% based onthin-layer chromatography (TLC) and/or high-performance liquid chro-matography (HPLC) analysis, performed as described by Barenholz andcoworkers (38,49,50).

pH Indicators: The quencher p-xylene-bis-pyridinium bromide (DPX) andthe pH-sensitive fluorophore pyranine (8-hydroxy-pyrene-1,3,6-trisulfonate)were purchased from Molecular Probes (Junction City, Oregon, U.S.A.). AO(hemizinc chloride salt) was purchased from Aldrich (Milwaukee, Wisconsin,U.S.A.).

Other Reagents: The ionophores nigericin and nonactine, Hepes,Sephadex G-50, Sepharose 6B (Pharmacia) and Dowex 50 WX-4 (Dow)400 mesh were obtained from Sigma. AS, 99.999% pure, was obtained fromAldrich. The Dowex 400 mesh was activated and purified as described byAmselem et al. (51).

tert-Butanol was obtained from BDH Laboratory Supplies (Poole, U.K.).

Preparation of 100-nm SSL Having TransmembraneAS Gradient

We used HSPC/cholesterol/2000PEG-DSPE 95.8:31.9:31.9 (weight ratio).All lipids were codissolved in tert-butanol, lyophilized overnight, andhydrated at 70�C in 250 mM (NH4)2SO4 to form multilamellar vesicles(MLV). Downsizing was performed by stepwise extrusion in two steps,firstly through two stacked 0.4-mm and then through 0.08-mm pore-diameterpolycarbonate filters obtained from Poretics (Livermore, California,U.S.A.). Each extrusion step was performed 8 to 11 times at 70�C usingthe high-pressure extrusion device supplied by Northern Lipids Inc. (pre-viously Lipex, Vancouver, British Colombia, Canada). The SSL wereanalyzed for vesicle size distributions by dynamic light scattering (49).

The transmembrane AS higher inside/lower outside gradient of max-imal magnitude used for DOX loading into liposomes was created using

14 Barenholz

either: (i) gel exclusion chromatography on Sephadex G-50 pre-equilibratedwith the desired salt or nonelectrolyte solution (dextrose or sucrose); or(ii) consecutive dialysis against at least 100-fold the liposome dispersionvolume (10). The external concentration of ammonium ions was determinedusing an ammonium electrode and, when the external medium contained anonelectrolyte, also by conductivity measurements (10). When needed, theconcentration of ammonia in the external compartment was controlled bydilution of the liposome suspension in 250 mM Na2SO4 or K2SO4 (for mea-surements that require the ionophores nonactine or nigericin).

All other steps, which include loading of these liposomes with DOX-HCl, removal of residual free drug, and characterization of the SSL duringthe various steps of preparation as well as of the final product, are describedbelow and summarized in Table 2.

Liposome Loading with DOX

A solution of DOX (0.5–20 mM) was added to the liposome dispersion(1–120 mM phospholipids) after the creation of an AS gradient. The loadingwas performed above the Tm of the liposome PC (>65�C for HSPC).The percent loading was followed with incubation time at the desired tem-perature by mixing an aliquot of the incubation mixture with washed andcleaned Dowex (50 mg/per mg total DOX) to remove the free (unloaded)DOX as described below (4).

Removal of Nonentrapped (Free) DOX

The separation and removal of nonliposome-associated DOX (free DOX)from the liposome-entrapped DOX (liposomal DOX) was achieved throughthe complete binding of free DOX (but not of the liposomal DOX) to thecation exchange resin Dowex (10,49–51), as described first by Storm et al.(52) and modified by Amselem et al. (51). When loading reached a plateau,free DOX was removed by dialysis, or gel exclusion chromatography orchromatography (batch or column) on Dowex cation exchanger asdescribed elsewhere (10). After removing all nonloaded DOX, a suspensionof DOX in SSL referred to as DOX-SSL (¼Doxil) was obtained.

Assays of Characterization and QC of DOX-SSL

Conductivity Measurements

Conductivity was measured as described (10) using a conductivity meter(Radiometer, Copenhagen, Denmark) type CDM3 equipped with a CDC304 immersion electrode with manual temperature compensator type CDA100. The instrument was calibrated as specified by the manufacturer. Thedetermination of the (NH4)2SO4 concentration from the conductivity mea-surements was done at constant temperature (4�C) using a calibration curve,in the range of 0.016 mM to 120 mM AS in glucose or sucrose (total


osmolarity 285 mOsmol). For this calibration curve, conductivity rangevaried from 7 mS to 22 S.

pH and Ammonia Measurements

pH and ammonia measurements were carried out as described elsewhere (10)using a Corning 250 pH/ion analyzer (Corning Science Products, Corning,New York, U.S.A.) equipped with an automatic temperature compensationstainless steel probe. For the determination, of ammonium ion concentra-tion, we used the Corning ammonia combination electrode (Corning476130). A 5-mL sample volume was used in all measurements. The calibra-tion curve was performed using NH4Cl and (NH4)2SO4 as described in themanufacturer’s operating instructions. Calibration curves were obtained atpH 7.0, 8.5, and 13.5. The relationship between the NH3-related electrodepotential in millivolts and the log of (NH4)2SO4 conductivity (mS) is linear;the slope and intercept are pH dependent. This calibration curve enabled usto determine the ammonium ion concentration over a broad pH range.

The extraliposomal ammonium ion concentration ½ðNH4Þþmedium� was

measured as ammonia with the ammonia electrode at pH 13.5. Under theseconditions, all ammonium ions are converted to ammonia and no leakageof intraliposomal ammonium ion occurred during the measurement. For mea-surements of total ammonium ion plus ammonia present in both intraliposomeaqueous phase and external medium ammonia ½ðNH4Þ

þmedium þðNH4Þ

þliposome�

the liposomes were sonicated under acidic conditions (pH 1.5–2.0) using theTransonic 460/H bath sonicator in sealed vials for 45 minutes. Then, in orderto convert ammonium ion to ammonia, NaOH was added to bring the pH to13.0 to 13.5, and the total ammonia concentration was measured by the ammo-nia electrode. The total ammonia concentration determined for the liposomedispersion after the complete replacement of the medium AS by nonelectrolytewas identical to the AS determined by conductivity meter after complete dis-ruption of the liposomes (see ‘‘Conductivity Measurements’’ above).

Doxorubicin Quantification

DOX concentration was determined spectrophotometrically based on themolar extinction coefficient of 125000 OD M�1(38) in a dual-beam spectro-photometer (either Perkin-Elmer Lambda 3B or Kontron Uvikon 860).The DOX quantification was confirmed by HPLC (49–51). Purity of DOXand its degree of degradation during the processes of liposome prepara-tion and liposome storage were determined by a combination of HPLCand TLC, as described by Barenholz leave et al. (38,49,50).

Level of Free Doxorubicin

Two approaches were used: (i) the selective adsorption of free (only) DOX toDowex cation exchanges either in polycarbonate tips of pipetors (range 0.1–1.0 mL) or in small glass columns; (ii) small gel-exclusion chromatography

16 Barenholz

columns containing 2 mL preswollen beaded 12% cellulose, having anexclusion limit of 5000 Da [Excellulose GF-5, 40–100mm, Pierce (Rockford,Illinois,U.S.A.)] (49–51).

Lipid Quantification and Chemical Stability

Phospholipid concentration was determined using our modification ofBartlett’s procedure (49,53). Cholesterol concentration and purity were det-ermined by HPLC or enzymatically by cholesterol oxidase (49,53). Purity ofphospholipids as raw materials, and the extent of their hydrolysis duringvarious steps of liposome preparation and liposome storage, were assessedby TLC and enzymatic determination of the increase in level of nonesterifiedfatty acids (10,38,49–51,53).

DOX/PL Mole Ratio

DOX/phospholipids (PL) mole ratio was determined from the phospholipidphosphorus and liposomal DOX content (after removal of the free drugreleased from liposomes by Dowex cation exchanger). It was used to assessefficiency of loading, to study the level and rate of drug leakage during stor-age, and to investigate effect of temperature on drug release (see ‘‘Level ofFree Doxorubicin’’ and ‘‘Lipid Quantification and Chemical Stability’’ above).

Size Distribution of Liposomes

Liposome size distribution was determined by photon correlation spec-troscopy (10,49), using either: the Malvern 4700 Automeasure laser lightscattering spectrometer system (Malvern Instruments, U.K.); a CoulterN4SD submicron particle analyzer with size distribution processor analysis(Coulter Electronics, Luton, U.K.); or ALV–NIBS/HPPS with ALV 5000/EPP multiple digital correlator (ALV-Laser Vertriebsgesellschaft GmbH,Langen, Germany). Size distribution analysis was performed using theCONTIN algorithm (49). All size distributions of LUV SSL were unimodal,having mean size of �100 nm.

Transmembrane pH and Ammonium Ion Gradients

Three different approaches were used:

Use of the pH-sensitive membrane-impermeable flurophore pyranine

based on the ratiometric method, which determines directly level of dissociation

of pyranine from the ratio between the charged (unprotonated) pyranine andtotal pyranine in the intraliposome aqueous phase: Addition of impermeableDPX, which acts as a quencher to pyranine fluorescence, into the liposomeexternal medium ensures lack of contribution of extraliposome medium pyr-anine fluorescence (18,22). This method is considered ‘‘invasive’’ as thepyranine has to be added in the hydration medium prior to liposome prepara-tion and cannot be used for pH determination of intraliposome aqueous phase


in preformed liposomes. Also it cannot be used in Doxil after DOX loading dueto partial spectral overlapping between pyranine and DOX. This approachgives direct pH measurements of the intraliposome aqueous phase.

Using pyranine (8-hydroxy-1,3,6-pyrene trisulfonate) as intraliposomepH indicator, the liposomes were prepared as above (as in section ‘‘Prepara-tion of �100 nm SSL Loaded with DOX via Transmembrane AS Gradient’’)with the exception that pyranine (0.5 mM) was included in the hydrationsolution. Removal of untrapped pyranine was achieved by gel filtrationon a Sephadex G-50 column, preequilibrated with either NaCl, KCl, sucroseor AS solution (according to need). All these solutions also contained10 mM Hepes buffer at the desired pH (usually pH 7.5).

We found that the ratiometric method is superior because it is notdependent on pyranine concentration and therefore free of error in pipeting(18,22,54). Calibration curves were constructed by preparing liposomes inwhich the hydration of the lipids to form MLV was done using solutions ofhigh concentration at the desired pH in the range of 3.0 to 10.0. Gel-exclusionchromatography on a Sephadex column, as mentioned above, yielded a seriesof liposome preparations with a fixed external pH (pH 7.5), but differentinternal pH values determined by the buffer used for lipid hydration. NeitherKI nor DPX, which quench the fluorescence of aqueous solutions of pyranine,has much effect on the fluorescence intensity of pyranine in the void volumeafter gel-exclusion chromatography, which indicates the complete removal ofthe pyranine from the extraliposomal medium.

In the ratiometric method, the fluorescence intensity of the liposomescontaining pyranine (F) and in the presence of the quencher DPX was deter-mined at 520 nm upon excitation at two wavelengths 460 nm (of the chargedunprotonated pyranine) and 415 nm (of the pH-independent isosbesticwavelength that describe the total pyranine concentration). The ratio ofF460F415 is described as F. The ratiometric measurement is used to determinethe intraliposome aqueous phase pH (18,22). Then nigericin (or nonactine)at final concentration of 5 mM was added to disrupt the pH and/or ammo-nium ion gradient that induce complete gradient collapse and the measure-ment at the above two excitations was repeated, and indeed it demonstrateda shift of the intraliposome aqueous pH to be identical to the extraliposomemedium pH (10).

Recently we found that the presence of ions introduces artifacts in thedetermination of pH by pyranine (54). This effect is related to the relativeposition of the ion (both cations and anions) in the Hoffmeir series (54).Compared with other ions, AS was found to have only a minimal effecton this shift, which agrees well with the location of NH4

þ in the cationHoffmeir series and of sulfate in the anion series (54).

Use of AO as an amphipathic weak base, the fluorescence intensity of

which is considered to be pH dependent: Being an amphipathic weak

18 Barenholz

base, AO distributes between the intraliposome aqueous medium and theextraliposome aqueous medium according to the transmembrane pH gradient.When there is an inner liposome high/outer liposome low Hþ gradient, AOwill accumulate inside the liposomes. The distribution is proportional to thepH gradient (10,55). In addition, AO liposome to medium distribution isaffected by all factors described above for DOX (10,12). However, AOhas a very high octanol/buffer partition coefficient (>100-fold higher thanof DOX) and high pKa of 10.45 compared with �8.25 of DOX. Therefore,when loaded into liposomes via transmembrane AS gradient above lipo-some-forming lipid Tm, it permeates through the lipid bilayer at a fast rateand the determination of its distribution can be done ‘‘on line’’ using a spec-trofluorometer in less than five minutes. However, no distribution into theliposome aqueous phase occurs when medium pH is below 5.3. At thispH (�5 pH units lower than the AO pKa) the level of uncharged AO istoo low. This method has the advantage that it can be used to characterizepreformed liposomes and therefore it is considered noninvasive. It deter-mines the transmembrane liposome inner liposome high/outer liposomelow proton gradient. However, it cannot be used for Doxil characterizationdue to spectral overlapping between AO and DOX. This method alsosuffers from being unable to differentiate between the contribution of trans-membrane pH gradients and effects related to AO precipitation (55).Comparison of the AO distribution method with the third method, whichmakes use of 14C methylamine liposome/medium distribution (described in‘‘Use of radioactive methylamine between intraliposome aqueous phase andthe external liposome aqueous medium’’ below), suggests that under certainconditions the AO gives reliable results on the transmembrane gradients(Wasserman et al.). In both fluorescent approaches (i and ii) a large dilutionis required due to limitation of the fluorescent determination. Such dilution isnot required for the pH gradient determination by 14C methylamineliposome/medium distribution described below in section ‘‘Use of radio-active methylamine between intraliposome aqueous phase and the externalliposome aqueous medium.’’

Using AO: 1 mM final concentration of AO was added to the desiredsolution (3 mL) containing various ratios of potassium chloride to AS at afinal concentration of 1 mM, then an aliquot of the liposomes loaded withAS was added to the spectroflurometer cuvette, and the decrease of fluores-cence intensity at 525 nm (excitation 490 nm) due to distribution into theliposomes was monitored continuously by the spectrofluorometer underconditions of continuous mixing. After reaching a plateau that indicatesequilibration, the pH and/or ammonium ion gradients were abolished by addi-tion of nigericin or nonactine to a final concentration of 5mm, and the increasein fluorescence due to dequenching was monitored. The ratio F/Fn� 100 wasused to calculate the percentage of AO distributed into the liposomes due to thepH and/or ammonium ion gradient (10,18,22,55). Calibration curves were


prepared by preparing SSL having a range of different defined proton or ASgradients. The above F/FN� 100 ratio was used to plot the calibrationcurve relating fluorescence intensity and pH or ammonium ion gradients(10,18,22,55 and references listed therein).

Use of radioactive methylamine between intraliposome aqueous phase

and the external liposome aqueous medium: The basic idea was developedby Schuldiner and Padan (56) and was later adopted for liposome use in drugdelivery (57). As the methylamine is not fluorescent, this method can beapplied to preformed liposomes even when they are loaded with fluorescentmolecules. It also seems (although not fully proven) that this assay, whenperformed above the liposome-forming lipid Tm, is the most direct one todetermine transmembrane pH gradients as it is not affected by binding andself-association of the methylamine (Garbuzenko et al., in preparation). Itcan also be performed and adopted for a broad range of lipid concentrationsincluding that of the product itself without the need for dilution.

Briefly, liposomes (10 mM) were incubated for 30 minutes at 37�Cfor egg phosphatidylcholine (EPC) and at 60�C for HSPC-based liposomeswith �50� 103 dpm of [14C] methylamine (1� 108 dpm/mole). At the end ofincubation an aliquot of this mixture was passed down a Sephadex G-50minispin column equilibrated in 10 mM histidine–sucrose buffer 10%, pH6.7 buffer. Liposomes were eluted at the column void volume and separatedfrom the unencapsulated methylamine. The concentration of liposomesin the original liposomal dispersion and in the void volume fraction wasdetermined from the organic phosphorus (phospholipid) concentration(see section ‘‘Lipid Quantification and Chemical Stability’’ above) (10,49,53).

The magnitude of the transmembrane liposome pH gradients was deter-mined before and after TMN loading into the liposomes. The distributionof methylamine is determined as percentage of methylamine encapsulationas follows:

X¼ ratio between [14C]-methylamine radioactivity (dpm) and phos-pholipids concentration (mM) in the original liposome dispersionafter the incubation and before Sephadex G-50 separation.

Y¼ ratio between [14C]-methylamine counts (dpm) and phospholipidconcentration (mM) in the void volume fraction after the separationby gel-exclusion chromatography. Percentage of encapsulation(%)¼Y/X� 100.

Collapse of Liposomal Transmembrane Ammonium Ionand Proton Gradient and Release of EncapsulatedDOX by Ionophores

Nonactine and nigericin are two ionophores used to collapse the liposomaltransmembrane ammonium and pH gradients, respectively. Nonactine is an

20 Barenholz

ionophore that exchanges NH4þ with Kþ (Wasserman V et al.). In the presence

of Kþ nonactine will induce collapse of the transmembrane ammonium gradi-ent which will be followed by proton gradeint collapse (Wasserman V et al.)and (Garbuzenko C et al., unpublished results). Nigericin is an ionophorethat abolishes the inside liposome high/medium low proton gradient byexchanging Kþ from medium for Hþ in the liposomes. Therefore, in thepresence of Kþ, nigericin leads to the release of protons from the liposomalaqueous phase, followed by the collapse of the proton gradient, leading tothe collapse of the ammonium ion gradient (1,10) and (Wasserman V et al.),For measuring the ionophore effect, 0.6mmol liposome phospholipids wasadded to 3 mL of 150 mM KCl, 10mL of either nigericin (final conc5mM) or nonactine (final conc 4mM) was added to the loaded liposome pre-paration. The transmembrane gradients were then determined by the AO and14C-MA distribution assays as described above (sections ‘‘Use of AO as anamphipathic weak base, the fluorescence intensity of which is considered tobe pH dependent’’ and ‘‘Use of radioactive methylamine between intralipo-some aqueous phase and the external liposome aqueous medium’’). ForHSPC-based liposomes, the assay was performed above the HSPC Tm at60�C. To check how the ionophore-induced effect compared with the situationof complete liposome solubilization, the same measurements were performedafter complete liposome solubilization by Triton X-100 or hydrogenatedTriton X-100 (which lacks ultraviolet absorbance). The comparison indica-tes that the ionophores are as efficient as complete liposome solubilizationby detergents.

All fluorescence intensity measurements described here were per-formed using a Perkin-Elmer LS-50B luminescence spectrometer. Some ofthe methods were adapted to much smaller volumes using 96-well platesand the Bio-Tek Synergy HT multiwell plate reader (equipped with KC-4software) (Bio-Tek Instruments, Winoaski, Vermont, U.S.A.).

SUMMARY OF THE CHARACTERIZATION OF 100 NMDOX–SSL REMOTE LOADED WITH DOX VIA TRANSMEMBRANEAS GRADIENT

Table 2 demonstrates that for a gradient in which NH4ð Þ2SO4

� �liposome

½ NH4ð Þ2SO4�medium �1000, the pH in the intraliposome aqueous phaseis below pH 5.25 and the pH gradient is above 3 pH units. This gradientcollapsed by each of the two ionophores, nigericin and nonactine, in thepresence of Kþ ions. Such a transmembrane ammonium gradient enablesbetter than 90% loading of DOX into SSL. The loading did not exhaustall the transmembrane ammonium ion gradient and the transmembranepH gradient. The residual transmembrane AS gradient is an importantfactor in the DOX–SSL stability and performance.


ACKNOWLEDGMENTS

This work was supported in part by grants from Liposome Technology Inc.,later SEQUUS Pharmaceuticals (LTI, Menlo Park, California, U.S.A.),then ALZA Corporation, Mountain View, California, and the BarenholzFund. This manuscript is based on studies done since 1988 and still ongoingby many students and researchers of the Laboratory of Membrane andLiposome Research at the Hebrew University–Hadassah Medical School,Jerusalem, Israel, headed by the author. The most important contributorsare Drs. G. Haran, R. Cohen, O. Garbuzenko, and S. Clerc.

All studies on the application of Doxil were done in collaborationwith Professor Alberto Gabizon, Oncology, Shaare Zedek Hospital,Jerusalem, Israel (3–5,8).

The help of Mr. Sigmund Geller in editing this manuscript and ofMrs. Beryl Levene in typing it is acknowledged with pleasure.

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2

Encapsulation of Drugs Within Liposomesby pH-Gradient Techniques

David B. Fenske

Department of Chemistry, University College of the Fraser Valley,Abbotsford, British Columbia, Canada

Pieter R. Cullis

Department of Biochemistry and Molecular Biology, University ofBritish Columbia, Vancouver, British Columbia, Canada

INTRODUCTION

It is now recognized that drug delivery systems can improve the pharmaco-logical properties of many drugs, resulting in increased circulation lifetimesand enhanced efficacy (1). This is often due to altered pharmacokinetic andbiodistribution properties of the drugs, which result from their encapsulationwithin a specific drug carrier system. In recent years, a variety of lipid- orpolymer-based nanoparticles have been developed and characterizedincluding liposomes (2), micelles (3), dendritic unimolecular micelles (4), andpolymeric nanospheres (3,5), to name a few. Of these many systems, perhapsthe best characterized, and certainly one of the first to be developed, were theliposomes, small artificial lipid bilayers with diameters in the nanometer-to-micrometer size range. Although first discovered over 35 years ago, when itwas observed that lipids dispersed in water spontaneously formed largemultilamellar vesicles (6), a significant amount of technological developmentwas required before their full potential as drug delivery systems could berealized. In addition to a thorough knowledge of the physical properties

27

of lipids in membranes (such as the effect of lipid composition on membranepermeability), this included techniques for the rapid generation of uni-lamellar vesicles possessing an optimal size and narrow size distribution, andfor the encapsulation of drugs and macromolecules within them. The firstrequirement was met by the development of extrusion technology, and thelatter by the use of pH gradients as a driving force for the accumulationof weakly basic drugs in the interior of acidic vesicles.

Most of the early methods for the formation of liposomes (7) did notgenerate liposomes of optimal size and polydispersity, and were often tech-nically demanding and time consuming. In contrast, the extrusion methodallowed for the rapid generation of monodisperse populations of unilamellarvesicles (8–10), which in turn allowed characterization of the physical prop-erties and in vivo characteristics of a wide variety of liposomal systems.From this work came an understanding of several key features that wouldbe shared by all optimized liposomal drug delivery systems: (i) an appropri-ate rate of release of drug; (ii) a small size (on the order of 100 nm), and (iii) along circulation lifetime (half-life > five hours in mice). Drug retention isimportant because the drug has to stay with its carrier long enough to reachits target, at which point some drug leakage becomes acceptable and per-haps even necessary. Drug retention can be regulated by careful choice ofthe liposome membrane composition (see below). As far as size is concerned,most liposomal systems are based on the large unilamellar vesicle (LUV)with a diameter close to 100 nm. These systems possess sufficiently largeinternal volumes for the transport of encapsulated material, but are themselvessmall enough to circulate for a time sufficient to reach sites of disease such astumors or sites of inflammation. Vesicles that are much larger or smaller arerapidly cleared from the circulation. However, several other factors besidessize also affect circulation lifetime. The lipid composition of an LUV willaffect both circulation lifetime and drug retention, both of which were foundto be greatly enhanced in systems made from phosphatidylcholine (or sphin-gomyelin) and cholesterol (11–14). Further improvements in circulationlongevity have been achieved by the inclusion of ganglioside GM1 in thevesicle formulation (15–17), or by grafting water-soluble polymers such as(polyethylene) glycol onto the vesicle surface, thereby generating what havecome to be known as ‘‘stealth’’ liposomes (16,18–20).

A major step forward in the design of the first generation of drugtransport systems came with the development of methods for achievingthe encapsulation and retention of large quantities of drug within liposomalsystems. Perhaps the most important insight in this area was the recognitionthat many chemotherapeutic drugs (and other drugs such as antifungals andantibiotics) could be accumulated within vesicles in response to transmem-brane pH gradients (DpH) (21–23). It had long been recognized that pHgradients could influence transmembrane distributions of certain weak acidsand bases (see Ref. 23 and references therein), and the fact that manychemotherapeutics were weak bases made this an obvious area of inquiry.

28 Fenske and Cullis

Subsequent studies led to considerably broader applications involving thetransport and accumulation of a wide variety of drugs, biogenic amines,aminoacids, peptides, lipids, and ions in LUVs exhibiting a DpH (for areview, see Ref. 23). To date, the application of this technology has led tothe development of several liposomal anticancer systems that exhibitimproved therapeutic properties over free drug. Our initial efforts led tothe development of a liposomal version of doxorubicin, the most commonlyemployed chemotherapeutic agent, which is active against a variety of asciticand solid tumors, yet exhibits a variety of toxic side effects. The pH-gradientapproach (11,12,24–26) was expected to provide significant improvements inoverall efficacy, or reductions in side effects, due to high drug-to-lipid (D/L)ratios and excellent retention observed both in vitro and in vivo. This hasbeen realized in liposomal doxorubicin preparations that are currently eitherin advanced clinical trials (27,28), or have been approved by the U.S. Foodand Drug Administration (FDA) for clinical use (29). Other liposomaldoxorubicin formulations (30–39) are in various Phase I or II clinical trials,often with promising results. A variety of other liposomal drugs are cur-rently in preclinical or clinical development: these include vincristine(13,14,40–42), mitoxantrone (22,43–46), daunorubicin (22,29,47,48), cipro-floxacin (49,50), topotecan (51), and vinorelbine, to name a few. Of these,our group has been prominent in devising methods for the encapsulationof doxorubicin, vincristine, and ciprofloxacin.

Liposomal delivery systems are finally reaching a stage of developmentwhere significant advances can reasonably be expected in a short term. Thefirst of the conventional drug carriers are reaching the market, whereas newliposomal drugs are being developed and entered into clinical trials. Theseadvances stem from the fact that the design features required of drug deliv-ery systems that have systemic utility are becoming better defined. Based onthe studies indicated above, we now know that liposomal systems thatare small (diameter � 100 nm) and that exhibit long circulation lifetimes(half-life� five hours in mice) following IV injection exhibit a remarkableproperty termed ‘‘disease site targeting’’ or ‘‘passive targeting’’ that resultsin large improvements in the amounts of drug arriving at the disease site.For example, liposomal vincristine formulations can deliver 50- to 100-foldhigher amounts of drug to a tumor site relative to the free drug (11,13–15).This can result in large increases in efficacy (15). These improvements stemfrom the increased permeability of the vasculature at tumor sites (52,53) orsites of inflammation, which results in preferential extravasation of small,long-circulating carriers in these regions.

Over the past 20 years, our laboratory has played a major role in thedevelopment of liposomal systems optimized for the delivery of conventionaldrugs, almost all of which are encapsulated by pH-gradient techniques. Ourinitial studies led to the development of several liposomal drug delivery sys-tems in which uptake was driven by the ‘‘citrate’’ method of generating pHgradients (15,21–23,27,54–58). This was followed by the development of new

Encapsulation of Drugs Within Liposomes by pH-Gradient Techniques 29

approaches for the loading of drugs via generation of DpH (57,58). In thispaper, we will describe these methods and their applications, and in the processprovide a rationale for the development of new loading methods for the future.

THE FORMATION OF LARGE UNILAMELLARVESICLES BY EXTRUSION METHODS

Many research questions in membrane science require liposomal systemsthat are unilamellar and which possess a specific size distribution, usually inthe nanometer range. This is particularly true for questions involving thepresence of pH or ion gradients, which cannot be adequately modeled usinglarge, multilamellar systems. In our case, investigations relating ion and pHgradients to lipid asymmetry (23,59) were the driving force for the develop-ment of extrusion technology. Early methods available for the generationof unilamellar vesicles, which included dispersion of lipids from organicsolvents (60), sonication (61), detergent dialysis (62), and reversed phaseevaporation (63), had serious drawbacks (59). However, Papahadjopoulosand coworkers (8) had observed that sequential extrusion of multilamellarvesicles (MLVs) through a series of filters of reducing pore size under lowpressure gave rise to LUV systems. Further development of this methodin our laboratory led to an approach involving direct extrusion of MLVs,at relatively high pressures (200–400 psi), through polycarbonate filters witha pore size ranging from 30 to 400 nm. This allowed generation of narrow,monodisperse vesicle populations with diameters close to the chosen poresize (Fig. 1) (9,10). The method is rapid and simple, and can be performedfor a wide variety of lipid compositions and temperatures. As it is necessaryto extrude the lipid emulsions at temperatures 5 to 10�C above the gel-to-liquid crystalline phase transition temperature, the system is manufacturedso that it may be attached to a variable-temperature circulating water bath.

We have previously (2) described the formation of LUVs from MLVs bythe freeze-thaw extrusion method. Here, we describe a modification of thismethod, which involves extrusion in the presence of ethanol. This methodallows for easier extrusion of highly saturated phospholipid preparations,and gives rise to vesicles that are more spherical and possess a slightly largertrapped volume. We will describe in detail the formation of a 20 mM solu-tion of 100 nm LUVs composed of sphingomyelin/cholesterol, a highlyordered lipid mixture that is frequently chosen for drug delivery applicationsdue to its good circulation lifetime and drug retention properties.

Preparation of Sphingomyelin:Cholesterol (55:45) LargeUnilamellar Vesicle by Extrusion

When several experiments will be performed using the same lipid formula-tion, greater consistency will be achieved if each LUV sample is prepared


from the same lipid stock solution, in this case sphingomyelin:cholesterol(55:45) in ethanol. For example, if LUV with a concentration of 20 mM isdesired, the lipid stock should be in excess of 100 mM. In one case, 500 mmolof lipid [275 mmol egg sphingomyelin (Avanti Polar Lipids; Northern Lipids)and 225 mmol cholesterol (Sigma-Aldrich; Northern Lipids)] were dissolvedin 3.75 mL of ethanol, giving a solution with a lipid concentration of 133 mM.If desired, an aliquot of [3H]cholesterol hexadecyl ether (Perkin-Elmer LifeSciences Canada) (a nonexchangeable lipid marker) can be added to the solu-tion. In our case, addition of approximately 0.9 mCi of [3H] cholesterylhexadecyl ether (CHE) gave rise to a lipid solution with a specific activity of3600 dpm/mmol. This can be adjusted as needed. The specific activity can be

Figure 1 Freeze-fracture electron micrographs of egg phosphatidylcholine largeunilamellar vesicles prepared by extrusion through polycarbonate filters with poresizes of (A) 400 nm, (B) 200 nm, (C) 100 nm, (D) 50 nm, and (E) 30 nm. The bar inpanel (A) represents 150 nm. Source: From Ref. 7.


measured by diluting the ethanol solution and measuring both activity andlipid concentration, or by assaying the final LUV preparation. Wheneverpossible, lipid concentrations should be verified by an appropriate assay.Phospholipid concentrations are verified using the assay of Fiske andSubbarow for the quantitative determination of inorganic phosphate (64).The assay, which combines simplicity, accuracy, and reproducibility, isgenerally used for determining the concentration of phospholipid stock solu-tions or LUV preparations. Detailed protocols for this assay have recentlybeen described by us (65) and will not be repeated here.

For the formation of vesicles with a final concentration of approxi-mately 20 mM, 100 mmol of lipid (0.75 mL) is removed from the ethanol stockand placed in a scintillation vial with a tiny stir bar. While stirring rapidly,4.25 mL of appropriate buffer (the internal buffer or hydration buffer) isadded such as 350 mM citrate pH 4.0 (pH-gradient loading), 350 mM ammo-nium sulfate (for amine loading), or 350 mM MgSO4 pH 6.5 (for ionophoreloading) (these will all be discussed below). This will give internal buffer/saltconcentrations of 300 mM. This lipid emulsion is then used for extrusion.

The extruder (Northern Lipids) is assembled with two polycarbonatefilters (Nuclepore polycarbonate membranes; Whatman) with pore size of0.1 mm and diameter of 25 mM, and connected to a circulating water bathequilibrated at 65�C. The lipid emulsion is extruded 10 times through thefilters under a pressure of approximately 400 psi. For larger LUVs (200–400 nm), lower pressures will be adequate (100–200 psi). After each pass, thesample is cycled back to the extruder. It is important to start at a low pres-sure and gradually increase until each pass takes less than one minute.

Following extrusion, the LUVs are dialyzed against the hydrationbuffer (2� 1 L) overnight to remove ethanol, followed by dialysis for afurther 24 hours against the desired external buffer, usually 20 mM N-[2-hydroxyethyl] piperazine-N0-[2-ehtanesulfonic acid] (HEPES) 150 mM NaClpH 7.5 (citrate loading), 150 mM NaCl (ammonium sulfate loading), or20 mM HEPES 300 mM sucrose pH 6.5 (ionophore loading). Following dia-lysis, the lipid concentration of the final LUVs is determined by a phosphateassay (64,65) or by liquid scintillation counting, and is usually in the rangeof 18 to 19 mM.

GENERATION OF pH GRADIENTS VIA INTERNALCITRATE BUFFER

Early studies in our laboratory on membrane potentials and the uptake ofweak bases used for the measurement of DpH led to the recognition thata variety of chemotherapeutic drugs could be accumulated within LUVsexhibiting transmembrane pH gradients (59). This ‘‘remote-loading’’ tech-nique, so named because drug is loaded into preformed vesicles, is based onthe membrane permeability of the neutral form of weakly basic drugs such


as doxorubicin. When doxorubicin (pKa¼ 8.6) is incubated at neutral pHin the presence of LUVs exhibiting a DpH (interior acidic), the neutral formof the drug will diffuse down its concentration gradient into the LUVinterior, where it will be subsequently protonated and trapped (the chargedform is membrane impermeable). As long as the internal buffer (300 mMcitrate pH 4) is able to maintain the DpH, diffusion of neutral drug willcontinue until either all the drugs have been taken up, or the bufferingcapacity of the vesicle interior has been overwhelmed. This process isillustrated in Figure 2 for the uptake of doxorubicin into EPC/Chol andegg phosphatidylcholine (EPC) LUVs, where it is seen that uptake isdependent on time, temperature, and lipid composition (21). If conditionsare chosen correctly, high D/L ratios can be achieved (D/L¼ 0.2 mol:mol)with high trapping efficiencies (98% and higher) and excellent drug reten-tion. A diagrammatic illustration of this process is given in Figure 3A (alsosee insert). Interestingly, much higher levels of doxorubicin can be loadedthan would be predicted on the basis of the magnitude of DpH (23,66).This would appear to result from the formation of doxorubicin precipi-tates within the LUV interior, which provides an additional driving forcefor accumulation (67,68). Doxorubicin forms fibrous precipitates that areaggregated into bundles by citrate (69) or sulfate (67,68) counteranions,and which affect the rate of doxorubicin release from LUVs (70). Theseprecipitates can be visualized by cryoelectron microscopy, where they areseen to give the LUVs a ‘‘coffee-bean’’ appearance (Fig. 4). Some LUVscontain several bundles of fibers. This has been corroborated by recent obser-vations that very high levels of uptake can be achieved in the absence of apH gradient by the formation of doxorubicin–Mn2þ complexes (71–74).

The experimental procedure described below for the accumulation ofdoxorubicin within DSPC/Chol LUVs represents our ‘‘basic’’ pH-gradientmethod for drug loading. This basic system can be used for the uptake of awide variety of drugs (22) and all the remote-loading methods, which followare based on similar principles involving the generation of DpH, eventhough this may not always be immediately obvious.

Remote Loading of Doxorubicin into DSPC:Cholesterol(55:45) Large Unilamellar Vesicle

DSPC/Chol (55:45) LUVs (diameter ¼ 100 nm) are prepared as described insection ‘‘Preparation of Sphingomyelin/Cholesterol (55:45) Large Uni-lamellar Vesicle by Extrusion’’ [(Lipid) ¼ 20 mM, volume ¼ 5 mL], using350 mM citrate pH 4.0 as the hydration buffer, and 20 mM HEPES150 mM NaCl pH 7.5 (HEPES-buffered saline) as the external buffer. In thiscase, the pH gradient is formed during the final dialysis step. It would alsobe possible to omit the final dialysis step and form the pH gradient by oneof two common column methods. This could be desirable if the LUV


Figure 2 (A) Effect of incubation temperature on uptake of doxorubicin into200 nm EPC/cholesterol (55:45 mol/mol) large unilamellar vesicles (LUVs) exhibi-ting a transmembrane pH gradient (pH 4 inside, 7.8 outside). Doxorubicin was addedto LUVs (D/L ¼ 0.3 wt:wt) equilibrated at 21�C, 37�C, and 60�C. (B) Effect ofcholesterol on the uptake of doxorubicin at 20 into 100 nm LUVs exhibiting a trans-membrane pH gradient (pH 4.6 inside, 7.5 outside). Lipid compositions were EPC andEPC/cholesterol (1:1 mol/mol). The initial drug-to-lipid ratio was 100 nmol/mmol.Source: From Refs. 12 (A), 21 (B).


preparation will not be used in a fairly short time, or if the lipid compositionis such that pH gradients are not stable over longer periods of time. The firstalternate method is passing the LUVs down a column of Sephadex G-50(Amersham Pharmacia Biotech) equilibrated in HEPES-buffered saline

Figure 3 (Caption on next page)


(the method described below), and the second method involves the use ofspin columns. Spin columns permit the rapid separation of LUVs from theirhydration buffer (or from unencapsulated drug) on hydrated gel at lowcentrifugation speeds. They are particularly useful for monitoring druguptake with time (as described below). On the day prior to drug loading,a slurry of Sephadex G-50 in HEPES-buffered saline (HBS) is preparedby adding a small volume (2–3 mL) of dry G-50 powder to 200 to 300 mLHBS with frequent swirling. Small quantities of gel are added as necessaryuntil the settled G-50 occupies about half the aqueous volume. The hydratedgel is allowed to swell overnight. To prepare the spin columns, a tiny plug ofglass wool is packed into the end of a 1-mL disposable syringe (without theneedle), which is then placed in a 13� 100 mm glass test tube. The G-50slurry is swirled, and the syringes are immediately filled using a Pasteur pip-ette. The syringes (in test tubes) are then placed in a desktop centrifuge, andthe gel is packed (to 0.6 or 0.7 mL) by bringing the speed to 2000 rpm(�670 g) momentarily. More G-50 slurry is added, and the centrifugationis repeated. When finished, the moist G-50 bed should be 0.9 to 1.0 mL.The spin columns are covered with parafilm to prevent drying, and are usedwithin an eight-hour period.

If the second dialysis step against external buffer is omitted during theformation of LUV, transmembrane pH gradients can be formed by running

Figure 3 (Figure on previous page) Diagrammatic representations of drug uptake inresponse to transmembrane pH gradients. Prior to drug loading, it is necessary toestablish the primary pH gradient or the primary ion gradient, which will generatea DpH. Lipid films or powders are initially hydrated and then extruded in the internal(or hydration) buffer, giving rise to a vesicle solution in which both the external andthe internal solutions are the same, as indicated by the grey shading in the upperframe of the insert (top right). The vesicles are then passed down a gel exclusion col-umn (Sephadex G-50) hydrated in the external buffer, giving rise to vesicles with apH or ion gradient (lower frame of insert). (A) The standard pH-gradient method.The internal buffer is 300 mM citrate pH 4, and the external buffer is 20 mM HEPES150 mM NaCl pH 7.5. The precipitation of certain drugs such as doxorubicin, whichprovides an addition driving force for uptake, is not indicated in the figure. (B) Asecond method for generating DpH involves the initial formation of a transmem-brane gradient of ammonium sulfate, which leads to an acidified vesicle interior asneutral ammonia leaks from the vesicles. Here, the internal buffer is 300 mM ammo-nium sulfate and the external buffer is 150 mM NaCl. Possible drug precipitation isnot indicated. (C) Transmembrane pH gradients can also be established by iono-phores (such as A23187) in response to transmembrane ion gradients (e.g., Mg2þ,represented as solid circles). A23187 couples the external transport of one Mg2þ ion(down its concentration gradient) to the internal transport of two protons, resultingin acidification of the vesicle interior. An external chelator such as EDTA is requiredto bind Mg2þ ions as they are transported out of the vesicle. Other divalent cationssuch as Mn2þ can also be used. See text for further details. Source: From Ref. 2.


an aliquot (400 mL) of the LUVs down a column (1.5� 15 cm) of SephadexG-50 eluted in HBS. The LUV fractions, which will elute at the void volumeand are visible to the eye, are collected and pooled. The final volume will beapproximately 2 mL and the lipid concentration will be around 5 mM. Alter-natively, the pH gradient can be formed using spin columns prepared inHBS (spin 4� 100 mL) and pooling the fractions.

Doxorubicin (Sigma-Aldrich) is often loaded at a D/L ratio of0.2 mol:mol. A doxorubicin standard solution is prepared by dissolving 1.0 mgof drug in 0.5 mL of saline (150 mM NaCl). The concentration is verified onthe spectrophotometer using the doxorubicin extinction coefficient e¼ 1.06�104 M�1 cm�1 (75). Aliquots of lipid (5mmol) and doxorubicin (1mmol,approximately 0.5 mg) are combined in a glass test tube (or plastic Ependorftube) with HBS to give a final volume of 1 mL (5 mM lipid concentration).Drug uptake occurs during a 30-minute incubation at 65�C. This is verifiedat appropriate time points (0, 5, 15, and 30 minutes) by applying an aliquot(50–100mL) to a spin column and centrifuging at 2000 rpm for two minutes.LUVs containing entrapped drug will elute off the column, while free doxo-rubicin will be trapped in the gel. An aliquot (50 mL) of the initial lipid–drugmixture is saved for determination of initial D/L ratio.

Figure 4 Cryoelectron micrograph of 100 nm DSPC/cholesterol large unilamellarvesicles (LUVs) containing doxorubicin (drug-to-lipid ratio ¼ 0.05 wt:wt) loadedin response to a transmembrane pH gradient (inside acidic). The precipitated drugis clearly visible, and gives the LUV the appearance of a ‘‘coffee-bean.’’ The pH gra-dient was generated by the ionophore A23187 in response to a transmembrane Mg2þ

gradient. Abbreviation: DSPC, distearoylphosphatidylcholine. Source: Johnston, un-published results.


The initial mixture and each time point are then assayed for doxoru-bicin and lipid. Lipid concentrations can be quantified by the phosphateassay (see above) or by liquid scintillation counting of an appropriateradiolabel. Doxorubicin is quantified by an absorbance assay (see below).The percent uptake at any time point (e.g., t¼ 30 minutes) is determinedby %-uptake ¼ [(D/L) t¼30minutes]� 100/[(D/L) initial]. Doxorubicin can beassayed by both a fluorescence assay and an absorbance assay, but we findthe latter to be more accurate. The standard curve consists of four to fivecuvettes containing 0 to 150 nmol doxorubicin in a volume of 0.1 mL; sam-ples to be assayed are of the same volume. To each tube is added 0.9 mL of1% (v/v) Triton X-100 (in water) solution. For saturated lipid systems suchas DSPC/Chol, the tubes should be heated in a boiling water bath for 10 to15 seconds, until the detergent turns cloudy. Samples are allowed to cool,and absorbance is read at 480 nm on a UV/Visible spectrophotometer.

GENERATION OF pH GRADIENTS VIA TRANSMEMBRANEAMMONIA GRADIENTS

Despite its successful application in several drug delivery systems (22), thepH-gradient approach utilizing internal citrate buffer does not provide ade-quate uptake of all weakly basic drugs. A case in point is the antibioticciprofloxacin, a commercially successful, quinolone antibiotic widely usedin the treatment of respiratory and urinary tract infections (56). Ciproflox-acin is a zwitterionic compound that is charged and soluble under acidic andalkaline conditions, but is neutral and poorly soluble in the physiologicalpH range, precisely the external conditions of most drug-loading techniques.This low solubility results in low levels of uptake (<20%) when the drug isloaded using the standard citrate technique. However, high levels of encap-sulation can be achieved using an alternate DpH-loading method that isbased on transmembrane gradients of ammonium sulfate (56,67,76). If agiven drug is incubated with LUVs containing internal ammonium sulfatein an unbuffered external saline solution, a small quantity of neutral ammo-nia will diffuse out of the vesicle, creating an unbuffered acidic interior witha pH �2.7 (56). Any neutral drug that diffuses into the vesicle interior willconsume a proton and becomes charged and therefore trapped. If con-tinued, drug uptake leads to consumption of the available protons and thediffusion of additional neutral ammonia from the vesicles will create moreprotons to drive drug uptake. This will continue until all the drugs have beenloaded, or until the internal proton supply is depleted, leaving a final inter-nal pH of about 5.1 (56). The technique is ideal for ciprofloxacin, becausethe drug is supplied as an HCl salt, and thus is acidic and soluble when dis-solved in water. In addition, the amine method results in higher uptakelevels for other drugs such as doxorubicin. A diagrammatic scheme of themethod is given in Figure 3B.


This technique has been applied to a variety of drugs including doxoru-bicin (57,67,68,76), epirubicin (57), ciprofloxacin (56,57,67,77), and vincristine(57). The method is equally effective using a range of alkylammonium salts(e.g., methylammonium sulfate, propylammonium sulfate, or amylammoniumsulfate) to drive uptake (57). Some drugs such as doxorubicin precipitate andform a gel in the vesicle interior (67,68,70), whereas others such as ciprofloxacindo not (67,77). Ciprofloxacin can reach intraliposomal concentrations as highas 300 mM, and while the drug does form small stacks as shown by 1H-NMR, itdoes not form large precipitates (77), even though its solubility in buffer cannotexceed 5 mM. As a result of the lack of precipitation and rapid exchangeproperties, ciprofloxacin can respond rapidly to changes in electrochemicalequilibria such as depletion of the pH gradient. This explains the observedrapid leakage of ciprofloxacin from LUVs in response to serum destabilizationor loss of pH gradient. In contrast, doxorubicin, which is known to form inso-luble precipitates within LUVs in the presence of both citrate (69,70) andammonium sulfate (67,68), is retained within vesicles in the presence of serum.This is a clear illustration of how the physical state of encapsulated drugs willaffect retention and therefore may impact efficacy.

The experimental procedure below describes the uptake of ciprofloxa-cin into sphingomyelin (SPM)/Chol LUVs. Drug delivery vehicles preparedfrom SPM/Chol often exhibit greater efficacy than those prepared fromDSPC/Chol (13). Included is a description of the Bligh–Dyer extractionprocedure (78), which involves partitioning the lipid and water-soluble druginto organic solvent and aqueous layers, respectively. This is necessarybecause lipid interferes with the ciprofloxacin assay.

Remote Loading of Ciprofloxacin into SPM/CholesterolLarge Unilamellar Vesicles

SPM/Chol (55:45) LUVs (diameter ¼ 100 nm) are prepared as described inthe section ‘‘Preparation of Sphingomyelin/Cholesterol (55:45) Large Unilam-ellar Vesicle by Extrusion’’ [(Lipid)¼ 20 mM, volume¼ 5 mL], using 350 mMammonium sulfate ((NH4)2SO4) as the hydration buffer, and 150 mM NaClas the external solution. In this case, the amine and pH gradients are formedduring the final dialysis step. It would also be possible to omit the final dialysisstep, and form the amine and pH gradients by one of two common columnmethods, as described above for the citrate method. The first alternate methodis passing the LUVs down a column of Sephadex G-50 (Amersham PharmaciaBiotech) equilibrated in 150 mM saline (the method described below), andthe second involves the use of spin columns. Spin columns are prepared(using saline rather than HBS) for monitoring drug uptake with time.

If the second dialysis step against external buffer is omitted during theformation of LUV, the amine and pH gradients are formed by running analiquot (200 mL) of the LUVs down a column (1.5� 15 cm) of Sephadex


G-50 eluted in saline (150 mM NaCl), as described above. Alternatively, thegradient could be formed using spin columns (spin 2� 100 mL), and poolingthe fractions.

Ciprofloxacin (Bayer Corporation) is often loaded at a D/L ratio of0.3 mol:mol. After preparation of a ciprofloxacin standard solution (4 mMin water), 5 mmol of lipid and 1.5 mmol of ciprofloxacin are pipetted into aglass test tube (or plastic Ependorf tube), adding saline to give a finalvolume of 1 mL (5 mM lipid concentration). This solution is incubated at65�C for 30 minutes, with aliquots (50–100 mL) withdrawn at appropriatetime points (0, 5, 15, and 30 minutes) and applied to a spin column [centri-fuge at 2000 rpm (�670 g) for two minutes]. An aliquot (50 mL) of the initiallipid–drug mixture is saved for determination of initial D/L.

The initial mixture and each time point are then assayed for ciproflox-acin and lipid. Lipid can be quantified using the phosphate assay (64,65) or byliquid scintillation counting. Ciprofloxacin is quantified by an absorbanceassay following removal of drug from lipid by a Bligh–Dyer extraction pro-cedure (78) (see below). The percent uptake is determined as described in thesection ‘‘Remote Loading of Doxorubicin into DSPC/Cholesterol (55:45)Large Unilamellar Vesicle.’’

To perform the ciprofloxacin assay, a standard curve is prepared con-sisting of six glass test tubes containing 0, 50, 100, 150, 200, and 250 nmolciprofloxacin (in water). The volume is made up to 1 mL with 200 mM NaOH.For the blank, 1 mL of 200 mM NaOH is used. Each LUV sample to beassayed should contain less than 250 nmol ciprofloxacin in a volume of 1 mL.

To each standard and assay sample, 2.1 mL of methanol and 1 mL ofchloroform are added and vortexed gently. Only one phase should bepresent (if two phases form, 0.1 mL methanol is added and the solution isvortexed again). One milliliter of 200 mM NaOH and 1 mL chloroform arethen added to each tube, which are then vortexed at high speed. Two phasesshould form, an aqueous phase containing the ciprofloxacin with a volumeof 4.1 mL (top), and an organic phase containing the lipid with a volume of2 mL (bottom). If a clean separation is not obtained, the tubes can be centri-fuged at 2000 rpm for two minutes in a desktop centrifuge.

After carefully removing the aqueous phase, the absorbance at 273.5 nmis read to obtain nmol Cipro present in the original sample volume(in microliters), thereby yielding the sample drug concentration (mM).

IONOPHORE-MEDIATED GENERATION OF pH GRADIENTSVIA TRANSMEMBRANE ION GRADIENTS

The observation that improved remote loading of ciprofloxacin could beachieved using ammonium sulfate solutions rather than sodium citratebuffers highlighted the need for further investigation and development ofdrug-loading methodologies. In this section, we examine an approach in


which ionophores, responding to transmembrane ion gradients (involvingNaþ, Mn2þ, or Mg2þ), generate a secondary pH gradient that providesthe driving force for drug uptake.

Recently, we have developed a new method of remote loading that isbased on the ionophore-mediated generation of a secondary pH gradient inresponse to transmembrane gradients of monovalent and divalent cations (58).The process is diagrammed in Figure 3C. A primary ion gradient is gen-erated when LUVs formed by extrusion in K2SO4, MnSO4, or MgSO4

solutions are passed down a column equilibrated in a sucrose-containingbuffer. The use of sulfate salts is important, because chloride ion can dissi-pate pH gradients by forming neutral HCl that can diffuse out of the vesicle.Likewise, sucrose is chosen as a component of the external buffer ratherthan saline, because chloride ion can interfere with some ionophores (79).After establishing the primary ion gradient, the drug (which to date includesdoxorubicin, mitoxantrone, ciprofloxacin, vincristine, and other vincaalkaloids) is added. If the LUVs contain a potassium salt, the ionophorenigericin is added, whereas if the LUVs contain either Mn2þ or Mg2þ,the ionophore A23187 and the chelator ethylenediaminetetraacetic acid(EDTA) are used. Under the current conditions, nigericin couples the out-ward flow of a potassium ion (down its concentration gradient) to theinward flow of a proton. Likewise, A23187 couples the outward flow ofa single divalent cation to the inward flow of a pair of protons. In bothcases, ionophore-mediated ion transport is electrically neutral and resultsin acidification of the vesicle interior, thereby creating a pH gradient thatdrives drug uptake. For systems containing divalent cations, EDTA che-lates manganese or magnesium as they are transported out of the vesicles,and is required to drive drug uptake. Both ionophore methods result inhigh levels of encapsulation for the drugs ciprofloxacin and vincristine(80–90%), and excellent in vitro retention (58). However, the A23187-loaded systems exhibit excellent in vivo circulation and drug retentionproperties, which are comparable to systems loaded by the citrate or aminemethods, whereas the nigericin-loaded systems do not.

Ionophore-Mediated Loading of Vincristine, Vinblastine, orVinorelbine into SPM/Chol Large Unilamellar Vesicles

LUVs (diameter¼ 100 nm) are prepared from SPM/Chol (55:45) as descri-bed above [(Lipid)¼ 20 mM]. It has been shown that liposomal vincristineprepared from SPM/Chol exhibits greater efficacy than systems preparedfrom DSPC/Chol (13). The hydration solution is 350 mM MgSO4, whichwhen diluted with the ethanol used in extrusion will give an internal salt con-centration of 300 mM. The external buffer is 20 mM HEPES 300 mMsucrose pH 7.5 containing 15 mM Na2EDTA. In this case, the Mg2þ gradi-ent is formed during the final dialysis step. It would also be possible to omit


the final dialysis step, and form the Mg2þ gradient by one of two commoncolumn methods, as described above for the other methods. The firstalternate method is passing the LUVs down a column of Sephadex G-50(Amersham Pharmacia Biotech) equilibrated in HEPES-buffered sucrosecontaining Na2EDTA (the method described below), and the secondinvolves the use of spin columns.

Spin columns for monitoring drug uptake with time are hydrated in300 mM sucrose. Assuming the final dialysis step against HEPES-bufferedsucrose (20 mM HEPES 300 mM sucrose pH 7.5) containing Na2EDTA isomitted, the Mg2þ gradient can be formed by running an aliquot (200 mL)of the LUVs down a column (1.5� 15 cm) of Sephadex G-50 eluted inHEPES-buffered sucrose containing 15 mM Na2EDTA. Alternatively, thegradient can be formed using spin columns prepared in the same buffer (spin2� 100 mL), and pooling the fractions.

Vincristine sulfate, vinblastine sulfate (Fine Chemicals, Australia), orvinorelbine ditartrate (OmniChem Sa, Belgium) is added to the LUVs togive a D/L ratio of 0.1 wt:wt. If desired, the drugs can be labeled with traceamounts of [14C]vincristine sulfate (Chemsyn Laboratories, Lenexa, Kansas,U.S.A.), [3H]vinorelbine, or [3H]vinblastine (Moravek Biochemicals, Inc.,Breas, California, U.S.). The sample is diluted to a final lipid concentrationof 5 mM with addition of HEPES-buffered sucrose containing 15 mMNa2EDTA. Ionophore A23187 (Sigma-Aldrich) is added to a concentrationof 2 mg/mg lipid. The mixture is incubated at 60�C for two hours, saving analiquot (50 mL) for determination of initial D/L ratio. Aliquots (100 mL) arewithdrawn at appropriate time points (0, 10, 20, 30, 60, and 120 minutes) andapplied to spin columns (2000 rpm for two minutes). The initial mixtureand each time point are then assayed for drug and lipid to monitor druguptake. When maximum uptake has been achieved, the remainder of thesample is dialyzed against HEPES-buffered sucrose for two hours to removeunencapsulated drug and traces of ionophore.

A representative uptake experiment is shown in Figure 5, where theeffect of temperature is apparent (80). For both vinorelbine and vincristine,little uptake is observed at 25�C, far below the gel-to-liquid crystalline phasetransition temperature of egg SPM (about 44�C), whereas 80% uptake isobserved at 60�C where the membrane is fluid. Interestingly, the uptakeof vinblastine at 25�C yields an end point nearly identical to that obtained

Figure 5 (Figure on next page) Effect of temperature on the uptake of (A) vinbla-stine, (B) vinorelbine, and (C) vincristine into 100 nm ESM/Chol large unilamellarvesicles (55:45) containing 300 mM MgSO4. After addition of ionophore A23187(2 mg/mg lipid), 15 mM EDTA, and drug solution to the liposome preparation(5 mM lipid; final drug-to-lipid ratio¼ 0.1 wt:wt), samples were incubated at 25�C(&) and 60�C (�). Aliquots were removed at different time points for determinationof lipid and drug concentrations as described in the text. Abbreviation: ESM, eggsphingomyelin. Source: From Ref. 80.


Figure 5 (Caption on previous page)


at higher temperature, although the rate of uptake is somewhat reduced.This was attributed to the higher hydrophobicity of this drug relative tovinorelbine and vincristine.

COMPARISON OF LOADING METHODS

We have already noted one example that demonstrates the benefits ofpossessing more than a single method for loading drugs via pH-gradientmethods. Drugs such as ciprofloxacin that have low solubilities atnear-neutral pH values will not exhibit efficient loading when the standardcitrate method is used (due to the requirement for external pH values near 7,a condition that is in turn dictated by citrates optimal buffering pH near 4).In that case, the use of the ammonium sulfate method, where the externalsolution can be unbuffered saline, allowed for much higher D/L ratios tobe obtained. Such general considerations were behind the development ofthe ionophore method as well, and our initial studies showed that ciproflox-acin could be as efficiently loaded by this new method as by that involvingammonium sulfate. These observations, coupled with the growing recogni-tion that certain drugs formed insoluble precipitates within liposomes inthe presence of the correct counteranion(s), and a developing appreciationof the role of drug retention in liposomal drug efficacy, have led us andothers to investigate whether specific loading methods will result in opti-mized formulations for specific drugs. Although these studies are currentlyin progress and thus the final word cannot yet be made, a number of earlystudies point toward the superiority of certain methods over others in spe-cific applications. We will note a few of these here, looking forward toresearch in this area as it unfolds in the near future.

Zhigaltsev et al. (80) have recently examined the in vitro and in vivoloading and retention properties of three closely related vinca alkaloids(vincristine, vinblastine, and vinorelbine) in liposomal systems composed ofSPM/Chol. Results were obtained for both the citrate and the ionophore(using Mg2þ) methods of loading. Interestingly, it was noted that whenloading was accomplished using an ionophore method, drug retention wassignificantly improved by increasing the D/L ratio (from 0.1 to 0.3 wt:wt).However, drug retention remained the same following an increase in D/Lratio when the citrate method of loading was used. It was suggested thatthe differences in retention observed for the two methods could be relatedto differences in the intravesicular forms of the drugs in the two systems.

Similar results have been described in a recent study comparing theencapsulation of topotecan within DSPC/Chol liposomes using four differ-ent loading methods (81). The four methods included the citrate method, theammonium sulfate method, and two variations of the ionophore A23187method (one using MnCl2 and the other MnSO4). Both the citrate and theA23187-MnCl2 methods exhibited reduced uptake (final D/L ratios inthe range of 0.1), whereas the ammonium sulfate and A23187-MnSO4


methods exhibited higher final D/L ratios (in the range of 0.2; close to 100%)and much better retention. In fact, with the two superior loading systems, D/Lratios as high as 0.3 could be achieved. These two systems also exhibitedimproved retention as the D/L ratio was increased from 0.1 to 0.2, retainingfive times as much drug at 36 hours at the higher ratio. The amount of drugretained by the ammonium sulfate and MnSO4 methods was 10 times as greatas that retained by the citrate and MnCl2 methods as well. Cryoelectronmicroscopy studies revealed the presence of topotecan precipitate within thetwo sulfate-containing systems. Together, these results demonstrate the im-provements in drug retention that can be achieved using a pH gradient incombination with the correct counteranion (in this case, sulfate).

It is clear that very different rates of drug retention are observed whenencapsulation is achieved by different methods of loading, even though thedriving force for uptake in all cases is a pH gradient of approximatelythe same magnitude. These differences may be due to interactions of thedrugs with different counteranions, which can result in the formation of pre-cipitate and have a profound effect on the rate of release. The fact that drugefficacy is highly sensitive to drug release rates, especially for drugs that onlyact at a certain point in the cell cycle (such as vincristine), demonstrates theimportance of having a variety of methods available for regulating the rateof drug release. This highlights the need for developing new methods of drugencapsulation, and for meticulously investigating each existing technologyfor each drug of interest. Of particular interest at this time will be in vivoanimal studies that compare drug retention rates to antitumor efficacy fordifferent loading systems. Such studies could lead to significant improve-ments in the treatment of human disease.

CONCLUSIONS

The rapid development of liposomal technology over the past 15 years has ledto a wide variety of delivery systems for conventional drugs, some of which arein clinical trials or have been approved for use by the U.S. FDA. At least threedifferent methods now exist for loading weakly basic drugs into liposomes inresponse to transmembrane pH gradients. By appropriate selection of loadingmethod, drug, and other parameters such as D/L ratio, delivery systems exhib-iting a range of drug retention properties can be generated. The question ofwhether these will lead to delivery systems exhibiting optimized antitumorefficacy is an exciting current and future topic of investigation.

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39. Northfelt DW, Dezube BJ, Thommes JA, et al. Pegylated-liposomal doxorubicinversus doxorubicin, bleomycin, and vincristine in the treatment of AIDS-relatedKaposi’s sarcoma: results of a randomized phase III clinical trial. J Clin Oncol1998; 16:2445.

40. Gelmon KA, Tolcher A, Diab AR, et al. Phase I study of liposomal vincristine.J Clin Oncol 1999; 17:697.

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43. Lim HJ, Masin D, Madden TD, Bally MB. Influence of drug release character-istics on the therapeutic activity of liposomal mitoxantrone. J Pharmacol ExpTher 1997; 281:566.

44. Chang CW, Barber L, Ouyang C, Masin D, Bally MB, Madden TD. Plasmaclearance, biodistribution and therapeutic properties of mitoxantrone encapsu-lated in conventional and sterically stabilized liposomes after intravenousadministration in BDF1 mice. Br J Cancer 1997; 75:169.

45. Adlakha-Hutcheon G, Bally MB, Shew CR, Madden TD. Controlled destabili-zation of a liposomal drug delivery system enhances mitoxantrone antitumoractivity. Nat Biotechnol 1999; 17:775.

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47. Gill PS, Wernz J, Scadden DT, Cohen P, et al. Randomized phase III trial ofliposomal daunorubicin versus doxorubicin, bleomycin, and vincristine inAIDS-related Kaposi’s sarcoma. J Clin Oncol 1996; 14:2353.

48. Pratt G, Wiles ME, Rawstron AC, et al. Liposomal daunorubicin: in vitro and invivo efficacy in multiple myeloma. Hematol Oncol 1998; 16:47.

49. Webb MS, Boman NL, Wiseman DJ, et al. Antibacterial efficacy against anin vivo Salmonella typhimurium infection model and pharmacokinetics of aliposomal ciprofloxacin formulation. Antimicrob Agents Chemother 1998;42:45.

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53. Brown JM, Giaccia AJ. The unique physiology of solid tumors: opportunities(and problems) for cancer therapy. Cancer Res 1998; 58:1408.


54. Boman NL, Mayer LD, Cullis PR. Optimization of the retention properties ofvincristine in liposomal systems. Biochim Biophys Acta 1993; 1152:253.

55. Bally MB, Mayer LD, Loughrey H, et al. Dopamine accumulation in large uni-lamellar vesicle systems induced by transmembrane ion gradients. Chem PhysLipids 1988; 47:97.

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58. Fenske DB, Wong KF, Maurer E, et al. Ionophore-mediated uptake ofciprofloxacin and vincristine into large unilamellar vesicles exhibiting transmem-brane ion gradients. Biochim Biophys Acta 1998; 1414:188.

59. Cullis PR. Liposomes by accident. J Liposome Res 2000; 10:ix.60. Batzri S, Korn ED. Single bilayer liposomes prepared without sonication.

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65. Fenske DB, Maurer N, Cullis PR. Encapsulation of weakly-basic drugs, anti-sense oligonucleotides and plasmid DNA within large unilamellar vesicles fordrug delivery applications. In: Torchilin VP, Weissig V, eds. Liposomes: A Prac-tical Approach. 2nd ed. Oxford: Oxford University Press, 2002, Chapter 6.

66. Harrigan PR, Wong KF, Redelmeier TE, Wheeler JJ, Cullis PR. Accumulation ofdoxorubicin and other lipophilic amines into large unilamellar vesicles in responseto transmembrane pH gradients. Biochim Biophys Acta 1993; 1149:329.

67. Lasic DD, Ceh B, Stuart MC, Guo L, Frederik PM, Barenholz Y. Transmem-brane gradient driven phase transitions within vesicles: lessons for drug delivery.Biochim Biophys Acta 1995; 1239:145.

68. Lasic DD, Frederik PM, Stuart MC, Barenholz Y, McIntosh TJ. Gelation ofliposome interior. A novel method for drug encapsulation. FEBS Lett 1992;312:255.

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72. Abraham SA, Edwards K, Karlsson G, et al. Formation of transition metal-dox-orubicin complexes inside liposomes. Biochim Biophys Acta 2002; 1565:41.

73. Abraham SA, McKenzie C, Masin D, et al. In vitro and in vivo characterizationof doxorubicin and vincristine coencapsulated within liposomes through use oftransition metal ion complexation and pH gradient loading. Clin Cancer Res2004; 10:728.

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3

Incorporation of Lipophilic Antitumorand Antiviral Drugs into the Lipid Bilayer

of Small Unilamellar Liposomes

Reto Schwendener

Institute of Molecular Cancer Research, University of Zurich, Zurich, Switzerland

Herbert Schott

Institute of Organic Chemistry, Eberhard-Karls University, Tuebingen, Germany

INTRODUCTION

Since the discovery of vesicular structures, termed liposomes, by AlecBangham, a tremendous amount of work on applications of liposomes hasemerged. The use of small unilamellar liposomes as carriers of drugs fortherapeutic applications has become one of the major fields in liposomeresearch. The majority of these applications are based on the encapsulationof water-soluble molecules within the trapped volume of the liposomes.Long circulating poly(ethylene glycol) (PEG) modified liposomes with cyto-toxic drugs doxorubicin, paclitaxel, vincristine, and lurtotecan are examplesof clinically applied chemotherapeutic liposome formulations (1,2).

In contrast to the extensive exploitation of the trapped aqueousvolume of the liposomes that serves as nanocontainer for water-solublemolecules, the phospholipid bilayer has not been given the same attentionfor its use as carrier matrix for lipophilic drugs. An exploratory survey ofthe number of cited literature references in Medline performed in February2005 gave the following result. With the general keywords ‘‘drug’’ and

51

‘‘liposome’’ 23,246 entries were found, whereas ‘‘aqueous’’ and ‘‘drug’’and ‘‘liposome’’ gave 1537 entries and ‘‘lipophilic’’ and ‘‘drug’’ and ‘‘lipo-some’’ resulted in 429 references. Thus, the development of liposomal drugformulations with lipophilic drugs is less popular. This discrepancy mayhave several reasons, the main probably consisting in the chemistry requiredto transform water-soluble molecules into lipophilic compounds for theincorporation into the lipid bilayer matrix of liposomes. The most favor-able chemical modifications to obtain a molecule that intercalates in a stablefashion into the lipophilic moiety of a lipid bilayer consist in the attachmentof long chain fatty acyl or alkyl residues, for example saturated or unsatu-rated fatty acids (palmitic or stearic acid) and alkylamines (hexadecyl- oroctadecylamine) to a suitable functional group (hydroxy or amino groupsor direct linkage to carbon atoms) of the hydrophilic part of the molecule.Some recent examples of lipophilic modifications of antitumor drugs andtheir formulation in liposomes are gemcitabine, 5-iodo-20-deoxyuridine,methotrexate, paclitaxel, and cytosine arabinoside (3–11).

Drugs that are highly lipophilic by their own nature, e.g., taxanes andepothilones, can only be used therapeutically by addition of possibly toxicsolubilizing agents (e.g., Cremophor EL) in complex pharmaceutical formu-lations (12–14). One of several feasible ways of obtaining parenterallyapplicable formulations of such drugs without eliciting toxic effects originat-ing from the formulation vehicles is their incorporation into the bilayermatrix of phospholipid liposomes.

Since the beginning of our work with liposomes that dates back morethan 20 years, we chose the approach of the chemical transformation ofwater-soluble nucleosides of known cytotoxic and antiviral properties intolipophilic drugs or prodrugs (see references summarized in Table 1).

As lipophilic anchors, we chose modifications with long acyl and alkylchains, preferably of similar chain lengths as the phospholipids to allow opti-mal alignment with the phospholipids molecules of the liposome bilayers.

The first cytotoxic nucleoside we modified was 1-b-D-arabinofurano-sylcytosine (ara-C) because of its major clinical disadvantages of a veryshort plasma half-life and rapid degradation by deamination to the inactivemetabolite 1-b-D-arabinofuranosyluracil, a shortcoming that also hindersthe oral application of ara-C. To reduce these limitations, a large numberof 50- and N4-substituted ara-C derivatives have been synthesized andcharacterized in the past (50). In our first studies, we showed thatN4-acyl-derivatives of ara-C were active in vivo as liposomal formulations atsignificantly lower concentrations than ara-C (15–18,51). However, the pro-tection of the N4-acyl-ara-C derivatives (e.g., N4-palmitoyl-ara-C) againstenzymatic deamination was only partially achieved and suggested to beinsufficient for a significant improvement of the cytotoxic and pharmaco-kinetic properties. Based on these findings, we synthesized a new class ofN4-alkyl-ara-C derivatives with alkyl chain lengths ranging between 6 and 22

52 Schwendener and Schott

C-atoms (19,20). These compounds showed a typical structure–activity corre-lation between the length of the alkyl side chain and their antitumor activityprofile (19). The pharmacokinetic, cytotoxic, and pharmacological propertiesof N4-hexadecyl-ara-C were intensively examined, followed by several studieson the most effective derivative, N4-octadecyl-ara-C (NOAC), which is highly

Table 1 Liposome Formulations of Lipophilic Drugs and Corresponding LiteratureReferences

Lipophilic antitumor and antiviral drugs References

Dipalmitoyl-50-fluoro-20-deoxyuridine 1516

Cytosine arabinoside acyl prodrugs 1718

N4-alkyl derivatives of ara-C 1920

N4-hexadecyl-ara-C 21N4-octadecyl-ara-C 22

23242526272829303132333435

Phospholipid-N4-acyl- and N4-alkyl-ara-C conjugates 363738

Antiviral dinucleoside derivatives of AZT, ddC, ddI 394041424344

Amphiphilic antitumor dinucleosides 4546474849

Abbreviations: AZT, azidothymidine; ddC, dideoxycytidine; ddI, dideoxyinosine.

Incorporation of Lipophilic Antitumor and Antiviral Drugs 53

lipophilic and extremely resistant toward deamination (21–36). NOAC exertsexcellent antitumor activity after oral and parenteral therapy in mouse tumormodels (25,26,28,32,33). In contrast to the parent drug ara-C, the cellularuptake of NOAC is nucleoside-transporter independent and only insignificantamounts are phosphorylated to ara-C triphosphate. Furthermore, NOAC iscytotoxic in ara-C–resistant leukemia cells and treatment of multidrug-resistant tumor cells did not induce P-170 glycoprotein expression,suggesting that the N4-alkyl-ara-C derivatives are able to circumvent multi-drug resistance 1 (33). Owing to the fact that the N4-alkyl-ara-C drugs haveexcellent cytotoxic activities in solid tumors, we conclude that the mech-anisms of action of the N4-alkyl-ara-C derivatives are obviously distinctfrom ara-C (34,35).

More recently, we further modified NOAC by the synthesis of a newgeneration of lipophilic/amphiphilic duplex drugs that combine the clinicallyused antitumor drugs ara-C and 5-fluorodeoxyuridine (5-FdU) with NOAC,yielding the heterodinucleoside phosphates arabinocytidylyl-N4-octadecyl-1-b-D-arabinofuranosylcytosine (ara-C-NOAC) and 20-deoxy-5-fluorouridylyl-N4-octadecyl-1-b-D-arabinofuranosylcytosine (5-FdU-NOAC) (Fig. 1).Depending on the synthesis conditions, the duplex drugs are linked either via50!50 or 30!50 phosphodiester bridges (45,52). Due to the combination of theeffects of both active molecules that can be released in the cells as monomers oras the corresponding monophosphates, the cytotoxic activity of the duplex drugsis expected to be more pronounced as compared to the monomeric drugs.Further, it can be anticipated that monophosphorylated (MP) ara-C and5-FdU (5-FdU-MP), respectively, are directly released in the cell after enzymaticcleavage of the duplex drugs. Thus, MP molecules would not have to pass thefirst phosphorylation step, which is known to be rate limiting (46–49).

Ethynylcytidine [1-(3-C-ethynyl-b-D-ribopentafuranosyl)-cytosine] (ETC)is a novel nucleoside that was found to be highly cytotoxic (53–55). By com-bination of ETC with NOAC, we synthesized the new lipophilic derivativeNOAC-ETC [30-C-ethynylcytidylyl-(50!50)-N4-octadecyl-1-b-D-arabinofura-nosylcytosine]. The chemical structure of ETC-NOAC is shown in Fig. 1.

The chemical modification of the cytotoxic nucleosides ara-C, 5-FdU,ETC, and correspondingly of the antiviral nucleosides azidothymidine(AZT), dideoxycytidine (ddC) and dideoxyinosine (ddI) and their formulationin liposomes render these new heterodinucleoside compounds interestingcandidates for further developments.

MATERIALS AND METHODS

Chemicals

Soy phosphatidylcholine (SPC) was obtained from L. Meyer, Hamburg,Germany. Cholesterol (Fluka AG, Buchs, Switzerland) was recrystallized


from methanol. PEG-dipalmitoylphosphatidyl ethanolamine (PEG-DPPE,Mr 2750) was obtained from Sygena (Genzyme, Liestal, Switzerland).DL-a-Tocopherol, all buffer salts, and other chemicals used were of analyti-cal grade and obtained from Merck (Darmstadt, Germany) or Fluka.

Figure 1 Chemical structures of the 50!50 phosphodiester duplex drugsAra-C-NOAC, mol. wt. 801 g/mol, 5-FdU-NOAC, mol. wt. 804 g/mol, andETC-NOAC, mol. wt. 825 g/mol. Abbreviations: Ara-C-NOAC, arabinocytidylyl-(50!50)-N4-octadecyl-1-b-D-arabinofuranosylcytosine; 5-FdU-NOAC, 20-deoxy-5-fluorouridylyl-(50!50)-N4-octadecyl-1-b-D-arabinofuranosylcytosine; ETC-NOAC,30-C-ethynylcytidylyl-(50!50)-N4-octadecyl-1-b-D-arabinofuranosylcytosine.


Synthesis of Lipophilic Arabinofuranosyl Cytosine N4-AlkylDerivatives and Heterodinucleoside Phosphate Duplex Drugs

The heterodinucleoside duplex drugs ara-C-NOAC [arabinocytidylyl-(50!50)-N4-octadecyl-1-b-D-arabinofuranosylcytosine, mol. wt. 801 g/mol],5-FdU-NOAC [20-deoxy-5-fluorouridylyl-(50!50) -N4-octadecyl-1-b-D-ara-binofuranosylcytosine, mol. wt. 804 g/mol] and NOAC-ETC [30-C-ethynyl-cytidylyl-(50!50) -N4-octadecyl-1-b-D-arabinofuranosylcytosine, mol. wt.825 g/mol] were synthesized by condensation of NOAC via a 50!50

phosphodiester linkage to ara-C, 5-FdU or ETC, respectively, using themethod as described (45,52).

Preparation of Small Unilamellar Liposomes Containingara-C-NOAC, 5-Fdu-ara-C-NOAC, and NOAC-ETC

Stable lyophilized liposome preparations containing the lipophilic duplexdrugs were prepared as follows. The lipids SPC (40 mg/mL), cholesterol(4 mg/mL), D,L-a-tocopherol (0.2 mg/mL) and the drug (5–10 mg/mL) weredissolved in methanol/methylene chloride (1:1, v/v) in a round bottom flask.PEG-modified liposomes were obtained by addition of PEG(2000)-DPPE(14 mg/mL) to the basic lipid mixtures. After thorough removal of theorganic solvent on a rotatory evaporator (Buchi, Flawil, Switzerland) at40�C during 30 to 60 minutes, lipids and drugs were solubilized by additionof an appropriate volume of phosphate buffer (10 mM, pH 7.4) containing230 mM mannitol (Fluka, PB-mannitol) that serves as a cryoprotectant. Smallunilamellar liposomes were prepared by high-pressure filter extrusion through0.1-mm filters (Nuclepore, Sterico AG, Dietikon, Switzerland). The resultingliposomes were sterile filtrated (0.2mm, Gelman Sciences, Ann Arbor, Michi-gan, U.S.), aliquoted at appropriate volumes (e.g., 10 mL liposomes with 5 mgdrug/mL) into sterile vials, frozen in liquid nitrogen and lyophilized during 28to 48 hours (Dura-DryTM lyophilizer, FTS Systems, New York, U.S.A.). Thelyophilized liposomes can be reconstituted shortly before use with sterile wateror 0.9% sodium chloride. Liposome size and homogeneity were routinelymonitored by laser light scattering (Submicron Particle Sizer Model 370,Nicomp, Santa Barbara, California, U.S.A.).

The concentration of the duplex drugs ara-C-NOAC, 5-FdU-NOACor ETC-NOAC in the liposomes can be varied from 1 mg/mL to about10 mg/mL, depending on the concentration of phospholipids, the lipid com-position and the method of liposome preparation. The concentrations ofincorporated duplex drugs can be determined by reverse phase high pressureliquid chromatography (29,45). Reconstituted lyophilized liposomes retaintheir size and homogeneity longer than 72 hours after reconstitution. Thedrugs remain chemically stable during the freeze-drying process and reconsti-tution. Liposome preparations are ready for parenteral use within one to twohours after reconstitution.


RESULTS

In Vitro Cytotoxic Activity the Duplex Drugs

The cytotoxic activity of the duplex drugs was tested in vitro on B16F1mouse melanoma cells using a dye-reduction assay (47,48). The IC50 valuesfor the duplex drugs are shown in Figure 2. Furthermore, the antitumoractivities of ara-C-NOAC, 5-FdU-NOAC and ETC-NOAC were confirmedby the National Cancer Institute in vitro drug screening program where theyexerted cytotoxic activities in several tumors out of a panel of 60 humantumor cell lines of different types (data not shown).

Mechanisms of Action of Liposomal Duplex Drugs

In earlier studies, the duplex drugs 5-FdU-NOAC and ara-C-NOAC werefound to be strong inhibitors of the cell cycle, mainly arresting tumor cellsin the early S-phase (33,46–48). However, with liposome formulations ofthe duplex drugs cell uptake, intracellular distribution, biodistribution,and metabolism were not yet investigated in details. Due to their similarityto NOAC, it can be assumed that they have comparable pharmacological

Figure 2 In vitro cytotoxic activity on B16F1 melanoma cells of the duplex drugs inliposome formulations and of ETC dissolved in phosphate buffered saline. The cor-responding IC50 values in mM are shown on the bars. Abbreviations: ara-C-NOAC,arabinocytidylyl-N4-octadecyl-1-b-D-arabinofuranosylcytosine; 5-FdU-NOAC, 20-deoxy-5-fluorouridylyl-N4-octadecyl-1-b-D-arabinofuranosylcytosine; ETC, ethynylcy-tidine [1-(3-C-ethynyl-b-D-ribopentafuranosyl)-cytosine]; NOAC, N4-octadecyl-ara-C;PBS, phosphate buffered saline.


properties. In a study with 5-FdU-NOAC, we found that the drug was ableto overcome 5-FdU resistance in p53-mutated and androgen-independentDU-145 and PC-3 cells (48). It can be expected that the heterodinucleosidedimer is cleaved to 5-FdUMP and NOAC, resulting in sustained intracellu-lar drug concentrations over an extended period, consequently increasingduration and magnitude of the cytotoxic effect. This hypothesis is supportedby the fact that 5-FdU-NOAC specifically inhibits thymidylate synthetaseactivity and that it exerts a cell cycle phase-dependent cytotoxicity, twocharacteristic action mechanisms of 5-FdU (48). In contrast to the free drugs5-FdU and ara-C, the higher concentrations and longer incubation periodsthat are required in vitro to obtain similar cytotoxic effects of 5-FdU-NOAC can possibly be explained by a slow cell uptake and the prodrugnature of the duplex drug. This might result in persisting intracellularconcentrations of the active metabolites that lead to an enhanced cytotoxiceffect in vivo. The slow hydrolysis of the duplex drugs by phosphodi-esterase-I and human serum analyzed in vitro give further indications tothe prodrug nature of the compounds (33). Thus, due to expected changesof the pharmacokinetic properties and the prodrug character of 5-FdU-NOAC, the lipophilic duplex drug may have more favorable in vivo proper-ties than the individual compounds 5-FdU and NOAC. In a recent study,we demonstrated the superior antitumor effects of 5-FdU-NOAC contain-ing immunoliposomes targeted to the ED-B isoform of fibronectin, aprotein which is specifically expressed in the extracellular matrix of solidtumors (49).

CONCLUSIONS AND PROSPECTS

With the chemical transformation of water-soluble nucleosides into lipophi-lic compounds and their incorporation into the lipid membrane part ofliposomes as their carriers, we developed a new class of cytotoxic and anti-viral drug formulations that can be applied for the treatment of tumorsand viral infections by parenteral and, at least in the case of NOAC, possi-bly also by oral routes. Lipophilic ara-C derivatives, particularly thecomprehensively studied drug NOAC and the duplex drugs composed ofNOAC and ara-C, 5-FdU and ETC represent very promising new anti-cancer drugs of high cytotoxic activity, ability to circumvent resistancemechanisms, and strong apoptosis-inducing capability. Based on our find-ings, we conclude that due to the chemical modification of ara-C, 5-FdUand ETC to molecules that possess new physicochemical properties suchas high lipophilicity and stability against enzymatic degradation, togetherwith the possibility to prepare lyophilized liposome formulations of thesecompounds, more potent antitumor drugs are made available.

In previous studies performed with similar heterodinucleoside phos-phate dimers composed of the antivirally active nucleosides AZT, ddC and


ddI and formulated in liposomes, we found significantly different pharmaco-kinetic properties and superior antiviral effects in comparison to the parenthydrophilic nucleosides (39–41,55).

We conclude that the chemical modification of water-soluble mole-cules by attachment of long alkyl chains and their stable incorporation intothe bilayer membranes of small unilamellar liposomes represent a very pro-mising example of taking advantage of the high loading capacity lipidbilayers offer for lipophilic drugs. The combination of chemical modificationsof water-soluble drugs with their pharmaceutical formulation in liposomes is avaluable method for the development of novel pharmaceutical preparationsnot only for the treatment of tumors or infectious diseases, but also for manyother disorders.

ACKNOWLEDGMENTS

The authors thank Rosanna Cattaneo-Pangrazzi and Nils Schaffner fortheir valuable contributions.

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4

Liposome-Encapsulated Hemoglobin asan Artificial Oxygen Carrier

Vibhudutta Awasthi, Beth A. Goins, and William T. Phillips

Department of Radiology, University of TexasHealth Science Center at San Antonio,

San Antonio, Texas, U.S.A.

INTRODUCTION

Safety and adequate availability of blood is still a major concern in transfu-sion medicine. The requirement of pretransfusion processing, storage, andcrossmatching of blood are other factors that have given impetus to thesearch for safe, shelf-stable, and efficacious oxygen-carrying fluids. Severalfluids containing modified hemoglobin as an oxygen carrier are in advancedstages of preclinical or clinical trials (Table 1). Such preparations would savemany lives in situations of acute blood loss or when persons refuse homolo-gous blood transfusion on religious grounds. The earliest attempts to usepurified hemoglobin were met with several problems. Hemoglobin has atendency to break down into constituent a- and b-chains and has signifi-cant toxic effects upon administration (10–13). A simple approach ofcross-linking hemoglobin chains, diaspirin-cross-linked or a-a–cross-linkedhemoglobin, was the first attractive option to prevent its dissociation(6,14–16). Subsequently, hemoglobin was conjugated to poly(ethyleneglycol) or PEG to impart prolonged circulation (17). These small-sizedmodified hemoglobins extravasate through the endothelium and sequesternitric oxide (NO), causing a significant vasoconstriction (14,18–20). An alter-native design of recombinantly mutating hemoglobin to prevent NO-binding

63

and reduce oxygen affinity of hemoglobin is also currently under investiga-tion (21). All these modified hemoglobin preparations are essentially insolution phase and are not spatially isolated in the same manner as is thehemoglobin inside the red blood cells (RBCs). A more complete oxygencarrier would be hemoglobin that is encapsulated and supplemented withthe oxidoreductive system of RBC.

The encapsulation of hemoglobin within an artificial membrane wasfirst conceptualized by Chang in 1964 (22). Hemoglobin encapsulation hasseveral important advantages over free hemoglobin. Encapsulated hemoglo-bin has reduced vasoactivity compared to free hemoglobin (23–25). It alsomimics oxygen diffusivity of the RBCs (26) and is mostly metabolized bythe reticuloendothelial system (RES) similar to the red cells. Molecularlymodified free hemoglobins not only have short circulation half-life but alsobecome functionally inactive after a few hours in circulation. The latter ismainly driven by a gradual oxidation of hemoglobin in circulation. Closejuxtaposition of enzymes/cofactors that prevent irreversible damage to thehemoglobin molecule has a potential to circumvent this problem. Such anintimacy between enzymes, cofactors and hemoglobin is easily possible ifall ingredients are coencapsulated together. It is possible to coencapsulatenot only antioxidants, but also allosteric factors to modify oxygen affinityof hemoglobin. Toxicity-wise, encapsulation has a potential to mitigate pro-inflammatory and neurotoxic effects of free hemoglobin that can manifestduring resuscitation of subjects with traumatic brain injury (27–29).

One approach to compartmentalize hemoglobin is to encapsulatehemoglobin in biodegradable polymer-PEG-polylactide (30). These nano-capsules have a diameter of 80–150 nm and contain superoxide dismutase,catalase, carbonic anhydrase, and other enzymes of Embden-Meyerhofpathway that are needed for long-term function of an oxygen carrier (31,32).The polylactide capsules are metabolized in vivo to water and carbon

Table 1 Hemoglobin-Based Oxygen Carriers

Company Product Clinical trials Literature

Baxter, Illinois, U.S.A. Recombinant hemoglobin Phase I (1,2)Biopure,

Massachusetts,U.S.A.

Hemopure1 cross-linkedbovine polyhemoglobin

Approved inSouth Africafor human use

(3–5)

Northfield, Illinois,U.S.A.

Polyheme1 cross-linkedhuman polyhemoglobin

Phase III (6,7)

Sangart, California,U.S.A.

Hemospan1

MalPEG-hemoglobinPhase I/II

(Sweden)(8)

Hemosol, Ontario,Canada

Hemolink1 O-raffinosecross-linked hemoglobin

Phase III (9)

64 Awasthi et al.

dioxide; therefore, they do not accumulate in the RES. Another elegantapproach to hemoglobin encapsulation is based on liposomes (33–39). Thisencapsulated product has been variably termed hemoglobin vesicles, neo-redcells, or liposome-encapsulated hemoglobin (LEH) and contains highlyconcentrated (>36 g/dL) purified hemoglobin within the phospholipidmembranes (Fig. 1).

FORMULATION FACTORS INFLUENCING THECOMPOSITION OF LEH

The principal formulation goal for LEH is to use the least possible ingredi-ents of simple and well-defined characteristics without undermining theoxygen-carrying capacity of hemoglobin. Beside hemoglobin and lipid, afew additional components are added to the formulation for improvingstability and modifying oxygen affinity of the encapsulated hemoglobin.

Lipid Composition

LEH is primarily composed of a combination of saturated high-carbon phos-pholipids and cholesterol. Synthetic phospholipids replaced hydrogenatedsoy lecithin when the latter was found to induce several untoward biologicalresponses (40). Current choice of a saturated high-carbon phospholipid ismostly between distearoyl phosphatidylcholine (DSPC, Tm 55�C) and

Figure 1 Schematic illustration of liposome-encapsulated hemoglobin. Abbreviations:PEG, polyethylene gylcol; HB, hemoglobin.

Liposome-Encapsulated Hemoglobin as an Artificial Oxygen Carrier 65

dipalmitoyl phosphatidylcholine (DPPC, Tm 41�C). The phase transitiontemperature dictates the manufacturing method for LEH as well as its in vivobehavior. Although DSPC-LEH is more stable in vivo, it needs processing ata higher shear force as compared to DPPC-LEH. Like conventional lipo-somes, cholesterol (40–50 mol%) in LEH is added to impart rigidity to thebilayer. Lipids that polymerize upon ultraviolet (UV) irradiation have alsobeen tested for encapsulation of hemoglobin. The potential of hemoglobinoxidation by UV light is minimized by using carbonyl-hemoglobin as a pre-cursor (41). Polymerized LEH is thought to have a more stable physicalstructure and maintains particle size even after repeated freeze-thaw (42).Novel non-phospholipid LEH (polyoxyethylene-2-cetyl ether:cholesterol,3:1 M ratio) has also been reported, but it has not been studied further (43).

Hemoglobin interacts with the phospholipid bilayer by both hydro-phobic and ionic forces (44). The potential exists for mutual detrimentalprocesses, such as aggregation, peroxidative decomposition of unsaturatedfatty acids, unfolding of hemoglobin, oxidation of the heme iron, displace-ment of the heme relative to globin, and deconjugation of the porphyrinring. Ionic interaction seems stronger than the hydrophobic interactionand is dependent on pH and ionic strength of the medium (45). Hemoglo-bin encapsulated in egg-PC liposomes converts to methemoglobin morequickly than that encapsulated in saturated lipids (46). Anionic phospholi-pids enhance the rate of hemoglobin oxidation and displace heme relativeto globin (47). Cholesterol inhibits both the hydrophobic and ionic interac-tions between hemoglobin and lipid (44). Oxidation of lipids by hemoglobinis retarded by vitamin E and cholesterol (48). Any lysophospholipid impur-ity favors oxidative degradation of hemoglobin (49).

Hemoglobin encapsulation in the liposomes is best measured by aratio of hemoglobin to the total phospholipid content in a preparation(hemoglobin-to-lipid ratio, Hb/L). Although it has not been possible tomatch hemoglobin content of RBCs, it is desirable to encapsulate largeamounts of hemoglobin within a minimum amount of lipid. To improveencapsulation efficiency, anionic phospholipids, such as dimyristoyl- anddipalmitoyl-phosphatidylglycerol (DMPG and DPPG), are usually incorpo-rated in the formulation (50–52). Charged lipids enhance encapsulation byinteracting with oppositely charged domains of proteins. Using about9 mol% of DPPG in conjunction with optimal encapsulating conditions,Tsuchida et al. achieved an Hb/L of 1.61 (37,53). In addition to this bene-ficial interaction with hemoglobin, anionic lipids are known to undesirablyenhance interaction of liposomes with complement and other opsonizingproteins in vivo (54–56). Such interactions result in a rapid uptake ofLEH by the RES and toxic effects manifested as vasoconstriction, pulmon-ary hypertension, dyspnea, etc. It is possible to reduce the toxicity of anionicphospholipids by PEG modification of LEH surface (57). In view of thedrawbacks of anionic phospholipids, an amino acid–based synthetic anionic

66 Awasthi et al.

lipid, 1,5-dipalmitoyl-L-glutamate-N-succinic acid, has been used in LEH(58). It is believed that this amino acid–based lipid is better tolerated thananionic phospholipids, though a concern about its metabolic fate stillremains unresolved.

Surface Modification of LEH

LEH composed of phosphatidylcholine/cholesterol is rapidly eliminatedfrom the body by the RES. Often the elimination process is preceded by inter-action of particulate LEH, with complement and other opsonizing proteins,resulting in a form of pseudoallergic reactions. One way to circumvent thephysiological responses to LEH transfusion and to prolong its circulationis to conceal the liposome surface by steric modification. It is believed thata hydrophilic coating on the liposome surface creates a steric barrier,enabling liposomes to circulate longer (59). PEG-linked phosphatidylethano-lamines (PEG-PE) are the hydrophilic polymers most widely used. The otherbenefits of PEG-modification are the reduction of particle aggregation andmodification of LEH viscosity. These effects improve the flow propertiesof LEH through narrow capillaries (60). Ganglioside GM1 is another lipidwidely reported to increase the circulation time of liposomes, but in LEHit was found to be of little benefit in prolonging circulation (61).

Incorporation of PEG-PE in the liposome bilayer is most easily donewhen preparing the lipid phase just prior to its hydration with an aqueousphase (33). However, this technique results in the PEG brush or mushroomoccupying the limited space inside the liposomes. The same steric hindrancethat makes PEG useful may inhibit the encapsulation of substances by thisexclusion phenomenon (62). The smaller the size of the liposomes, thegreater is the impact of PEG on total usable space for encapsulated material.This conventional method of PEGylation also requires more PEG-lipid thanis needed for useful stealthing of a liposome. In the case of multi-lamellarliposomes, the magnitude of PEG-PE wastage is more (57). Realization ofthe problems associated with conventional PEGylation lead to the develop-ment of a technique where PEG-PE is inserted in the outer layer of liposomesafter the final manufacturing stages (63). This technique, called postinser-tion, is especially useful in the case of LEH. Technically, an aqueous solutionof PEG-PE is mixed with preformed LEH in such a volume that PEG-PE isbelow its critical micelle concentration (cmc). Below cmc, amphiphilic PEG-PE exists as a monomeric dispersion and it intercalates into the outer lipidlayer of LEH. The degree of incorporation is a function of PEG-chainlength, fatty acid, temperature, and concentration of lipids (64). Besides dou-bling the circulation T1/2 of LEH and reducing its accumulation in the RES,this postinsertion technique improves the encapsulation efficiency of hemo-globin (57). Postinsertion requires that the lipid bilayer remains in a rela-tively fluid state during the process. Therefore, reaction temperature is


maintained near Tm of the bilayer. Although this requirement poses a pro-blem with high-melting lipids (DSPC), hemoglobin oxidation and denatura-tion at high temperatures may be prevented by using carbonyl-hemoglobininstead of oxyhemoglobin.

Hemoglobin: Source and Properties

Hemoglobin is the major oxygen carrier in all vertebrates, but in practice,the source of hemoglobin for LEH is limited to that from human or bovineorigin. With rapid advances in biotechnology, it is fair to acknowledgerecombinant hemoglobin as a potential source of hemoglobin for LEH. Ithas also been possible to synthetically transform the protoheme moiety ofthe hemoglobin molecule into an amphiphilic compound, which enablesit to interact with phospholipid membranes of liposome for efficient encap-sulation and protects it from oxidative degradation (65). These innovativepreparations have not yet been used in LEH technology.

Currently, stroma-free human hemoglobin from outdated RBCs isthe source of hemoglobin for most LEH research and developmentaround the world. However, during early stages of development, purifiedbovine hemoglobin has been investigated for preparing LEH (66–69).Stability of hemoglobin during LEH manufacturing is a significant prob-lem. Through cold processing (4–8�C) and prevention of foam formation,hemoglobin is stabilized. Oxidation and denaturation of oxyhemoglobin isinevitable if it is exposed to high temperature. Use of deoxyhemoglobinis not recommended because of its tendency to bind oxygen and impractical-ity of maintaining an inert atmosphere during manufacturing. To preventoxidative damage, sometimes hemoglobin is first converted into carbonyl-hemoglobin before initiating the encapsulation procedure. Carbonylation isperformed by saturating hemoglobin with pure carbon monoxide. Carbonyl-hemoglobin is more stable than oxyhemoglobin and resists oxidativedenaturation, even at temperatures as high as 60�C for several hours (37).In practice, carbonyl-hemoglobin within the liposomes is readily convertedback to oxyhemoglobin by reoxygenating dilute LEH suspension underbright light (37).

Bovine hemoglobin appears to be a convenient and abundant sourcefor LEH preparation. Converting from a human to a bovine source of hemo-globin could have significant advantages in terms of the economics of LEHproduction. The deoxygenated and carbonylated bovine hemoglobin pre-parations have denaturation transition temperatures at 83�C and 87�C,respectively, which are higher than those of human hemoglobin and enablepasteurization (70). It has been shown that antibody response does not affectoxygen-binding properties of bovine hemoglobin in dogs (71); however,immunological response to multiple infusions of bovine hemoglobin-basedLEH is still a concern.

68 Awasthi et al.

The ability of hemoglobin to pick up oxygen in the lung and release it intissues is somewhat dependent on the allosterically controlled affinity ofhemoglobin for oxygen. Oxygen affinity of hemoglobin is measured as thepartial pressure of oxygen to saturate 50% of hemoglobin (p50). Normalp50 of human hemoglobin in RBCs is about 27 mmHg (72). Certain substan-ces called allosteric modifiers, such as 2,3-diphosphoglycerate, affect oxygenaffinity of hemoglobin (73). Other substances with hemoglobin-modifyingproperties similar to diphosphoglycerate are pyridoxal 50-phosphate andinositol phosphate (74). A synthetic compound, RSR13, is also capable ofchanging hemoglobin oxygen affinity (75). Bovine hemoglobin, on the otherhand, is responsive to chloride ion as an oxygen affinity modifier (76,77).Oxygen affinity of hemoglobin in LEH is easily altered by coencapsulationof one of these allosteric modifiers along with hemoglobin (58,78,79). Recentexperimental evidence has challenged a paradigm that the artificiallyassembled hemoglobin-based oxygen carriers should have a p50 close to thatof RBCs. It has been demonstrated that under mild hypoxia, a high p50 maybe helpful, whereas in severe hypoxia a low p50 may be beneficial (80). Ani-mal experiments also support the view that in severe blood loss, a low oxygenaffinity (8–12 mmHg) is beneficial (81–84). Interestingly, LEH with a low affi-nity also improves oxygen delivery and functional capillary density (85).

Antioxidants and Methemoglobin Reduction

Hemoglobin has a tendency to undergo auto-oxidation. Oxidation of hemo-globin increases the rate of heme loss, resulting in denaturation andprecipitation. Pure oxyhemoglobin is prone to auto-oxidation even at refri-gerated temperature. Deoxyhemoglobin does not undergo auto-oxidationand is known to be more stable against thermal and chemical denaturation(86). However, preparing oxygen-free hemoglobin is technically difficult be-cause partially saturated hemoglobin is more susceptible to auto-oxidationthan the fully oxygenated hemoglobin (87). Molecular oxygen binds toferrous atom of deoxyhemoglobin and can acquire one of the unpaired elec-trons of the ferrous’s outer shell to produce methemoglobin and superoxidefree radical [Eq. (1)]. Auto-oxidation also is a regular phenomenon insideRBCs; however, native hemoglobin is kept in functional form by a highlyspecialized enzymatic system. Cellular methemoglobin reductase, super-oxide dismutase, catalase, glutathione peroxidase, and glutathione reductasehelp keep the end products in control (88).

HbFe2þ þO2 ¼> HbFe2þ �O2 , HbFe3þ �O�2 ¼> HbFe3þ þO�2ð1Þ

Activities of the RBC enzymes depend on a constant supply of reducednicotinamide dinucleotides. Purified hemoglobin is not associated withRBC-like protection because most of the enzymes and cofactors are lost


during the separation process. Early attempts of using enzyme systems forin vitro reduction of methemoglobin were based on reductases (89,90). Thefirst application of such systems in the development of oxygen carriers wasreported by Hayashi et al. (91). In LEH, it is possible to coencapsulate com-ponents of the enzyme system with hemoglobin. LEH containing 0.1 mMb-NAD, 100 mM D-glucose, 2 mM adenine, 2 mM inosine, 1 mM MgCl2,1 mM KCl, 9 mM KH2PO4, and 11 mM Na2HPO4 has been reported toreduce methemoglobin formation from 1%/hr to 0.4%/hr at 37�C (92).The use of reductants (thiols, ascorbate, methylene blue, etc.) and enzymes(catalase and superoxide dismutase) has also been investigated (93–95).The effectiveness of the reductants depends upon their redox potential rela-tive to that of the ferrous-ferric system. Thus, homocysteine and glutathioneare more efficient than the easily oxidizable cysteine. Cysteine demonstratespro-oxidative behavior because of its rapid auto-oxidation and generation offree radicals (95). Coencapsulation of catalase (5.6� 104 U/mL) within LEHreduces peroxide-dependent methemoglobin formation (94). Coencapsula-tion of catalase and superoxide dismutase together with homocysteine(5–10 mM) in LEH improves the reduction process over homocysteine alone(95). These systems work well in vitro, but have a tendency to fail in vivo (93).In vivo, hemoglobin is saturated and desaturated continuously dependingupon the pO2, which ranges from 100 mmHg in the arterial blood to about20 mmHg in tissue capillaries. Considering that deoxyhemoglobin is proneto rapid oxidation and is produced at tissue pO2, its repeated encounters withoxygen in lung may be the reason for this observation (93). It is possible toreduce hemoglobin oxidation by adding reductants, such as methyleneblue and ascorbate in the external phase of LEH, but the external reductantsare rapidly consumed (93).

From a manufacturing viewpoint, a photochemical method of reducingmethemoglobin in the presence of flavin electron acceptor (96) has alsobeen applied to the reduction of methemoglobin in LEH (97). Visible lightirradiation (435 nm) of LEH containing flavin mononucleotide and ethylene-diamine tetraacetic acid rapidly reduces methemoglobin. But, there arepractical limitations to this method for achieving methemoglobin reduc-tion. First, the ethylenediamine tetra-acetic acid–based reaction producesunwanted side products, and second, the reaction requires significant dilu-tion of LEH suspension to allow visible light to penetrate and perform.

Particle Size

As an oxygen carrier, it is desirable that LEH circulates in blood withoutsignificant accumulation in any other organ. In general, the circulationhalf-life (T1/2

) of conventional liposomes decreases with increasing size, anio-nic charge density, and bilayer fluidity (98). Large liposomes (>200 nm)have short circulation T1/2

as they are rapidly eliminated from circulation

70 Awasthi et al.

by the RES through a complement-mediated phenomenon (56,99,100). Aliposome of large size may facilitate multiligand interactions or may beopsonized by a protein specific for large liposomes (101). Smaller liposomes(<200 nm) have lower RES uptake due to their reduced recognition bycirculating opsonins. On the other extreme, very small liposomes (�60 nm)accumulate in liver more than the intermediate-sized liposomes (102–104). Itappears that elevated liver accumulation of very small liposomes is due totheir access to the hepatocytes through the fenestrated hepatic endothelium(105). Incorporation of PEG-lipids in the liposomes inhibits liposome-induced complement activation and reduces RES uptake (99,106).

Besides influencing biodistribution, liposome size is also an importantdeterminant of encapsulation efficiency because as the size of the liposomesincreases, the entrapped volume increases for constant lamellarity (107).PEG-lipids reduce the requirement of small size for long circulation, buttheir influence is restricted within a size range. The ideal LEH formulationhas a size that is as large as possible while still retaining a PEG-mediatedprolonged circulation T1/2

. Beyond a certain size, the stealth property ofPEG-liposomes becomes insignificant and the distribution is characterizedby proportionately high RES accumulation (Fig. 2). It seems that a sizerange of 210–275 nm is the optimum size where PEG-liposomes still retainprolonged circulation (103). Above this size range, the circulation T1/2

islimited, whereas below this range the captured volume is considerablyreduced. Earlier, Maruyama et al. have also shown that circulation T1/2

ofDSPC vesicles drops off rapidly over 300 nm (108).

0.35

0.30

0.25

0.20

0.15

0.10

0.05

0.00

Inje

cted

dos

e pe

r gr

am (

%)

136.2 ± 5.9 nm165.5 ± 4.3 nm209.2 ± 9.5 nm275.0 ± 23.6 nm318.0 ± 6.0 nm

Liver Blood

Figure 2 Accumulation of liposomes of different sizes in liver and their retention inblood 24 hours after intravenous injection of Tc-99m-liposomes (distearoyl phos-phatidylcholine:Chol:PEG5000-DSPE:a-tocopherol: 90:80:4.5:3.9 M ratio) in rabbits.About 11–17 mg phospholipid was administered.


Colloid Oncotic Pressure and Isotonicity

Blood is a complex system with a variety of circulating proteins that notonly enhance the viscosity of blood, but also exert oncotic pressure in thevasculature. Oncotic pressure acts in opposition to the hydrostatic pressureand balances the fluid distribution between blood and interstitial fluidcompartments. Normal plasma has colloid oncotic pressure (COP) of about28 mmHg (109,110). Unencapsulated hemoglobin solutions exert oncoticpressure by virtue of their high hemoglobin concentration and any modi-fication of hemoglobin has a significant impact on its oncotic property(111). On the other hand, encapsulated hemoglobin has little impact onboth overall osmolarity and oncotic activity of LEH. Therefore, LEH byitself has a poor oncotic pressure. An adequate oncotic activity in LEH isnecessary to correct and maintain vascular volume deficit. It is made oncoti-cally active by adding albumin, pentastarch, or other such substances to thedispersion medium. LEH preparation with 5% extravesicular albumin has aCOP close to that of plasma.

Like COP, osmotic pressure is also a colligative property and dependson the number of species in solution. Osmotic properties of LEH dependupon the dispersion medium that usually contains salts and oncotic agents.Intuitively, a resuscitative fluid containing LEH should be isotonic withplasma because it will be administered in large quantities. However, in thecontext of recent evidence in support of hypertonic saline as a resuscitativefluid, it may not be always true (112–114). The widespread salutary effectsof hypertonic saline have made this fluid an attractive choice, especially whencombined with hyperoncotic colloid (112,115). Indeed, a combination ofLEH and hypertonic saline was found to be associated with improved bloodpressure, reduced acidosis, and increased survival in a rat model of 70% hypo-volemic shock (116). It is of interest to investigate the resuscitative propertiesof improved LEH formulations available today in combination with a hyper-tonic dispersion phase. The effect of hyperosmotic external medium on thelong-term physicochemical stability of LEH also needs further investigation.

Endotoxin

Contamination with endotoxin is an important and frustrating problem inLEH manufacturing for two reasons. Firstly, hemoglobin has a strongtendency to bind endotoxin, where one hemoglobin molecule binds to fourendotoxin molecules (Kd 3.1� 10�8 M) (117). Secondly, endotoxin hasamphiphilicity that enables its stable insertion into lipid bilayer. Such aninteraction not only presents contamination and stability problems, but alsohampers accurate quantitation of endotoxin. The best possible way to pre-vent endotoxin contamination is to use aseptic precautions with utmostcare. All the machinery, filters, and water should be endotoxin-free. Glassand metallic components may be dry-heat sterilized at about 200�C for three

72 Awasthi et al.

to four hours, while components that cannot be subjected to dry heat areautoclaved, washed with 0.2–0.5 M sodium hydroxide solution, andflushed with endotoxin-free water. For endotoxin testing in LEH, the com-mon limulus amebocyte lysate (LAL)-based methods are not applicable,unless the assay is modified to account for the presence of hemoglobin(118–120). By compensating for hemoglobin absorbance and by dilutingthe sample to maximum valid dilution (MVD), it is sometimes possible touse chromogenic methods for endotoxin testing in samples with smallamounts of hemoglobin. For highly concentrated preparations of hemo-globin, however, dilution to MVD may not work and a kinetic gel-clotmethod based on LAL may be the method of choice. The gel-clot methodis relatively insensitive to the presence of hemoglobin at MVD and canbe used effectively to monitor endotoxin (118). Even further modificationsof the gel-clot assay for endotoxin determination in LEH have been propo-sed, which use a proper detergent to solubilize the lipid bilayer (121) and ahistidine-immobilized agarose gel to concentrate LPS (122).

CURRENT MANUFACTURING TECHNOLOGY

Hemoglobin encapsulation has been attempted by most of the traditionalmethods of liposome manufacturing including freeze-thaw, sonication (123),reverse-phase evaporation (124), dehydration-rehydration (125), and detergentdialysis (126). Conservation of the oxygen-carrying property of hemoglobin andpreservation of particle size are the factors governing the method of LEH pre-paration. In addition, the ability to scale-up production of LEH in largevolumes is also an important determinant. The two techniques that have sur-vived these requirements are extrusion through membrane filters of uniformpore size and high-shear homogenization in a microfluidizer. Figure 3 showsa general scheme of LEH manufacturing involving these two techniques.

In the conventional extrusion method, the lipid-hemoglobin suspen-sion is sequentially passed through filters of decreasing pore size. Althoughpolycarbonate filters provide uniform-sized pores, they have a tendency torapidly clog when a highly concentrated lipid-hemoglobin suspension isextruded. Cellulose ester membranes are easier to work with, but they donot provide a sharp cutoff in pore size. To improve the rate of extrusionand encapsulation efficiency, and to impart reduced lamallarity to the LEH,anionic lipids are added to the formulation. For practical purposes, extru-sion temperature needs to be maintained close to the Tm of the lipid, butnot so high as to damage the hemoglobin. This condition limits the choiceof saturated phosphatidylcholine in LEH composition. Although hemoglo-bin can be stabilized by carbonylation, the successful laboratory method ofconverting CO-hemoglobin back to oxyhemoglobin is limited in scale-up.To ease the process of extrusion and to prevent the filter from clogging, aproliposome approach is sometimes recommended. In this approach, about


250 nm precursor liposomes are prepared by repeated extrusion, freeze-drying, and rehydration. This additional step produces a proliposomepowder with a stabilized and controlled size, which upon reconstitution withhemoglobin solution attains a particle size close to that of the proliposomes.Such a method is reported to yield highly uniform LEH, with large encapsu-lation efficiency, and is exemplified by a reproducibly high-quality LEH (58).Judicious selection of encapsulation conditions—such as pH, ionic strength,temperature, and content of anionic lipid—results in the maximization ofhemoglobin encapsulation (53). The extruder-produced LEH has beencharacterized in detail (37). Modern extruders have the capacity to producelarge amounts of LEH, in both continuous as well as batch operations.

Another useful method to produce LEH is based on homogenizationof lipid phase with hemoglobin solution in a high-shear microfluidizer(Microfluidics Corp., Newton, Massachusetts, U.S.A.). Simplicity andscalability are the major advantages of this technique. The capacities ofthe microfluidizers vary from few milliliters to several liters. The actual pro-cess of microfluidization and parameter setup has not changed too muchsince it was first used for LEH production (38,127). The microfluidizerforces the lipid-hemoglobin mixture at high pressure through an interactionchamber where two high-velocity streams of liquid collide against eachother, resulting in particle size reduction. The pressure is applied by a pneu-matically-driven pump. The final particle size of LEH depends upon the

Figure 3 Manufacturing scheme for liposome-encapsulated hemoglobin (LEH).Lipid phase is mixed with hemoglobin and the mixture is homogenized in an extruderor a microfluidizer. Unencapsulated hemoglobin is separated by filtration, beforePEGylation is performed by postinsertion. The resulting PEG-LEH is converted intooxyhemoglobin form and concentrated to obtain final product. Abbreviation: IXC,interaction chamber.

74 Awasthi et al.

channel size of the chamber, the number of times the suspension is passedthrough the chamber, lipid composition, the processing temperature, andpressure. Processing the material several times through the interactionchamber ensures uniformity in particle size; however, homogenous andcompact size distribution is difficult to obtain. It has been reported thatthe addition of 5–10 mg/mL albumin offers a limited control over postmi-crofluidization size distribution (128). In the current LEH manufacturingtechnique, Awasthi et al. use M110T model microfluidizer and pass the mix-ture 15 times through an interaction chamber (200–400 mm) under 20 to40 psi. It is possible to produce 175–250 nm LEH under these conditionswith optimization of the lipid formulation (57). Even with optimal proces-sing methods, the hemoglobin encapsulation efficiency is usually no morethan 5% to 25%. To improve the utilization of hemoglobin, recycling ofunencapsulated hemoglobin has been successfully investigated (129). Moreadvanced homogenizers that combine the high shear process of microflui-dizers and the size control of extruders have recently become availablefrom Avestin Inc. (Ottawa, Canada). Although not yet used for LEH man-ufacturing, in principle these combination-machines may offer a significantadvancement.

FREEZE-DRYING LEH

A long-recognized goal in LEH technology is to freeze-dry LEH for ease oftransportation and enhanced stability. It is a complex problem where oxi-dation and denaturation of hemoglobin as well as restoration of liposomesize upon reconstitution is a major challenge. Freeze-drying of pure hemoglo-bin alone results in large amounts of methemoglobin (130). Deoxygenationimproves its stability, especially if the oxygen is kept away after lyophiliza-tion also. Substances that are known to reduce hemoglobin oxidation duringfreeze-drying include macromolecules (Ficoll 70 and 400), amine buffers(Tris), amino acids (glutamate, b-alanine, etc.), and polyhydroxyls (manni-tol, glucose, trehalose, etc.). For example, at 0.25 M, glucose retains over95% of freeze-dried hemoglobin as oxyhemoglobin (131). With these protec-tive agents, freeze-drying hemoglobin is not an issue, but to keep it functionalis a significant challenge. The role of residual moisture in the function of pro-tective agents is not clear. Labrude et al. provided evidence that the protec-tive agents enhance hemoglobin stability by holding or reinforcing thecritical number of hydration molecules around hemoglobin (132). On theother hand, Pristoupil et al., reported that the presence of water moleculesfavors the oxidation and denaturation of hemoglobin (133).

Another concern with freeze-drying LEH is the instability of liposomestructure upon lyophilization. Vesicle formation occurs in the presence ofbulk water and when water is removed, loss of structural integrity is inevi-table. Fusion, crystal formation, and phase transition are observed, resulting


in leakage and increase in particle size upon rehydration. Liposomes reformwhen reconstituted with aqueous phase, but membrane rupturing and reseal-ing results in a loss of encapsulated contents. The leakage is primarily drivenby the repacking of bilayers after rehydration, not necessarily by vesicle fusion(134,135). Leakage after freeze-drying and rehydration also depends on vesiclesize, number of bilayers, and the presence of cholesterol. A small amount ofnegative charge in the bilayer significantly improves the stability of liposomes.Lyoprotection can also be afforded by adding sugars, which form an amor-phous, glassy matrix during freezing and exhibit low molecular mobility afterdrying. Besides glucose and sucrose, trehalose is the most widely investigatedsugar as cryoprotectant (136). Osmotic and volumetric properties of thesesugars and their interaction with phospholipid head groups reduce the Tm

of the bilayers in dry state. The liposome protection is not sugar-specific(136–138); however, the utility of cryoprotective sugars is influenced by thelipid composition of the liposomes (134) and the dry-mass ratio between thestabilizing sugar and the lipid (139). The choice of cryoprotectant is mainly dri-ven by the acceptable concentration of sugar to provide equivalent stabiliza-tion (136). Trehalose works well for freeze-drying liposomes under less thanoptimal conditions of processing and storage (136). Another important factorthat influences the ultimate particle size is the rate of freezing during lyophili-zation. It has been shown that slow freezing improves content retention insideliposomes (138,140).

First attempts to freeze-dry LEH were made in the late 1980s usingtrehalose and sucrose as cryoprotectants (141). More sucrose (0.5 M) wasneeded than trehalose (0.25 M) to achieve a similar degree of hemoglobinretention when the sugars were present both inside as well as outside theLEH. Hemoglobin oxidation also decreased, possibly due to the free-radicalscavenging property of the sugars (141). LEH with trehalose (0.15–0.25 M)was also reported to be structurally and functionally acceptable after threeand six months of dry storage at room temperature (142,143). The size ofLEH, methemoglobin concentration, p50, and hemoglobin retention afterrehydration were all within the functional range. When investigated in miceand rabbits, circulation persistence of lyophilized LEH was not significantlydifferent from that of the unlyophilized LEH (142,144). No hemodynamicor biochemical response was observed in rats administered with freeze-driedLEH, except a transient thrombocytopenia and marginal increase in serumthromboxane B2 (142,144). Since these early attempts with relatively prelimi-nary LEH formulations, no attempts at developing lyophilized formulationshave been made with improved LEH formulations available today.

STORAGE STABILITY

Shelf life of an LEH preparation is dependent on the change in particle size andoxidative damage to the lipid and hemoglobin upon storage. LEH tends to

76 Awasthi et al.

sediment on storage unless it is inhibited by a steric factor or charge-basedrepulsion. PEG-lipid provides steric inhibition while incorporation of anionicphospholipid improves storage stability by repulsion among liposome parti-cles in suspension. Empirically, about 2 mol% of anionic lipid or 0.3 mol%of postinserted PEG-lipid (78) is capable of preventing LEH aggregation. Inregard to oxidative degradation of LEH, both hemoglobin and phospholipidsare sensitive to oxidation and their oxidative products are mutually destructive(145). The question of lipid oxidation in liposomes has been reviewed severaltimes and it has been shown that the inclusion of a-tocopherol retards lipidperoxidation by free radicals (146–148). Hemoglobin oxidizes to methemoglo-bin in a temperature-sensitive reaction; about 50% of stroma-free hemoglobinturns into methemoglobin within 24 hours when incubated in ambient air at37�C. The reaction is slowed if the LEH is completely deoxygenated bypurging it with an inert gas (nitrogen) during storage. It should be kept inmind that partial deoxygenation increases methemoglobin formation. LEHpreparations, absolutely free of oxygen, have been shown to be amenable tostorage even at room temperature (25�C) for a prolonged duration (78).

EVALUATION TECHNIQUES

Considering that LEH is still in a developmental phase, it is imperative thateach batch of LEH is fully characterized for physicochemical and biologicalproperties. Typically, lipid content, particle diameter, oxygen affinity, hemo-globin, methemoglobin, carbonyl-hemoglobin, oncotic pressure, viscosity,endotoxin, and osmolality are determined by conventional methods. Severalissues that need specific attention in the case of LEH are as follows:

1. Turbidity caused by the presence of lipid results in significant lightscattering. If not taken into consideration, in spectrophotometry-based hemoglobin estimation, the latter leads to erroneousinterpretation.

2. Methemoglobin estimation in LEH is particularly problematicbecause it entails separation of encapsulated hemoglobin from thelipid phase while preserving the oxyhemoglobin-to-methemoglobinratio. Unfortunately, the current methods are prone to convertoxyhemoglobin into methemoglobin or are unable to extract hemo-globin and methemoglobin completely from the liposomes. Apossible solution is to employ electron paramagnetic resonanceto determine Fe3þ iron in LEH (149).

3. The fact that PEGylation of LEH is done by postinsertion neces-sitates the estimation of incorporated PEG-lipid in the outer layerof LEH.

4. The presence of extravesicular hemoglobin may also significantlyinfluence the apparent size determined by dynamic light scattering


because of its interaction with anionic lipid in the liposomebilayer (150).

5. Rheologically, LEH behaves as a non-Newtonian fluid and theviscosity of the product depends upon the particle density and pre-sence of solutes and macromolecules in the dispersion phase ofLEH (37,151).

6. As discussed previously, endotoxin is a serious issue in LEHmanufacturing and a modified LAL-based gel clot assay is recom-mended to determine endotoxin in LEH (122).

Upon administration in animal models of shock, LEH is expected tocorrect volume deficit, salt imbalance, and tissue hypoxia for a prolongedperiod of time. Although LEH is formulated to provide these capabilities,in vivo results are not always ideal. LEH should be evaluated for biodistri-bution, circulation, immunological effects, survival, toxicity, histopathol-ogy, physiology, and oxygen delivery in animal models. Depending uponthe goals of the study, the animal species that have been tested in variouslaboratories include mouse, rat, rabbit, hamster, dog, pig, and baboon. Itis not the object of this chapter to chronologically review the literature inthis respect. Continuous improvements in all aspects of LEH technologynecessitate that only recent advances be discussed.

Biodistribution

LEH is taken up by the organs of RES. Opsonization of the vesicles byimmunoproteins is the most likely path of LEH clearance; however, anopsonin-independent pathway of LEH clearance has been reported wheremonocytic uptake of LEH is blocked by anti-CD14, anti-CD36, and anti-CD51/61 antibodies (152). Like conventional liposomes, the extent of RESuptake depends on several factors including size, lipid composition, dose,surface modification, and the animal species. The development of stable la-beling of liposomes with the gamma-emitting radionuclide, technetium-99m(Tc-99m), opened the possibility of utilizing nuclear imaging to follow LEHdisposition (153). This method has been extensively used to evaluateLEH biodistribution in small animals (Fig. 4). When Tc-99m-LEH(DPPC, cholesterol DPEA, PEG-distearoylphosphatidylethanolamine,5:5:1:0.033 M ratio) was administered (top-load, 14 mL/kg), RES organsaccounted for about 27% and 14% in rats and rabbits, respectively, after48 hours; circulating LEH was 33.3% (rat) and 51% (rabbit) (154). It isimportant to note that the RES is also the normal route for eliminationof senescent RBCs. The accumulated LEH disappears from liver and spleenwithin a week of infusion (155). In a slightly different formulation of LEH(DSPC, cholesterol, vitamin E, 51.4:46.4:2.2), administration of a small dose(2 mL) of LEH in rabbits yielded 14% circulating LEH and enormousaccumulation in liver (52.1%) after 24 hours. PEGylation of the same

78 Awasthi et al.

formulation improved the circulating LEH to 40% and reduced the liveruptake to 19.1% of administered dose at 24 hours (57). In contrast to theliver uptake, PEGylation increased the spleen uptake by 8.5-fold. Whenthe same formulation of LEH was doped with 10% of anionic lipid(DMPG), the liver uptake of LEH dropped from 35% (un-PEGylated) to11.5% (PEGylated). A larger dose (25% of circulating blood) of the sameLEH in a rodent model of 25% hypovolemia demonstrated 18% and 31%of circulating LEH after 48 hours in rats and rabbits, respectively (129).Liver uptake (rats, 10.3% and rabbits, 5.4%) and spleen (rats, 2.4% and rab-bits, 0.8%) showed a corresponding drop. Using nuclear imaging, it has beenpossible to visualize the temporal accumulation of LEH in liver and spleen(33,156,157). The relative contribution of these organs to the LEH metabo-lism is a function of liposome size, composition, and the presence of surfacePEG-coating. These recent studies demonstrate a significant improvement inthe handling of LEH in animal models, mostly attributed to the advances informulation and manufacturing technology.

Circulation T1/2 vs. Functional T1/2

It is desired that the intravascular persistence of LEH in humans should be atleast five to seven days, which is the time the body needs to regenerate RBCs(158,159). Although circulation T1/2

of LEH is governed by its RES-basedelimination from circulation, functional capacity of circulating LEH is deter-mined by the rate of hemoglobin-to-methemoglobin conversion. Onceadministered, methemoglobin is generated at a rate of greater than 1% to2% per hour (92). Although it is not possible to prevent this process, it ispossible to retard hemoglobin oxidation by coencapsulating catalase and

Figure 4 A set of gamma camera images of a rabbit intravenously injected withTc-99m-LEH. Twenty-five percent of blood was exchanged with liposome-encapsulated hemoglobin (LEH) (hypovolemic) and the animal was imaged usinga gamma camera at various times after infusion. The images clearly show a pro-longed circulation of LEH as evidenced by the continued grayscale intensity in heart.


cofactors (94). Circulation T1/2of LEH is also affected by the amount admi-

nistered (160). A large dose of LEH exhausts the endocytotic or the plasmaopsonizing capacity and tends to increase the circulating liposomes in blood(51). On the basis of circulation kinetics in 25% hypovolemic exchange mod-els, the half-life of 99mTc-LEH in blood was 30 and 39.8 hours in rats and rab-bits, respectively. Although it is difficult to accurately estimate circulation T1/2

of LEH in humans on the basis of animal experiments, it has been roughly esti-mated that a circulation T1/2

of 12 to 20 hours in rats translates into 40 to 60hours in humans (161). Based on this relationship, 30 hours T1/2

of currentformulation of LEH in rats would likely have a T1/2

of over 90 hours in humans.

Toxicity and Immunological Effects of LEH

Encapsulation of hemoglobin subdues the toxicity observed with free hemo-globin, but the liposome itself has a typical toxicity profile. The lipidcomposition of LEH has a significant impact on LEH toxicity. LEH madeup of hydrogenated soy lecithin induces several transient untoward biologicalresponses, such as hypertension, tachycardia, thrombocytopenia, hemocon-centration, and elevation of thromboxane B2. Use of synthetic DSPC in placeof soy lecithin significantly improves this toxicity profile (40). The fact thatplatelet activating factor (PAF) antagonist BN 50739 prevents these hemody-namic changes indicates that PAF is partially responsible for these effects ofLEH (162). Liposomes also have a tendency to interact with complement pro-teins and cause pulmonary hypertension and other hypersensitivity reactionsin patients (100,163,164). A marked reduction in circulating thrombocyteshas also been observed in animals (165). Incorporation of anionic phospho-lipid in the LEH composition aggravates thrombocytopenic reaction. Theseeffects have been grouped within complement activation-related pseudoal-lergy or CARPA (56,100). LEH-mediated complement activation is depen-dent on the particle size, size distribution, charge, and surface coating. Itmay be partially prevented by PEGylation, uniform size distribution, andby reducing the content of anionic lipids.

In an attempt to investigate the effect of LEH on host immune res-ponse to infection, Sherwood et al. injected phosphatidylinositol or PEG-PEcontaining LEH in mice challenged with Listeria monocytogenes (166). LEHenhanced mortality and reduced in vitro phagocytic activity of rat alveolarmacrophages (166). With regard to the potential of generating antibodyresponse, LEH with homologous hemoglobin injected subcutaneously in ratswith or without Freund’s adjuvant were found to be nonantigenic. Heterolo-gous hemoglobin showed an insignificant increase in antibody titers withFreund’s adjuvant (167). How these results affect the possible use of bovinehemoglobin in LEH preparation is not yet clear.

Administration of large doses of LEH may also result in RES toxicity.It has been shown that LEH causes only a transient change in plasma

80 Awasthi et al.

enzyme profiles in rats without any irreversible damage to the RES (155,168).A similar observation was made earlier when LEH was administered in mice(25% top-load); liver and spleen abnormalities were transient and disappearwithin one to two weeks with no significant alteration of blood chemistry(142). The animals receiving LEH (20 mL/kg body weight, phospholipid4 g/dL) show splenomegaly for several days after infusion, but the total bodyweight tends to remain normal (155). The phagocytic index initially dropsbefore reversibly increasing for several days beyond the basal level (155).

Physiological and Survival Studies in Animal Models of Shock

The ultimate object of LEH is to globally improve physiology of shock andenhance survival in acute blood loss. Cardiovascular parameters—such asmean arterial pressure, cardiac output, heart rate, blood gas, and mixedvenous oxygen tension—are usually measured to evaluate the physiologicalresponses to LEH infusion in animal models of shock. The most compre-hensive assessment of LEH efficacy has been performed by Tuschida andcolleagues. Unlike albumin solution, LEH (10 g/dL in 5% albumin) main-tained mean arterial blood pressure, heart rate, aortic blood flow, andperipheral resistance near basal levels when up to 90% of circulating bloodin rats was exchanged with LEH (169). The LEH also preserved oxygendelivery and consumption similar to the homologous RBCs for at least30 minutes after the completion of the exchange; however, renal corticaloxygen tension and skeletal muscle oxygenation were significantly below thelevel seen in animals transfused with RBCs (169). Lower tissue oxygenationwith LEH may be due to the loss of shear-induced vasodilation or thecomplement-mediated vasoconstriction (60). Exchange transfusion (90%)with PEGylated LEH shows higher blood flow in abdominal aorta thanthat with LEH without PEG (60). It is possible that the nonaggregatingbehavior of PEG-LEH improves its rheological properties in small capil-laries. PEG-LEH was also shown to supply oxygen better than the LEHwithout PEG (60). At the microvascular level, LEH was found to improvesubcutaneous blood flow and tissue oxygenation in a conscious hamstermodel of 50% shock (25). With regard to the efficacy of LEH to improvesurvival, rats with 50% hypovolemic shock receiving LEH suspended inrecombinant albumin showed 100% six-hour survival as compared to only75% six-hour survival of rats infused with albumin alone (170). Althoughimportant, not many long-term survival studies are reported in the literature.

SUMMARY

Spatial separation of hemoglobin confers many desirable properties to theLEH. Yet, like other hemoglobin-based oxygen carriers, it is not a completeresuscitation fluid for hypovolemic shock. Intervention in severe hemorrhage


requires more than what is provided by enhanced oxygen-carrying capacityin the resuscitation fluid. At the same time, pathophysiology related tohemorrhage calls for additional components, such as oncotic and hemostaticagents. Based on these facts, the artificially assembled hemoglobin prepara-tions are rightly described as oxygen therapeutics rather than as bloodsubstitutes. A complex LEH formulation that is stable over a wide rangeof temperatures, contains inhibitors of complement activation, reperfusioninjury, oncotic substance, and has long circulation and functional T1/2

withminimal toxicity, needs further development. At the same time, many ofthe important milestones have been accomplished and LEH continues toshow promise as part of an advanced oxygen-carrying resuscitative fluid.

ACKNOWLEDGMENT

The authors acknowledge the grant support from the Office of NavalResearch, Washington, D.C.

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116. Rabinovici R, Rudolph AS, Vernick J, et al. A new salutary resuscitative fluid:liposome encapsulated hemoglobin/hypertonic saline solution. J Trauma 1993;35:121.

117. Jurgens G, Muller M, Koch MH, et al. Interaction of hemoglobin with enter-obacterial lipopolysaccharide and lipid A. Physicochemical characterizationand biological activity. Eur J Biochem 2001; 268:4233.

118. Cliff RO, Kwasiborski V, Rudolph AS. A comparative study of the accuratemeasurement of endotoxin in liposome-encapsulated hemoglobin. Artif CellsBlood Substit Immobil Biotechnol 1995; 23:331.

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119. Roth RI, Levin J. Measurement of endotoxin levels in hemoglobin prepara-tions. Meth Enzymol 1994; 231:75.

120. Vanhaecke E, Pijck J, Vuye A. Endotoxin testing. J Clin Pharm Ther 1987;12:223.

121. Harmon P, Cabral-Lilly D, Reed RA, et al. The release and detection of endo-toxin from liposomes. Anal Biochem 1997; 250:139.

122. Sakai H, Hisamoto S, Fukutomi I, et al. Detection of lipopolysaccharide inhemoglobin-vesicles by Limulus amebocyte lysate test with kinetic-turbidimetricgel clotting analysis and pretreatment of surfactant. J Pharm Sci 2004; 93:310.

123. Djordjevich L, Miller IF. Synthetic erythrocytes from lipid encapsulated hemo-globin. Exp Hematol 1980; 8:584.

124. Hunt CA, Burnette RR, MacGregor RD, et al. Synthesis and evaluation of aprototypical artificial red cell. Science 1985; 230:1165.

125. Shew RL, Deamer DW. A novel method for encapsulation of macromoleculesin liposomes. Biochim Biophys Acta 1985; 816:1.

126. Jopski B, Pirkl V, Jaroni HW, et al. Preparation of hemoglobin-containing lipo-somes using octyl glucoside and octyltetraoxyethylene. Biochim Biophys Acta1989; 978:79.

127. VidalNaquet A, Gossage JL, Sullivan TP, et al. Liposome-encapsulated hemo-globin as an artificial red blood cell: characterization and scale-up. BiomaterArtif Cells Artif Organs 1989; 17:531.

128. Moynihan KL. United States Patent and Trademark Office, NeXstar Pharma-ceuticals, Inc.: USA, Patent no. 5,589,189 (1996).

129. Awasthi VD, Garcia B, Klipper R, et al. Kinetics of liposome-encapsulatedhemoglobin after 25% hypovolemic exchange transfusion. Int J Pharm. 2004;283:53.

130. Labrude P, Chaillot B, Vigneron C. Influence of physical conditions on the oxi-dation of hemoglobin during freeze-drying. Cryobiology 1984; 21:33.

131. Chaillot B, Labrude P, Vigneron C, et al. Freeze-drying of hemoglobin solu-tions without adjuvant and in presence of glucose, tris, and beta-alanine: astudy by electron spin resonance of the oxidized compounds produced. Am JHematol 1981; 10:319.

132. Labrude P, Chaillot B, Vigneron C. Problems of haemoglobin freeze-drying:evidence that water removal is the key to iron oxidation. J Pharm Pharmacol1987; 39:344.

133. Pristoupil TI, Kramlova M, Fortova H, et al. Haemoglobin lyophilized withsucrose: the effect of residual moisture on storage. Haematologia (Budap)1985; 18:45.

134. van Winden EC, Crommelin DJ. Short term stability of freeze-dried, lyopro-tected liposomes. J Control Rel 1999; 58:69.

135. Zhang W, van Winden EC, Bouwstra JA, et al. Enhanced permeability offreeze-dried liposomal bilayers upon rehydration. Cryobiology 1997; 35:277.

136. Crowe JH, Crowe LM, Oliver AE, et al. The trehalose myth revisited: introductionto a symposium on stabilization of cells in the dry state. Cryobiology 2001; 43:89.

137. Strauss G, Schurtenberger P, Hauser H. The interaction of saccharides withlipid bilayer vesicles: stabilization during freeze-thawing and freeze-drying. Bio-chim Biophys Acta 1986; 858:169.


138. van Winden EC, Zhang W, Crommelin DJ. Effect of freezing rate on the stabilityof liposomes during freeze-drying and rehydration. Pharm Res 1997; 14:1151.

139. Crowe JH, Crowe LM. Factors affecting the stability of dry liposomes. BiochimBiophys Acta 1988; 939:327.

140. van Winden EC. Freeze-drying of liposomes: theory and practice. MethEnzymol 2003; 367:99.

141. Rudolph AS. The freeze-dried preservation of liposome encapsulated hemoglo-bin: a potential blood substitute. Cryobiology 1988; 25:277.

142. Cliff RO, Ligler F, Goins B, et al. Liposome encapsulated hemoglobin: long-term storage stability and in vivo characterization. Biomater Artif Cells Immo-bil Biotechnol 1992; 20:619.

143. Rudolph AS, Cliff RO. Dry storage of liposome-encapsulated hemoglobin: ablood substitute. Cryobiology 1990; 27:585.

144. Rudolph AS, Cliff RO, Klipper R, et al. Circulation persistence and biodistri-bution of lyophilized liposome-encapsulated hemoglobin: an oxygen-carryingresuscitative fluid. Crit Care Med 1994; 22:142.

145. Takeoka S, Teramura Y, Atoji T, et al. Effect of Hb-encapsulation with vesicleson H2O2 reaction and lipid peroxidation. Bioconugate Chem 2002; 13:1302.

146. Chatterjee SN, Agarwal S. Liposomes as membrane model for study of lipidperoxidation. Free Radic Biol Med 1988; 4:51.

147. Grit M, Crommelin DJ. Chemical stability of liposomes: implications for theirphysical stability. Chem Phys Lipids 1993; 64:3.

148. McCay PB. Vitamin E: interactions with free radicals and ascorbate. Annu RevNutr 1985; 5:323.

149. Abugo OO, Balagopalakrishna C, Rifkind JM, et al. Direct measurements ofhemoglobin interactions with liposomes using EPR spectroscopy. Artif CellsBlood Substit Immobil Biotechnol 2001; 29:5.

150. Pitcher WH III, Huestis WH. Preparation and analysis of small unilamellarphospholipid vesicles of a uniform size. Biochem Biophys Res Commun2002; 296:1352.

151. Djordjevich L, Kashani A, Miller IF, et al. Measurements of viscosity of syn-thetic erythrocyte suspensions. Biorheology 1987; 24:207.

152. Shibuya-Fujiwara N, Hirayama F, Ogata Y, et al. Phagocytosis in vitro ofpolyethylene glycol-modified liposome-encapsulated hemoglobin by humanperipheral blood monocytes plus macrophages through scavenger receptors.Life Sci 2001; 70:291.

153. Phillips WT, Rudolph AS, Goins B, et al. A simple method for producing atechnetium-99m-labeled liposome which is stable in vivo. Nucl Med Biol1992; 19:539.

154. Sou K, Klipper R, Goins B, et al. Circulation kinetics and organ distribution ofhb-vesicles developed as a red blood cell substitute. J Pharmacol Exp Ther2005; 312:702.

155. Sakai H, Horinouchi H, Tomiyama K, et al. Hemoglobin-vesicles as oxygencarriers: influence on phagocytic activity and histopathological changes in reti-culoendothelial system. Am J Pathol 2001; 159:1079.

156. Goins B, Klipper R, Sanders J, et al. Physiological responses, organ distribu-tion, and circulation kinetics in anesthetized rats after hypovolemic exchange

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transfusion with technetium-99m-labeled liposome-encapsulated hemoglobin.Shock 1995; 4:121.

157. Goins BA, Phillips WT. The use of scintigraphic imaging as a tool in the devel-opment of liposome formulations. Prog Lipid Res 2001; 40:95.

158. Hughes GS Jr., Francome SF, Antal EJ, et al. Hematologic effects of a novelhemoglobin-based oxygen carrier in normal male and female subjects. J LabClin Med 1995; 126:444.

159. Sehgal LR, Gould SA, Rosen AL, et al. Polymerized pyridoxylated hemoglo-bin: a red cell substitute with normal oxygen capacity. Surgery 1984; 95:433.

160. Laverman P, Brouwers AH, Dams ET, et al. Preclinical and clinical evidencefor disappearance of long-circulating characteristics of polyethylene glycol lipo-somes at low lipid dose. J Pharmacol Exp Ther 2000; 293:996.

161. Woodle MC, Newman MS, Working PK. Biological properties of sterically sta-bilized liposomes. In: Lasic DD, Martin F, eds. Stealth Liposomes. BocaRaton: CRC Press, 1995:103.

162. Rabinovici R, Rudolph AS, Yue TL, et al. Biological responses to liposome-encapsulated hemoglobin (LEH) are improved by a PAF antagonist. CircShock 1990; 31:431.

163. Chanan-Khan A, Szebeni J, Savay S, et al. Complement activation followingfirst exposure to pegylated liposomal doxorubicin (Doxil): possible role inhypersensitivity reactions. Ann Oncol 2003; 14:1430.

164. Parnham MJ, Wetzig H. Toxicity screening of liposomes. Chem Phys Lipids1993; 64:263.

165. Phillips WT, Klipper R, Fresne D, et al. Platelet reactivity with liposome-encapsulated hemoglobin in the rat. Exp Hematol 1997; 25:1347.

166. Sherwood RL, McCormick DL, Zheng S, et al. Influence of steric stabilizationof liposome-encapsulated hemoglobin on Listeria monocytogenes host defense.Artif Cells Blood Substit Immobil Biotechnol 1995; 23:665.

167. Chang TM, Lister C, Nishiya, et al. Immunological effects of hemoglobin,encapsulated hemoglobin, polyhemoglobin and conjugated hemoglobin usingdifferent immunization schedules. Biomater Artif Cells Immobil Biotechnol1992; 20:611.

168. Sakai H, Horinouchi H, Masada Y, et al. Metabolism of hemoglobin-vesicles(artificial oxygen carriers) and their influence on organ functions in a rat model.Biomaterials 2004; 25:4317.

169. Izumi Y, Sakai H, Kose T, et al. Evaluation of the capabilities of a hemoglobinvesicle as an artificial oxygen carrier in a rat exchange transfusion model.ASAIO J 1997; 43:289.

170. Sakai H, Masada Y, Horinouchi H, et al. Hemoglobin-vesicles suspended inrecombinant human serum albumin for resuscitation from hemorrhagic shockin anesthetized rats. Crit Care Med 2004; 32:539.


5

An Original Lipid Complex Systemfor Amphotericin B

Malika Larabi

Department of Radiology/Nuclear Medicine, Lucas MRS Imaging Center,Stanford, California, U.S.A.

Philippe Legrand and Gillian Barratt

Universite Paris-Sud, Chatenay-Malabry, France

INTRODUCTION

Amphotericin B (AmB) is a broad-spectrum antifungal agent that is theantibiotic of choice for disseminated fungal infections, particularly in immu-nocompromised patients. AmB is also used for the treatment of Leishmaniasisas a second-line treatment. However, its toxicity toward mammalian cells isoften dose limiting, whatever its indication.

AmB is a large, amphiphilic molecule (Fig. 1). It is generally acceptedthat its antimicrobial action can be attributed to complexation of ergosterolin the fungal or parasite membrane, leading to pore formation (2) at concen-trations below the critical micellar concentration of the antibiotic. However,AmB also undergoes self-association to form first dimers followed by largeraggregates at higher concentrations, which is a major cause of its toxicitytoward mammalian cell membranes.

AmB has very low bioavailability by the oral route and so has to begiven intravenously for the treatment of systemic infections. Due to its in-solubility in aqueous media, the traditional formulation is mixed micelles withdeoxycholate (Fungizone1, Bristol-Myers Squibb). However, the micelles

93

are unstable on dilution in the bloodstream and release AmB in the self-associated toxic form. AmB can also be transferred from micelles tolipoproteins, and the uptake of these particles by host cells, particularly inthe kidney, is a contributing factor to toxicity.

For over two decades, research has been directed toward incorporatingAmB into lipid-based drug delivery systems in order to reduce its toxicity.Based on pioneering work in the 1980s (3), a large number of systemshave been developed and three of them are now commercially available.Lipid-based systems are able to retain AmB better than the surfactantmicelles and release it slowly in the form of monomers, thus reducing toxicityto mammalian cells (2). Among other formulations that have been proposedwe can cite emulsions (4) and nanoparticles (5,6), as well as chemical modi-fication of the AmB molecule (7,8) and associations with other drugs.

The three commercially available AmB formulations differ in theirmorphology, their composition, and, consequently, their biological activity.AmBisome1 (developed by Nexstar Pharmaceuticals, commercially availablefrom Gilead Sciences) is a true liposome formulation, consisting of small,unilamellar vesicles (9). Their small size confers a prolonged circulation time

Hydrophobic stretch

Hydrophobic stretch

Hydrophilic stretch

Hydrophilic stretch

Myc

osam

ine

ring

OH

CO

OH

OH

OH OH

OH

OH

OH

OH

OH

OH

CH

3

NH

3

CH

3

CH

3

CH

3

H

HH

H

H

HH

H H H

H

H O

O

OO

H

H

H

HH

O

Figure 1 Structure of amphotericin B. Source: From Ref. 1.

94 Larabi et al.

and favors penetration into the tissues, but the AmB loading is relativelylow, representing only 5 mol%. The other formulations are lipid complexesand achieve higher payloads. Amphotec1 (developed by Liposome Technol-ogy Inc. and commercially available from Intermune) is composed of smalldisc-like particles combining AmB with cholesterol sulfate in equimolar pro-portions (10). On the other hand, Abelcet1 (developed by The LiposomeCompany Ltd. and commercially available from Zedeus Pharma) combinesAmB with two synthetic phospholipids in ribbon-like assemblies that can beup to 5 mm in length. The antibiotic represents about a third in molar pro-portions (11). The large size of Abelcet particles could be a limitation in thetreatment of disseminated disease (12).

All three of these new AmB formulations improve the therapeuticindex of the drug considerably compared with Fungizone. Larger cumu-lative doses can be given, leading to cures that were hitherto impossible.However, they are expensive and thus inaccessible in developing countrieswhere their use would be of most benefit (13).

The aim of the research carried out in our laboratory was to develop alipid formulation that could be manufactured by a simple and reproducibleprocedure. In particular, we wanted to produce phospholipid-based parti-cles with a high AmB loading coupled to a small particle size.

The procedure chosen for the preparation of lipid complexes of AmBwas nanoprecipitation. This procedure has been developed in our laboratoryfor a number of years and can be applied to the formulation of a number ofdifferent colloidal systems: liposomes, microemulsions, polymeric nanoparti-cles (nanospheres and nanocapsules), complexes, and pure drug particles(14–16). Briefly, the substances of interest are dissolved in a solvent A and thissolution is poured into a nonsolvent B of the substance that is miscible withthe solvent A. As the solvent diffuses, the dissolved material is ‘‘stranded’’ assmall particles, typically 100 to 400 nm in diameter. The solvent is usually analcohol, acetone, or tetrahydrofuran and the nonsolvent A is usually water oraqueous buffer, with or without a hydrophilic surfactant to improve colloidstability after formation. Solvent A can be removed by evaporation undervacuum, which can also be used to concentrate the suspension. The concen-tration of the substance of interest in the organic solvent and the proportionsof the two solvents are the main parameters influencing the final size of theparticles. For liposomes, this method is similar to the ethanol injection tech-nique proposed by Batzri and Korn in 1973 (17), which is however limited to40 mM of lipids in ethanol and 10% of ethanol in final aqueous suspension.

AmB was formulated with C14 lipids—dimyristoyl phosphatidyl-choline (DMPC) and dimyristoyl phosphatidylglycerol (DMPG)—as inAbelcet. Different formulation parameters were investigated: the AmB/lipid ratio, the starting concentration of AmB, and the lipid composition(ratio DMPC/DMPG). The resulting colloids were characterized in termsof size, polydispersity, and zeta potential (reflecting surface charge). The

An Original Lipid Complex System for Amphotericin B 95

morphology of the objects was investigated by electron microscopy aftercryofracture and cryodeposition. The interaction of AmB with lipids wasinvestigated by spectroscopic studies. Complementary information was ob-tained by differential scanning calorimetry (DSC) and X-ray diffraction.In particular, an original experimental setup developed by our laboratory (18)coupling X-ray diffraction with DSC was used, allowing us to follow chan-ges in the lipid bilayer structure as a function of temperature. Because theaim of formulating AmB with lipids is to reduce its toxicity to mammaliancells and organs, the toxicity of the optimized lipid formulation was assessedin a number of in vitro and in vivo systems, and compared with the com-mercially available formulations. The immunomodulating effects of AmBtoward macrophages and the influence of the formulation on these effectswere investigated because of their possible roles in the antimicrobial activityor the toxicity of the drug. Finally, the efficacy of the formulations againstLeishmania parasites was evaluated, both in vitro, using infected macro-phages, and in vivo, in mice.

PREPARATION OF LIPID COMPLEX AmB

Materials

AmB was purchased from Sigma (Saint-Quentin-Fallavier, France), andFungizone from Squibb (Neuilly, France). DMPC and DMPG were pur-chased from Avanti Polar Lipids Inc., (Alabama, U.S.A.). Solvents andother reagents were obtained from Carlo Erba reagenti (Val de Reuil, France).

Mother solutions of AmB were prepared in dimethylsulfoxide (DMSO)(or methanol). All drugs and lipids were stored at�20�C, protected from light.

For the purposes of comparison, commercial AmB-lipid formulationswere obtained as follows. Amphotec was obtained from Intermune (U.S.A.).AmBisome and Abelcet were kind gifts from Gilead Sciences (Foster City,California, U.S.A.) and Zedeus Pharma (Versailles, France), respectively.Ampholiposomes (cationic oligolameller liposomes containing AmB) weresupplied by the Pharmacie Centrale des Hopitaux (Paris, France).

Procedure

Nanoprecipitation was carried out according to the procedure described byStainmesse et al. (14) (Fig. 2). AmB was dissolved in methanol (10 mL), usuallyat a concentration of 0.35 mg/mL (366mM). This solution was mixed with asolution of phospholipids at different concentrations (typically 1 mg/mL,1.3 mM), also in methanol (5 mL), at 40�C. This organic phase (15 mL) wasthen added to a water phase (MilliQ

1

water, 15 mL, Waters Millipore, France)under gentle magnetic stirring at 20�C. The methanol was then removed bylow-pressure solvent evaporation to yield an aqueous dispersion in a volumeof 15 mL.

96 Larabi et al.

The AmB/phospholipid ratio was varied between 2.5% and 50% (w/w)and, at a fixed AmB/phospholipid ratio, the phospholipid composition wasvaried from 100% DMPC to 100% DMPG. As a control, AmB was alsoprecipitated alone, without lipid.

The quality of the colloidal preparation could be assessed after themixing step. If small particles were formed, the mixture was transparentyellow with a blue–green Tyndall effect at the top of the flask. The particlesize was not affected by the evaporation step.

It should be noted that concentration of phospholipids and AmB inmethanol is limited by the solubility of these two substances in the organicphase. A high final concentration of methanol in the mixture with water isalso necessary to form submicronic particles. Thus, the suspensions had tobe concentrated by rotary evaporation before testing in biological systems.

PHYSICAL CHARACTERIZATION

Colloidal Properties—Size, Zeta Potential, and Morphology

The mean particle diameter was measured by photon correlation spectroscopy(PCS) with a Nanosizer N4 (Coultronics, Margency, France). The size andpolydispersity of AmB lipid preparations depended on both the AmB/phospho-lipids ratio and the phospholipid composition. At a DMPC/DMPG molar ratioof 7/3, when the AmB content was below 10% w/w, large polydisperse particleswere formed. At AmB ratios of 20% to 50% of total weight of phospholipids, amajority of submicronic particles were obtained. The smallest size, around300 nm, and minimal polydispersity were achieved with AmB at 35% w/w; that

Figure 2 Schematic representation of the nanoprecipitation procedure. Abbrevia-tion: AmB, amphotericin B.


is a DMPC/DMPG/AmB molar ratio of 7/3/5 (Table 1). The proportions ofthe two phospholipids (DMPC/DMPG) also influenced the size and thepolydispersity, the optimum being the ratio 7/3, the same as that in Abelcet (1).In contrast, when AmB was treated in the same way without lipids, a polydis-perse suspension was obtained, whatever the concentration.

The optimal formulation [DMPC/DMPG/AmB in molar ratio 7/3/5,referred to as lipid complex of amphotericin B (LC-AmB)] was stable inaqueous suspension for six months after preparation when stored at þ 4�C,with no change in size. Other formulations increased in size a few days afterthe preparation with or without precipitation.

The zeta potential of the formulations was determined by Dopplervelocimetry and PCS on a Zetasizer 4 (Malvern Instruments, U.K.), withoutfurther dilution. The zeta potential of LC-AmB under these conditions was�44 mV, slightly lower than that measured for the same lipid compositionwithout AmB, �55 mV, but remaining consistent with colloidal stability.This reduction in the absolute value of the zeta potential could be due tothe presence of AmB at the surface, because free AmB dispersed in waterunder the same conditions had a less negative zeta potential; about �27 mV.

The morphology of AmB lipid complexes was examined by electronmicroscopy. Two different preparation techniques were used: freeze-fracturing and air-drying.

Table 1 Size, Polydispersity, and Zeta Potential of Complexes as a Function ofAmB Content (w/w) with a Lipid Composition DMPC/DMPG 7/3

AmB/lipid ratio(w/w) (%)

Mean diameter(nm)

Polydispersityindex

Zeta potential(mV)

0 >1000 0.68 –55.12.5 >1000 0.51 –67.15 >1000 0.45 –60.810 >1000 1.20 –55.020 690� 129: 79% 0.51 –46.3

184� 57: 21%30 559� 161: 66% 0.56 –46.3

157� 46: 34%35 303� 53 0.16 –43.840 368� 115 0.28 –42.845 248� 158: 87% 0.35 –43.2

>1000: 13%50 275� 78: 93% 0.45 –45.9

>1000: 7%

Abbreviations: AmB, amphotericin B; DMPC, dimyristoyl phosphatidylcholine; DMPG,

dimyristoyl phosphatidylglycerol.

Source: From Ref. 1.

98 Larabi et al.

For freeze-fracture, a drop of the formulation containing 30% glycerolwas deposited on a thin copper planchet and rapidly frozen in liquid pro-pane. Fracturing and shadowing using Pt-C were performed in a BalzersBAF 310 freeze-etch unit. Other samples were simply deposited on a freshlycleaved mica plate and air-dried before shadowing as above. Replicas wereexamined with a Philips 410 electron microscope.

AmB prepared by the solvent displacement method without lipidsappeared to be aggregated like a bunch of grapes after air drying and shad-owing. Freeze-fracture electron microscopy of the same preparation yieldeda string-of-pearls structure of several microns (1). The LC-AmB composi-tion (7/3/5) examined after air-drying showed discs of about 250 nm indiameter that did not fuse when they were deposited one on top of the other(Fig. 3A). The thickness of the disc was evaluated from the angle of shadow-ing and the length of the shadow to be about 29 A. A similar thickness, 21 A,was obtained by small-angle X-ray diffraction experiments at temperaturesbetween 4�C and 40�C (Larabi et al., unpublished data, 2000).

When this sample was freeze-fractured, fracture did not occur in theplane of the disc, but at right angles so the micrograph showed thin,dumbbell-like structures (Fig. 3B). These observations suggest that the phos-pholipids are arranged in an interdigitated, rather than bilayer, structure inthe discs. This arrangement is not found when phospholipids were precipi-tated alone by the same protocol, showing that the AmB–lipid interactionsdetected by physicochemical techniques are at the origin of this particularstructure. A periodicity at 4 A that increased in intensity with the proportionof AmB was detected by wide-angle X-ray diffraction at 17�C. A similarresult with ribbon-like structures of DMPC/DMPG/AmB 7/3/5, hadalready been observed by Janoff et al., who suggested that this could repre-sent an ordered state of the fatty acid chains induced by AmB (11).

Modification of the ratio of the two phospholipids yielded differentstructures in freeze-fracture electron microscopy. Without DMPG (molar ratioDMPC/DMPG/AmB 10/0/5), large lamellar structure were observed. Thepresence of DMPG favored the formation of shorter lamellar structures, bothstacked and fused in places or a predominantly disc morphology.

Analysis of AmB–Lipid Interactions

In order to obtain more information about the interactions between AmBand lipids at the molecular level, we chose to use spectroscopic measure-ments: electronic absorption and circular dichroism (19). The particularstructure of the AmB molecule, with several conjugated double bonds inthe hydrophobic stretch, means that these techniques can be used to studyits aggregation state under different conditions. Of the two, circular dichro-ism spectroscopy is the more sensitive way of detecting the aggregated formsof AmB.


Absorbance measurements were made by using a Cary 1E UV-visiblespectrometer (Varian, France), while circular dichroism spectra were recordedwith a Jobin-Yvon Mark V dichrograph, both at 20�C, after dilution in water.

The UV spectra of AmB varied according to its proportion in the lipidformulation, indicating that its self-aggregation was limited by its complexa-tion with the phospholipids. These results were confirmed by circulardichroism (CD). When the AmB–lipid complexes containing 10% to 50%w/w of AmB were examined (Fig. 4), the dichroic doublet characterizingthe aggregation state of AmB, centered on 335 nm, was still observed but

Figure 3 Electron microscopy of lipid complex of amphotericin B. (A) Air-dryingand shadowing, bar¼ 200 nm; (B) freeze-fracture, bar¼ 200 nm. Source: From Ref. 1.

100 Larabi et al.

the intensity of the dichroic band varied according to the percentage ofAmB. For ratios above 20%, the intensity was lower and the negative bandswere progressively replaced by three positive bands (420, 392, 375 nm) thathad a similar intensity whatever the ratio. The minimum doublet intensitywas reached with the 35% ratio. These positive bands are characteristic ofinteractions of AmB with lipids (20). In contrast to LC-AmB, the CD spec-trum of Abelcet showed a more intense dichroic doublet, although thisspectrum also showed evidence of reduced AmB self-association (21).

The AmB aggregates in LC-AmB are different from those formed byfree AmB aggregates in terms of their ability to dissociate on dilution belowthe critical micellar concentration of AmB. In contrast to free AmB aggre-gates, the interaction between the lipid and the aggregated AmB was verystable because the absorption and CD spectra of the preparation containing35% AmB with lipid were not affected by dilution down to 5�10�8 M AmB.Incubation for one hour at 37�C did not affect its spectrum either. Thestrong interaction between AmB and lipids was confirmed by DSC

Figure 4 Evolution of circular dichroism spectra [DeL-R� 10�2 (M�1 cm�1)] withdifferent percentages (w/w) of amphotericin B (AmB) in AmB/lipid preparationswith molar ratios DMPC/DMPG 7/3 (20%: -�-�-�-�-; 30%: -&-&-&-; 35%: _____;40%: -D-D-D-; 50%: __ __ __). Source: From Ref. 1.


analysis. In fact, as observed by Janoff et al. with Abelcet (11) when a highproportion of AmB was associated with DMPC/DMPG (7/3) the mainphase transition peak of the lipids was broadened and the enthalpy of thetransition was reduced (Larabi et al., unpublished data, 2000). This indi-cates that lipid–lipid interactions are diminished because the lipids interactwith AmB.

This reduction in self-aggregated AmB and the stability of the formula-tion suggested that LC-AmB could be a useful pharmaceutical formulationbecause it is generally accepted that the origin of toxicity toward mammaliancell membranes is free, self-associated AmB (2).

The ratio of the two phospholipids also affected the absorbance andCD spectra. In particular, a high proportion of the negatively charged lipidDMPG led to an inversion of the dichroic doublet, suggesting a drasticchange in AmB organization.

These results clearly show that our solvent displacement process leadsto the formation of AmB-lipid structures that are different are from the‘‘ribbon-like’’ ones described by Janoff et al. (11,21) for the same composi-tion. It was therefore interesting to investigate the toxicity and efficacy ofthis formulation.

EVALUATION OF TOXICITY

Toxicity In Vitro

Toxicity Toward Macrophages

Mouse peritoneal macrophages were obtained by peritoneal lavage fromthioglycolate-stimulated CD1 mice (Charles River, St-Aubin-les-Elbeuf,France). These were plated in 96-well plates with 105 cells/well in RPMI1640 Glutamax with 10% of fetal bovine serum (FBS). After adherence,the medium was removed and replaced by medium containing the differentformulations of AmB. The plates were incubated for 4, 24, 48, and 72 hoursat 37�C in humidified 5% CO2 incubator. Control cells were incubated withculture medium alone. Cell viability was determined by a colorimetric assayusing the tetrazolium salt 3-[4,5-dimethylthiozole-2-yl]-2,5,-biphenyl tetra-zodium bromide (MTT).

Table 2 shows the results obtained for four-hour and 24-hour exposureto AmB. LC-AmB had an IC50 above 100 mg/L after 24-hour exposure. Thisindicates that it has very low toxicity, comparable to the commercial formu-lation AmBisome (9). The toxicity increased with the time of exposure for allformulations (after a 48-hour exposure, the IC50 of LC-AmB was 86 mg/L,data not shown). Very similar results were obtained in the presence ofpolymyxin B, eliminating the possibility that the toxicity was due to thecontamination of the formulations with lipopolysaccharide (LPS) (22).

102 Larabi et al.

Hemolytic Properties

AmB formulations were dispersed in phosphate-buffered saline (PBS) at dif-ferent concentrations (0.1–100 mg/mL) and incubated for five minutes at37�C. Freshly isolated human erythrocytes were then added to a final hema-tocrit of 2% and incubated at the same temperature for 30 minutes. Aftercentrifugation, the supernatant was removed and the RBC pellet was lysedwith sterile water. The hemoglobin remaining in the pellet was estimated fromits absorption at 560 nm recorded with a spectrophotometer. The percentagehemolysis was calculated from the difference between the hemoglobinremaining in the test samples and the control incubated with PBS alone.

AmB solubilized in DMSO and dispersed in PBS provoked 50%hemolysis of human erythrocytes at 3.5 mg/L of AmB. Fungizone andAmB prepared by the same process as LC-AmB but without lipids wereslightly less toxic (Hb50 5 mg/L). All the lipid formulations caused less than50% hemolysis at the highest concentration tested (100 mg/L).

One factor determining toxicity of AmB formulations is the form inwhich the antibiotic is released—monomeric or aggregated—because onlyself-associated AmB can complex cholesterol in eukaryote membranes (25).The differential toxicity of the lipid formulations toward macrophages couldbe related to their stability in the culture medium. For example, the Ampho-liposome formulation, which is destabilized in the presence of serum (24), has

Table 2 Toxicity of LC-AmB In Vitro and In Vivo

Formulation

In vitro(mg/mL of AmB)

In vivo(mg/kg of AmB)

Hb50

IC50

(macrophages) LD50

30 min 4 hr 24 hrIn thisstudy Literature

Fungizone 5 57 4.5 3.5 2.5 (23)AmBisome >100 >100 >100 >175 (23)Ampholiposomes >100 >100 46 22 (24)LC-AmB >100 >100 >100 >200Abelcet >100 >100 84 40 50–70 (23)Amphotec >100 >100 91 >100 (10)

Hb50: concentration causing 50% lysis of human erythrocytes.

IC50: concentration causing 50% loss of viability of mouse peritoneal macrophages, using the

MTT test, after the incubation times stated.

LD50: acute toxicity assessed in CD1 male mice after a single intravenous bolus injection.

Values are calculated from the number of mice surviving the injection.

Abbreviation: LC-AmB, lipid complex of amphotericin B.



a greatly reduced IC50 after a longer incubation time. Another factor thatcould be important in the toxicity toward macrophages is the uptake ofAmB, which could occur by phagocytosis of intact particles, by transferbetween the particles and the membrane, and via the intermediary of lipopro-teins. In previous work, we compared the association of different AmBformulations with mouse peritoneal macrophages (26). The association ofAmB presented as LC-AmB was less than that of Abelcet but greater thanof the Fungizone. Therefore, there is no direct correlation between uptakeand toxicity, but different intracellular trafficking may explain the results.

Toxicity In Vivo

Acute Toxicity

A single bolus injection (200 mL) containing various doses of AmB of differ-ent formulations was given intravenously to groups of 10 male CD1 mice(Charles River, France), weighing 25 to 30 g. Mouse survival was monitoreddaily for 30 days and the LD50 was determined by the method of Litchfieldand Wilcoxon (27).

The acute toxicity observed for Fungizone and Abelcet in this study wasin accordance with the data reported in the literature (23). LC-AmB was lesstoxic than Abelcet. The concentrations of LC-AmB necessary to determine theLD50 without increasing the injection volume were higher than those thatcould be obtained by the process as described above, and the preparationwas concentrated further by rotary evaporation, leading to an increase in visco-sity at concentrations above 10 g/L of AmB, corresponding to 80 mg/kg.Although all the mice given 200 mg/kg of AmB as LC-AmB survived theinjection, three mice in this group died a few days later. Therefore, the maxi-mum tolerated dose was 100 mg/kg of AmB for this new formulation. Themice that received this dose behaved normally and no visible organ anomalieswere found at autopsy. There is a strong contrast between the acute toxicity ofLC-AmB and Abelcet, despite their similar composition. In fact, the toxicityof LC-AmB was comparable to that of AmBisome.

Toxicity After Repeated Doses

The low acute toxicity of LC-AmB would be expected to allow higher cumu-lative doses of the antibiotic to be administered. This hypothesis was tested inCD1 mice, which were given various doses of LC-AmB daily for three weeks.Groups were also treated with Fungizone (0.5 mg/kg) and Abelcet (10 mg/kg)according to the same regimen. At the end of the treatment period, mice weresacrificed and various biochemical parameters were measured (28).

In general, the treatments were well supported by the mice. There wereno significant changes in total body weight, kidney weight, or liver weight,except the liver weight in the group given the highest dose of LC-AmB(20 mg/kg). This dose also caused a small but significant reduction in the

104 Larabi et al.

hematocrit, as did Fungizone and Abelcet. However, parameters of kidneyfunction (uremia and creatinemia) were not affected. The probable uptakeof these formulations by the liver was indicated by a significant increasein transaminase activity in the blood for all formulations (28).

Cytochrome P450 was also affected. The overall level of hepatic micro-somal cytochrome P450 was slightly increased in the groups treated withAbelcet and LC-AmB at 20 mg/kg, while the expression of some isoforms,especially 3A1 in both the liver and kidney, decreased significantly. This isan intriguing and potentially important result, which is in accordance withobservations by Inselmann et al. in the rat (29). There have been noreports of AmB metabolism by cytochrome P450–based systems, or indeedby any other enzymes. However, the isoenzymes that were affected by AmBare those frequently implicated in the metabolism of xenobiotics, so this sideeffect would need to be taken into account during combination therapy.

The results of the chronic administration study indicate that LC-AmBdoes not induce any new toxicity and that its side effects are the same asthose produced by the conventional formulation (Fungizone) and a com-mercial lipid formulation (Abelcet) but that they appear at higher doses.This difference is probably due to both the stability of the formulation, pre-venting rapid release of AmB as aggregates or transfer to lipoproteins, andits size difference with Abelcet, which could lead to less rapid uptake byphagocytic cells. These encouraging results with respect to toxicity promp-ted us to test the efficacy of the formulation. For this, we chose to look atin vitro and in vivo models of Leishmaniasis, as well as the immuno-modulating properties of AmB.

EVALUATION OF ACTIVITY

Activity In Vitro

Activity Against Leishmania donovani Strains

The parasites were cultivated as promastigotes within thioglycolate-elicitedmouse peritoneal macrophages. The wild-type strain, MHOM/IN/80/DD8, and an AmB-resistant strain (AmB-R) derived from it by drug pres-sure (30) were used. The macrophages were plated in Labtek eight-chamberslides for four hours before adding the parasites at a 1:20 cell/parasite ratiofor the AmB-R strain and 1:10 for the wild-type strain, in order to infectabout 80% of the cells. Twenty-four hours later, the formulations wereadded at various concentrations for four days. Thereafter, the slides werefixed and stained with Giemsa before counting parasites. The IC50 concen-trations for each formulation were calculated according to the method ofNeal and Croft (31).

The results are summarized in Table 3. LC-AmB showed a good activ-ity toward the wild-type strain, with the lowest IC50 of all the formulations


tested. The IC50 observed for the AmB-R strain was also one of the lowest,but was still much higher than that of the wild type, indicating that the resis-tance had not been overcome. It has been shown that the resistance in thisstrain is due to the replacement of ergosterol by another sterol in the para-site membrane, thus removing the main target of AmB, as well as increasingmembrane fragility (30).

These results indicate that despite the strong interactions betweenAmB and lipids in LC-AmB that reduce its toxicity, the antibiotic can be madeavailable over a four-day incubation period.

Immunomodulating Properties

There is a great deal of evidence that AmB can exert a number of effectsdirectly on cells of the immune system, and particularly on macrophagesto increase nonspecific defense mechanisms against pathogens and cancercells. These mechanisms include the production of nitric oxide (NO) (32)and tumor necrosis factor alpha (TNF-a) (33), which could contribute tothe antifungal and antiparasitic activity of AmB. However, excess TNF-aproduction could also be responsible for some of the side effects associatedwith AmB treatment, such as fever and chills.

In the light of this, we investigated NO and TNF-a production by thio-glycolate-elicited mouse peritoneal macrophages after exposure to variousAmB formulations, including LC-AmB (26). NO was measured by a colori-metric assay for its stable end product, nitrite, whereas TNF-a productionwas assessed by a bioassay involving L929 cells. Briefly, we confirmed thatNO production by the inducible nitric oxide synthase (NOS) II enzyme couldbe stimulated in these cells by combinations of AmB and gamma interferon,but not by AmB alone or AmB combined with bacterial endotoxin. When

Table 3 Activity of LC-AmB Against Leishmani donovani In Vitro

IC50 (mg/mL) after 4 days

Formulation Wild-type AmB-R

AmB (in DMSO) 0.045� 0.006 0.723� 0.081Fungizone 0.041� 0.010 0.751� 0.006AmBisome 0.042� 0.005 0.657� 0.078Amphotec 0.075� 0.025 0.209� 0.031Abelcet 0.032� 0.007 0.622� 0.034LC-AmB 0.008� 0.003 0.221� 0.031Ampholiposomes 0.075� 0.022 0.204� 0.019

Wild-type and AmB-resistant (AmB-R) strains of L. donovani MHOM/IN/80/DD8 growing in

mouse peritoneal macrophages were used. IC50 values calculated according to Ref. 31 were

obtained after four days of incubation with the formulations.

Abbreviation: LC-AmB, lipid complex of amphotericin B.


106 Larabi et al.

compared at the same dose, lipid formulations stimulated less NO productionthan free AmB (dispersed in culture medium from a mother solution inDMSO); however, when LC-AmB was added at a concentration of 10 mg/L,which would have been toxic in the case of free AmB, the NO production washigher than that observed with 1 mg/L free AmB.

AmB also induced TNF-a production without any costimulator.Again, the lipid formulations induced less of this cytokine than free AmBat equivalent concentration, but higher concentrations of LC-AmB gave sig-nificantly higher levels (26). However, the doses necessary to stimulate theproduction of these mediators were much higher than those needed to arrestthe growth of L. donovani promastigotes in macrophages. We thereforeconcluded that the immunomodulating effects of AmB do not contributegreatly to the results reported in section ‘‘Activity Against Leishmaniadonovani Strains.’’

Activity In Vivo

A preliminary test of the efficacy of LC-AmB was made in a model ofvisceral leishmaniasis in the BALB/c mouse (22). The mice were infectedintravenously with amastigotes of the MHOM/ET/67/H43 strain from ahamster spleen. Treatment was started seven days after injection and con-sisted of intravenous injections of AmB formulations for three consecutivedays, or subcutaneous injections of sodium stibogluconate (Pentostam) at15 mg/kg for five consecutive days as a positive control. Mice were killed14 days after infection and the parasite load in the liver was estimated fromGiemsa-stained smears. ED50 and ED90 values were determined by sigmoi-dal regression analysis.

In one experiment, LC-AmB was compared with AmBisome (small uni-lamellar liposomes). LC-AmB was found to have an ED50 of 0.19 mg/kg andan ED90 of 0.51 mg/kg, whereas for AmBisome both these parameters werebelow 0.20 mg/kg, the lowest dose tested. In another experiment, LC-AmBwas compared with Abelcet and showed a better reduction of parasite burdenafter three injections of 1 mg/kg, but there were not sufficient data to allowED50 values to be calculated (22). Therefore, we can conclude that this newAmB formulation retains antileishmanial activity in vivo, but it is difficult toposition it with respect to other formulations.

CONCLUSION

These results clearly show that our solvent displacement process leads to theformation of AmB-lipid structures that are different from the ‘‘ribbon-like’’ones described by Janoff et al. (11,21) for the same composition. Ourobservations are consistent with a model of the antibiotic intercalatedbetween the phospholipids in an interdigitated structure for the molar ratioDMPC/DMPG/AmB 7/3/5. The strong interaction between AmB and


lipids resulted in a reduction of free self-associated AmB, which is one expla-nation for the low toxicity of this formulation leading to a considerableimprovement in the therapeutic index. The small size of the complexes, inparticular in contrast to Abelcet, is certainly also a factor determining theirdifferent biological activities. We have not yet performed any biodistribu-tion studies with our formulation, but we could predict that the smallerparticles might remain in the circulation longer than the Abelcet ‘‘ribbons.’’On the other hand, they probably do not have the long-circulating proper-ties of the small unilamellar liposomes constituting AmBisome.

The preliminary findings in a murine model of leishmaniasis show thatthis formulation has potential for treating this disease. However, its anti-fungal efficacy remains to be tested.

The nanoprecipitation technique is an interesting one for the produc-tion of colloids on a large scale because it is simple to put into practice anddoes not involve chlorinated solvents. However, the evaporation of theorganic solvent (in this case methanol) is still an energy-requiring step, whichcould be replaced by tangential filtration to exchange the dispersing phase.

This technique allowed us to prepare small colloids with a high pro-portion of AmB (33% molar proportion compared with 5% in AmBisome).However, at present we have used expensive, synthetic phospholipids. Itmay be possible to replace DMPC and DMPG by natural or partiallyhydrogenated natural phospholipids such as those from egg yolk or soy.This sort of economic consideration must be taken into account for thefuture development of the formulation.

ACKNOWLEDGMENTS

We thank all our collaborators in these studies: M. Appel, C. Bories, S. Bouvet,M. Cheron, S. Croft, J.P. Dedieu, J.P. Devisssaguet, S. Gil, A. Gulik,M. Lepoivre, P. Loiseau, N. Pages, F. Pons, F. Puisieux, J. Schlatter, andV. Yardley.

This work was supported by the Centre National de la RechercheScientifique (CNRS) and the Universite Paris-Sud. Malika Larabi receivedpersonal grants from the Chancellerie des Universites de Paris and theAcademie de Pharmacie.

REFERENCES

1. Larabi M, et al. New lipid formulation of amphotericin B: spectral and micro-scopic analysis. Biochim Biophys Acta 2004; 1664:172.

2. Bolard J, et al. One-sided action of amphotericin B on cholesterol-containingmembranes is determined by its self-association in the medium. Biochemistry1991; 30:5707.

108 Larabi et al.

3. Lopez-Berestein G, et al. Liposome-encapsulated amphotericin B for treatmentof disseminated candidiasis in neutropenic mice. J Infect Dis 1984; 150:278.

4. Tabosa do Egito ES, et al. In vitro and in vivo evaluation of a new amphotericinB emulsion-based delivery system. J Antimicrob Chemother 1996; 38:485.

5. Espuelas MS, et al. Polymeric carriers for amphotericin B: in vitro activity, toxi-city and therapeutic efficacy against systemic candidiasis in neutropenic mice.J Antimicrob Chemother 2003; 52:419.

6. Loiseau PM, et al. Design and antileishmanial activity of amphotericin B-loadedstable ionic amphiphile biovector formulations. Antimicrob Agents Chemother2002; 46:1597.

7. Al-Abdely HM, et al. Efficacies of KY62 against Leishmania amazonensis andLeishmania donovani in experimental murine cutaneous leishmaniasis andvisceral leishmaniasis. Antimicrob Agents Chemother 1998; 42:2542.

8. Golenser JS, et al. Efficacious treatment of experimental leishmaniasis withamphotericin B-arabinogalactan water-soluble derivatives. Antimicrob AgentsChemother 1999; 43:2209.

9. Alder-Moore JP, Proffitt R. Development, characterisation, efficacy and modeof action of AmBisome, a unilamellar liposomal formulation. J Liposome Res1993; 3:429.

10. Guo LSS, Working PK. Complexes of amphotericin B and cholesteryl sulfate.J Liposome Res 1993; 3:437.

11. Janoff AS, et al. Unusual lipid structures selectively reduce the toxicity ofamphotericin B. Proc Natl Acad Sci USA 1988; 85:6122.

12. Mullen AB, Carter KC, Baillie AJ. Comparison of the efficacy of various formu-lations of amphotericin B against murine visceral leishmaniasis. AntimicrobAgents Chemother 1997; 41:2089.

13. Guerin PJ, et al. Visceral leishmaniasis: current status of control, diagnosis andtreatment, and a proposed research and development agenda. Lancet Infect Dis2002; 2:494.

14. Stainmesse S, et al. Procede de preparation de systeme colloidaux dispersibles delipides amphiphiles sous forme de liposomes submicroniques, European PatentN 894018571, 1989.

15. Fessi H, Devissaguet JP, Puisieux F. Process for the preparation of disper-sible colloidal substance in the form of nanoparticles, US Patent, N 5,118,528,1992.

16. Fessi H, et al. Nanocapsule formation by interfacial polymer deposition follow-ing solvent displacement. Int J Pharm 1989; 55:R1.

17. Batzri S, Korn ED. Single bilayer liposomes prepared without sonication.Biochim Biophys Acta 1973; 298:1015.

18. Lopez C, et al. Thermal and structural behavior of milk fat: 3. Influence of creamcooling rate and droplet size. J Coll Interf Sci 2002; 254:64.

19. Bolard J, Seigneuret M, Boudet G. Interaction between phospholipid bilayermembranes and the polyene antibiotic amphotericin B: lipid state and cholesterolcontent dependence. Biochim Biophys Acta 1980; 599:280.

20. Jullien S, Vertut-Croquin A, Bratjburg J, Bolard J. Circular dichroism for thedetermination of amphotericin B binding to liposomes. Anal Biochem 1988;172:197.


21. Janoff AS, et al. Amphotericin B lipid complex (ABLCTM): a molecular ratio-nale for the attenuation of AmB related toxicities. J Liposome Res 1993; 3:451.

22. Larabi M, et al. Toxicity and antileishmanial activity of a new stable lipidsuspension of amphotericin B. Antimicrob Agents Chemother 2003; 47:3774.

23. Lasic DD, Papahadjopoulos D. Medical Applications of Liposomes. Amsterdam,The Netherlands: Elsevier, 1998.

24. Paul M, et al. Activity of a new liposomal formulation of amphotericin B againsttwo strains of Leishmania infantum in a murine model. Antimicrob Agents Che-mother 1997; 41:1731.

25. Brajtburg J, Bolard J. Carrier effects on biological activity of amphotericin B.Clin Microbiol Rev 1996; 9:512.

26. Larabi M, et al. Reduction of NO synthase expression and TNF alpha produc-tion of macrophages by amphotericin B lipid carriers. Antimicrob AgentsChemother 2001; 45:553.

27. Litchfield JT, Wilcoxon F. A simplified method of evaluating dose-effect experi-ments. J Pharmacol Exp Ther 1949; 96:99.

28. Larabi M, et al. Study of the toxicity of a new lipid complex formulation ofamphotericin B. J Antimicrob Chemother 2003; 53:81.

29. Inselmann G, Volkmann A, Heidemann H. Comparison of the effects ofliposomal amphotericin B and conventional amphotericin B on propafenonemetabolism and cytochrome P450 in rats. Antimicrob Agents Chemother2000; 44:131.

30. Mbongo N, et al. Mechanism of amphotericin B resistance in Leishmaniadonovani promastigotes. Antimicrob Agents Chemother 1998; 42:352.

31. Neal RA, Croft SL. An in-vitro system for determining the activity of com-pounds against the intracellular amastigote form of Leishmania donovani.J Antimicrob Chemother 1984; 14:463.

32. Mozaffarian N, Berman JW, Casadevall A. Enhancement of nitric oxide syn-thesis by macrophages represents an additional mechanism of action for ampho-tericin B. Antimicrob Agents Chemother 1997; 41:1825.

33. Rogers PD, et al. Amphotericin B activation of human genes coding forcytokines. J Infect Dis 1998; 178:1726.

110 Larabi et al.

6

Coupling of Peptides to the Surfaceof Liposomes—Application to

Liposome-Based Synthetic Vaccines

Francis Schuber and Benoıt Frisch

Laboratoire de Chimie Bioorganique, Faculte de Pharmacie,Universite Louis Pasteur, Strasbourg-Illkirch, and

Chimie Enzymatique, Illkrich, France

Fatouma Said Hassane

Laboratoire de Chimie Bioorganique, Faculte de Pharmacie,Universite Louis Pasteur, Strasbourg-Illkirch, France

INTRODUCTION

Liposomes are versatile drug delivery systems that can be surface-modifiedwith a variety of molecules that carry out a number of functions such as pro-moting the targeting of the vesicles to specific cell types and/or modulatingtheir biodistribution and pharmacokinetic properties (e.g., polyethyleneglycol (PEG)ylated liposomes). Targeting of liposomes, which represents amajor issue to increase the specificity and efficiency of bioactive moleculesdelivery, has been a much-studied approach during these last decades (1–3).It involves, in most cases, the use of ligands that are recognized by receptors(over)expressed at the surface of target cells. These ligands, which areconjugated to the surface of liposomes according to well-established biocon-jugation methods, are either small molecules, such as, e.g., folic acid orcarbohydrate clusters that trigger receptor-mediated endocytosis, or proteinssuch as monoclonal antibodies that are directed against specific antigens.

111

Besides drug delivery, liposomes have also gained wide acceptance in otherfields such as diagnostic imaging (4,5) and vaccines. In this chapter we willfocus our attention specifically on the conjugation of peptides to liposomes.We will outline the major techniques involved, present some applications ofliposomes-peptides constructs, and mainly discuss their use as vaccines.

TECHNIQUES FOR COUPLING PEPTIDES TO THESURFACE OF LIPOSOMES

Numerous methods have been developed for attaching ligands to the surface ofliposomes; for reviews, see Refs. (3,6–8). For peptides, they fall into two majorcategories: (i) covalent coupling of the peptides to preformed liposomes thatcontain functionalized hydrophobic anchors such as, e.g., derivatives of phos-phatidylethanolamine (PE); or (ii) incorporation of lipopeptides, obtained byconjugation of peptides to hydrophobic anchors (fatty acids, phospholipids),into liposomes either during the preparation of the vesicles or by postinsertioninto preformed vesicles. In this section, we will briefly discuss these techniquesand focus on the ones we have been using in our own work.

Covalent Coupling of Peptides to Preformed Liposomes

Among the chemical conjugation strategies that lead to the coupling ofpeptides to the surface of liposomes, the most efficient ones involve theformation of stable thioether bonds or bioreducible disulfide linkages (8)(Fig. 1A–C). Accordingly, a HS-peptide, i.e., a peptide that was extendedat its N- or C-terminus by a linker containing a free thiol group such as,for example, a cysteine residue, is allowed to react with a thiol-reactive deri-vative of a (phospho)lipid that was incorporated into liposomes during theirformation. These conjugation reactions are generally very chemoselective,i.e., specific for the thiol function, and they can be performed with high yieldunder mild conditions in aqueous media; moreover, they give access to wellcontrolled ligand/epitope densities at the surface of the liposomes. Theirpotential drawback resides in the introduction of reactive groups in the interiorof the liposomes; however, in most cases this can be alleviated by appropriatemeans. Other conjugation reactions have also been used, such as coupling ofpeptides via hydrazone, amide, or carbamate bonds (Fig. 1D–F).

Conjugation of Peptides via Thioether and Disulfide Bonds

A frequently used strategy to couple peptides to the surface of liposomesconsists in the use hydrophobic/amphipathic anchors that are function-alized with maleimide or bromoacetyl groups, i.e., thiol-reactive functions,which give by reaction with HS-peptides very stable thioether linkages.These functions are conveniently introduced into hydrophobic anchors suchas phospholipids, e.g., PE (9,10), the adjuvant Pam3CAG (11) or cholesterol

112 Schuber et al.

(12–14) by reaction with heterobifunctional reagents (Fig. 2); the function-alized anchors, some of which are commercially available, are then mixedwith the other constituents of the liposomes and become inserted into thebilayers during the formation of the vesicles. Reaction of HS-peptides withmaleimide functions (Fig. 1A) exposed at the surface of the liposomesoccurs generally quite readily when performed at pH 6.5 to 7.5 under inertatmosphere. Of note, because some HS-peptides undergo oxidation andproduce, e.g., (peptide-S)2, it is advisable to treat the peptides prior to theconjugation step with a reducing agent. To that end, we preferentially usetris(2-carboxyethyl)phosphine, an efficient nonpermeating reagent (15). Afterthe coupling step, to eliminate the residual maleimides, especially thosepresent in the interior of the vesicles, a treatment of the liposomes withan excess of thiol (such as mercaptoethanol) is advisable. Reaction ofHS-peptides with bromoacetyl groups similarly affords, in excellent yield,stable thiother bonds. However, this coupling reaction occurs only verysluggishly at neutral pHs and is much accelerated at higher pH values such

Figure 1 Conjugation reactions for coupling peptides to the surface of preformedliposomes. Functionalized lipophilic anchors were incorporated into liposomes andreacted with the peptides in aqueous media. Reactive endgroup functions:(A) maleimide; (B) bromo- or iodoacetyl; (C) 2-pyridyldithio; (D) carboxylic acid;(E) p-nitrophenyl carbonate and (F) hydrazide. Abbreviations: EDC, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; NHS, N-hydroxysuccinimide.

Coupling of Peptides to the Surface of Liposomes 113

as 8.5 to 9.5 (Fig. 1B). These coupling reactions allowed our group toprepare monoepitope liposomal vaccination constructs, which contained alsomonophosphoryl lipid A (MPL) as adjuvant (16–19). Full experimentaldetails are found in the cited references.

The vast difference in reactivity of HS-peptides with bromoacetyl andmaleimide groups, as a function of pH, prompted us to design a second

Figure 2 Structure of functionalized anchors and heterobifunctional reagents. DPPEis given as an example. Abbreviations: DPPE, 1,2-dipalmitoyl-sn-glycero-3-phos-phoethanolamine; DPPE-AcBr, bromoacetyl dipalmitoyl phosphatidylethanolamine.

Figure 3 Design of a diepitope liposomal construct. Small unilamellar liposomes(PC/PG/Chol; 55/25/50 molar ratio; diameter: �100 nm) containing 10 mol% ofbromo-acetyl 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine and 10 mol% of thethiol-reactive lipopeptide adjuvant anchor Pam3CysAlaGly-Mal were reacted, at25�C successively at pH 6.5, with the T-helper epitope QYI, derivatized with a C-linkerat its N-terminus, followed at pH 9.0 by the B-epitope TPE derivatized with a CGlinker at its N-terminus. Abbreviations: PC, phosphatidylcholine; PE, phosphatidyletha-nolamine; SUV, small unilamellar vesicles. Source: From Refs. 11, 20, 21.

114 Schuber et al.

generation of peptide-based vaccines: the liposomal diepitope constructs(see section ‘‘Potent Humoral Response Elicited By Liposomal DiepitopeConstructs’’ and Fig. 3). Accordingly, two different hydrophobic anchors, PEand S-[2,3-bis (palmitoyloxy)-(2-RS)-propyl]-N-palmitoyl-(R)-cysteinylalanyl-glycine (Pam3CAG) (Fig. 2) functionalized, respectively, with a bromoacetyland a maleimide function were incorporated into liposomes. At pH 6.5, thefirst epitope, i.e., HS-peptide (1), reacted exclusively with the maleimidegroup, leaving the bromoacetyl function intact. The second epitope, i.e.,HS-peptide (2), was coupled to the latter group after the pH was raisedto 8.5–9.0. This strategy allowed the conjugation on a single vesicle oftwo different peptides, each one on a specific anchor, that might have unre-lated roles in the immune response (21,22). This technique is actually moregeneral and could also be applied to conjugate two different HS-ligands/peptides to any carrier functionalized with both thiol-reactive groups.

An alternative and straightforward technique to conjugate a HS-peptide to liposomes is to generate a disulfide linkage with a derivative ofPE, incorporated into the vesicles, that carries a 2-pyridyldithio linkage(Fig. 1C). Such functionalized analogs of PE, which are easily obtainedby reaction of the phospholipid with a heterobifunctional reagent such asN-hydroxysuccinimidyl 3-(2-pyridyldithio) propionate (Fig. 2), were classi-cally used to prepare immunoliposomes (10). Despite the fact that suchbonds could be reduced in biological environments, we have found that,compared to thioether linkages, no changes in the intensity of the immuneresponse to a model peptide-liposomal construct could be observed (20,23).

Although commercially available heterobifunctional reagents aremostly used to prepare thiol-reactive functionalized hydrophobic anchors,it is sometimes advisable to synthesize specific ones containing, for example,polyethylene glycol spacers of various lengths (20). This might be of impor-tance when steric hindrance problems arrive (ligand recognition/availability)or when the spacer-arms, acting as haptens, are able to trigger an immuneresponse of their own. For example, we have found that the phenyl-maleimide moiety introduced by the reagent succinimidyl-4-(p-maleimido-phenyl)-butyrate into maleimidophenylbutyrate phosphatidylethanolamine(MPB-PE) (Fig. 2) can be quite immunogenic (23).

Miscellaneous Coupling Reactions BetweenPeptides and Preformed Liposomes

Several alternative techniques can be exploited to conjugate peptides topreformed liposomes. They all involve hydrophobic anchors incorpo-rated into the bilayers of vesicles that are able to react with modified orunmodified peptides. Their principles will be listed below, accompanied bypertinent references.

Amide and carbamate bonds: Unprotected peptides can be attached to thesurface of liposomes by engaging, for example, their N-terminus into an amide


bond with the carboxylic function of fatty acids (24) or phospholipid deriva-tives (25) anchors. These conjugation reactions, which are performed in thepresence of coupling reagents, might be hampered by a lack of regiospecificitywhen several reacting groups are present within the peptide sequence. Activeesters such as PE-PEG2000-NHS (Fig. 2) (26,27) or carbonates such asPE-(PEG)n-OCO-pNO2-Phenyl (28) (Fig. 2) were also incorporated intoliposomes and reacted with the N-terminal amine of peptides to give, respectively,amide or carbamate linkages at the distal end of the PEG chains (Fig. 1D and E).

Hydrazone and a-oxohydrazone linkages (29): Amphiphilic a-oxoaldehydes were synthesized recently and incorporated into liposomes. Thesefunctions react smoothly, with high chemoselectivity, with a-hydrazinoacetylpeptides yielding an a-oxohydrazone linkage (30,31). This ligation could be par-ticularly interesting for polyfunctional peptides that carry a multiplicity of amineand/or thiol groups and are therefore not amenable to the other couplingreactions. A comparable strategy was developed earlier by Zalipsky et al. (32).Accordingly, a PE-(PEG)2000 derivative end-functionalized with a hydrazidegroup was incorporated into liposomes and reacted with a Na-glyoxylyl pep-tide, obtained by oxidative treatment of N-terminus seryl- or threonyl-peptideswith sodium periodate, to yield a hydrazone linked conjugate (Fig. 1F).

Coupling of Peptides to Liposomes by Postinsertion Techniques

In a different approach, peptides are conjugated in a first step to lipophilicanchors such as long chain fatty acids or (phospho)lipids, extended forexample with PEG chains, followed by their insertion into liposomes accord-ing to the techniques summarized below. To synthesize lipid conjugates ofpeptides, a convenient strategy consists in performing, in the penultimatestep, an acylation on the N-terminus of the peptide still attached to the solid-phase resin, followed by the release of the lipopeptide. Some authors haveadded a lysine residue at the N-terminus that permits the introduction oftwo acyl chains (e.g., lauryl, and palmitoyl) on the peptide. Alternatively,peptides can also be ‘‘lipidated’’ by reaction with functionalized (phospho)-lipids according to conjugation reactions very similar to the ones discussedabove. The mode of association of lipidated peptides with liposomes dependson several parameters, including their physicochemical properties such as thespontaneous formation of micelles. Essentially two major techniques areavailable: (i) inclusion of the lipopeptides in the (phospho)lipid mixturefollowed by the preparation of the liposomes (33–35), and (ii) micelle-transferor postinsertion techniques. This is the most straightforward method thathas been used to associate lipidated peptides/proteins to liposomes undermild conditions that, for example, do not provoke the leakage of the vesicles.This approach is based on the formation of micelles by peptide-PEG-PEconjugates which, on incubation with preformed liposomes, leads underoptimized conditions, into a spontaneous transfer of the peptide conjugate

116 Schuber et al.

into the outer membrane leaflets of the vesicles (33,36,37). In contrast to thefirst method, this technique allows the conjugation of the peptide only onthe outer surface of the vesicles. One potential limitation here, as comparedto the covalent coupling strategies, might be a relatively lesser control onthe ligand density present at the surface of the lipsomes.

TARGETED LIPOSOME-PEPTIDE CONSTRUCTS

Selective targeting of endothelial cells of tumor neovasculature and tumorcells that overexpress, for example, avb3 and avb5 integrins has been muchinvestigated using synthetic peptides (38). Accordingly, controlled deliveryof therapeutic agents by liposomes conjugated to such ligands was mainlydeveloped with a variety of linear and constrained cyclic forms of RGD(Arg-Gly-Asp)-containing peptides that interact with high affinity with thesecell adhesion proteins. In such studies, the RGD-peptides, whose affinity andspecificity could also be optimized by phage-display technology (39), wereconjugated to the surface of liposomes via spacer-arms of variable lengthsincluding PEG–chains that provide long circulation half-lives to the targetedliposomes (39). Other conjugates between liposomes and small peptides thatshow, for example, a high affinity for the endothelium growth factor recep-tor, an attractive target for tumor therapy, have also been developed (26,40).Peptides such as vasoactive intestinal peptide grafted to sterically stabilizedliposomes have also been applied to targeted imaging of, e.g., breast cancer(41). In the particular context of targeting, the important technique of phagedisplay should not be overlooked. It allows the identification of peptide-ligands in the absence of any a priori knowledge of a cell surface receptor.These ligands can be further improved for affinity and specificity, and selectedfor, e.g., mediating the internalization of the complex with the targeted drugdelivery system (42). For example, liposomes bearing Langerhans cellstargeting peptides that were selected according to this approach (43) couldbe very valuable for the design of vaccination constructs. Somewhat related,liposomes carrying peptides that are intrinsically active on immunocompe-tent cells have also been described as delivery systems. They include themacrophage activator tetrapeptide Thr-Lys-Pro-Arg (tufstin) (44,45) andchemotactic agonist peptides such as fMet-Leu-Phe (46) that are alsorecognized by receptors at the surface of their target cells.

Because of the particular challenge that poses the cytosolic delivery ofbioactive molecules encapsulated in (targeted) liposomes after endocytosis, agreat interest was drawn recently by the so-called ‘‘cell-penetrating peptides’’such as the basic peptides derived from protein transduction domains, i.e.,homeodomain of antennapedia or TAT protein of HIV-1 (47). Theserelatively short peptides were first believed to trigger the transport of drugdelivery cargoes directly across the plasma membrane, bypassing endocytosis


and lysosomal degradation. Although still under investigation, it seemsmore realistic to assume, according to recent studies (48), that the uptakeof these peptides does not escape cell internalization via absorptiveendocytosis after interaction with, e.g., surface-expressed heparan sulfateglycosaminoglycans (49). TAT-peptide–tagged liposomes were pioneeredby Torchilin et al. (50,51); these authors and others (49,52) have shown thatcell uptake of liposomes decorated with these peptides are indeed greatlyincreased compared to conventional liposomes. In line with these studies,it was recently shown that the cationic tandem repeat peptide (140–150)2

derived from apolipoprotein E, coupled to the distal end of PEG chainscarried by liposomes, triggered an efficient uptake of the vesicles into pri-mary brain capillary endothelial cells that was mediated by cell-surfaceheparan sulfate proteoglycans (53).

APPLICATION OF LIPOSOME-PEPTIDECONSTRUCTS TO VACCINATION

Peptides representative of protein epitopes are attractive for the design ofsubunit vaccines (54). Compared to traditional approaches, synthetic peptide-based vaccines present many advantages; they lead to selective immuneresponses and, because peptides are chemically defined and can be producedat relatively large scales devoid of biological contaminants, they are consideredto be safe. Peptide-based constructs are also flexible because they can incor-porate a variety of different epitopes, e.g., from the same and/or differentpathogens. A major limitation of small peptides is their lack of immunogeni-city; however, this can be circumvented by their conjugation to appropriatecarriers such as proteins (e.g., tetanus toxin) or natural/synthetic polymersand by a coadministration with adjuvants. Liposomes were found to beexcellent peptide antigen carriers (19,55–57) that are devoid of toxicity and,importantly, have a very low intrinsic immunogenicity. This latter point canbe crucial because carriers such as proteins can trigger immune responses oftheir own and introduce unwanted ‘‘carrier suppression’’ effects. A majoradvantage of liposomes is their flexibility; the same vesicles can carry: (i) anti-gens, either encapsulated or surface bond, and (ii) immunoadjuvants—that arerecognized by Toll-like receptors (TLRs)—either within their bilayers such asMPL or diacylated [e.g., 2-kDa macrophage-activating lipopeptide (MALP-2)]and triacylated (Pam3Cys derivatives) lipopeptides (58,59), or encapsulatedsuch as CpG oligodeoxynucleotides (60). The enhancement of the immunogeni-city of antigens associated to liposomes was first ascribed to the tropism of thesevesicles for antigen-presenting cells (APCs) such as macrophages or dendriticcells (DCs). However, recent studies have helped to better understand at cellu-lar levels the specific contribution of liposomes to the immune response (61).Thus liposomes, after internalization, were shown to have the ability to channelprotein and peptide antigens into the major histocompatibility complex (MHC)

118 Schuber et al.

class II pathway of phagocytic APCs, resulting in the induction of antibodiesresponses. Moreover, and less predictably, liposomes can also serve asefficient delivery systems for entry of exogenous antigens into the majorhistocompatibility complex (MHC) class I pathway, via intracellular proces-sing (proteasome, etc.) and transporter associated with antigen processing(TAP) complex (cross presentation), and thus help to induce CD8þ T cellresponses (62). These observations are of great importance for promoting acytotoxic T lymphocytes (CTL)-mediated immunity by, e.g., antiviral andantitumoral vaccines.

Frisch et al. have observed that, in contrast to encapsulated peptides,the expression of small B-epitope peptides at the surface of vesicles contain-ing MPL as adjuvant was able to induce strong and specific humoralresponses (production of antibodies) (16,55). Consequently, for the produc-tion of our vaccination constructs (see Sections ‘‘Potent Humoral ResponseElicited by Liposomal Diepitope Constructs’’ and ‘‘Potent AntitumoralResponse Elicited by Structurally Defined Liposomal Diepitope Con-structs’’) we have associated peptide epitopes to the surface of liposomes. Thereare several methods for associating peptides to such vesicles; in most cases theyinvolve a ‘‘lipidation’’ of the antigen either before its incorporation into lipo-somes (i.e., synthesis of lipopeptides) or by coupling to preformed liposomescontaining functionalized lipophilic anchors (see Section ‘‘Techniques for Cou-pling Peptides to the Surface of Liposomes’’). In fact, it was shown some timeago that the conjugation of fatty acids, such as palmitic acid, to synthetic pep-tide antigens largely enhanced their immunogenicity (63); however, comparedto their free forms, this effect can be much increased by incorporation of theacylpeptides into liposome carriers to which lipid-soluble adjuvants, such asMPL or muramyl tripeptide-PE, can also be associated (64–66). The same resultwas observed by conjugation of peptides to the surface of preformed liposomes(16,17,67). Interestingly, whereas in many studies the liposomal constructs wereadministered intraperitoneally, or subcutaneoulsy, it was shown that liposomescontaining lipid-anchored synthetic peptides were also active when givenintranasally and were able, e.g., to elicit long-lasting immunity and effective pro-tection against an influenza virus challenge (68).

The mode of association of peptides to liposome carriers might alsobe critical to induce a preferential immune response either humoral or cellmediated. For example, using a human mucin MUC1 20-mer peptide, itwas found that only the physical association of the peptide to liposomes(either encapsulated or surface exposed after anchoring) was necessary toobserve a cell-mediated response (34). In line with this observation, it wasrecently shown that a soluble peptide, representing a Melan-A/MART-1tumor-associated antigen, when encapsulated into sterically stabilized lipo-somes, was able to stimulate a CTL response and this construct representeda suitable formulation for a specific tumor immunotherapy (69). In contrast,and in agreement with other studies (16), only the liposome surface exposed


MUC1 peptide was able to trigger a humoral immune response generatingspecific antiMUC1 antibodies (34).

Because conformational epitopes are not easily mimicked with linearpeptides, which can elicit nonspecific antibodies, several alternative strate-gies such as synthetic cyclic peptides have been developed [see e.g., (18)].A similar conformational restriction was seemingly achieved with a b-amyloidpeptide that was anchored to the surface of liposomes via hydrophobic tailsintroduced at its both N- and C-termini. The reconstituted peptide provedhighly immunogenic and elicited antibodies that could significantly preventamyloid plaque formation in a model system (70).

Finally, besides conventional liposomes that are made from natural(e.g., egg yolk and soybean) or synthetic phospholipids, novel liposomescalled ‘‘archaeosomes’’ that are prepared from the polar ether lipidsextracted from various archaeobacteria proved also interesting for thedesign of vaccines as peptide antigen carriers (71) and as powerful self-adjuvanting vaccine delivery vesicles that promote both humoral andcell-mediated immunity (72). Related to this, one can mention that pseudo-peptides, which are less prone to proteolysis when conjugated to liposomes,were also competent in triggering a humoral immune response (73).

The two examples from our work we are going to describe below are thedesign and study of liposomal diepitope constructs combining either: (i) B andT-helper (Th) peptide epitopes, which induced particularly powerful humoralresponses (21) (Fig. 3); or (ii) CTL and Th epitopes, which provided a power-ful antitumor vaccine (74) (Fig. 4). For the production of these constructs wehave conjugated peptides that contain a cysteine residue either at the N- or C-terminus, to the surface of preformed liposomes by reaction with thiol reactivefunctionalized phospholipids and/or Pam3Cys lipopeptide anchors (Fig. 2).To that end, we have developed strategies that give, in aqueous media, high

Figure 4 Design of a chemically defined diepitope liposomal anticancer vaccine.Small unilamellar liposomes (PC/PG/Chol; 75/20/50 molar ratio; diameter:�65 nm) containing 5 mol% of the synthetic thiol-reactive lipopeptide adjuvantanchor Pam3CSS-Mal were reacted, at 25�C and pH 6.5, with equimolar quantitiesof the peptides ErbB2 (p63–71), derivatized with a CG linker at its N-terminus, andHA307–319, derivatized with a C-linker at its C-terminus. Abbreviations: PC,phosphatidylcholine; PE, phosphatidylethanolamine; SUV, small unilamellarvesicles. Source: From Refs. 11, 74.

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yield and chemically well defined (i.e., chemoselective and regiospecific) conju-gation steps and also controlled epitope densities and ratios. This a posterioricoupling on lipophilic anchors whose hydrophobic moieties are masked by thebilayers circumvents the necessity to produce lipopeptide conjugates that canotherwise be quite difficult to handle and purify. In view of the developmentof vaccines, it should be stressed that liposomes are particularly amenableto such controlled chemistries; this is in contrast with other carriers whereill-defined conjugation steps can perturb the antigen and thus offset the useof structurally defined peptides.

Potent Humoral Response Elicited by LiposomalDiepitope Constructs

Major advances in molecular and cellular immunology, including the unrav-eling of the intricacies of the antigen presentation processes and morerecently of the roles of adjuvants, have provided the opportunity to designvaccines on much more rational bases. It is thus considered that diepitopeconstructs that combine both B and Th epitopes are the minimal built-insubunit vaccines that can be obtained, for example, by coupling the epitopesto MAP (75) or by producing chimeric peptides by colinearization of theepitopes (76). These latter constructs have, however, shown some limita-tions; thus, because the respective roles of the B and the Th epitopes arevastly different at cellular levels during the humoral immune response,and that consequently there is no a priori necessity for them to be covalentlylinked, we have designed constructs in which a single vesicle acts as asupramolecular carrier for the two types of epitopes that remain structurallyindependent (21). This was made feasible by devising a chemical strategythat allowed the controlled coupling of two different peptides—each on adifferent lipophilic anchor—to the surface of the same preformed liposome(22) (see Section ‘‘Covalent Coupling of Peptides to Preformed Lipo-somes’’). We reasoned that the B epitopes present on the liposomes wouldtarget our constructs to antigen-specific B lymphocytes; therefore itscoupling to the surface of the vesicles via a phospholipid anchor might besuitable. In contrast, after uptake of the constructs—triggered by theclustering of the B-cell receptors (BCRs)—the Th epitopes would need tobe targeted intracellularly to MHC class II–containing compartments andmight therefore need a specific, and different, type of anchor. To that endwe have selected the amphiphilic triacylated lipopeptide Pam3CAG(Fig. 2), which can be easily incorporated into liposomes (58). This moleculeand other synthetic analogs of the Escherichia coli lipopeptide are known fortheir built-in adjuvanticity, due to their interaction with the TLR2/TLR1heteromers (77), and also for their ability to efficiently elicit humoral aswell as cell-mediated immune responses against peptide antigens that areconjugated to them (78).


We have designed a model diepitope liposome constructs (PC/PG/Chol, 55/25/50; 100 nm dia.) carrying at their surface: (i) a B epitope ofsequence CG-TPEDPTDPTDPQDPSS (TPE; protein I/IIf 1495–1510)originating from a Streptococcus mutans cell surface adhesin (79) that med-iates the attachment of this bacterium to the host cells and to tooth surfaces;this peptide which was extended at its N-terminus by a CG spacer was con-jugated to DPPE-COCH2Br (Fig. 2), a thiol-reactive phospholipid anchor(20), and (ii) a tetanus toxin–derived ‘‘promiscuous’’ Th epitope (80) withthe sequence C-QYIKANSKFIGITEL (QYI; TT 830–844), in which anadditional cysteine was introduced at the N-terminus, which was conjugatedto Pam3CAG-Mal a thiol-reactive derivative of the triacylated lipo-peptide (11). This synthetic construct (Fig. 3), when administered toBALB/c mice, induced highly intense (titers> 20,000), anamnestic andlong-lasting (animal life span) immune responses indicating that thisapproach is quite successful (21). Two parameters were found of primeimportance to elicit this response with our diepitope constructs: (i) thesimultaneous expression of B and Th epitopes on the same vesicle, rulingout any bystander effect due to the T-helper epitope and underlining the effi-cacy of the immune response when both epitopes are presented simulta-neously to the same cells, and (ii) the lipopeptide Pam3CAG anchor ofthe Th epitope could not be replaced by a phospholipid anchor, i.e., a lesserimmune response of mostly T-independent nature was observed. Analysisof the antibody response revealed a complex pattern; thus, besides thehumoral response (production of IgG1, IgG2a, IgG2b), a superposition ofa T-independent (TI-2 type) response was also found (IgM and IgG3). Theseresults indicate that these liposomal diepitope constructs could be attractivein the development of synthetic peptide-based vaccines.

Potent Antitumoral Response Elicited by StructurallyDefined Liposomal Diepitope Constructs

Active specific immunotherapy involving host CD8þ CTL responses totumor-associated antigens (81) is increasingly pursued in the treatmentof cancer (82). Of particular importance in this field is the development ofinnovative vaccination formulations (carriers, adjuvants) capable to effi-ciently target, in vivo, APCs such as DCs, and to deliver CTL antigens tothe appropriate cellular compartments resulting in the induction of potentand long-lasting cellular immune responses. Recently we have designed mul-tivalent liposomal constructs, i.e., vesicles known for their tropism forAPCs, that codeliver two different peptide epitopes: (i) CG-TYLPTNAL(p63–67), a HLA-A24–restricted CTL epitope (83) derived from the humanproto-oncogene ErbB2 (Her2/neu) which is overexpressed in many tumors(breast, ovarian, etc.), and (ii) PKYVKQNTLKLAT-C (HA307–319), a‘‘promiscuous’’ T-helper epitope derived from influenza hemagglutinin

122 Schuber et al.

(84). Both thiol-derivatized peptides were conjugated to the surface of smallunilamellar vesicles (PC/PG/Chol; 75/20/50; 65 nm dia.) containing athiol-reactive functionalized Pam3CSS anchor (11,59), i.e., an amphipathictriacylated lipopeptide chosen for its adjuvanticity, its activation of DCs(85), via interaction with TLR2/TLR1, and its capacity to channel its pep-tide cargo to antigen processing and MHC class I/II presentation pathways(Fig. 4). Immunization studies were performed to evaluate the capacity ofthese liposomal constructs to protect BALB/c (H-2Kd) mice against tumorgrowth in a model system using syngenic renal carcinoma (Renca) cells thatstably express human ErbB2. Importantly for our approach, the p63–67peptide is also a CTL epitope in BALB/c mice (86). Subcutaneous challengewith Renca-ErbB2þ cells of subcutaneous vaccinated animals resulted in acomplete rejection of tumors, indicating the induction of a potent protectiveimmunity. In contrast, the same vaccinated mice were not protected againsta challenge with ErbB2-negative Renca cells demonstrating the specificity ofthe immune responses induced by the liposomal vaccine. Interestingly, lipo-somal constructs that lacked the Th epitope were somewhat less efficient;this was correlated with experiments indicating that in ex vivo restimulationwith an ErbB2-derived peptide of splenocytes from animals vaccinated withthe diepitope CTL/Th-liposomes resulted in a higher interferon-c produc-tion by T cells than with the only CTL epitope-liposomes. Intravenousrechallenge of vaccinated, tumor-free animals two months after the firsttumor challenge did not result in the formation of lung tumor nodules, sug-gesting that long-lasting immune responses had been induced. Therapeuticvaccination of mice bearing established Renca-ErbB2þ tumors led to a com-plete tumor rejection in two thirds of the animals and delayed tumor growthin the remaining ones. Taken together, our liposomal diepitope constructsthat conveniently combine CTL/Th peptide antigens and lipopeptide adju-vants efficiently enhance the immunogenicity of ErbB2-associated epitopesand represent very promising synthetic delivery systems for the design ofspecific antitumor vaccines (74).

CONCLUSIONS

Suitable liposome surface coupling methods are available for the prepara-tion of peptide-targeted vesicles and vaccination constructs. Many of themare adaptations of chemical conjugation strategies that were extensivelydeveloped during these last decades for other ligands such as proteins (anti-bodies, etc.) or small molecular weight molecules. Among the differentapproaches, coupling of HS-peptides to preformed liposomes containingthiol-reactive functionalized (phospho)lipid derivatives is particularlyappealing because of the high yields that are generally observed under mildconditions. Moreover, these highly chemoselective reactions give reproduc-ibly access to vesicles with well-controlled epitope densities, an important


parameter for the design of vaccination constructs. Manipulation of thecoupling conditions also gave access to synthetic biepitope constructs whereeach type of epitope is conjugated to a different anchor, each one playinga specific role in the immune response. Such constructs participate in thegeneral trend toward increasingly sophisticated liposomal vaccinationconstructs that might also contain TLR-interacting adjuvants, such asdiacylated and triacylated lipopeptides, MPL or CpG-type oligodeoxynu-cleotides, and/or carry targeting devices such as mannosyl clusters (87).Although the immune response to liposome-based vaccination strategy isparticularly complex, it should be kept in mind that the high lateral mobilityof liposome-associated peptides might facilitate their formation of subdo-mains and enable multivalent interactions between the vesicles and cells,favoring, e.g., the BCR clustering in B-lymphocytes, a phenomenon ofprime importance in the humoral response. Major topics of investigationin this field, already tackled by the group of Alving (88–91) and that shouldyield information of great importance, are the mechanisms of internalizationof the liposomal constructs by APCs, such as DCs, the processing of thelipidated peptides, and the routes of intracellular transport and presentationof the epitopes. Finally, the conjugation of peptide epitopes to the liposomalbilayers via hydrophobic anchors should also be considered from practicalviewpoint; such constructs are amenable to storage in a lyophilized formand are easily reconstituted in their original size (92).

Finally, although many peptides could be covalently conjugated tothe surface of liposomes, it should be kept in mind that this could bevery difficult with some types of peptides. For example, there are notyet straightforward techniques that allow the association of hydrophobicpeptides, such as some CTL and T-helper epitopes, to liposomes. A pro-gress in this area would be welcomed. Moreover, in contrast to its freeform, a peptide on coupling might sometimes trigger the aggregationand fusion of the vesicles, e.g., (93), and apart from the well known‘‘fusion peptides’’ this phenomenon is not always easily predictable(94). Nevertheless, the recent successes in the use of liposomal vaccinationconstructs, including a vaccine that has been brought to the market, ishighly encouraging (57).

ACKNOWLEDGMENTS

We gratefully thank all our coworkers whose names are cited inthe references. We are also much indebted to the laboratories headed byProf. D. Wachsmann and Dr. W. Wels with whom we have collaboratedover the last years and who were in charge of the immunochemical andthe in vivo studies. The Centre National de la Recherche Scientifique, theRegion Alsace, and the Ligue Nationale Contre le Cancer are also thankedfor their financial support.

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55. Alving CR, et al. Liposomes as carriers of peptide antigens: induction of antibo-dies and cytotoxic T lymphocytes to conjugated and unconjugated peptides.Immunol Rev 1995; 145:5.

56. Chikh G, Schutze-Redelmeier MP. Liposomal delivery of CTL epitopes to den-dritic cells. Biosci Rep 2002; 22:339.


57. Felnerova D, et al. Liposomes and virosomes as delivery systems for antigens,nucleic acids and drugs. Curr Opin Biotech 2004; 15:518.

58. Fernandes I, et al. Synthetic lipopeptides incorporated in liposomes: in vitrostimulation of the proliferation of murine splenocytes and in vivo induction ofan immune response against a peptide antigen. Mol Immunol 1997; 34:569.

59. Roth A, et al. Synthesis of thiol-reactive lipopeptide adjuvants. Incorporationinto liposomes and study of their mitogenic effect on mouse splenocytes.Bioconjugate Chem 2004; 15:541.

60. Engler OB, et al. A liposomal peptide vaccine inducing CD8(þ) T cells inHLA-A2.1 transgenic mice, which recognise human cells encoding hepatitis Cvirus (HCV) proteins. Vaccine 2004; 23:58.

61. Leserman L. Liposomes as protein carriers in immunology. J Lipos Res 2004;14:175.

62. Rao M, Alving CR. Delivery of lipids and liposomal proteins to the cytoplasmand Golgi of antigen-presenting cells. Adv Drug Deliver Rev 2000; 41:171.

63. Hopp TP. Immunogenicity of a synthetic HBsAg peptide: enhancement byconjugation to a fatty acid carrier. Mol Immunol 1984; 21:13.

64. Brynestad K, et al. Influence of peptide acylation, liposome incorporation, andsynthetic immunomodulators on the immunogenicity of a 1–23 peptide ofglycoprotein D of herpes simplex virus: implications for subunit vaccines.J Virol 1990; 64:680.

65. Tosi PF, Radu D, Nicolau C. Immune response against the murine MDRI pro-tein induced by vaccination with synthetic lipopeptides in liposomes. BiochemBiophys Res Commun 1995; 212:494.

66. Babu JS, et al. Priming for virus-specific CD8þ but not CD4þ cytotoxicT lymphocytes with synthetic lipopeptide is influenced by acylation units andliposome encapsulation. Vaccine 1995; 13:1669.

67. Krowka J, et al. Lymphocyte proliferative responses to soluble and liposome-conjugated envelope peptides of HIV-1. J Immunol 1990; 144:2535.

68. Levi R, et al. Intranasal immunization of mice against influenza with syntheticpeptides anchored to proteosomes. Vaccine 1995; 13:1353.

69. Adamina M, et al. Encapsulation into sterically stabilised liposomes enhancesthe immunogenicity of melanoma-associated Melan-A/MART-1 epitopes. Br JCancer 2004; 90:263.

70. Nicolau C, et al. A liposome-based therapeutic vaccine against beta-amyloid pla-ques on the pancreas of transgenic NORBA mice. Proc Natl Acad Sci USA 2002;99:2332.

71. Conlan JW, et al. Immunization of mice with lipopeptide antigens encapsulatedin novel liposomes prepared from the polar lipids of various Archaeobacteria eli-cits rapid and prolonged specific protective immunity against infection with thefacultative intracellular pathogen, Listeria monocytogenes. Vaccine 2001;19:3509.

72. Krishnan L, et al. The potent adjuvant activity of archaeosomes correlates tothe recruitment and activation of macrophages and dendritic cells in vivo.J Immunol 2001; 166:1885.

73. Benkirane N, et al. Antigenicity and immunogenicity of modified syntheticpeptides containing D-amino acid residues. J Biol Chem 1993; 268:26279.

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74. Roth A, et al. Induction of effective and antigen-specific antitumor immunityby a liposomal ErbB2/HER2 peptide-based vaccination construct. Br J Cancer.In press.

75. Tam JP, Lu YA. Vaccine engineering: enhancement of immunogenicity ofsynthetic peptide vaccine related to hepatitis in chemically defined modelsconsisting of T- and B-cell epitopes. Proc Natl Acad Sci USA 1989; 86:9084.

76. Partidos C, Stanley C, Steward M. The influence of orientation and number ofcopies of T- cell and B-cell epitopes on the specificity and affinity of antibodiesinduced by chimeric peptides. Eur J Immunol 1992; 22:2675.

77. Buwitt-Beckmann U, et al. Toll-like receptor 6-independent signaling by diacy-lated lipopeptides. Eur J Immunol 2005; 35:282.

78. Bessler WG, et al. Bacterial lipopeptides constitute efficient novel immunogens andadjuvants in parenteral and oral immunization. Behring Inst Mitt 1997; 98:390.

79. Lett E, et al. Immunogenicity of polysaccharides conjugated to peptidescontaining T- and B-cell epitopes. Infect Immun 1994; 62:785.

80. Panina-Bordignon P, et al. Universally immunogenic T cell epitopes: promiscu-ous binding to human MHC class II and promiscuous recognition by T cells. EurJ Immunol 1989; 19:2237.

81. Novellino L, Castelli C, Parmiani G. A listing of human tumor antigens recog-nized by T cells: March 2004 update. Cancer Immunol Immunother 2005;54:187.

82. Finn OJ. Cancer vaccines: between the idea and the reality. Nat Rev Immunol2003; 3:630.

83. Okugawa T, et al. A novel human HER2-derived peptide homologous to themouse K-d-restricted tumor rejection antigen can induce HLA-A24-restrictedcytotoxic T lymphocytes in ovarian cancer patients and healthy individuals.Eur J Immunol 2000; 30:3338.

84. O’Sullivan D, et al. On the interaction of promiscuous antigenic peptides withdifferent DR alleles. Identification of common structural motifs. J Immunol1991; 147:2663.

85. Espuelas S, et al. Effect of synthetic lipopeptides formulated in liposomes on thematuration of human dendritic cells. Mol Immunol. In press.

86. Nagata Y, et al. Peptides derived from a wild-type murine proto-oncogenec-erbB-2/HER2/neu can induce CTL and tumor suppression in syngeneic hosts.J Immunol 1997; 159:1336.

87. Espuelas S, et al. Synthesis of an amphiphilic tetraantennary mannosyl conjugateand incorporation into liposome carriers. Bioorg Med Chem Lett 2003; 13:2557.

88. Rao M, et al. Intracellular processing of liposome-encapsulated antigens bymacrophages depends upon the antigen. Infect Immun 1995; 63:2396.

89. Rao M, et al. Visualization of peptides derived from liposome-encapsulated pro-teins in the trans-Golgi area of macrophages. Immunol Lett 1997; 59:99.

90. Rao M, et al. Trafficking of liposomal antigen to the trans-Golgi of murinemacrophages requires both liposomal lipid and liposomal protein. Exp CellRes 1999; 246:203.

91. Rothwell SW, Wassef NM, Alving CR, Rao M. Proteasome inhibitors block theentry of liposome-encapsulated antigens into the classical MHC class I pathway.Immunol Lett 2000; 74:141.


92. Friede M, Van Regenmortel MH, Schuber F. Lyophilized liposomes as shelfitems for the preparation of immunogenic liposome-peptide conjugates. AnalBiochem 1993; 211:117.

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94. deSouza DL, et al. Membrane-active properties of alpha-MSH analogs: aggrega-tion and fusion of liposomes triggered by surface-conjugated peptides. BiochimBiophys Acta 2002; 1558:222.

130 Schuber et al.

7

Encapsulation of Nucleic Acid–BasedTherapeutics

Norbert Maurer, Igor Zhigaltsev, and Pieter R. Cullis

Department of Biochemistry and Molecular Biology,University of British Columbia, Vancouver, British Columbia, Canada

INTRODUCTION

Liposomes represent one of the most clinically advanced drug-deliverysystems with the range of medical applications extending from chemotherapyof cancer and infectious disease to vaccines and gene therapy (1,2). However,liposomal formulations of genetic drugs such as antisense oligonucleotidesand plasmid DNA (pDNA) for systemic applications are difficult to achieve (3).The large size and highly-charged nature of these molecules mitigates againstthe formation of small, neutral, serum-stable carriers, which are required toachieve the long circulation times necessary for efficient accumulation at diseasesites such as sites of tumor growth and inflammation. In particular, passiveencapsulation of pDNA in liposomes is very inefficient due to the large sizeof these molecules. Efficient entrapment requires interaction between the lipidcomponents of the carrier and the nucleotide-based drugs. However, this inter-action is very difficult to control. For example, complexes formed throughelectrostatic interaction between negatively-charged polynucleotides andcationic liposomes exhibit broad size distributions. These complexes efficientlytransfect cells in vitro; however, in vivo their large size and/or positive chargetriggers rapid clearance from the circulation (4–8). They can also be highly toxic(8). Therefore, substantial effort has been focused on constructing lipid-basedcarriers with improved in vivo characteristics.

131

In this chapter, the recent advances in the development of small serum-stable carriers for nucleic acid therapeutics such as antisense and immunestimulatory oligonucleotides and pDNA are reviewed with the emphasis onour work. Two basic approaches for encapsulation will be described. The firstof these employs incubation of large unilamellar vesicle (LUV) containinga cationic lipid and a polyethyleneglycol (PEG) coating with oligo- orpolynucleic acids in the presence of ethanol (9–11). The use of membrane-destabilizing agents such as ethanol in conjunction with PEG-lipids offersa way to control the interaction between negatively-charged polyelectrolytesand cationic liposomes and results in the entrapment of the nucleic acids insmall liposomes. The resulting particle will be referred to as ‘‘stabilizednucleic acid-lipid particle,’’ or SNALP, and the approach of making it aspreformed vesicle approach (PFV). The second method involves a detergent-dialysis procedure for the encapsulation of pDNA, resulting in the formationof ‘‘stabilized plasmid-lipid particles’’ (SPLP) (11–13). These SNALP andSPLP systems demonstrate long circulation lifetimes and preferentially accu-mulate at tumor sites and sites of inflammation following IV administrationdue the ‘‘enhanced permeability and retention’’ effect associated with themore permeable vasculature found in these disease sites (14). Highly specifictransgene expression at distal tumor sites has been observed following IVinjection of SPLP (15,16). It is also important to note that these systems natu-rally target antigen-presenting cells in vivo as they, like all other liposomal orparticulate systems, are removed from the blood by the fixed and free macro-phages of the mononuclear phagocyte system (reticuloendothelial system),resulting in accumulation in organs such as the liver and the spleen. Encap-sulation of oligonucleotides containing immune stimulatory CpG motifs inliposomes (SNALP) results in an immune response that is enhanced com-pared to either lipid or oligonucleotide alone (17). This forms the basis forthe application of liposome-encapsulated CpG oligonucleotides in immunetherapy (18).

Both approaches are simple and allow efficient encapsulation ofnucleic acid-based molecules such as oligonucleotides (9,10) and pDNA(8,10,12) in liposomes that are small in size (about 100 nm diameter) andstable in circulation, protecting the cargo from degradation. In the sectionsto follow, we will provide a brief overview of these methods.

METHODOLOGY

Entrapment of Polynucleotides Using the PreformedVesicle Approach

The preformed vesicle (PFV) approach involves incubation of liposomescontaining a cationic lipid and a PEG coating with polynucleotides in thepresence of ethanol. Typically, LUV composed of distearoyl-phosphatidyl-choline (DSPC), cholesterol (Chol), 1-O-(20-(x-methoxy-polyethylene-glycol)

132 Maurer et al.

succinoyl)-2-N-myristoyl-sphingosine (PEG-CerC14), and 1,2-dioleoyl-3-dimethylammoniumpropane (DODAP) are used. However, any zwitterioniclipid including dioleoylphosphatidyl-ethanolamine (DOPE) alone or in combi-nation with Chol can be substituted for DSPC/Chol (10). DODAP was chosenas the cationic lipid because it has a protonable amino group. Its apparent pKa isestimated to be between 6.6 and 7 (19). Entrapment can therefore be performedat low pH (pH 4) where DODAP is positively-charged and nonentrapped poly-nucleotides can be dissociated from the cationic lipid by neutralizing the pH andremoved by subsequent anion exchange chromatography. Adjusting the pH to7.5 also renders the surface charge of the liposomes neutral.

Liposome preparation: Ethanolic suspensions of LUVs composed ofDSPC/Chol/PEG-CerC14/DODAP (20:45:10:25 mol%) were either pre-pared by addition of ethanol to extruded liposomes or by addition of lipidsdissolved in ethanol to an aqueous buffer solution and subsequent extru-sion. Both methods give the same entrapment results and will be describedin greater detail in the following: (i) After hydration of a lipid film in 50 mMpH 4 citrate buffer and five freeze/thaw cycles LUVs were generated byextrusion through two stacked 100-nm filters (10 passes). Ethanol was sub-sequently slowly added under rapid mixing to a concentration of 40% (v/v).Slow addition of ethanol and rapid mixing are important as liposomesbecome unstable and coalesce into large lipid structures as soon as the etha-nol concentration exceeds a certain upper limit. (ii) LUVs were prepared byslow addition of the lipids dissolved in ethanol (0.4 mL) to citrate bufferat pH 4 (0.6 mL) followed by extrusion through two stacked 100-nm filters(two passes) at room temperature. Dynamic light scattering measurementsperformed in ethanol and after removal of ethanol by dialysis show no sig-nificant differences in size, which is typically 75� 18 nm. The extrusion stepcan be omitted if ethanol is added very slowly under vigorous mixing toavoid high local concentrations of ethanol.

Entrapment of oligo- and polynucleotides: The oligo- or polynucleotidesolution was slowly added under vortexing to the acidic ethanol-containingliposome dispersion, which typically contained 10 mg/mL of lipid. Theresulting dispersion was incubated at 40�C for one hour, and then dialyzedfor two hours against a 1000-fold volume excess of citrate buffer to removemost of the ethanol and twice against a 1000-fold volume excess of HBS[20 mM 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES)/145 mM NaCl, pH 7.5]. At pH 7.5 DODAP becomes charge-neutral andpolyelectrolyte bound to the external membrane surface is released fromits association with the cationic lipid. Unencapsulated polyelectrolytes weresubsequently removed by anion exchange chromatography on diethylami-noethyl (DEAE)-sepharose CL-6B columns equilibrated in HEPES- bufferedsaline (HBS) (pH 7.5). Finally, it should be noted that liposome formationand encapsulation could be combined in a single step by mixing the lipids dis-solved in ethanol with an aqueous solution containing the polynucleotide.

Encapsulation of Nucleic Acid–Based Therapeutics 133

Determination of trapping efficiencies: Trapping efficiencies weredetermined after removal of external polyelectrolytes by anion exchangechromatography. Oligonucleotide concentrations were determined by UV-spectroscopy. The absorbance at 260 nm was measured after solubilizationof the samples in chloroform/methanol at a volume ratio of 1:2.1:1 chlo-roform/methanol/aqueous phase (sample/HBS). Alternatively, absorbancewas read after solubilization of the samples in 100 mM octylglucoside. Theantisense concentrations were calculated according to: c (mg/mL)¼A260�1OD260 unit (mg/mL)� dilution factor (mL/mL), where the dilution factoris given by the total assay volume (mL) divided by the sample volume (mL).OD260 units were calculated from pairwise extinction coefficients forindividual deoxynucleotides, which take into account nearest neighborinteractions. The Oligreen assay was used in some instances, obviating theneed for prior removal of unencapsulated oligonucleotide. Fluorescenceemission was read at 520 nm with the excitation set at 480 nm before andafter addition of n-octyl b-D-glycopyranoside (OGP) to a final concentrationof 50 mM. The addition of detergent results in slight (10%) quenching of theOligreen fluorescence and this accounted for the calculation of trapping effi-ciencies. Oligonucleotide concentrations were determined relative to a stan-dard curve. Plasmid DNA encapsulation efficiencies were determined byA260 as described above (1OD¼ 50 mg/mL) and/or with the PicoGreenassay (10). Adenosine triphosphate (ATP) encapsulation was determinedby A260 with 1OD corresponding to 35.8 mg/mL ATP. Lipid concentrationswere determined by the inorganic phosphorus assay after separation of thelipids from the oligonucleotides by a Bligh and Dyer extraction.

Encapsulation of pDNA Using the Detergent Dialysis Procedure

The encapsulation of pDNA can also be accomplished with the use of adetergent dialysis procedure (12). In contrast to the PFV approach, the deter-gent dialysis procedure starts off with a micellar system and leads toencapsulation of pDNA in unilamellar liposomes called SPLP after detergentremoval. Plasmid entrapment relies on a delicate balance between cationiclipid content and ionic strength of the solution.

Encapsulation of pDNA: Typically pDNA was encapsulated in SPLPcomposed of DOPE, dioleoyldimethylammonium chloride (DODAC), and1-O-(20-(x-methoxy-polyethylene-glycol)succinoyl)-2-N-dodecanoylsphingo-sine (PEG-CerC20). Lipids (DOPE:DODAC:PEG-CerC20, 84:6:10 mol%)were dissolved in ethanol or chloroform and dried to a lipid film. The lipidmixture was resuspended in HBS (5 mM HEPES, 150 mM NaCl, pH 7.5)containing 200 mM OGP and 0.4 mg/mL pDNA [e.g., Plasmid DNA Con-taining the cytomegalovirus promotor and coding for luciferase (pCMV)-luc]. The final lipid concentration was 10 mg/mL. The mixture of lipid,plasmid, and OGP was dialyzed for 16 to 18 hours against three changes

134 Maurer et al.

of a 1000-fold volume excess of HBS. Unentrapped plasmid was removedby DEAE-Sepharose CL-6B chromatography. Entrapment efficiencieswere determined as described above for the PFV approach.

Separation of encapsulated plasmid from empty liposomes by sucrosedensity gradient centrifugation: The plasmid samples were applied to the topof a discontinuous sucrose gradient in 12.5-mL ultracentrifuge tubes. Thegradient was formed with 3 mL each of 10% sucrose, 2.5% sucrose, and 1%sucrose in HBS layered consecutively from bottom to top. The gradients werecentrifuged at 160,000� g for two hours at 20�C. The lipid-encapsulatedpDNA banded tightly at the interface between 2.5% and 10% sucrose,while the unassociated lipid was present as a smear from the top of thegradient to the interface between 1% and 2.5% sucrose. The SPLP band wascollected. Buffer-exchange and concentration was performed by diafiltration.

RESULTS

Preformed Vesicle Approach

The following paragraphs describe the factors that are important for encap-sulation and summarize the physicochemical and in vivo characteristics ofthe resultant liposomal systems using oligonucleotides as an example. How-ever, it should be noted that the PFV approach could be generally applied tothe entrapment of negatively-charged polyelectrolytes including pDNAand nonnucleotide-based polyelectrolytes.

Encapsulation and Physicochemical Properties

Ethanol is required for entrapment to occur. Addition of increasing amountsof ethanol to 100 nm DSPC/Chol/DODAP liposomes leads to the for-mation of large lipid structures following oligonucleotide addition and aconcomitant increase in oligonucleotide entrapment levels (Table 1). The

Table 1 Entrapment of Antisense Oligonucleotidein the Absence of Polyethyleneglycol-Ceramidea

% EtOH (v/v) % Encapsulation

0 4.5� 0.520 20.5� 1.530 32.5� 2.5

aEncapsulation efficiencies are listed as a function of ethanol

concentration for distearoyl-phosphatidyl-choline/choles-

terol/1,2-dioleoyl-3-dimethylammoniumpropane large unila-

mellar vesicle (LUVs). The initial oligonucleotide-to-lipid

ratio was 0.034 mol/mol (0.3 mg/mg). The LUVs used for

these experiments were 99� 22 nm in size. The encapsulation

values are given as mean� SD.


increase in size and entrapment reflect a progressive reorganization ofthe LUVs into large multilamellar liposomes, which can be seen in the trans-mission electron microscope (TEM) micrograph presented in Figure 1C. At40% (v/v) ethanol and higher, liposomes become unstable and fuse.

Incorporation of PEG-ceramide allows control of liposome size. Theaddition of ethanol was required for oligonucleotide encapsulation to occur.However, the size of the liposomes formed was large and could not becontrolled. Therefore, regulatory components, which allow the control offusion and aggregation processes responsible for the size increase, arerequired. It is known that incorporation of PEG-lipid conjugates into theliposomal membrane can inhibit liposome fusion and aggregation (20).PEG-lipids were therefore an obvious choice for regulating liposome size.In the presence of 2.5 to 10 mol% of PEG-Cer, oligonucleotides could beentrapped in liposomes that were not significantly larger than the parent lipo-somes from which they originated. Figure 2 depicts encapsulation efficienciesas a function of ethanol concentration for liposomes containing 10 mol%PEG-Cer. Maximum entrapment was reached at 40% ethanol and ethanolconcentrations in excess of 25% (v/v) were required for entrapment to occur.No entrapment was found in the absence of ethanol. The amount of ethanolrequired for entrapment to occur was dependent on the PEG-Cer content ofthe liposomes, decreasing with decreasing amount of PEG. It should be notedthat detergents such as octylgucoside could be substituted for ethanol.

Liposome size and entrapment efficiency. Both the size of the lipo-somes entrapping oligodeoxyonucleoliele (ODN) and the entrapmentefficiency depend on the initial oligonucleotide-to-lipid ratio. Figure 3 showsthat oligonucleotides can be efficiently entrapped at high oligonucleotide-to-lipid ratios. The entrapment efficiency is plotted as a function of the initialoligonucleotide-to-lipid ratio. The binding level at maximum entrapment is0.16 mg oligonucleotide per mg of lipid (0.023 mol/mol, negative-to-positivecharge ratio¼ 1.5). This corresponds to approximately 2200 oligonucleotidemolecules per 100 nm liposome and demonstrates the high efficiency of thisentrapment procedure. Entrapment efficiencies are about three orders ofmagnitude higher than obtained by passive encapsulation based on thetrapped volume.

Upon increasing the oligonucleotide-to-lipid ratio, the size as well as thepolydispersity of the samples increased slightly from 70� 10 nm for liposomesalone to 110� 30 nm for an initial oligonucleotide-to-lipid weight ratio of0.2 mg/mg. Freeze-fracture electron microscopy showed an increase in the num-ber of larger liposomes with increasing initial oligonucleotide-to-lipid ratios.

Morphology. Structural details were visualized by cryo-TEM. Figure 1Ais a cryo-TEM image of a sample with an entrapped oligonucleotide-to-lipidratio of 0.13 mg/mg. It confirms the coexistence of unilamellar liposomeswith bi- and multilamellar liposomes. The membranes of the latter are inclose contact. The inset of Figure 1A is an expanded view of a multilamellar

136 Maurer et al.

Figure 1 (A) Cryo-TEM picture of distearoyl-phosphatidyl-choline/cholesterol/1-O-(20-(x-methoxy-polyethylene-glycol)succinoyl)-2-N-myristoyl-sphingosine/1,2-dioleoyl-3-dimethylammoniumpropane liposomes entrapping oligonucleotides. Theinset is an expanded view of a multilamellar liposome showing two initially separatemembranes forced into close apposition by bound oligonucleotides (indicated bythe arrow). The entrapped antisense-to-lipid weight ratio was 0.125 mg/mg. Emptyliposomes prepared the same way as the oligonucleotide-containing liposomes canbe seen in (B). (C) TEM electron micrograph of a sample prepared in 30% ethanolin the absence of polyethyleneglycol-ceramide prior to anion exchange chromatogra-phy. The concentric bilayers of multilamellar liposomes can be clearly seen. Theentrapment was 32% at an initial oligonucleotide-to-lipid weight ratio of 0.3 mg/mg.Throughout the figure, the bars represent 100 nm. Abbreviation: TEM, transmissionelectron microscope.


liposome and shows two initially separate membranes forced into close appo-sition by bound oligonucleotides. The number of multilamellar liposomesincreases with increasing initial oligonucleotide-to-lipid ratio. The initial lipo-somes in the absence of antisense were unilamellar (Fig. 1B). The existence ofmultilamellar liposomes can only mean that more than one liposome partici-pates in their formation and points to an adhesion-mediated mechanism offormation (2,10).

Encapsulation is not dependent on a particular oligonucleotide or on lipidcomposition. Entrapment is a general feature of the interaction of negatively-charged polyelectrolytes with cationic liposomes. Figure 4 (black bars)shows that different oligonucleotides as well as pDNA can be efficientlyentrapped in DSPC/Chol/DODAP/PEG-CerC14 liposomes. Encapsulationof pDNA in preformed vesicles was less efficient than encapsulation of shortoligonucleotides. The maximum DNA-to-lipid ratio obtained with pDNAwas about four times lower than that achieved for the oligonucleotides.Other nonnucleotide-based polyelectrolytes such as polyanetholsulfate anddextransulfate can also be encapsulated (unpublished results). In contrast to

Figure 2 Encapsulation as a function of ethanol concentration. Oligonucleotideswere added to distearoyl-phosphatidyl-choline/cholesterol/1-O-(20-(x-methoxy-poly-ethylene-glycol)succinoyl)-2-N-myristoyl-sphingosine/1,2-dioleoyl-3-dimethylam-monium propane liposomes in varying concentrations of ethanol at an initialoligonucleotide-to-lipid ratio of 0.24 mg/mg. Abbreviations: AS, antisense oligonu-cleotide; %EtOH(v/v), percentage of ethanol in volume/volume.

138 Maurer et al.

the efficient encapsulation of large molecules, entrapment of ATP, a smallmolecule with three negative charges, was less than predicted based ontrapped volume calculations (10% at an initial ATP-to-lipid ratio of0.2 mg/mg and 50 mM citrate buffer). This indicates that there is a criticalsize (length) and number of charges required for entrapment to occur. Theentrapment procedure can be extended to other lipid compositions includingDOPE systems (Fig. 4, white bars).

Pharmacokinetics

SNALP systems can exhibit plasma half-lives of up to 12 hours, significantlylonger than the circulation half-life of free oligonucleotides and cationicliposome oligonucleotide complexes (Fig. 5). Increasing surface charge andPEG-coatings that dissociate from the liposome carrier can reduce thecirculation half-life (9). For example, replacement of PEG-CerC20 withPEG-CerC14 results in a reduction of the half-life from 10 to 12 hours to5 to 6 hours. This demonstrates a strong dependency of the circulationhalf-life on the length of the acyl chain contained in the hydrophobicceramide group, which anchors the PEG coating to the membrane. ThePEG-CerC14 lipid is able to rapidly exchange out of the lipid bilayer, withan in vitro half-life of approximately 1.1 hours. Under the same conditions,the exchange rate of the PEG-CerC20 is much slower (approximately 13 days),and this lipid is therefore able to provide steric protection against

Figure 3 Plot of the entrapment efficiency expressed as the entrapped oligonucleo-tide-to-lipid ratio (full circles) and percent entrapment (open circles) as a function ofthe initial oligonucleotide-to-lipid ratio. The ratios are given in w/w. Abbreviation:AS, antisense oligonucleotide.


interaction with plasma proteins and allows extended circulation times com-parable to classical sterically stabilized liposomes containing PEG2000-distearoylphosphatidylethanolamine (DSPE) (21,22). In summary, thecirculation half-life of SNALP can be adjusted by selecting PEG-lipids with

Figure 4 (Black bars) Entrapment of different antisense oligonucleotides as well aspDNA (pCMV-luc) in distearoyl-phosphatidyl-choline (DSPC)/cholesterol (Chol)/1-O-(20-(x-methoxy-polyethylene-glycol)succinoyl)-2-N-myristoyl-sphingosine (PEG-CerC14)/1,2-dioleoyl-3-dimethylammoniumpropane (DODAP) liposomes. The initialoligonucleotide-to-lipid weight ratio was 0.1 mg/mg and 300 mM citrate buffer wasused for oligonucleotide entrapment. The pDNA entrapment was performed in50 mM citrate buffer at a pDNA-to-lipid weight ratio of 0.03 mg/mg. (White bars)Entrapment of anti-c-myc DSPC/Chol/PEG-CerC14/DODAP (20/45/10/25 mol%)liposomes and dioleoylphosphatidyl-ethanolamine (DOPE)/PEG-CerC14/DODAP(45/10/45 mol%) liposomes. The initial oligonucleotide-to-lipid weight ratio was0.12 mg/mg for the DSPC/Chol system and 0.11 mg/mg for the DOPE system. Theinitial lipid concentration was 13 mM. The mRNA targets and sequences of the oligonu-cleotides are as follows: human c-myc (16-mr), 50-AACGTTGAGGGGCAT-30,human ICAM-1, 50-GCCCAAGCTGGCATCCGTCA-30 and human EGFR,50-CCGTGGTCATGCTCC-30. Abbreviations: DOPE, dioleoylphosphatidyl-ethanola-mine; DSPC, distearoyl-phosphatidyl-choline; Chol, cholesterol; pDNA, plasmid DNA.

140 Maurer et al.

different dissociation rates or by incorporation of cationic lipids with per-manent charge (9).

Detergent Dialysis Approach

A different approach for the encapsulation of pDNA starts off with amicellar system (12). It involves solubilization of lipids and pDNA inoctylglucoside-containing buffers and subsequent removal of the detergentby dialysis. This is in contrast to the preformed vesicle approach thatemployed subsolubilizing concentrations of ethanol or detergent renderingthe liposomes morphologically intact. The physicochemical characteristicsand in vivo properties of the resultant liposomal system, also called SPLP,will be described in the following.

Encapsulation and Physicochemical Properties

Plasmid DNA can be efficiently entrapped in liposomes. Encapsulation usingthe SPLP approach relies on the presence of a cationic lipid and a steric

Figure 5 Circulation profiles in mice. The influence of surface charge and presence ofa steric polymer surface coating on the circulation of SNALP was evaluated follow-ing IV administration in ICR mice. Formulations contained 3H-labeled mICAMODN and were administered at ODN doses of 15 mg/kg and approximate lipid dosesof 100 mg/kg body weight. Formulations evaluated were: distearoyl-phosphatidyl-choline (DSPC)/CH/1,2-dioleoyl-3-dimethylammoniumpropane/1-O-(20-(x-meth-oxy-polyethylene-glycol)succinoyl)-2-N-dodecanoylsphingosine (�), DSPC/CH/dioleoyldimethylammonium chloride (DODAC)/1-O-(20-(x-methoxy-polyethylene-glycol)succinoyl)-2-N-myristoyl-sphingosine (&), DODAC/dioleoylphosphatidyl-ethanolamine (1/1)/ODN complexes (¤), and free ODN (~). The lipid ratios were20/45/25/10 (mol/mol/mol/mol). The data points represent the mean� SD fromfive animals.


barrier lipid, as in the case of the PFV approach. Figure 6A demonstratesthat pDNA can be efficiently entrapped in DOPE/DODAC/PEG-ceramide(84:6:10 mol%) liposomes (12,13,23). Encapsulation efficiencies are comparableto those obtained using the PFV approach for pDNA. The trapping efficienciesare a very sensitive function of the relative amounts of cationic lipid andPEG-ceramide and the ionic strength of the medium (3,12,13). With increasingsize of the plasmid encapsulation, the efficiency decreased, coming down from

Figure 6 Encapsulation of plasmid DNA (pDNA) in small sterically stabilizedliposomes [stabilized plasmid-lipid particles (SPLP)] using a detergent dialysisprocedure. (A) Entrapped pDNA-to-lipid ratio as a function of the initial pDNA-to-lipid ratio (mg/mg). The initial lipid concentration was 10 mg/mL. (B) Cryo-electron micrograph showing the structure of SPLP. The location of the plasmidis indicated by the striated pattern superimposed on the liposomes. The bar repre-sents 100 nm.

142 Maurer et al.

80% for a 2.9 kb plasmid to 35% for a 15.6 kb plasmid at an initial plasmid-to-lipid ratio of 0.02 mg/mg (13).

Size and morphology. The cryo-EM picture in Figure 6B shows thatthese plasmid-lipid systems have the morphological features of LUV.The encapsulated pDNA can be seen as a striated pattern superimposedon the liposomes. The average diameter from dynamic light scatteringmeasurements is 70 nm. It should be noted that empty liposomes have beenremoved by ultracentrifugation as described in the ‘‘Methodology’’ section.

The mechanism of SPLP formation is not completely understood.Detergent dialysis involves a progression through different aggregatestructures including spherical micelles, disk-like micelles and liposomes asmore and more of the detergent is removed (24–26). In the presence of acationic lipid, the surface charge density on these aggregates will increasein going from micelles to liposomes. For entrapment to occur, pDNA hasto interact at a distinct point along this route. Both bilayer disks as wellas liposomes can act as intermediate structure as both could form unilamel-lar liposomes internalizing the pDNA in response to a reduction of thesurface area of one of their monolayers following DNA binding (27).

Pharmacokinetics, Tumor Accumulation, and TumorTransfection of SPLP

The SPLP system is one of a few systems that have been directly comparedto lipoplexes. The pharmacokinetics and biodistribution of the lipid as wellas the pDNA was followed together with the levels of gene expression at adistal tumor site (8). Figure 7A shows the pharmacokinetics of SPLP intumor-bearing mice in comparison to DODAC/DOPE lipoplexes. Theclearance of SPLP from circulation can be described by a first-order processwith a half time of 6.4� 1.1 hours. Relatively low levels of uptake by thelung and liver have been observed. Approximately 3% of the injected lipiddose accumulated at the tumor site. In contrast to SPLP, lipoplexes wererapidly cleared from circulation (t1/2

�15 minutes) and accumulated pre-dominantly in the lung and liver. Less than 0.5% of the injected dose wasfound at the tumor site after one hour and decreased at later timepoints.

The administration of SPLP results in reporter gene expression at thetumor site (Fig. 7B). Injection of free plasmid or lipoplexes resulted in nodetectable gene expression at the tumor site. However, transfection wasobserved in the lung, liver, and spleen. SPLP, on the other hand, did notshow detectable levels of gene expression in these organs.

CONCLUSIONS

Polynucleotides have been encapsulated by a variety of methods (12). How-ever, none of these procedures has yielded small, serum-stable particles in


combination with efficient encapsulation at high nucleic acid-to-lipid ratiosthat are required for clinical utility as a systemic drug carrier. We havedeveloped two procedures, the preformed vesicle approach and the detergentdialysis procedure, that allow efficient encapsulation of nucleic acid-basedmolecules in liposomes that are small in size (about 100 nm diameter) andstable in circulation. The preformed vesicle approach can be generally

Figure 7 Pharmacokinetic properties and in vivo gene expression of stabilized plas-mid-lipid particles (SPLP). (A) The levels of intact plasmid DNA (pDNA) in thecirculation resulting from IV injection of naked plamid pDNA (&), lipoplexes (�),and SPLP (�) were determined by Southern blot analysis of plasma samples(100mg pDNA/mouse). (B) Transgene expression at a distal tumor site resultingfrom IV injection of naked plamid pDNA (&), plamid pDNA-cationic liposomecomplexes (�), and SPLP (�).

144 Maurer et al.

applied to the entrapment of negatively-charged polyelectrolytes includingnonnucleotide-based molecules. Encapsulation by the detergent dialysis pro-cedure is more difficult to control as it relies on a delicate balance of cationicand PEG-lipid content and the ionic strength of the solution. Plasmid DNAcan be readily encapsulated using this approach; however, encapsulation ofoligonucleotides in small liposomes is difficult to achieve. The entrapmentof nucleic acids was found to be highly dependent on chain length, requiringa minimum length to occur and decreasing as the chain length became toolong (10,13).

The development of procedures that allow efficient encapsulation ofpDNA and oligonucleotides in small serum-stable liposomes has been a majoradvance toward systemic delivery of such drugs. Two of these systems haveshown promising results and have progressed into formal preclinical andclinical testing, respectively (16,18): SNALP, consisting of immune stimula-tory oligonucleotides encapsulated in a liposome also called Oligovax, andthe SPLP system containing a therapeutic plasmid. Liposome-encapsulatedimmune stimulatory oligonucleotides promise great potential for the treat-ment of cancer and inflammatory and infectious diseases. Encapsulationprotects these oligonucleotides from degradation, allowing the use of the nat-ural, more specific phosphodiester sequences instead of synthetic backbone-modified oligonucleotides that exhibit a variety of nonspecific and toxiceffects. Encapsulation can significantly enhance the immune stimulatorypotency of these molecules, naturally targeting them to antigen, presentingcells such as the macrophages of the liver and spleen, which are responsiblefor removal of particulate systems from circulation (17). Intravenous admini-stration of SNALP containing immune stimulatory CpG oligonucleotideresulted in significantly enhanced plasma cytokine levels and immune cell acti-vation as compared to free oligonucleotide (17). The liposome-encapsulatedoligonucleotides form a multimodal technology platform (18). For example,liposome-encapsulated oligonucleotides can be combined with a specific diseasemarker, for example, a tumor antigen, to direct a specific immune responseagainst a particular disease, in this case against cancer. This technology can alsobe applied to the development of cancer vaccines, infectious disease vaccines oras an adjuvant to existing vaccines or alone to stimulate a protective immuneresponse. In addition, liposome-encapsulated immune stimulatory oligonucleo-tides can enhance the potency of tumor antibodies such as Herceptin byenhancing antibody-dependent cellular cytotoxicity.

The plasmid-containing SPLP system can achieve highly selective pro-tein expression at sites of disease after systemic administration, resulting inlocal therapeutic effects while minimizing systemic exposure. Different genetherapy approaches have been tested including delivery of a plasmid thatencodes an enzyme that converts a prodrug into its active cytotoxic formand plasmids that express immune stimulatory proteins and toxins. The firstof these approaches has progressed into clinical trials (16).


In summary, the preformed vesicle approach and detergent dialysisprocedure have enabled development of nucleic acid-based therapeuticswith clinical utility. Further applications of these liposomal systems withnew nucleic acid-based therapeutics such as small interfering RNA for genesilencing are being developed and have demonstrated promising results (28).

REFERENCES

1. Allen TM, Cullis PR. Drug delivery systems: entering the mainstream. Science2004; 303:1818.

2. Maurer N, Fenske DB, Cullis PR. Developments in liposomal drug delivery sys-tems. Exp Opin Biol Ther 2001; 1:923.

3. Maurer N, Mori A, Palmer L, et al. Lipid-based systems for the intracellulardelivery of genetic drugs. Mol Membr Biol 1999; 16:129.

4. Felgner PL, Gadek TR, Holm M, et al. Lipofection: a highly efficient, lipid-mediated DNA transfection procedure. Proc Natl Acad Sci USA 1987; 84:7413.

5. Gao X, Huang L. Cationic liposome-mediated gene transfer. Gene Ther 1995; 2:710.6. Felgner PL. Nonviral strategies for gene therapy. Sci Am 1997; 276:102.7. Chonn A, Cullis PR. Recent advances in liposome technologies and their

applications for systemic gene delivery. Adv Drug Del Rev 1998; 30:73.8. Tam P, Monck M, Lee D, et al. Stabilized plasmid-lipid particles for systemic

gene therapy. Gene Ther 2000; 7:1867.9. Semple SC, Klimuk SK, Harasym TO, et al. Efficient encapsulation of antisense

oligonucleotides in lipid vesicles using ionizable aminolipids: formation of novelsmall multilamellar vesicle structures. Biochim Biophys Acta 2001; 1510:152.

10. Maurer N, Wong KF, Stark H, et al. Spontaneous entrapment of polynucleo-tides upon electrostatic interaction with ethanol destabilized cationic liposomes:formation of small multilamellar liposomes. Biophys J 2001; 80:2310.

11. Fenske DB, Maurer N, Cullis PR. Encapsulation of weakly-basic drugs, anti-sense oligonucleotides, and plasmid DNA within large unilamellar vesicles fordrug delivery. In: Torchilin VP, Weissig V, eds. Liposomes: A PracticalApproach. 2nd ed. Northants, U.K.: Oxford University Press, 2003:167–191.

12. Wheeler JJ, Palmer L, Ossanlou M, et al. Stabilized plasmid-lipid particles:construction and characterization. Gene Ther 1999; 6:271.

13. Saravolac EG, Ludkovski O, Skirrow R, et al. Encapsulation of plasmid DNA instabilized plasmid-lipid particles composed of different cationic lipid concentra-tion for optimal transfection activity. J Drug Target 2000; 7:423.

14. Maeda H, Wu J, Sawa T, Matsumara Y, Hori K. Tumor vascular permeabilityand the EPR effect in macromolecular therapeutics: a review. J Control Rel2000; 65:271.

15. Fenske DB, MacLachlan I, Cullis PR. Stabilized plasmid-lipid particles: a systemicgene therapy vector. In: Phillips MI, ed. Methods in Enzymology: Gene TherapyMethods. Vol. 346. San Diego, CA, U.S.A.: Academic Press Inc., 2002:36–71.

16. http://www.protivabio.com17. Mui B, Raney SG, Semple SC, Hope MJ. Immune stimulation by a CpG-

containing oligodeoxynucleotide is enhanced when encapsulated and deliveredin lipid particles. J Pharmacol Exp Ther 2001; 298:1185.

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18. http://www.inexpharm.com19. Bailey AL, Cullis PR. Modulation of membrane fusion by asymmetric trans-

bilayer distributions of amino lipids. Biochemistry 1994; 33:12573.20. Holland JW, Hui C, Cullis PR, Madden TD. Poly(ethylene glycol)-lipid

conjugates regulate the calcium-induced fusion of liposomes composed of phos-phatidylethanolamine and phosphatidylserine. Biochemistry 1996; 35:2618.

21. Webb MS, Saxon D, Wong FM, et al. Comparison of different hydrophobicanchors conjugated to poly(ethylene glycol): effects on the pharmacokineticsof liposomal vincristine. Biochim Biophys Acta 1998; 1372:272.

22. Semple SC, Chonn A, Cullis PR. Interactions of liposomes and lipid-basedcarrier systems with blood proteins: Relation to clearance behaviour in vivo.Adv Drug Deliv Rev 1998; 32:3.

23. Mok KW, Lam AM, Cullis PR. Stabilized plasmid-lipid particles: factors influ-encing plasmid entrapment and transfection properties. Biochim Biophys Acta1999; 1419:137.

24. Ollivon M, Eidelman O, Blumenthal R, Walter A. Micelle-vesicle transition ofegg phosphatidylcholine and octyl glucoside. Biochemistry 1988; 27:1695.

25. Lasic DD. The mechanism of vesicle formation. Biochem J 1988; 256:1.26. Almog S, Litman BJ, Wimley W, et al. States of aggregation and phase

transformations in mixtures of phosphatidylcholine and octyl glucoside.Biochemistry 1990; 29:4582.

27. Lasic DD. Liposomes in Gene Delivery. Boca Raton: CRC Press, 1997, Chapter 7.28. Morrissey DV, Blanchard K, Shaw L, et al. Activity of systemically administered

stabilized siRNAs in a mouse model of HBV replication. Presented at the Amer-ican Association for the Study of Liver Disease (AASLD) Conference, Boston,Massachusetts, 2004.


8

Intraliposomal Trapping Agents forImproving In Vivo Liposomal Drug

Formulation Stability

Daryl C. Drummondy, Mark E. Hayes, Charles O. Noble IV, andDmitri B. Kirpotin

Hermes Biosciences, Inc., South San Francisco,California, U.S.A.

John W. Park

University of California at San Francisco Comprehensive Cancer Center,San Francisco, California, U.S.A.

Zexiong Guo

First Affiliated Hospital of Jinan University,Guangzhou, P.R. China

INTRODUCTION

Controlling the rate of drug release from liposomal carriers is essential foroptimum drug delivery (1,2). Liposome formulations that are too unstablerelease their drug while still in the general circulation, thus reducing the ben-efits of site-specific drug delivery resulting from the enhanced permeability

yDaryl C. Drummond is supported in part by a New Investigator Award from the California

Breast Cancer Research Program of the University of California, Grant Number 7KB-0066.

149

and retention effect and allowing for many of the systemic toxicitiesassociated with the unencapsulated agent. Formulations that are too stablerisk not making the drugs bioavailable, and thus able to act on theirmolecular targets. Furthermore, delivery strategies that rely on moleculartargeting of solid tumors, including immunoliposomes, require that the lipo-somal drug formulation arrive at the cancer cell intact to take full advantageof the benefits of targeting (3,4).

A wide range of drug-entrapment strategies based on various iongradients have been hailed for their ability to quantitatively load certainweakly basic or weakly acidic drugs into the liposomal lumen (5–12). Of equalimportance is the ability of these remote-loading strategies to stabilize thedrug, so that upon administration the drug is retained inside the liposomeuntil reaching its site of action. Although several drugs in the anthracyclineclass of anticancer agents have been stabilized with relative ease, this stabilityhas been replicated less successfully when alternative classes of drugs havebeen entrapped. For example, vincristine (VCR) (13–16) and various camp-tothecin (17–19) liposome formulations are substantially more unstable thandoxorubicin formulations (14) prepared using similar lipid compositions anddrug-loading methodologies.

This chapter discusses the use of intraliposomal trapping agents tomaximize the retention of weakly basic amphipathic drugs while in thecirculation. We have employed gradients of substituted ammonium saltsof poly(anionic) polymers and polyols to encapsulate and retain drugsmore stably inside liposomes. To date, we have used these strategies toencapsulate and stabilize a number of both standard and novel anticancerchemotherapeutic agents inside liposomes (Fig. 1). The polyanionic trap-ping agents form stable intraliposomal complexes with the weakly basicdrug, possibly forming precipitates or gels inside the liposome. A dia-gram depicting the stabilization process is shown in Figure 2A. Here,the novel histone deacetylase inihibitor, LAQ824 (Novartis Pharmaceuti-cals; East Hanover, New Jersey, U.S.A.), is shown in a complex with asulfated polyol, sucrose octasulfate. The disruption of the complex andthe subsequent transmembrane diffusion of the drug govern the apparentin vivo stability of the liposomal formulation. Chemical structures for someof the anionic trapping agents employed for liposomal drug stabilizationare shown in Figure 2B. The large majority of formulations that haveemployed remote-loading strategies have used either citrate or sulfateas the counterion for protons, Mn2þ, or ammonium (6,8,16,19,20). Ourexperience has been that liposomal drug formulations prepared using theseanions as trapping agents often result in poor in vivo stability for a numberof well-established and novel anticancer agents. This chapter describes ourefforts to improve the in vivo stability of these liposomal drugs usingpolyanionic trapping agents, including polyphosphate, sucrose octasulfate,and inositol hexaphosphate.

150 Drummond et al.

METHODS

Preparation of Trapping Solutions and Liposomes

Many of the salts employed in the preparation of remote-loading gradientsare commercially available and thus involve nothing more than dissolving

Figure 1 Chemical structures of some amphipathic weak bases that have been loadedand stabilized in liposomes using trialkylammonium salts of polyanionic trappingagents in our lab. (A) Doxorubicin, (B) epirubicin, (C) vinorelbine, (D) vincristine,(E) vinblastine, (F) topotecan, (G) irinotecan, (H) swainsonine, (I) 2-diethylami-noethyl-ellipticinium, (J) 6-(3-aminopropyl)ellipticine, and (K) LAQ824.

Intraliposomal Trapping Agents 151

Figure 2 (Continued on facing page) Intraliposomal drug stabilization using polyanio-nic trapping agents. (A) Depiction of intraliposomal stabilization of LAQ824 using thepolyanionic polyol, sucrose octasulfate. Upon sequestration in the liposomal lumen,the drug forms a stable complex with sucrose octasulfate, possibly forming a gel or pre-cipitate. The rates of dissolution of the precipitate, disruption of the complex, andtransmembrane diffusion of the drug all contribute to the in vivo stability of the lipo-somal drug formulation. (B) Chemical structures of poly(anionic) trapping agents:(I) sulfate, (II) citrate, (III) sucrose octasulfate, (IV) poly(phosphate), (V) suramin,and (VI) dextran sulfate.

152 Drummond et al.

the salt in water to the desired concentration and, in some instances, adjust-ing the pH into a range acceptable for the chemical and physical stability ofthe liposome formulation (pH 4–8, but preferably 5–7). Manganese sulfate,ammonium sulfate, ammonium citrate, and citric acid are a few of the saltsthat are readily available, and perhaps noncoincidentally represent the largemajority used in remote-loading strategies. Other polyanionic trapping agentsrequire exchange of the counter ion for one more suitable for drug loading. Weprefer the use of substituted ammonium salts, including triethylamine, diethy-lamine, 2-diethylaminoethanol, and 4-(2-hydroxyethyl)-morpholine for thispurpose. However, we should emphasize that it is also possible to load weaklybasic amphipathic drugs into liposomes using other cationic species, including

Figure 2 (Continued from previous page)


ammonium and even sodium (21). Other pharmaceutically acceptable substi-tuted ammonium salts are described in Handbook of Pharmaceutical Salts (22).

If the trapping agent is available in the acid form, then simple titrationwith the chosen amine gives rise to the desired salt. However, many poly-anionic compounds are not stable in this form and only available as saltswith other cations; Naþ, Kþ, Ca2þ, or Mg2þ. Ion-exchange chromatographycan be used to prepare the most suitable salt. Typically, an appropriatecation-exchange resin (e.g., Dowex 50Wx8-200, Dow Chemical Co.) iswashed with 1 N solutions of NaOH and HCl, and subsequently equili-brated with higher concentrations of HCl to maintain the resin in the hydro-gen form. A concentrated solution of the polyanionic compound is thenadded to the column and eluted with water, using a conductivity meter todetect elution of the acidic form of the polyanion. The polyanion is thenimmediately titrated with the substituted amine of choice to give the desiredsalt. An electrode specific for the initial cationic species (e.g., Naþ electrode)can be used to measure the efficiency of exchange. The salt is then dilutedto a concentration, preferably chosen to maximize the drug load, whilepreventing unusually high osmotic imbalances that might result in the lipo-somes bursting during drug loading or in the presence of plasma (23,24).

Liposomes can be prepared using a wide range of methods that havebeen thoroughly reviewed in a previous edition of this series (Vol I, 2nd ed.)and elsewhere (25); therefore, it will not be described in great detail here.Our preference for liposome formation involves dissolving the lipids inethanol at an elevated temperature followed by rapid mixing with an aque-ous solution of the trapping agent (typically corresponding to 0.5–0.75 Msubstituted ammonium salt) equilibrated at the same temperature, followedby sizing of the liposomes using high-pressure extrusion (Vol I, Chapter 4 ofthis series). The liposomes are typically characterized with regard to particlesize to ensure the liposome size is acceptable for the desired applicationbefore proceeding with the generation of the gradient.

Gradient Generation and Drug Loading

The gradient for the polyanionic trapping agent is generated by removalof the extraliposomal salt using gel filtration chromatography, dialysis,ion-exchange chromatography, or a combination of these approaches. Typi-cally, gel filtration chromatography is utilized for bench-scale preparations,whereas dialysis is preferred for large-scale production. Ion-exchange chro-matography is particularly useful for removing trace amounts of polyanio-nic trapping agents that may precipitate drugs outside the liposomes prior totheir loading. The external solution is then exchanged for one that containsboth an isotonicity agent (sucrose, dextrose, saline) and an appropriatebuffer. The concentration of the isotonicity agent used is selected to mini-mize the potential for osmotic shock resulting in liposome lysis during drug

154 Drummond et al.

loading at elevated temperatures. The specific agent employed is chosen toallow for drug solubilization. For example, although doxorubicin is solubleand can be loaded in saline solutions, many other drugs are salted out whensaline is present, thus requiring the use of a nonionic isotonicity agent, suchas sucrose or dextrose. The buffer is chosen to support the pH optimum fordrug loading. A study of drug loading at different pHs can be used to deter-mine this optimum. Although many drugs are loaded under a pH widerange, some drugs have multiple titratable groups or are affected by neigh-boring substituents such that only a narrow pH range can be used to obtainloading, while avoiding the extremes of pH that may result in chemicaldegradation of either the drug or liposomal lipid components.

Drug loading is initiated by adding the drug at the desired drug-to-lipid ratio and raising the temperature to above the phase transition ofthe phospholipid (PL) component. Some loading may proceed below thephase transition, but it is generally less efficient than that observed at highertemperatures. Upon addition of the drug, the pH of the extraliposomal solu-tion may require further adjustment. Many drugs are available as acidicsalts and, depending on the amount of drug added, their addition mayadversely affect the pH of the solution, so that it falls out of the optimumrequired for loading. The samples are then incubated for a determinedamount of time, in our hands typically 30 minutes at 60�C. Others haveshown loading to be complete in as short as 10 to 15 minutes for phospha-tidylcholine-containing formulations (12,26). Finally, the loading reaction isquenched rapidly by lowering the temperature rapidly below the phase tran-sition. For research-scale preparations, this simply involves incubation onice for 15 to 20 minutes.

Assessing Drug Entrapment and Retention

Assessing Drug Entrapment

To assess drug entrapment or drug retention, the drug-to-lipid ratio is deter-mined and compared to an initial ratio, either a preloading ratio in the caseof drug-entrapment determinations or a preincubation ratio in the case ofdrug-retention assays. For research-scale liposome preparations, the drug-loaded liposomes are commonly purified to remove unencapsulated drugfollowing drug loading. Purification can be accomplished by gel filtrationchromatography, dialysis, or ion-exchange chromatography. For large-scale–manufactured liposomes, it is often not necessary or even desirableto purify the final product, as a result of the high efficiency of drugencapsulation observed with many agents prepared using remote-loadingstrategies. However, in such cases, quality control often involves taking asample of the batch, purifying it, and determining the efficiency of encapsu-lation by comparing the drug-to-lipid ratio of the purified liposomes to thoseprior to drug-loading. The drug can be analyzed by a variety of methods,


depending on the chemical nature of the drug itself, including high-performanceliquid chromatography, fluorimetry, or simply UV/Vis spectrophotometry.The lipid is commonly determined by phosphate analysis (27), but can also bedetermined using a radioactively labeled and nonexchangeable lipid marker,such as [3H]cholesterylhexadecylether. Two parameters are important in char-acterizing drug encapsulation in liposomes. The first is the drug load, expressedcommonly as grams of drug/gram or mol of lipid, and the second is entrapmentefficiency, expressed as the percentage of drug encapsulated as a function of theinitial preload ratio [Eq. (1)]:

Entrapment efficiency (%) = 100�½drug=lipid�p½drug=lipid�i

ð1Þ

where [drug/lipid]p refers to the determined drug-to-lipid ratio following puri-fication of the loaded liposomes and [drug/lipid]i refers to the drug-to-lipid ratioof the initial preparation prior to loading or purification.

The drug load can provide some information about the amount ofdrug that can be loaded into each liposome. This amount may be limitedby the size of the liposome, the chemical nature of the trapping agent, thephysicochemical properties of the drug, or the magnitude of the gradientprepared. The second parameter gives an indication of how efficient thedrug-encapsulation process was under a specific set of conditions (tempera-ture, pH, input drug-to-lipid ratio, liposome size, and lipid composition).

Drug Retention

The characterization of drug retention is important for determining thestability of the liposomal drug formulation during storage and while in thegeneral circulation. In order to effectively characterize any liposome drug-trapping method, it is important to determine the stability using conditionsthat mimic both situations. For stability during storage, the liposomal drugshould be concentrated to a concentration suitable for injection and stored inthe presence of the excipient (i.e., isotonicity and buffering agents) to be usedduring storage. Because the excipients may influence the gradients used toretain the drug in liposomes, it is important to mimic the conditions to beused in the final product as precisely as possible. Stability studies are bestperformed under the conditions employed during storage, typically 4�C to6�C for most gradient-loaded liposomal drugs. Accelerated stability studiesat elevated temperatures have also been performed, but with the importantqualification that the elevated temperature may affect drug formulation sta-bility in an indirect fashion by altering the physical state of the liposomalmembrane. Although discrete phase transitions are often reported for lipidmembranes, there are sometimes pretransitions, phase transitions that areobscured by the presence of cholesterol (chol), or transitions that are less wellunderstood (28), which may indeed affect the membrane permeability of the

156 Drummond et al.

drug or ions used to create the gradients essential for their entrapment. Thus,without careful modeling, it may be difficult to predict what would in factoccur at the actual temperature used during storage. Drug retention duringstorage can be determined by removing an aliquot or a vial of liposomal drugat prescribed times and then measuring the drug and PL components follow-ing purification as described above. The amount of drug that remainsentrapped is then compared to the amount of drug associated with the lipo-some at time ¼ 0 to determine the drug retention.

Drug retention in the blood is a considerably more complicated under-taking. Multiple formulations prepared using different drug-trapping agentscan be initially screened by simple incubation in the presence of human plasmato get a general idea of the effect of plasma proteins on formulation stability.Incubation in the presence of saline or other isotonic buffers does not provide asufficient reservoir of drug-binding sites for the liposome-associated drugs andthus may result in a false sense of security with regard to the stability of theformulation. Unfortunately, it is often common practice to initially describethe stability of a liposomal drug by its degree of drug retention in one of thesesimple media. Our preference is to screen formulation methods initially using amicrodialysis assay where small wells containing liposomes are separated froma significantly larger reservoir of human plasma by a filter with pore sizes of30 nm. The large dilution factor provides a more stringent test of the liposome’sstability than a simple 1:2 to 1:5 dilution with plasma. At prescribed times, analiquot of the sample is then removed and purified by gel filtration chromato-graphy. The lipid is then measured by either scintillation counting of [3H]CHEor phosphate analysis of PLs. The drug is determined by fluorimetry or HPLCand the drug-to-lipid ratio of the purified liposome is calculated and comparedto the initial liposome preparation to determine the amount of drug leakage.A representative study is shown in Figure 3A for multiple liposome formula-tions of liposomal 4-(3-aminopropyl)ellipticine. Purification is not absolutelyrequired if using [3H]CHE, but is necessary to remove phosphate-containingspecies in the plasma if a simple phosphate assay is used.

Although these assays allow for rapid screening of multiple formulationmethods to remove rapidly leaking formulations from further consideration,they are not necessarily an accurate predictor of the liposomal drug’s stabi-lity in vivo. The most rigorous test of liposomal drug retention is to measurethe change in drug-to-lipid ratio in vivo using small rodent models (13,16).Small molecular weight free drugs are commonly cleared at a considerablyfaster rate than same drugs encapsulated in liposomes. Thus, a reductionin the drug-to-lipid ratio is an excellent indicator of the degree of drug leak-age from the liposome in vivo. If further screening is required, then single ordual time point (e.g., 8 and 24 hours) studies in mice have allowed us to reducefurther the liposomal drug formulations being considered. A completepharmacokinetic study in rats, measuring both lipid ([3H]CHE) and drug,concentrations will give a complete data set, including information about


Figure 3 In vitro (A) and in vivo (B) stability of APE encapsulation in liposomesprepared using a wide range of intraliposomal trapping agents. All formulations werecomposed of 1,2-distearoyl-3-sn-phosphatidylcholine: cholesterol:N-(polyethyleneglycol)distearoylphosphatidylethanolamine (DSPC:Cho:PEG-DSPE) (3:2:0.015,mol:mol:mol). Cholesterylhexadecylether ([3H]CHE) was added at a ratio of 0.5mCi/mmol PL for the in vivo study. All liposomes were loaded at a APE-to-PL ratioof 100 g/mol. All formulations had an identical triethylammonium or ammoniumconcentration of 0.55 M. (A) Liposomes were prepared using the following trappingagents; poly(phosphate) (&), linear triphosphate (&), trimetaphosphate (~), andsulfate (~). Liposome samples were incubated with human plasma in a microdialysisassay at prescribed time points, purified by gel filtration chromatography, and ana-lyzed for both drug (fluorimetry) and lipid (liposomal PO4). The APE-to-PL ratiowas then calculated and compared to the initial ratio, prior to incubation, to deter-mine the amount of APE retained in the liposomes. (B) Liposomal APE was preparedusing ammonium salts of various polyanionic trapping agents and administeredintravenous to Swiss–Webster mice. At 24 hours, the mice were sacrificed and theblood collected and analyzed for both APE and lipid ([3H]CHE) scintillation count-ing. The %ID in the blood at 24 hours for liposomal lipid and APE are depicted bythe white and black bars, respectively. The corresponding APE-to-PL ratio (normal-ized to the ratio of the administered liposomes) is shown using the hatched bars.Abbreviations: %ID, percentage injected dose; APE, 6-(3-aminopropyl)ellipticine;PL, phospholipid.

158 Drummond et al.

the pharmacokinetics of the liposome carrier itself, its encapsulated drug, andthe rate of drug leakage from the formulation.

FACTORS INFLUENCING IN VIVO DRUG RETENTION

Anion or Polyanion Used as Trapping Agent

Our experience and that of others is that the use of pH, Mn2þ, or ammo-nium gradients to load various anticancer drugs can result in quantitativeloading of a wide range of drugs (6,8,9,12,13,19,26,29). However, with theexception of anthracycline class of drugs, many drugs from other classesleak rapidly from liposomes in the blood. Our experience has been thatthe anionic counterion plays an important role as a trapping agent, forminga stable complex with the drug inside the liposome and limiting is trans-membrane diffusion once in the circulation. However, different counterionsdisplay varying levels of effectiveness in their ability to stabilize the drug.An example of this is shown in Figure 3. Initially an in vitro microdialysisassay was performed as described above, incubating liposome formulationsloaded with 6-(3-aminopropyl)ellipticine (APE), but prepared using triethyl-ammonium (TEA) salts of various anions, with human plasma at 37�C andthen measuring the amount of drug retained over time (Figure 3A). As canbe observed, sulfate was a relatively poor trapping agent for this particulardrug, with greater than 25% of the drug having leaked by 24 hours and 50%by 72 hours. Liposomes formed with trimetaphosphate, a cyclical phosphatederivative, also leaked but less readily than the sulfate. Liposomes formedusing tripolyphosphate or polyphosphate (n¼ 13–18) were considerablymore stable in this assay. It should be noted that when the liposomes pre-pared using the sulfate salt were examined in vivo, the drug leaked evenmore rapidly (data not shown).

A study in mice considering the concentrations of drug and lipid inthe blood at 24 hours, as well as the relative drug-to-lipid ratios, is shownin Figure 3B. It was observed that phosphate was the poorest stabilizer ofAPE in liposomes, followed by citrate, poly(vinylsulfonate), and poly-(phosphate). When poly(phosphate) was used as the stabilizing anion, theamount of drug retained in the liposomes was greater than 95% at 24 hours.We have recently developed liposomal formulations of the histone deacety-lase inhibitor LAQ824 (29), vinorelbine (VNB) (30), and irinotecan (31) in acomplex with sucrose octasulfate that demonstrate remarkable in vivo sta-bility. These results demonstrate that the chemical nature of the trappingagent employed in drug loading can dramatically affect the in vivo stabilityof the subsequent liposome preparation.

Some questions have also been raised about the activity of liposomalagents prepared using polyanions because highly stable liposomes loaded with


either VCR or a weakly basic camptothecin derivative, and encapsulatedusing dextran sulfate or suramin, displayed either decreased activity (11)or increased toxicity (32). We have encapsulated a wide range of agentsand observed considerably activity both in vitro (Fig. 4) and in vivo (29).Targeted liposomal therapeutics are particularly dependent on formulationstability, as they must reach their target intact in order to optimally takeadvantage of the molecular targeting. A liposomal ellipticine analog (APE)was shown to have considerable HER2-specific cytotoxic activity in HER2-overexpressing breast cancer cells (Fig. 4), demonstrating that the drug couldbe made bioavailable in a relevant time period. Our preference has been for theuse of the high charge density polyols, most notably sucroseoctasulfate. The useof these agents has resulted in a number of highly stable and active liposomeformulations of anticancer drugs (29–31). In addition, sucrose octasulfate is

Figure 4 Cytotoxicity of HER2-directed liposomal APE in HER2-overexpressingBT474 human breast carcinoma cells. Cells were plated at a density of 5000 cells/welland incubated for four hours with varying concentrations of unencapsulated (�), lipo-somal (�), or antiHER2 (F5)-immunoliposomal (~) Cells were assayed forviability using a standard tetrazolium-based assay three days later. Abbreviation: APE,6-(3-aminopropyl)ellipticine.

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readily manufactured in a defined form that is more difficult to control withpolymers. It is also free of serious systemic toxicities and does not display theanticoagulant activity (33) associated with many other polyanionic polymers.

Precipitation or Gelation of Drug

Doxorubicin loaded into liposomes using ammonium sulfate gradients wasshown to form intraliposomal crystals that were thought to stabilize the druginside the liposome (7). Similar structures were later observed with lipo-somes loaded with doxorubicin using the pH gradient method with citrateas the counterion (34). It is possible that intraliposomal gelation or precipi-tation plays an important role in stabilizing the drug inside the liposomes,and that various counterions precipitate or gelate the drug more efficiently.Indeed, we have observed using microscopy that APE forms precipitateswith either poly(phosphate) or poly(vinylsulfonate), but not with sulfateor citrate (unpublished observation). In contrast, doxorubicin forms precip-itates with both citrate and sulfate under the same conditions. Althoughprecipitation or gelation may play a role in the stabilization process, it doesnot ensure in vivo drug retention similar to that seen with doxorubicin-citrateor doxorubicin-sulfate formulations. Bally et al. have recently demonstratedthat topotecan similarly forms what appears to be intraliposomal precipitates(Fig. 5) (19). However, these liposomes release topotecan at a considerablyfaster rate than observed for similar doxorubicin formulations, suggesting thatthe precipitation is not solely sufficient for in vivo stability.

Figure 5 An electron micrograph of unloaded liposomes or liposomal topotecan sta-bilized in a SO4 complex following loading using MnSO4 gradients in the presence of theionophore A23187 at a ratio of topotecan-to-lipid of 0.2 (wt:wt). Source: From Ref. 35.


Drug-to-Lipid Ratio

The concentration of drug loaded into the liposome can effect the stabilityof the final formulation. An early study demonstrated that using drug-to-lipid ratios that were too high resulted in less stable formulations, possiblyresulting from dissipation of the pH-gradient used for drug loading(15,16,36). However, more recent studies (19), including those of our own,have shown that liposomes with higher drug loads have markedly increasedstability. The higher intraliposomal drug concentrations could drive theformation of stable intraliposomal precipitates as the drug surpasses the aque-ous solubility of drug inside the liposome. Thus, there may exist a balancebetween the formation of stable precipitates and gels inside the liposomesand the dissipation of a pH gradient that may help keep the drug in a lessmembrane permeable charged form inside the liposome. We have observedthat the use of polyanionic trapping agents, such as sucrose octasulfate, allowfor liposome drug loading at remarkably high drug-to-lipid ratios. An exam-ple of this is shown in Figure 6. Here the vinca alkaloid, VNB, is shown toload quantitatively up to a drug-to-PL ratio of 450 g VNB/mol PL. The highdrug loads result in part from the increased concentrations of anionic groupsthat can be loaded into liposomes when present as a polyanion, withoutcausing a destabilizing osmotic imbalance.

Lipid Composition, Size, and Osmolarity

The lipid composition of the liposome membrane plays an important role incontrolling the rate of drug release. The phase transition temperature of theliposomal lipids in part determines the rate of drug leakage, with liposomescontaining lipids of shorter lengths or unsaturations displaying increasedrates of drug leakage (37–40). In these studies, doxorubicin leaked fromliposomes composed of unsaturated phosphatidylcholines significantlyfaster than liposomes prepared from hydrogenated phosphatidylcholinesor distearoylphosphatidylcholine. The presence of chol also helps reduce thepermeability of PLs vesicles to small molecular weight drugs or ions (41–43).Finally, the inclusion of sphingomyelin into liposome formulations contain-ing chol has also been shown to reduce membrane permeability to drugs orsmall molecular weight ions (13,44). This possibly results from an increasedmembrane cohesiveness due to intermolecular hydrogen bonding betweenneighboring chol hydroxyl groups and sphingomyelin amide nitrogens(45,46). However, sphingomyelin is presently a costly lipid to use as a majorcomponent of a liposomal therapeutic. Fortunately, it appears that drugretention can be in large part controlled through modulation of intraliposo-mal drug complexes as described above, although further improvements inthe drug-release profile upon inclusion of sphingomyelin are possible.

The size of the liposome determines the entrapped volume (47) andthus limits the amount of drug that can be entrapped in a single liposome.

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However, a high radius of curvature at very small liposome sizes (i.e.,~50–70 nm) may also result in membrane defects that increase the per-meability of drugs or gradient-forming ions, thus also adversely affectingliposomal drug retention. We have observed with both topotecan andVNB, loaded under specific conditions (drug load and specific trappingagents), that decreasing vesicle size below 80 nm resulted in an increased rateof drug release from the carrier.

Osmotic imbalances are also important to control when designing lipo-somal drug formulations. In the presence of plasma or high temperatures,significant osmotic imbalances can result in liposome lysis and release ofthe internal contents (23,24,48). As described above, it is possible to reducethese imbalances and thus encapsulate higher concentrations of anionic sitesfor drug-binding with the use of high-density polyanionic compounds fortrapping drugs inside liposomes.

Figure 6 Determination of the loading capacity for VNB in liposomes prepared usinggradients of TEA sucroseoctasulfate (0.65 M TEA). Liposomal loading efficiency as afunction of input vinorelbine (VNB)-to-phospholipid (PL) ratio. 1,2-distearoyl-3-sn-phosphatidylcholine/cholestrol/N-(polyethylene glycol)distearoylphosphatidylethano-lamine (DSPC/chol/PEG-DSPE) 3:2:0.015, mol:mol:mol) liposomes were loaded withVNB, with the initial amount of VNB added to the liposomes varying from 150 to550mg VNB/mmol PL. Following incubation for 30 minutes at 60�C, the loadingmixture was quenched on ice and unencapsulated drug was removed by gel filtrationchromatography using a Sephadex G-75 column eluted with Hepes-buffered saline(pH 6.5). The resulting VNB-to-PL ratio, following loading, was determined by quan-titating both VNB and PL in the resulting purified liposomal VNB formulation, andthe loading efficiency by comparing this ratio to the input ratio. Abbreviations: VNB,vinorelbine; PL, phospholipid; TEA, triethylammonium.


COLLOIDAL AND CHEMICAL STABILITY CONSIDERATIONS

The chemical and colloidal stability of liposomal drug formulations preparedusing remote-loading gradients is an important concern for maintaininghighly stable and active liposomal therapeutics. A detailed discussion ofthe colloidal stability of lipid vesicles is beyond the scope of this chapterand the reader is referred to several excellent reviews on the subject(49,50). However, in general, aggregation can be minimized by inclusion ofanionic lipids or polymer-coated surfaces. Because aggregated vesiclesmay have a greater propensity for increased drug leakage, it is importantto minimize the level of aggregation during both storage and while in cir-culation. The concentration of liposomes in the vial, as well as the chosenexcipient, may also help determine the degree of colloidal stability for thefinal liposome preparation.

Because the gradients prepared can result in extremes of pH undersome circumstances, it is important to characterize the stability of boththe encapsulated agent during storage and the liposomal lipids. Fortunately,many drugs demonstrate increased stability at low pH and thus are notadversely affected by entrapment using these methods. However, somedrugs such as VCR can be inactivated at low pH. VCR is deformylated atlow pH, resulting in an inactive byproduct (Fig. 7A). When liposomes areprepared that result in a relatively low internal pH, the inactive by-productappears over time during storage (Fig. 7B). Fortunately, this reaction can becontrolled to some extent by modulating the trapping agent used. Forexample, citrate has a reasonable buffering capacity; therefore, under cer-tain conditions, it can keep the pH in a range where this inactivation isminimized. The use of sulfate as a trapping agent is more problematicdue to its poor buffering ability; therefore, it is more difficult to controlthe resulting deformylation. Although typically less sensitive to extremesin pH, lipids also have sensitive bonds, most notably the sn-2 ester bondof PLs. If not carefully controlled, the resulting lysolipids and fatty acidscan destabilize liposome membranes, resulting in increased drug leakage.Mayer et al. have shown that the substitution of sphingomyelin forphosphatidylcholine can help alleviate this problem, as the amide bondspresent in sphingomyelin are considerably less sensitive to acid (13). Theuse of reverse pH gradients (5) where the interior is alkalinized is more ofa concern as more chemical entities are sensitive to alkaline inactivationwhen compared to acidic inactivation.

CONCLUSIONS

This chapter reviews the methodology and characterization of novelintraliposomal drug stabilization strategies. In order to achieve highly stable

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liposomal drug formulations where the drug is retained in the liposomewhile in the general circulation, drugs were complexed with polyanionictrapping agents, and preferably polyanionic polyols, inside the liposomes.The resulting formulations were considerably more stable than lipo-somes prepared using traditional remote-loading strategies that employcitrate or sulfate. We have also demonstrated that these formulations arehighly active, and thus able to release the drug at a rate reasonable enoughto achieve cytotoxicity of targeted formulations in vitro and efficacy in vivo.Finally, the stabilization strategies must be optimized depending on theagent to be entrapped to provide for chemical stability of both the lipid anddrug components. We believe these improvements in liposome technology willhelp the field move beyond its initial success with delivery of anthracyclines toa wider range of drugs.

Figure 7 The stability of liposomal VCR in liposomes prepared using differenttriethylammonium ion gradients. VCR can be deformylated under acidic conditions,similar to those found in the intraliposomal lumen of remote-loaded liposomes. (A)The deformylated product is inactive compared to the parent drug. (B) HPLC chro-matograms show peaks for both VCR (rt¼ 9.5 minutes) and deformylated VCR(rt¼ 11.1 minutes) for liposomes prepared with either sulfate or citrate as the intra-liposomal trapping agent and stored for three months at 4�C to 6�C. Abbreviation:VCR, vincristine.


REFERENCES

1. Drummond DC, Meyer O, Hong K, et al. Optimizing liposomes for delivery ofchemotherapeutic agents to solid tumors. Pharmacol Rev 1999; 51(4):691.

2. Allen TM, Cullis PR. Drug delivery systems: entering the mainstream. Science.2004; 303(5665):1818.

3. Sapra P, Allen TM. Ligand-targeted liposomal anticancer drugs. Prog Lipid Res2003; 42(5):439.

4. Noble CO, Kirpotin DB, Hayes ME, et al. Development of ligand-targeted lipo-somes for cancer therapy. Expert Opin Ther Targets 2004; 8(4):335.

5. Clerc S, Barenholz Y. Loading of amphipathic weak acids into liposomes inrepsonse to transmembrane calcium acetate gradients. Biochim Biophys Acta1995; 1240:257.

6. Haran G, Cohen R, Bar LK, et al. Transmembrane ammonium sulfate gradientsin liposomes produce efficient and stable entrapment of amphiphathic weakbases. Biochim Biophys Acta 1993; 1151:201.

7. Lasic DD, Ceh B, Stuart MCA, et al. Transmembrane gradient driven phasetransitions within vesicles: lessons for drug delivery. Biochim Biophys Acta1995; 1239:145.

8. Fenske DB, Wong KF, Maurer E, et al. Ionophore-mediated uptake ofciprofloxacin and vincristine into large unilamellar vesicles exhibiting transmem-brane ion gradients. Biochimica Biophysica Acta 1998; 1414(1–2):188.

9. Cullis PR, Hope MJ, Bally MB, et al. Influence of pH gradients on the transbi-layer transport of drugs, lipids, peptides and metal ions into large unilamellarvesicles. Biochim Biophys Acta 1997; 1331:187.

10. Abraham SA, McKenzie C, Masin D, et al. In vitro and in vivo characterizationof doxorubicin and vincristine coencapsulated within liposomes through use oftransition metal ion complexation and pH gradient loading. Clin Cancer Res2004; 10:728.

11. Zhu G, Oto E, Vaage J, et al. The effect of vincristine-polyanion complexes inSTEALTH liposomes on pharmacokinetics, toxicity and anti tumor activity.Cancer Chemother Pharmacol 1996; 39:138.

12. Madden TD, Harrigan PR, Tai LCL, et al. The accumulation of drugs withinlarge unilamellar vesicles exhibiting a proton gradient: a survey. Chem PhysLipids 1990; 53:37.

13. Webb MS, Harasym TO, Masin D, et al. Sphingomyelin-cholesterol liposomessignificantly enhance the pharmacokinetic and therapeutic properties of vincris-tine in murine and human tumour models. Br J Cancer 1995; 72:896.

14. Sapra P, Moase EH, Ma J, et al. Improved therapeutic responses in a xenograftmodel of human B lymphoma (Namalwa) for liposomal vincristine versusliposomal doxorubicin targeted via anti-CD19 IgG2a or Fab fragments. ClinCancer Res 2004; 10:1100.

15. Mayer LD, Nayar R, Thies RL, et al. Identification of vesicle properties thatenhance the antitumor activity of liposomal vincristine against murine L1210leukemia. Cancer Chemother Pharmacol 1993; 33:17.

16. Boman NL, Mayer LD, Cullis PR. Optimization of the retention properties ofvincristine in liposomal systems. Biochim Biophys Acta 1993; 1152(2):253.

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17. Messerer CL, Ramsay EC, Waterhouse D, et al. Liposomal irinotecan: formula-tion development and therapeutic assessment in murine xenograft models of col-orectal cancer. Clin Cancer Res 2004; 10(19):6638.

18. Liu JJ, Hong RL, Cheng WF, et al. Simple and efficient liposomal encapsulationof topotecan by ammonium sulfate gradient: stability, pharmacokinetic andtherapeutic evaluation. Anti-Cancer Drugs 2002; 13(7):709.

19. Abraham SA, Edwards K, Karlsson G, et al. An evaluation of transmembraneion gradient-mediated encapsulation of topotecan within liposomes. J ControlRelease 2004; 96(3):449.

20. Allen TM, Newman MS, Woodle MC, et al. Pharmacokinetics and anti-tumoractivity of vincristine encapsulated in sterically stabilized liposomes. Int JCancer 1995; 62:199.

21. Kirpotin DB. Compound-loaded liposomes and methods for their preparation.United States Patent,6,110,491,2000.

22. Stahl PH, Wermuth CG, eds. Handbook of Pharmaceutical Salts. Weiheim:Wiley-VCH, 2002.

23. Mui BLS, Cullis PR, Evans EA, et al. Osmotic properties of large unilamellarvesicles prepared by extrusion. Biophys J 1993; 64:443.

24. Mui BLS, Cullis PR, Pritchard PH, et al. Influence of plasma on the osmotic sen-sitivity of large unilamellar vesicles prepared by extrusion. J Biol Chem 1994;269:7364.

25. Woodle MC, Papahadjopoulos D. Liposome preparation and size characteriza-tion. Meth Enzymol 1989; 171:193.

26. Harrigan PR, Wong KF, Redelmeier TE, et al. Accumulation of doxorubicinand other lipophilic amines into large unilamellar vesicles in response to trans-membrane pH gradients. Biochim Biophys Acta 1993; 1149(2):329.

27. Bartlett GR. Phosphorous assay in column chromatography. J Biol Chem 1959;234:466.

28. Tristram-Nagle S, Nagle JF. Lipid bilayers: thermodynamics, structure, fluctua-tions, and interactions. Chem Phys Lipids 2004; 127(1):3.

29. Drummond DC, Marx C, Guo Z, et al. Enhanced pharmacodynamic and anti-tumor properties of a histone deacetylase inhibitor encapsulated in liposomesor ErbB2-targeted immunoliposomes. Clin Cancer Res 2005; 11:3392–3401.

30. Mamot C, Drummond Dc, Noble CO, et al. Epledermal growth factor receptor-targeted immunoliposomes significantly enhance the efficacy of multiple antican-cer drugsin vivo. Cancer Res 2005; 65:1631–1638.

31. Drummond DC, Noble CO, Guo Z, Hong K, Park JW, Kirpotin DB. Develope-ment of a highly active nanoliposome irinotecan using a novel intraliposomalstabilization strategy. Cancer Res 2006; 66:3271–1638.

32. Colbern GT, Dykes DJ, Engbers C, et al. Encapsulation of the topoisomerase Iinhibitor GL147211C in pegylated (STEALTH) liposomes: pharmacokineticsand antitumor activity in HT29 colon tumor xenografts. Clin Cancer Res 1998;4:3077.

33. Fisher RS. Sucralfate: a review of drug tolerance and safety. J Clin Gastroenterol1981; 3(Suppl 2):181.

34. Li X, Hirsh DJ, Cabral-Lilly D, et al. Doxorubicin physical state in solution andinside liposomes loaded via a pH gradient. Biochim Biophys Acta 1998; 1415(1):23.


35. Abraham SA, Edwards K, Karlsson G, Hudon N, Mayer LD, Bally MB. Anevaluation of transmembrane gradient-mediated encapsulation of topotecanwithin liposomes. J Control Release 2004; 96:449–461.

36. Mayer LD, Tai LCL, Bally MB, et al. Characterization of liposomal systemscontaining doxorubicin entrapped in response to pH gradients. Biochim BiophysActa 1990; 1025:143.

37. Gabizon AA, Barenholz Y, Bialer M. Prolongation of the circulation timeof doxorubicin encapsulated in liposomes containing polyethylene glycol-derivatized phospholipid: pharmacokinetic studies in rodents and dogs. PharmRes 1993; 10(5):703.

38. Charrois GJ, Allen TM. Drug release rate influences the pharmacokinetics, biodis-tribution, therapeutic activity, and toxicity of pegylated liposomal doxorubicinformulations in murine breast cancer. Biochim Biophys Acta 2004; 1663(1–2):167.

39. Bally MB, Nayar R, Masin D, et al. Liposomes with entrapped doxorubicinexhibit extended blood residence times. Biochim Biophys Acta 1990; 1023(1):133.

40. Mayer LD, Cullis PR, Bally MB. Designing therapeutically optimized liposomalanticancer delivery systems: lessons from conventional liposomes. In: Papahad-jopoulos L, ed. Medical Applications of Liposomes. New York: Elsevier Science,1998.

41. Papahadjopoulos D, Nir S, Oki S. Permeability properties of phospholipidmembranes: effect of cholesterol and temperature. Biochim Biophys Acta 1972;266(3):561.

42. Papahadjopoulos D, Jacobson K, Nir S, et al. Phase transitions in phospholipidvesicles. Fluorescence polarization and permeability measurements concerningthe effect of temperature and cholesterol. Biochim Biophys Acta 1973; 311:330.

43. Mayhew E, Rustum YM, Szoka F, et al. Role of cholesterol in enhancing theantitumor activity of cystosine arabinoside entrapped in liposomes. Cancer TreatRep 1979; 63:1923.

44. Kirby C, Gregoriadis G. The effect of lipid composition of small unilamellarliposomes containing melphalan and vincristine on drug clearance after injectioninto mice. Biochem Pharmacol 1983; 32(4):609.

45. Smaby JM, Momsen M, Kulkarni VS, et al. Cholesterol-induced interfacial areacondensations of galactosylceramides and sphingomyelins with identical acylchains. Biochemistry 1996; 35:5696.

46. Schmidt CF, Barenholz Y, Thompson TE. A nuclear magnetic resonance studyof sphingomyelin in bilayer systems. Biochemistry 1977; 16:2649.

47. Perkins WR, Minchey SR, Ahl PL, et al. The determination of liposome cap-tured volume. Chem Phys Lipids 1993; 64:197.

48. Allen TM, Mehra T, Hansen C, et al. Stealth liposomes: an improved sustainedrelease system for 1-b-D-arabinofuranosylcytosine. Cancer Res 1992; 52:2431.

49. Lasic DD, Papahadjopoulos D. Liposomes and biopolymers in drug and genedelivery. Curr Opin Solid State Mater Sci 1996; 1:392.

50. Heurtault B, Saulnier P, Pech B, et al. Physico-chemical stability of colloidallipid particles. Biomaterials 2003; 24(23):4283.

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9

Radiolabeling of Liposomesfor Scintigraphic Imaging

Peter Laverman

Department of Nuclear Medicine, Radboud University Nijmegen Medical Centre,Nijmegen, The Netherlands

Gert Storm

Department of Pharmaceutics, Utrecht Institute for Pharmaceutical Sciences,Utrecht University, Utrecht, The Netherlands

William T. Phillips, Ande Bao, and Beth A. Goins

Department of Radiology, University of Texas Health Science Center at San Antonio,San Antonio, Texas, U.S.A.

INTRODUCTION

Since their discovery, liposomes have been labeled with radionuclides inorder to trace these nanoparticles in vivo. A variety of radionuclides andlabeling techniques have been used, ranging from weak beta-radiation emit-ters such as tritium (3H) and carbon-14 (14C) for tissue distribution andpharmacokinetic studies, to gamma-radiation emitters such as technetium-99m (99mTc), indium-111 (111In), and gallium-67 (67Ga), for both biodistribu-tion and scintigraphic studies [reviewed in (1–3)]. Preferably, liposomes shouldbe labeled after their preparation and just prior to the experiments. For thispurpose, so-called ‘‘afterloading’’ or ‘‘remote-labeling’’ methods are suitable.In this situation, the preformed liposomes are labeled prior to the start of theexperiment. These methods are almost indispensable when using relativelyshort-lived radionuclides, such as 99mTc or 111In (physical half-lives of 6.0 and

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68 hours, respectively). The main advantage of the use of gamma-radiationemitting radionuclides is the possibility of using whole-body scintigraphicimaging techniques to visualize the biodistribution of the radiolabel. Pre-clinically, this technique is a valuable tool in addition to the so-called‘‘ex vivo’’ studies, comprising the dissection of tissues and subsequentcounting for radioactivity. Small-animal imaging permits the noninvasivemonitoring of the biodistribution in the same animals for long periodsof time, thus eliminating the need of sacrificing animals at several time-points postinjection to quantify the disposition profiles in time. In addition,dynamic scans can be recorded to monitor the pharmacokinetic behavioron second- or minute-based intervals. In general, gamma radiation can bemeasured more easily than the weak b radiation, providing the opportunityto avoid tissue solubilization with scintillation liquid.

This chapter will focus on several techniques for liposome labelingwith gamma-radiation emitting radionuclides and their use in scintigraphicimaging. For more detailed information concerning the scintigraphic ima-ging techniques, see Volume III, Chapter 11 in this Liposome Technologybook series and Goins and Phillips (3).

SCINTIGRAPHIC IMAGING

Scintigraphic imaging is a noninvasive imaging technique commonly appliedin nuclear medicine. Radiolabeled compounds (called radiopharmaceuticalsor radiotracers) are administered intravenously to patients for diagnostic or,in certain cases, therapeutic purposes. The in vivo distribution can provideimportant physiological information about tissue function.

For diagnostic applications, the tracer is labeled with a radionuclideemitting photons with energies ranging from 100 to 500 keV. These photonenergies are high enough to allow detection outside the body by a gammacamera. The photons are collimated by a lead collimator and then strike aNaI crystal where scintillations are produced, which are converted into elec-tronic signals by photomultiplier tubes. Radionuclides with a very lowgamma energy will be attenuated by the subject and unable to reach theNaI crystal. On the other hand, when radionuclides with a higher gammaenergy are used, septa of the lead collimator have to be thicker, and the imageswill have lower resolution. Therefore, radionuclides with an intermediateenergy of approximately 120 to 150 keV, such as 99mTc, are favorable forscintigraphic imaging.

Photons with detectable energy differences that are emitted by variousradionuclides can be quantified simultaneously, but independently fromeach other. This allows the use of dual-labeling approaches (4). These experi-ments will reveal information regarding both the liposomal carrier—labeledwith one radionuclide—and the encapsulated compound—labeled with adifferent radionuclide—after a single injection in the same animal. However,

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the two radionuclides have to be chosen carefully, because the gamma cam-era has to detect the gamma energy peaks of the two nuclides separately. Theenergy peaks from the photons of the different radionuclides should besufficiently different so that they do not overlap.

THE CHOICE OF THE RADIONUCLIDE

Several factors have to be considered when choosing the radiolabel. Firstly,the radiolabeled liposomal formulation should have a high stability from aradiochemical point of view. Release of radiolabel from the liposomes dur-ing or after injection will result in the presence of free radiolabel and thuslead to underestimation of the injected dose of radiolabeled liposomes.Moreover, this will complicate interpretation of the scintigraphic images,because the fate of the radiolabel no longer merely reflects the fate ofthe liposome nanoparticles. Depending on its physical characteristics, theunbound radiolabel will be excreted via the hepatobiliary route or inthe urine, or taken up by organs such as the thyroid glands and the stomach.This same problem in image interpretation will eventually occur with someof the most stable liposome labels following metabolism of the liposome bythe reticuloendothelial cells and release of the radiolabel from the liposome.

Secondly, the circulation time of the liposomal formulation is animportant factor to consider. When using long-circulating liposomes, a radio-label with a relatively long physical half-life (one to five days) is required toallow imaging at late time-points. For a short-circulating liposome, the useof a short-lived radiolabel is sufficient. Using 99mTc is suitable for imagingup to 44 hours postinjection, whereas 111In can be used for studies lasting72 hours or more.

The commonly used isotopes for scintigraphic imaging—67Ga, 99mTc,111In and iodine-123 (123I)—are all widely available (Table 1). However,generally 99mTc is preferred over the other isotopes, due to its optimal

Table 1 Physical Characteristics of Some Commonly UsedGamma Radiation Emitters

Radionuclide Half-life (day)Photons (keV),[abundance (%)]

67Ga 3.3 93 (38)185 (21)300 (17)

99mTc 6.01 141 (89)111In 2.8 171 (91)

245 (94)123I 13.2 159 (83)125I 59.4 36 (7)

Radiolabeling of Liposomes for Scintigraphic Imaging 171

imaging characteristics with an ideal photon energy. Moreover, in a nuclearmedicine department setting,99mTc is readily available because it can beeluted daily from a commercially available 99Mo/99mTc-generator. Because67Ga,111In, and 123I are cyclotron products, they are more expensive and notalways available in every nuclear medicine department. Although iodine-125(125I) is used widely for labeling proteins and peptides, this radionuclide isunsuitable for whole-body clinical scintigraphic imaging, due to its lowgamma energy. However, there has been a recent report describing theimaging of 125I-sodium iodide in a mouse metastatic lung tumor model usinga high-resolution pinhole single photon emission tomography cameraespecially designed for small animals (5).

LABELING METHODS

Several liposome-labeling methods have been developed in the last decades.These methods can be distinguished based on their labeling mechanism.Probably the first method developed for liposome labeling is encapsulationof the radiolabel in the aqueous interior during the manufacturing of theliposomes. However, this method is rather laborious and therefore unsuit-able for routine use. A second, relatively simple, method is the reductionof the radiolabel in the presence of the liposomes, resulting in association ofthe label with the outside of the lipid bilayer. This approach appeared toyield very unstable radiolabeled preparations (6) and is therefore not pref-erable. Two approaches that have proven to yield radiolabeled liposomeswith high efficiency and good radiochemical stability are the so-called‘‘afterloading methods.’’ The radiolabel is either trapped in the aqueousphase after the manufacturing of the liposomes or the radiolabel can bechelated to a lipid–chelator conjugate incorporated in the lipid bilayer ofpreformed liposomes. The usefulness of liposome-labeling methods greatlyimproved with the development of the afterloading methods. The lattertwo methods will be discussed in more detail.

Afterloading Methods Using Aqueous Space Trapping

There are two major ways in which liposomes have been radiolabeled by sta-bly trapping the radionuclide in the liposome interior: (i) use of secondmolecule encapsulated in liposome and (ii) chemical gradient with pH orammonium sulfate. In the first method, a radionuclide is incubated with alipophilic chelator and then mixed with an aliquot of liposomes encapsulatinga second molecule. Once the lipophilic chelator carries the radionuclide acrossthe lipid bilayer, the second molecule interacts with the radionuclide-chelator causing the radionuclide to become trapped within the interior ofthe liposome. This interaction may be due to the second molecule having ahigher affinity for the radionuclide than the original lipophilic chelator. An

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alternative labeling mechanism is that the lipophilic chelator is converted fromthe lipophilic form to a hydrophilic form after interaction with the secondmolecule. Because the hydrophilic form cannot cross back through the lipidbilayer, it becomes trapped in the liposome interior.

In the second method, the radionuclide is incubated with a chelatorthat has sufficient lipophilicity to cross the lipid bilayer at around pH 7.0and amine groups. When this chelator crosses the lipid bilayer and entersthe lower pH environment (typically pH 5.0) of the liposome interiorcontaining ammonium sulfate, the amine groups become protonized. Theseprotonized groups convert the lipophilic chelator into a charged hydrophilicchelator that is unable to cross back through the lipid bilayer, thus leadingto trapping of the radionuclide by pH gradient. The most common chemicalused to provide the pH gradient is ammonium sulfate.

111In- and 67Ga-Trapping Methods

Mauk and Gamble were among the first who labeled preformed liposomeswith 111In using a trapping method (7). They incorporated the ionophoreA23187 into the lipid bilayers to facilitate the transport of charged speciesacross the bilayer and enclosed the chelator nitrilotriacetic acid (NTA,1 mM) in the aqueous interior to trap the 111In inside the liposomes. Radio-activity was added to the liposomes in the form of 111InCl3 and liposomeswere then heated to approximately 80�C. The reported labeling efficiencywas higher than 90%. In vivo stability was not tested. A disadvantage of thismethod is the need to label at high temperatures, which might negativelyaffect the liposome integrity.

Hwang et al. improved the afterloading trapping method by first con-verting 67Ga and 111In to the lipophilic forms (67Ga-8-hydroxyquinoline(67Ga-oxine) or 111In-8-hydroxyquinoline (111In-oxine). Due to this highlipophilicity of the radionuclide chelator, the radionuclides were able tocross the lipid bilayers (8). Once inside the liposome, the radiolabeled com-plex is chelated by the encapsulated NTA and consequently trapped insidethe liposome. Biodistribution studies with sphingomyelin/cholesterolliposomes labeled with either 111In or 67Ga via this method showed that thecirculating liposomes are stable in vivo (8). Because NTA is a relatively weakchelator, metal translocation to serum proteins such as transferrin and othermetal-binding proteins may occur after liposome disruption. Gabizon et al.studied the use of deferoxamine (Df) as a chelator for 67Ga (9). Liposomescontaining 25 mM Df in the aqueous interior were incubated with 67Ga-oxineovernight at 4�C. Labeling efficiency ranged from 57% to 88%, irrespective oftheir lipid composition. Main differences between the biodistribution of 67Ga-NTA and 67Ga-Df, once released from the liposomes, is their route ofclearance; following metabolism of the liposome,67Ga-Df is cleared rapidlyvia the kidneys to the urine, whereas the 67Ga of the 67Ga-NTA complex


translocates to transferrin and thus stays in the circulation for extended times(9,10). This long circulation time of the 67Ga radionuclide when attached toNTA can interfere with imaging of the biodistribution of the radiolabeledlong-circulating liposomes, because both the 67Ga-NTA and the 67Ga-lipo-somes show a long circulation time. When interpreting the image, it isimpossible to distinguish between these two forms of the radiolabel. Disadvan-tage of this method is the rather long incubation time, apparently required forthe labeling.

Similar to these methods, diethylenetriaminepentaacetic acid (DTPA) canbe used as a chelator in the aqueous phase of the liposomes for labeling with111In. This results in a stable radiolabeled preparation with a labeling efficiencyof approximately 80%. During the preparation of the liposomes, DTPA isencapsulated and the free DTPA is removed by gel permeation chromatographyon a disposable column (11). Subsequently, these liposomes are labeled with111In-oxine. Liposomes are incubated for 30 minutes at room temperature inthe presence of 111In-oxine. The nonencapsulated 111In-oxine is then removedby gel permeation chromatography. When using relatively rigid liposomes,the incubation with the 111In-oxine should be carried out at a higher tempera-ture to facilitate transport of 111In-oxine through the bilayer. Harrington et al.used this method to determine the distribution of pegylated liposomes similar toCaelyx1/Doxil1 in both tumor-bearing animals (12) and patients with solidtumors (13).

Recently, this method was adapted to label two commerciallyavailable liposomal formulations: doxorubicin encapsulated in polyethyleneglycol (PEG)-coated liposomes (Caelyx1/Doxil1) (14) and daunorubicinencapsulated in small distearoyl-phosphatidyl-choline/cholesterol liposomes(Daunoxome1) (15). Although no DTPA was encapsulated in these lipo-somes, the labeling efficiency was typically between 70% and 80% and theradiolabeled preparations were stable in vivo during the time course of theexperiment (four hours). Most likely, the lipophilic 111In-oxine avidly associ-ates with the lipid bilayer and encapsulation of DTPA might not be necessarywhen the experimental observation period does not exceed four to six hours.

99mTc-Trapping Methods

99mTc-Liposome–Labeling Method UsingHexamethylpropyleneamine Oxime-Glutathione

One method for the labeling of liposomes with 99mTc using the lipophilicchelator, hexamethylpropyleneamine oxime (HMPAO) was developed byPhillips et al. (16). Lipophilic HMPAO enters the liposome where it interactswith glutathione and becomes converted to the hydrophilic form, which istrapped in the liposome. A detailed protocol for radiolabeling liposomesusing 99mTc-HMPAO has been reported (3). In a typical experiment, 10 to15 mCi (370–555 MBq) of 99mTc in the form of sodium pertechnetate

174 Laverman et al.

(TcO4�) in 5 mL saline is added to a commercially available HMPAO kit

(Ceretec1, GE Healthcare, Chalfont St. Giles, U.K.). The HMPAO kit con-tains the HMPAO chelator as well as stannous chloride for reduction of the99mTc and other stabilizers. After five minutes incubation, an aliquot (2 mL)of the 99mTc-HMPAO is added to an equal volume (2 mL) of liposomes (50mmol/mL phospholipids) containing reduced glutathione. The mixture isincubated at room temperature for 15 to 30 minutes. After incubation, the99mTc-liposome mixture is typically loaded onto a disposable SephadexG-25 column (PD-10, GE Healthcare Biosciences, Piscataway, New Jersey,U.S.) to remove any free 99mTc from the 99mTc-liposomes. This separation isnot necessary when labeling efficiencies are >90%, but is normally per-formed to provide a radiochemically purer 99mTc-liposome product forinjection. This separation can be especially useful in research situationsbecause any free 99mTc or 99mTc-HMPAO can potentially distribute differ-ently from the 99mTc-liposomes of interest. Radiochemical purity can also bedetermined using paper chromatography with 0.9% saline as the mobilephase. In this system, the 99mTc-liposomes remain at the origin whereas free99mTc and hydrophilic 99mTc-HMPAO migrate.

An advantage of this method is the fact that HMPAO is commerciallyavailable and does not require a liposome researcher to synthesize the chem-ical in house. Second, the method can be used to label a wide variety ofpreformed liposome formulations. Third, the preformed liposomes can belabeled rapidly and demonstrate good in vitro and in vivo stability, espe-cially when compared with earlier encapsulation and surface associationmethods (16). One disadvantage of the HMPAO method is the high costof HMPAO kits. It is also very important that all untrapped glutathionebe removed prior to the labeling procedure because the untrapped glu-tathione can prematurely convert the lipophilic HMPAO to its hydrophilicform, which cannot cross the lipid bilayer, resulting in poor labeling yields.Another disadvantage is the requirement that the liposomes must coencap-sulate reduced glutathione in addition to the drug of interest, requiring aseparate special batch of liposomes containing the drug be prepared. Theneed for glutathione coencapsulation can also be a problem if the drug isunstable in the presence of reduced glutathione. The HMPAO glutathionemethod probably should not be chosen by investigators planning toperform a chemical surface modification to the preformed liposomes thatcould cause leakage of glutathione from the liposome interior as this wouldlead to poor labeling. The HMPAO-glutathione method can be used byinvestigators when postinsertion of a lipid conjugate such as PEG-lipid isdesired (17,18).

The HMPAO-glutathione method has been used in a number ofpreclinical animal studies (19). An example is shown in Figure 1, where thebiodistribution of 99mTc-HMPAO-labeled PEG liposomes is compared tothat of PEG-liposomes labeled with 99mTc via the surface chelator


hydrazino nicotinamide (HYNIC). In addition, the clinical performanceof 99mTc-HMPAO-labeled PEG liposomes was investigated in a series ofpatients with possible infectious and inflammatory disease (20). The imagesobtained with 99mTc-PEG-liposomes were directly compared to those obtainedwith 111In-IgG-scintigraphy, the standard infection imaging modality at theRadboud University Nijmegen Medical Center. Thirty-five patients sus-pected of having infectious or inflammatory disease received 740 MBq99mTc-HMPAO-PEG-liposomes. In this group of patients with predomi-nantly musculoskeletal pathology, 99mTc-HMPAO-PEG-liposome scintigraphyshowed high sensitivity (94%) and specificity (89%). All infectious andinflammatory foci were detected, missing only one case of endocarditis,which was also not seen on the 111In-IgG scan. False-positive results werenoted in two patients with (noninfected) pseudoarthrosis. An example ofan 99mTc-HMPAO-labeled PEG-liposome scan is depicted in Figure 2.One patient experienced flushing and chest tightness during liposomaladministration; both symptoms rapidly disappeared by lowering the infusionrate. This clinical evaluation of 99mTc-HMPAO-PEG liposomes showed thatfocal infection and inflammation can be adequately imaged with this agent.This clinical study with 99mTc-PEG-liposomes indicated that this new ima-ging agent can offer an effective and convenient scintigraphic method tovisualize focal infection and inflammation. The particular liposome formula-tion that was used in humans was associated with side effects most likely dueto complement activation. However, the encountered side effects observed infour out of 44 patients impede further use of this formulation in patients anda new formulation lacking these side effects needs to be developed.

Figure 1 Images of rats with unilateral Staphylococcus aureus abscess in calfmuscle, recorded at 0 (five minutes), 1, 6, and 24 hours postinjection of 99mTc-labeledhydrazino nicotinamide-liposomes (A) and hexamethylpropyleneamine oxime-liposomes (B).

176 Laverman et al.

99mTc-Liposome–Labeling Method UsingBMEDA-Glutathione

A new method for radiolabeling glutathione-containing liposomes with 99mTcusing a different chelator based on SNS/S pattern complexes has been recentlyreported (21). One particular chelator, N,N-bis(2-mercaptoethyl)-N0,N0 diethyl-ethylenediamine (BMEDA), was shown to efficiently label the liposomes.

The BMEDA method shares some of the same features as theHMPAO method such as good in vitro and in vivo stability with a varietyof preformed liposome formulations, and the need for coencapsulation ofglutathione. In addition, an advantage of the BMEDA-labeling method isthat it can also be used for labeling liposomes with therapeutic rheniumradionuclides (22). Currently, for the BMEDA method, there is no commer-cially available kit. Also the BMEDA chemical is not currently commerciallyavailable and must be synthesized.

Typically for this method, a BMEDA solution is prepared by mixing5.6 mg (5.0 mL) of BMEDA with 5.0 mL degassed water and four drops of0.05 M NaOH. This BMEDA solution is stirred at room temperature for40 minutes. At around the same time, a solution of 99mTc-glucoheptonate isprepared for use as a coligand in the preparation of 99mTc-BMEDA. The99mTc-glucoheptonate solution is prepared by mixing 1.0 mL of a 10 mg/mLfreshly made degassed glucoheptonate solution containing 0.16 mg/mL stan-nous chloride with 99mTc (15 mCi, 555 MBq) in the form of TcO4

� in saline,and stirring the mixture for 20 minutes at room temperature. The 99mTc-BMEDA solution is then prepared by mixing 1.0 mL of the BMEDA solutionwith 0.65 mL of 99mTc-glucoheptonate, adjusting the pH to 8.0 and stirring the

Figure 2 A true positive 99mTc-hydrazino nicotinamide-polyethylene glycol-liposomescintigram (left) and a false-negative 111In-labeled polyclonal immunoglobulin Gscintigram (right) of a patient with painful swelling and redness at the level of anold tibial fracture.


mixture for 25 minutes at room temperature. Radiochemical purity of the99mTc-BMEDA can be determined using instant thin-layer chromatography(ITLC) with methanol as the mobile phase and paper chromatography usingsaline as the mobile phase. To prepare the 99mTc-liposomes, 0.65 mL 99mTc-BMEDA solution is then adjusted to pH 7.0 and added to 1.0 mL ofglutathione-liposomes (50 mg/mL phospholipid). This mixture is stirred forone hour at room temperature. As described above for 99mTc-HMPAO-lipo-somes, the 99mTc-BMEDA-liposomes can be separated from any free 99mTcand 99mTc-BMEDA by Sephadex G-25 column chromatography.

BMEDA Method Using pH Gradient Liposomes

Aqueous phase trapping of 99mTc inside liposomes can be accomplishedusing a pH or ammonium gradient and 99mTc-BMEDA (23). In this method,liposomes are prepared using the same procedure used to load high levels ofdrugs in liposomes (24–26). Preformed liposomes are prepared in the pre-sence of ammonium sulfate. Immediately before radiolabeling, the ammoniumsulfate in the external liposomal environment is removed by centrifugation orcolumn chromatography creating a pH gradient. The 99mTc-BMEDA is pre-pared as described in previous section for glutathione-containing liposomes,and then added to the washed ammonium sulfate-liposomes (50 mg/mLphospholipid) and incubated for one hour at room temperature. Any free99mTc-BMEDA is separated from the 99mTc-liposomes by Sephadex G-25column chromatography. Labeling efficiencies were typically 70% to 80% withgood in vitro serum stability.

This method has a significant advantage over glutathione-liposomeslabeled with HMPAO or BMEDA in that commercial liposome drugs suchas Doxil1 can be directly radiolabeled for pharmacokinetic and distributionstudies during preclinical development (23). This is possible because thesedrugs are loaded into liposomes using the pH gradient methodology.Figure 3 depicts images of a normal rat acquired at various times after intra-venous injection of commercial Doxil1 directly labeled with 99mTc-BMEDA. The amount of 99mTc-Doxil1 injected was based on the clinicaldosage. The blood clearance of 99mTc-Doxil1 was similar to that reportedfor unlabeled Doxil1 with a gradual removal from the blood over 44 hours(27). With this labeling method, there was also no significant excretionthrough the bowel and bladder, indicating a stable association of the radio-nuclide with the liposomes. On the 44-hour image,99mTc-Doxil1 can still bevisualized in the heart due to ~20% of the 99mTc-activity still remaining inthe blood. Performance of a 44-hour image is still possible with the 99mTcradionuclide even though its physical half-life is six hours. To obtain this44-hour image, a 20-minute acquisition time is required. The quality ofthe 44-hour image is decreased in comparison to the 4- and 20-hour images,due to the low number of counts acquired. Even though it is of decreased

178 Laverman et al.

quality, the 44-hour image can still provide useful information. On this44-hour image, it is interesting to observe that a uniformly high level of99mTc-activity was observed in the intestines and abdominal tissues. Thisintestinal uptake may be related to the cutaneous and digestive system tox-icities reported in clinical trials with Doxil1 (28–31). The mechanism ofuptake in the intestinal region may be related to the long circulation timeof the PEG-modified liposomes because this type of distribution has notbeen observed previously with shorter circulating non-PEG liposomes thatare more readily cleared by the liver and spleen.99mTc-Doxil1 could be usedin the clinic to verify that there is sufficient uptake of the actual liposomaldrug in the tumor or metastases by performing noninvasive imaging eitherduring or before treatment. As previously described as a drawback for theuse of the BMEDA-glutathione loading method, the pH-gradient loadingmethod also requires the synthesis of BMEDA, and development of acommercially available kit.

Afterloading Methods Using External Surface Chelation

111In-Liposomes

The first method utilizing the presence of a chelator on the surface of theliposome was based on the conjugation of DTPA to octodecylamine, asingle-chain fatty acid (32). This conjugate was incorporated in the lipidbilayer. Although a high labeling efficiency could be achieved, a majordrawback was the relatively poor in vivo stability, most likely due to thelipid exchange with blood components. More recently, DTPA has been

Figure 3 Planar scintigraphic images acquired at various times of a normal ratintravenously injected with 99mTc-Doxil1 (17 MBq) 99mTc, 2 mg doxorubicin,and 16 mg total lipid labeled using 99mTc-N,N-bis(2-mercaptoethyl)-N0,N0 diethyl-ethylenediamine method. Abbreviations: H, heart; L, liver; K, kidney; B, bowel.


conjugated to phospholipids with a phosphatidylethanolamine (PE)headgroup and the DTPA-PE conjugate incorporated into liposomes for111In-labeling studies. The in vivo stability has been shown to be better whena phospholipid is used compared with a single fatty acid chain. A detaileddescription of the preparation and application of radiolabeled DTPA-PEliposomes has been published (33).

99mTc-Liposomes

The DTPA-PE conjugates were also investigated for labeling liposomes with99mTc (34,35). DTPA-PE liposomes were found to be less useful for 99mTclabeling than for 111In labeling because it was difficult to maintain precisecontrol of the amount of stannous chloride needed to reduce the 99mTc toachieve a high labeling efficiency. Also, in vivo stability of the 99mTc-lipo-somes was lower due to the dissociation of 99mTc nonspecifically bound tothe liposome surface.

More recently, a new chelation method based on the technetium che-lator, HYNIC, was developed by Laverman et al. (36). HYNIC is wellknown for its use in labeling peptides and proteins with high efficiencyand excellent stability (37). N-hydroxysuccinimidyl hydrazino nicotinatehydrochloride was conjugated to the free amino group of distearoylpho-sphatidyl-ethanolamine (DSPE) and subsequently incorporated in the lipidbilayer during the liposome preparation.

PEG-liposomes were prepared essentially as follows: a lipid mixture(egg phosphatidylcholine:PEG-DSPE:HYNIC-DSPE:cholesterol) in metha-nol/chloroform (10:1) was prepared with a molar ratio of 1.85:0.15:0.07:1.After evaporation of the organic solvents, the resulting lipid film wasdispersed in PBS at room temperature. After sizing by extrusion, the suspensionwas dialyzed extensively against PBS overnight at 4�C, with four buffer changesto remove unconjugated HYNIC. Liposomes were stored in PBS at 4�C andcould be labeled efficiently for over a period of approximately three to fourmonths. Lypohilization of the liposomes greatly improved the shelf life ofthe HYNIC-PEG-liposomes (>1 year) without affecting their in vivo behavior(38). Radiolabeling of HYNIC-liposomes was essentially performed asdescribed previously (39). The 99mTc labeling of HYNIC is based on reductionof the 99mTc and stabilization of the binding with HYNIC using coligands.Briefly, the 99mTc in the form of 99mTcO4

� is reduced by stannous sulphateand N-[Tris(hydroxymethyl) -methyl]glycine (tricine) is added as a coligand.The mixture is incubated at room temperature for 20 minutes. Radiochemicalpurity was determined using ITLC on ITLC-SG strips with 0.15 M sodiumcitrate (pH 5.0) as the mobile phase.

This method is relatively inexpensive and easy to apply, although thereis no commercially available kit. However, recently succinimidyl-HYNICbecame commercially available (Solulink, Inc., San Diego, California, U.S.).Liposomes are labeled rapidly and with a labeling efficiency generally higher

180 Laverman et al.

than 95%.99mTc-HYNIC-PEG-liposomes showed high in vitro and in vivo sta-bility (36). The in vivo biodistribution was compared to that of PEG-liposomeslabeled with 99mTc-HMPAO in rats with an intramuscular Staphylococcusaureus abscess (Fig. 1). Besides a decreased kidney uptake as compared toHMPAO-liposomes—reflecting an advantage in favor of the HYNIC-liposomes—the in vivo behavior was similar. A potential disadvantage ofthis method is that the surface of the liposome is modified, which couldpossibly interfere with the use of other surface ligands in a targetedliposome system.

Subsequently, the clinical performance of 99mTc-HYNIC-PEG-liposomeshas been studied. A study was initiated in patients suspected of having anexacerbation of Crohn’s disease to assess the role of 99mTc-HYNIC-PEG-liposomes to determine the extent and severity of active disease (39). Althoughinflamed colon segments were visualized with this agent in seven patients, only amoderate relation between 99mTc-HYNIC-PEG-liposome scan grading and theconventional verification procedures (endoscopy or radiology) was found. Moreimportantly, the study was prematurely terminated because of unacceptable sideeffects (tightness in chest and/or stomach region, mild hyperventilation, anderythema of the face and upper extremities) in three out of nine patients, mostlikely due to complement activation (40).

The same liposomal preparation was used to investigate the effectof the administered dose on the biodistribution and pharmacokinetics (41).The effect of the lipid dose of 99mTc-HYNIC-PEG-liposomes was investi-gated in the low-dose range (0.02–1.0 mmol/kg), typically for noninvasiveimaging applications. The biodistribution and pharmacokinetics of 99mTc-HYNIC-PEG-liposomes at various dose levels were studied in rats andrabbits with a focal Escherichia coli infection. Moreover, the pharmacoki-netics of 99mTc-HYNIC-PEG-liposomes at two lipid dose levels were studiedin four patients. In rabbits, enhanced clearance was observed at a dose level of0.02 mmol/kg. The circulatory half-life decreased from 10.4 to 3.5 hours (at1.0 and 0.02 mmol/kg, respectively). At the lowest dose level, liposomes weremainly taken up by the liver and to a lesser extent by the spleen. Most impor-tantly, the rapid clearance of low-dose PEG liposomes was also observedin humans when relatively low lipid doses were administered as is shown inFigure 4. This study showed that, at very low lipid doses, the biodistributionof PEG liposomes is dramatically altered.

CONCLUDING REMARKS

Scintigraphic techniques have proven to be useful when investigating thein vivo behavior of liposomes. The major advantage of scintigraphy isthe ability to obtain noninvasive quantifiable images at many time pointsin either humans or animals. This rapidly provides the in vivo behavior ofthe liposomes.


Several techniques for labeling liposomes have been developed in thepast decades. The most promising methods are the afterloading methods.A well-established method is based on the entrapment of the radiolabel inthe aqueous space of preformed liposomes. This method has proven usefulin several studies and is widely used. A reducing agent (glutathione) isentrapped during preparation and the 99mTc is added in a lipophilic formto the liposomes just prior to the experiment. Recently, a new method basedon the entrapment of ammonium sulfate has been developed to label commer-cially available liposome products that use a pH-gradient drug-loading

Figure 4 Anterior whole-body scintigram of a patient injected with 0.1mmol/kg(left) and a patient injected with 0.5mmol/kg (right) four hours postinjection of740 MBq 99mTc-hydrazino nicotinamide-polyethylene glycol-liposomes.

182 Laverman et al.

mechanism. These methods generally yield radiolabeled liposomes with goodin vivo stability. A second afterloading method is based on the chelation ofthe radiolabel by a lipid-chelator conjugate in the lipid bilayer. This methodalso yields liposomes with high radiochemical purity and high stability.

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2. Phillips WT. Delivery of gamma-imaging agents by liposomes. Adv Drug DelivRev 1999; 37:13.

3. Goins B, Phillips WT. Radiolabelled liposomes for imaging and biodistributionstudies. In: Torchilin VP, Weissig V, eds. Liposomes: A Practical Approach.Oxford, U.K.: Oxford University Press, 2003:319.

4. Awasthi VD, Goins B, Klipper R, Phillips WT. Dual radiolabeled liposomes:biodistribution studies and localization of focal sites of infection in rats. NuclMed Biol 1998; 25:155.

5. Marsee DK, Shen DHY, MacDonald LR, et al. Imaging of metastatic pulmo-nary tumors following NIS gene transfer using single photon emissioncomputed tomography. Cancer Gene Ther 2004; 11:121.

6. Love WG, Amos N, Williams BD, Kellaway IW. Effect of liposome surfacecharge on the stability of technetium (99mTc) radiolabelled liposomes. J Micro-encapsul 1989; 6:105.

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8. Hwang KJ, Merriam JE, Beaumier PL, Luk KF. Encapsulation, with high effi-ciency, of radioactive metal ions in liposomes. Biochim Biophys Acta 1982; 716:101.

9. Gabizon A, Huberty J, Straubinger RM, Price DC, Papahadjopoulos D. Animproved method for in vivo tracing and imaging of liposomes using a gallium67-deferoxamine complex. J Liposome Res 1988; 1:123.

10. Ogihara-Umeda I, Kojima S. Increased delivery of gallium-67 to tumors usingserum-stable liposomes. J Nucl Med 1988; 29:516.

11. Corvo ML, Boerman OC, Oyen WJ, et al. Intravenous administration of super-oxide dismutase entrapped in long circulating liposomes. II. In vivo fate in a ratmodel of adjuvant arthritis. Biochim Biophys Acta 1999; 1419:325.

12. Harrington K, Rowlinson-Busza G, Syrigos KN, Uster PS, Vile RG, StewartJSW. Pegylated liposomes have potential as vehicles for intratumoral and subcu-taneous drug delivery. Clin Cancer Res 2000; 6:2528.

13. Harrington KJ, Mohammadtaghi S, Uster PS, et al. Effective targeting of solidtumors in patients with locally advanced cancers by radiolabeled pegylatedliposomes. Clin Cancer Res 7, 243, 2001.

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16. Phillips WT, Rudolph AS, Goins B, Timmons JH, Klipper R, Blumhardt R.A simple method for producing a technetium-99m-labeled liposome which isstable in vivo. Nucl Med Biol 1992; 19:539.

17. Awasthi VD, Garcia D, Klipper R, Goins BA, Phillips WT. Neutral and anionicliposome-encapsulated hemoglobin: effect of postinserted poly(ethylene glycol)-distearoylphosphatidylethanolamine on distribution and circulation kinetics. JPharmacol Exp Ther 2004; 309:241.

18. Sou K, Klipper R, Goins B, Tsuchida E, Phillips WT. Circulation kinetics andorgan distribution of Hb-vesicles developed as a red blood cell substitute.J Pharmacol Exp Ther 2005; 312:702.


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22. Bao A, Goins B, Klipper R, Negrete G, Phillips WT. 186Re-Liposome labelingusing 186Re-SNS/S complexes: in vitro stability imaging biodistribution in rats.J Nucl Med 2003; 44:1992.

23. Bao A, Goins B, Klipper R, Negrete G, Phillips WT. Direct 99mTc labeling ofpegylated liposomal doxorubicin (Doxil) for pharmacokinetic and non-invasiveimaging studies. J Pharmacol Exp Ther 2004; 308:419.

24. Haran G, Cohen R, Bar LK, Barenholz Y. Transmembrane ammonium sulfategradients in liposomes produce efficient and stable entrapment of the amphi-pathic weak bases. Biochim Biophys Acta 1993; 1151:201.

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27. Gabizon A, Shmeeda H, Barenholz Y. Pharmacokinetics of pegylated liposomaldoxorubicin review of animal and human studies. Clin Pharmacokinet 2003; 42:419.

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30. Tsavaris N, Kosmas C, Vadiaka M, et al. Pegylated liposomal doxorubicin in theCHOP regimen for older patients with aggressive (stage III/V) non-Hodgkin’s lymphoma. Anticancer Res 2002; 22:1845.

31. Skubitz KM. Phase II trial of pegylated-liposomal doxirubicin (Doxil) insarcoma. Cancer Investig 2003; 21:167.

32. Hnatowich DJ, Friedman B, Clancy B, Novak M. Labeling of preformed lipo-somes with Ga-67 and Tc-99m by chelation. J Nucl Med 1981; 22:810.

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33. Torchilin VP, Weissig V, Martin FJ, Heath TD, New RRC. Surface modificationof liposomes. In: Torchilin VP, Weissig V, eds. Liposomes: A PracticalApproach. Oxford, U.K.: Oxford University Press, 2003:193.

34. Ahkong QF, Tilcock C. Attachment of 99mTc to lipid vesicles containing thelipophilic chelate dipalmitoylphosphatidylethanolamine-DTTA. Nucl Med Biol1992; 19:831.

35. Tilcock C, Ahkong QF, Fisher D. 99mTc-labeling of lipid vesicles containing thelipophilic chelator PE-DTTA: effect of tin-to-chelate ratio chelate contentsurface polymer on labeling efficiency biodistribution behavior. Nucl MedBiol 1994; 21:89.

36. Laverman P, Dams ET, Oyen WJ, et al. A novel method to label liposomes with99mTc by the hydrazino nicotinyl derivative. J Nucl Med 1999; 40:192.

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38. Laverman P, Van Bloois L, Boerman OC, Oyen WJ, Corstens FH, Storm G.Lyophilization of Tc-99m-HYNIC labeled PEG-liposomes. J Liposome Res2000; 10:117.

39. Brouwers AH, De Jong DJ, Dams ET, et al. Tc-99m-PEG-liposomes for the eva-luation of colitis in Crohn’s disease. J Drug Target 2000; 8:225.

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41. Laverman P, Brouwers AH, Dams ET, et al. Preclinical and clinical evidence fordisappearance of long-circulating characteristics of polyethylene glycolliposomes at low lipid dose. J Pharmacol Exp Ther 2000; 293:996.


10

Liposomal Bisphosphonates for theTreatment of Restenosis

Hila Epstein, Eyal Afergan, Nickolay Koroukhov, Galit Eisenberg,Dikla Gutman, and Gershon Golomb

Department of Pharmaceutics, School of Pharmacy, Faculty of Medicine,The Hebrew University of Jerusalem, Jerusalem, Israel

INTRODUCTION

Percutaneous coronary interventions (PCI) are widely used to treat patientswith symptomatic coronary artery disease, which usually presents as anginaor myocardial infarction (1). PCI procedures include balloon dilation, endo-luminal stenting, excisional atherectomy, intravascular brachytherapy, andlaser ablation. Successful treatment of stenotic coronary arteries by PCI islimited by the occurrences of acute vessel occlusion and late restenosis. Reste-nosis is characterized by reobstruction of the lumen by 50% or more (2).Although the restenosis rates decrease with stenting (3–5), restenosis remainsa serious clinical problem, particularly in multivessel disease. This is becauseof increasing case complexity and aggressive neointimal proliferation due tothe inflammatory reaction triggered by the injury and the implanted stent (6).

A large number of clinical trials have investigated various drugs in anattempt to reduce the rate of restenosis. Pharmacological therapies can bedivided into categories based on mechanisms of action: prevention ofthrombus formation, prevention of vascular recoil and remodeling, andprevention of inflammation and cell proliferation (1). Systemic pharmacol-ogy approaches to reduce restenosis have failed. In the past, due to poorunderstanding of restenosis pathophysiology, choosing the right drug and

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lack of correlation between animal and human studies were the mostprobable reasons for the numerous failures. A major drawback in the treat-ment of restenosis was the inability to achieve adequate drug levels at theangioplasty site (7).

The recognition of this fact has led to the development of local drug-delivery systems and drug-eluting stents (DESs). Local drug delivery usingperfusion balloons has proven difficult, primarily because of a low uptakeof drug at the angioplasty site (8,9). In contrast, DESs have been shown tobe highly effective in providing an adequate concentration of drug (10,11).Local treatment, delivering medication directly to the site of vascular injuryvia polymeric-coated stents is a rational approach to achieve adequate localdrug delivery while minimizing systemic toxic effects. In the past severalyears, novel stent-based devices for drug delivery have been developed,aimed at reduction of neointimal proliferation in response to stent place-ment. The first approved and commercially available DES was Cypher(Cordis, a Johnson & Johnson Company, New Brunswick, New Jersey,U.S.A.). Rapamycin (Sirolimus) is a naturally occurring macrolide antibioticand a potent immunosuppressive agent. Sirolimus halts the cell cycle in thelate G1 phase and blocks the transition between the d and S phases by inhi-biting the activation of a specific target protein (mTOR) and migration ofsmooth muscle cells (SMCs) (12,13). Taxus (Boston Scientific Corporation,Natick, Massachusetts, U.S.A.) was the second approved DES containingPaclitaxel, a microtubule-stabilizing agent with potent antiproliferative activ-ity (14,15). The binding of Paclitaxel to tubulin results in a blockade of celldivision in the G0/G1 and G2/M phases, leading to reduced cell proliferation,migration, and signal transduction (16,17). Paclitaxel, a potent antiprolifera-tive agent, is not a cell-specific drug that inhibits endothelial and quiescentSMC, raising the concern of long-term untoward effects. Additional concernis focal restenosis at treatment margins (10). Nevertheless, DESs represent asuccessful example of a widely accepted implantable drug delivery system.

Other agents such as actinomycin D, C-Myc antisense, dexamethasone,and matrix metalloproteinase inhibitors, aimed at altering inflammatory andsmooth muscle actions in the biological repair response to vascular injury,are being evaluated. The success of these devices depends upon multipleissues, including stent platform, carrier, drug properties, and pharmaco-kinetic profile (18–26). Large randomized, controlled trials, demonstrate arestenosis rate of 5% to 10% with DES (27).

Although the mechanism of arterial injury during angioplasty appearsto be widely accepted, there has been some debate over the mechanism ofrestenosis. In general, most investigators agree that the process of resteno-sis is associated with the healing response to the arterial injury from theangioplasty. The healing response to mechanical injury comprises fourprocesses: elastic recoil, thrombus incorporation, neointimal hyperplasia(i.e., SMC migration/proliferation, extracellular matrix deposition), and

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vessel remodeling. Stenting results in an exaggerated neointimal prolifera-tive response with both nonelastic recoil and remodeling due to the rigidscaffolding effect of the stent.

INFLAMMATION AND RESTENOSIS

Inflammation plays an essential role in the initiation and progression ofatherosclerosis (28). Emerging experimental and clinical data indicate thatleukocytes may be central to intimal growth after mechanical arterial injury(i.e., angioplasty or PCI). In animal models of vascular injury, neutrophil andmonocyte recruitment precedes intimal thickening (29–31), and inflammatorycell number within the vessel wall is a powerful predictor of the extent of cel-lular proliferation and intimal thickening (32). Infiltration and accumulationof monocytes/macrophages play a major role in the pathophysiologicalresponse after stent-induced arterial injury, with inflammatory cells compris-ing up to 60% of neointimal cells in rabbit, porcine, and nonhuman primatemodels and in human autopsy specimens (31). Meticulous examination ofballoon-injured arteries demonstrates that neutrophils are present in abun-dance within hours of balloon injury and accumulate in the arterial mediafor several days after injury with a paucity of monocytes/macrophages (30).Clinical studies have shown that angioplasty is associated with leukocyte acti-vation and increased expression of the 2-integrin Mac-1 (CD11b/CD18) bothsystemically and locally across the injured vessel that predicts clinical resteno-sis (33–35) and angiographic late lumen loss (36). Leukocyte recruitment andinfiltration occur rapidly at sites of vessel injury following balloon angio-plasty or stenting where the lining endothelial cells (EC) have been denudedand, consequently, platelets and fibrin have been deposited. In addition topromoting the accumulation of leukocytes at sites of platelet coverage withinthe vasculature, the binding of platelets to leukocytes induces neutrophils andmonocyte activation, upregulates cell adhesion molecule expression, andgenerates signals that promote integrin activation, chemokine synthesis,and production of reactive oxygen intermediates (37–43).

The mechanisms by which leukocytes modulate vascular repair arelikely to be multifactorial (44). These inflammatory cells contribute to neo-intimal thickening due to their direct bulk within the intima, generation ofinjurious reactive oxygen intermediates, elaboration of growth and chemo-tactic factors, or production of enzymes [e.g., matrix metalloproteinases(MMPs), cathepsins G and S] capable of degrading extracellular constitu-ents and thereby facilitating cell migration (45). Fukuda et al. (46) haverecently shown that circulating monocytes count in human patients in-creased and reached its peak two days after stent implantation and thatthe maximum monocytes count after stent implantation showed significantpositive correlation with in-stent neointimal volume at six-month follow-upin patients after stent implantations.

Liposomal Bisphosphonates for the Treatment of Restenosis 189

Hypothesis: Systemic Depletion of Macrophages toPrevent Restenosis

Macrophages belong to the mononuclear phagocyte system (MPS). Theyoriginate from pluripotent stem cells of bone marrow that are precursorsfor all hematopoietic cells, i.e., lymphocytes, erythrocytes, osteoclasts, neu-trophils and mononuclear phagocytes (47). After certain differentiationsteps in the bone marrow, the committed stem cells give rise to monocytes,which move to the circulation, migrate to distinct tissue compartments, anddifferentiate into macrophages. In normal steady state in the body, macro-phages form a constant population with a balance between renewal and celldeath. In inflammation, the distribution and development of macrophageschanges significantly (48). Upon inflammatory stimuli, the amount of circu-lating monocytes and their migration to the site of inflammation increasesdramatically. The increased amount of monocytes in circulation is a conse-quence of the enhanced production of monocytes in bone marrow, whichstems from the shorter cell cycle time of the precursor cells. In addition,the half-life of the cells in the circulation decreases and a major proportionof the cells leaving the blood migrate to the site of inflammation. Theseevents lead to a 10-fold increase in inflammatory macrophages at theinflamed tissue (49,50).

We hypothesized that macrophages play a pivotal role in the patho-genesis of restenosis. We further hypothesized that systemic depletion ofmonocytes would decrease macrophage recruitment in the arterial wall, andconsequently may attenuate neointimal formation. Monocyte depletion canbe achieved with systemic injection of liposomes containing bisphosphonates(BPs). Van Rooijen et al. have shown that monocyte and macrophage deple-tion of the liver, spleen and bone marrow can be achieved by liposomal BPs(clodronate) (51). The BPs, bone-seeking agents, are a family of drugs thatinhibit bone resorption via osteoclast inactivation and are used clinically inseveral calcium-related disorders such as tumor osteolysis and osteoporosis.Osteoclasts and macrophages share a common hematopoietic progenitorcell in the bone marrow. BPs have poor cell membrane permeability. It iswell known that liposomes are readily taken up by cells of the MPS (formerlyknown as RES), macrophages in particular and to some extent neutrophils,by the process of phagocytosis. Encapsulation of BPs (such as clodronate andalendronate) in liposomes increases the magnitude of potency. Thus, a cell-specific delivery system of BPs that is capable of depleting macrophagesmay be beneficial for both the underlying atherosclerotic and restenotic pro-cesses while proving minimally toxic to nonphagocytic cells (52).

In this chapter, we will review studies on formulation variables affect-ing monocyte and macrophage targeting (e.g., size and number of vesicles),in vitro characterization in cell cultures, and in vivo immunomodulation andanti-inflammatory responses.

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MACROPHAGE/MONOCYTE INHIBITION BY LIPOSOMALDELIVERY SYSTEM OF BPs

BPs are hydrophilic charged molecules that cannot readily enter cells bypassive diffusion (53). After administration to patients or animals, theyaccumulate mainly in the bone tissue, and are cleared rapidly from the circu-lation into urine (53–55). Several studies have shown that the uptake of BPinto osteoclasts is mediated by their binding to bone mineral (45,56–58).This binding is essential for the effective antiresorptive activity of the drugon osteoclasts. BPs bound to the bone surface are internalized by osteoclastsin the course of bone resorption (56,57). A particulate dosage form such asliposomes can be used to enhance the intracellular delivery of BPs intophagocytic cells in cell culture and in animals (51,59). The use of liposomesas a delivery system for BPs was first introduced by van Rooijen and vanNieuwmegen (51), who used MLV-liposomes to deliver clodronate intophagocytic cells in vivo. Earlier methods for depletion of macrophages werebased on the administration of other particulates, silica, asbestos (60), andcarrageenan (61). These methods resulted in partial depletion as well asunwanted effects on nonphagocytic cells. Negatively charged liposomes arenontoxic, and after phagocytosis by monocytes/macrophages, the lipidbilayers of the liposomes are disrupted under the influence of the lysosomalphospholipases in the macrophage. The drug, which is dissolved in aqueouscompartments, is released into the cell. On the other hand, free BP, releasedby leakage from liposomes or released from dead macrophages, will notenter cells in amounts that are able to disturb their metabolism. Thisapproach, named the liposome-mediated macrophage ‘‘suicide’’ technique,was intensively used to eliminate macrophages from different compartmentsof the body in animals to study the role of macrophages in pathological andimmunological conditions (62).

Liposomal Encapsulation of BPs

Liposomes were prepared by thin lipid film hydration. BPs were dissolvedin deionized water at a concentration of 110 mM clodronate and 150 mMalendronate. The pH of the solutions was adjusted to 7.2 with sodiumbicarbonate. All solutions were filter sterilized before use (0.2-mm filter).1,2-Distearoyl-sn-glycerol-3-phosphocholine (DSPC), 1,2-Distearoyl-sn-glycerol-3[Phosphor-rac-(1-glycerol)] (DSPG), and cholesterol (3:1:2) weredissolved in t-butanol and lyophilized overnight. The lyophilized cake washydrated with an aqueous solution containing the BPs (alendronate orclodronate) at 55�C to 60�C and left to stand for one hour at the same tem-perature. The suspension was then extruded three times through doublepolycarbonate membranes of 0.8-, 0.4-, 0.2-, and 0.1-mm pore sizes (Nucleo-pore), by means of extruder. Liposomes were passed through a Sephadex


G-50 column and eluted in 2-(N-Morpholino) ethansulfonic acid hydrate(MES)/N-(2-Hydroxyethyl)piperazine-N-(2-ethane-sulfonic acid) (HEPES)buffer pH 7.2 (50 mM MES, 50 mM HEPES, 75mM NaCl) to removeunencapsulated BPs. Several formulations with different sizes were obtained(0.6, 0.4, 0.2, and 0.1 mm). Liposome size and morphology was determinedby dynamic light scattering and cryo-TEM microscopy (Fig. 1).

The mean sizes obtained were 500� 150, 350� 77, 192� 25, and100� 16, for 0.6, 0.4, 0.2, and 0.1 mm liposomes, respectively. Drugconcentration was determined by spectrophotometric assay of chromophoriccomplex between the BP and copper (II) ions (63) or by high performanceliquid chromatography (HPLC) (64). Lipid concentration was determinedby Bartlett method (65). Stability of the liposomes was determined by exam-ining drug leakage. Then 400 mL of liposomal formulations were centrifuged

Figure 1 Cryo-TEM microscopy of DSPC:DSPG:CHOL liposomes obtained bythin-film hydration method and extruded through 0.2-mm polycarbonate mem-branes. Abbreviations: DSPC, 1,2-distearoyl-sn-glycerol-3-phosphocholine; DSPG,2,2-distearoyl-sn-glycerol-3[phosphor-rac-(1-glycerol)]; CHOL, cholesterol.

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with a Centricon separation filter (Millipore, 30,000 MWCO) at 3500 g for 60minutes at 4�C. The liposomes were retained in the upper chamber, and 100to 150 mL of the filtrate was recovered from the lower chamber; drug and lipidconcentration were determined by HPLC. Leakage was evaluated everymonth for two years.

Effects of Charge and Size on Liposomes Uptake

The half life (t1/2) clearance of liposomes from the blood, which may range

from minutes to hours, and the distribution of liposomes to the organs, canbe controlled in part by altering the physical properties of the liposomessuch as their size, fluidity, and surface charge (66,67). In fact, ultra-smallliposomes with neutral charge are sometimes considered as ‘‘stealth lipo-somes’’ (long-circulating) (68). Studies with highly endocytotic cells, suchas RAW 264 macrophages and human monocytes (primary culture), haverevealed that encapsulation of BPs in liposomes enhances their inhibitoryactivity 20- to 1000-fold compared with free drug (Fig. 2). SMC and ECare insensitive to the liposomal drug delivery (52,59,70). Moreover, BPsin unilamellar liposomes seem to be more potent and less toxic thanmultilamellar liposomes (71). This was attributed to the negative chargeon the liposome surface and better encapsulation efficiency of unilamellar

Figure 2 Effect of liposomal formulations: BPs (alendronate and clodronate), emptyand free drugs on RAW 264 cell survival. Curves represent percentage of cell inhibi-tion with different BP concentrations. Cell count in buffer only was determined to be100% (n¼ 3). Abbreviation: BPs, bisphosphonates. Source: From Ref. 69.


liposomes (71). Negative and positive charges are known to enhance thedelivery of liposomes into cells through adsorptive endocytosis (66,72,73).However, positively charged lipids are not approved by the Food and DrugAdministration for clinical use. On the other hand, negative charge may induceleakage of encapsulated contents in biological fluids. Therefore, the surfacecharge density of the liposomal BPs (LBPs) has been optimized for minimalleakage and effective intracellular delivery of encapsulated drugs (74).

Liposomal size has a pivotal role in macrophage uptake; as the size ofthe liposome increases, there is enhancement of liposome uptake by macro-phages, as also reported by others previously (66,67). However, although thetrend remains the same, the clearance of liposomes is affected to differingextents depending on the composition (75).

Monocyte and Macrophage Depletion In Vivo

We have examined blood monocytes, tissue macrophages [fluorescenceactivated cell sorting (flow cytometry) (FACS)], and total white bloodcells (WBC) (Coulter) count before and after treatment with LBPs. Therabbit model of balloon angioplasty with and without stenting was utilized(52,76). WBC count increased slightly 48 hours after surgery, with no signif-icant difference between controls and the liposomal alendronate (LA) andliposomal clodronate (LC) dose groups (76). Monocyte number at 24 and48 hours after balloon injury and stenting was significantly lower inLA- and LC-treated animals. WBC and monocyte counts at six days aftersurgery returned to baseline levels (Fig. 3). Liver and spleen macrophagenumbers were reduced by LBPs at six days after treatment. Similarly,decreased arterial macrophage numbers were observed in LBPs treatedrabbits three and six days after injury (52).

Further support to the notion that liposomal BPs exert their effectssystemically was achieved through the use of liposomes loaded with thefluorescent marker, Rhodamine, with or without a BP (52,69). Markedreduction of the fluorescent signal was observed in blood monocytes (as well asreduced number) and in the liver and spleen of LBP-treated animals. Fluo-rescent liposomes (FL) were detected in injured but not in intact arteries. FLcoadministered with LBPs significantly reduced the fluorescent signal in theinjured arterial wall. The inactivation of monocytes after systemic administra-tion of LBPs results in reduction of tissue macrophages in the injured artery.Thus, the outcome of systemic administration was manifested as local treat-ment for the injured artery.

As mentioned, Fukuda et al. (46) have recently shown that circulat-ing monocytes count increased and reached its peak two days after humanstent implantation (from 350� 167 to 515� 149/mm3). These data supportthe correlation between monocytes depletion and prevention of restenosis.Inactivation of systemic monocytes immediately after injury suppresses

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the inflammatory cascade, resulting in decreased neointimal formation. Thetransient depletion suffices to significantly reduce long-term experimentalneointimal formation.

Cytokine Activity

BPs have numerous biochemical effects on cellular metabolism ranging fromthe inhibition of general cell metabolism, such as glycolysis (77), geranyl-geranyl inhibition of the mevalonate pathway, to the release of inflammatorymediators. The inflammatory mediators affected by BPs include prosta-glandins, cytokines, and reactive oxygen metabolites. Clodronate inhibitsprostaglandin E2 synthesis in cultured mice calvaria cells treated in vitro orin vivo (78,79). Clodronate and alendronate were found to inhibit inter-leukin (IL)-6 secretion by human osteoblastic cells (80). In contrast to thenon-amino-BPs, clodronate, and etidronate, the amino-containing BPs,alendronate and pamidronate showed proinflammatory properties on macro-phage functions by inducing the secretion of cytokines from macrophages(81). Although at high concentrations pamidronate was a potent inhibitorof cytokine secretion from RAW 264 macrophages, at low concentrations it

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Figure 3 Time response of CD14þmonocytes, expressed as percentage of total bloodleukocytes, in LC-treated and control balloon-injured rabbits and control. Abbrevia-tions: WBC, white blood cells; LC, liposomal clodronate. Source: From Ref. 52.


augmented the secretion of IL-6 from LPS-treated cells. Except for one study,which showed inhibition of cytokine release from LPS-stimulated humanmonocytes (82), the induction of cytokine secretion seems to be common tothe amino-BPs. It has been shown that BPs also diminish secretion of reactiveoxygen species by human neutrophils, polymorphonuclear leukocytes, andmacrophages in vitro (83–85).

The effect of different BPs (alendronate and clodronate) in free form orencapsulated in liposomes on cytokine secretion was evaluated using RAW264 macrophage activated with LPS. LC and free clodronate reduce cytokinesecretion; LC is over 10 times a more potent inhibitor of cytokine secretionfrom RAW 264 than the free drug (86). The amino-BP alendronate augmentsthe release of IL-6 and IL-1b. Liposome encapsulation shifted this inductioneffect to lower concentrations of alendronate (87). The results of TNFa werevery similar to the IL-1b data. Regulatory regions of murine genes for cyto-kines and inducible nitric oxide synthase (iNOS) have sequences, which havebinding affinity genes for the transcription factors, NFkB and AP-1 (88–90).Binding of LPS to its receptor on plasma membrane leads to the activationof a protein kinase cascade, which ultimately induces the nuclear activity ofthese transcription factors. This activation results in increased expressionof inflammatory proteins in the activated macrophages. Therefore, the inhi-bition or induction of the cytokines and iNOS may be reflected in the DNA-binding activity of NFkB and/or AP-1. In RAW 264 cells, LPS inductionfor four hours caused a marked increase in the nuclear localization of threeNFkB complexes and one AP-1 complex (81). The inhibitory effect of LC oncytokine and NO secretion was seen in the DNA binding activity of NFkB.

Effect on PDGFbR and on Platelet Derived Growth Factor Receptorb Tyrosine Phosphorylation

Platelet derived growth factor (PDGF)-BB is a strong chemoattractant forvascular SMCs involved in neointima formation secondary to vascularinjury (91,92). In vivo studies conducted in our laboratory revealed thatPDGFbR activation (i.e., tyrosine phosphorylation) markedly increasedto 135% of baseline levels in balloon-injured arteries of untreated rats,whereas it was barely detectable in LC-treated rats (i.e., below baselineactivity) (69). Balloon-injury upregulated PDGFbR in control (121%)and LC-treated rats (233%). Injury resulted in a strong accumulation ofPDGF-B protein within the vessel wall on days 1 and 3 after injury, reach-ing 333% and 219% of the baseline level, respectively. In LC-treated rats,PDGFb accumulation was strongly reduced (181% and 168%, on day 1,and day 3, respectively), which was in good correlation with the reducedactivation of PDGFbR at these time points.

Macrophages are a rich source of growth factors including PDGF-BB(28), which facilitates SMC migration to the injured vessel. Suppression of

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PDGFbR activation in treated animals (rat and rabbit) probably corres-ponds to a substantial reduction of PDGF-BB protein levels in the lesion,which can explain the reduced SMC migration and neointimal formationin treated animals. Our data are in conjunction with reduction in arterialand blood cytokines, IL-1b, TNFa, NFkB, and MMP-2 activity followinginjury (52). The systemic inactivation results in reduced expression of localinflammatory mediators leading to reduced activation and proliferation ofSMC and decreased neointimal formation.

INHIBITION OF RESTENOSIS

Rat Vascular Injury Model

The rat carotid artery injured by a balloon catheter has been widely used as amodel of angioplasty. The rat model is a ‘‘proliferation’’ model without foamcells (93). This form of injury causes immediate coagulation and thrombosiscascade in which platelets adhere, spread, and degranulate on the denudedsurface of the artery, and approximately 24 hours later SMC begin to prolif-erate. Liposomal BPs, clodronate, and alendronate were injected to malesabra rats, 15 and 3 mg/kg, respectively (52,69,76). Marked neointimalformation and decreased luminal area were observed in control animals.Neointima/media (N/M) ratio was 1.3� 0.2, and luminal stenosis was44� 3%. LC and LA suppressed intimal growth when administered intrave-nously on day�1 and dayþ6. N/M ratios were reduced by 60% and 69% forLC and LA, respectively.

Hypercholesterolemic Rabbit Model

In this model, rabbits are fed an atherosclerotic diet for 30 days, and undergoarterial denudation by a Fogarty balloon and/or stent deployment in the iliacor carotid arteries. Liposomal BP, clodronate, and alendronate were injectedintravenously to the rabbits (52,76). Massive proliferation of SMCs and extra-cellular matrix formation was observed in control animals after balloon injury.No significant differences were found between treatments of empty liposomes,saline, or free BP (15 mg/kg). Luminal stenosis was significantly reduced from75� 8% in the control to 41� 8% with LC (15 mg/kg) treatment and 68� 5%with LA (3 mg/kg) treatment. After the stenting procedure in the iliac artery,there was abundant concentric neointimal formation composed of SMC andfoam cells, with both intraluminal and outward neointimal growth. Luminalstenosis was 58� 11%. LA (3 mg/kg) significantly reduced neointimal forma-tion at 28 days (Fig. 4). There was no significant difference between animalstreated with 3 or 6 mg/kg LA or 15 mg/kg LC. Clodronate is several ordersof magnitude less potent than the amino BP, alendronate, in inhibitingosteoclasts and consequently bone-related disorders such as tumor osteolysisand osteoporosis (53). The difference in potency stems from the mechanism of


action of these BPs on osteoclast inhibition: nonhydrolyzable adenosinetriphosphate (ATP) analogue formation by clodronate and geranyl-geranylenzyme inhibition in the mevalonate pathway induced by alendronate (94).The more potent alendronate induces apoptosis of both osteoclasts andmacrophages at relatively lower concentrations. The dose of alendronaterequired for the reduction of neointima after balloon injury was lower thanthat required by clodronate, 3 mg/kg versus 15 mg/kg, respectively. Inhibi-tion of restenosis even in the highly cellular hypercholesterolemic modelwas achieved by a single intravenous (IV) liposomal application of the potentBP alendronate at the time of injury (52). Other amino BPs such as pami-dronate and 2-(2-Aminopyrimidino)ethlidene-1,1-bisphsphonic acid betaine(ISA)-13-1 suppressed intimal growth when administered intravenously,but to lesser extent than LA (69). A drug potency effect relationship was

Figure 4 Hypercholesterolemic rabbit carotid artery 30 days after balloon injury.Photomicrographs of Verhoff’s tissue elastin staining of (A,C) full-size and (B,D)higher magnification sections from (A,B) control, (C,D) liposomal alendronatetreated (3 mg/kg intravenous, at the time of injury). Control animals were treatedwith buffer, free BP (alendronate), or empty liposomes and grouped as control(n¼ 20 arteries/group, �P< 0.05). Abbreviation: BP, bisphosphonate.

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established, alendronate > pamidronate >ISA-13-1>clodronate (55,95).These data correlate with the rank of BP potency in inhibiting osteoclasts.

Different dosage regimens were examined; multiple doses of LC(15 mg/kg) or LA (1.5 mg/kg) at day�1 and dayþ6 were found to have sameeffect as one dose at day�1. A single dose at dayþ6 had no effect. Treatmentof LA, a single dose, at the time of injury required dose adjustment, and anincrease of dose from 1.5 mg/kg to 3 mg/kg (76). LA administration at thetime of injury caused transient suppression of monocytes, lasting less thanone week, consistent with previous studies on monocyte and macrophagedepletion after administration of LBP (52,96).

Mechanism of Restenosis Inhibition

Adhesion of monocytes occurs shortly after stenting (20,32). The number ofneointimal macrophages after stenting varies with time: macrophages com-pose almost 40% of neointimal cells at first week, less than 7% three weekslater, and almost equal to 1% at 12 weeks, correlating with SMC prolifera-tion in the neointima: 30%, 8%, and 1% at those time points, respectively (97).Thus, the immediate infiltration of macrophages is associated with earlySMC proliferation, which leads to neointimal formation and late restenosis.Innate immunity triggers the healing response that leads to neointimal for-mation. Early transient inactivation of this inflammatory response reducesearly SMC proliferation and late arterial narrowing. The neointima in thismodel is composed of two semidiscrete layers: a deeper layer composedprimarily of macrophages and a luminal layer composed primarily ofSMCs (98). The effect of LBP treatment on proliferating SMCs, whichcomprise the major component of human restenotic lesions, is indirectlymediated via the inhibitory effect on macrophages; in vitro studies showedunequivocally that macrophages are much more sensitive (52,97) to LBPsthan SMCs. Our treatment did not affect SMC directly; SMC were affectedindirectly by reducing cytokines secretion due to monocytes depletion. Theimpact of monocytes and macrophages on vascular repair of stented bloodvessels is even more significant. Macrophage content in the vessel wall ismarkedly higher and prolonged in stented versus balloon-injured arteries(32,97), and it correlates with neointimal formation (32). The reduction ofboth SMC proliferation and content supports the major role of monocytesin neointimal formation after stent injury and the potential use of macro-phage depletion in its modulation.

CONCLUSION

Innate immunity plays a central role in vascular injury and repair. Sup-pression of monocyte numbers by IV infusion of LBPs inhibits intimalhyperplasia after balloon injury (52,99,100). The preprocedural activation


status of innate immunity correlates with late restenosis (101–104), enablingpreprocedural risk stratification. Although initial studies with glucocorti-coids failed to show reduction in post balloon restenosis (105,106), prolongedimmunosuppressive therapy with prednisone was found to reduce in-stentrestenosis in high-risk high-C-reactive protein patients [Immunosuppressivetherapy for the prevention of restenosis after coronary artery stent implanta-tion (IMPRESS) study] (107). Thus, systemic immunomodulation effectivelysuppresses the reparation that leads to neointimal formation in a high-risk‘‘inflammatory’’ subset of patients. Glucocorticoids suppress both innateand adaptive immunity, with a possible acute stimulatory affect on innateimmunity (108,109). Specific targeting of monocytes, addressed by LBPtherapy, differs from glucocorticoids in specifically targeting innate immu-nity. Furthermore, it has a favorable pharmacodynamic profile and thuspossesses high potential for clinical use. Is there any rationale to examinesystemic therapy in the ‘‘era of DESs’’? Most systemic pharmacologicaltherapies have been unsuccessful in preventing arterial restenosis in humans,and the preliminary results of DESs are remarkable in reducing in-stentrestenosis. Nevertheless, LBPs offer a systemic therapy to a systemic process(110,111), regardless of the procedure and the device(s) used. If effective in aclinical setting, it may be an easily administered, cost-effective modality thatallows flexibility in choosing the type and number of stents to be deployed,may serve as an adjunct therapy in high-risk patients, and may even reducethe need for stenting altogether (7).

ACKNOWLEDGMENT

G. Golomb is a member of the David R. Bloom Center of Pharmacy atThe Hebrew University of Jerusalem.

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206 Epstein et al.

11

Development of a Liposomal VaccinationSystem for Immunity-Modulating

Antitumor Therapy

Andreas Graser

Pharmaceutical Technology Development Formulation Liquids, F. Hoffmann-LaRoche Ltd., Basel, Switzerland

Abdo Konur

Klinikum Geb. 302T/TVZ Johannes Gutenberg-Universita€t Mainz,Mainz, Germany

Alfred Fahr

Lehrstuhl fu€r Pharmazeutische Technologie, Friedrich-Schiller-Universita€t Jena,Jena, Germany

INTRODUCTION

About a 100 years ago, Coley observed tumor regression after applicationof a mixture of bacterial toxins. This experiment made the foundation ofimmune therapy for cancer, which only recently became a promisingtreatment and an efficient alternative to chemotherapy. In contrast to che-motherapy (1), this new treatment does not kill tumor cells directly; ratherit enhances the sensitivity of the patient’s own immune system against tumorcells with all the potential positive aspects like high selectivity of treatmentand much less side effects (2).

However, after numerous attempts in the last decades, vaccinationagainst tumors, as it can be named, is not easy to achieve (3) in contrast to

207

vaccination against infectious diseases. This may be caused by tolerancemechanisms of the cancer cells, as it can happen, when T-lymphocytes getantigens presented without costimulatory signals (4). Another tolerancemechanism inhibiting the immune response is an observed dysfunction ofT-cells by the expression of low-affinity T-cell receptors, which might becaused by factors produced by tumor cells (5).

After identification of those and many other immunological processes,several strategies were tested in the meantime to influence these processes. Forexample, administration of interleukin-2 was thought to activate cytotoxiccells and T-helper cells (6). However, clinical studies using this approachshowed a disastrous outcome by displaying a bunch of serious side effects.

A much more promising approach is the active specific immune ther-apy, which tries to induce a specific immune response of the immune systemagainst the tumor cells. It is possible to inject either autogenic or allogenictumor cells or also a lysate thereof (7).

Another milestone in this research area was the isolation and identifi-cation of antigens from tumor tissue [e.g., tyrosine-related proteins (TRP)-1and (TRP)-2]. After the administration of these antigens, it became clearthat a sufficient success was only possible by the concomitant administrationof adjuvant-acting substances like CpG-oligonucleotides (8).

One of the important relay stations in the immune system activation isthe dendritic cell. Activation of these cells causes the most efficient stimula-tion of T-cells, which in turn attack tumor cells (9). This turned the dendriticcells (DCs) into the focus of modern immune research and therapy. How-ever, another difficulty appeared that became a problem recently. In orderto activate the dendritic cell, the antigen has to be present in the cytosoland has to be processed there further; this is not an easy task to perform.

Here, liposomes come into play as modern drug-carrier systems. It iswell known from earlier studies that dioleoyl phosphatidylethanolamine(DOPE)-containing liposomes administered to somatic cells may lead toan uptake of the liposomal content into cells (10,11). However, this wasnot demonstrated up to now for DCs. The use of liposomes would also havethe advantage—if they work—that at the same time an antigen and an adju-vant could be given concomitantly.

The aim of this study was the development of a liposomal carrier sys-tem able to deliver antigen and adjuvant into DCs in order to activate theimmune system for killing tumor cells.

METHODOLOGY

Starting point of our investigation was the construction of a TRP2-peptidecontaining liposome. One peptide epitope of TRP2 (SVYDFFVWL; aa180–188) is not only presented by human HLA-A�0201 but also by murineMHC class I molecule H2-Kb on B16 tumor cells making it an attractivemodel for preclinical anti-tumor studies in C57BL/6 (12).

208 Graser et al.

Materials

All phospholipids used were of synthetic quality and purchased from AvantiPolar Lipids (Alabama, U.S.A.). Cholesterol was purchased from Calbiochem(California, U.S.A.)

� TRP-2 peptide aa sequence 180 to 188 (SVYDFFVWL) was pur-chased from Bachem (Weil am Rhein, Germany).

� CpG-oligodesoxynucleotide 1826 (50-TCCATGACGTTCCTGAC-GTT-30, 20 mer, phosphorothioate) was obtained from Eurogentec(Seraing, Belgium). RPMI 1640 medium was purchased fromGibcoBRL (Eggenstein, Germany) and fetal calf serum (FCS)from Bio Whittaker (Verviers, Belgium).

� Buffers used: phosphate buffered saline (PBS): 6.5 mM Na2HPO4,1.5 mM KH2PO4, 2.5 mM KCl, 140 mM NaCl, H2O, adjusted topH 7.25 and autoclaved.

� ACK lysis-buffer: 1.00 g KHCO3, 37.2 mg Na2-EDTA, ad 1000 mLH2O, adjusted to pH 7.4 and filtrated to sterility.

� HBSS: Hanks balanced salt solution (GibcoBRL, Eggenstein,Germany).Rhodamine-phycoerythrin (PE) was purchased from MolecularProbes (Eugene, Oregon, U.S.A.).All other reagents used were of at least analytical purity (p.A.).

� HPLC of CpG was performed with an Agilent HPLC System series1100, equipped with a two wavelength UV detector and a quartern-ary gradient pump (Agilent Technologies Germany, Waldborn,Germany).

� For CpG analysis, a DNAPac-PA-100 analytical column 4� 250 mm(Dionex Corp., Sunnyvale, U.S.A.) operated at 55�C and a flowrate of 1 mL/min was used.

� For TRP-2 analysis, a reversed phase gradient method already devel-oped (13) was slightly modified for our purpose. We used a WatersAlliance 2695 separations module equipped with a Waters 996 PDAdetector and a CC 125/3 Lichrospher 100-5 RP-18 column (Machereyund Nagel, Duren, Germany) at 30�C and a flow rate of 0.5 mL/min.

� Photon correlation spectroscopy (PCS) for size measurement wasperformed using a Zetasizer HS3000 (Malvern, Herrenberg,Germany). Samples were diluted until a count rate of 50 to 250kilocounts was achieved.

� Murine melanoma cell line B16F1 was obtained from ATCC(Manassas, VA, U.S.A.).

� Mice (breed C57BL/6; Harlan Winkelmann, Borchen, Germany)weighing 20–35 g were kept at room temperature of 28�C, a relativeair humidity of 60%, and a day/night rhythm of 12 hours underpathogen-free conditions. The animals were fed with water andAltromin 1324 (Altromin, Lage, Germany) adlibitum.

Development of a Liposomal Vaccination System 209

DCs were generated (BmDC) from thigh bone marrow of na€��veC57BL/6 mice according to established procedures. In a spleen cell mixtureof the same mice breed, DCs were identified using a fluorescent antibody.

FACS-Binding Studies of Liposomes

Spleen cells or BmDCs were diluted using RPMI containing 10% FCS to aconcentration of 5 � 106 cells/mL. Liposomes were prepared for these inves-tigations with 0.3 mol% Rhodamine-PE as fluorescent marker and added tothe cell suspension at a concentration of 10 mmol lipid/mL. Samples wereincubated with anti-CD11c-FITC (Pharmingen, Hamburg, Germany), whichbinds specifically to DCs. After removing unbound liposomes with fluores-cence activated cell sorter (FACS) washing buffer (1% BSA in PBS) thesample was analyzed in a FACS-device (FACSCalibur, Becton-Dickinson,Heidelberg, Germany) at 488 nm excitation wave length, 585 and 530 nmdetection wavelength.

Dimer X Assay for the Measurement of CirculatingAntigen-Specific Cytotoxic T-Cells

For measuring the amount of circulating CTL, the following protocol wasperformed. C57BL/6 mice were immunized with the vaccine. In the daysafter immunization, 100 mL blood was taken retro-orbitally, incubated with1.4 units heparin in PBS and stored at 4�C. For final analysis, 100 mg of aDimer X mouse H-2Kb:Ig (BD Pharmingen, Hamburg, Germany) weremixed with 42 mg of TRP-2 peptide for 24 hours at 4�C. The blood samplesfor analysis were centrifuged (200 g, 4�C, five minutes) resuspended in 50 mLFACS washing buffer and incubated with 1 mg of the prepared Dimer X forone hour. After washing twice with 2 mL FACS washing buffer, 1 mg of thesecondary antibody goat-antimouse-phycoerythrin [GaM-PE F(ab) 2-frag-ment, Dako, Glostrup, Denmark] was added and incubated for 15 minutesat 0�C. The samples were then treated with ACK lysis-buffer, washed andresuspended in 500 mL PBS containing 1% para-formaldehyde. FACS anal-ysis was performed at 530 nm (CD8 bearing T-cells ¼ CTL) and 585 nm(TRP-2MHCI specific T-cell receptors, GaM-PE).

Construction of a Liposomal Carrier for Tyrosine-RelatedProtein-2 Peptide at Lab Scale

As the solubility of this peptide in water is very low, the peptide can be asso-ciated with the liposomal membrane. As the peptide is only sparingly solublein methanol or chloroform, DMSO had to be used as dissolution mediumfor mixing the peptide with the lipids for liposome formation. A 10 mg/mLstock solution in DMSO of the peptide could be obtained. Appropriateamounts of lipid stock solutions and the peptide stock solution were mixed(lipid:peptide ratio ¼ 95:5) and processed as thoroughly described in the

210 Graser et al.

literature (14,15) (drying of the solution in a round-bottomed flask by rotaryevaporation at 10 mbar vacuum for 60 minutes). The resulting lipid filmshowed crystalline inclusions, which could be removed by resolvation ofthe film with a chloroform/methanol (1:1) mixture with subsequent rotaryevaporation. This was repeated three times. The final lipid film was homo-genous. Buffer was added in the usual manner and agitated, the resultinglipid suspension could be extruded through polycarbonate membrane filterswith a pore size of 100 nm using a standard device (Liposofast1) (16).

The liposome lipid composition was varied in order to get (i) a highimmunological response, (ii) to increase the dose, and (iii) to increase thestability of the formulation. A complete list of all investigated liposomeformulations is given in Table 1. Liposomes were made with or withoutactive ingredients.

Upscaling of the Liposome Production Process

The procedure described previously yielded liposomes of sufficient qualityup to a process volume of 5 mL. Larger quantities (about 30 mL) necessaryfor intensive studies made with the described protocol were not feasible.

Therefore, a modified protocol was used. The appropriate amount oflipid and peptide powder was weighted in a 50-mL glass bottle, buffer wasadded and for 30 minutes prehomogenized using an Ultra-Turrax T8 (IKAGmbH & Co. KG, Staufen, Germany) operating at 25,000 rpm. The glassbottle containing the lipid suspension was thermostatted during this processto 55�C. The resulting particle size (measured by PCS) was 390 nm.

After this, the obtained suspension was processed in a homoge-nizer (Emulsiflex-C5, Avestin Inc., Ottawa) operated at a pressure at thehomogenization valve of 300 kPa. The particle size was 230 nm after a homo-genization period of 30 minutes. The final process step was extrusion usingpolycarbonate membrane filters with a pore size of 100 nm installed in the

Table 1 Lipid Composition of All Liposomal Formulations Mentioned in the Text

FormulationChol

(mol%)DOPS(mol%)

DLPE(mol%)

POPC(mol%)

DOPG(mol%)

SM(mol%)

AVE 3 33.3 33.3 33.3AVE 5 33.3 33.3 33.3AVE 6 48 13 12 12 15AVE 14 33.3 33.3 33.3AVE 43 33.3 33.3 33.3AVE 44 33.3 33.3 33.3

Abbreviations: DOPS, dioleoylphosphatidylserine; DLPE, dilauroylphosphatidylethanolamine;

POPC, palmitoyloleoylphosphatidylcholine; DOPG, dioleoyl phosphatidylglycerol; SM,

sphingomyeline.


Emulsifexl-C5 (pressure 14,000–30,000 kPa, 10 minutes in cycling mode,finally five times in single mode). The final particle size was about 130 nm.

The obtained liposome dispersion was made sterile by filtrationthrough 200-nm pore-sized filters and stored in sterile bottles till usage at4�C. The liposomes were stable in size for at least 120 days.

Tyrosine-Related Protein-2 Peptide Incorporationin Liposomal Bilayer

Incorporation of greater peptide amounts into the liposomal bilayer is lim-ited by the disturbance of the bilayer integrity by the peptide itself, the lipidcomposition, and the production process conditions.

At a molar ratio of peptide to lipid of 0.002 (0.2 mol%) and 0.005 (0.5mol%) at process start, all of the peptide was incorporated into the liposo-mal membrane bilayer. At a 5 mol% value of the peptide in relation to thelipid amount at the start of the experiments, only 33% of the total peptidewas recovered in the final liposomal formulation after extrusion; a concomi-tant loss in lipid content was also observed. This led us to the conclusionthat at higher TRP-2 peptide ratios lipid-peptide aggregates may be formed,which cannot be extruded.

Construction of a Liposomal Carrier for CpG Oligonucleotides

For the ease of preparation and analytical simplification, we formulatedCpG in a separate liposome formulation and mixed it together with theTRP-2 peptide containing liposomes before administration. The hydrophilicCpG was added to the hydration medium used for liposome formation at aconcentration of 10 mg/mL. After hydration (lipid content 40 mmol/mL)and extrusion, liposomally entrapped CpG was separated from free CpGby size exclusion chromatography. The resulting diluted dispersion wasconcentrated using Vivaspins (Vivascience AG, Hannover, Germany) at1000 rpm. The size of the liposomes was on average 96.4� 12.4 nm. TheCpG content of the liposomes was measured by HPLC and had an averagevalue of 527.4� 29.1 mg/mL.

RESULTS AND DISCUSSION

Production of the Liposomal Formulation

The upscaling process of the TRP-2 peptide formulation delivered a signifi-cantly increased loading capacity for the peptide (Fig. 1), compared to thelaboratory process using film drying for a given lipid composition. Alsothe variability for the process was lower for the upscaling process [sD

(homogenization)¼ 11.5%, sD (extrusion)¼ 33.3%]. The lipid compositionsinvestigated played only a minor role for the loading capacity.

212 Graser et al.

In Vitro Characterization and Optimization of the LiposomalVaccine System by FACS Analysis

In order to get an effective stimulation of CTL, an efficient presentation ofthe antigen peptide on MHC molecules in synergism with costimulatingreceptors on the antigen-presenting cells (APC) is necessary. DCs (‘‘Introduc-tion’’) are the most prominent APCs for activating T-cells. The first criticaltest was therefore binding and uptake of our liposomes (without peptide)by DCs, as only taken up peptides will be processed by the dendritic cell.In Figure 2, it is evident that the liposomal formulations AVE 3 and AVE5 show a higher binding and a higher uptake than all other formulations.This is presumably due to the presence of strong negative charges (Table 1)on these liposomes, as AVE 14 does not have any negative charges on thesurface. The reduced binding and uptake of AVE 43 and AVE 44 comparedto AVE 3 and AVE 5 can be attributed to the lack of phosphatidylethanola-mine in those liposomes. Uptake of liposomes is most pronounced in thecase of AVE 3. This might be due to the special lipid composition, as theseliposomes resemble closely to the main lipid composition of HIV (17) andit may be speculated that DCs might therefore take these liposomes upmore eagerly.

Especially phosphatidylserine (PS) seems to deliver a good liposomebinding signal for DCs; therefore the influence of PS content on the bindingto DCs was studied in greater detail in a second set of experiments (data notshown in figure 2). At a PS content of 50% in the liposomes, about 80% of

Figure 1 Influence of liposome formation method on TRP2 content of liposomes.


the DCs showed via FACS-analysis binding of liposomes, whereas T-cellsderived from spleen showed only a 5% binding of these liposomes.

The same type of experiments performed for phosphatidylglycerol(PG) content of liposomes showed a similar trend, but not as pronouncedas in the case of PS. Here about 40% of the DCs and 8% of the T-cellsshowed binding to liposomes containing 50% PG. Influence of PE-contenton the binding to these cells was not of great significance (data not shown).

Incorporation of TRP-2 peptides into the liposome formulationshad an influence on the uptake of these liposomes by DCs. All relevantformulations (AVE 3 and AVE 5) showed a moderate reduction inuptake (� 20%).

Microscopic Investigations of Liposome Uptake by Dendritic Cells

Liposomes (without peptide) were labelled with rhodamine-PE in the lipidbilayer and in the inner compartment using FITC-dextrane 9000 and incu-bated with DCs for one hour. Figure 3 shows clearly that only in the case ofAVE 3 and AVE 43 could a significant uptake be observed. The fluorescenceinside the DCs is in the case of AVE 3 homogenously distributed in thecytosol, whereas in the case of AVE 43 the liposomes seem to be caughtin granular structures, presumably endosomes. The PS causes the liposomes

Figure 2 Influence of different lipid compositions on binding to DCs. Binding anduptake was performed for 90 min at 4�C (binding only) or 37�C (uptake). DC werevisualized by CD11c stain. Shown is the percentage of CD11cþ cells binding lipo-somes (% binding). Binding to DC was specific, as no binding was detectable toCD8þ T cells and only a weak binding to B220þ B cells (data not shown).

214 Graser et al.

to be taken up by DCs; the function of the PE in the PS-containing lipo-somes seems to be the positive influence on the release from the endosomes.

Activation of Dendritic Cells by Liposomes ContainingTyrosine-Related Protein-2 Peptides

In order to study in vitro the activation potential of the vaccine formulation,4� 106 BmDCs were incubated either with 400 mg free TRP-2 or 40 mg TRP-2in liposomal formulations. In addition, CpG-ODNs were added in this seriesof experiments concomitantly either in its free form (20 nmol) or in its lipo-somal form (5.2 nmol). The incubation took place in 4 mL RPMI (with 5%FCS) for 48 hours at 37�C. After completion of the incubation, the cells werestained with antibodies specific for CD80, CD86 and MHC II molecules,which are expressed in high amounts on the surface of DCs after activation.

Figure 3 Binding and internalization of different liposomal formulations by bonemarrow–derived dendritic cells (BmDC) from mice. BmDC were incubated forone hour with liposomes at 37�C and liposome uptake was analyzed by confocallaser scanning microscopy.


In order to detect only DCs, the cells were counterstained with anti-CD11c-PE. Figure 4 shows clearly that incubation of the cells alone withfree TRP-2 does not show any stimulation of the DCs. Free CpG showsalone a faint activation; the concomitant incubation with free TRP-2 andfree CpG, however, does not give any significant activation of the DCs.In contrast to these results, an evident DC activation was observed afterincubation with TRP-2 incorporated in AVE 3 combined with free orliposomal CpGs.

Influence of the Liposomal Vaccine System on the Number ofActivated Circulating Cytotoxic T Cells

In order to judge the efficiency of the vaccine system, the number of speci-fically activated CTL was measured. In case of a successful activation, thenumber of these cells should rise significantly in the timeframe of days afterimmunization.

For these experiments, the optimal formulation in comparison to freeantigen was tested. The mice were immunized three times with 10mg liposomalAVE 3 TRP-2 with a concomitant administration of 13.3 nmol lipo-somal AVE 3 CpG. The controls were done with 100 mg free TRP-2 and5 nmol free CpG. On the days following the third injection, 100 mL blood

Figure 4 Activation of BmDCs in vitro. 4� 106 BmDC were cultured for 48 h at37�C in 4 mL RPMI/5%FCS with the indicated formulations. BmDC with a strongexpression of MHC-class-II, CD80 and CD86 were quantified by FACS.

216 Graser et al.

was taken retro-orbitally and the percentage of activated CTL was measuredby Dimer X assay.

It can be clearly seen in Figure 5 that the liposomal vaccination had adramatic effect on the number of activated CTL at day 1 (5.4% cells acti-vated) and day 2 (4.1% cells activated). Free TRP-2 together with freeCpG showed at even 10 times higher concentration no visible vaccinationpower (comparable to pure buffer administration).

Test for the Prophylactic and Therapeutic Efficiency of theLiposomal Vaccine System

The prophylactic antitumor efficiency was tested by injecting 12 mice twicein seven days with AVE 3 TRP-2 (10 mg TRP-2) and AVE 3 1826 CpG(1.3 mg CpG) intradermally; the control group remained untreated. Sevendays after the last immunization 2� 105 B16 tumor cells in 200 mL HBSSwere injected into the tail vein of each mouse. Twenty days after tumorinoculation, the animals were sacrificed and the metastases in the preparedlungs counted. As Table 2 shows, the liposomal vaccination has a significanteffect on the tumor growth in comparison to untreated animals. This is alsoreflected by the visual appearance of the lungs (data not shown).

Figure 5 Relative amount of circulating TRP2 specific cytotoxic T-lymphocytes inthe blood of mice. After three rounds of vaccination with the indicated formulationsblood was collected and TRP2-specific CD8þ T cells were quantified by TRP2-loaded DimerX by FACS analysis. SIINFEKL-loaded DimerX used as controlwas a background level (data not shown).


In order to prove the efficiency of the liposomal system in tumor ther-apy (administration of the liposomes after tumor induction), seven animalswere treated with 2� 106 B16 tumor cells (injection of a suspension in200 mL HBSS into tail vein). After four and seven days the formulation[AVE 3 TRP-2 (10 mg TRP-2) and AVE 3 1826 CpG (1.3 mg CpG)] was giveninto the foot pad of the hind legs of the mice intradermally. Twenty-onedays after the injection of the tumor cells, the animals were sacrificed andthe metastases in the prepared lungs were counted. A second group of sevenanimals received the tumor cells, but no liposomal treatment was applied.Table 2 indicates the high antitumor potency of the formulation.

SUMMARY

We could demonstrate, that liposomal formulations with negative surfacecharges, especially efficient is here PS, are binding to and taken up byDCs. After optimization of the liposomal formulation and the productionprocess, by which the incorporation of tumor antigens into the liposomalbilayer could be increased significantly, an applicable and stable formulationcould be developed. Together with a liposomal formulation of an adjuvant,experiments in vitro and in vivo clearly showed the superior effect of thisformulation in vaccination against tumors. Even a therapeutic treatmentwas effective using this kind of formulation. The beneficial effect of a lipo-somal formulation for antigen and adjuvant is also demonstrated by otherrecently published work (18–20) and may have important implications forcancer therapy.

ACKNOWLEDGMENTS

We thank Dr. Jerome and Dr. Nahde for helping with the cell cultureand animal experiments and Dr. Merdan for the confocal laser scanningmicroscopy experiments. We would also like to thank vectron therapeutics

Table 2 Prophylactic and Therapeutic Efficiency of the Liposome VaccinationSystem

Number of lungmetastases (average)

prophylactic treatment

Number of lungmetastases (average)

tumor therapy

Immunization by AVE 3TRP-2 and AVE 3 1826 CpG

7.7 (n¼ 12) 10.5 (n¼ 7)

Nonimmunized 50.7 (n¼ 7)a 150.5 (n¼ 7)

aFive animals died during the experiment due to development of high tumor mass.

218 Graser et al.

AG, Marburg for financial support of these studies and Dr. Muller and Dr.Kontermann for continuous interest and discussion during this work.

REFERENCES

1. Coley WB. The treatment of malignant tumors by repeated inoculations of ery-sipelas. With a report of ten original cases. Am J Med Sci 1983; 105:487–511.

2. Mitchell MS. Cancer vaccines, a critical review—Part II. Curr Opin InvestigDrugs 2002; 3(1):150–158.

3. Rosenberg SA, Yang JC, Schwartzentruber DJ, et al. Immunologic and thera-peutic evaluation of a synthetic peptide vaccine for the treatment of patients withmetastatic melanoma. Nat Med 1998; 4(3):321–327.

4. Lee PP, Yee C, Savage PA, et al. Characterization of circulating T cells specificfor tumor-associated antigens in melanoma patients. Nat Med 1999; 5(6):677–685.

5. Fink J, Ferrone S, Frey A. Where have all the T-cells gone? Mechanisms ofimmune evasion by tumors. Immunol Today 1999; 20:158.

6. O’Garra A. Cytokines induce the development of functionally heterogeneous Thelper cell subsets. Immunity 1998; 8(3):275–283.

7. Mitchell MS, Harel W, Kan-Mitchell J, et al. Active specific immunotherapy ofmelanoma with allogeneic cell lysates. Rationale, results, and possible mecha-nisms of action. Ann NY Acad Sci 1993; 690:153–166.

8. Hartmann G, Weiner GJ, Krieg AM. CpG DNA: a potent signal for growth,activation, and maturation of human dendritic cells. Proc Natl Acad Sci USA1999; 96(16):9305–9310.

9. Armstrong AC, Eaton D, Ewing JC. Cellular vaccine therapy for cancer. ExpertRev Vaccines 2002; 1(3):303–316.

10. Connor J, Yatvin MB, Huang L. pH-sensitive liposomes: acid-induced liposomefusion. Proc Natl Acad Sci USA 1984; 81(6):1715–1718.

11. Reddy R, Zhou F, Nair S, Huang L, Rouse BT. In vivo cytotoxic T lymphocyteinduction with soluble proteins administered in liposomes. J Immunol 1992;148(5):1585–1589.

12. Bloom MB, Perry-Lalley D, Robbins PF, et al. Identification of tyrosinase-related protein 2 as a tumor rejection antigen for the B16 melanoma. J ExpMed 1997; 185(3):453–459.

13. Gyongyossy-Issa MI, Muller W, Devine DV. The covalent coupling ofArg-Gly-Asp-containing peptides to liposomes: purification and biochemicalfunction of the lipopeptide. Arch Biochem Biophys 1998; 353(1):101–108.

14. Mayer LD, Hope MJ, Cullis PR. Vesicles of variable sizes produced by a rapidextrusion procedure. Biochim Biophys Acta 1986; 858(1):161–168.

15. Szoka F, Papahadjopoulos D. Comparative properties and methods of prepara-tion of lipid vesicles (liposomes). Annu Rev Biophys Bioeng 1980; 9:467–508.

16. MacDonald RC, MacDonald RI, Menco BPM, Takeshita K, Subbarao NK,Hu LR. Small-volume extrusion apparatus for preparation of large, unilamellarvesicles. Biochim Biophys Acta 1991; 1061:297–303.

17. Chander R, Schreier H. Artificial viral envelopes containing recombinant humanimmunodeficiency virus (HIV) gp160. LifeSci 1992; 50:481–489.


18. Li WM, Dragowska WH, Bally MB, Schutze-Redelmeier MP. Effective induc-tion of CD8þ T-cell response using CpG oligodeoxynucleotides and HER-2/neu-derived peptide co-encapsulated in liposomes. Vaccine 2003; 21(23):3319–3329.

19. Storni T, Ruedl C, Schwarz K, Schwendener RA, Renner WA, Bachmann MF.Nonmethylated CG motifs packaged into virus-like particles induce protectivecytotoxic T cell responses in the absence of systemic side effects. J Immunol2004; 172(3):1777–1785.

20. Jerome V, Graser A, Muller R, Kontermann RE, Konur A. Cytotoxic Tlymphocytes responding to low dose TRP2 antigen are induced against B16melanoma by liposome-encapsulated TRP2 peptide and CpG DNA adjuvant.J Immunther 2006; 29(3):294–305.

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12

Influenza Virosomes as Adjuvants inCancer Immunotherapy

Reto Schumacher, Giulio C. Spagnoli, and Michel Adamina

Department of Surgery, Institute for Surgical Research and Hospital Management,University of Basel, Basel, Switzerland

INTRODUCTION

Immunopotentiating reconstituted influenza virosomes (IRIV) are spherical150-nm sized particles consisting of a phospholipid bilayer in which influenzavirus A/Singapore strain–derived hemagglutinin (HA) and neuraminidase(NA) are intercalated. As such, they resemble and mimic the influenza virusenvelope. The difference from conventional liposome formulations lies in theinclusion of the viral envelope proteins HA and NA as well as viral phospho-lipids. Especially, the inclusion of influenza virus HA provides IRIV withdelivery and immunogenic capacities. IRIV are licensed for human use asadjuvant in hepatitis A vaccination and as influenza subunit vaccine (1).

IRIV adjuvance in hepatitis A vaccination has been demonstratedas enhancement of humoral responses (1). There are only few adjuvantslicensed for human use and they predominantly enhance humoral immuneresponses (2–4). In view of chronic viral diseases, infections linked to intracel-lular pathogens, and cancer immunotherapy, there is a need for appropriateadjuvants that have the capability to enhance cellular immune responses, inparticular cytotoxic T-cell (CTL) responses (4,5). Here, we addressed IRIV-elicited immune responses and IRIV capacity to enhance CTL responses.

221

PRODUCTION OF IRIV

The production of IRIV includes solubilization and admixture of phospholip-ids and influenza HA followed by sterile filtration and detergent removal (6).Egg phosphatidylcholine (PC, 32 mg)(Lipoid GmbH, Ludwigshafen,Germany) and phosphatidylethanolamine (PE, 8 mg), (R. Berchtold, Biochem-isches Labor, Bern, Switzerland) are dissolved in 2.66 mL of PBS containing100 mM octaethylenglycol (PBS-OEG) (Fluka Chemicals, Switzerland). Theinfluenza A/Singapore HA is purified as described previously (7). A solutioncontaining 2 mg HA is centrifuged for 30 minutes at 100,000� g and the pelletis dissolved in 1.33 mL of PBS-OEG. The phospholipids and the HA solutionare mixed and sonicated for one minute. This mixture is then centrifuged forone hour at 100,000� g and the supernatant sterile filtered (0.22 mm). Viro-somes are then formed by detergent-removal using SM Bio-Beads (Bio Rad,Hercules, Pennsylvania, U.S.A.). Final influenza HA content is determinedby single radial diffusion (8). Control liposomes (L) are similarly produced,in the absence of influenza virus components.

IN VITRO CHARACTERIZATION OF IRIV

To characterize IRIV-elicited immune responses in vitro, we addressed cellproliferation and cytokine expression in peripheral blood mononuclearcell (PBMC) cultures, as well as IRIV effects on dendritic cells (DC). In allexperiments, PBMC were obtained from healthy donors and, if needed,further separated into different cell subsets. Finally, cells were cultured inthe presence or absence of IRIV as indicated.

IRIV Induce CD41 T-Cell Proliferation

Cell proliferation was addressed by conventional 3H-thymidine incorporationassays. Briefly, PBMCs were cultured in the presence of IRIV and liposomes,and in absence of any stimuli. On day 5 of culture, cells were pulsed with3H-thymidine for 18 hours, then harvested, and cell proliferation was deter-mined by tracer incorporation measurement.

Proliferation assays performed with PBMC cultures from a number ofhealthy donors demonstrated that IRIV indeed elicited cell proliferation in alltested cultures to an extent variable from donor to donor (Fig. 1A). Further pro-liferation assays with CD4þ T-cells or CD8þ T-cells in coculture with CD14þcells demonstrated that IRIV induce CD4þ T-cell proliferation, whereas noproliferation of CD8þ T-cells could be observed (Fig. 1C). Dissection of theCD4þ T-cell population into CD4þCD45ROþ and CD4þCD45RAþ cellsubsets pointed to CD4þCD45ROþ cells as proliferative responders(Fig. 1D). Importantly, culture of cord blood mononuclear cells in presenceof IRIV did not result in major cell proliferation (Fig. 1B), underliningantigen experience as prerequisite for IRIV-induced cell proliferation.

222 Schumacher et al.

Figure 1 (Continued on next page) Immunopotentiating reconstituted influenzavirosomes (IRIV) induced antigen specific proliferation of CD4þCD45ROþ cells.(A) Peripheral blood mononuclear cells (PBMC) from healthy donors (n¼ 3) were cul-tured in the absence of stimuli (Neg), in the presence of IRIV (V), and in the presence ofcontrol liposomes (L) at the indicated dilutions. Proliferation was measured on day 6 ofculture by 3H-thymidine incorporation. (B) Cord blood mononuclear cells from twodonors were cultured in the absence of stimuli (Neg) or in the presence of phytohaemag-glutinin (PHA), concanavalin A (ConA), IRIV (V) or L at the indicated concentrations.Proliferation was measured on day 3 of culture for PHA and ConA cultures and on day 6for IRIV and L stimulated cultures. (C) Purified CD4þ or CD8þ cells were coculturedwith autologous irradiated PBMC in the absence of stimuli (Neg) and in the presence ofIRIV (V) at the indicated concentrations. Proliferation was measured on day 6 of cultureby 3H-thymidine incorporation. (D) Purified CD4/CD45RAþ cells and CD4/CD45ROþcells were isolated from PBMC of one healthy donor and cocultured with autologous irra-diated PBMC in the presence of IRIV (V) or L at the indicated concentration. Proliferationwas measured on day 6 of culture by 3H-thymidine incorporation. Source: From Ref. 6.

Influenza Virosomes as Adjuvants in Cancer Immunotherapy 223

IRIV Induce a Cytokine Expression Profile Consistentwith a T Helper 1 Response

Because CD4þ T-cells can favor humoral, cellular, or even regulatoryresponses, we first addressed the nature of IRIV-elicited CD4þ T-cellresponses by investigating the expression of cytokines. Real time polymerasechain reaction (RT-PCR) (Fig. 2) and enzyme-linked immunosorbent assays(Fig. 3) demonstrate that IRIV induce gene expression and secretion ofinterferon-c, granulocyte monocyte-colony stimulating factor (GM-CSF),and tumor necrosis factor-alpha (TNF-a) in PBMC cultures, whereas nointerleukin-4 gene expression or cytokine production could be observed. Thispattern suggests that IRIV induce a CD4þ T helper 1 response. Phenotypingof CD4þ T-cells for the expression of CXCR3, a chemokine receptor charac-teristic for inflammatory and T helper 1 immune responses (10), furthercorroborates this indication. CD4/CXCR3 double staining demonstratedincreased percentages of CXCR3-expressing CD4þ T-cells in PBMC cultures



upon IRIV stimulation (Fig. 4C). Taken together, cytokine expression patternand increased percentages of CXCR3þ CD4þ T-cells upon IRIV stimulationof PBMC cultures strongly indicate that IRIV induce a CD4þ T helper 1response in vitro.

IRIV Induce Expression and Secretion of Chemokines

In addition to above-mentioned cytokines, IRIV were also shown to induceexpression and secretion of various chemokines, such as IP-10, MIG, andRantes (Schumacher R, unpublished). Secretion of chemokines is importantfor the recruitment of immune cells; however, the relevance of this in vitrofinding has not been addressed by migration assays or by in vivo studies so far.

IRIV-Induced Cytokine Secretion Favors Maturationof Dendritic Cells

We also addressed IRIV effects on antigen-presenting cells (APC). We inves-tigated IRIV effects on DC, some of the most effective professional APC.

Figure 2 Cytokine gene expression in immunopotentiating reconstituted influenzavirosomes (IRIV) stimulated peripheral blood mononuclear cells (PBMC). PBMCwere cultured in the presence or absence of IRIV. On days 1 and 2, culture cells wereharvested and total cellular RNA was extracted and reverse transcribed. The cDNAsthus obtained were tested in real time polymerase chain reaction assays in the presenceof primers specific for the indicated cytokine genes. Source: From Refs. 6 and 9.


First, we incubated immature dendritic cells (iDC) in the presence or absenceof IRIV and could not observe upregulation of defined maturation markers.However, iDC incubation with supernatants harvested from IRIV stimu-lated PBMC cultures resulted in upregulation of CD86, human leukocyteantigen (HLA)-class I molecules, and, in most cases, also of CD83 (6). Theseresults demonstrate that IRIV-induced cytokine secretion of PBMC indeedfavors maturation of DC.

IN VITRO EVALUATION OF IRIV CYTOTOXIC T-CELLADJUVANCE

The induction of CD4þ T helper 1 responses suggests that IRIV could pro-vide adjuvance to the generation of HLA class I–restricted CTL responses.Thus, we addressed the capacity of IRIV to enhance the induction of CTLspecific for influenza matrix (IM) 58–66 epitope and Melan-A/Mart-127–35

melanoma-associated epitope. Briefly, CD14-cells isolated from healthydonor’s peripheral blood were cocultured with autologous iDC in presenceof peptide and empty IRIV or in presence of peptide alone.

Figure 3 Cytokine secretion in immunopotentiating reconstituted influenza viro-somes (IRIV)-stimulated peripheral blood mononuclear cells (PBMC). PBMC froma healthy donor were cultured in the absence of stimuli (Neg) or in the presenceof IRIV (V, 1:50 diluted) or control liposomes (L, 1:50 diluted). On days 1, 2,and 4 supernatants were harvested and the concentrations of interferon-c (A),GM-CSF (B), TNF-a (C), and interleukin-4 (D) were determined by ELISA.Abbreviations: GM-CSF, granulocyte monocyte colony stimulating factor; TNF-a,tumor necrosis factor-a. Source: From Ref. 6.


IRIV-Enhanced Induction of HLA ClassI–Restricted CTL Responses

Tetramer staining showed increased percentages of IM58–66 (Fig. 5) orMelan-A/Mart-127–35 (Fig. 6) specific CD8þ cells upon stimulation withpeptide and IRIV (C) as compared to stimulation with peptide and lipo-somes (B) or with peptide alone (A). Limiting dilution analysis performedto address the frequency of IM58–66 specific CTL demonstrated that1/22000 CD8þ T-cells specifically recognized the target peptide in culturesstimulated with IM58–66 peptide in the presence of IRIV (6). In contrast, nocytotoxicity was detectable in cultures stimulated with IM58–66 alone.

IRIV-Mediated CTL Adjuvance Requires CD41 T cells

The demonstration of increased numbers of HLA class I–restricted CTL spe-cific for target peptides and of IRIV-induced CD4þ T-cell proliferation

Figure 4 Increased percentages of CXCR3þCD4þ T cells in immunopotentiatingreconstituted influenza virosomes (IRIV) stimulated peripheral blood mononuclearcells (PBMC). Healthy donors’ PBMC were cultured in the absence of stimuli (A),in the presence of liposomes [1:50 final dilution, (B)], or IRIV [1:50 final dilution,(C)]. After six days, culture cells were phenotyped for the expression of CXCR3and CD4 by phosphatidylethanolamine and fluorescein isothiocyanate labelledmonoclonal antibodies respectively. Source: From Ref. 6.


raised the question as to whether IRIV mediate CTL adjuvance throughCD4þ T-cell activation. We performed CTL induction experiments incocultures with irradiated and nonirradiated CD4þ cells. Briefly, CD14þcells, CD8þ cells, and CD4þ cells (irradiated or nonirradiated), isolatedfrom healthy donors’ peripheral blood, were cocultured in presence ofIM58–66 and IRIV, and in presence of IM58–66 alone. Tetramer staining

Figure 5 Immunopotentiating reconstituted influenza virosomes (IRIV) adjuvance oncytotoxic T-cell (CTL) induction. PBMC from a healthy donor were cultured in the pre-sence of influenza matrix (IM)58–66 (A), IM58–66 and control liposomes (B) or IM58–66

and IRIV (C). After a seven-day culture, percentages of IM58–66 specific CTL withincultured cells were quantified by HLA-A0201/IM58–66 phosphatidylethanolaminetetramer staining (fluorescence 2) and anti CD8 fluorescein isothiocyanate staining(fluorescence 1). CTL precursor frequencies detected in IM58–66 and IRIV stimulatedcultures within the same experiment are shown in (D). Source: From Ref. 6.


demonstrated IRIV CTL adjuvance in cocultures performed in the presenceof nonirradiated but not of irradiated CD4þ cells (Fig. 7), thereby suggestingthat IRIV mediated CTL adjuvance acts through CD4þ T-cell activation.

DISCUSSION

Adjuvants enhancing HLA class I–restricted CTL responses are especiallyneeded for treatment or prevention of chronic viral diseases and infectionslinked to intracellular pathogens, and for cancer immunotherapy. Amongthe very few adjuvants licensed for human use, we evaluated the capacityof IRIV to enhance HLA class I–restricted CTL responses in vitro. We ad-dressed IRIV-elicited immune responses and the induction of CTL specificto IM58–66 and Melan-A/Mart-127–35 epitopes. Proliferation assays, cytokineexpression studies, and phenotypes of CD4þ T-cells demonstrated that IRIV

Figure 6 Immunopotentiating reconstituted influenza virosomes (IRIV) adjuvanteffects in the induction of tumor associated antigen-specific cytotoxic T cell.CD14-negative cells from a healthy donor peripheral blood mononuclear cells werecocultured with autologous immature dendritic cells (iDC) in the presence of Melan-A/Mart-127–35, alone (A) or supplemented with either control liposomes (B) orIRIV (1:50, C). On day 7, culture cells were restimulated with Melan-A/MART-127–35

pulsed iDC and cultured for six further days [see ‘‘Materials and Methods’’]. On day 7after restimulation cells were stained with fluorescein isothiocyanate-conjugated anti-CD8andphosphatidylethanolamine-conjugatedHLA-A0201/Melan-A/MART-127–35

tetramers. Source: From Ref. 6.


induce a CD4þ T helper 1 response in PBMC culture. Furthermore, cyto-kines released upon IRIV stimulation of PBMC favored maturation of DC.

CTL induction experiments consistently demonstrate that IRIV indeedenhance induction of HLA class I–restricted CTL specific for IM58–66 andMelan-A/Mart-127–35 epitopes. CTL induction in presence of irradiated ornonirradiated CD4þ cells showed that IRIV CTL adjuvance requiresCD4þ T-cell activation. Remarkably, IRIV CTL adjuvance observed inour in vitro studies is solely due to IRIV immunogenicity and independentof peptide delivery and protection capacities, as peptides were not encapsu-lated in nor attached to IRIV. Further studies are warranted to clarifywhether and to what extent delivery, protection, and immunogenic capacitiesof IRIV synergize in CTL adjuvance. The fact that IRIV adjuvance wasobserved in relation to the tumor-associated epitope Melan-A/Mart-127–35

encourages further evaluation of IRIV as potential adjuvants in cancer

Figure 7 Immunopotentiating reconstituted influenza virosomes (IRIV) mediatedadjuvance in cytotoxic T-cell induction requires CD4þ T cells. CD8þ and CD14þcells were cultured in the presence of autologous intact or irradiated CD4þ cells.These cultures were stimulated with influenza matrix (IM)58–66 (1mg/mL) alone (A) orsupplemented with IRIV (1:50) (B). After seven days of incubation both cocultures wererestimulated with irradiated IM58–66 pulsed CD14þ cells and cultured for six furtherdays in the presence of interleukin-2 [see ‘‘Materials and Methods’’]. Six days afterrestimulation, cultures were stained with HLA-A0201/IM58–66 PE-specific tetramersand anti-CD8 fluorescein isothiocyanate monoclonal antibodies. Source: From Ref. 6.


immunotherapy. Finally, considering IRIV-mediated CTL adjuvance, therelevance of these in vitro findings should be validated by in vivo studies.

ACKNOWLEDGMENTS

This work was partially supported by a grant from the Kommission furTechnologie und Innovation (Berne, Switzerland) to Michael Heberer. TheIRIV used in this work were provided by courtesy of Mario Amacker andRinaldo Zurbriggen, from Pevion Biotech, Berne, Switzerland.

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2. Singh M, O’Hagan D. Advances in vaccine adjuvants. Nature Biotechnol 1999;17:1075.

3. Raychaudhuri S, Rock KL. Fully mobilizing host defense: building better vac-cines. Nat Biotechnol 1998; 16:1025.

4. Seder RE, Hill AVS. Vaccines against intracellular infections requiring cellularimmunity. Nature 2000; 406:793.

5. Moingeon P, Haensler J, Lindberg A. Towards the rational design of Th1 adju-vants. Vaccine 2001; 19:4363.

6. Schumacher R, Adamina A, Zurbriggen R, et al. Influenza virosomes enhanceclass I restricted CTL induction through CD4þ T cell activation. Vaccine2004; 22:714.

7. Skehel JJ, Schild GC. The polypeptide composition of influenza A viruses.Virology 1971; 44:396.

8. Guesdon JL, Avrameas S. An immunoenzymatic method for measuring low con-centrations of antigens by single radial diffusion. Immunochemistry 1974; 11:595.

9. Filgueira L, Zuber M, Juretic A, et al. Differential effects of interleukin-2 andCD3 triggering on cytokine gene transcription and secretion in cultured tumorinfiltrating lymphocytes. Cell Immunol 1993; 150:205.

10. Qin S, Rottman JB, Myers P, et al. The chemokine receptors CXCR3 and CCR5mark subsets of T cells associated with certain inflammatory reactions. J ClinInvest 1998; 101(4):746.


13

Liposome-Based DNA/Protein Vaccines:Procedures for Entrapment and

Immunization Studies

Gregory Gregoriadis

The School of Pharmacy, University of London, and Lipoxen PLC,London, U.K.

Andrew Bacon, Brenda McCormack, and Peter Laing

Lipoxen PLC, London, U.K.

Benoıt Frisch and Francis Schuber

Laboratoire de Chimie Bioorganique, Faculte de Pharmacie, Universite LouisPasteur, Strasbourg-Illkirch, and Chimie Enzymatique, Illkirch, France

INTRODUCTION

Intramuscular injection of naked plasmid DNA is known to elicit humoraland cell-mediated immune responses against the encoded antigen (1–3).Promotion of such immunity is the result of DNA uptake by muscle cells,leading to the expression and extracellular release of the antigen, which is thentaken up by antigen-presenting cells (APCs) (2,3). It is also believed (3) thatsome of the injected DNA is taken up directly by APCs, either locally at theinjection site or after its migration into the lymph nodes. There are two maindisadvantages of immunization with naked DNA. First, DNA enters only aminor fraction of muscle cells, which, at any rate, are not professional APCs.Second, injected naked DNA is exposed to and digested by deoxyribonucleasein the interstitial fluid, thus necessitating its use in relatively large quantities.

233

It is not unusual that injection into regenerating muscle (previously treatedwith muscle-damaging agents) is required in order to enhance transfectionand immunity. It has been proposed (1,4) that immunization by the use ofliposomes with entrapped DNA could circumvent the need for muscle invol-vement and facilitate (5) instead the uptake of the DNA by APCs infiltratingthe site of injection or in the lymphatics (where many liposomes will end up),at the same time protecting DNA from deoxynribonuclease attack (6). More-over, transfection of APCs with liposomal DNA and subsequent immuneresponses to the expressed antigen could be promoted by the judicious choiceof vesicle surface charge, size, and lipid composition, or by the coentrap-ment of DNA with plasmids expressing appropriate cytokines [e.g., inter-leukin 2 and interferon-c (IFN-c)] or immunostimulatory sequences.

Methods have been now developed by which plasmid DNA can bequantitatively entrapped into large (6) or small (7) neutral, anionic, or cationicliposomes that are capable of transfecting cells in vitro with varyingefficiency (6). Using this technology (1,4,8–10), immunization of inbred oroutbred mice by a variety of routes, including the oral route (10), with (cationic)liposomal DNA led to much greater humoral and cell-mediated (as evidencedby splenic IFN-c levels) immune response, including cytotoxic T lymphocyte(11) immune responses to the encoded antigen than those obtained with nakedDNA or DNA complexed to preformed similar liposomes. Entrapment ofDNA within the liposomes (as opposed to complexing) was verified (8) by gelelectrophoresis in the presence of the anionic sodium dodecyl sulphate (SDS):the latter competed with and replaced DNA from the surface of liposomes withwhich it was complexed. In contrast, DNA entrapped in identical liposomesremained with its carrier, presumably because SDS had no access to the DNA.

More recently, we have observed (12) that coentrapment of the plasmidDNA vaccine together with the protein vaccine it encodes in the same lipo-some by the use of the same technology leads, after only one injection, toeven stronger immune responses than those seen with liposomes containingthe DNA or the protein vaccine alone (section ‘‘Immunization Studies’’).This approach to genetic immunization mimics the way by which immunityis achieved in viral infections where both the viral DNA and the envelopeproteins it encodes contribute to the immune responses against the virus.Our technology has been further advanced by the finding (section ‘‘Immu-nization Studies’’) that coating liposomes containing the DNA and proteinvaccines with mannose residues (via the incorporation into the bilayers of amannosylated lipid) further potentiates immune responses to the vaccine,presumably by the targeting of such liposomes to the mannose receptorson the surface of APCs (13). Here, we describe the methodology for theincorporation of plasmid DNA and/or protein into liposomes of varyinglipid composition, vesicle size, and surface charge, as well as immunizationstudies with cationic liposomes (with or without incorporated mannosylatedlipid) coentrapping DNA and the protein it encodes.

234 Gregoriadis et al.

MATERIALS

Egg phosphatidylcholine (PC), distearoyl phosphatidylcholine, egg phos-phatidylethanolamine (PE), phosphatidic acid (PA), phosphatidyl glycerol(PG), and phosphatidylserine (PS) (more than 99% pure) were from LipoidGmbH, Ludwigshaten, Germany. Dioleoyl phosphatidylcholine (DOPE),stearylamine (SA), and cholesterol were from Sigma Chemical Co., Poole,Dorset, U.K. The sources of 1,2-bis (hexadecylcycloxy)-3-trimethylaminopropane (BisHOP), N-[1-(2,3-dioleyloxy) propyl]-N,N,N-triethylammo-nium (DOTMA), 1,2-dioleyloxy-3-(trimethylamonium propane) (DOTAP),1,2-dioleyl-3-dimethyl-ammonium propane (DODAP), and 3b(N,N,-dimethy-laminoethane)-carbamyl cholesterol (DC-CHOL) have been describedelsewhere (4,6,8,9). N-[2-(2-{2-[-(2,3-bis-octadec-9-enyloxy-propoxy)-ethoxy]-ethoxy}-ethoxy)-ethyl]-3-(3,4,5-trihydroxy-6-hydroxymethyl-tetrahydro-pyran-2-ylsulfanyl)-propionamide (DOGMann) was prepared synthetically (14).Sepharose (CL) 4B and polyethyleneglycol 6000 were obtained from Pharma-cia. Plasmid DNAs used were pRc/CMV HBS encoding the S (small) proteinof the hepatitis B virus surface antigen (HBsAg, subtype ayw) (4), pGL2-encoding luciferase (6), pRSVGH-encoding human growth hormone, pCMV4.65-encoding Mycobacterium leprosy protein (a gift from Dr R. Tasconof the National Institute of Medical Research), CMV 4.EGFP-encodingenhanced fluorescent green protein, VR 1020-encoding Schistosome protein,pCI-OVA encoding ovalumin and p1.17/SichHA encoding the hemaggluti-nin antigen of influenza virus (Sichuan strain) [obtained from the NationalInstitute of Biological Standards and Control (South Mimms)]. HepatitisB surface antigen (purity >95% by SDS-PAGE) from yeast was from Aldev-ron (Fargo, U.S.). A/Sichuan/2/87 influenza virus was inactivated withb-propiolactone. All other reagents were of analytical grade.

ENTRAPMENT OF PLASMID DNA AND PROTEIN VACCINESINTO LIPOSOMES BY THE DEHYDRATION–REHYDRATIONPROCEDURE

The dehydration–rehydration procedure is characterized by its mildnessand is thus compatible with most labile materials. The amounts of lipids andvaccine materials described below are typical for the preparations made foranimal work described here but could be scaled up or down depending onthe number of animals and the amount of antigens used.

Solutions

Solution A

PC (16 mmol) and DOPE (or PE) (8 mmol) are dissolved in about 2 to 5 mLchloroform (molar ratio 4:2). For charged liposomes, 4 mmol of PA, PG, or

Liposome-Based DNA/Protein Vaccines 235

PS (anionic) or 4 mmol of SA, BisHOP, DOTMA, DOTAP, DODAB, orDC-CHOL (cationic) are also added (molar ratio 4:2:1). For cationic man-nosylated liposomes, 4 mmol of DOGP-4 a Man was included at the molarratio of 4:2:1:1. Greater amounts of charged lipids can be added dependingon the amount of vesicle surface charge required.

Solution B

Up to 500 mg of plasmid DNA (for the amount of PC shown above) is dis-solved in 2 mL distilled water, or 10 mM sodium phosphate buffer (PB) ofpH 7.2 if needed. For liposomes containing both the plasmid DNA andthe vaccine protein it encodes (or only the protein), up to 1 mg of the proteinis included. The nature of buffer with respect to composition, pH, andmolarity can be varied as long as this does not interfere with liposome for-mation or DNA and protein entrapment yield. Amounts of added DNA andprotein can be increased proportionally to the total amount of lipid used.For cationic liposomes, the amount of added DNA can also be increasedby employing more cationic lipid.

Procedure Steps

Entrapment of plasmid DNA and/or protein into liposomes entails the pre-paration of a lipid film from which multilamellar vesicles and, eventually,small unilamellar vesicles (SUVs) are produced. SUVs are then mixed withthe plasmid DNA and/or protein destined for entrapment and dehydrated.The dry cake is subsequently broken up and rehydrated to generate multi-lamellar ‘‘dehydration–rehydration’’ vesicles (DRV) containing the plasmidDNA and/or protein. On centrifugation, liposome-entrapped vaccines areseparated from nonentrapped materials. When required, the DRV are redu-ced in size by microfluidization in the presence or absence of nonentrappedmaterials or by employing an alternative method (7) of DRV production,which utilizes sucrose (see below).

Preparation of Small Unilamellar Vesicles

The chloroform solution of lipids (Solution A) is placed in a 50-mL round-bottomed spherical Quick-fit flask. Following evaporation of the solvent ina rotary evaporator at about 37�C, a thin lipid film is formed on the walls ofthe flask. The film is flushed for about 60 seconds with oxygen-free nitrogen(N2) to ensure complete solvent removal and to replace air. Two millilitersof distilled water and a few glass beads are added into the flask, the stopperis replaced, and the flask shaken vigorously by hand or mechanically untilthe lipid film has been transformed into a milky suspension. This processis carried out above the liquid-crystalline transition temperature (Tc) ofthe phospholipid component of liposomes (>Tc) by prewarming the water


before its placement into a prewarmed flask. The suspension is allowed tostand at a temperature greater than the Tc for about one to two hours,whereupon multilamellar liposomes are formed. The milky suspension(without the glass beads) is then sonicated at a temperature greater thanthe Tc (with frequent intervals of rest) using a titanium probe slightly imm-ersed into the suspension, which is under N2 (achieved by the continuousdelivery of a gentle stream of N2 through thin plastic tubing). This step ismeant to produce a slightly opaque to clear suspension of SUVs of up to80 nm in diameter. The time required to produce SUVs varies, depending onthe amount of lipid used and the diameter of the probe. For the amountsof lipid mentioned above, a clear or slightly opaque suspension is usuallyobtained within up to four sonication cycles, each lasting 30 seconds with30-second rest intervals in between, using a probe of 0.75-inch diameter. Theprocess of sonication is considered successful when adjustment of the settingson the sonicator is such that the suspension is agitated vigorously. The soni-cated suspension of SUVs is centrifuged for two minutes at 3000 rpm toremove titanium fragments and the supernatant is allowed to rest at a tem-perature greater than the Tc for about one to two hours.

Preparation of Vaccine-Containing DRV

SUVs are mixed with Solution B, and rapidly frozen in liquid nitrogen whilethe flask is rotated and freeze-dried overnight under vacuum (< 0.1 Torr) ina freeze-dryer. If necessary, the suspension can be transferred into an alter-native Pyrex container prior to freezing and drying. To the freeze-driedmaterial, 0.1 mL H2O ( per 16 mmol of PC) prewarmed at a temperaturegreater than the Tc is added and the mixture is swirled vigorously at a tem-perature greater than the Tc. The volume of H2O added must be kept at aminimum, i.e., enough H2O to ensure complete hydration of the powderunder vigorous swirling. The sample is kept at a temperature greater thanthe Tc for about 30 minutes. The process is repeated with 0.1 mL H2O and,30 minutes later at a temperature greater than the Tc, with 0.8 mL PB( prewarmed at a temperature greater than the Tc). The sample is thenallowed to stand for about 30 minutes at a temperature greater than theTc. The liposomal suspension, now containing multilamellar DRV withentrapped and nonentrapped plasmid DNA and/or protein, is centrifugedat 40,000� g for 60 minutes (4�C). The pellet obtained (DNA- and/orprotein-containing DRV) is suspended in H2O (or PB) and centrifugedagain under the same conditions. The process is repeated at least once toremove the remaining nonentrapped material. The final pellet is suspendedto an appropriate volume (e.g., 2 mL) of H2O or PB. When the liposomalsuspension is destined for in vivo use (e.g., intramuscular or subcutaneousinjection), NaCl is added to a final concentration of 0.9%. The z-averagediameter of the suspended vesicles measured by photon correlation spectro-scopy (PCS) is about 600 to 700 nm (8).


Estimation of Vaccine Entrapment in DRV Liposomes

DNA and/or protein vaccine entrapment in DRV liposomes is monitored bymeasuring the vaccine in the suspended pellet and combined supernatants.The most convenient way to monitor DNA entrapment is by using radio-labelled (32P or 35S) DNA. For protein entrapment, the use of 125I-labelledprotein tracer is recommended. If a radiolabel is not available or cannot beused, appropriate quantitative techniques should be employed. To determineDNA or protein by such techniques, a sample of the liposome suspension ismixed with Triton X-100 (up to 5% final concentration) or, preferably, withisopropanol (1:1 volume ratio) so as to liberate the entrapped materials.However, if Triton X-100 or the solubilized liposomal lipids interfere withthe assay of the materials, liposomal lipids or the DNA must be extractedusing appropriate techniques (6). Entrapment values for protein and DNA,whether alone or coentrapped, range between about 20% to 80% (protein)and 30% to 100% (DNA) of the initial material depending on the DNAor protein used and, in the case of DNA, the presence or absence of cationiccharge. Values are highest for DNA when it is entrapped into cationic DRV(typical values in Table 1).

Generation of Vaccine-Containing Small Liposomes fromDRV by Microfluidization

This procedure and the ‘‘sucrose’’ method are used when vaccine-containingsmaller vesicles (down to about 100–200 nm z-average diameter) are required.

The DRV liposomal suspension obtained in section ‘‘Preparation ofVaccine-Containing Dehydration–Rehydration Vesicles’’ (prior to the sepa-ration of entrapped from nonentrapped vaccine) (‘‘unwashed liposomes’’) isdiluted to 10 mL with H2O and passed for a number of full cycles through aMicrofluidizer 110S (Microfluidics, Newton, Massachusetts, U.S.A.). The pres-sure gauge is set at 60 psi throughout the procedure to give a flow rate of35 mL/min. The number of cycles used depends on the vesicle size required(6,15) or the sensitivity of DNA when present. For instance, in the case ofpGL2, microfluidization for more than three cycles resulted in progressivesmearing of the DNA and failure to transfect cells in vitro (6). It is likelythat other plasmid DNAs will behave similarly on extensive microflui-dization. Microfluidization of the sample can also be carried out after theremoval of nonentrapped materials in section ‘‘Preparation of Vaccine-Containing Dehydration–Rehydration Vesicles’’ (‘‘washed liposomes’’),although vaccine retention in this case may be reduced: the presence ofunentrapped vaccine as mentioned above during microfluidization (a processthat destabilizes liposomes, which then reform as smaller vesicles) isexpected (6) to diminish vaccine leakage, perhaps by reducing the osmoticrupture of vesicles (15,16). However, with cationic DRV, DNA is unlikelyto leak significantly because it is associated with the cationic charges of


Tab

le1

Inco

rpo

rati

on

of

Pla

smid

DN

Aa

nd

Pro

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into

Lip

oso

mes

by

the

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dra

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nM

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rate

dp

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PC

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44

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55

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5.6

28

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C,

DO

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2.1

11

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C,

DO

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57

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PC

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E,

PS

c1

2.6

PC

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E,

PG

b5

3.5

PC

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E,

PG

c1

0.2

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SA

b7

4.8

d

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69

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(Co

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Tab

le1

Inco

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the bilayers. When the number of cycles required has been completed, themicrofluidized sample (about 10 mL) can, if needed, be reduced in volumeby placing it in dialysis tubing, which is then covered in a flat container withflakes of polyethyleneglycol 6000. Removal of excess H2O from the tubing isrelatively rapid and it is therefore essential that the sample be inspectedregularly. When the required volume has been reached, the sample is trea-ted for the separation of entrapped in free materials. This is carried out eitherby molecular sieve chromatography using a Sepharose CL 4B column or bycentrifugation as in the section ‘‘Preparation of Vaccine-ContainingDehydration–Rehydration Vesicles’’ (e.g., in the case of cationic liposomes).

Preparation of Vaccine-Containing Small Liposomesby the ‘‘Sucrose’’ Method

Quantitative entrapment of vaccines into small (up to about 200 nm diame-ter) liposomes in the absence of microfluidization (which can damage DNAand other labile materials when extensive) can be carried out by a novelone-step method (7) as follows: SUVs (e.g., cationic) prepared as in section‘‘Preparation of Small Unilamellar Vesicles’’ are mixed with sucrose to givea range of sucrose-to-lipid weight/weight ratio of 1.0 to 5.0 and the appro-priate amount of plasmid DNA (e.g., 10–500 mg) and/or protein (e.g., upto 1 mg). The mixture is then rapidly frozen and subjected to dehydrationby freeze-drying, followed by rehydration as in section ‘‘Preparation ofVaccine-Containing Dehydration–Rehydration Vesicles.’’

Estimation of Vaccine Entrapment in Small Liposomes

The content of vaccine within the small liposomes is estimated as in thesection ‘‘Estimation of Vaccine Entrapment in Dehydration–RehydrationVesicles Liposomes’’ for both microfluidized and ‘‘sucrose’’ liposomes andexpressed as percentage of DNA and/or protein in the mixture subjectedto freeze drying as in the section ‘‘Preparation of Vaccine-ContainingSmall Liposomes by the ‘‘Sucrose’’ Method’’ in the case of ‘‘sucrose’’ smallliposomes or in the original DRV preparation (obtained in the section‘‘Estimation of Vaccine Entrapment in DRV Liposomes’’) for microflui-dized liposomes. Vesicle size measurements are carried out by PCS asdescribed elsewhere (6,8,17). Liposomes can also be subjected to micro-electrophoresis in a Zetasizer to determine their zeta potential. This isoften required to determine the net surface charge of DNA-containingcationic liposomes.

IMMUNIZATION STUDIES

In two separate immunization studies, the liposome-based codelivery app-roach (i.e., liposomes coentrapping plasmid DNA and the protein encodedby the DNA) was tested in female Balb/c mice in groups of four to six


animals. In the first study, mice were injected subcutaneously with a singledose (0.2 mL) of DRV liposomes (mixed with Alum) composed of PC,DOPE, and DOTAP (molar ratios 4:2:1) containing 10 mg p1.17/SchHADNA encoding the hemagglutinin (HA) influenza antigen (A/Sichuan/2/87) and 1.5 mg killed influenza virus (A/Sichuan/2/87), or with the sameliposomes containing either 10 mg p1.17/SichHA DNA or 1.5 mg killed influ-enza virus. Animals were bled at time intervals and sera assayed by enzymelinked immunosorbent assay (ELISA) (4) for total anti-HA IgG. Figure 1shows that immunization with liposomes coentrapping the DNA and thekilled influenza virus led to much greater end point titres than liposomesentrapping either the DNA or the virus alone (1000-fold and 100-foldincrease respectively). Such a response persisted for at least 120 days.

In the second study (Fig. 2), mice were given a single subcutaneousinjection of small DRV liposomes prepared in the presence of sucrose (3:1sucrose-to-lipid mass ratio; see text) from PC, DOPE, DOTAP, andDOGMann (molar ratios 4:2:1:1) and containing 10 mg pRc/CMV HBSDNA encoding the HBsAg and 0.5 mg HBsAg, or a single subcutaneousinjection of HBsAg in the form of Engerix

1

, a licensed commercial pre-paration adsorbed to Alum. Results (Fig. 2) clearly demonstrate a superioranti-IgG HBsAg response for the liposomal formulation (a nearly eightfoldincrease of anti-HBsAg titres measured by ELISA), which persisted for atleast 90 days. As with the influenza results (Fig. 1), immunization with

Figure 1 Anti-HA IgG titres (�SD) (Y-axis) in mice immunized with a single sub-cutaneous injection of (Alum-adsorbed) DRV liposomes composed of PC, DOPE,and DOTAP (molar ratios 4:2:1) and containing p1.17/SichHA DNA and killedinfluenza virus (&), killed influenza virus only (~), or DNA only (!). For other de-tails see the text. Abbreviations: HA, hemagglutinin; DRV, dehydration–rehydrationvesicles; PC, phosphatidylcholine; DOPE, dioleoyl phosphatidylcholine; DOTAP,1,2-dioleyloxy-3-(trimethylamonium propane).


liposomes containing the DNA or the HBsAg antigen alone led to muchlower responses (data not shown).

It can therefore be concluded that immunization with liposomes con-taining both DNA and the encoded antigen leads to superior immuneresponses when compared with liposomes entrapping the DNA or proteinvaccine alone.

REFERENCES

1. Gregoriadis G. Genetic vaccines: strategies for optimization. Pharm Res 1998;15:661–670.

2. Davis HL, Whalen RG, Demeneix BA. Direct gene transfer in skeletal musclein vivo: factors influencing efficiency of transfer and stability of expression.Hum Gene Ther 1993; 4:151–156.

3. Lewis PJ, Babiuk LA. DNA vaccines: A Review. Adv Virus Res 1999; 54:129–188.

4. Gregoriadis G, Saffie R, de Souza JB. Liposome-mediated DNA vaccination.FEBS Lett 1997; 402:107–110.

5. Gregoriadis G. Engineering targeted liposomes: progress and problems. TrendsBiotechnol 1995; 13:527–537.

6. Gregoriadis G, Saffie R, Hart SL. High yield incorporation of plasmid DNAwithin liposomes: effect on DNA integrity and transfection efficiency. J DrugTarget 1996; 3:469–475.

Figure 2 Anti-HBsAg IgG titres (�SD) (Y-axis) in mice immunized with a singlesubcutaneous injection of small DRV liposomes composed of PC, DOPE, DOTAP,and DOGMann (molar ratios 4:2:1:1) containing pRc/CMV HBS DNA and theencoded antigen HBsAg (&) or with HBsAg in the form of Engerix1 (�). For otherdetails, see the text. Abbreviations: DRV, dehydration–rehydration vesicles; PC,phosphatidylcholine; DOPE, dioleoyl phosphatidylcholine; DOTAP, 1,2-dioleyloxy-3-(trimethylamonium propane).


7. Zadi B, Gregoriadis G. A novel method for high-yield entrapment of solutes intosmall liposomes. J Liposome Res 2000; 10:73–80.

8. Perrie Y, Gregoriadis G. Liposome-entrapped plasmid DNA: characterizationstudies. Biochim Biophys Acta 2000; 1475:125–132.

9. Perrie Y, Frederik PM, Gregoriadis G. Liposome-mediated DNA vaccination:the effect of vesicle composition. Vaccine 2001; 19:3301–3310.

10. Perrie Y, Obrenovic M, McCarthy D, Gregoriadis G. Liposome (Lipodine J)mediated DNA vaccination by the oral route. J Liposome Res 2002; 12:185–197.

11. Bacon A, Caparros-Wanderley W, Zadi B, Gregoriadis G. Induction of a cyto-toxic T lymphocyte (CTL) response to plasmid DNA delivered by Lipodine.J Liposome Res 2002; 12:173–183.

12. Bacon A, Hreczuk-Hirst DH, McCormack B, et al. In: Proceedings of 30thAnnual Meeting of the Controlled Release Society, Abstract 441, 2003:884.

13. Garcon N, Gregoriadis G, Taylor M, Summerfield J. Targeted immunoadjuvantaction of tetanus toxoid-containing liposomes coated with mannosylated albu-min. Immunology 1988; 64:743–745.

14. Espuelas S, Haller P, Schuber F, Frisch B. Synthesis of an amphiplilic tetra-antennary mannosyl conjugate and incorporation into liposome carriers. BioorgMed Chem Lett 2003; 13:2557–2560.

15. Gregoriadis G, da Silva H, Florence AT. A procedure for the efficient entrap-ment of drugs in dehydration-rehydration liposomes (DRV). Int J Pharm1990; 65:235–242.

16. Kirby C, Gregoriadis G. Dehydration-rehydration vesicles (DRV): a newmethod for high yield drug entrapment in liposomes. Biotechnology 1984;2:979–984.

17. Skalko N, Bouwstra J, Spies F, Gregoriadis G. The effect of microfluidization ofprotein-coated liposomes on protein distribution on the surface of generatedsmall vesicles. Biochim Biophys Acta 1996; 1301:249–254.


14

Liposome-Polycation-DNA: A NonviralGene Vector Turned into a Potent

Vaccine Carrier

Lisa M. Shollenberger

University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, U.S.A.

Leaf Huang

University of Pittsburgh School of Pharmacy, Pittsburgh, Pennsylvania, U.S.A.

LIPOSOME-POLYCATION-DNA COMPLEXES

Liposome-polycation-DNA (LPD) nanoparticles (1) are formed by sponta-neous rearrangement of a lipid shell around a polycation-condensed bacterialplasmid DNA core to form a virus-like structure (2). The LPD complexesconsist of liposomes that are either made of cationic (LPDI) or anionic(LPDII) lipids and are sometimes referred to as lipopolyplexes, a broadercategory that also includes other lipid-based vectors (2).

The weakly immunogenic protamine sulfate USP (1) condenses DNAto form a toroid structure of super-coiled DNA about 50 nm in diameter (2).The DNA in this form or in the preformed LPDI complex cannot be dis-placed from the protamine by polycations such as spermidine and histonesor by other nucleic acids like genomic DNA (2). DNA in this toroid struc-ture is transcriptionally inactive and this conformation allows for protectionof DNA from enzymatic degradation by nucleases and other environmentalassaults such as mechanical stress (1,2). After the liposome surrounds thetoroid, the resulting homogenous LPDI nanoparticles are slightly less than

245

100 nm in diameter (1). These LPDI nanoparticles are stable, meaning theycan be stored at 4�C for four weeks with no loss of activity (3) or lyophilizedand stored at room temperature without significant change in particle size(no aggregation) or loss of transfection efficiency (2).

Initial vaccination studies with LPDI nanoparticles were complet-ed using liposomes prepared with both 1,2-dioleyltriammonium propane(DOTAP) and cholesterol. After it was determined that cholesterol playedonly a small structural role and was not necessary for activity, the liposomeswere then prepared using only DOTAP to become an LPDI type of formula-tion. Regardless of the lipid used, the ratio of cationic lipid, polycation, andDNA must be maintained to have all properties associated with LPDIparticles (2).

Uptake and Delivery

Uptake of LPDI nanoparticles by cells occurs mainly through the endocyticpathway (2). The small, homogenous LPDI particles are taken up mucheasier through the endocytic pathway than other liposome/DNA complexessuch as lipoplex particles, which are larger and heterogeneous (2). Studies oflipoplex particles in COS and HeLa cells show that the lipoplex enters theendocytic compartment and moves to the perinuclear region within 24 hoursafter internalization, but does not fuse with lysosomes (4). Contrastingly,other groups show that endocytic vesicles directly fuse with lysosomes afterinternalization of the lipoplex particles (4). Occasionally, the lipoplex parti-cles can escape the early endosomes and be released into the cytoplasm (4).Unless DOPE is incorporated into the liposome, it is likely that early endo-some escape of the lipoplex occurs mainly through local destabilizationeffects of the cationic liposomes (4).

When discussing the ability of nucleic acid to enter the nucleus, size isthe most important consideration (4). Oligonucleotides less than 100 basepairs in length can freely diffuse into the nucleus and oligos of 20 to 30base pairs in length will accumulate in the nucleus when administered byeither cationic lipoplex or cytoplasmic injection (5).

Transfection

LPDI nanoparticles, with their net positive charge, have the ability to trans-fect all cell types because of the negative charges on cell surfaces (2) butshould, however, be pure for optimal activity (4). The greatest limitationof LPDI nanoparticles is their sensitivity to serum due to the net positivecharge of the particle (6). Because DNA in the LPDI particles is completelyprotected, the relative amount of active DNA delivered is higher for theseparticles than for other lipid-based transfection methods (2). The increasein DNA delivered by LPDI particles causes two outcomes distinct from lipo-plex DNA delivery, including a higher toxicity as well as higher transfection

246 Shollenberger and Huang

efficiency (1). Transfection efficiency is charge ratio (þ/�) dependent andagain must be ideal for optimum gene expression (2). These LPDI nanoparticleswere originally designed for gene therapy applications as a liposome-basedDNA delivery system (1), but are now being exploited as potent peptidevaccine carriers.

LPDI AND THE IMMUNE SYSTEM

Bacterial DNA (unmethylated CpG motifs) is the ligand for toll-likereceptor 9 (TLR9) and subsequent tumor necrosis factor (TNF)-alpha pro-duction is indicative of this TLR signaling in professional antigen presentingcells (APCs) (7). Indeed, DNA alone induces the production of TNF-alphain both dendritic cell line DC2.4 or bone marrow–derived dendritic cells(BMDC), but this level is not as high as when LPDI particles are used (7).As shown by in vitro experiments, protamine in the LPDI nanoparticlesacts as a structural component only and is not involved in immunostimula-tory activity (8). Similarly, it has been shown that cationic liposomes do notinduce TNF-alpha production in either DC2.4 or BMDC, but, interestingly,the cationic liposomes do, in fact, activate both these types of murine den-dritic cells (7). Both the DNA and cationic liposome components of LPDInanoparticles are necessary for stimulating the expression of cell surfacemarkers CD80 and CD86 on DC2.4, which is indicative of dendritic cellmaturation and activation [(9), Han SJ, et al. Subcutaneous antigen loadingof dendritic cells by liposome-protamine-DNA (LPD) nanoparticles resultsin their activation and induction of specific antitumor immune response(unpublished)]. Moreover, to fully activate the dendritic cells through TLRsignaling, both DNA and cationic liposomes are required (7). Althoughwork is under way, no information is currently available about the modeof action of cationic liposomes in immunostimulation. It is most likely thatcationic liposomes stimulate dendritic cells through an NFjB-independentpathway (10) considering the liposomes themselves do not induceTNF-alpha when administered to dendritic cells.

Vaccines and Adjuvants

Successful vaccines have several important properties such as safety, effec-tiveness, low cost per dose, and ease of preparation. Vaccines, whetherpeptide, protein, or DNA, have limited potency without codelivery withan adjuvant and/or a specialized delivery system (11). There is currentlya lack of safe, nontoxic, effective, Food and Drug Administration (FDA)-approved vaccine adjuvants capable of stimulating cellular (Th1) immunity (12).Most potent immune activators are also toxic at relatively low doses andcannot, therefore, be successfully used as adjuvants (12).

Liposome-Polycation-DNA 247

In order to be an effective vaccine adjuvant or delivery system, antigenmust be presented in the lymph node (11). This process involves the activa-tion and migration of professional APCs such as dendritic cells, as well asthe appropriate presentation of the antigen of interest to primary T-cellsin that lymph tissue (7). Dendritic cells can take up antigen in the peripheryand migrate to the lymph node upon activation or the antigen can be deliv-ered to the lymph node and dendritic cells activated there. When injectedsubcutaneously in the mouse hind footpad, LPDI can move to local lymphnode [Han SJ, et al. Subcutaneous antigen loading of dendritic cells by liposome-protamine-DNA (LPD) nanoparticles results in their activation and inductionof specific antitumor immune response (unpublished)] and is better than otherlipid particles at activating dendritic cells in that tissue (7). LPDI nanoparticleshave several unique properties that confer the strong adjuvanticity includingthe ability to move to the local draining lymph node and to activate dendriticcells in that lymph node (7).

LPDI nanoparticles have several features that make it an ideal vaccineadjuvant as well as an antigen carrier. When administered intravenously,cationic liposome-based nonviral vectors, including LPDI, induce a systemic,Th1-like innate immune response (5). The immune-activating, adjuvantproperties of LPDI nanoparticles is especially suitable for delivering tumor-specific antigens in the context of vaccination (13). In our model, theunmethylated (CpG) DNA in the LPDI complex induces the productionof Th1 cytokines and stimulates antitumor natural killer (NK) activity(13). When the LPDI nanoparticles are administered systemically in largedoses, a strong and rapid Th1 cytokine response [TNF-alpha, interferon(IFN)-gamma, interleukin (IL)-12] is initiated and this cytokine productionis related to the tumoristatic effects (13). It is thought that NK cells activatedby proinflammatory cytokines may kill some tumor cells in a nonspecificmanner. Once tumor cells are killed, the cellular debris can be taken up byAPCs to elicit further killing via a specific cytotoxic T lymphocyte (CTL)response and, in fact, depletion of NK cells in vivo abolishes the nonspecifictumoristatic effect seen by LPDI treatment (7,13). As stated previously,recent research from our laboratory demonstrates that both the cationicliposome and DNA components are required for full immunostimulatoryactivity by LPDI particles, including induction of proinflammatory cytokinesand production of costimulatory molecules by dendritic cells (7). When bothof these responses share spatial and temporal location, they direct anacquired, tumor-specific CTL response, which, in conjunction with the otheractivities, can inhibit the growth of established tumors in mice (12). In light ofthese interesting aspects of the LPDI particles, one would expect the applica-tion of these particles as FDA-approved adjuvants. Potential issues withmanufacturing and quality control, as well as particle stability, have beenimplicated as reasons for the procrastinated registration of liposome-basedadjuvants for human use (14).


LPDI as a Peptide Vaccine Carrier

In addition to having an overabundance of several self-antigens, tumor cellsexpress unique antigens, which can be recognized by the host immune sys-tem, provided that the immune system is simultaneously activated. Withoutthis activation, the immune system will become tolerized to the unique anti-gens known as tumor-associated antigens (TAAs), which are usually smallpeptides of 8 to 10 amino acids. The potential exists for the eradication ofcancers by injection of TAAs and the subsequent immune response. Indeed,there have been many tumor-reactive CTLs identified that recognize specificTAAs (15).

As stated previously, the potency of peptide vaccines alone is poor, butantigenic peptides derived from TAAs will be a powerful tool in the preven-tion and treatment of cancer (11). A strong nonspecific antitumor responseis elicited when LPDI containing noncoding DNA is injected intravenously(16) due to the induction of potent Th1 cytokines that stimulate the tumorlytic activity of NK cells and CTLs. It has been established that LPDIparticles can efficiently and effectively deliver TAAs in the context of a pep-tide vaccine. As is true with the encapsulated DNA of the LPDI particle,encapsulated peptide is also protected from extracellular assaults such asproteases, which would normally degrade the antigenic peptide during vacci-nation delivery and is thereby efficiently delivered to APCs for presentationon major histocompatibility complex (MHC) molecules. Peptide vaccines alsohave other advantages, including chemical stability and definition, becauseonly the most important epitope needs to be used (17). DC vaccination issometimes used to overcome obstacles associated with peptide vaccinationsuch as the degradation of peptide when not encapsulated in a carrier suchas the LPDI nanoparticle. In this strategy, dendritic cells from a patient areremoved for receipt of peptide ex vivo and are then reintroduced into thebody. Time of MHCI presentation is increased and CTL response is enhancedwhen peptide is delivered to the cytoplasm during DC vaccination (18).

Our laboratory uses a mouse model to study preventative and thera-peutic vaccination strategies for human papilloma virus (HPV)-associatedtumors. For preventative vaccination studies, six- to eight-week-old femaleC57BL/6 mice are injected subcutaneously with LPDI on days 0 and 5 andare challenged on day 10 with a subcutaneous injection of 105 E7-expressingTC-1 tumor cells with tumor growth measured three times weekly.

Using this murine tumor model, our laboratory has demonstrated theability of LPDI particles to efficiently deliver TAA and thereby generate pro-tective immunity as well as elimination of established tumors. TC-1 murinetumor cells expressing HPV E7 are used to generate tumors in naive mice aswell as to challenge mice vaccine against the E7 peptide. Indeed, mice immu-nized with LPDI containing a nine-amino acid peptide corresponding to thedominant MHC Class I epitope of E7 show strong antigen-specific antitumor


responses against TC-1 tumor establishment including an E7-specific CTL res-ponse (11). Naive mice immunized with E7 using LPDI particles generated astronger CTL response than E7 immunization using other liposome/peptide immunization strategies (19). Mice with TC-1 tumors that are sub-sequently immunized with LPDI/E7 cause complete tumor regression(7,11). Moreover, mice immunized intravenously or subcutaneously withLPDI/E7 could not establish E7-expressing TC-1 tumors up to 50 daysafter vaccination (11), whereas treatment with any combination other thanLPDI/E7 causes TC-1 tumors to grow rapidly in mice (20–22).

To review, in an experimental mouse model, LPDI/E7 vaccination bothprevents the establishment of metastatic E7-expressing tumors in naive micethrough an induced E7-specific T-cell immune response and, in mice with pre-viously established E7-expressing tumors, causes tumor regression with onesubcutaneous injection of LPDI/E7 [Han SJ, et al. Subcutaneous antigenloading of dendritic cells by liposome-protamine-DNA (LPD) nanoparticlesresults in their activation and induction of specific antitumor immune response(unpublished)]. A robust immune response follows administration of LPDI/peptide particles, which can be used as either a preventative or therapeuticcancer vaccination strategy due to the ability of the particles to prevent andeliminate tumors, respectively, in mouse models.

SUMMARY

LPDI nanoparticles are homogenous, self-forming spheres between 100 and200 nm in diameter that are formed from the spontaneous rearrangement ofa lipid bilayer around a polycation condensed DNA core. The LPDI parti-cles (lipopolyplexes) have benefits over lipoplexes, which are composed ofliposomes and DNA. Homogenous particles are formed during preparationand thus allow a more consistent production of particles, as required bythe FDA for clinical use. The LPDI particles also have a lower toxicityassociated with them as opposed to lipoplexes, which can generate severe sys-temic inflammatory responses, most likely to the increased DNA content onthe surface of the particles. The internalization of DNA inside the LPDI alsohas a benefit of DNA protection. The DNA is not nearly as accessible tonuclease attack and mechanical stress. Therefore, a lower quantity ofDNA is used because it is protected inside of the LPDI for delivery.

The LPDI can cause release of Th1 cytokines, most notably TNF-alpha, IFN-gamma, and IL-12 but to a much lower degree than lipoplexes.Also, LPDI nanoparticles can activate the professional APCs, macrophages,and dendritic cells.

It has been demonstrated that the unmethylated CpG motifs in theDNA activate DCs through TLR9, but association with the cationic liposomeis required for full activation of these cells. Because the liposomes alone donot generate a cytokine response, we can suppose there is a second receptor


for the cationic liposome that does not signal through the TLR signalingpathway as the TLRs do. Because the LPDI particles themselves can activatethe immune system, they can be used as an adjuvant with a peptide vaccine.

LPDI nanoparticles hold great promise in the area of vaccination.These particles can be designed in such a way as to target specific cells inthe body. Without any modifications, these particles, when administeredsystemically, localize to several organs, most notably the lungs, liver, andspleen. The particles are also readily taken up by APCs, which are also vitalin the development of an adaptive immune response. All vaccination strate-gies require the induction of an adaptive immune response. In addition,valuable characteristics of vaccines include low cost per dose, stability, easeof administration, and a potent and long-term effect. Some of the morevaluable aspects of LPDI nanoparticles of a nonviral vector for vaccinationinclude the ability to produce homogenous particles on a large scale with arelatively low cost per dose. The LPDI-based vaccine can be a cheap vaccinesolution that is shelf stable for indefinite periods of time when lyophilized.

REFERENCES

1. Li S, Huang L. In vivo gene transfer via intravenous administration of cationiclipid-protamine-DNA (LPD) complexes. Gene Ther 1997; 4:891.

2. Li S, Huang L. Functional polymorphism of liposomal gene delivery vectors:lipoplex and lipopolyplex. In: Janoff AS, ed. Liposomes: Rational Design.New York: Marcel Dekker, Inc., 1999:89.

3. Li B, et al. Lyophilization of cationic lipid-protamine-DNA (LPD) complexes.J Pharm Sci 2000; 89:355.

4. Miller AD. Cationic liposomes for gene therapy. Angew Chem Int Ed Engl 1998;37:1768.

5. Whitmore M, Li S, Huang L. LPD lipopolyplex initiates a potent cytokineresponse and inhibits tumor growth. Gene Ther 1999; 6:1867.

6. Li S, et al. Characterization of cationic lipid-protamine-DNA (LPD) complexesfor intravenous gene delivery. Gene Ther 1998; 5:930.

7. Cui Z, et al. Immunostimulation mechanism of LPD nanoparticle as a vaccinecarrier. Mol Pharm 2005; 2:22.

8. Muzio M, et al. IRAK (Pelle) family member IRAK-2 and MyD88 as proximalmediators of IL-1 signaling. Science 1997; 278:1612.

9. Hemmi H, et al. A Toll-like receptor recognizes bacterial DNA. Nature 2000;408:740.

10. Cui Z, Han SJ, Huang L. Coating of mannan on LPD particles containingHPV E7 peptide significantly enhances immunity against HPV-positive tumor.Pharm Res 2004; 21:1018.

11. Dileo J, et al. Lipid-protamine-DNA-mediated antigen delivery to antigenpresenting cells results in enhanced anti-tumor immune responses. Mol Ther2003; 7:640.

12. Petrovsky N, Aguilar JC. Vaccine adjuvants: current state and future trends.Immunol Cell Biol 2004; 82:488.


13. Whitmore MM, et al. Systemic administration of LPD prepared with CpGoligonucleotides inhibits the growth of established pulmonary metastases bystimulating innate and acquired antitumor immune responses. Cancer ImmunolImmunother 2001; 50:503.

14. Plotkin SA. Vaccines, vaccination, and vaccinology. J Infect Dis 2003; 187:1349.15. Parmiani N, et al. T-cell response to unique and shared antigens and vaccination

of cancer patients. Cancer Immunol 2002; 2:6.16. van Driel WJ, et al. Vaccination with HPV16 peptides of patients with advanced

cervical carcinoma: clinical evaluation of a phase I-II trial. Eur J Cancer 1999;35:946.

17. Wang RF, Wang HY. Enhancement of antitumor immunity by prolonging anti-gen presentation on dendritic cells. Nat Biotechnol 2002; 20:149.

18. Dow SW, et al. Lipid-DNA complexes induce potent activation of innateimmune responses and antitumor activity when administered intravenously.J Immunol 1999; 163:1552.

19. Tan Y, et al. The inhibitory role of CpG immunostimulatory motifs in cationiclipid vector-mediated transgene expression in vivo. Hum Gene Ther 1999;10:2153.

20. Ochsenbein AF, et al. Immune surveillance against a solid tumor fails becauseof immunological ignorance. Proc Natl Acad Sci USA 1999; 96:2233.

21. Zinkernagel RM, et al. Antigen localisation regulates immune responses in adose- and time-dependent fashion: a geographical view of immune reactivity.Immunol Rev 1997; 156:199.

22. De Smedt T, et al. Regulation of dendritic cell numbers and maturation bylipopolysaccharide in vivo. J Exp Med 1996; 184:1413.


15

Automated Screening of Cationic LipidFormulations for Transfection

Ulrich Massing and Peter Jantscheff

Department of Clinical Research, Tumor Biology Center,Freiburg, Germany

INTRODUCTION

Many of the most serious diseases are caused by gene defects. A very impor-tant example is cancer, having its origin in a cascade of acquired gene defects(1,2). Others are the result of inherited, mostly single-gene defects, and aprominent example is cystic fibrosis (3). As first suggested in the 1970s byFriedmann and Roblin (4), it should be possible to correct the inheritedor acquired gene defects by replacing the defective gene and/or by overcom-ing the malfunction by introducing a correct gene. Today—more than 30years later—gene therapy, the use of therapeutic genes as drugs, is a verypromising approach. During the last one and a half decades, more than1,000 gene therapy studies have been performed (5). But at this point, genetherapy is still far from being a standard therapy.

The problems averting gene therapy to become a standard therapy arediverse, but one of the most important obstacles is the lack of techniquesthat allow the transfer of genes into cells (transfection) in a biologically safe,nontoxic, selective, and efficient way. This problem of ‘‘drug delivery,’’where the drug is a gene, is particularly challenging for genes, which arelarge and complex, and which require targeting to the nuclei of cells. Mostof the vectors currently in use for clinical gene therapy trials are based onattenuated or modified versions of viruses (70.4%) (5,6). Although efficient,

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there are serious safety problems associated with viral vectors including pos-sible activation of the patient’s immune system, risk of infection with traces ofthe wild-type virus, or insertion mutagenesis (7,8). For example, the risk of asevere immune response to the attenuated virus particles was dramaticallydemonstrated by the death of an 18-year-old man in an adenovirus-based genetherapy study in 1999 (9,10). Unfortunately, also the risk of insertional muta-genesis and subsequent oncogene activation became real (11). In a recentclinical trial children suffering from X-linked severe combined immunodefi-ciency, retroviruses were used for gene transfer, and 2 out of 10 patients devel-oped T-cell leukemia as a consequence of this treatment (12).

A promising alternative to viral gene transfer is lipofection, the transferof the negatively charged DNA material by cationic lipids (13–18). There isno restriction on the size of the therapeutic gene and no risk of immunogeni-city or infection (19). Thus, lipofection in vivo can be principally performedseveral times (20). Furthermore, cationic lipids can be synthesized in largequantities with relatively little effort.

However, in contrast to viral gene transfer, the efficiency of lipofectionis still limited (21–23) and the finer features of the mechanism of lipofectionare only partly understood. Thus, only 8.6% of all gene therapy studies per-formed so far are based on lipofection (5). This percentage is decreased from13% in 2001, clearly indicating that not the safety, but transfection efficiency(TE), is the most urgent problem in gene therapy. An overview of gene ther-apy studies based on lipofection as well as a discussion of lipoplex behavior invivo is given by Audouy et al. (24).

Mechanistical Aspects of Lipofection

Despite the fact that many different cationic lipids have been synthesizedand tested for transfection (25–34), relatively few systematic structure–activity–TE-relationship studies have been performed (35–39). As aresult, no general relationship between chemical structure and TE could bedrawn from these studies. One reason for this is that the chemical structureof a cationic lipid is not directly responsible for TE. TE rather depends on thebiophysical characteristics of the cationic lipid aggregate (e.g., liposomes andlipoplexes), which, for its part, is dependent on the chemical structure of thelipids. In a previous study with analogs of the transfection lipid N-[1-(2,3-dioleoyloxy) propyl]-N,N,N-trimethylammoniumchloride (DOTAP) (40) whichdiffer in their nonpolar hydrocarbon chains, it could be shown that the TEstrongly depended on the biophysical properties of the resulting liposomesand lipoplexes (35). Minimal alterations of biophysical properties by using lipidswith different hydrocarbon chains or by mixing the lipid with different neutralhelper lipids could completely allow or prevent transfection.

As indicated in Figure 1, the process of lipofection can be divided intoindependent steps: (i) preparation of a lipofection reagent, (ii) formation oflipoplexes, and (iii) the transfection itself.

254 Massing and Jantscheff

Preparation of a Lipofection Reagent

Cationic lipids cannot be dissolved in water and form aggregates in aqueoussolution, such as bilayers. To prepare a homogeneous reagent, in most casesliposomes were made from cationic lipids in a first step. When it is notpossible to form stable lipid bilayers (i.e., liposomes) using a single lipid,then it may be necessary to combine the cationic lipid with one or moreso-called helper lipids like cholesterol (Chol) (41) or 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE) (42).

Formation of Lipid/DNA Complexes (Lipoplexes)

Cationic lipids have the advantage of directly interacting with the negativelycharged DNA, thereby forming lipid-DNA complexes. For this, the cationicliposomes are added in excess to the DNA to be introduced into the cells.A decisive factor for the shape and structure of the resultant lipoplexesand, therefore, the success of transfection is the ratio of lipid/DNA. Mixingexperiments and electron microscopy have revealed that lipid/DNA chargeratios greater than 1 result in positively charged lipoplexes in which the

Figure 1 The principles and variant parameters of lipofection. (i) Preparation of alipofection reagent: cationic liposomes were prepared from cationic lipids and helper(if required). (ii) Formation of positively charged lipoplexes by addition of DNA (e.g.,reporter plasmid carrying the firefly luciferase gene) to the cationic liposomes.(iii) Transfection (lipofection) by incubation cells with the preformed lipoplexes.The efficiency of gene transfer (lipofection efficiency) can be determined from reportergene amount or activity (e.g., luciferase activity). Most of the steps of a lipofectionexperiment can be varied and optimized (grey spots).

Automated Screening of Cationic Lipid Formulations for Transfection 255

DNA is present in a highly condensed form (43–45). The strong condensa-tion of DNA also explains why it is protected in the lipoplexes fromnucleases (46). X-ray diffraction studies also provided evidence that lipo-plexes have regular structures. Radler et al. proposed a lipoplex modelconsisting of lamellar lipid layers, whereby DNA layers are surrounded bylipid bilayers like a sandwich, producing a regular grid (47). The occurrenceof lamellar lipid layers in lipoplexes probably explains why bilayer (liposome)forming lipids tend to have higher transfection efficiencies. The structure andassembly of lipoplexes were discussed in detail by Safinya (48).

Transfection of Cells

Passage of lipoplexes into cells: Due to their positive charge, the lipo-plexes added to the cells can interact with their negatively charged cellmembranes (49). In contrast to earlier speculations that lipoplexes pass intothe cell by fusing with the cell membrane (50), it appears certain today thatpassage into the cell takes place primarily via endocytosis. This has beendemonstrated using various cells by taking electron microscopic images ofthe passage of gold-labeled lipoplexes into the cells (49,51).

Once inside the cell, the lipoplexes are located in the endosomes, whichapparently do not fuse with lysosomes. Rather, a considerable number ofendocytotic vesicles accumulate in the vicinity of the nucleus after a fewhours (52,53). Investigations carried out using fluorescence-labeled lipo-plexes show that lipoplexes can be detected in the cytosol in almost every cellthat has been treated.

Passage of DNA into the nucleus: Direct insertion of lipoplexes intothe nucleus does not induce expression of the proteins for which the DNAis encoded (51). Apparently the DNA—when it is complexed with cationiclipids—cannot be detected by the transcription apparatus of the cell. Itappears that the DNA is not released by the lipoplexes in the nucleus. TheDNA must therefore break free of the protective lipid envelope in the cyto-sol before it can pass into the nucleus. An interesting model of themechanism of DNA release from lipoplexes is based on the results of fusionexperiments using cationic and anionic liposomes (54). After endocytosis ofthe lipoplexes into the endosomes, interactions take place between the posi-tively charged lipids in the lipoplexes and negatively charged lipids in theendosomal membranes. In this process, anionic lipids diffuse into the lipo-plex, form close lipid pairs with the cationic lipids, thereby neutralizing thepositive charge. This weakens the interactions of the cationic lipids withthe DNA. The DNA is released from the lipoplexes in the cytosol and canenter the nucleus.

It is discussed that the addition of the helper lipid DOPE (see above)increases the release of DNA from the lipoplexes in the endosomes and


enhances transfection efficiencies. DOPE has fusogenic properties, and it couldbe demonstrated that it supports the necessary membrane perturbationprocess (42,55).

However, the passage of DNA into the nucleus is the most ineffectivestep in transfection procedure using lipoplexes (51). This could be caused bythe DNA being released ineffectively from the lipoplexes and/or the freeDNA being broken-down before it reaches the nucleus. However, it hasto be remarked that transport within the nucleus is not necessary for thetherapeutic action of RNAi-approaches. siRNA directly interferes withmRNA within the cytosol; thus, transfer into the nucleus is not necessaryfor its activity. Thus, lipofection (siFection) seems to be a promising toolfor siRNA-transfer and the first papers of siFection are now being published(56,57).

Cationic Lipids Suitable for Lipofection

The structure of a cationic lipid can be broken-down into three structuralelements: a lipophilic lipid anchor comprising one or—mostly—two longalkyl chains or Chol, a spacer, and a polar, positively charged head groupconsisting of one or more quaternised or protonatable amino groups.Figure 2 shows a few of the well-known, ‘‘older’’ cationic lipids, whichcan be classified as either monocationic or polycationic lipids. A series ofrecently synthesized cationic lipids will be discussed later.

Monocationic lipids: These lipids contain primary, secondary, tertiary, orquaternary amino groups as polar head groups. Primary, secondary, or tertiaryamino groups are usually protonated under physiological conditions (pHaround 7.4) and, are therefore cationic, quaternary amino groups carry a per-manent positive charge. Permethylated (quaternary) amino functions as withN-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammoniumchloride (DOTMA)(50) and DOTAP (25) have been described, as well as quaternisations viaintroduction of an additional hydroxyethyl group as in N-(1,2-dioleoyloxy-propyl)-N,N-dimethyl-N-hydroxyethylammoniumbromide (DORI) (27).

Cholesterol as well as unsaturated or saturated hydrocarbon chainsare used as lipophilic lipid anchors. Although C18-hydrocarbon chains(oleoyl or oleyl unit) are only used in unsaturated compounds, structuralvariations with C14-, C16-, or even C18-hydrocarbon chains in saturatedcompounds are known (27). The lipophilic units are linked with a parentstructure (usually glycerol) via ether (e.g., DOTMA) or ester bridges (e.g.,DOTAP). Ester bridges are often used to create the linkage to avoid cyto-toxicity, because ether bonds are more difficult to break down biologicallythan ester bonds (58). Substances that are easy to decompose andare therefore often used as a spacer are carbamate units (29) [e.g., 3b-[N-(N0,N0-dimethylaminoethyl)carbamoyl]-cholesterol (DC-Chol)], amide units,or phosphate esters. However, a direct correlation between toxicity and the


type of bond has never been definitely demonstrated due to the variety ofpossible causes of toxic side effects. The Chol unit was first used to synthesizeDC-Chol (30) which was already tested in clinical trials (22).

Polycationic lipids: These lipids have head groups with more than onequaternary or protonatable, primary, secondary, or tertiary amino function.Many of these compounds have head groups that are derived fromnaturally occurring polyamines. The examples shown in Figure 2 carrythe spermine N,N-dioctodecyl-amidoglycylspermin [DOGS (31)] or spermi-din–cholesterol [SpdC (32)] unit, respectively. In these examples, thedistance between the amino groups is three or four methylene groups,respectively. Such ‘‘natural’’ structures should be minimally toxic due totheir ability to be broken down biologically. Additionally, these lipidsshould be able to bind tightly to DNA due to the natural ability ofpolyamines to bind well with DNA.

Figure 2 Examples of cationic lipids, differing in the head group structure (mono/polycationic) and the nonpolar lipid anchor (Chol/hydrocarbon chains). Abbrevia-tions: DOTAP, N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethyl-ammoniumchloride;DOTMA, N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammoniumchloride; DC-Chol,3b-[N-(N0,N0-dimethylaminoethyl)carbamoyl]-cholesterol; DOGS, N,N-dioctodecyl-amidoglycylspermin; DORI, N-(1,2-dioleoyloxypropyl)-N,N-dimethyl-N-hydroxyethyl-ammoniumbromide; SpdC, spermidin-cholesterol.


SCREENING FOR IMPROVED CATIONIC LIPIDS

Due to the limitations of predicting TE from lipids chemical structure anddue to the observation that lipofection efficiency toward different cells (celllines) can vary to a great extent, our strategy for the optimization of lipo-fection comprises systematic testing of novel cationic lipids for theirtransfection behavior toward various target cells in an automated screeningapproach (59,60). The goal is to identify promising new transfection lipidsand to develop optimized lipofection protocols for different cell types. Usingthis screening approach, we tested well-known cationic lipids (as describedabove) as well as new cationic lipids which were synthesized by means ofcombinatorial solid phase chemistry (61–63).

Robot System

The establishment of a rapid screening system for the identification of new lipo-fection reagents needs both usual and unusual functional requirements on arobotic platform (Fig. 3). The system required a liquid handling system withdynamic spacing capable of accessing tubes and microplates in various formats,a robotic arm capable of moving plates as well as oddly shaped vials and tools,as well as integrated modules for micorfilter plate (MTP)-washing, MTP-absorbance, and luminescence reading, and the ability to integrate additionaldevices such as a sonicator and a CO2 incubator. We developed a suitable sys-tem based on a standard pipeting robot (Tecan Genesis) and the 96-well plateformat (Fig. 3A). The robot was linked to a CO2 incubator by a robotic con-veyor, which transports the cell culture plates to the worktable (Fig. 3B). Toassure semi-sterile conditions for cell culture work, a customized hood contain-ing 0.5-mm filter elements was placed on the top of the robot (Spetec, Erding).

Screening Procedure

For automation, the lipofection process was split of into four independentparts as follows: (i) preparation of cationic liposomes, (ii) formation of lipo-plexes, (iii) transfection of the cells, and (iv) quantification of the lipofectionefficiency and lipofection-induced cytotoxicity. As shown in Figure 1, thissubdivision corresponds to the typical lipofection procedure and each partcan be performed separately.

Preparation of Cationic Liposomes

For transfection screening, candidate lipids, dissolved in organic solvents, aretransferred from vials into glass test tubes using the liquid handling arm. Inthis first step, defined mixtures of cationic lipids with helper lipids are also pre-pared. The organic solvent is removed under a stream of nitrogen to create athin lipid film on the surface of the glass tube. For this purpose, the roboticarm grips a comb-shaped device with four needles. The gripping action opens


a valve permitting the nitrogen to stream through the needles and the roboticarm moves over the test tubes, drying four lipids at a time. Buffer is added andthe tubes are transported into a water bath sonicator where a dispersion ofsmall cationic liposomes is formed.

Figure 3 (A) Robot system for lipofection screening: (A) Worktable with racks formicroplates, buffer reservoirs, plastic, and glass vials. (B) Four tip liquid handlingarm. (C) Gripper for transport of microplates and glass test tubes. (D) High powerwater bath sonicator. (E) Nitrogen evaporator. (F) Microplate washer. (G) Absor-bance reader. (H) Luminescence reader. (I ) Transparent hood. (J ) CO2 incubatorwith pneumatic door (from the rear, front view in B). (B) Self-constructed roboticconveyor for the transport of cell culture plates from the incubator to the worktable.


Formation of Lipoplexes

For lipoplex formation, the liposomal dispersions are diluted to there finalconcentration and appropriate amounts of the dispersions are placed intothe wells of a 96-deep-well-plate. Afterwards, the plasmid DNA (CMVluc;a plasmid carrying the firefly luciferase gene) is diluted and similar amountsare pipetted into the vials containing the different amount of cationic lipo-somes. For each lipid or lipid/helper–lipid mixture, eight different DNA:lipid-charge ratios from 1:1 to 1:15 are prepared. Directly after addingthe reporter plasmid, the mixture was thoroughly mixed by aspirating anddispensing the liquid by the robot’s pipetting needles. Because we found thata minimal lipoplex formation time is critical to TE, lipoplexes were allowedto maturate for one hour.

Cell Transfection

COS-7 or CHO cells (for initial transfection screening) or cells of therapeuticinterest (e.g., dendritic cells and various cancer cells) at a confluence of 50%,grown in 96-well culture plates, were placed into the robot by the roboticconveyor. In a fully automated process, the robot removes the lid from thecell culture microtiter plate, dispenses lipoplexes into the wells (triplicates),replaces the lid and returns the plate to the incubator. After four hours,the cells are automatically retrieved, the cell monolayers are carefully washedusing a special drop mode of the integrated plate washer, fresh medium isadded, and the cells are incubated for further 42 hours before harvesting.

Quantification of Transfection Efficiency

For quantification of TE, a reporter plasmid carrying the firefly luciferasegene under the control of the CMV promoter is used to form lipoplexes (part2). Forty-two hours after transfection, the growth medium is removed, andthe cells are washed using the drip mode of the microplate washer and lysed.Cell lysates are diluted and aliquots transferred to white MTPs for the lucifer-ase activity assay and transparent MTPs for the bicinchoninic acid (BCA)protein assay. Assay specific standards and controls are added to the micro-plates, and luciferase activity and protein content of the lysates are measured.TE is calculated by dividing luciferase activity by protein content. Dividingprotein content of transfected cells by protein content of control nontrans-fected cells results in a relative measure of cytotoxicity (compare Fig. 1). Adetailed description of the development and use of the automated transfectionscreening system was given by Regelin et al. (59). A typical result of a stan-dard screening for one lipid is shown in Figure 4 (64) (lipofection profile).As such, a profile gives information about lipofection efficiency, the bestlipid:DNA-ratio, and toxicity of the lipofection reagent at a glance; lipofec-tion profiles for comparison of new lipofection reagents is used routinely.


Screening Capacity

With the system described above, nine microplates each with three lipids andeight ‘‘lipid-to-DNA-ratios’’ per lipid can be assayed in one run. In this way,the throughput can be reached a maximum of 108 different lipids or lipidmixtures (in eight different lipids/DNA ratios, respectively) in five days.In addition to the screening approach, the automated assay system also offersthe opportunity to vary several parameters simultaneously in one experiment(Fig. 1, grey shaded boxes). For example, instead of testing several lipids at oneDNA concentration, different concentrations of liposomes at different DNAconcentrations can be tested to determine optimal lipid–DNA combinationsfor distinct cell lines. This gives detailed information on the transfection proper-ties of distinct lipids or mixtures, and can be used to further characterize ascreening hit and/or to optimize transfection protocols.

Figure 4 Standard transfection profile of the cationic lipid 10-(cholesteryloxycarbo-nyl-methyl)-1,4,10-triazadecane acetate (CholAc43) (64) on COS-7-cells is shown.A standard transfection experiment comprised eight different lipid/DNA-chargeratios from 1:1 to 1:15 (x-axis). TE (luciferase activity) is expressed in relationto the TE of a standard lipid (DOTAP), determined in the same experiment (DOTAP¼ 100%, left bar). As a measure of toxicity, the protein content after the transfectionexperiment is shown in the same diagram (left y-axis). Abbreviation: DOTAP, N-[1-2,3-dioleoyloxy)propyl-N,N,N-trimethyl-ammoniumchloride.


COMBINATION OF THE SCREENING APPROACH WITHCOMBINATORIAL SOLID PHASE SYNTHESISOF CATIONIC LIPIDS

In this section, an example will be given in which a (small) library of a newtype of cationic lipids was synthesized and screened for TE (63). For synthesis,combinatorial solid phase chemistry was used. All cationic lipids of the exam-ple library are structurally based on 3-methylamino-1,2-dihydroxy-propane asthe polar, cationic lipid part. As nonpolar lipid part, different hydrocarbonchains are bound to the amino group of the scaffold and the amino groupwas further methylated to get constantly cationic-charged lipids. Lipids weresynthesized in both configurations and as racemats, and the counterions werevaried as well. Table 1 summarizes the structural features of these lipids.

Synthesis

The solid-phase synthesis strategy was based on the utilization of 4-methoxy-trityl chloride resin. To gain access to a large number of compounds, onlycommercially available building blocks were used and protective groupswere omitted if possible. The synthesis strategy resulted in a new class ofcationic lipids as shown in Figure 5 (compound 6). The structure bases on

Table 1 Cationic Lipid Library of N,N-Dialkyl-N-Methyl-Amino-2,3-Propandiols with Alkyl Groupsof Different Length

Name R1 R2

KL-1-1 C10 C8

KL-1-2 C12 C8

KL-1-3 C14 C8

KL-1-4 C16 C8

KL-1-5 C18 C8

KL-1-6 C10 C10

KL-1-7 C10 C12

KL-1-8 C10 C14

KL-1-9 C16 C10

KL-1-10 C18 C10

KL-1-11 C12 C12

KL-1-13 C18 C12

KL-1-14 C14 C14

KL-1-15 C14 C16

KL-1-16 C18 C14

KL-1-17 C16 C12

All lipids were synthesized in R- or S-configuration or as

racemat. Chloride, sulfate, methylsulfate, and acetate were

used as counterions. (R1 and R2; Fig. 5).


3-methylamino-1,2-dihydroxy-propane as the polar, cationic lipid part. Asnonpolar lipid part, different hydrocarbon chains are bound to the aminogroup of this scaffold. The amino group is further methylated to get a con-stantly cationic-charged lipid. This synthesis strategy allows to synthesizethe new lipids in different configuration, and with different counterions.

The synthesis started with the immobilization of (R) -2,3-epoxy-1-propanol2 on the 4-methoxytrityl chloride resin 1 (65). Reaction of the epoxide 3 with along-chained amine yielded the polymer-bound secondary amine 4, which is con-verted to the tertiary amine 5 by reductive amination (66). Quaternization of thetertiary amine by methyl iodide (67) and cleavage from the solid phase gavethe cationic lipid 6, which was further purified by prep. high performance liquidchromoatography (HPLC). Lipids with different alkyl chain length, differentchirality, and different counterions have been prepared by this synthetic route(Table 1). Larger amounts of racemic lipid KL-1-14 containing two tetradecylhydrocarbon chains were synthesized by alkylation of 3-methylamino-1,2-pro-pandiol with 1-bromotetradecane.

Screening/Lipofection Studies

Screening the Combinatorial Lipid Libraryfor Lipofection Properties

This first screening of the cationic lipid library was aimed to identifythe cationic lipid with the optimal hydrocarbon chain composition for the

Figure 5 Combinatorial solid phase synthesis of N,N-dialkyl-N-methyl-amino-2,3-propandiol with alkyl groups of different length.


development of suitable lipofection reagents and protocols. This initialscreening was performed using the COS-7 cell line, because COS-7 is easyto transfect and the differences between the lipids are easy to see. All lipidswith different hydrocarbon chains (lipids KL-1-1 to KL-1-17, Table 1) thatare R-configurated and having chloride as counterion were tested using astandard protocol. From each lipid, eight different lipoplexes containingequimolar amounts of the helper lipid DOPE were formed using eight dif-ferent DNA:lipid-charge ratios from 1:1 to 1:15. For comparison, the TEof the well-know cationic lipid DOTAP N-[1-(2,3-dioleoyloxy) propyl]-N,N,N-trimethylammonium methylsulfate) at its best lipid/DNA chargeration of 2.5 was also determined in each run.

The results of this first screening are shown in Figure 6. All new lipidsshowed higher transfection efficiencies ranging from about 11,000 to170,000 lu/mg protein, which corresponds to 250% to 3860% of the DOTAPvalues. The transfection efficiencies strongly depended on the hydrocarbonchains of the lipids. In general, increasing the overall length of the hydrocar-bon chains resulted in higher transfection efficiencies. An overall length of atleast 28 CH-units seems to be necessary for transfection efficiencies 10-foldhigher than that of DOTAP. Also, the combination of the hydrocarbonchains seems important. Comparing the lipids 10, 14, and 17 all having atotal of 28 CH-units, the lipid 14 bearing two C-14 hydrocarbon chainswas the most effective.

The transfection profiles of the most effective lipids of this group aresimilar, showing a peak (highest TE) at lipid/DNA ratios from 2 to 5. Forthe most effective lipids, the viability of the cells at maximum TE usuallydecreased to roughly 50%. An exception is the lipid 14, which shows the high-est TE as well as only a minor toxicity of about 70% viability. We chose lipidKL-1-14 for further development of a versatile transfection reagent.

Influence of Helper Lipids on Transfection Efficiencies

The previous screening experiments were performed with lipoplexes contain-ing equimolar amounts of the helper lipid DOPE. Here, the influence ofdifferent ratios of the helper lipids DOPE and Chol on TE of KL-1-14were tested. The transfection behavior of KL-1-14 without any helper lipidwas tested as well.

TE of KL-1-14 without helper lipids was very low and reached onlyabout twice the TE, which was found for the standard lipid DOTAP. Inde-pendent of the amount of DOPE incorporated in the lipoplexes (ratio ofDOPE/KL-1-14: 0.3, 0.5, 0.6, 0.7, 0.8, 0.8, 1.0, and 1.2), transfection behav-iors (maximum transfection efficiencies and transfection profiles) of allmixtures were similar and comparable to the profile of KL-1-14/DOPE(1:1) as shown in Figure 2 (individual data for all mixtures are not shown).

Using Chol as helper lipid for KL-1-14, the transfection efficiencieswere no longer similar for the different Chol/KL-1-14-ratios (Fig. 7).


Highest transfection efficiencies were found for Chol/KL1-14-ratios of0.5 to 0.7. Higher or lower ratios led to lower transfection efficiencies,which were similar to that of the KL-1-14/DOPE mixtures. Toxicity of all

Figure 6 Lipofection results (lipofection profiles) of lipoplexes from the R-configu-rated cationic lipids KL-1-1 to KL-1-17 (Table 1) in a mixture with equimolaramounts of 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE) (counter-ion: chloride) and the pCMVluc-plasmid. Each bar represents the mean (� S.D.)of three wells of a 96-well microtiter plate. X-axis (left) represents the transfectionefficiencies expressed in relative light units (RLU) (lu/mg protein). X-axis (right)represents the viability of the cells compared to nontreated control cells. Y-axisrepresents the different cationic lipid/plasmid DNA-charge ratios from 1 to 15.


mixtures was similar and moderate (about 70% survival at maximumTE, data not shown).

Influence of the Configuration, Methylation, andthe Counterions on Transfection Efficiency

We compared the TE and toxicity of KL-1-14 synthesized in the R- andS-configuration [with 0.6 mol% Chol as helper lipid (see above)].The transfection efficiencies for both lipids were statistically similar. Thus,for further experiments, KL-1-14 was synthesized as racemat.

Methylation of KL-1-14 was an important prerequisite for itstransfection properties. A KL-1-14-analog, which was not methylated, didnot transfect at all. It could be assumed that the nonmethylated KL-1-14was not sufficiently protonated at physiological pH, so that the formationof a bilayer structure from these lipids is not possible. As previously shown forDOTAP-analogs, formation of lipid bilayer is an important prerequisitesfor a cationic lipid to be a transfection lipid (35,47).

We further investigated the influence of four different counterions ontransfection behavior of KL-1-14 (methylsulfate, sulfate, chloride, and ace-tate). Chloride as counterion resulted in the highest TE. Using methylsulfate

Figure 7 Maximum lipofection efficiencies [RLU (lu/mg protein)] of the most effec-tive lipoplexes of R-configurated KL-1-14 in a mixture with Chol in different Chol/KL-1-14 ratios from 0.3 to 1.2 (counterion:chloride) and the pCMVluc-plasmid(charge ratio: 7 or 9). Each bar represents the mean (� S.D.) of three wells of a96-well microtiter plate. Abbreviations: Chol, cholesterol; DOPE, 1,2-dioleoyl-sn-glycero-3-phosphatidyl-ethanolamine; RLU, relative light units.


or sulfate as counterion, TE was reduced to 70% to 73% of the values foundfor chloride. Acetate as counterion led to the lowest TE, which was only57% of the chloride values.

Transfection Properties Toward Different Cell Lines

For testing the transfection properties of KL-1-14 toward the mammacarcinoma cell lines MDA-MB-468 and MCF-7, the polarized cell lineMDCK-C7, and the primary dendritic cells KL-1-14 was used in its racemicform with chloride as counterion and as a mixture with 60 mol% Chol. Incomparison to the transfection efficiencies found for COS-7-cells (see above),the transfection efficiencies were generally lower (Table 2). For MDA-MB-468 and MCF-7, transfection efficiencies were reduced by a factor of about10, the MDCK-C7-cells by a factor of about 80, and the dendritic cells bya factor of about 500. Nevertheless, the transfection efficiencies found forKL-1-14/Chol (1:0.6) were generally higher than for DOTAP, respectively.For the mamma carcinoma cell lines, transfection efficiencies with KL-1-14/Chol (1:0.6) were four times higher than for DOTAP. For MDCK-C7and dendritic cells, the increase was from 1.9- to 2.5-fold. We also testedKL-1-14 as equimolar mixture with DOPE for its transfection efficienciestoward the mamma carcinoma cell lines and the dendritic cells. Again, trans-fection efficiencies were greatly reduced even for the KL-1-14/DOPE mixtureand were similar to the values found for KL-1-14/Chol (1:0.6).

Table 2 Transfection Efficiencies of KL-1-14 in a Mixture with Chol (Ratio: 0.7)or DOPE (Ratio: 1.0) Toward the Mamma Carcinoma Cell Lines MDA-MB-468and MCF-7, the Polarized Cell Line MDCK-C7, and the Primary Dendritic Cells

Lipofectionreagent

Mamma carcinoma cellsPolarized cells

MDCK-C7Primary cellsDC (5 days)MDA-MB-468 MCF-7

KL-1-14/Chol(0.6)

19.640� 8.725(n¼ 6), 424%a

19.985� 19.295(n¼ 5), 393%a

2.490� 175(n¼ 3),

187%a

395� 135(n¼ 3),247%a

KL-1-14/DOPE (1.0)

17.515� 5.760(n¼ 5), 378%a

9.250� 1.865(n¼ 5), 182%a

— 410� 115(n¼ 3),258%a

DOTAP 4.630� 910(n¼ 9)

5.085� 3.005(n¼ 7)

1.330� 165(n¼ 3)

160� 295(n¼ 3)

Transfection efficiencies of the KL-1-14 lipoplexes were compared to the TE achieved with the

standard transfection lipid DOTAP. Results were given in RLU (lu/mg protein) and, for easier com-

parison, standardized on the lipofection efficiency of DOTAP-lipoplexes, which was set to 100%aCompared to the respective DOTAP-value.

Abbreviations: DOPE, 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine; DOTAP, N-[1-(2,

3-dioleoyloxy)propyl]-N,N,N-trimethylammoniumchloride; TE, transfection efficiency; DC,

dendritic cells; RLU, relative light units; chol, cholesterol.


CONCLUSION AND FUTURE DIRECTIONS

Improving and easing the process of transfection is one of the most impor-tant prerequisites for pushing gene therapy in clinical practice. Today,compared to viral transfection techniques, lipofection is less effective but hasstrong advantages concerning safety aspects, versatility, and also themanufacturing aspects. To become a method routinely used, lipofection hasto be improved. One successful strategy is the screening of a large numberof new cationic lipids, systematically synthesized by means of combinatorialchemistry, for their transfection properties to find those with better transfec-tion properties. Furthermore, performing a fully automated screening, manyother parameters like ratio of cationic lipid to DNA, amount of lipoplexesadded to cells, duration of transfection, the cell type, and many others couldbe varied systematically (Fig. 1). Thus, transfection protocols of new as wellas existing cationic lipids can be improved or adapted to distinct cells ordistinct transfection problems, e.g., transfection in a serum environment.

ACKNOWLEDGMENTS

Special thanks to all the PhD and Diploma Students Involved in this project(Anne Regelin, Heike Wursthorn, Caria Kusters. Stefan Fankhaenel,Thomas Fichert).

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30. Gao X, Huang L. A novel cationic liposome reagent for efficient transfec-tion of mammalian cells. Biochem Biophys Res Commun 1991; 179(1):280–285.


31. Behr JP, Demeneix B, Loeffler JP, Perez-Mutul J. Efficient gene transfer intomammalian primary endocrine cells with lipopolyamine-coated DNA. Proc NatlAcad Sci USA 1989; 86(18):6982–6986.

32. Guy-Caffey JK, Bodepudi V, Bishop JS, Jayaraman K, Chaudhary N. Novelpolyaminolipids enhance the cellular uptake of oligonucleotides. J Biol Chem1995; 270(52):31391–31396.

33. Lee ER, Marshall J, Siegel CS, et al. Detailed analysis of structures and formula-tions of cationic lipids for efficient gene transfer to the lung. Hum Gene Ther1996; 7(14):1701–1717.

34. Byk G, Dubertret C, Escriou V, et al. Synthesis, activity, and structure–activityrelationship studies of novel cationic lipids for DNA transfer. J Med Chem 1998;41(2):229–235.

35. Regelin AE, Fankhaenel S, Gurtesch L, Prinz C, von Kiedrowski G, Massing U.Biophysical and lipofection studies of DOTAP analogs. Biochim Biophys Acta2000; 1464(1):151–164.

36. Huang CY, Uno T, Murphy JE, et al. Lipitoids—novel cationic lipids forcellular delivery of plasmid DNA in vitro. Chem Biol 1998; 5(6):345–354.

37. Ren T, Liu D. Synthesis of diether-linked cationic lipids for gene delivery. BioorgMed Chem Lett 1999; 9(9):1247–1250.

38. Wang J, Guo X, Xu Y, Barron L, Szoka FC Jr. Synthesis and characterization oflong chain alkyl acyl carnitine esters. Potentially biodegradable cationic lipidsfor use in gene delivery. J Med Chem 1998; 41(13):2207–2215.

39. Fichert T, Regelin A, Massing U. Synthesis and transfection properties of novelnon-toxic monocationic lipids. Variation of lipid anchor, spacer and head groupstructure. Bioorg Med Chem Lett 2000; 10(8):787–791.

40. Massing U, Kley JT, Gurtesch L, Fankhaenel S. A simple approach to DOTAP andits analogs bearing different fatty acids. Chem Phys Lipids 2000; 105(2):189–191.

41. Crook K, Stevenson BJ, Dubouchet M, Porteous DJ. Inclusion of cholesterol inDOTAP transfection complexes increases the delivery of DNA to cells in vitro inthe presence of serum. Gene Ther 1998; 5(1):137–143.

42. Litzinger DC, Huang L. Phosphatidylethanolamine liposomes: drug delivery,gene transfer and immunodiagnostic applications. Biochim Biophys Acta 1992;1113(2):201–227.

43. Eastman SJ, Siegel C, Tousignant J, Smith AE, Cheng SH, Scheule RK. Biophy-sical characterization of cationic lipid: DNA complexes. Biochim Biophys Acta1997; 1325(1):41–62.

44. Gershon H, Ghirlando R, Guttman SB, Minsky A. Mode of formation andstructural features of DNA-cationic liposome complexes used for transfection.Biochemistry 1993; 32(28):7143–7151.

45. Sternberg B, Sorgi FL, Huang L. New structures in complex formation betweenDNA and cationic liposomes visualized by freeze-fracture electron microscopy.FEBS Lett 1994; 356(2–3):361–366.

46. Bhattacharya S, Mandal SS. Evidence of interlipidic ion-pairing in anion-induced DNA release from cationic amphiphile-DNA complexes. Mechanisticimplications in transfection. Biochemistry 1998; 37(21):7764–7777.

47. Radler JO, Koltover I, Salditt T, Safinya CR. Structure of DNA-cationic lipo-some complexes: DNA intercalation in multilamellar membranes in distinctinterhelical packing regimes. Science 1997; 275(5301):810–814.


48. Safinya CR. Structures of lipid-DNA complexes: supramolecular assembly andgene delivery. Curr Opin Struct Biol 2001; 11(4):440–448.

49. Wrobel I, Collins D. Fusion of cationic liposomes with mammalian cells occursafter endocytosis. Biochim Biophys Acta 1995; 1235(2):296–304.

50. Felgner PL, Gadek TR, Holm M, et al. Lipofection: a highly efficient, lipid-mediated DNA-transfection procedure. Proc Natl Acad Sci USA 1987; 84(21):7413–7417.

51. Zabner J, Fasbender AJ, Mozninger T, Poellinger KA, Welsh MJ. Cellular andmolecular barriers to gene transfer by a cationic lipid. J Biol Chem 1995;270(32):18997–19007.

52. Escriou V, Ciolina C, Helbling-Leclerc A, Wils P, Scherman D. Cationiclipid-mediated gene transfer: analysis of cellular uptake and nuclear import ofplasmid DNA. Cell Biol Toxicol 1998; 14(2):95–104.

53. Escriou V, Ciolina C, Lacroix F, Byk G, Scherman D, Wils P. Cationic lipid-mediated gene transfer: effect of serum on cellular uptake and intracellular fateof lipopolyamine/DNA complexes. Biochim Biophys Acta 1998; 1368(2):276–288.

54. Xu Y, Szoka FC Jr. Mechanism of DNA release from cationic liposome/DNAcomplexes used in cell transfection. Biochemistry 1996; 35(18):5616–5623.

55. Farhood H, Serbina N, Huang L. The role of dioleoyl phosphatidylethanol-amine in cationic liposome mediated gene transfer. Biochim Biophys Acta1995; 1235(2):289–295.

56. Sioud M, Sorensen DR. Cationic liposome-mediated delivery of siRNAs in adultmice. Biochem Biophys Res Commun 2003; 312(4):1220–1225.

57. Spagnou S, Miller AD, Keller M. Lipidic carriers of siRNA: differences in theformulation, cellular uptake, and delivery with plasmid DNA. Biochemistry2004; 43(42):13348–13356.

58. Obika S, Yu W, Shimoyama A, et al. Properties of cationic liposomes composedof cationic lipid YKS-220 having an ester linkage: adequate stability, high trans-fection efficiency, and low cytotoxicity. Biol Pharm Bull 1999; 22(2):187–190.

59. Regelin AE, Fernholz E, Krug HF, Massing U. High throughput screening methodfor identification of new lipofection reagents. J Biomol Screen 2001; 6(4):245–254.

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16

Incorporation of Poly(Ethylene Glycol)Lipid into Lipoplexes: On-Line

Incorporation Assessmentand Pharmacokinetics Advantages

Nathalie Mignet, Mamonjy Cadet, Michel Bessodes,and Daniel Scherman

Unite Pharmacol. Chim. Genet.,Universite Rene Descartes Paris, Paris, France

INTRODUCTION

Cationic lipids have been widely studied as a means of nucleic acid delivery.These amphiphilic dialkyl molecules allow for high association with DNA,thanks to electrostatic interactions. DNA has been shown to locate at theinterlamellar spacings, interacting strongly with the cationic polar groupof the lipids (1). Release of phosphate counterions and circular dichroismstudies the nature of supercoiled DNA in these structures (2). These com-plexes efficiently deliver DNA to the cells. They are actively used in vitro totest new DNA clones or transiently express a protein of interest. In contrast,their in vivo applications have been limited due to numerous nonspecificinteractions of the cationic lipid/DNA complexes, which are called lipople-xes, upon intravenous injection. These interactions either render lipoplexesunavailable for the target of interest, in particular because of their interac-tion with lung endothelium, or lead to fast elimination through seric protein

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association or complement activation. Moreover, toxicity has been attribu-ted to platelet aggregation and complement activation by cationic lipoplexes.

Liposomes, which had been widely developed earlier, also sufferedfrom high elimination from the blood stream. However, they gained a lot bybeing shielded by a poly(ethylene glycol) (PEG) coat (3). This was achievedby inserting PEG-lipid into the lipid bilayer. Pharmacokinetic profile ischaracterized by extended circulation time and reduced volume of distribu-tion leading to enhanced tumor uptake (4). Hence, this strategy was appliedto lipoplexes. The aim was simpler than for liposomes, i.e., to achieve stericrepulsion from plasma proteins by the ‘‘mushroom’’ formed around thesurface of the lipoplexes. This was partially achieved as shown by limitationof lipoplex aggregation in serum containing biological media (5).

However, the shielding effect did not completely reach the expecta-tions and worse, inhibited transfection. First, the shielding effect does notcompletely mask the cationic charges at the surface of the lipoplexes (6). Thehydrated PEG-configuration at the surface of the particle allows for holesleading to limited but maintained interaction with cell membranes and sericprotein (7). Hence, intravenously injected lipoplexes do not gain in circu-lation time as much as conventional liposomes previously did (8). Second,insertion of PEG-lipid into lipoplex cationic bilayer stabilizes the complexesin a lamellar phase limiting the release of the nucleic acid (9). Even lipo-plexes, made of dioleoylphosphatidylethanolamine, which are subject tostructural changes due to pH reduction (from lamellar to hexagonal phase),remain in lamellar phase when PEGylated (10). Thus, original PEG-lipid hadto be designed to circumvent these problems. We will particularly describeanionic PEG-lipids, degradable PEG-lipids, and exchangeable PEG-lipids.These systems, as well as a postgrafting method as another possible strategyto be evaluated further, bring original avenues and strategies for in vivoplasmid delivery.

WHY LIPOPLEX PEGYLATION IS NEEDED

Serum Protein Interaction

Serum inactivation of lipoplexes is one of the hurdles PEGylation proposed toface. The interaction of positively charged complexes with negatively chargedplasma components highly reduced the chance for the lipoplexes to reach thecell membranes of interest. Among serum proteins attracted to cationic par-ticles, albumin is obviously involved, and has even been adsorbed to cationicparticles prior injection to render them stealth (11). Binding of lipoplexes withlipoproteins also contributes to the inactivation of these particles. In additionto a simple binding, a fusion takes place between the cationic bilayers and theproteins, hence reducing the transfection efficacy of the complexes (12).

Independent of the nature of serum protein absorbed on the lipo-plexes, the resulting effect is the formation of large aggregates, inducing

274 Mignet et al.

or not the dissociation of the complexes. Oleic acid and heparin effectswould be to displace the mixture oligonucleotide N-[1-(2,3-Dioleoyloxy)]-N, N, N-trimethylammoniumpropane (DOTAP), whereas immunoglobulinor bovine serum albumine (BSA) would not (13).

Numerous studies reported the in vitro interaction of lipoplexes withserum (14). The parameters checked, such as maintained lipid–DNA interac-tions, aggregate formation, zeta potential changes, or DNA morphologicalchanges, did not allow for consistent prediction of in vitro transfection effi-ciency of lipoplexes in serum (15). However, it is obviously an indication ofpoor delivery of DNA at the target site.

As described above, lipoplex PEGylation limits the formation ofaggregates and zeta potential changes in serum.

Nonspecific Binding to Circulating Cells

The lipoplex positive charges might also promote nonspecific binding oflipoplexes to circulating cells such as erythrocytes, lymphocytes, and endo-thelial cells (16). Uptake by the phagocytic cells in the reticuloendothelialsystem might be responsible for inactivation and lipoplex-directed immuneresponse. Some protocols tend to reduce the inflammatory toxicity associa-ted with plasmid DNA uptake by the liver. For instance, sequential injectionof empty cationic liposome prior to lipoplexes could decrease the unspecificdelivery (17). Here again, the capacity of conventional liposomes to escape,at least temporarily, the reticuloendothelial system made it tempting toadapt the PEG-lipid strategy to lipoplexes.

Complement Activation

Opsonization by complement components also represents a potential barrierfor intravenous gene delivery. Cationic charges of the particles activate thecomplement, which then takes part in particle elimination. This hurdle ispossibly limited by using short hydrophobic chains, reducing the particlesize, and eventually PEG insertion into lipoplexes (18). The interactioneffect between the lipoplex and the complement might not be such a limita-tion. Indeed, it was reported that depletion of complement by injection ofcobra venom factor and anti-C3 antibodies in mice indicated no differencesupon intravenous injection of lipoplexes, neither in terms of tissue distribu-tion nor in lipofection efficiency (19).

Nonspecific Binding to Lung Endothelium

Interaction with other biological surfactants might add to clearance and lipo-plex inactivation. Size of the particles plays a major role because extensivemicrovasculature of the lung leads to pulmonary clearance of larger particlesby capillary bed deposition. Vascularization was identified as the main

Incorporation of Poly(Ethylene Glycol) Lipid into Lipoplexes 275

element leading to gene transfection in the lung cells by using transgenicmouse model with tissue-specific angiogenesis in the liver. Hypervasculari-zation of the liver resulted in increased lipoplex entrapment but did notcorrelate with increased gene expression (20).

Pharmacokinetic

Hence, following systemic administration, the major organ of accumulationis the lung, followed by the spleen, liver, heart, and kidney. Moreover, geneexpression is measured in the lung, then heart, spleen, and liver (21).

In the case of local administration, lipoplexes are generally retained atthe site of injection, with poor dispersion (22). In contrast to small emulsionsor neutral liposomes, which immediately appear in the venous outflow perfu-sate following intratumoral injection, the appearance of cationic liposomes ishighly restricted to the injection zone (22). The authors deduced that thedetermining factor altering the pharmacokinetic properties is not the rate oftransfer from the interstitial space to the vascular site but rather the rateof transfer from the injection site to the well-vascularized region (23).

Due to serum inhibition and opsonization, circulation lifetime can bevery short for numerous reported lipoplexes. The formulation content playsan important role in the biodistribution depending on the colipid invol-ved. Cholesterol, for instance, would increase the lipoplex circulation timeby reducing particle leakage in serum (24). Stabilizing the lipid bilayer alsocontributes to increased circulation time to a small extent. Reducing thesize of the particles dramatically increases the circulation time of the com-plexes, as was early shown for conventional liposomes. Hence, modifyingthe lipopolyamine-based lipoplex preparation method from the film to theethanolic injection method, basically reducing the lipoplex size, might helpgain a factor of 7 in the blood (25).

EXAMPLES OF PEG-LIPIDS SUITABLE FORLIPOPLEX INCORPORATION

Available PEG-Lipids

PEG is a widely used molecule as a component in pharmaceutical formula-tions. PEG is particularly useful thanks to its low cost and various simplesynthetic methods (26). PEG-lipid has been developed as a means of stabiliz-ing conventional liposomes. A lipid moiety has been linked to the largePEGylated head in order to anchor the molecule to the particles. Instead ofshielding a direct layer of polymer PEG around the particle, which would beless stable, the idea is to favor hydrophobic interactions between the PEG-lipid and the particle bilayer lipids. This anchor had led to two conformationsof the PEG on the particle surface commonly called mushroom and brushregimes (27), representing a more condensed or extended conformations

276 Mignet et al.

of the PEGylated head. These arrangements obviously depend on the PEGlength and the amount of PEG-lipid incorporated in the vesicle (28). Hence,brushes form high grafting density where PEG chains overlap laterally,whereas in the mushroom regimen, at weaker grafting density, lateralchains do not interact (29). An overload of PEG-lipid will inexorably leadto particle destabilization (30). Before reaching this stage, the availablePEG-lipids allow some latitude.

The choice of commercially available PEG-lipids is ultimately quitereduced. The mainly reported ones are the anionic polyethylene glycol-phosphoethanolamine (PEG-PE) and the neutral PEG-ceramide (Fig. 1).

Our laboratory mostly works with PEG-cholesterol. It is easilyobtained in one step by addition of cholesteryl chloroformate and amino-methoxy-PEG (31). Introduction of a linker between the cholesterol and thePEG part would induce higher membrane fluidity and reduce more effici-ently protein interactions as compared to PEG-cholesterol. A diaminobutanespacer was shown to improve significantly the sustained release of calceinfrom lipoplexes incubated in 30% serum (32). The spacer effect on bicatenarPEG-lipid has not been intensively studied because it can be expected thatit would induce less effect on PEG-dioleoyl than on PEG-cholesterol, thelipidic anchor being predominant in the bilayer stabilization (Fig. 2).

PEG-Lipid Bearing Anionic Charges Within the Spacer

An alternative to the conventional PEG-PE was to incorporate between thePEG and the lipidic chain, a linker bearing anionic charges convenientlypositioned in order to interact with the free amines of the cationic lipidwithin the lipoplexes (8).

Figure 1 Commercially available PEG-lipids. Abbreviation: PEG, poly(ethyleneglycol).


These PEG-lipids offer two main advantages:

� Keep the PEG more tightly bound to the hydrophobic bilayer.� Interact with the lipoplex amines still available for protein unspecific

interactions, reducing the holes left by the PEG moiety (Scheme 1).

The anionic charges of the linker were incorporated during the synthesisvia the coupling of amino acids on the dioctadecylamine moiety. Hence, thecontrolled PEG-lipid bore four glycine, whereas the other linkers were consti-tuted of glycine and aspartic acids, or glycine, aspartic acid, and glutamic acidas represented in Figure 3.

The zeta potential revealed different values upon incorporation of thedifferent cholesterol PEG as can be seen in Figure 3. Although the incorpora-tion of polyethylene glycol-dioctadecylamine (PEG-DODA) bearing no nega-tive charges induced a zeta potential reduction (from þ60 mV for cationiclipids to þ16 mV), the introduction of two negative charges in the PEG-lipidlinker allowed approaching a value close to neutrality (þ5 mV). These dataare comparative from one to another and do not refer to a real negativecharge value because the PEG moiety interferes with the electrophoretic

Figure 2 Poly(ethylene glycol) cholesterol.

Scheme 1 Anionic PEG-lipid: strategy. Abbreviation: PEG, poly(ethylene glycol).

278 Mignet et al.

mobility of the particles (6). Moreover, the incorporation of these PEG-lipidsinto lipoplexes resulted in a higher stability of the lipoplexes in the serum,which correlated with the zeta potential values obtained (8). In particular,the particle size remained stable over 24 hours in 10% serum at 37�C forthe lipoplexes bearing the bi-anionic PEG-lipid (Fig. 4). A slight increasein size was observed for the PEG and PEG bearing a single anionic chargeindicating that the presence of the PEG reduced the interaction between sericprotein and lipoplexes, but not as efficiently as the PEG-lipid bearing twonegatives charges for which diameter changes were extremely weak.

Cleavable PEG-Lipid

Grafting of PEG on the liposome surface interferes with the ability of theliposome to undergo membrane fusion and destabilization in the endosome.Meyer et al. observed this point (33). The stabilization of the lipoplexes intoa lamellar phase would be a possible reason for this transfection inhibition,by lack of destabilization into the endosome (34). Thus, cleavable PEG-lipidhas been designed to limit the nonspecific interaction with proteins,although restoring the ability of the particles to interact with the endosomalcellular membranes and to release their therapeutic payload.

Acid-Sensitive PEG-Lipid

With the aim to reduce unspecific protein interactions while controllingDNA release, acid-sensitive PEG has been designed. Several pH-sensitivesystems are described in Volume I, Chapter 8. Briefly, the objective is todevelop molecules for biological purposes, which means molecules ableto be destabilized at slight pH changes. Tumors or ischemia sites present

Figure 3 Effect on the zeta potential of the incorporation of 5% PEG-DODA intocationic lipoplexes [lipopolyamine RPR209120/DOPE 1/1, ratio (mol) lipid/DNA¼ 10 in 150 mM NaCl]. Abbreviation: PEG, poly(ethylene glycol); DODA,dioctadecylamine; DOPE, dioleoylphosphatidylethanolamine.


acidic pH as compared to physiological pH and would be the preferentialtargets for such targeting molecules.

Acid-sensitive cholesterol PEG as well as acid-sensitive Brij compoundshave been synthesized and incorporated into lipoplexes (Fig. 5) (41). Differentorthoester linkages incorporated within the PEG-lipid spacer allowed differ-ent sensitivity of the molecule to pH. The orthoester group (42) is advantageousnot only because of its sensitivity to pH changes but also because

� this sensitivity might be modulated by its structure, and� the hydrolysis is self-catalytic.

Figure 4 Effect on the serum stability of the incorporation of 5% cholesterol poly(ethy-lene glycol) (PEG) into cationic lipoplexes [lipopolyamine RPR209120/DOPE 1/1, ratio(mol) lipid/DNA¼ 10 in 150 mM NaCl]. Lipoplexes were incubated in DMEMþ 10%SVF, at 37�C, aliquots were regularly sampled and monitored by dynamic diffusion.Results represent a mean between three measurements. Error bars are not presented tosimplify the graph, but differences among PEG, PEG-1, and PEG-2 are significant.Abbreviations: PEG, poly(ethylene glycol); DOPE, dioleylphosphatidylethanolamine;DMEM, Dulbecco’s Modified Eagle Medium.

280 Mignet et al.

In addition to extracellular degradation in tissues, endosomal acidifi-cation might also trigger PEG-lipid cleavage. We showed that despite thepresence of the PEG, which slightly reduces lipoplex internalization intothe cells, DNA transfection level almost reaches the level of the cationic lipo-plex (31). Cholesterol PEG incorporation into lipoplexes not only reduceslipoplex internalization, but also inhibits the transfection efficiency.

Figure 5 Degradable PEG-lipid: degradability via the orthoester function, thevinylether, or the disulfide group. Abbreviation: PEG, poly(ethylene glycol). Source:From Refs. 31, 35–40.


In contrast, pH-sensitive cholesterol-PEG or pH-sensitive PEG-lipid helpsrestore the transfection efficacy by destabilization at the endosomal pH(31). A series of PEG-diorthoester with various PEG lengths or acyl chainswere synthesized and incorporated into DNA-based nanolipoparticles. Par-ticle stability was achieved at pH 8.5 but rapidly collapsed at pH 5 (35).According to the PEG moiety, DNA was released at different rates as afunction of the pH and transfection activity was obtained (43). Low-pH–sensitive stabilized plasmid-lipid nanoparticles (SPLP) have been studied byincorporation of the PEG-diorthoester in the SPLP and were shown to givehigher level of DNA transfection as compared to the non–pH-sensitive SPLP(44). Other PEG-lipids sensitive to pH have been reported; they contain avinyl ester group between the PEG and the lipid with different spacers(Fig. 5). Incorporated into liposomes, they provide faster leakage rates ascompared to the nonsensitive PEGylated lipoplexes (36).

PEG-Lipid Bearing a Disulfide Function Within the Linker

Other cleavable PEG-lipids have been developed (Fig. 5). Bearing a disulfidebond, they are sensitive to thiolytic agents. Thiolysis results in the liberationof separate PEG and lipid, and may regenerate the original lipid when linkedthrough a dithiobenzylurethane group (37). Liposomes with detachable polymercoating have been prepared. Recovery of lipid fusion capability after PEG clea-vage was evidenced by fluorescence measurements. Surprisingly, upon cleavageof this link with a reductive agent like dithiothreitol, the fluorescent markerentrapped into the liposome was released. It is noteworthy that other phenom-enon, like aggregation, occurred after PEG cleavage, which could provoke therelease. More interesting was the fact that acid-sensitive cholesterol hemisucci-nate based formulations were able to transfect when grafted with detachablePEG, which was not the case in presence of PEG-distearoylphosphoethanola-mine (DSPE) (38). Restoration of pH sensitivity to liposomes depends on theamount of PEG-lipid incorporated in the formulation and on the stability ofthe disulfide linkage (39). Nevertheless, pharmacological studies based on thesecleavable acid-sensitive formulations did not allow doxorubixin release uponPEG cleavage (40).

Exchangeable PEG-Lipid

Instead of referring to natural trigger, such as pH changes or enzymehydrolysis, Cullis and coworkers looked into exchangeable PEG derivatives.Lipoplex steric stabilization should be transient to restore lipoplex interac-tion with endosomal membrane and required for nucleic acid release andtransfection. Time-dependent stabilization occurs until an exchange happenswith the membrane of interest, depending on the acyl chain length and thesize of the PEG part (45). As could be expected, the shorter the acyl chain,the more exchangeable the PEG-ceramide (46). Several SAINT PEG of

282 Mignet et al.

different acyl chain length were also synthesized (47). Addition of anionicliposomes to lipoplexes bearing different acyl chain length PEG-inducedDNA released. This release, monitored by accessibility of picogreen, wastime dependent and occurred faster with lipoplex bearing the PEG-lipidwith the shortest acyl chain.

PEG-LIPID INCORPORATION INTO LIPOPLEXES: PROTOCOLSAND MONITORING

Few systems allow for the incorporation measurements of PEG-lipids into lipo-plexes. First of all, because the nonincorporated PEG-lipid should be removedfrom the liposome PEG mixture, prior to determination of the associated PEGconcentration, it is not always easy for positively charged particles. They glue tothe exclusion membrane used to eliminate the free PEG-lipid and do not easilyultracentrifuge if highly charged. Membrane exclusion is, however, facilitatedby the presence of PEG-lipid, as compared to free lipoplexes.

PEG-Lipid Insertion in Liposomes

As for conventional liposomes, temperature and time of incubation areimportant factors for PEG-lipid insertion into cationic bilayers (48). Transi-tion temperature of cationic lipids has not always been determined, althoughit would be interesting data to have. The incorporation of PEG-lipid into thefilm before hydration is usually more efficient than its postinsertion intothe particles. However, postincorporation allows to work with limitedamounts of materials, and to test more easily multiple conditions.

Determination of PEG-Lipid Concentration

A colorimetric estimation of inserted PEG-phospholipids was developedbased on the two-phase system used to quantify phospholipids (49). Theformation of a complex between the phospholipids and Fe(SCN)3 transfersthe chromophore Fe(SCN)3 to the organic phase, allowing quantificationof the phospholipids present in the solution. This system was applied toPEG-phospholipids (50). It is quite sensitive but obviously limited to PEG-phospholipids. The PEG-lipids, which bear no phosphate group, cannot bequantified by this method.

Another colorimetric method of the associated PEG-lipid might beperformed using the method described by Baleux (51). This assay consistsof the formation of a complex between the PEG moiety and iodine forminga solution absorbing at 500 nm. This method is less sensitive than the pre-vious one because it does not rely on a specific chemical function on thePEG, although it allows for wider type of PEG-lipid to be quantified.

In all cases, the other components present in the medium should pre-cautiously be tested for their interference with the system.



284 Mignet et al.

PEG-Lipid-Induced Lipoplex Stabilization

One indirect method to test the internalization of PEG-lipids is to workwithin conditions where the lipoplexes aggregate, and point out the stabili-zation induced by the PEG-lipid.

At a charge ratio between the amines of the cationic lipid and thephosphates of DNA around 1, depending on the medium, particles willaggregate (52). To search for the conditions where PEG-lipid will stabilizethe lipoplexes, different amounts of PEG-lipid are added before adding theDNA under the above conditions. Spectrometry (measure of turbidity byultraviolet) or dynamic diffusion (size of the particles) will allow testingthe PEG-lipid effect. Typically, the lipoplexes taken as control (withoutPEG-lipid) will aggregate. The samples bearing PEG-lipid will also aggre-gate until the amount of PEG-lipid necessary to sterically stabilize theparticles is reached. In this case, the turbidity will decrease and/or the sizeof the particles will approach the original size of the particles without PEG-lipid. Masson, et al. (31) reported this technique to prove the liability of theacid-sensitive PEG-lipid, which is described in Volume I, Chapter 8.

First, to test the pertinence of our test, we applied our technique to evi-dence the influence of the PEG length. For the same amount of PEG-lipidintroduced into a particle, we expect a higher stabilization with the one bear-ing the larger PEG moiety. That is what we obtained (Fig. 6). We also testedthis point with PEG-cholesterol, using PEG75 and PEG110, and reached thesame conclusion (Fig. 6).

This technique was also applied to select the lipid part of the PEG-lipid thatwould most efficiently anchor the PEG-lipid into the liposomes. As observed,PEG-cholesterol induces a better stabilization than the PEG-PE (Fig. 6).

PHARMACOKINETIC PROPERTIES OF PEG-LIPOPLEXES

Insertion of PEG-lipid into conventional liposome phospholipid bilayerhad substantially increased their circulation half-life. Pharmacokinetic ofPEGylated liposome is clearly modified by the presence of the PEG-lipid;extended circulation time was reported as reviewed (4).

This strategy applied to lipoplexes did not reach the expectations.Complete masking of the cationic charges was not achieved by PEG shield-ing on the surface of the cationic particles. Hence, the distribution profilethat can be found in the literature do not impress much as compared to

Figure 6 (Figure on facing page) Incorporation of different poly(ethylene glycol)(PEG) lipid into lipoplexes. PEG-lipid is incubated with the appropriate amountof lipids RPR209120/DOPE (1/1), then plasmid is added to reach a charge ratio¼ 1in 150 mM NaCl. Abbreviations: PEG, poly(ethylene glycol); DOPE, dioleylphospha-tidylethanolamine.


non-PEGylated lipoplexes, even though a little gain might be obtained,thanks to diminished accumulation in the lung following intravenous injec-tion (53). Passive accumulation to tumor sites is ultimately low and fewexamples of lipoplexes reaching this target have been reported so far (54).

Biodistribution studies are usually performed via radioactive labelingof the lipids or the liposomes.111Indium or 14C-labeled lipids are used asliposome markers to measure circulating plasma levels of liposomes. Label-ing of liposomes with 67Ga has also been described (55).

To evaluate the circulation profile of our newly designed lipoplexes, weoptimized the method reported by Takeuchi et al. using phosphatidyletha-nolamine lissamine rhodamine as the marker (56). Introduced as low as0.5% to 2% (percentage of total lipid) in the formulation, it does not modifythe structure of the lipoplexes, although allows following their tissue distri-bution. We verified that the marker was not lost during these experimentsby incorporating the label in conventional liposomes. Comparison of thecirculation time obtained with reported data on conventional liposomeradioactively labeled indicated similar results. We found that the calibrationcurves, obtained upon extraction of this lipid from tissue homogenates, wereindependent from the tissue, opposite to phosphatidylethanolamine fluores-cein. Thus, phosphatidyl ethanolamine lissamine rhodamine derivative,incorporated into lipoplexes, allowed reproducible quantification of lipo-plex circulation time and biodistribution. We applied the protocol asdescribed by Nicolazzi et al. to all our PEGylated lipoplexes (8). Figure 7represents a biodistribution comparison between lipoplexes bearing the dif-ferent PEG described earlier: cholesterol PEG110, anionic PEG110 DODA(bearing two negative charges), and the acid-labile PEG110 (cholesteryl-orthoester hexacycle-PEG).

As can be seen in Figure 7, different functional improvements wereobserved according to the PEG-lipid used. PEGylated lipoplexes did notincrease the circulation time of the lipoplexes but reduced by a factor of 3nonspecific accumulation in the lung. In contrast, use of negatively chargedDODA-PEG improved significantly the lipoplex circulation time. Nonspeci-fic accumulation in the lung was also reduced in this case.

As expected, acid-labile PEG did not increase the circulation time ofthe lipoplexes as compared to PEGylated lipoplexes but allowed for thesame level to be reached, indicating the stability of the acid-sensitivePEG-lipid moiety in the blood.

PEG-LIPOPLEXES: WHAT MORE IS NEEDED?

As of today, the insertion of PEG-lipid into lipoplexes helped in reducing thenonspecific interaction of lipoplexes with serum protein. However, the PEGy-lation did not totally reach its goal into improving lipoplex circulation time.

286 Mignet et al.

Figure 7 (Continued on next page) Comparison of the biodistribution of PEGylatedlipoplexes in vivo. Abbreviation: PEG, poly(ethylene glycol).


Two problems remain:

� An incomplete charge masking� The increased particle stabilization, which limits DNA release and

subsequent transfection


288 Mignet et al.

New avenues could come from reacting small chemical entities withthe remaining amines at the surface of the lipoplexes, to obtain completelyneutralized DNA-loaded particles (57). Gain in circulation time was signifi-cant by this so-called postgrafting method.

The second limitation, however, remains: i.e., lipoplex stabilization,which prevents DNA release. A combination of solutions should be envisionedfor further improvement. For instance, we could combine the postgraftingmethod with exchangeable PEG. We indeed combined the use of acid-sensitivePEG-lipid and the postgrafting method. Results tend to show an improvedDNA release cumulated with higher circulation time (unpublished). However,the differences in tumor growth and vascularization render difficult the obten-tion of significant and reproducible results.

Combined systems still remain a high objective, such as both targetingand cleavable lipoplexes. Identification of suitable ligand is probably the mainlimitation for specificity, and the PEG-lipid bearing the ligand should be inthe extended conformation for the highest possible ligand interaction with thetarget receptor, which is not as often as the case on the surface of the liposo-mes (58). After reaching the target, removal of the PEG layer to allow forinternalization, destabilization, and DNA release looks like a magic bullet.

REFERENCES

1. Radler J, et al. Structure of DNA-cationic liposome complexes: DNA intercala-tion in multilamellar membranes in distinct interhelical packing regimes. Science1997; 275:810.

2. Ghirlando R, et al. DNA packaging induced by micellar aggregates: a novelin vitro DNA condensation system. Biochemistry 1992; 31:7110; Wagner K, et al.Direct evidence of the counterion release upon cationic lipid-DNA condensation.Langmuir 2000; 16:303.

3. Lasic D, Papahadjopoulos D. Liposomes revisited. Science 1995; 267:1275.4. Gabizon A, Shmeeda H, Barenholz Y. Pharmacokinetics of pegylated liposomal

Doxorubicin: review of animal and human studies. Clin Pharmacokinet 2003;42:419.

5. Nicolazzi C, et al. Cationic lipids for Transfection. Curr Med Chem 2003;10:1263.

6. Woodle M, et al. Sterically stabilized liposomes. Reduction in electrophoreticmobility but not electrostatic surface potential. Biophys J 1992; 61:902.

7. Miller C, et al. Liposome-cell interactions in vitro: effect of liposome surfacecharge on the binding and endocytosis of conventional and sterically stabilizedliposomes. Biochemistry 1998; 37:12875.

8. Nicolazzi C, et al. Anionic polyethyleneglycol lipids added to cationic lipoplexesincrease their plasmatic circulation time. J Control Rel 2003; 429.

9. Shi F, et al. Interference of polyethylene glycol-lipid analogues with cationiclipid-mediated delivery of oligonucleotides roles of exchangeability and non-lamellar transitions, Biochem J 2002; 336:333.


10. Song L, et al. Characterization of the inhibitory effect of PEG-lipid conjugateson the intracellular delivery of plasmid and antisense DNA mediated by cationiclipid liposomes. Biochim Biophys Acta 2002; 1558:1.

11. Faneca H, Simoes S, Pedroso de Lima MC. Association of albumin or prota-mine to lipoplexes: enhancement of transfection and resistance to serum. J GeneMed 2004; 6:681.

12. Tandia B, et al. Lipid mixing between lipoplexes and plasma lipoproteins is amajor barrier for intravenous transfection mediated by cationic lipids. J BiolChem 2005; 280:12255.

13. Zelphati O, et al. Effect of serum components on the physico-chemical propertiesof cationic lipid/oligonucleotide complexes and on their interactions with cells.Biochim Biophys Acta 1998; 1390:119.

14. Li B, et al. Lyophilisation of Cationic Lipid-Protamine-DNA (LPD) complexes.J Pharm Sci 2000; 89:355.

15. Zhang Y, Anchordoquy T. The role of lipid charge density in the serum stabilityof cationic lipid/DNA complexes. Biochim Biophys Acta 2004; 1663:143.

16. Ferrari M, et al. Trends in lipoplex physical properties dependent on cationiclipid structure, vehicle and complexation procedure do not correlate with biolo-gical activity. Nucl Acids Res 2001; 29:1539.

17. Zhang J, Liu F, Huang L. Implications of pharmacokinetic behavior of lipoplexfor its inflammatory toxicity. Adv Drug Deliv Rev 2005; 57:689.

18. Plank C, et al. Activation of the complement system by synthetic DNA com-plexes: a potential barrier for intravenous gene delivery. Hum Gene Ther1996; 7:1437.

19. Barron L, Meyer K, Szoka F. Effects of complement depletion on the pharma-cokinetics and gene delivery mediated by cationic lipid-DNA complexes. HumGene Ther 1998; 9:315.

20. Simberg D, et al. The role of organ vascularization and lipoplex-serum initialcontact in intravenous murine lipofection. J Biol Chem 2003; 278:39858.

21. Liu F, et al. Factors controlling the efficiency of cationic lipid-mediated transfec-tion in vivo via intravenous administration. Gene Ther 1997; 4:517.

22. Leclercq F, et al. Design, synthesis and evaluation of gadolinium cationic lipidsas tools for biodistribution studies of gene delivery complexes Bioconj Chem2003; 14:112.

23. Nomura T, et al. Effect of particle size and charge on the disposition of lipid car-riers after intratumoral injection into tissue-isolated tumors. Pharm Res 1998;15:128.

24. Allen T, Cleland L. Serum-induced leakage of liposome contents. Biochim Bio-phys Acta 1980; 597:418.

25. Tranchant I, et al. Physicochemical optimisation of plasmid delivery by cationiclipids. J Gene Med 2004; Suppl 1(Rev.):S24.

26. Yuda T, Maruyama K, Iwatsuru M. Prolongation of liposome circulationtime by various derivatives of polyethyleneglycols. Biol Pharm Bull 1996;19:1347.

27. Needham D, Stoicheva N, Zhelev D. Exchange of monooleoylphosphatidyl-choline as monomer and micelle with membranes containing poly(ethyleneglycol)-lipid. Biophys J 1997; 73:2615.

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28. Kaasgaard T, Mouritsen O, Jorgensen K. Screening effect of PEG on avidinbinding to liposome surface receptors. Int J Pharm 2001; 214:63.

29. Kenworthy AK, et al. Structure and phase behavior of lipid suspensionscontaining phospholipids with covalently attached poly(ethylene glycol).Biophys J 1995; 68:1903.

30. Ross P, Hui SW. Polyethylene glycol enhances lipoplex-cell association and lipo-fection. Biochim Biophys Acta 1999; 1421:273.

31. Masson C, et al. pH-sensitive PEG lipids containing orthester linkers: newpotential tools for non viral gene delivery. J Control Rel 2004; 99:423.

32. Carrion C, Domingo JC, de Madariaga MA. Preparation of long-circulatingimmunoliposomes using PEG-cholesterol conjugates: effect of the spacer armbetween PEG and cholesterol on liposomal characteristics. Chem Phys Lipids2001; 113:97.

33. Meyer O, et al. Cationic liposomes coated with polyethylene glycol as carriers foroligonucleotides. J Biol Chem 1998; 273:15621; Shi G, et al. Efficient intracellu-lar drug and gene delivery using folate receptor-targeted pH-sensitive liposomescomposed of cationic/anionic lipid combinations. J Control Release 2002;80:309.

34. Holland JW, Cullis PR, Madden TD. Poly(ethyleneglycol)-lipid conjugates pro-mote bilayer formation in mixtures of non-bilayer-forming lipids. Biochemistry1996; 35:2610.

35. Guo X, Szoka F. Steric stabilization of Fusogenic Liposomes by a low-pHsensitive PEG-diortho ester-lipid conjugate. Bioconj Chem 2001; 12:291.

36. Shin J, Shum P, Thompson D. Acid-triggered release via dePEGylation ofDOPE liposomes containing acid-labile vinyl ether PEG-lipids. J Contr Rel2003; 91:187.

37. Zalipsky S, et al. New detachable poly(ethylene glycol) conjugates: cysteine-clea-vable lipopolymers regenerating natural phospholipid, diacyl phosphatidyletha-nolamine. Bioconj Chem 1999; 10:703.

38. Kirpotin D, et al. Liposomes with detachable polymer coating: destabilizationand fusion of dioleoylphosphatidylethanolamine vesicles triggered by cleavageof surface-grafted poly(ethylene glycol). FEBS Lett 1996; 388:115.

39. Ishida T, et al. Targeted delivery and triggered release of liposomal doxorubicinenhances cytotoxicity against human B lymphoma cells. Biochim Biophys Acta2001; 1515:144.

40. Zhang J, et al. Pharmaco attributes of dioleoylphosphatidylethanolamine/cholesterylhemisuccinate liposomes containing different types of cleavable lipo-polymers. Pharmacol Res 2004; 49:185.

41. Bessodes M, et al. Acid sensitive compounds for delivering drugs to the cells.PCT Int Appl WO 02/0510, 2002:73, Mars 14.

42. Li S, Dory P. Hydrolysis of cyclic orthoesters: experimental observations andtheoretical rationalization. Tetrahedron 1996; 52:14841.

43. Li WL, et al. Low-pH-sensitive poly(ethylene glycol) (PEG)-stabilized plasmidnanolipoparticles: effects of PEG chain length, lipid composition and assemblyconditions on gene delivery. J Gene Med 2005; 7:67.

44. Choi J, MacKay J, Szoka F. Low-pH-sensitive PEG-stabilized plasmid-lipidnanoparticles: preparation and characterization. Bioconj Chem 2003; 14:420.


45. Webb MS, et al. Comparison of different hydrophobic anchors conjugated topoly(ethylene glycol): effects on the pharmacokinetics of liposomal vincristine.Biochem Biophys Acta 1998; 1372:272.

46. Mok K, Lam A, Cullis P. Stabilized plasmid-lipid particles: factors influencingplasmid entrapment and transfection properties. Biochim Biophys Acta 1999;1419:137.

47. Rejman J, et al. Characterization and transfection properties of lipoplexes stabi-lized with novel exchangeable polyethylene glycol-lipid conjugates. BiochimBiophys Acta 2004; 1660:41.

48. Uster P, et al. Insertion of poly(ethylene glycol) derivatized phospholipid intopre-formed liposomes results in prolonged in vivo circulation time. FEBS Lett1996; 386:243.

49. Stewart JC. Colorimetric determination of phospholipids with ammoniumferrothiocyanate. Anal Biochem 1980; 104:10.

50. Nag A, Mitra G, Ghosh P. A colorimetric estimation of polyethyleneglycol-con-jugated phospholipid in stealth liposomes. Anal Biochem 1997; 250:35.

51. Baleux B. Colorimetric determination of nonionic, poly(oxyethylene) surface-active agents using an iodine-iodide solution. CR Acad Sci: Sci Chim 1972;274:1617.

52. Pitard B, et al. Virus-sized self-assembling lamellar complexes between plasmidDNA and cationic micelles promote gene transfer. Proc Natl Acad Sci USA1997; 94:14412.

53. Hofland H, et al. Folate-targeted gene transfer in vivo. Mol Ther 2002; 5:739.54. Krasnici S, et al. Effect of the surface charge of liposomes on their uptake by

angiogenic tumor vessels. Int J Cancer 105, 561, 2003; Hood J, et al. Tumorregression by targeted gene delivery to the neovasculature. Science 2002;296:2404.

55. Gabizon A, Papahadjopoulos D. Liposome formulations with prolonged circu-lation time in blood and enhanced uptake by tumors. Proc Natl Acad Sci USA1988; 85:6949.

56. Takeuchi H, et al. Polymer coating of liposomes with a modified polyvinyl alco-hol and their systemic circulation and RES uptake in rats. J Contr Rel 2000;68:195.

57. Thompson B, et al. Neutral post-grafted colloidal particles for gene delivery.Bioconj Chem 2005; 16:608.

58. Jeppesen C, et al. Impact of polymer tether length on multiple ligand-receptorbond formation, Science 2001; 293:465.

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17

Efficient Gene Transfer by Lipid/PeptideTransfection Complexes

Scott A. Irvine and Stephen L. Hart

Molecular Immunology Unit, Institute of Child Health, London, U.K.

Jean R. McEwan and Faiza Afzal

Centre for Cardiovascular Genetics, University College London,Rayne Institute, London, U.K.

INTRODUCTION

The use of cationic liposomes is an established methodology for the nonviraltransfer of DNA into cells. Gene therapy, the transfer of therapeutic genesinto cells, is viewed as having great potential for the intervention of numer-ous pathogenic conditions. This transfer can be facilitated by either viral ornonviral vectors. Viral vectors confer considerable transfection efficiency,yet the therapeutic use of these vectors has generated safety concerns, dueto incidences of immunogenicity and oncogenesis, whereas nonviral vectorshave the advantage of lacking these serious side effects. However, theygenerally fail to match the level of transfection efficiency attributed to viralvectors by a significantly large degree.

To bridge this gap, liposomal transfection efficiency can be dramati-cally enhanced by the inclusion of peptides into the complex withoutincreasing immunogenicity. Peptides can be selected to assist lipofection ateach key stage of the process: complex formation, cell targeting and uptake,endosomal disruption, and nuclear targeting. The purpose of this chapter is

293

to discuss the role of peptides in aiding the passage of liposome-complexedDNA from the outside of the plasma membrane to the nucleoplasm.

THERAPEUTIC GENE TRANSFER

Gene therapy is a long-established concept, described in 1970 as the use ofrecombinant DNA for the ‘‘treatment or cure of inherited disease in man’’(1). In effect, the pathology of an inherited disorder can be alleviated by theexpression of an introduced gene to compensate for the defective gene product.

The potential use of gene therapy has since expanded as conditionssuch as cancer, atherosclerosis, transplant operations, and infectious diseaseare now viewed as suitable targets for intervention. For example, HIV andparasitic infection (2–5). Furthermore, the ability to transfer genes into cellin vitro is also an important tool in the research of gene expression.

Genes can be introduced by the application of naked DNA alone; how-ever, better efficiency is achieved when the DNA is incorporated into a deliveryvector. These delivery vectors consist of viral, those utilizing modified virus par-ticles for DNA delivery, and nonviral, for which various chemicals are used toaid DNA packaging and delivery. Viral vectors confer significantly better trans-fection efficiency than nonviral vectors; however, recently the toxicity andoncogenic side effects of viral vectors have become a major concern (6). Non-viral vectors do not have such serious side effects but lack the efficiency (7).

One of the principal forms of nonviral delivery is liposome-mediatedgene transfer, in which the DNA is enveloped in a cationic lipid that actsas a shield against the degradation or inactivation of the DNA during theprocess of gene transfer.

LIPOSOME AND PEPTIDES

The first synthetic gene delivery vectors—such as Lipofectin, an equimolarformulation of—DOTMA (N-[2,3-(dioleyloxy) propyl]-N,N,N-trimethylam-monium chloride) and dioleoylphosphatidylethanolamine (DOPE)—depend-ing on the cell line, transfects cells from 5-fold to greater-than-100-fold moreeffectively than either the calcium phosphate or the diethylaminoethyl(DEAE)-dextran transfection technique (8). Since then, an extensive array ofnovel cationic lipids have been developed for both in vitro and in vivo uses,including clinical trials (9–12). Despite the many successes in the developmentof liposome technologies, there remains a need for systems of improved genetransfer efficiency, particularly for in vivo and clinical applications. Oneapproach to enhancing liposome function is to incorporate accessory nonlipidelements. For example, proteins such as transferrin have been incorporatedinto liposomes to target cells to a specific receptor and thus aid receptor-mediated uptake (3), and DNA-binding proteins such as protamine have beenincorporated to enhance DNA packaging within the complex (13). The aim of

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this chapter is to review the contribution and role of peptides to aid genedelivery by liposomes.

Peptides have many desirable properties as components of syntheticvectors. Peptide synthetic chemistry is well established, with the convenienceof automated synthesis resulting in a well-defined, high-purity product oflow toxicity and immunogenicity for in vivo use. Furthermore, even shortpeptides of 7 to 30 amino acids can accommodate enormous structuraldiversity, functionality, and combinations of properties.

Lipid components of liposomes must perform a number of functions.First, it is important that the DNA is packaged into a small particle toprotect the DNA and condense it into a smaller particle compatible withcellular uptake. Next, the lipids must enable the transfection process, whichinvolves three key stages: cell attachment and uptake into the cell, endoso-mal escape, and then movement into the nucleus for the expression of thetransferred gene (Fig. 1) (14).

Peptide modification of liposomes offers the potential for enhancingthe packaging process and for enhancing each stage of the lipofectionprocess, to ultimately improve transfection efficiency.

COMPLEX FORMATION

Packaging of plasmid DNA (pDNA) and other nucleic acids into liposomalor other synthetic gene transfer vectors involves a balance between the sta-bility of the vector complex in the extracellular, endosomal, and cytoplasmicenvironments, and its potential to unpackage in the nucleus, releasingthe DNA for decoding to mRNA. The rate of dissociation of nucleicacid–binding elements, or unpackaging, is a major factor that can limittransfection efficiency (15). Lipids used in isolation may be deficient in theirability to form stable, homogeneous particles. For example, freshly formedcationic liposome DNA complexes were shown by freeze-fracture electronmicrographs to adopt elongated structures, implying poor condensation ofDNA (16). Incorporation of polycationic peptide sequences such aspoly-L-lysine and protamine reduced the size of liposome DNA complexby sixfold (17,18). This assists endocytic internalization because theclathrin-coated vesicles are approximately 100 nm in diameter (19). Polyly-sine-condensed DNA complexes show an increased resistance to nucleasedegradation, potentially promoting their survival in the endosome, with69% to 89% of poly-L-lysine–complexed pDNA surviving DNase I treat-ment, while only 19% of uncomplexed DNA remained undegraded (20).

Poly-L-lysine was one of the original reagents used to condense DNA intoa transfecting particle (21) and numerous studies have been widely reportedwith these reagents. Oligolysine peptides as short as (K)16 will also packagepDNA, alone (polyplex) or in combination with lipids (lipopolyplex) (22–26).In both lipopolyplex and polyplex formulations, the DNA is condensed

Gene Transfer by Lipid/Peptide Transfection Complexes 295

within a particle of 100–150 nm (25). The advantages are that shorter poly-mers of lysine have lower cytotoxicity and form smaller particles comparedto high-molecular-weight polylysine derivatives, and have higher transfec-tion efficiency (27).

Peptides with (K)16 domains can also form stable transfection particleswith large DNA molecules including P1 artificial chromosomes of 110 kb(28) and bacterial artificial chromosome (BAC) DNA constructs in a rangeof sizes up to 250 kb (29). The size, determined by atomic forces microscopy, oflipopolyplex particles formed with BAC DNA is directly proportional tothe size of the BAC and transfections performed with equimolar amountsof 100-kb BACs and 8-kb pDNA are similar in efficiency.

Figure 1 Potential points for the enhancement of liposome-mediated gene transfer.The above diagram illustrates the characteristic lipofection pathway demonstratingthe four key stages (bold, underlined), complex formation, targeting and internalization,endosomal escape, and nuclear translocation. Indicated alongside (italic) are the peptidesthat can be used to augment the transfection potential of the liposome. Abbreviation:pDNA, plasmid DNA.

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Other polycationic amino acids have been assessed for gene transferproperties. A comparison of transfection efficiencies of homo-polyaminoacid peptides reported the order of efficiency of polyornithine>poly-L-lysine ¼ poly-D-lysine>polyarginine (30). The study of Ramsay et al. (31)supported these findings, demonstrating that poly-L-ornithine mediatestransfection at least fivefold more efficiently than poly-L-lysine vectors.Ornithine differs from lysine in its shorter side chain of –(CH2)3NH2

compared to –(CH2)4NH2 of lysine. The enhanced efficiency of polyor-nithine reagents may be explained by the higher binding affinity of theornithine reagents compared to lysine polymers and by the smaller, morestable complexes formed with cationic polymers bearing primary aminogroups on shorter side chains (32).

The kinetics of cationic peptide-mediated pDNA condensation andunpackaging have been studied recently (33). Two cationic peptides derivedfrom adenovirus were studied, the 19–amino acid core peptide m (mu), andpepV, a 23–amino acid peptide derived from adenovirus core protein Vcombined with a 7.52 kb plasmid. It was reported that processes of plasmidpacking and expansion resembled protein folding and unfolding, respec-tively. At suboptimal ratios of peptide/pDNA, i.e., partially condensedplasmid, a multitude of complex conformations were observed as it searchedfor the most thermodynamically stable form, i.e., fully condensed pDNA.At higher ratios, thermodynamically stable, condensed pDNA moleculesresulted, which were more difficult to reverse or unpackage. It was proposedthat stable states could limit unpackaging of pDNA in the nucleus and thatmore controlled methods of packaging are required (33).

TARGETING

A great number of peptide-based strategies have been developed to promotecell targeting and binding of liposomes.

Integrins and Receptor-Targeted Vectors

Integrins are a family of more than 20 heterodimeric membrane proteins.The interaction of ligands and integrins allows cellular signal transductionevents, both inward and outward, regulating cellular activities such as theattachment of cells to the basement membrane, cell-to-cell interactions,and motility (34). A number of invasive intracellular pathogens utilize integ-rin receptor uptake to invade cells, such as echovirus (35), foot and mouthdisease virus (36), the enterobacterium Yersina pseudotuberculosis (37), andadenovirus. Approximately half of the integrins bind ligands that containa common tripeptide the residues RGD motif, indicating its importancefor integrin–ligand interaction (38).


Experiments on internalization by Y. pseudotuberculosis demonstratedthat high-affinity integrin-binding ligands are required to achieve both bind-ing and internalization (37). The variable binding affinities of RGDsequences to different integrin receptors depends on the peptide sequencesurrounding the RGD. This effect can be further enhanced by producinga cyclized peptide rather than a linear one in which the peptide adopts acyclic form due to intramolecular cysteine bridging. This reduces the free-dom of movement within the peptide, in doing so increasing the specificityof the required match between the ligand and the receptor (24,38,39).

The level of receptor expression also influences the ability of the ligandto promote internalization with a high surface density of cell surfacereceptors promoting greater levels of uptake (40). The profile of integrinexpression and activation may be altered during pathogenic processes suchas lung inflammation, wound healing, and cancer (41,43–46), opening up awindow of opportunity for integrin-mediated targeting specificity.

Integrin receptor–binding peptides have been used to enhance lipo-some binding, uptake, and expression (25,47–49). The inclusion of ana5b1 integrin–targeted peptide into a liposomal complex enhanced transfec-tion efficiency four- to five-fold in Jurkat cells and 10- to 13-fold in TF-1cells (48). Confocal and electron microscopy revealed that the mechanismof cell entry conferred by RGD peptides on liposomes is predominantlyby clathrin-coated endocytosis rather than by phagocytosis (50).

Phage Panning

Ligands that bind cell surfaces can be tested for their affinity to mediate cellularinternalization and can be assessed using filamentous phage (fd) display (22,51).The coding sequence of the peptide being tested is fused with that of the majorcoat protein gene VIII within the genome of the fd phage. This results in the tubu-lar capsid of the virus being coated in numerous subunits of the fusion protein,thus allowing substantial opportunity for the cell and ligand to interact (22,51).

Libraries of random peptide sequences are commercially available andcan be used to screen or ‘‘biopan’’ for candidate ligands for internalization.The screen can initially involve 109 potential peptides, each expressed exclu-sively on the surface of a phage. The specifics of the methodology can varybut a typical method of phage screening is as follows. The phages are addedto the cells and allowed to adhere to the cell surface but are prevented frominternalizing by incubation at 4�C (52–54). The cells then undergo a series ofwashes to remove unbound and low-affinity phages. Finally, the boundphages are eluted from the cells and amplified in bacteria (52–54). Bindingaffinity does not always correlate with the ability of the ligand to induceinternalization because this is also related to the nature and rate of activityof the receptor it interacts with. The uptake of phage can be assayed byimmunofluorescence using confocal microscopy (22,51).

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The panning can then be repeated with the phages selected from theprevious round and along with increased stringency washes peptides of evengreater binding affinity can be selected (54). The advantage of using thephage display is that prior knowledge of the receptor is unnecessary andthe peptide can be selected for a specific cell type. High-affinity ligands havebeen identified for a number of cell types using phage screen includingendothelial cells (55,56), smooth muscle cells (57), and human airway epithe-lial cells (54,58–60). A similar screening technique can be done in vivo,through rounds of phage injection, purification, and amplification to selectpeptide ligands specific to particular organs (61).

A peptide selected by phage screening on human airway epithelialcells demonstrated a 10-fold higher transfection efficiency compared to anintegrin-targeted lipopolyplex vector (24,54). The scrambled version of thispeptide had substantially reduced receptor-binding activity; hence the activ-ity was sequence specific (54). Of interest, it has emerged that a number ofthe peptides selected in this particular study resembled receptor-bindingmolecules expressed by viral and bacterial pathogens such as herpes simplexvirus, rotavirus, Mycoplasma pneumoniae, and rhinovirus (54).

Uptake

The entry of liposome-based gene delivery vectors has been suggestedto occur through direct fusion with the plasma membrane (8). However,experimental evidence has failed to support this notion (62). It has beenhypothesized that the universal cell ancestor would require the ready entryof external DNA through the plasma membrane to allow for fast adaptation;however, the unregulated transfer of genetic material would endanger geneticdiversity as evolution progressed (63). Furthermore, the membrane wouldhave to protect the specialized constituents of the intracellular environmentfrom being corrupted by that of the extracellular (63). Because the cell has ademand for external compounds for signaling and metabolic purposes, car-rier mechanisms are required to internalize essential macromolecules (63).DNA-bearing vectors also exploit these pathways and the two principalpathways utilized and mentioned throughout this chapter are the clathrin-mediated and the caveolae-mediated mechanisms of endocytosis (Fig. 2) (62).

Clathrin-mediated endocytosis involves the internalization of trans-membrane receptor–ligand complexes stimulating the formation of a coatedpit that eventually buds off the membrane to form an intracellular endocy-totic vesicle. This process is dependent on the protein clathrin that isrecruited to the membrane and forms a cage-like structure around the form-ing pit. Internalization via clathrin-dependent pathway allows the uptakeof particles approximately 120 nm in size (63–65). Once internalized, theclathrin coating disassociates from the endosome to be recycled and toallow the endosome to fuse with an intracellular compartment, usually a


lysosome (66). Examples of these pathways include the uptake of low-den-sity lipoproteins and transferrin (64).

Caveolae-dependent endocytosis occurs in cholesterol- and sphingo-myelin-rich flask-shaped invaginations of the cell membrane known ascaveolae (67). The shape is determined by a framework constructed by theprotein caveolin. The protein binds plasma cholesterol, inserting into

Figure 2 Proposed pathways for liposomal entry into the cell enhanced by peptides.These include direct cell entry suggested as the mechanism of entry by cell-penetrat-ing peptides and receptor-mediated endocytosis by caveolae- and clathrin-dependentendocytosis.

300 Irvine et al.

the membrane as a loop into the inner leaflet of the plasma membrane and thenself-associates, giving a striated appearance on the surface of the membraneinvaginations (64). It is thought that this protein stabilizes the caveolae to pre-vent unregulated budding from the membrane (65) and that the caveolae areinvolved in the uptake of serum albumin and lipids (68). Internalization is pro-moted by the receptor tyrosine phosphorylation of the caveolae, such as theserum albumin receptor gp60 (69), leading to the formation of a cytoplasmicvesicle known as a caveosome. Caveosomes can internalize particles with adiameter of approximately 60 nm (64). Experiments have demonstrated a rolefor caveolae-dependent uptake in the transportation of particles to organellessuch as the Golgi apparatus and in the regulation of receptor turnover bytargeting receptors for degradation. Both these processes are energy dependent,requiring the action of the GTPase dynamin (70).

Cell-Penetrating Peptides

Cell-penetrating peptides (CPPs), also referred to as protein-transductingdomains, facilitate the entry of cargo molecules through the cell membrane,allowing their efficient translocation and migration into the perinuclearzone. CPPs have been used to facilitate the cellular uptake of a range ofmolecular cargos such as DNA, tumoricidal antibodies, imaging agents,and liposomes (71–73).

An example of this class of peptide is the 86–amino acid trans-activatingtranscriptional activator (TAT) of HIV-1 (74,75). Following incubation withcultured cells, TAT protein is internalized and subsequently transactivatesviral promoters (75). The protein has multiple facets: invasion, nuclear trophism,and DNA binding (76–81). An invasion domain of TAT has been identifiedwithin amino acids 37 to 72 with the critical basic region from amino acids(49 to 57), also known as the ‘‘minimal transduction domain,’’ which consistsof the sequence –Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg–. Any deletion inthe sequence caused a reduction in translocating activity (82–84). Other promi-nent CPPs are reviewed in References 73 and 85.

A common characteristic of CPPs is that they contain a minimal motifof less than 20 amino acids rich in basic residues. There are two subclasses ofCPPs; the first class consists of amphipathic helical peptides, where lysine isthe predominant supplier of the positive charge, for example model amphi-pathic peptide (MAP) and transportan, whereas the second group, whichincludes TAT and penetratin, is rich in arginine residues (48–60,86).

Substantial work has been undertaken on the potential of the CPPpeptides TAT and Antp/penetratin for gene delivery. Some workers havefound that TAT alone has the ability to promote DNA transfer (80,87)but their combination with liposomes appears to produce a more robustvector system. For example, a 15- to 25-fold increase in uptake was reportedin liposomes modified with either TAT or the drosophila CPP, Antp, in


comparison to unmodified liposomes across a diverse panel of cell types(88). Other groups have also demonstrated the combination of TAT withcationic liposomes to be an effective vector system (89–91) while Antp-derivatized liposomes demonstrated a 50-fold increase in internalization (92).

It is likely that the predominantly positively charged amino acids of TATand other CPPs will interact with anionic components on the surface of the cellmembrane (85). This idea is supported by the observation that cell associationwith CPP liposomes in glycosaminoglycan-deficient chinese hamster ovary(CHO) cells is greatly reduced and is competitively inhibited by the presenceof heparin (88,93). Furthermore, the removal of the heparan sulfate chainsby the action of glycosaminoglycan lyase also suppressed the transduction ofthe TAT protein (94).

TAT liposomes remain intact within one hour of translocation andslowly migrate through the cell, bypassing the endocytic pathway, to theperinuclear zone where they disintegrate (95). The mechanism utilized byTAT to migrate across the membrane was thought to be energy independentbecause it operates at similar rates at both 4�C and 37�C (95,96). Cell entryby TAT is also unhindered by metabolic inhibitors such as sodium azide oriodoacetamide (97). Peptides constructed of both the D and L amino acids ofAntp can be detected intracellularly, the inference of which is that no speci-fic receptor was required because both isomers had equal potential (98,99).

However, the view that CPP uptake is energy independent and bypassesthe endosomal pathway is not universally accepted. Chloroquine, an inhibitorof endosomal acidification, increases TAT liposomal uptake and low tempera-tures reduce the transactivation potential of the complex (85–91). Moreover,experiments on green fluorescent protein (GFP)-conjugated TAT and anotherCPP, VP22, suggested that the CPPs were taken up by classical endocytosisand the authors suggested that the countertheory of nonendocytic uptake isa result of artifacts generated during fixing and staining of samples (85,91).Further studies on the fluorochrome-labeled unconjugated peptide demon-strated that TAT enters the cell predominantly by clathrin-dependentendocytosis via heparan sulfate receptors, although inhibition of this pathwayonly partially halts the uptake, indicating other pathways are also involved(91). TAT-modified liposomes observed in living confocal microscopy studiesrevealed that binding of the TAT liposome to the plasma membrane is fol-lowed by intracellular uptake into endocytic vesicles (89).

It has also been proposed that CPP are taken up by clathrin-independent mechanisms such as caveolin-mediated endocytosis (100).TAT-containing liposomes were colocalized with caveolin 1, a marker forcaveolar endocytosis, but not with markers for clathrin (101). An inhibitorof caveolin and nystatin reduced TAT peptide reporter in HepG2 and CHOcells by 50% (80). However, it was pointed out by Brooks et al. that HepG2cell lines do not contain caveolin 1 (102,103). The caveolin pathways’ rele-vance to CPP uptake may depend on the cell type because nystatin inhibits

302 Irvine et al.

by 50% in some cells but has no detectable effect on others, such as buffalogreen monkey cells (80). Furthermore, caveolin-null or -deficient cells havebeen found to support efficient uptake of TAT, hence indicating that thispathway is nonessential (91).

Therefore, evidence exists to support numerous possible mechanismsfor the uptake promoted by TAT peptides. An alternative explanation is thatTAT is a ‘‘sticky opportunistic peptide’’ that has the ability to bind to the cellsurface and exploit multiple mechanisms in order to enter the cytoplasm (92).Clearly, the mechanism of internalization requires further study. Neverthe-less, it has been observed that transfection with CPPs requires less lipid andtherefore proves to be less cytotoxic to cells in vitro and in vivo, making ita promising vector system for future gene therapy (95,104).

Haptides

The enhancement of lipofection via non–receptor-mediated, energy-inde-pendent pathways has also been ascribed to haptotactic peptides (haptides)derived from sequences on the C-termini of the fibrinogen b chain (Cb),c chain (preCc), and the extended aE chain of fibrinogen (CaE) (105). Thesepartially hydrophobic, cationic peptides of 19 to 21 amino acids coat theouter surface of anionic liposomes, changing their zeta potential (106).Premixing fluorescent rhodamine-containing liposomes or doxorubicin-containing liposomes with Cb, preCc, and, to a lesser extent, CaEsignificantly enhanced their cellular uptake (106).

The amino acid sequences of haptides comprise hydrophobic and cat-ionic residues with a net charge of þ4 to þ5 per 19 to 21 amino acids. It wasproposed that haptides could be attracted to the anionic liposomes as wellas the anionic cell membrane and that the hydrophobic properties of thehaptide facilitate membrane translocation (106). Haptide uptake wasreported to be energy independent, occurring at 4�C. The advantage of thispeptide compared to CPP such as TAT and Antp, is that, unlike the virus-derived peptides, the haptides are not recognized as foreign antigens anddo not induce cell transformation (106). However, haptides have also beenfound to accelerate fibrin clot formation and lack cell specificity (106).

Endosome Disruption

The inhibition of endosomal degradation or enhancement of endosomalescape by liposomes is an established strategy to enhance lipid-mediatedtransfection (Fig. 3).

Fusogenic peptides, derived from viral sources, are particularly wellcharacterized (107–111). The amphipathic peptide from the N-terminalregion of the hemagglutinin (HA)-2 subunit of HA was one of the first suchpeptides described, and, subsequently, a range of influenza-derived peptides


were described (110). HA peptides have been exploited for liposomal genetransfer (112,113).

A synthetic peptide has been designed to mimic the effects of viralfusogenic properties (114,115). It consists of 30 amino acids with the majorrepeat of Glu-Ala-Leu-Ala; so, it is referred to as a GALA peptide. It under-goes a conversion from an aperiodic conformation at neutral pH and becomesan amphipathic alpha helix at pH 5. In the more acidic environment,the peptide interacts with lipid bilayers (114,115). GALA has been incorpo-rated into transferrin-targeted liposome, with the effect of significantly

Figure 3 Endosomal escape assisted by fusogenic peptides. These peptides assist therelease of DNA from the endosome, avoiding degradative damage from the bindingwith the lysosome.

304 Irvine et al.

increasing the release of plasmid into the cytosol (116). Through mechan-isms involving the receptor-mediated uptake of ligand-bound liposomeinto a clathrin-coated vesicle, there is the risk that the strong associationbetween ligand and receptor within the endosome will prevent the releaseof the complex because, in the natural state, both are recycled to the surface.Hence in such circumstances, a fusogenic peptide might be of considerablebenefit (117,118).

An example of a potential fusogenic peptide to facilitate lipofection isthat of the N terminal region of surfactant protein B (SPB). This protein is acomponent of the pulmonary surfactant mix of phospholipids and proteinswith the function of reducing surface tension at the air–liquid interface, withthe primary purpose of preventing alveolar collapse (119). SPB is expressedas a 381–amino acid form that is processed to a mature 79–amino acid form(120). The interaction with lipid bilayers occurs through five amphipathica helices (121). Sequence alignments have revealed that SPB has homologyto a number of membrane-interacting saposin-like proteins (122).

The positively charged amino acids promote the interaction betweenthe peptide and the negatively charged head groups of the phosphatidylgly-cerol (123–125). The purpose of this particular property has been proposedto facilitate the transition of surfactant phospholipid membranes from thelamellar body to the alveolar spaces (123).

This interaction with liposomes results in destabilization and fusion(126–128). Following membrane binding of the SPB protein, there is a lossof vesicular contents in a dose-dependent manner, suggesting a loss of vesic-ular integrity (129–132).

It was found that the minimal region capable of supporting maximalfusogenic activity was contained within residues 1 to 37, which consists ofthe N-terminal and helices 1 and 2. Any addition or reduction to thissequence has the effect of reducing the fusogenic properties of the peptide(123). The incorporation of a 25–amino acid sequence of the N terminusif SPB into a liposomal vector significantly raised the efficiency of genetransfer upon microinjection into chicken embryo in vivo, in that lessDNA complex was required to obtain maximal luciferase activity (133).With low concentrations of the SPB peptide, such as 50:1 lipid:peptide ratio,8 mg of DNA was required per well, whereas at 5:1 ratio only 4 mg of DNAwas required (133). It may be the case that, as reported by Ref. 123, thelonger 37–amino acid peptide may have conferred an even greater enhance-ment of gene transfer.

NUCLEAR LOCALIZATION SEQUENCE

The end stage of liposomal gene transfer is the entry of the DNA complexinto the nucleus of the cell to allow gene transcription (134,135), and entry tothe nucleus can by attained following the breakdown of the nuclear envelope


during mitosis. This limits the use of lipofection to the gene therapy of con-ditions involving proliferating cells, while hampering genetic interventiondiseases of nonmitogenic cells such as neurons (136). It has been observedalso that uptake of DNA into a cell does not always correlate with eventualreporter gene activity. Hence the ability of the plasmid to access the nucleusis an important consideration in designing a vector complex (Fig. 4) (137).

Nontargeted entry into the nucleoplasm of a cell is limited to com-plexes or molecules of less than 50 KDa in diameter due to the size of thenuclear pore complex, which is 9 nm (138–140). Nevertheless, larger mole-cules can also enter the nucleus, but through energy-dependent transportmechanisms. This system is necessary for selectively transporting nucleopro-teins after their synthesis in the cytosol, into the nucleus. These proteins aresynthesized with a nuclear localization sequence (NLS) allowing the recogni-tion and activation of the pathway (138–140). The sequence is recognized byand binds to the nuclear pore receptor consisting of Importin a and breceptor, which carries the NLS-associated molecule through the nuclearpore into the nucleosome by an energy-dependent process (141).

Inclusion of an NLS consensus peptide into a lipoplex renders non-dividing cells susceptible to gene transfection. There is dramatic improvement

Figure 4 Transfer of DNA from cytoplasm into the nucleoplasm. The DNA-containing complex can enter the nucleus by (1) crossing the membrane duringmitotic nuclear membrane breakdown; (2) diffusion through nuclear pore for smallparticles; and (3) targeted uptake through the nuclear pore, facilitated by a nuclearlocalization sequence. Abbreviations: NLS, nuclear localization sequence; IMP,importin.

306 Irvine et al.

in lipofection of confluent bovine aortic endothelium by the use of SV40concensus T-antigen (Sct), an NLS constructed from the M9 sequence ofheterogeneous nuclear riboprotein to the scrambled sequence of the SV-40large tumor antigen (T-ag). This complex can result in a transfection effi-ciency of 83% and a 63-fold increase in reporter gene expression (142).Vaysse et al. recorded an increase in lipofected reporter gene expressionbetween 4- and 16-fold when employing an SV-40–derived NLS peptidein both primary and growth-arrested cells. This was a mitosis-independentbut energy-dependent process that led to increased accumulation of DNAin the nucleus, indicating NLS activity (143).

There are a large variety of NLS proteins, both cellular and viral (144).A common feature of a number of these peptides is the high presence oflysine residues, in particular the SV-40 T-ag, which consists of the residuesPKKKRKV (144–148). The involvement of lysine-rich residues in nucleartargeting developed the hypothesis that part of the transfection-enhancingpotential of the DNA-condensing peptide poly-L-lysines may be due NLSproperties of the peptide. The addition of an oligolysine-RGD peptide intoa LipofectAMINE DNA complex led to an increase in pDNA movementto the nucleus, which was inhibited by wheat germ agglutinin, an inhibitorof the NPC, and also blocked by an antibody to the NPC, thus indicating anadditional role for the peptide (148).

In common with the polylysine DNA-condensing peptide, the m peptidehas also been shown to have nuclear localization properties. Confocal micro-scopy revealed that m peptide in a complex containing fluorescent lipid- anddye-labelled DNA associates with the nuclei and nucleoli of both dividingand nondividing cells within 15 minutes of exposure to the complex, thus sug-gesting an NLS function. However, this property was not detectable when thepeptide became incorporated into a 3b-[N-(N0,N0-dimethylaminoethane)-carbamoyl]-cholesterol (DC-chol)/DOPE cationic liposome (149). It maybe the case that the lipids may mask the critical residues.

SUMMARY

Liposomes can be modified in numerous fashions by the addition of peptidesequences. The benefit of this is that the inclusion of the peptide allowsthe lipoplex to be optimized for its task such as the targeting a specific celltype with a specific receptor ligand sequence or the transfection of nondivid-ing cells, with an NLS.

Peptides can also help overcome the most significant drawback tousing liposome vectors when compared to viral vectors, which is lowertransfection efficiency. Additional benefits include promotion of compac-tion, assisting cellular uptake of the DNA. Even peptides derived fromviruses themselves can be used to compensate this deficit (e.g., adenovirusm protein and the HIV TAT).


Peptide modification of liposomes has been found to enhance genetransfer up to a 1000-fold (150). They have potential as an equally activeand yet safer alternatives to the viral vectors.

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95. Torchilin VP, Levchenko TS, Rammohan R, et al. Cell transfection in vitro andin vivo with nontoxic TAT peptide-liposome-DNA complexes. Proc Natl AcadSci USA 2003; 100(4):1972–1977.

96. Thoren PE, Persson D, Isakson P, et al. Uptake of analogs of penetratin,Tat(48–60) and oligoarginine in live cells. Biochem Biophys Res Commun2003; 307(1):100–107.

97. Torchilin VP, Rammohan R, Weissig V, et al. TAT peptide on the surface ofliposomes affords their efficient intracellular delivery even at low temperatureand in the presence of metabolic inhibitors. Proc Natl Acad Sci USA 2001;98(15):8786–8791.

98. Futaki S, Suzuki T, Ohashi W, et al. Arginine-rich peptides. An abundantsource of membrane-permeable peptides having potential as carriers for intra-cellular protein delivery. J Biol Chem 2001; 276(8):5836–5840.

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100. Ferrari A, Pellegrini V, Arcangeli C, et al. Caveolae-mediated internalization ofextracellular HIV-1 tat fusion proteins visualized in real time. Mol Ther 2003;8(2):284–294.

101. Fittipaldi A, Ferrari A, Zoppe M, et al. Cell membrane lipid rafts mediatecaveolar endocytosis of HIV-1 Tat fusion proteins. J Biol Chem 2003;278(36):34141–34149.


102. Brooks H, Lebleu B, Vives E. Tat peptide-mediated cellular delivery: back tobasics. Adv Drug Deliv Rev 2005; 57(4):559–577.

103. Fujimoto T, Kogo H, Nomura R, et al. Isoforms of caveolin-1 and caveolarstructure. J Cell Sci 2000; 113(Pt 19):3509–3517.

104. Torchilin VP, Levchenko TS. TAT-liposomes: a novel intracellular drugcarrier. Curr Protein Pept Sci 2003; 4(2):133–140.

105. Gorodetsky R, Vexler A, Shamir M, et al. New cell attachment peptidesequences from conserved epitopes in the carboxy termini of fibrinogen. ExpCell Res 2003; 287(1):116–129.

106. Gorodetsky R, Levdansky L, Vexler A, et al. Liposome transduction into cellsenhanced by haptotactic peptides (haptides) homologous to fibrinogen C-ter-mini. J Control Release 2004; 95(3):477–488.

107. Subramanian A, Ma H, Dahl KN, et al. Adenovirus or HA-2 fusogenic pep-tide-assisted lipofection increases cytoplasmic levels of plasmid in nondividingendothelium with little enhancement of transgene expression. J Gene Med2002; 4(1):75–83.

108. Wharton SA, Martin SR, Ruigrok RW, et al. Membrane fusion by peptide ana-logues of influenza virus haemagglutinin. J Gen Virol 1988; 69(Pt 8):1847–1857.

109. Plank C, Oberhauser B, Mechtler K, et al. The influence of endosome-disruptive peptides on gene transfer using synthetic virus-like gene transfersystems. J Bio Chem 1994; 269(17):12918–12924.

110. Wagner E. Application of membrane-active peptides for nonviral gene delivery.Adv Drug Deliv Rev 1999; 38:279–289.

111. Wagner E, Plank C, Zatloukal K, et al. Influenza virus hemagglutinin HA-2N-terminal fusogenic peptides augment gene transfer by transferrin-polylysine-DNA complexes: toward a synthetic virus-like gene-transfer vehicle. Proc NatlAcad Sci USA 1992; 89(17):7934–7938.

112. Simoes S, Slepushkin V, Duzgunes N, et al. On the mechanisms of internaliza-tion and intracellular delivery mediated by pH-sensitive liposomes. BiochimBiophys Acta 2001; 1515:23–37.

113. Simoes S, Slepushkin V, Gaspar R, et al. Gene delivery by negatively chargedternary complexes of DNA, cationic liposomes and transferrin or fusigenic pep-tides. J Biol Chem 1998; 5(7):955–964.

114. Parente RA, Nir S, Szoka FC Jr. pH-dependent fusion of phosphatidylcholinesmall vesicles. Induction by a synthetic amphipathic peptide. J Biol Chem 1988;263:4724–4730.

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132. Poelma DL, Zimmermann LJ, Scholten HH, et al. In vivo and in vitro uptakeof surfactant lipids by alveolar type II cells and macrophages. Am J PhysiolLung Cell Mol Physiol 2002; 283(3):L648–L654.

133. Longmuir KJ, Haynes SM, Dickinson ME, et al. Optimization of a peptide/non-cationic lipid gene delivery system for effective microinjection into chickenembryo in vivo. Mol Ther 2001; 4:66–74.

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143. Vaysse L, Harbottle R, Bigger B, et al. Development of a self-assemblingnuclear targeting vector system based on the tetracycline repressor protein.J Biol Chem 2004; 279(7):5555–5564.

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146. Reed MW, et al. Synthesis and evaluation of nuclear targeting peptide-antisense oligodeoxynucleotide conjugates. Bioconjug Chem 1995; 6:101–108.

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148. Colin M, Moritz S, Fontanges P, et al. The nuclear pore complex is involved innuclear transfer of plasmid DNA condensed with an oligolysine-RGD peptidecontaining nuclear localisation properties. J Biol Chem 2001; 8(21):1643–1653.

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18

Phospholipid- and Nonphospholipid-Based Vesicles for Drug and DNADelivery to Mitochondria in Living

Mammalian Cells

Volkmar Weissig, Sarathi V. Boddapati, Shing-Ming Cheng,Gerard G. M. D’Souza, and Vladimir P. Torchilin

Department of Pharmaceutical Sciences, School of Pharmacy,Bouve College of Health Sciences, Northeastern University,


INTRODUCTION

Mitochondria as Pharmacological Targets

The mitochondrion is an essential organelle for all eukaryotic cells. Thenumber of mitochondria per single cell depends on its energy demand. Meta-bolically active organs such as the liver, the brain, and cardiac and skeletalmuscle tissues may contain up to several thousands of mitochondria per cell,whereas somatic tissues with a lower demand for energy contain only a fewdozen of these organelles. Each mitochondrion is composed of two mem-branes, which together create two separate compartments, the matrix spaceand the intermembrane space. The outer membrane is permeable to mole-cules smaller than 5 kDa, whereas the inner membrane is highly impermeableand characterized by an unusually high content of membrane proteins aswell as a unique lipid composition. The mitochondrial inner membraneproteins are components of the respiratory chain and a large number of

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transport proteins. The impermeability of the inner membrane is a prerequi-site for the establishment of an imbalance in the distribution of protonsbetween the mitochondrial matrix and the cytosol, which in turn is the driv-ing force for the synthesis of adenosine triphosphate. Mitochondria areunique in comparison to all other organelles as they contain their owngenome (mtDNA) and the necessary transcription and translation systems.Human mtDNA is a circular molecule 16,569 bp in size; it encodes tworibosomal RNAs, all 22 tRNAs necessary for protein synthesis at mitochon-drial ribosomes, and 13 polypeptides that are all subunits of the oxidativephosphorylation (OXPHOS) enzyme complexes.

As the ‘‘power house’’ of the cell, the mitochondrion is essential forenergy metabolism. As the ‘‘motor of cell death’’ (1), this organelle is centralto the initiation and regulation of apoptosis. In addition, mitochondria arecritically involved in the modulation of intracellular calcium concentrationand the mitochondrial respiratory chain is the major source of damagingreactive oxygen species. Mitochondria also play a crucial role in numerouscatabolic and anabolic cellular pathways.

Mitochondrial dysfunction either causes or at least contributes to alarge number of human diseases. Malfunctioning mitochondria are foundin several adult-onset diseases, including diabetes, cardiomyopathy, inferti-lity, migraine, blindness, deafness, kidney and liver diseases, and stroke. Theaccumulation of somatic mutations in the mitochondrial genome has beensuggested to be involved in aging, in age-related neurodegenerative diseases,as well as in cancer. Also, an increasing number of xenobiotics and pharma-ceuticals are being recognized to manifest their toxicity by interfering withmitochondrial functions.

Sparked off by key discoveries in 1988 revealing the link betweenmtDNA mutations and human diseases (2,3) and in the early 1990s invo-lving the role mitochondria play in programmed cell death (4–7), thisorganelle is increasingly recognized as a prime target for pharmacologicalintervention (8). In particular, the mitochondrial permeability transitionpore complex, a multiprotein complex formed at the contact site betweenthe mitochondrial inner and outer membranes, is widely accepted as beingcentral to the process of cell death and therefore presents a privileged phar-macological target for cytoprotective and cytotoxic therapies (4).

Significant pharmacological and pharmaceutical efforts toward thetreatment of mitochondrial diseases undertaken during the last decade haveled to the emergence of ‘‘mitochondrial medicine’’ as a whole new field ofbiomedical research (9,10). Technologies that allow the targeting of bothsmall drug molecules and large macromolecules to and into mitochondriawill eventually lead to a large variety of cytoprotective and cytotoxictherapies. The delivery of therapeutic DNA and RNA such as antisense oli-gonucleotides, ribozymes, plasmid DNA expressing mitochondrial encodedgenes, and wild-type mtDNA may provide the basis for treatment of

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mitochondrial DNA diseases; the targeting of antioxidants into the mito-chondrial matrix may protect mitochondria from oxidative stress andperhaps even contribute to slowing down the natural aging process; themitochondria-specific targeting of naturally occurring toxins or syntheticdrugs such as photosensitizers may open up avenues for new anticancertherapies. Moreover, delivering molecules known to trigger apoptosis bydirectly acting on mitochondria may overcome the apoptosis resistanceof many cancer cells. Drugs designed to target mitochondrial uncouplingproteins may become a basis for treating obesity.

The Mitochondrial Membrane Potential andMitochondriotropic Molecules

During OXPHOS, electrons from the hydrogens on nicotinamide adeninedinucleotide (NADH) and FADH2 are carried along the respiratory chainat the mitochondrial inner membrane, thereby releasing redox energy that isused to translocate protons across the inner membrane from the mitochondrialmatrix into the intermembrane space. This process creates a transmembraneelectrochemical gradient, which includes contributions from both a mem-brane potential (negative inside) and a pH difference (acidic outside). Themembrane potential of mitochondria in vitro can be as high as 180 mV, whichis the maximum a lipid bilayer can sustain while maintaining its integrity (11).Although metabolic processes reduce this potential in living cells to about130 to 150 mV (9), it is by far still the largest within cells.

Given appropriate physicochemical properties, positively charged mole-cules can accumulate inside mitochondria in response to the highly negativemembrane potential. The mitochondrial accumulation of such molecules,generally referred to as ‘‘mitochondriotropics,’’ is mainly governed by theirlipophilicity (log Kow), their acid/base dissociation constant (pKa), and theirelectric charge (z). Using a theoretical model for the accumulation of chemi-cals in tumor mitochondria, Trapp and Horobin (12) recently determinedthe optimal parameters for, and possible mechanisms of, mitochondrialuptake. They found that both cations and anions can accumulate in mito-chondria. In general, accumulation of cations required the compounds to belipophilic and a permanent cation, or have a pKa value >11. Uptake involvedelectrical attraction and, for the more lipophilic compounds, partitioning.However, selective uptake into mitochondria of transformed cells occurreddue to electrical effects with slightly hydrophilic or lipophilic (log Kow between�2 andþ2) monocations of strong bases. Uptake of anions can also occur withlipophilic weak acids, by an ion trap mechanism. Trapp and Horobin’s model,which was based on the Fick–Nernst–Planck equation , correctly predicted themitochondrial accumulation of well-known mitochondriotropic molecules.

Figure 1 shows the chemical structure of representative mitochondrio-tropic molecules. The most widely used among them is Rhodamine 123

Phospholipid- and Nonphospholipid-Based Vesicles 319

(Fig. 1, compound A), which has been used extensively as a stain for mito-chondria in living cells since its introduction in 1982 (13). As early as in 1969methyltriphenylphosphonium salts (Fig. 1, compound B) were demon-strated to be taken up rapidly by mitochondria in living cells (14) andthe mitochondrial accumulation of dequalinium chloride (Fig. 1, com-pound C) was established during the 1980s (15). As can be seen fromFigure 1, another typical structural feature mitochondriotropics share,and which has not been included into the Trapp and Horobin model, is thatin many structures the p-electron charge density extends over at least threeatoms or more instead of being limited to the internuclear region betweenthe heteroatom and the adjacent carbon atom. This causes a distributionof the positive charge density between two or more atoms, i.e., the positivecharge is delocalized. Such molecules are referred to as ‘‘delocalizedcations.’’ Sufficient lipophilicity, combined with delocalization of their posi-tive charge to reduce the free energy change when moving from an aqueousto a hydrophobic environment, have been thought already 20 years ago tobe prerequisites for their mitochondrial accumulation in response to themitochondrial membrane potential (15).

Mitochondria-Specific Delivery Systems

Strategies for the design of mitochondria-targeted drug and DNA deliverysystems and the principles such systems are based upon have been reviewedearlier by us comprehensively (16–18). Therefore, the scope of this chaptershall be limited exclusively to approaches involving mitochondriotropicmolecules–mediated drug and DNA delivery to mammalian mitochondriain response to the mitochondrial membrane potential.

The term ‘‘stoichiometric carriers’’ in Figure 2 refers to covalentconjugates, which are composed of biologically active molecules and themitochondriotropic triphenylphosphonium (TPP) cation. This mitochondria-targeted drug delivery system has been pioneered by Murphy and coworkers.Since the middle of the 1990s, they have synthesized a large variety ofstoichiometric conjugates by linking for example vitamin E, ubiquinol, anti-biotics, or peptide nucleic acids to an alkyl derivative of the TPP cation

Figure 1 Chemical structures of commonly used typical mitochondriotropic mole-cules: (A) rhodamine 123; (B) methyltriphenylphosphonium; (C) dequalinium chloride.

320 Weissig et al.

(for methyl-TPP see Fig. 2) in order to probe, prevent, or alleviate mitochon-drial dysfunction (19–23). In a series of extensive in vitro studies performedby Murphy and coworkers, bioactive molecules linked to TPP were shown toaccumulate up to several hundredfold inside mitochondria in comparisonto the corresponding native, i.e., free bioactive molecules. More recently theyalso tested the potential of TPP as a mitochondria-specific drug carrier forin vivo administrations by investigating the mode of delivery, tissue distribu-tion, and clearance of three different TPP conjugates within mice (19). Theycould show that relatively high doses of TPP conjugates can be fed safely tothe animals over long periods of time resulting in steady-state distributionswithin heart, brain, liver, and muscle. Moreover, TPP conjugates were alsodetectable in fetuses and neonates following oral administration to pregnantor lactating dams. The intramitochondrial accumulation of TPP conjugatesin vivo was demonstrated following intravenous injection of a thiol reactiveTPP derivative, which was able to bind covalently to protein thiols inside themitochondrial matrix. In summary, as result of a remarkable line of workover the last decade, Murphy and coworkers have shown the feasibility ofdelivering by simple oral administration small molecules selectively tomitochondria in organs mostly affected by mitochondrial diseases, i.e., brain,heart, and muscle. Detailed pharmacokinetic studies of TPP conjugates areongoing (19).

A potential (but general) drawback of the use of stoichiometric carriers isthe need for covalent linkage between carrier and bioactive molecule, whichmay influence its biological activity. Also, on its way to the mitochondria,

Figure 2 Schematic overview of membrane potential–based strategies for the tar-geted delivery of bioactive molecules to mitochondria in living mammalian cells.


the bioactive entity remains accessible to enzymatic degradation or any othernonspecific interactions with tissue or cell components. These problems can beavoided by the use of ‘‘vesicular carriers’’ (Fig. 2). However, whilst encapsula-tion of bioactive molecules into mitochondria-targeted colloidal vesicles wouldprovide protection and would be without impact on the biological activity, anymitochondrial uptake of vesicular drug carriers appears highly improbableand not even desirable in the interest of maintaining mitochondrial integrity.The overall strategic goal for the development of mitochondria-specific vesiclesis therefore the selective delivery of encapsulated drugs or DNA to the site ofmitochondria, while at the same time protecting the bioactive entities frompremature systemic elimination, metabolism, or any other interactions withtissue- and cell-specific biomolecules. Such highly selective organelle-specifictargeting should significantly increase the therapeutic index of any drugintended to act on mitochondrial targets. Upon reaching the mitochondrialouter membrane, the carrier system then has to become destabilized or hasto disintegrate in order to release its cargo. We have described such vesicularmitochondria-specific carriers for the first time in 1998 (24) and we havedemonstrated their suitability for mitochondria-specific drug and DNA deli-very during the last five years. In the following, mitochondria-specific vesicularcarriers and their application for drug and DNA delivery to mitochondriawithin living mammalian cells will be described and discussed.

MITOCHONDRIOTROPIC LIPOSOMES

For the design of mitochondriotropic liposomes, we have used a method, thathas been a standard procedure in liposome technology for over 30 years:the lipid-mediated anchoring of artificially hydrophobized water-solublemolecules into liposomal membranes (25–28). We have hydrophobizedmitochondriotropic TPP cations by conjugating them to long alkyl residues;specifically, we have synthesized stearyl TPP (STPP) salts (29). Followingliposome preparation in the presence of STPP, the liposomal surface becamecovalently modified with TPP cations, thereby rendering these liposomes mito-chondriotropic as verified in vitro by fluorescence microscopy (30).

Synthesis of Stearyl Triphenylphosphonium Bromide

Stearyl bromide (5.5 mmol) and triphenylphosphine (5.8 mmol) were heatedunder reflux for 20 hours in 30 mL anhydrous xylene. Upon completion ofthe reaction (as monitored by thin-layer chromatography) the solvent wasremoved followed by purifying the obtained crude yellowish oil by silicagel column chromatography using methanol:chloroform (5:95) as an eluent.Purified STPP, obtained as colorless oil, crystallized on standing and wasrecrystallized from ether to yield pure STPP in 35% to 45% yields.

STPP can be identified by 1H nuclear magnetic resonance (NMR)(CDCl3) and 31P NMR (with external 85% H3PO4 as reference): 1H NMR:

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7.68 to 7.88 (m, 15H), 3.7 to 3.8 (m, 2H), 1.55 to 1.65 (t, 4H), 1.2 to 1.35 (m,28H), 0.7 to 0.8 (t, 3H); 31P NMR d: 25.34.

Preparation of Liposomes with Incorporated STPP

Liposomes (SUVs) were prepared by probe sonication according to standardprocedures (31) in the presence of STPP. A mixture of lecithin, cholesterol,and STPP (PC/Ch/STPP ¼ 65/15/20, molar ratio; final total lipid 25 mg/mL) was dissolved in chloroform followed by removal of the organic solventusing a rotary evaporator. After adding 5 mM HEPES (pH 7.4) to the dry lipidfilm, the sample was probe sonicated with a Sonic Dismembrator (Model100,Fischer Scientific) at a power output of approximately 10 W for 30 minutes.To remove any titanium particles, which have been shed from the tip of theprobe during sonication, the sample was centrifuged for 10 minutes at3000� g. The formed liposomes were separated from free, i.e., nonincorpo-rated, STPP by gel filtration chromatography on a Sephadex G-15 column.

The obtained STPP liposomes were characterized by size distributionanalysis, 31P NMR spectroscopy (Fig. 3), and by zeta potential measurements(Fig. 4). The size of liposomes with 20 mol% incorporated STPP was deter-mined to be 132� 59 nm, which did not change significantly upon storage at4�C over several days. The 31P NMR spectrum of STPP liposomes showstwo chemical shifts correlating to the phosphorus in the lipid’s phosphategroups and to the positively charged phosphorus of STPP. No differences inboth chemical shifts between the free compounds (i.e., free STPP and free

Figure 3 31P NMR spectrum of SUV liposomes with 20 mol% incorporated STPP.Spectrum was taken using a VARIAN Mercury 300 NMR spectrometer, dP as indi-cated in the figure. Abbreviations: NMR, nuclear magnetic resonance; STPP, stearyltriphenylphosphonium; SVV, small unilamellar vesicle. Source: From Ref. 30.


phospholipid) and the liposomal incorporated molecules could be found,which may indicate that the TPP group of STPP does not interact with thebilayer membrane, i.e., is sufficiently exposed to the aqueous environment.The zeta-potential of STPP liposomes increases linearly with increasingamounts of incorporated STPP until it reaches a plateau between 15 and20 mol% STPP. Whether this observed plateau is due to a limitation of themaximal amount of STPP incorporable in phosphatidylcholine (PC)/Ch lipo-somes has not yet been investigated. For in vitro studies, liposomes with aninitial amount of 20 mol% STPP were used.

In Vitro Applications of Stearyl Triphenylphosphonium Liposomes

The cellular uptake and intracellular distribution of 20 mol% STPP contain-ing liposomes was studied in breast cancer cells (BT 20) using epifluorescence

Figure 4 Zeta potential of liposomes with varying amounts of incorporated STPP.The zeta potential was determined at 2.5 V, 657 nm, 2.00 Hz and 25�C using the ZetaPotential Analyzer Version 3.26 from Brookhaven Instruments Corporation. Foreach measurement, 10mL liposome solution (total lipid 25 mg/mL; STPP contentvarying between 0 and 25 mol%) were added into 2 mL HBS, pH 7.4 and incubateduntil temperature equilibration was attained. Abbreviations: STPP, stearyl triphenyl-phosphonium. HBS, HEPES-buffered saline. Source: From Ref. 30.

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microscopy. The cells were incubated with fluorescence-labeled STPP lipo-somes for one hour in serum free medium, thoroughly washed, and allowedto grow for another hour in complete medium. Typically obtained epifluores-cence microscopic images are shown in Figure 5. Panel A displays cellsincubated with STPP liposomes, which have been labeled by incorporationof 0.5 mol% Rhodamine-PE, whereas panel B shows cells, the mitochondria ofwhich have been specifically stained with Mitotracker red. On comparingboth panels in Figure 5, it can be seen that cells incubated with STPP lipo-somes display the same distinct punctate fluorescence pattern as cells stainedwith the mitochondria-specific dye. Such a comparison of staining patternshas been used earlier to reveal the localization of labeled thiol proteins insidemitochondria (32) and also to show the localization of labeled oligonu-cleotides at and inside mitochondria within living mammalian cells (33).Considering that the fluorophore in our STPP liposomes was covalentlylinked to phospholipids and not to the mitochondriotropic entity, i.e., toSTPP, we concluded from Figure 5A that at least partially intact phospholipidvesicles seem to have accumulated at or near the site of mitochondria (30).

BOLA-LIPID–BASED MITOCHONDRIA-SPECIFICDELIVERY SYSTEMS

Symmetric amphiphilic molecules, in which two hydrophilic residues arelinked by hydrophobic segments, are generally known as ‘‘bola-lipids’’based on their resemblance to an old South American hunting weapon.Well-characterized bola amphiphiles are archaebacterial lipids, whichusually consist of two glycerol backbones connected by two hydrophobic

Figure 5 Epifluorescence microscopic images of BT 20 cells (A) Cells incubated withRhodamine–PE labeled stearyl triphenylphosphonium liposomes; (B) mitochondriain BT 20 cells stained with Mite Tracker Red. Abbreviation: PE, phosphatidylethano-lamine. Source: From Ref. 30.


chains. The self-assembly behavior of such bipolar archaeal lipids has beenextensively studied and it has been shown that they can self-associate intomechanically very stable monolayer membranes (34,35).

During the middle of the 1990s, while screening mitochondriotropicdrugs potentially able to interfere with the mitochondrial DNA metabolism(36) in Plasmodium falciparum, we found (by accident) that dequaliniumchloride, a bola-amphiphilic drug (Fig. 6, top), tends to self-associate intocolloidal structures when sonicated as an aqueous suspension. Employingtransmission electron microscopy as well as photon correlation spectro-scopy, we found that dequalinium forms upon probe sonication sphericappearing aggregates with a diameter between about 70 and 700 nm.Freeze-fracture electron microscopic (EM) images showed both convexand concave fracture faces, thereby demonstrating the liposome-like aggrega-tion of dequalinium. At the time of their discovery, these vesicles were termedDQAsomes (for dequalinium-based liposome-like vesicles; pronounced‘‘dequasomes’’) (24).

A structural difference between dequalinium and archaeal lipids, how-ever, lies in the number of bridging hydrophobic chains between the polarhead groups. In contrast to common arachaeal lipids, in dequalinium thereis only one alkyl chain that connects the two cationic hydrophilic headgroups. Therefore, theoretically two different conformations within a self-assembled layer structure are imaginable (Fig. 6). While the stretchedconformation would give rise to the formation of a monolayer, assumingthe horseshoe conformation would result in the formation of a bilayer.While analyzing the self-assembly behavior of dequalinium salts employingthe Monte Carlo Computer Simulations (37) it was also found that bothconformations, i.e., bola and horseshoe, could theoretically coexist,although the balance between them appeared to be temperature dependent(H.J. Mogel, M. Wahab, unpublished).

Following the discovery of ‘‘DQAsomes,’’ we have explored these vesi-cles as the first available mitochondria-targeted colloidal drug and DNAdelivery system. We were able to demonstrate in a series of papers thatDQAsomes and DQAsome-like vesicles are well suited for the delivery ofplasmid DNA and of small molecules specifically to mitochondria withinliving mammalian cells (38–43).

Preparation of DQAsomes and DQAsome-Like Vesicles

Dequalinium salts can be purchased from Sigma Chemical Co., St. Louis,Missouri, U.S.A. Analogues of dequalinium, however, are not commerciallyavailable; they can be synthesized according to protocols by Galanakiset al. (44–46). In a structure–activity relationship study (38), we foundthat the methyl group in ortho position to the quaternary nitrogen atthe quinolinium ring system seems to play an essential role in the

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self-assembly behavior of these bola amphiphiles. Although removal of thismethyl group significantly impairs stability of formed vesicles, replacing themethyl group by an aliphatic ring system confers unexpected superior vesicleforming properties to this bola amphiphile. Vesicles made fromthis cyclohexyl derivative of dequalinium have in contrast to vesiclesmade from dequalinium a very narrow size distribution (169� 50 nm),which hardly changes at all even after storage at room temperature forover five months.

Figure 6 (Top) Chemical structure of dequalinium; (bottom) possible conformationsof single–chain bola amphiphiles. Amphiphiles in a stretched conformation (bola)would form monolayers, while amphiphiles in a bended conformation (horseshoe)would form bilayers.


To prepare DQAsomes or vesicles composed of dequalinium deriva-tives, the appropriate amount of bola-lipid (10 mM final) was dissolved inmethanol, dried using a rotary evaporator, suspended in 2.5 mL 5 mM N-2-hydroxyethylpiperazine-N’-2-ethane sulfonic acid (HEPES), pH 7.4, bathsonicated for about one hour followed by probe sonication for 45 minutes(10 W). The sample was then centrifuged for 30 minutes at 3000 rpm, theclear, or in some cases, opaque supernatant collected and the remaining non-solubilized residue lyophilized. The concentration of solubilized bola-lipidcan be determined spectrophotometrically or can be inferred from theamount of recovered compound after lyophilization.

Incorporation of DNA into DQAsomes

DQAsome/DNA complexes (‘‘DQAplexes’’) can be prepared by simply mix-ing DNA with the appropriate amount of preformed DQAsomes in salt-free5 mM HEPES buffer at pH 7.4. To choose the correct ratio between DNAand DQAsomes, the DNA-binding capacity of each new batch of DQAsomesshould be determined. The quantitative DQAsome–DNA-binding assay,which has been routinely used in our laboratory, employs SYBRTM GreenI. The fluorescence signal of this dye is greatly enhanced when bound toDNA. Displacement of the dye from DNA results in loss of fluorescence.

About 500 ng pDNA dissolved in 1 mL distilled water was mixed with2.5 mL 5 mM HEPES, pH 7.4 and 0.5 mL SYBR (1:5000 diluted in 5 mMHEPES). While stirring with a magnetic stirrer, fluorescence at 520 nmwas continuously monitored in a Hitachi Fluorescence Spectrophotometer(excitation wavelength of 497 nm; 2.5 mm excitation and emission slits).After the fluorescence signal of the DNA–SYBR complex became stabilized(within less than two minutes) the minimal amount of DQAsomes necessaryto completely bind the DNA was determined. To that end 2 mL DQAsomes(10 mM) were added stepwise in one-minute intervals until the fluorescencesignal reached background level. Approximately 10 mL 10 mM DQAsomesare sufficient to bind 625 ng DNA and to block its interaction with SYBR,resulting in the almost complete loss of fluorescence.

Delivery of DNA to Mitochondria in Living Mammalian Cells

Based on the intrinsic mitochondriotropism of dequalinium and its uniqueself-assembly behavior, we have developed a strategy for direct mitochondrialtransfection (47–49), which involves the transport of a DNA-mitochondrialleader sequence (MLS) peptide conjugate to mitochondria using DQAsomes,the liberation of this conjugate from the cationic vector upon contactwith the mitochondrial outer membrane followed by DNA uptake viathe mitochondrial protein import machinery. We have demonstrated thatDQAsomes fulfill all essential prerequisites for a mitochondria-specificDNA delivery system: they bind and condense pDNA (24), protect it from

328 Weissig et al.

DNAse digestion, and mediate its cellular uptake (39). DQAsome/DNAcomplexes (‘‘DQAplexes’’) do not release the DNA upon contact with anio-nic liposomes mimicking cytoplasm membranes, but do release DNA whenin contact with liposomes mimicking mitochondrial membranes (42). TheDNA release from DQAplexes at natural mitochondrial membranes wasconfirmed by incubating DQAplexes with isolated rat liver mitochondria (41)and it was also shown that MLS peptides linked to DNA do not interferewith DQAsomal binding and release (50). Utilizing a newly developedprotocol for selectively staining free pDNA inside the cytosol, we havedemonstrated that DQAsomes, upon their endosomal escape, selectivelydeliver pDNA to and release the pDNA exclusively at the site of mitochondriain living mammalian cells (40). Free pDNA was not detectable anywhereelse in the cytoplasm of cells treated with DQAplexes. Finally, utilizingconfocal fluorescence microscopy and a DNA-MLS peptide conjugate,we have shown that DQAsomes mediate the delivery of DNA into mito-chondria within living mammalian cells (51).

It should be emphasized at this point that the use of physicochemicalmethods is so far the only way to demonstrate the import of transgene DNAinto the mitochondrial matrix in living mammalian cells. The unavailabilityof a mitochondria-specific reporter plasmid designed for mitochondrialexpression severely hampers current efforts toward the development ofeffective mitochondrial expression vectors. Although any new nonviraltransfection system (i.e., cationic lipids, polymers, and others) aimed atthe nuclear-cytosolic expression of proteins can be systematically testedand subsequently improved by utilizing anyone of many commercially avail-able reporter gene systems, such a methodical approach to develop mito-chondrial transfection systems is currently impossible.

Synthesis of DNA–Mitochondrial Leader SequencePeptide Conjugates

GeneGripTM pDNA bearing 6 to 10 maleimide residues along a specific100 bp region (Gene Therapy Systems), was prelabeled with fluoresceinusing a Label-IT nucleic acid labeling kit (Mirus Corp, Wisconsin, U.S.A.)and then coupled to the leader sequence of the mitochondrial matrix proteinmalate dehydrogenase, via a C-terminal cysteine residue. The sulphydrylgroup of the peptide was first freshly reduced by 0.1 mM tris(2 carboxyethyl)phosphine hydrochloride (TCEP) in 100 mM sodium phosphate buffer, pH7.0 at room temperature for two hours. Without the removal of TCEP, thereduced ligand was then added in 20 molar excess to the maleimide-labeledplasmid and incubated overnight at 4�C to yield the circular conjugate.The circular conjugate was digested with the restriction enzyme BamHIwhich acts upon a single site [at position 3018 immediately adjacent tothe 100 bp peptide nucleic acid (PNA)-binding region] to generate the line-arized conjugate bearing the MLS peptides at one end.


Cell Exposure and Confocal FluorescenceMicroscopic Analysis

BT 20 cells incubated in serum free medium for 10 hours with the vector/DNA complexes (DQAplexes, C-DQAplexes). For control, cells wereexposed to naked DNA and empty vesicles. The cells were then stained withMitotracker Red CMXRos (Molecular Probes) for five minutes to enablethe visualization of mitochondria followed by confocal fluorescence micro-scopic analysis on a Zeiss Meta 510 Laser Scanning Microscope.

Figure 7 shows confocal fluorescence micrographs of cells incubatedwith MLS-pDNA conjugates, which were vectorized with vesicles madefrom the cyclohexyl derivative of dequalinium (C-DQAsomes). For the cellexposures imaged in the left column (panels A, C, and E) the non-restricted,i.e., circular form of pDNA was used, whereas for the experiments picturedin the right column (panels B, D, and F) the plasmid DNA was linearizedbefore DQAplex formation. The characteristic mitochondrial staining pat-tern (panels A and B) shows the functional viability of the imaged cellsand the intracellular fluorescence (panels C and D) demonstrates efficientcell internalization of the fluorescein labeled DNA. Panels A and B werethen overlaid with panels C and D, respectively to produce the compositeimage seen is panels E and F. Strikingly, in the overlaid images, there ishardly any fluorescence detectable. Almost all areas of fluorescence linkedto DNA in panels C and D overlap with the stained mitochondria in panelsE and F, strongly suggesting that almost the entire DNA has been deliverednot only towards mitochondria but also into the organelle (Please note thatmixed color pixels resulting from overlaying the mitochondrial and theDNA fluorescence cannot be visualized in black and white print). However,whether all the pDNA or at least a portion of it has actually entered themitochondrial matrix, i.e., has crossed both mitochondrial membranes,and therefore would potentially be accessible to the mitochondrial transcrip-tion machinery remains to be determined.

Incorporation of Small Molecules into DQAsomes

Besides exploring DQAsomes as a mitochondrial transfection vector, we alsohave been working on utilizing DQAsomes as a mitochondria-targeted carrier

Figure 7 (Figure on facing page) Confocal fluorescence images of BT20 cells stainedwith mitotracker after exposure for 10 hrs to fluorescence-labeled DNA complexedwith C-DQAsomes. (Left column) Circular MLS-pDNA conjugate, (right column)linearized MLS-pDNA conjugate. Top row (A and B): stained mitochondria (originalcolor red, shown here in white), middle row (C and D): fluorescence-labeled DNA(original color green, shown here in white) bottom row (E and F): correspondingoverlaid images (note that the black and white print used in this edition does notallow for depiction of mixed color pixels).

330 Weissig et al.



for small drug molecules, in particular of anticancer drugs known to triggerapoptosis via direct action on mitochondria. Dysregulation of the apoptoticmachinery is generally accepted as an almost universal component of thetransformation process of normal cells into cancer cells and a large body ofexperimental data demonstrates that mitochondria play a key role in the com-plex apoptotic mechanism (52–55). Consequently, any therapeutic strategyaimed at specifically triggering apoptosis in cancer cells is believed to havepotential therapeutic effect (56–60). Several clinically approved anticancerdrugs such as paclitaxel (61–63), VP-16 (etoposide) (64), and vinorelbine (63),as well as an increasing number of experimental anticancer drugs (65) such asbetulinic acid, lonidamine, ceramide, and CD437, have been found to actdirectly on mitochondria to trigger apoptosis. The therapeutic potential ofsuch anticancer drugs, which are known to act at or inside mitochondria,should be greatly enhanced by a drug-delivery system that specificallytargets mitochondria.

Preparation of Paclitaxel-Loaded DQAsomes

Dequalinium chloride (10 mM final) and paclitaxel (10 mM final) weredissolved in methanol in a round-bottom flask followed by removingthe organic solvent with a rotary evaporator. After adding 5 mM HEPES,pH 7.4, the suspension was sonicated with a probe sonicator until a clearopaque solution of DQAsomes with encapsulated paclitaxel was obtained(usually for about one hour). To remove undissolved material, the prepara-tion was centrifuged for 10 minutes at 3000 rpm.

The solubility of paclitaxel in water at 25�C at pH 7.4 is 0.172 mg/L (0.2mM), extremely low, making any separation procedure of nonencapsulatedpaclitaxel from DQAsomes unnecessary; i.e., in an aqueous environment,only paclitaxel encapsulated in DQAsomes would stay in colloidal solution.However, for control, a paclitaxel suspension was probe sonicated underidentical conditions used for the encapsulation of paclitaxel into DQAsomes,but in the complete absence of dequalinium chloride. As expected, uponcentrifugation, no paclitaxel was detectable in the supernatant using ultravio-let (UV) spectroscopy at 230 nm.

Determination of the Paclitaxel/Dequalinium Ratio

The amount of dequalinium in DQAsomes was measured using fluorescencespectroscopy (ex. 335 nm, em. 360 nm). At these wavelengths, paclitaxel doesnot display any fluorescence and therefore does not interfere with thedetermination of dequalinium. For measurements, 3 mL DQAsomes weredissolved in 3 mL methanol, resulting in a concentration of dequalinium,which lies within the linear range of a previously determined standard curve.

Because the UV spectra of paclitaxel and dequalinium in methanolstrongly overlap between 200 and 240 nm, before being able to measure

332 Weissig et al.

the amount of paclitaxel encapsulated into DQAsomes, dequalinium has tobe quantitatively removed from the preparation. To this end, a solid phaseextraction (SPE) column (J.T. Baker Bakerbond Octadecyl 40 mm Prep LCPacking) was equilibrated with methanol and loaded with 1 mL water fol-lowed by the application of 0.02 mL DQAsomes previously dissolved inmethanol/water¼ 10/1, v/v. Dequalinium was quantitatively eluted fromthe column by washing with a discontinued methanol/water gradient[1 mL methanol/water (1:4, v/v), followed by 1 mL methanol/water (3:2,v/v)]. Paclitaxel was eluted from the SPE column by washing with 1 mL100% methanol and measured via UV spectroscopy at 230 nm. The lackof any absorption at 315 nm demonstrates the complete absence of dequali-nium in the sample used for the determination of paclitaxel.

In a reproducible way, paclitaxel can be incorporated into DQAsomesat a molar ratio paclitaxel to dequalinium of about 0.6. In comparison to thefree drug, encapsulation of paclitaxel into DQAsomes increases the drug’ssolubility by a factor of about 3000.

Physicochemical Characterization of Paclitaxel-LoadedDQAsomes

Considering the known spherical character of DQAsomes, the results of anEM analysis of paclitaxel-loaded DQAsomes were rather surprising. Thetransmission EM image (Fig. 8, left panel) and the cryo-EM image (Fig. 8,right panel) of an identical sample show with a remarkable conformity rod-like structures roughly around 400 nm in length, the size of which could alsobe confirmed by size distribution analysis shown in Fig. 8 (middle panel).These complexes may represent the formation of worm-like micelles asrecently described for self-assembling amphiphilic block copolymers (66).

Tumor Growth Inhibition Study with DQAsomalEncapsulated Paclitaxel (43)

Ten million COLO-205 tumor cells were inoculated subcutaneously into theleft flank of nude mice after an appropriate state of anesthesia. All miceformed palpable tumors about 1 to 2 mm in diameter within seven days aftercell injection. Empty DQAsomes and paclitaxel-loaded DQAsomes were pre-pared as described above. For controls with free paclitaxel, the drug wasresuspended in 100% dimethylsulfoxide (DMSO) at 20 mM, stored at 4�C,and diluted in warmed medium immediately before use. In all controls, thedose of free paclitaxel and empty DQAsomes, respectively, were adjustedaccording to the dose of paclitaxel and dequalinium given in the paclitaxel-loaded DQAsome samples. In all groups, the dose was tripled after 1.5 weeks.From eight days after tumor inoculation, treatment was performed asfollows (each injection was given intraperitoneally in 0.15 mL volume).Untreated group (n ¼ 8), 5 mM HEPES, pH ¼ 7.4, was administered twice


a week for three weeks; Free paclitaxel (n ¼ 8), 2.52 mg/kg was administeredtwice a week for 1.5 weeks, followed by a dose increase to 7.56 mg/kg, whichwas administered also twice a week for another 1.5 weeks; empty DQAsomes(n¼ 8), 1.5 mg dequalinium/kg was administered twice a week for 1.5 weeks,followed by a dose increase to 4.5 mg dequalinium/kg, which was also admin-istered twice a week for another 1.5 weeks; Paclitaxel-loaded DQAsomes(n¼ 8), 1.5 mg DQA and 2.52 mg paclitaxel/kg was administered twice a weekfor 1.5 weeks, thereafter the dose was tripled to 4.5 mg DQA and 7.56 mgpaclitaxel/kg, also given twice a week for another 1.5 weeks. The tumorgrowth was determined by measurement of the diameter of the tumor nodulein two dimensions with a caliper twice a week.

Figure 9 shows that at concentrations where free paclitaxel and emptyDQAsomes do not show any impact on tumor growth, DQAsomes loadedwith equivalent amounts of paclitaxel appear to inhibit the tumor growth byabout 50%. Correspondingly, when the animals were sacrificed after 26 days,the average tumor weight in the treatment group was approximately half of

Figure 8 Paclitaxel encapsulated into DQAsomes. (A) Transmission electron micro-scopic image (uranyl acetate staining). (B) Size distribution. (C) Cryo–electronmicroscopic image. Source: From Ref. 43.

334 Weissig et al.

that in all controls (43). This preliminary in vivo study seems to suggest thatDQAsomes might indeed be able to increase the therapeutic potential ofpaclitaxel. However, more extensive studies need to be performed.

SUMMARY AND CONCLUSION

Mitochondriotropic liposomes as well as DQAsomes and DQAsome-likevesicles represent the first mitochondria-targeted colloidal drug and DNAdelivery systems. Their further exploration holds promise to open up newways for the treatment of cancer and a multitude of mitochondrial diseases.

ACKNOWLEDGMENT

The corresponding author (V. Weissig) is obliged to the MuscularDystrophy Association (Tucson, Arizona, U.S.A.), the United Mitochon-drial Disease Foundation (Pittsburgh, Pennsylvania, U.S.A.), MitoVec,

Figure 9 Tumor growth inhibition study in nude mice implanted with human coloncancer cells. The mean tumor volume from each group was blotted against the num-ber of days. Each group involved eight animals. For clarity, error bars were omitted.Note that after 1.5 weeks the dose, normalized for paclitaxel, was tripled in all treat-ment groups. Source: From Ref. 43


Inc. (Boston, Massachusetts, U.S.A.), and Northeastern University (Boston,Massachusetts, U.S.A.) for financial support received from these organiza-tions during the last four years.

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19

Spectral Imaging for the Investigation ofthe Intracellular Fate of Liposomes

Ulrich S. Huth, Rolf Schubert, and Regine Peschka-Suss

Department of Pharmaceutical Technology and Biopharmacy, Albert-LudwigsUniversity, Freiburg im Breisgau, Germany

INTRODUCTION

In principle, liposomes can enter (target) cells through different pathways:by direct fusion of liposomes and the plasma membrane (1) or by an endo-cytic uptake mechanism. Other liposome–cell interactions that have beendescribed in the literature are absorption, phospholipid and protein exchange,and cell-induced leakage of liposome contents (2,3).

Previous work has shown that the majority of cells internalize lipo-somes through an endocytic pathway (4,5). There are multiple pathwaysfor internalization involving vesicles of 50–300 nm in diameter. Theseinclude clathrin-mediated endocytosis, caveolae-mediated endocytosis,phagocytosis, macropinocytosis, and nonclathrin- noncaveolae-dependentendocytosis (6).

There are also multiple pathways for liposomes following cellularuptake. They may be delivered to lysosomes, recycled out of the cell,involved in transcytotic passage across an epithelial barrier, or deliveredto other cellular compartments such as the Golgi network. Each route offersopportunities for selective delivery of macromolecular drugs and nano-sized drugs; so the need to comprehend endocytic pathways has neverbeen more apparent (7). Figure 1 summarizes the different pathways ofendocytosis.

341

Endocytosis

Clathrin-Mediated Endocytosis

Clathrin-mediated (or clathrin-dependent) endocytosis normally occurs atspecialized sites, where complex structures called coated pits are assembledin order to concentrate surface proteins for internalization. The ‘‘coat’’ con-sists of many different proteins that are needed for stabilization of both thepit and the forming of the clathrin-coated vesicle. The two most abundantproteins found within these structures are clathrin and the adaptor proteinAP-2 (9).

The clathrin-coated vesicles bud continuously from the plasmamembrane and transport both the plasma membrane and the fluid contentof the vesicle into the cell. After entering the cytoplasm, the endocyticvesicle loses its clathrin coat and fuses quickly with other vesicles to formearly endosomes.

Figure 1 Endocytosis of liposomes: five different routes into the cell. Multiple path-ways can be used by the cell to internalize liposomes. Besides the well-characterizedclathrin-mediated endocytosis, other pathways can be applied by the cell. Possiblealternative pathways include phagocytosis or macropinocytosis—two pathways thatinternalize by an actin-driven protuberance of the plasma membrane. Other routesinclude the involvement of caveolae where substances are taken up into the cellbypass the traditional endosome/lysosome system (particles might escape from beingdegraded in lysosomes). Finally there exists an ill-defined mechanism that is neithermediated by caveolae nor by clathrin. In a single cell type, two or more of thesemechanisms can coexist. Source: Adapted from Ref. 8.

342 Huth et al.

Early endosomes are the main sorting station in the endocytic path-way. In their acidic interior (pH 5.9–6.0), the receptor and its ligand canbe released. The receptor may be recycled to the surface by vesicles that fusewith the plasma membrane. Material that cannot escape from the earlyendosomes is further transported via multivesicular bodies to late endo-somes and digesting lysosomes that contain a broad spectrum of peptidasesand hydrolases in an acidic surrounding [for reviews on endocytosis seeRefs. (10–12), for review on clathrin uptake see Refs. (9,13)].

Caveolae-Mediated Endocytosis andCaveolae-Like Endocytosis

Since their discovery in the early 1950s (14,15), caveolae had been consid-ered to be uninteresting static organelles that have no capability for theuptake of particles. However, in the past two decades, caveolae have movedinto the focus of many researchers because they seem to play an importantrole in the uptake of various agents, but this is not without controversy. In2002, Thomsen et al. published strong evidence that caveolae are static fixeddomains that are not involved in endocytosis (16), but it has also beenreported that caveolae and caveolin can clearly be internalized—at leastafter specific stimuli, as shown with simian virus 40 (SV40) (17), or by treat-ment with okadaic acid (18).

Caveolae are omega-shaped invaginations of the plasma membrane,with a diameter of 50–100 nm. They are found in a variety of cell types,especially endothelial cells, adipocytes, fibroblasts, muscle cells, and manytumor cells. Caveolae are rich in cholesterol and glycosphingolipids and theircoat is composed of a protein called caveolin (usually caveolin-1 or -2; in thecase of muscle cells, caveolin-3) (19). In contrast to the other pathways,substances taken up via caveolae-mediated endocytosis bypass the traditionalendosome/lysosome system (described above) and can be delivered to othercompartments such as the Golgi network and the endoplasmic reticulum(ER) (20) or mediate a delivery into the extracellular spaces (21). In addition,caveolae play an important role in transcytosis (22) through an endothelialbarrier, making them attractive targets for drug targeting systems, as is the casewith cancer therapeutics (23,24).

It should be noted that the protein caveolin-1 is not necessarily an inte-gral part of caveolae. The so-called lipid-raft–mediated pathways have beenstudied mainly in cells that do not express caveolin-1. Apparently, thesemechanisms are similar to caveolae uptake and are therefore discussedin the caveolae section. For detailed reviews on caveolae uptake, seeRefs. (25–30).

Macropinocytosis and Phagocytosis

Phagocytosis and macropinocytosis are actin-dependent and clathrin-independent processes that lead to the uptake of particles into large vesicles,

Spectral Imaging 343

often greater than 500 nm in diameter. Although phagocytosis is primarilyperformed by professional phagocytes such as neutrophils, macrophages,and dendritic cells, macropinocytosis can occur in a variety of cells upon cellstimulation induced by particles or growth factors (8).

After membrane ruffling and formation of the so-called pseudopodia,the material is engulfed by the cell and is further transported to vesicles(phagosomes/macropinosomes) that have the ability to become acidified.These vesicles fuse rapidly with late endosomes and/or lysosomes, exposingtheir contents to the hydrophilic enzymes.

Nonclathrin-Noncaveolae–Mediated Endocytosis

In addition to the well characterized roles of clathrin-caveolae-mediatedendocytosis and macropinocytosis/phagocytosis, an ill-defined route ofnonclathrin-noncaveolae mediated endocytosis still exists (31,32). It seemsthat all of the until now poorly understood mechanisms of internalizationcan be summarized in this topic.

Methods

Flow Cytometry and Spectral Bio-Imaging

A good approach to study endocytosis is a combination of flow cytome-try with a microscopic method such as spectral bio-imaging or confocalmicroscopy.

In our studies, we used flow cytometry to gain quantitative informationin terms of uptake kinetics and extent of uptake inhibition after treatmentwith several inhibitors.

To trace the intracellular routes of endocytosis, we applied differentfluorescently labeled markers, which are known to be internalized more or lessselectively via a single endocytic pathway. After incubation of cells togetherwith fluorescently labeled liposomes, we determined the colocalization—notby looking at merged colors but by analyzing the emitted spectra of the dyes.This spectral bio-imaging method enables the measuring of spectra of emittedlight at every pixel of the image (33,34). Spectral bio-imaging combines theadvantages of fluorescence spectroscopy with that of light microscopy.A mathematical algorithm separates the original view into its fluorescentcomponents by comparing the emission spectra of each pixel in the image withpreviously recorded reference spectra obtained from single color images. As aresult, the intensity of each dye is extracted at each pixel and this providesinformation on the localization of each dye in the image. Spectral bio-imagingenables the detection and separation of several, even spectrally overlapping,dyes in a single measurement (35).

Figure 2 describes the principle of spectral bio-imaging in more detail.

344 Huth et al.

INITIAL MODE OF INTERNALIZATION

The different mechanisms of uptake and cellular processing were studiedby the use of different inhibitors, which are summarized and reviewed in Table 1.Uptake mechanisms can be investigated by looking for colocalization offluorescently labeled liposomes and labeled markers for endocytosis. Thissection describes how to study the initial mode of internalization, whereas

Figure 2 Principle of spectral bio-imaging HUVEC incubated with pH-sensitiveDOPE:CHEMS liposomes (loaded with fluorescein isothiocyanate-dextran), chol-era toxin subunit B (Alexa Fluor 594 labeled), and diamidino-phenylindole-dihydrochloride.

(Text continues on page 351.)


Tab

le1

Agen

tsan

dIn

hib

ito

rsfo

rIn

ves

tigati

ng

En

do

cyto

sis

an

dIn

trace

llu

lar

Tra

ffick

ing

Pa

thw

ay

Inh

ibit

or/

ag

ent

Mec

ha

nis

mC

om

men

to

nse

lect

ivit

yR

efer

ence

s

Cla

thri

n-m

edia

ted

end

ocy

tosi

sC

hlo

rpro

ma

zin

eIn

tera

cts

wit

hcl

ath

rin

To

dis

tin

gu

ish

bet

wee

ncl

ath

rin

an

dca

veo

lae

36

,37

Imip

ram

ine

Inte

ract

sw

ith

cla

thri

n?

38

Sp

hin

go

sin

eIn

tera

cts

wit

hcl

ath

rin

?3

8H

yp

erto

nic

med

iaL

ead

tod

isru

pti

on

of

cla

thri

nfr

om

the

inn

ersi

de

of

the

pla

sma

mem

bra

ne

Ori

gin

all

yth

ou

gh

tto

be

sele

ctiv

e,b

ut

this

isn

ot

the

case

39

–4

2

Po

tass

ium

dep

leti

on

No

tsp

ecifi

c?L

ow

-pH

sho

cktr

eatm

ent

Ill-

defi

ned

mec

ha

nis

mN

ot

spec

ific?

43

–4

5M

eth

yl-b-

cycl

od

extr

inIn

clu

sio

nco

mp

lex

esw

ith

cho

lest

ero

lB

lock

sca

veo

lae

als

o16,4

2,4

6,4

7

Ph

eny

lars

ine

ox

ide

??

48

An

ticl

ath

rin

an

tib

od

ies

Ag

gre

ga

tio

nw

ith

cla

thri

nin

the

cyto

pla

smle

ad

sto

red

uce

dn

um

ber

of

coa

ted

pit

s

Hig

hly

spec

ific

49

Ta

cro

lim

us

cycl

osp

ori

nA

Blo

cks

dy

na

min

fun

ctio

n(s

eeb

elo

w)

No

tsp

ecifi

c5

0–

52

Rec

om

bin

an

tin

hib

ito

rsA

mp

hip

hy

sin

Th

ere

com

bin

an

tin

hib

ito

rsb

lock

dif

fere

nt

step

sin

coa

ted

pit

/v

esic

lefo

rma

tio

n

Hig

hly

spec

ific

53

AP

18

05

4E

psi

n5

5C

lath

rin

mu

tan

t5

6E

ps1

5m

uta

nt

57

,58

Ca

veo

lae-

med

iate

da

nd

cav

eola

e-li

ke

end

ocy

tosi

s

Fil

ipin

Bin

ds

to30 b

-hy

dro

xy

ster

ols

To

dis

tin

gu

ish

bet

wee

ncl

ath

rin

an

dca

veo

lae

8,2

7,4

2,

46

,59

,60

Gen

iste

inh

erb

imy

cin

Ty

rosi

ne

kin

ase

inh

ibit

ors

Are

des

crib

edto

dis

tin

gu

ish

bet

wee

ncl

ath

rin

an

dca

veo

lae

1,6

1–

63

346 Huth et al.

His

tam

ine

––

48,6

4,6

5In

do

met

ha

cin

Ara

chid

on

ate

acc

um

ula

tio

nR

are

lya

pp

lied

27

,66

Met

hy

l-b-

cycl

od

extr

inIn

clu

sio

nco

mp

lex

esw

ith

cho

lest

ero

lB

lock

scl

ath

rin

als

o16,4

2,4

6,4

7

N-e

thy

lma

lein

imid

eS

ulf

hy

dry

l-a

lky

lati

ng

ag

ent

Des

crib

edfo

rse

ver

al

pu

rpo

ses;

seem

sn

ot

tob

ese

lect

ive

62

,67

Ok

ad

aic

aci

dS

erin

e/th

reo

nin

ep

ho

sph

ate

inh

ibit

or

Lea

ds

to‘‘

sele

ctiv

e’’

stim

ula

tio

no

fca

veo

lae

up

tak

e

18

,64

PM

AP

rote

ink

ina

seC

act

iva

tor

Sh

ou

ldb

ea

n‘‘

sele

ctiv

e’’

inh

ibit

or

for

cav

eola

e,b

ut

als

ost

imu

late

ma

cro

pin

ocy

tosi

s

48

,68

Ny

sta

tin

Bin

dch

ole

ster

ol

Blo

ckch

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ster

ol-

sen

siti

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up

tak

e:v

iaca

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(an

dv

iacl

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?)

60

Dig

ito

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Sta

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kin

ase

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or

Des

crib

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dis

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dcl

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up

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53

Rec

om

bin

an

tin

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ito

rsC

av

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nm

uta

nt

Blo

cks

form

ati

on

of

the

cav

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at

Hig

hly

spec

ific

69

Ma

cro

pin

ocy

tosi

sA

mil

ori

de

dim

eth

yla

mil

ori

de

hex

am

eth

yla

mil

ori

de

(5-N

-eth

yl-

N-i

sop

rop

yl)

-a

mil

ori

de

Blo

ckN

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7Hþ

pu

mp

?A

mil

ori

de

on

lyd

escr

ibed

for

den

dri

tic

cell

s?

70

–7

2

LY

29

40

02

Blo

cks

PI-

3k

ina

seR

ever

sib

le;

spec

ific?

73

–7

7

(Co

nti

nu

ed)


Tab

le1

Ag

ents

an

dIn

hib

ito

rsfo

rIn

ves

tig

ati

ng

En

do

cyto

sis

an

dIn

tra

cell

ula

rT

raff

ick

ing

(Co

nti

nu

ed)

Pa

thw

ay

Inh

ibit

or/

ag

ent

Mec

ha

nis

mC

om

men

to

nse

lect

ivit

yR

efer

ence

s

Wo

rtm

an

nin

Blo

cks

PI-

3k

ina

seIr

rev

ersi

ble

;sp

ecifi

c?7

3–

78

Clo

stri

diu

md

iffi

cile

tox

inB

Ta

rget

sa

Rh

oG

TP

ase

Gen

era

lin

hib

ito

ro

fa

ctin

cyto

skel

eto

n7

9

No

ncl

ath

rin

-n

on

cav

eola

e-d

epen

den

ten

do

cyto

sis

4� C

Blo

cks

ener

gy

-dep

end

ent

pro

cess

esN

ot

spec

ific

80

Cy

toch

ala

sin

DB

ind

sto

act

inN

ot

spec

ific

16

,81

Aci

difi

cati

on

of

end

oso

mes

Ch

loro

qu

ine

Incr

ease

sen

do

som

al

pH

Ba

filo

my

cin

A1

issu

pp

ose

dto

sho

wst

ron

ger

effe

cts

tha

nch

loro

qu

ine

4,4

2,4

5,

82

,83

Ba

filo

my

cin

A1

,co

nca

na

my

cin

AS

pec

ific

inh

ibit

ors

of

v(Hþ

)AT

Pa

se4,6

1,8

4–87

Mo

nen

sin

Un

pro

ton

ate

dw

eak

ba

ses

bec

om

ep

roto

na

ted

inaci

dic

com

part

men

tsa

nd

incr

ease

thei

rp

H

–3

7A

mm

on

ium

chlo

rid

e4

5,8

2

Ea

rly

end

oso

mes

tola

teen

do

som

es

16� C

Blo

cktr

an

spo

rtfr

om

earl

yen

do

som

esto

late

end

oso

mes

76

,88

Act

infi

lam

ents

Cy

toch

ala

sin

D,

cyto

cha

lasi

nB

Bin

ds

toth

eg

row

ing

end

so

fa

ctin

fila

men

tsa

nd

pre

ven

tfu

rth

erfi

lam

ent

ass

emb

ly

Blo

cka

lla

ctin

-d

epen

den

tp

roce

sses

,e.

g.,

the

firs

tst

epo

fm

acr

op

ino

cyto

sis

16,4

4,8

0,

81

,89

La

tru

ncu

lin

A/

BB

ind

sto

act

inm

on

om

ers

an

dd

isru

pt

the

cyto

skel

eto

n8

9–

91

348 Huth et al.

Clo

stri

diu

mb

otu

lin

um

tox

inC

2D

isru

pts

act

infi

lam

ents

92

Mic

rotu

bu

les

Co

lch

icin

eIn

hib

its

tub

uli

np

oly

mer

iza

tio

nB

lock

sv

esic

letr

affi

ckin

ga

lon

gm

icro

tub

ule

s;IA

Ab

lock

sg

lyco

lysi

sa

sw

ell

93

No

cod

azo

leD

epo

lym

eriz

esm

icro

tub

ule

s1,1

8,8

1,

94

,95

Gri

seo

fulv

in,

IAA

,p

od

op

hy

llo

tox

in,

tax

ol,

vin

bla

stin

e,v

incr

isti

ne

Bin

dto

mic

rotu

bu

les

96

–9

8

Dy

na

min

dep

end

ence

Ca

lcin

euri

nin

hib

ito

rs:

tacr

oli

mu

s,cy

clo

spo

rin

A

Blo

ckth

ep

ho

sph

ata

seca

lcin

euri

nth

at

isin

vo

lved

inth

ed

eph

osp

ho

ryla

tio

no

fd

yn

am

in

Blo

ckd

yn

am

ind

epen

den

tp

roce

sses

;d

yn

am

inis

no

to

nly

inv

olv

edin

cla

thri

nu

pta

ke

(see

sect

ion

‘‘D

yn

am

inD

epen

den

ceo

nL

ipo

som

eU

pta

ke’

’)

50

–5

2

Rec

om

bin

an

tin

hib

ito

rsD

yn

am

inm

uta

nts

Do

min

an

tn

ega

tiv

em

uta

nt

99

,10

0

En

erg

yd

epen

den

ce4� C

Blo

cks

all

ener

gy

-dep

end

ent

pro

cess

esS

pec

ific

82,9

3,1

01

Met

ab

oli

ca

ctiv

ity

Gly

coly

sis

IAA

Inh

ibit

or

of

gly

cera

ldeh

yd

es-3

-p

ho

sph

ata

se;

blo

cks

als

om

icro

tub

ule

s

Fo

rth

eb

lock

of

met

ab

oli

ca

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ity

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tic

an

dm

ito

cho

nd

ria

lin

hib

ito

rssh

ou

ldb

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pp

lied

,a

sei

ther

inh

ibit

or

alo

ne

ha

sm

inim

al

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ct

96,9

7,1

02

(Co

nti

nu

ed)


Tab

le1

Ag

ents

an

dIn

hib

ito

rsfo

rIn

ves

tig

ati

ng

En

do

cyto

sis

an

dIn

tra

cell

ula

rT

raff

ick

ing

(Co

nti

nu

ed)

Pa

thw

ay

Inh

ibit

or/

ag

ent

Mec

ha

nis

mC

om

men

to

nse

lect

ivit

yR

efer

ence

s

2-d

eox

yg

luco

seN

on

met

ab

oli

zea

ble

carb

on

sou

rce

93

,10

1–

10

3

So

diu

mfl

uo

rid

eIn

hib

ito

ro

fp

ho

sph

op

yru

va

teh

yd

rata

sea

nd

py

ruv

ate

kin

ase

10

1,1

02

,10

4

Ox

ida

tiv

ep

ho

sph

ory

lati

on

DN

P,

po

tass

ium

cya

nid

eU

nco

up

les

the

ox

ida

tiv

ep

ho

sph

ory

lati

on

fro

mel

ectr

on

tra

nsp

ort

10

5

An

tim

yci

nA

Act

sa

tcy

toch

rom

eo

xid

ase

B1

01

,10

2

So

diu

mazi

de

?93,1

01,1

02

Fo

rma

ldeh

yd

eD

ecre

ase

sth

em

ito

cho

nd

ria

lm

emb

ran

ep

ote

nti

al

Tra

nsc

yto

sis

BF

AS

ever

al

effe

cts:

stim

ula

tes

tra

nsc

yto

sis

PM

A:

stim

ula

tio

nv

iap

rote

ink

ina

seC

BF

Ah

as

sev

era

lef

fect

so

ntr

affi

ckin

ga

nd

pro

tein

(re)

dis

trib

uti

on

61

,10

6P

MA

10

7,1

08

Ab

bre

via

tio

ns:

IAA

,io

do

ace

tam

ide;

BF

A,

bre

feld

inA

;P

MA

,p

ho

rbo

lm

yri

sta

tea

ceta

te;

DN

P,

din

itro

ph

eno

l;A

TP

ase

,a

din

osi

ne

trip

ho

sph

ata

se;

GT

Pa

se,

gu

an

osi

ntr

iph

osp

ha

tase

.

350 Huth et al.

the section ‘‘Intracellular Trafficking’’ focuses on different aspects of intracel-lular trafficking.

Clathrin-Mediated Endocytosis

Inhibition of clathrin function can be achieved by different approaches(Table 1). Here, we describe the most commonly used inhibitory drugsand the newer and more specific molecular inhibitors in the form ofdominant negative proteins.

Some inhibitors for this pathway, often described in the literature, do notdirectly affect the clathrin pathway but rather affect features involved withother pathways. For example, the acidification of endosomes is employed bythe other types of endocytosis as well—therefore, these inhibitors are less spe-cific and are described in the section ‘‘Intracellular Trafficking’’ The sameoccurs with dynamin dependence or metabolic activity (section ‘‘MetabolicActivity’’).

Pharmacological Inhibitors

CPZ and Other Cationic AmphiphilicDrugs (Imipramine, Sphingosine)

Cationic amphiphilic drugs such as chlorpromazine (CPZ), imipramine (38)(100 mM, 20–30 minutes), or sphingosine (5 mM) can be used to block clath-rin uptake. The phenothiazine CPZ seems to be an especially importanttool for studying clathrin-mediated endocytosis because it does not affectcaveolae-mediated endocytosis: the uptake of cholera toxin, the markerfor caveolae-mediated endocytosis (see section ‘‘Caveolae-Mediated Endo-cytosis and Caveolae-Like Endocytosis Pharmacological Inhibitors’’), isnot affected (36). The drug interacts with clathrin from the coatedpits and causes their loss from the surface membrane (37). CPZ is appliedin concentrations ranging from 5–15 mg/mL (corresponding to 14–42 mM)depending on the cell type and the incubation time [maximum effect after30 (to 60) minutes)]. We observed an interaction of CPZ with liposomemembranes, resulting in leakage of the contents (109) (for leakage testssee section ‘‘Interaction of Inhibitors and Liposomes: Leakage Tests’’). Thisinteraction is probably due to incorporation into the membrane. To circum-vent this problem, the incubation with liposomes can be started after firstremoving CPZ by extensive washing. It should be kept in mind that theeffects of CPZ are rapidly reversible (recovery occurs after 30 minutes) (37).

Hypertonic Media and Potassium Depletion

Both intracellular depletion of potassium and hypertonic treatment lead todisruption of clathrin from the inner side of the plasma membrane. Conse-quently, the formation of clathrin-coated pits and clathrin-coated vesicles is


blocked (39–41). Although it was originally thought that the inhibition withhypertonic media was a unique characteristic for the clathrin pathway, laterwork suggested that this was not the case (42). For treatment in hypertonicmedia, cells are incubated in normal saline adjusted to hypertonic conditionsby sucrose (0.45 M for 10 minutes). Upon returning the cells to normalmedium, these effects are quickly reversible.

For potassium depletion, cells are washed with potassium-free buffer(140 mM NaCl, 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid(HEPES), 1 mM CaCl2, 1 mg/mL D-glucose, pH 7.4) and then rinsed inhypotonic buffer (potassium-free buffer 1:1 diluted with distilled water)for five minutes. Then, cells are quickly washed three times in potassium-freebuffer followed by incubation for 20 minutes at 37�C in buffer. Controlexperiments are performed in the same manner, except all solutions addi-tionally contain 10 mM KCl.

Low-pH Shock Treatment

Acidification of the cytoplasm has been described as blocking clathrin-mediated endocytosis, but might affect cell morphology and viability (43,44).The mechanism of inhibition is still ill defined but it is clear that the pHof the medium has no effect on the intralysosomal pH (45). It should benoted that cytosolic acidification shows cell-type variations and might notbe highly specific.

For incubation, cells are preincubated for five minutes at 4�C in culturemedium without buffer substances and acidified with 20 mM succinicacid, pH 5.7 [Dulbecco’s modified Eagle’s medium (DMEM) withoutsodium bicarbonate, but containing 0.6% bovine serum albumin (BSA),20 mM 2-[N-morpholino]-ethanesulfonic acid (MES), 20 mM succinic acid,pH 5.7)]. For different methods useful for acidification of cells, see Ref. (43).

Methyl-b-Cyclodextrin

The water-soluble methyl-b-cyclodextrin (mbCD) is known to form solubleinclusion complexes with cholesterol, leading to depletion of cholesterolfrom the plasma membrane (16,46,47). As a result, cholesterol-rich micro-domains, which are involved in caveolae-mediated as well as clathrin-mediatedendocytosis, are destroyed. mbCD therefore decreases both clathrin- andcaveolae-mediated uptake. The two other well-known cyclodextrins [a-,and c-cyclodextrin (6 and 8 units of a-1,4 glucose)] do not bind cholesteroleffectively (both are not specific for cholesterol, but might remove phospholi-pids from the plasma membrane) and have no significant effect (46).

For inhibition, cells are incubated for 15 minutes with mbCD at 37�C(10 mM, in the absence of serum). The inhibition is reversible when cholesterolor serum is added. The inhibitory effect is still present after one hour, buttotal recovery occurs after three hours. Recovery can be delayed by adding

352 Huth et al.

a 3-hydroxy-3-methylglutary-CoA (HMG-CoA) inhibitor (1–5mg/mL lovas-tatin or 10mM simvastatin) that blocks endogen cholesterol synthesis (46).

Phenylarsine Oxide

Phenylarsine oxide (20 mM; 30 minutes) is used as an inhibitor for receptor-mediated (clathrin-mediated) endocytosis (48).

Anticlathrin Antibodies

Clathrin uptake can be inhibited by anticlathrin heavy chain antibodies.When applied to the cells, they lead to aggregation of clathrin in the cytoplasmand reduce the number of clathrin-coated pits on the plasma membrane (49).

Calcineurin Inhibition

The calcineurin inhibitors tacrolimus (FK 506) and cyclosporin A block thefunction of dynamin and are thought to be specific for clathrin-mediateduptake (50). The smal guanosine triphosphate (GTP)ase dynamin is alsoinvolved in other processes and is therefore described in section ‘‘DynaminDependence on Liposome Uptake.’’

Recombinant Inhibitors

For the formation of clathrin-coated vesicles, several proteins are required. Cur-rently, there are several recombinant inhibitors available, which block differentsteps of coated pit/vesicle formation: for example, amphiphysin (53), clathrinassembly protein AP180 (54), epsin (55), and clathrin mutant (56).

The use of recombinant inhibitors becomes easy when the protein isattached to green fluorescent protein (GFP), which eases the interpretationof the data [e.g., in the case of the Eps15 mutant GFP-EA95/295 (epidermalgrowth factor receptor (EGFR) pathway substrate clone 15 mutant), aregulatory protein of clathrin assembly (57,58)].

Marker for Clathrin Pathway

Epidermal growth factor (EGF), low-density lipoprotein (LDL), and trans-ferrin (Tfn) are often used as model peptides because all three are taken upvia clathrin-mediated endocytosis, but they are sorted into different path-ways after endocytosis.

Epidermal Growth Factor

Either fluorescent EGF or its fluorescent receptor (GFP-EGFR) can be used totrack the clathrin pathway (110). Following internalization, EGF is mainly tar-geted to early endosomes, then to lysosomes, and following this is subsequentlydegraded. After a 10-minute incubation period with EGF, the polypeptide hor-mone accumulates in ‘‘early endosomes,’’ and after 60 to 90 minutes, EGF


should accumulate in ‘‘lysosomes.’’ It should be considered that the use ofEGF can be difficult in cells that express low levels of the EGF receptor.

Low-Density Lipoprotein

The human LDL complex delivers cholesterol to cells by receptor-mediated,clathrin-mediated endocytosis. LDL is used to follow the ‘‘lysosomal’’ direc-ted pathway. Once internalized, LDL dissociates from its receptor andultimately accumulates in the lysosomes (111).

Human Transferrin

Serum Tfn belongs to a class of metal-binding glycoproteins of approxi-mately 80 kDa in size and is often used as an essential tool for studyingcellular uptake and the subsequent intracellular sorting and recycling.

Tfn binds to its receptor, is concentrated at clathrin-coated pits, andis further transported to the early endosome compartment from where it isbrought to the ‘‘recycling endosomes’’ and back to the plasma membranewhere the cycle starts again (1,20,37,61,112). To target early endosomes,cells should be incubated with Tfn (20–50 mg/mL) for about 10 minutes.However, we have never seen a significant change in distribution afterlonger incubation times [in COS-7 and human umbilical endothelial cells(HUVEC)], which could be explained by continuous cycling of the marker.

GFP–Clathrin Light Chain, Clathrin Light Chain–GFP

The plasmid-encoding human clathrin light chain A, with GFP attached atits NH2 terminus, can be used to label clathrin-coated invaginations at theplasma membrane (40,113). With this technique, it should be possible tomonitor the first step of this pathway—the binding and concentration atthe plasma membrane.

Shiga toxin is described as being transported after uptake via clathrin-coated pits from recycling endosomes to the Golgi apparatus (31).

Semliki Forest Virus

After attachment to the cell surface, the virus is located in clathrin-coatedpits and is further internalized by an endocytosis similar to the receptor-mediated endocytosis of LDL (114,115).

Other marker used to study clathrin-mediated uptake: a-2 macro-globulin (116).

Caveolae-Mediated Endocytosis and Caveolae-Like Endocytosis:Pharmacological Inhibitors

Filipin

The macrolide antibiotic filipin interacts with 3-b-hydroxysterols such ascholesterol in the plasma membrane to form filipin–sterol complexes (59).Subsequently, the filamentous caveolin-1-coat rapidly disassembles, which

354 Huth et al.

leads to inhibition of caveolae endocytosis. Filipin (5mg/mL for 45–60 min-utes or 10mg/mL for 15 minutes) cannot interact with the cholesterol, whichis a part of coated pits and does not influence clathrin pathways, makingfilipin a selective inhibitor for caveolae uptake (8,27). Effects of filipin arerapidly reversible (30 minutes) when incubated in cell culture media contain-ing 20% fetal calf serum (FCS) (60). When applying filipin, it should be kept inmind that it is also used as a stain for cholesterol and therefore increases thefluorescence of cells (stain with 0.05% filipin, 10 minutes; excitationmax¼378 nm; emissionmax¼ 450 nm).

Genistein and Herbimycin

The tyrosine kinase inhibitors genistein [4–50 mM dissolved in 0.05%dimethyl sulfoxide (DMSO), 30 minutes] and herbimycin (500 mM dissolvedin 0.05% DMSO, 30 minutes) are described as being useful inhibitors ofcaveolae uptake (61,62), which can be applied to discriminate this pathwayfrom clathrin-mediated uptake (63). It should be questioned if a block of theenzyme affects the uptake via caveolae uptake selectively. However, genistein isthought to inhibit the receptor-induced formation of caveolae (18).As for herbimycin, no comment can be given on its selectivity.

Histamine

Exposure to histamine [10 mM for 30–45 minutes; recovery (in the presence ofhistamine) after 90 minutes] specifically blocks the uptake of 5-methyltetra-hydrofolate, a substance that is used to study potocytosis/caveolae uptake(48,64). Histamine might also be used to increase the permeability of theendothelium: binding of histamine to its receptor leads to the contractionof the cytoskeleton and to the opening of intercellular junctions (65).

Indomethacin

Smart et al. discovered that indomethacin also blocks caveolae uptake andsuggest arachidonate accumulation as a potential inhibitor (66). Indometha-cin is described as inhibiting both the internalization of caveolae and thereturn of plasmalemma vesicles after an incubation time of 30 minutes inthe presence of 400 mM in MA104 cells (rhesus monkey kidney cells). Theeffects are rapidly reversible after removing the drug (66).

Methyl-b-Cyclodextrin

As described above, mbCD is a commonly used inhibitor for both theclathrin and the caveolae pathway. See the section on ‘‘Inhibiting Clathrin-Mediated Endocytosis’’ for details.

N-Ethylmaleinimide

The sulfhydryl-alkylating agent N-ethylmaleinimide (NEM) is used forseveral purposes: (i) it has been described that exocytosis and the regulationof membrane fusion by NEM-sensitive fusion factor can be inhibited by an


incubation with NEM (1 mM,<5 minutes) (62); (ii) NEM is often applied toinhibit transcytosis but one should be aware that even short incubation times(5–10 minutes) and low concentrations (0.3–1.0 mM) could damage theendothelium (67); and (iii) NEM is also described as interfering withthe caveolae mechanism but it does not seem to be appropriate for this use.

Phorbol 12-Myristate 13-Acetate

Phorbol esters are described as being protein kinase C activators andshould be selective inhibitors for caveolae uptake [1mM Phorbol 12-myristate13-acetate (PMA) for 30 minutes] (48,68). The inhibition occurs as quickly asfive minutes after adding PMA to cell culture medium and is not reversiblewithin five hours (MA104 cells). However, PMA has several other effects onseveral other pathways. An example of this is that it has been described tostimulate macropinocytosis and transcytosis (see the following).

Sterol-Binding Drugs: Nystatin, Digitoxin

The polyene antibiotic nystatin (5–25 mg/mL, 10 minutes) and the cardiacglycoside digitoxin (5 mg/mL, 10 minutes) bind cholesterol and can removeit from cell membranes, which leads to inhibition of caveolae uptake (60).Regarding its effect on clathrin uptake, no comment can be given at this time.

Staurosporine

Staurosporine (2–5 mM, 60 minutes), a general kinase inhibitor, has beenused to discriminate caveolae-mediated uptake from clathrin-mediateduptake (63).

Stimulation of Caveolae Uptake

Okadaic Acid

The serine/threonine phosphatase inhibitor okadaic acid (incubation timeand concentration vary from 10 nM, 30 minutes to 1 mM, 1 hour at 37�C)leads to ‘‘selective’’ stimulation of caveolae uptake (18,64).

Recombinant Inhibitors

Dominant Negative Caveolin Mutant

Specific inhibition can be achieved by transfecting cells with a dominantnegative mutant of caveolin (69).

Marker for Caveolae Pathway

To highlight the caveolae pathway, a couple of substances are discussed andapplied. The most commonly used markers besides the SV40 (17,20) areprobably cholera toxin B subunit (CtxB) and caveolin-1-GFP. Folate and

356 Huth et al.

folic acid are applied to study potocytosis, a special branch of the caveolaepathway (63,68,117). References 63 and 26 give an overview on differentmolecules and receptors internalized by caveolae and give examples of mole-cules that follow different caveolae pathways.

Caveolin-1-GFP

Probably the most appropriate marker for this pathway is caveolin-1 taggedwith GFP, which after gene expression, is located at sites of caveolae as wellas in the Golgi complex (20,26).

Cholera Toxin B Subunit

Caveolae can mediate the delivery of CtxB that binds to GM1 gangliosideat the plasma membrane and is delivered to intracellular compartments.Cholera toxin, produced by Vibrio cholerae, consists of five identicalsubunits B and one A chain. In addition to labeled SV40 and caveolin-1-GFP, CtxB is one of the most commonly used caveolae markers. However,two groups reported that the toxin is internalized by either a clathrin-independent caveolae pathway or a clathrin-dependent uptake, bringing itsselectivity/specificity into question (31,81,118). We controlled the suitabilityof this marker for COS-7 cells pretreated with CPZ, mbCD, and filipin and asexpected, the uptake was not influenced by CPZ treatment but was stronglydecreased by the latter two (data not shown).

5-Methyltetrahydrofolate, Folic Acid

In many studies, folate or folic acid (5 nM) is applied to study caveolae-mediated endocytosis resp. potocytosis (27,119,120).

Lactosylceramide

Bodipy-labeled glycosphingolipid lactosylceramide (LacCer) (120).

Labeled SV40

Uptake of SV40 occurs specifically by the caveolar pathway. Less than 5%of internalized particles are found to pass through clathrin-coated pits (26).Even though SV40 was ‘‘known’’ to be a highly specific marker for caveolaeuptake, the virus is now described as entering the cells via a differentmechanisms (121).

Other Materials Endocytosed via Caveolae or Lipid Rafts

Albumin, tetanus toxin, autocrine motility factor, interleukin-2, alkalinephosphatase, glycosyl-phosphatidylinositol (GPI)-GFP, polyoma virus,and echovirus 1 (26).


Macropinocytosis and Phagocytosis

Because of the strict requirement for actin, the most commonly used inhib-itors of macropinocytosis are the cytochalasins, especially cytochalasin D ortoxin C. These substances also block phagocytosis and intracellulartrafficking along actin filaments. Therefore, the results from these experi-ments are described in the trafficking section ‘‘Actin Dependence onLiposome Uptake.’’

The arrangements of actin filaments are regulated by the activity ofphosphatidylinositol-3 (PI-3) kinase and can be highly specifically inhibitedby wortmannin, which irreversibly binds to the catalytic subunit of the PI-3kinase (73). LY294002 also specifically inhibits this enzyme (74). Remark-ably, the enzyme is not necessary for the regulation of the extension toactin-rich pseudopods, but for preventing them from closing (75).LY294002 and wortmannin therefore seem to be specific inhibitors formacropinocytosis and phagocytosis.

Another blocking agent in use for this pathway is amiloride, an inhibitorof the sodium/proton pump (70). See Table 1 for an overview of used inhibitors.

As phagocytosis occurs only in specialized cells, for the purposes ofthis review, we do not differentiate macropinocytosis from phagocytosis.

Pharmacological Inhibitors

Amiloride, Dimethylamiloride, Hexamethylamiloride, and(5-N-Ethyl-N-Isopropyl) Amiloride

The uptake via macropinocytosis is described as being blocked by amilorideand its derivatives. These all inhibit the Naþ7Hþ exchange and EGF-Rtyrosine kinase (70,71). For inhibition, cells are incubated with amiloride(3 mM, five minutes), hexamethylamiloride, (100mM), or dimethylamiloride(0.1 mM, 5 minutes to 10mM, 60 minutes) (72).

LY294002 and Wortmannin

LY294002 and wortmannin inhibit the enzyme PI-3 kinase required for theclosure of pseudopodia to form intracellular vesicles (61,73–75,78,122).Compared to wortmannin, which is relatively unstable in aqueous media,the inhibitory effects of LY294002 are more specific, reversible (recoveryafter 10 minutes), and not light dependent. Therefore, LY294002 can beused for time-lapse experiments. Several studies have indicated that bothsubstances have little or no effect on the other pathways described (75).However, both substances might block the uptake of Tfn as well (76).

For inhibition, cells are incubated with LY294002 (3–100 mM,0.5–4 hours) or wortmannin (3–100 nM, 30 minutes to 4 hours). It shouldbe kept in mind that other enzymes might be inhibited by applying concen-trations that are too high.

358 Huth et al.

Because wortmannin is hydrophobic (on the day of the experiment wedissolve an aliquot in DMSO and never keep it for more than a day or two,away from light) and used at very low concentrations, it should not bemixed with serum or other protein-rich media too long before the experi-ment. The DMSO solution should be added to the medium immediatelybefore addition to the cells. If left in aqueous medium too long before addi-tion, it will bind to the walls of the tube.

Clostridium difficile Toxin B

Toxin B targets a Rho GTPase that is involved in the regulation of the actincytoskeleton (79).

Marker for Macropinocytosis and Phagocytosis

No specific cargo molecules have been identified to track macropinocytosis,which makes it difficult to follow the intracellular fate of macropinosomes.However, one substance that is applied to study macropinocytosis and totrack macropinocytic vesicles is labeled high-molecular-weight dextran(molecular weight 70,000) (77).

Nonclathrin-Noncaveolae–Mediated Endocytosis

No specific cargo molecules have been identified so far, which makes it dif-ficult to follow the intracellular fate of unspecific internalized material. Itappears that the uptake via noncoated vesicles is rapidly upregulated inresponse to inhibition of the clathrin-mediated pathway. Because of the lackof detailed information, no statements can be made about the role of thismode of internalization for the uptake of liposomes.

INTRACELLULAR TRAFFICKING

Several aspects of intracellular trafficking should be kept in mind in theintracellular trafficking section. The first is the dependence of acidification ofendosomes on the uptake of liposomes. This aspect is sometimes discussedwhen analyzing clathrin uptake. However, several other pathways are alsoin need of acidic compartments as a destination of uptake; so, we list thisfactor as an individual aspect. Other aspects of intracellular trafficking thatare of interest are the transport from early endosomes to late endosomes, thedependence of actin filaments and dynamin, and/or microtubules. Further-more, the energy dependence of liposome uptake is discussed.

Acidification of Endosomes

Molecules entering cells through endocytic pathways will rapidly (<2 min)experience a drop in pH from neutral to pH 5.9–6.0 in the lumen of


early/recycling vesicles, with further reduction from pH 6.0 to 5.0 duringprogression from late endosomes to lysosomes.

Because the intracellular pH at least partially regulates the intracellu-lar sorting of endocytosed ligands (61), a block of acidification leads to achange of intracellular distribution and diminished uptake (82).

Two different approaches are taken to prevent acidification: (i) severalsubstances are applied to block the acidification of endosomes by interferingwith endosomal enzymes such as H(þ)-ATPase and (ii) lysosomotropicreagents (generally weak bases) are known to accumulate within intracellu-lar organelles and increase their pH.

In both cases, the transport of some proteins to the lysosome isblocked, suggesting that acidification itself is required as part of this prog-ress. Regarding their effect on transcytosis there is a difference between bothgroups. Although bafilomycin and chloroquine have only a small effect, thelysosomotropic substances (ammonium chloride and monensin) suppresstranscytosis [mediated by clathrin (EGF); for details on transcytosis seesection ‘‘Transcytosis’’] (61,106).

Suppression of the Degradation Process

Chloroquine

The weak base chloroquine (30–300 mM, 30 minutes, either in the presence orabsence of serum) increases the endosomal pH (83) within a few minutes (45),leading to pH values close to 6.3 in both endosomes and lysosomes andtherefore preventing lysosomal degradation. As a mechanism of action, adirect inhibition of lysosomal hydrolases (cathepsin B1 and some phospho-lipases and lysophospholipases) is reported (82). In Kupffer cells, effects aretolerated at concentrations of 40 mM or less for up to four hours andare irreversible within two hours after medium replacement (82).

Bafilomycin A1 and Concanamycin A

The macrolide antibiotic bafilomycin A1 and concanamycin A are specificinhibitors of vacuolar-type H(þ)-ATPase, which prevents the acidificationof endosomes and lysosomes and increases the intralysosomal pH from about5.1–5.5 to about 6.3 (61,84–87). Bafilomycin A1 is applied in concentrationsranging from 25–1000 nM and is incubated for 30 to 60 minutes in the presenceor absence of serum. We observed maximal effects in COS-7 and HUVECwith concentrations of 100–200 nM and an incubation time of 60 minutes.

Lysosomotropic Reagents

We group reagents that are known to accumulate within acidic organelles inthis category. Unprotonated weak bases are capable of crossing biologicalmembranes and are concentrated in acidic compartments by protonation.Monensin and ammonium chloride suppress transcytosis, recycling, anddegradation.

360 Huth et al.

Monensin: Monensin (25 mM, 30 minutes) elevates the pH in acidiccompartments and inhibits receptor recycling by trapping receptors inendosomes (37).

Ammonium chloride (10–30 mM, 20 minutes preincubation): Ammoniaraises the intralysosomal pH to maximal levels within a few minutes. Effectsare rapidly reversible (five minutes) after removing NH4Cl from themedium (45,82).

Early or Late Endosomes, Lysosomes

It is reported that the transport from early to late endosomes can be blockedby incubating cells at 16�C (76,88).

Marker for Early or Late Endosomes, Lysosomes

As described above (section ‘‘Clathrin-Mediated Uptake’’), several ligandsfor clathrin-mediated endocytosis (see section ‘‘Clathrin-MediatedEndocytosis’’: EGF, Tfn, LDL) can be used to highlight early endosomesor lysosomes, depending on different incubation times.

Green fluorescent protein-RhoB: GFP-RhoB is localized in endocyticvesicles and has been shown to highlight early endosomes, recycling endo-somes, and multivesicular bodies, but is absent from lysosomes (123).Because RhoB is toxic when applied for long periods of time, the cellsshould be analyzed within 24 hours of transient transfection.

Enzyme detection: Lysosomes can also be stained via enzyme-catalyzedhydrolysis of heavily labeled and almost totally quenched substrates (124).

To detect the fate of liposomes, the nonfluorescent 4-methylumbelliferyl-p-D-glycoside [4MU-(3-D-glc)] can be encapsulated. 4MU-(3-D-glc) is a substratefor lysosomal 3-d-glycosidase that shows fluorescence after enzymatic hydro-lysis to methylumbelliferone (125).

Pyranine, 8-hydroxypyrene-1,3,6-trisulfonic acid: 8-Hydroxypyrene-1,3,6-trisulfonic acid (HPTS, also known as pyranine) can be used as a pHindicator. At low pH, HPTS fluorescence is greater with 405 nm excitation,whereas at neutral pH, fluorescence at 440 nm excitation is greater than at405 nm excitation. Therefore, a strong fluorescence signal at 405 nm excitationindicates HPTS in acidic compartments (endosomes and lysosomes); a strongsignal at 440 nm indicates HPTS in neutral compartments (cytosol) (126–128).

To investigate the fusion of liposomes with other organelles (endosomes),HPTS can be encapsulated along with its quencher p-xylene bispyridiniumdibromide (DPX). Upon destabilization of the liposomes and release of liposo-mal contents inside the endocytic compartment, HPTS is dequenched, resultingin increased emission signal. The same principle is used for leakage tests(see section ‘‘Interaction of Inhibitors and Liposomes: Leakage Tests’’).


LysoTracker and LysoSensor probes: Several commercially availableweakly basic amines can be used to stain lysosomes. They selectivelyaccumulate in acidic organelles when applied in very low concentrations(50 nM) and directly before imaging. For live cell imaging, keep in mindthat lysosomal probes can exhibit an alkalinizing effect on the lysosomes,such that longer incubation time can induce an increase in lysosomalpH (129).

Starvation and incubation with labeled dextran: For this method,cells are incubated under starvation [Earle’s balanced salt solution (EBSS)medium] conditions for two hours. To label early endocytic compartments,cells are subsequently incubated with 0.5 mg/mL dextran tetramethylrhoda-mine for five minutes. To label late endosomes, cells are washed and furtherincubated for an additional 10 minutes (130).

(Monoclonal) antibodies: Other characteristics of late endosomesare unique lipids or proteins associated with the different compartments.These can be used as a target for (monoclonal) antibodies. Often, also therecombinant GFP-fusion proteins are available (such as GFP-Rab5 andGFP-RhoB).

Early endosomes:

� Early endosomes antigen 1, which regulates fusion between endo-cytic vesicles

� RhoB [also enhanced green fluorescent protein (EGFP)-RhoB, seeabove]

� Rab4, which regulates recycling of proteins� Rab5C, which also regulates vesicle fusion (GFP-Rab5 is also

available)� Syntaxin 13, which regulates protein transport

Recycling endosomes:

� Rab11

Late endosomes:

� CD63� Rab7-GDP� Lysosome-associated membrane protein 1 (LAMP 1); also lysosomes� Mannose-6-phosphate receptor� Rab9 (also trans-Golgi network)� Lysobisphosphatidic acid (LBPA) also distinguishes late endo-

somes. LBPA is shaped like an inverted cone: it has a much largerhead than tail and enters highly curved membrane regions. Thelipid may help in the accumulation of molecules like cholesterolby specific lipid-protein interactions (131).

362 Huth et al.

Lysosomes:

� LAMP-1 or� LAMP-2� Rap1

For more detailed information on late endosomes and lysosome biogen-esis, refer to the articles by and Piper et al. and Luzio et al. (132–134).

Actin Dependence on Liposome Uptake

Both actin microfilaments and microtubules (see below) are involved inuptake as well as in the pathway to the lysosomes and the recycling path-way. Referring to clathrin-mediated uptake, Durrbach et al. showed actinparticipation in two steps of endocytosis: the initial uptake mechanismand the delivery to lysosomes (116).

The involvement of the actin cytoskeleton in liposome endocytosis isstudied by using cytochalasins, latrunculin, or toxin C2 to polymerize actinfilaments. For a review on actin assembly and endocytosis, see Ref. (135).

Cytochalasin D, Cytochalasin B

Cytochalasins are mold metabolites (from Zygospohum mansonii and relatedmolds), which inhibit microfilament polymerization by capping the growingend of the filaments and preventing further filament assembly and resultingin shortening (89). For disruption of the actin cytoskeleton, cells are pre-treated with CD [1–10 mM (added from a stock in DMSO)] in completeculture medium for 30 minutes to 5 hours (44,80) [cytochalasin B(CB)2 mg/mL].

Latrunculin A/B

The marine macrolides latrunculin A and the less potent variation latruncu-lin B (5–25 mg/mL, 60 minutes) bind to actin and disrupt the cytoskeletonat low concentrations (90,91). Their mechanism of action includes bindingto and sequestering actin monomers, resulting in filament depolymeriza-tion (89).

Clostridium botulinum Toxin C2

The binary Clostridium botulinum toxin C2 blocks the function of actin fila-ments similarly to cytochalasin D (92). Treatment is for one to four hours inconcentrations of 50 ng/100 ng or 100 ng/200 ng prior to adding liposomes.

Marker for Actin Movement

Labeled phallotoxines (phalloidins): The bicyclic peptides isolatedfrom Amanita phalloides mushroom bind selectively to F-actin in nanomolarconcentrations. They have advantages over antibodies for actin labeling


because they do not bind to monomeric G-actin and do not show unspecificstaining (129).

Microtubules Dependence on Liposome Uptake

Microtubules are used to cover relatively long distances in the cell. There-fore, the short distance between the cell surface and the early endosomesdoes not require microtubules (94,136). They are involved in the later stepsin endocytosis: early endosomes accumulate endocytic material for about10 minutes and then generate transport vesicles (0.5 mm in diameter), whichwill be taken to the cell center with a relatively high traveling speed of1 mm/sec (with velocities of up to 2.5 mm/sec) (95,137). These transportvesicle move on microtubule-tracks to the cell center (and to the lysosome) (94).When performing live cell imaging studies, it should be kept in mindthat microtubules are extremely sensitive to ultraviolet light, which causestheir polymerization.

Microtubules can be characterized as long hollow cylinders with outerdiameters ranging between 20 nm and 30 nm and an inner diameter of about14 nm. Their main constituent is alpha or beta tubulin, which is often thetarget of inhibitors and marker.

Colchicine

Colchicine inhibits tubulin polymerization when applied under the followingconditions: 30 mM for five minutes (0.1 mM, 30 minutes) (93). Colchicineconcentration should be maintained throughout the experiment.

Nocodazole

Nocodazole (1.5 mg/mL, one hour) depolymerizes microtubules, which pre-vents the transport vesicle from fusing with the prelysosomal compartments(late endosomes) and protects the contents of the transport vesicles fromlysosomal degradation (94).

Other Microtubule Reagents Used

Vinblastine (0.1 mM), vincristine (0.1 mM, 30 minutes preincubation) gri-seofulvin, iodoacetamide (IAA), podophyllotoxin, and taxol (96–98).

Marker for Microtubule Movement

Fluorescent tubulin, antitubulin antibody, and fluorescent taxol.

Dynamin Dependence on Liposome Uptake

Dynamin is a small motor protein with GTPase activity, which is involved invesicle formation and the pinch-off of coated vesicles from the plasmamembrane. Although it was originally thought that dynamin was specificto clathrin-mediated uptake, it has become clear that dynamin functions

364 Huth et al.

in other endocytic mechanisms such as caveolae-mediated endocytosis, pha-gocytosis (and possibly macropinocytosis) (51,52). The best way to studythe dependence of dynamin on the internalization of particles is the use ofdynamin mutants as described in (99,100) and with dynamin-EGFP.

Calcineurin Inhibition

The immune-suppressing drugs tacrolimus (FK 506) and cyclosporin Atarget the phosphatase calcineurin that is involved in the dephosphorylationof dynamin. It is reported that the uptake of liposomes was significantlydecreased after incubation with FK506 (1 mM, 10 minutes prior incubationwith liposomes) (1).

Energy Dependence on Liposome Uptake and Fusionwith Cell Membranes

At 4�C, endocytosis is inhibited, but binding to cell surface receptors andpassive fusion processes still can occur (82,93,101). Often cells are incubatedfor 60 minutes at 4�C. When applying this technique, it should be made surethat all buffers and substances are not warmer than 4�C because recoveryoccurs within seconds.

METABOLIC ACTIVITY

Endocytosis has been shown to be dependent on metabolic activity. A blockof metabolic activity might also be used to differentiate endocytosis fromfusion or cellular association. Either glycolysis or oxidative phosphorylationcan be affected, or both.

Cytosolic Glycolysis

Iodoacetamide

IAA (1–8 mM), an inhibitor of glyceraldehyde-3-phosphatase dehydro-genase selectively inhibits glycolysis. IAA is also known to modify thesulfhydryl groups in tubulin and affects the normal assembly of the cyto-skeleton (96,97,102).

2-Deoxyglucose

2-Deoxyglucose (2DG) is a nonmetabolizeable carbon source that blocksglycolysis in concentrations around 50 mM (103).

Sodium Fluoride

Sodium fluoride (104) (1–10 mM) inhibits two enzymes of glycolysis: theenolase (phosphopyruvate hydratase) and pyruvate kinase. Therefore, aero-bic glucose utilization and lactate formation are blocked.


Mitochondrial Oxidative Phosphorylation

� Dinitrophenol (1 mM) and potassium cyanide (KCN) (5 mM) areused as uncouplers of oxidative phosphorylation from electrontransport (105).

� Antimycin A (1 mg/mL) and sodium azide (NaN3, 10 mM, if usedalone; or 0.1%) act at cytochrome oxidase B (antimycin) and blockoxidative phosphorylation (101).

� Formaldehyde: treatment with low concentrations of formalde-hyde can lead to a series of effects, one example being the decreasein mitochondrial membrane potential and the inhibition of mito-chondrial respiration.

Combinations

It seems that both glycolytic and mitochondrial inhibitors must be present inorder to prevent endocytosis, as either inhibitor alone has minimal effect.Several combinations have been described in the literature, and a few arementioned here:

1. Combination of NaN3 (5 mM) and 2DG (50 mM): preincubationat 37�C for 30 minutes (93,101)

2. Mixture of antimycin A (1 mg/mL), NaF (10 mM) and NaN3

(0.1%) (4)3. Antimycin A plus NaF, no NaN3 (101).

TRANSCYTOSIS

Transcytosis is the transport of macromolecular cargo from one side of acell to the other within membrane-bound carriers (138). Multiple transcyto-tic mechanisms can be involved in this transport.

However, the initial step of uptake is a branch of the endocytic uptakeand corresponds to one of the mechanisms described in the section on‘‘Initial Mode of Internalization.’’ In most studies, clathrin-mediated orcaveolae-mediated mechanisms are discussed.

In polarized cells such as epithelial cells, the endocytic system is alsopolarized. Both endosomal systems (apical and basolateral) are spatiallyand functionally distinct: for example, apical endocytosis is highly depen-dent on microfilaments (137,139).

The apical clathrin-independent pathway is selectively stimulated byreagents that raise intracellular cAMP, such as mastoparan, fluoride, orcholera toxin. Apical endocytosis is also stimulated by brefeldin A (BFA)(106) or by PMA. For an excellent review on transcytosis, see Tuma andHubbard (138).

366 Huth et al.

Stimulation of Transcytosis

Brefeldin A

BFA is a fungal metabolite from Eupenicillium brefeldianum, which inducesmissorting of proteins. It highly specifically and reversibly blocks the mem-brane transfer from the ER through the Golgi apparatus by displacinga guanine nucleotide from adenosine diphosphate (ADP)-ribosylationfactor-1 (140,141). These effects are generally accompanied by a redistribu-tion of the trans-Golgi network (TGN) and endosomal and lysosomal com-partments (61,142). These effects can be used to determine whether aparticular protein or lipid redistributes precisely with a known compartmentmarker or in a different way (143).

BFA is often applied to increase the rate of transcytosis (variousconcentrations have been described, ranging from 1.6 to 10 mg/mL for 5 to10 minutes up to 50 mg/mL for one hour prior to labeling). However, itshould be noted that the effect depends on the type of targeted receptorbecause it enhances the transcytosis rate of Tfn, but not of EGF (61).

In other studies, BFA is used to study the recycling pathway becauseBFA prevents the formation of clathrin-coated buts in recycling endo-somes (76).

Phorbol 12-Myristate 13-Acetate

Phorbol esters are described to stimulate apical transcytosis probably viaprotein kinase C. These effects can be achieved by incubating cells withthe tumor promoter PMA [1 mM (to 2 mM)] for 30 minutes (to 60 minutes)(107,108). The total drug exposure time to about one hour because PMAshows an influence on tight junction permeability and thereby changes thetransepithelial resistance.

Inhibition of Transcytosis

As mentioned above, several endocytic systems have been discussed fortranscytosis. Therefore, in general, the inhibitors described in the section‘‘Initial Mode of Internalization’’ for the different endocytic pathwaysand inhibitors might be applied to influence transcytosis.

The following inhibiting agents are often used: NEM, the PI-3 kinaseinhibitors wortmannin and LY294002, filipin, nocodazole, colchicine, cyto-chalasin D, and the tyrosine kinase inhibitors herbimycin and genistein (alldescribed above).

Interestingly, lysosomotropic agents such as monensin and ammo-nium chloride suppress the rate of transcytosis, whereas bafilomycin A1 isdescribed as increasing transcytosis.


Marker for Transcytosis

Because transcytosis occurs in a number of different cell types, the nature ofcargo also varies. It is not limited to only macromolecules (immunoglobulinssuch as IgA and IgG) because several vitamins (Vitamin A, B12, and D) and ions(iron) also use an endocytic mechanism for their transcellular passage. For suit-able markers for transcytosis, we refer to the review from Tuma et al. (138).

GENERAL CONSIDERATIONS

Cellular Association or Uptake?

To discriminate the uptake of particles from cellular associated or bondedones, several methods have been developed:

1. NBD-PE liposomes: Fluorescence of liposomes that contain NBD-PE only in their outer monolayer can be quenched by addingsodium dithionite. Sodium dithionite reduces the nitro group of thefluorophor to an amino group, resulting in a quenching of fluores-cence (80,144,145).

2. Acid-wash treatment: mild treatment with acid buffer (e.g., 40 mMcitric acid, 120 mM NaCl, pH 3.0; incubation for 10 minutes)(80,146,147); treatment with 0.1 M glycine, 0.1 M NaCl, pH 3.0 isdescribed to remove uninternalized ligands (148).

3. Trypsin treatment is described to remove cell-associated lipidsafter treatment with 0.25% trypsin for a few minutes (better effectsare seen with increased incubation time and phosphatidylcholineconcentration) (149).

4. Protease treatment: After washing, cells are exposed to a solution of0.04% protease (pronase E) in Ca27Mg2þ free HEPES-containingethylene glycol tetra acetic acid (EGTA) (Sigma) for 15 minutesat 37�C as applied in (93). Cells are then washed three times withice-cold phosphate buffered saline (PBS) containing 0.1% BSA.

5. Trypan blue (0.4%), a substance that is usually applied to identifydead cells, can be used as a quencher for some of the external dextranconjugates [fluorescein isothiocyanate (FITC)-dextran] (72,129).

6. Incubation at 4�C (see section ‘‘Energy Dependence on LiposomeUptake and Fusion with Cell Membranes’’) and a block of meta-bolic activity (see section ‘‘Metabolic Activity’’) might also be usedto block endocytosis and to detect cellular association or fusion.

Toxicity of Applied Inhibitors

It should be kept in mind that high lipid concentrations and/or long incuba-tion times may be toxic for cells and mimic results seen with reduced uptake.Therefore, all experiments need to be accompanied by viability assays, for

368 Huth et al.

example, with 7-amino-actinomycin D, which penetrates cell membranes ofdying or dead cells (150). See Ref. 151 for a review on detection of dead cellsby flow cytometry.

Interaction of Inhibitors and Liposomes: Leakage Tests

Quenched fluorophores are encapsulated in liposomes and exposed to theinhibitors in the desired concentrations. If leakage of liposomes occurs,fluorescence intensity should increase significantly. Examples for appliedmarkers are: (i) self-quenching calcein (< 60 mM); (ii) HPTS (35 mM) withthe quencher DPX (50 mM) (126,128); and (iii) 8-aminonaphthalene-1,3,6-trisulfonic acid (ANTS) and DPX.

Suitability of Applied Inhibitor or Marker

Specificity

The use of the term ‘‘marker’’ is often misleading because it can be inter-preted to imply an absolute specificity that does not reflect the dynamicnature of intracellular compartments. It is important to remember thatthe predominant location of these markers reflects a balance of traffickingpathways into and out of each compartment. Marker can be internalizednot only by one but by different mechanisms. Even SV40, which is ‘‘known’’to be a specific marker for caveolae uptake, is now also described as enteringthe cells via a different mechanism (121). Therefore, not just one but severalinhibitors or markers for each mechanism should be applied and all resultsshould be taken into account before reaching a conclusion.

Quenching

The quantification of fluorescent particles in cellular systems is difficultbecause several aspects such as autofluorescence, bleaching (see below), andquenching hamper analysis. Keep in mind that many fluorophores show apH-dependent change in emission spectrum and intensity: fluorescein-labeled dextrans (FITC-dextran) and calcein are strongly quenched uponacidification. If available, one should read the fluorescence intensity at itsisosbestic point, where the intensity is not pH dependent.

Incubation Time and Concentration

Different incubation times and concentrations of applied marker might leadto different patterns of distribution. For example, LysoTracker Red is onlyselective for lysosomes when applied in low concentrations for a short time(10 minutes prior to imaging). The same has been described for other mark-ers such as EGF and Tfn (as described in the section ‘‘Caveolae-MediatedEndocytosis’’).


Bleaching

Fading of fluorochromes upon excitation is a photochemical process. Light-induced damage to the fluorochrome is often even more prominent in thepresence of oxygen (152). When fading, inter- and intramolecular processesrun off, causing a transmission of the excitation energy on molecules of theimbedding medium as translation or oscillation energy. The reduction offluorescence intensity can be decreased by an appropriate mounting medium(153–156). Mounting media prevent retardation of fluorescence intensity byseveral mechanisms: (i) removing oxygen from the medium, (ii) increasingviscosity to retard diffusion, and (iii) using reagents that reduce the amountof oxidized fluorochromes.

Autofluorescence

Autofluorescence of cells often complicates the studies with fluorescencemicroscopy (especially the application of green fluorescent substances). Thereare different reasons for the occurrence of this phenomenon (157): (i) thefluorescent pigment lipofuscin, which settles with rising age in the cytoplasmof cells; (ii) cell culture medium, which often contains phenol red thatincreases autofluorescence; (iii) endogen substances such as flavin coenzymes[flavin-adenine dinucleotide (FDA), flavin mononucleotide (FMN); absorp-tion/emission~450/515 nm], pyridine nucleotides [reduced nicotinamideadenine dinucleotide (NADH); absorption/emission~340/460 nm] orporphyrine; (iv) substances taken up by cells (as mentioned above: filipin);and (v) preparation of the cells: fixation with glutaraldehyde increasesautofluorescence.

To circumvent this problem, several methods have been developed(157). Probably, the most prominent method is treatment with sodium boro-hydride (0.1% in PBS, 30 minutes prior to staining). NaBH4 is known toneutralize Schiff’s bases through reduction of amine-aldehyde compoundsinto nonfluorescent salts.

Suitability of the Conditions

Presence of Serum

The presence of serum influences the incubation with liposomes in manyways. It has been shown in several cases that liposomes might interact withproteins present in serum, causing leakage of liposome contents (158,159).

Binding of highly lipophilic inhibiting agents to serum componentsmight decrease the concentration of the drugs (see section on ‘‘Wortmannin’’under ‘‘Macropinocytosis and Phagocytosis’’). Therefore, some inhibi-tors should rather be applied in the absence of serum or used in a higherconcentration.

370 Huth et al.

Artifacts from Fixation

Most commonly used fixation protocols for adherent cultured cells includetreatment with paraformaldehyde (PFA, up to 4%, 10 minutes), which isextremely efficient in cross-linking proteins. The majority of reactive sitessubject to cross-linking are primary amines, sulfhydryl groups, and guanidyland aromatic rings. Other protocols describe treatments with glutaraldehyde(0.1%) or formaldehyde (increases autofluorescence, see above), or a 5- to10-minute exposure to �20�C methanol. Keep in mind that methanol fixa-tion might lead to a loss of soluble proteins (160) and that cell-permeantsubstances might be redistributed by any fixation treatment [e.g., PFA treat-ment has been shown to redistribute doxorubicin conjugates (161)].

Exocytosis

Keep in mind that exocytosis might occur during sample preparation (e.g.,preparing cells for flow cytometric analysis). After the experiment (and priorto analysis) cells should be kept below 4�C to block all active processes (suchas exocytosis). Exocytosis might also be blocked with NEM (see section‘‘Caveolae-Mediated Endocytosis and Caveolae-Like Endocytosis Pharma-cological Inhibitors’’).

CONCLUSION

Cellular uptake is a diverse set of processes used by the cell. Often, the cellutilizes a variety of processes and not just a single mechanism. Therefore, dif-ferent approaches should be taken to investigate. In this review, we havefocused on several aspects of cellular internalization: mechanisms to studythe initial mode of internalization, and aspects of intracellular trafficking andmetabolic activity. These aspects should not be regarded individually butshould be considered together because often one aspect depends on the other.

It is important to know that the use of the different inhibitors has to beevaluated for each cell type, regarding applied concentrations and incuba-tion times. Concentrations that are too high might show toxic effects andmight mimic results seen with reduced uptake. Therefore, all experimentsneed to be accompanied by viability assays.

Furthermore, our own studies showed that not all inhibitors seem tobe useful for their suggested application in all cell types. Some inhibitors(amiloride and indomethacin) did not show an effect (not even on theuptake of the corresponding marker substances) in COS-7 and HUVEC.Consequently, not only one but a series of inhibiting agents with preferablydifferent modes of inhibition should be applied and combined with coloca-lization studies.

Regarding the uptake of liposomes, the mechanism of uptake is highlydependent on several additional factors: (i) desired cell type, (ii) liposome


composition, (iii) particle size, (iv) charge ratio, (v) time of incubation, (vi)activation of target cells, (vii) sterical stabilization, and (viii) specific charac-teristics of the homing devices.

Therefore, no detailed discussion on the interaction of any liposomeswith any particular cell type should be stated here. We refer to several recentpublications for a study of interaction of DOPE:CHEMS liposomes andCOS-7 and HUVEC (109) and for a study on size-dependent uptake ofparticles into B16-F10 (72). The combination of flow cytometry and amicroscopic method (e.g., spectral bio-imaging) turned out to be highlyuseful both to study the initial mode of internalization and to follow theintracellular fate of liposomes and other particulate carrier systems.

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Index

Abelcet1, 95, 104, 106circular dichroism spectrum of, 101

Acridine orange (AO), 7Adaptor protein AP-2, 342Adenosine triphosphate (ATP)

encapsulation, 134Adipocytes, 343Alkylammonium salts, 39Allosteric modifiers, 69a-a–cross-linked hemoglobin, 63Amanita phalloides, 363AmBisome, 94, 103, 104AmB–lipid interactions

analysis of, 100–102detection by physicochemical

techniques, 99AmB-resistant strain (AmB-R), 105AmB. See Amphotericin BAminomethoxy-PEG, 2776-(3-Aminopropyl)ellipticine (APE),

151, 159Ammonium glucuronate (AG)

gradient, 8Ammonium ion

amphipathic weakbases, loading of, 5–6

as anion, 6–8pharmacokinetic performance, 8role of, 6

[Ammonium ion]therapeutic performance, 8

concentration of, 15Doxil in, 3sulfate anion as of, 3transmembrane, 17

liposomal collapse of, 5, 20Ammonium sulfate (AS)

transmembrane, 2Ammonium sulfate method,

in drug loading, 44Amphipathic weak base, 3

in liposome loading, 4nigericin, effect of, 6nonactine induced release of, 6

Ampholiposome formulation, 103Amphotec, 95, 106Amphotericin B (AmB)

activityin vitro, 105in vivo, 107–108

acute toxicity of, 103, 104critical micellar concentration of, 101doses of, 104immunomodulating effects of, 96lipid complexes of, 95

air-drying technique, 98electron microscopy, 99freeze-fracturing technique, 98

383

[Amphotericin B (AmB)]materials for, 96morphology of, 96nanoprecipitation procedures,

96–97preparation of, 96

metabolism, 105in methanol, 96, 97physical characterization of, 97polydispersity of, 97–99preparation by solvent displacement

method, 99properties of

hemolytic, 103immunomodulating, 106

size, 97–100structure of, 94in TNF-a production, 107toxicity, 102–105

in vitro, 102–104in vivo, 104–105

UV spectra of, 100zeta potential of, 97–99

Anchors, hydrophobic, 112, 114, 115AnthracyclinesAnticlathrin antibodies, 353Antigen presenting cells (APCs), 118,

213, 233Antigenic peptides, 249Antioxidants, 69–70Antisense oligonucleotides, 131Antixodiant tempamine (TMN), 4APCs. See Antigen presenting cellsAqueous phase, intraliposome, 4, 6Archaeobacteria, 120Archaeosomes, 120Auto-oxidation of hemoglobin, 69AZT. See Azidothymidine

Bacterial artificial chromosome(BAC), 296

Bartlett methodlipid concentration, 192phospholipid concentration, 17

B-cell receptors (BCRs), 121clustering in B-lymphocytes, 124

b-amyloid peptide, 1191-b-D-arabinofuranosylcytosine

(ara-C), 523b(N,N,-dimethylaminoethane)-

carbamyl cholesterol(DC-CHOL), 235

3b-[N-(N0,N0-dimethylaminoethyl)carbamoyl]-cholesterol(DC-Chol), 257

1,2-Bis (hexadecylcycloxy)-3-trimethylamino propane(BisHOP), 235

Bisphosphonates (BPs)cell-specific delivery system

of, 190in deionized water, 191encapsulation of, 190, 191inflammatory mediators affected

by, 195liposomal delivery system of,

191, 193macrophage/monocyte of, 191

Bola-amphiphilic drug, 328Bone marrow–derived dendritic cells

(BMDC), 247Bovine hemoglobin, 68, 69Breast cancer cells, epifluorescence

microscopic images of, 325Brefeldin (BFA), 367

Caelyx1, 174Cancer cells, breast

epifluorescence microscopic imagesof, 325

Cancer immunotherapy, 221Carbonyl-hemoglobin, 67Cardiovascular parameters, 81Catalase, coencapsulation, 70Cationic lipids, 254Cationic liposomes, 293Caveolae

inhibitors of, 357stimulation of, 358

Caveolae-like endocytosis, 343Caveolae-mediated

endocytosis, 341, 343

384 Index

Caveolae pathway, 358caveolin-1-GFP, 359cholera toxin B, 359folic acid, 359lactosylceramide, 359marker for, 3585-methyltetrahydrofolate in, 359

Caveosome, 303CD4+ cells, CTL induction of, 231CD8+ cells, 223Cell exposure and confocal

fluorescence, 330Cell-penetrating peptides (CPPs), 117,

303–305applications of, 301cell association with, 304characteristic of, 303subclasses of, 303uptake of, 304

Cellulose ester membranes, 73Chlorpromozine CPZ interaction

with, 351lipid-raft–mediated pathways in, 343macropinocytosis in, 344transfection of, 255, 261, 268

Chlorpromazine (CPZ), 353Cholesteryl chloroformate, 277Chronic viral diseases, 221Ciprofloxacin, 38, 39, 40Cisplatin, 2Clathrin-coated pits, Semliki forest virus

in, 342, 354Clathrin-coated vesicles, 342Clathrin-mediated endocytosis, 301, 341Clathrin pathway

epidermal growth factor(EGF) and, 355

human transferrin (Tfn) and, 356hypertonic media for, 353low-density lipoprotein

(LDL) and, 356marker for, 355–356

Clathrin uptake, inhibitors for, 353Clostridium botulinum toxin C2, 365Colloid oncotic pressure (COP), 72CONTIN algorithm, 17CpG-oligodesoxynucleotide, 209

CPPs. See Cell-penetrating peptidesCritical micelle concentration

(CMC), 67Cryoelectron microscopy, 33, 45Cyclotron products, 172Cystic fibrosis, 253Cytochrome P450, hepatic microsomal,

104, 105Cytokine

activity, 195production, 248

Cytosolic glycolysis, 367Cytotoxicity, 295Cytotoxic nucleoside, 52Cytotoxic T-cells (CTL), 210, 221

Daunorubicin, 29Daunoxome1, 174Deferoxamine (Df), 173Dehydration–rehydration vesicles

(DRV), 236measured by photon correlation

spectroscopy (PCS), 237preparation of vaccine-containing,

237vaccine entrapment estimation, 238

Dendritic cells (DCs), 118, 208, 343activation of, 215–216

Deoxyhemoglobin, applicationsof, 68

Dequalinium chloride, 320, 332chemical structure of, 327cyclohexyl derivative of, 330mitochondriotropism of, 328vesicles, 327

Dextran sulfate, 138Dextran sulfate ammonium salt

(DSAS), 7Dideoxycytidine (ddC), 54Dideoxyinosine (ddI), 54Diepitope liposomal construct

antitumoral response elicitedby, 122–123

design of, 114, 120humoral response elicited by, 121parameters for, 122

Index 385

2-Diethylaminethyl-ellipticinium, 151Diethylenetriaminepentaacetic acid

(DTPA)as chelator, 174encapsulated in liposomes, 174

Dimethylammoniumpropane(DODAP), 133

Dimyristoyl phosphatidylcholine(DMPC), 66, 95

Dinitrophenol (DNP), 350Dioleoyldimethylammonium chloride

(DODAC), 134Dioleoylphosphatidyl-ethanolamine

(DOPE), 133, 255, 2941,2-Dioleyl-3-dimethyl-ammonium

propane (DODAB), 2351,2-Dioleyloxy-3-(trimethylamonium

propane) (DOTAP), 2351,2-Dioleyltriammonium propane

(DOTAP), 2461,5-Dipalmitoyl-L-glutamate-N-succinic

acid, 672,3-Diphosphoglycerate, 69Dipalmitoyl phosphatidylcholine

(DPPC), 66Dipalmitoyl-phosphatidylglycerol

(DPPG), 66Distearoyl phosphatidylcholine

(DSPC), 65, 132Distearoylphosphatidyl-ethanolamine

(DSPE), 180DNA, 245

complexes, 295protection of, 245toroid structure of, 245transfer of, 308

DNA complexes (DQAplexes), 328–329DNA-mitochondrial leader sequence

(MLS) peptide conjugates, 328synthesis of, 329

Doppler velocimetry, 98DOX. See DoxorubicinDoxil1, 2, 174

demonstration of, 13side effects of, 8

Doxorubicin (DOX), 29, 155, 282ammonium sulfate (AS) in, 2

[Doxorubicin (DOX)]in interstitial fluid, 11loading mechanism, 5partition coefficient of, 10release of, 11–13removal of, 15

Doxorubin-sterically stabilizedliposome, 15–23

characterization of, 16chemical stability of, 17conductivity measurements of, 15pH and ammonia measurements

of, 17quantification of, 16transmembrane AS gradient

in, 22DQAsomes (dequasomes)

dequalinium in, 335DNA incorporation into, 328paclitaxel-loaded, 332–335

physicochemical characterizationof, 333-335

preparation of, 332plasmid DNA delivery, 328preparation of, 326, 328tumor growth inhibition study,

333–335Drug

bola-amphiphilic, 326cationic amphiphilic, 353chemotherapeutic, 28duplex, 54

chemical structures of, 55concentration of, 56heterodinucleoside phosphate, 56hydrolysis of, 58in vitro cytotoxic effect of, 57mechanisms of, 57–58

encapsulation of, 28, 155entrapment efficiency, 155–156lipophilic, 51liposomal formulations, 29, 131loading of

citrate and ionophore methods, 32,40, 44

loading methods, comparison of,42–45

386 Index

[Drug]pH-gradient method for, 33

macromolecular, 341precipitation or gelation of, 161retention

rates of, 45role of, 44

spin columns for monitoring, 42therapeutic genes as, 253therapeutic index of, 322

Drug delivery systems, 27, 111development of, 29liposomal features of, 28–29

Drug-eluting stents (DESs), 188Drug-entrapment strategies, based on

ion gradients, 150

Emulsifexl-C5, 212Endocytosis, 256, 341

caveolae-mediated andcaveolae-like, 343

marker for, 358-359pharmacological inhibitors

for, 356clathrin-mediated, 342–343, 351

marker for, 355pharmacological inhibitor of, 351

endosomes in, 342intracellular routes of, 344macropinocytosis and

phagocytosis, 360marker for, 361pharmacological inhibitors

for, 360metabolic activity, 367methods of, 344nonclathrin-noncaveolae-

mediated, 344spectral bio-imaging method

for, 344–345Endocytotic cells, 193Endoplasmic reticulum (ER), 343Endosomes, 342

acidification of, 361disruption, 305marker for, 363

Endotoxin, 72–73Enzyme linked immunosorbent assay

(ELISA), 242Epidermal growth factor fluorescent

receptor (EGFR) pathway, 355Epirubicin, 151Epitope, 115Ergosterol, 106Escherichia coli

infection, 181lipopeptide, 121

Ethanoldynamic light scattering

measurements, 133encapsulation, 138

lipids dissolved in, 133TEM electron micrograph of, 137

Ethynylcytidine 1-(3-C-ethynyl-b-D-ribopentafuranosyl)-cytosine(ETC), 54

Eupenicillium brefeldianum, 369Exocytosis, 373

Fetal calf serum (FCS), 209Fibroblasts, 343Fick–Nernst–Planck equation, 319Filters, polycarbonate,

30, 73Fluoresceinlabeled-dextrans

(FITC-dextran), 371Fluorescent liposomes (FL), 194Fungizone1, 93, 104, 106

Gamma radiation emitters, 169advantage of , 170physical characteristics of, 171

Ganglioside GM1, 67Gel

exclusion chromatography, 18filtration chromatography, 154

Gemcitabine, 52Gene

therapy, 253, 293potential use of, 296

transfection, 275transfer, 254

Index 387

[Gene]polycationic amino acids for, 297therapeutic, 294

Golgi apparatus, 303, 341Green fluorescent protein (GFP), 355Green fluorescent protein-RhoB, 361

Hanks balanced salt solution(HBBS), 209

Haptides, 305Hemagglutinin (HA), 221, 242Hematopoietic cells, 190Hemoglobin

auto-oxidation of, 69bovine, 68, 69efficiency of, 67encapsulation of, 64, 66

in egg-PC liposomes, 65oxidation, 64, 69

by UV light, 65oxidation and denaturation, 68oxygen affinity of, 68as oxygen carrier, 63–64, 65postinsertion technique for, 67preparation of, 64properties of, 68–69in red blood cells (RBCs), 64retention, 76source of, 68–69stability of, 68

Hemoglobin-to-methemoglobinconversion, 79

Hemolysis, 103Hexamethylpropyleneamine oxime

(HMPAO), 174advantage and disadvantage of, 175

Human leucocyte antigen (HLA), 226Human papilloma virus (HPV), 249Hydrazino nicotinamide

(HYNIC), 176applications, 180

Hydrazone, 112Hydrogenated soybean PC

(HSPC), 148-Hydroxypyrene-1,3,6-trisulfonic acid

(HPTS), 363

Hypercholesterolemic rabbit model,197–198

Hypertonic saline (HS), 72Hypertonic saline (HS)–peptide,

112–115reaction of, 113with bromoacetyl functions, 114with maleimide functions, 113

Imipramine, 353Immature dendritic cells (iDC), 226Immunization, 241–243

liposome-based codelivery approachof, 241–242

studies of, 241Immunopotentiating reconstituted

influenza virosomes (IRIV), 221adjuvant in hepatitis, 221chemokines secretion in, 225cytokine gene expression in, 224–225cytokine secretion in, 225–226cytotoxic T-cell adjuvant, 226effects on

antigen-presenting cells (APC), 225dendritic cells (DC), 225

in vitro characterization of, 222in vitro evaluation of, 226production of, 222

Immunoproteins, 78Immunotherapy, cancer, 221Indium, 169Influenza matrix (IM), 226Inhibitors

pharmacological, 353amiloride, 360bafilomycin A1 and

concanamycin A, 362calcineurin, 355cardiac glycoside digitoxin, 358chloroquine, 362filipin, 356genistein and herbimycin, 357histamine, 357HMG-CoA, 355indomethacin, 357LY294002 and wortmannin, 360

388 Index

[Inhibitors]methyl-b-cyclodextrin (mbCD),

354, 357N-ethylmaleinimide (NEM), 357okadaic acid, 358phenylarsine oxides, 355phorbol esters, 358polyene antibiotic nystatin, 358staurosporine, 358toxin B, 361

recombinant, 355, 358Interferon-c (IFN-c), 234Intraliposomal drug stabilization

of LAQ824, 152using polyanionic trapping

agents, 152Intraliposomal osmotic pressure, 10Iodoacetamide (IAA), 3505-Iodo-20-deoxyuridine, 52Irinotecan, 151Isotonicity agent, uses of, 154–155

LAQ824, 150–151Large unilamellar vesicle (LUV),

28, 132ethanolic suspensions of, 133fiber bundles in, 33formation of, 30fractions, 37freeze–thaw extrusion method, 30

from multilamellar vesicles, 30lipid composition of, 28preparation, 30

Leishmania donovani strains, 105–107Leishmania parasites, 96Leishmaniasis, treatment of, 93Ligands, 111, 117

and integrins, interaction of, 297Limulus amebocyte lysate (LAL), 73Lipid(s)

cationic, 273biophysical characteristics of, 254classification of, 257screening of, 259, 263solid phase synthesis of, 263–264structural elements of, 257, 263

[Lipid(s)]transfection profile of, 262

concentration of, 31, 37dispersed in water, 27emulsion, 32formulation

in vitro and in vivo systems, 96toxicity of, 103

hemoglobin suspension, 73lipofection properties, 264in membranes, 28monocationic, 257oxidation of, 66polycationic, 257–258transfection efficiencies, 265zwitterionic, 133

Lipid-based systems, Amphotericin B(AmB) into, 95

Lipofection, 254, 293cationic lipids, 257characteristics, 295enhancement of, 305process of, 254reagent, preparation of, 255robot system for, 259–260screening

capacity, 262procedure, 259–262

steps of, 254Lipoid, 14Lipopeptide, amphiphilic triacylated,

121–122Lipophilic arabinofuranosyl cytosine

N4-alkyl derivatives,synthesis of, 56

Lipophilic drugs, 51liposome formulations of, 53

Lipoplexes, 254, 255, 273binding, 274

to circulating cells, 275to lung endothelium, 275–276

complement activation of, 275in COS and HeLa cells, 246formation of, 261in vitro interaction of, 275pharmacokinetics of, 276

Lipopolyplex, 245, 294

Index 389

Liposomal alendronate (LA), 194Liposomal anticancer systems, 29Liposomal clodronate (LC), 194Liposomal doxorubicin

formulations, 29Liposomal drug

effect of, 192formulations

chemical and colloidal stabilityof, 164

development of, 52polyanionic trapping

agents in, 150release of, 12, 13retention of, 159

Liposomal ellipticine analog(APE), 160

Liposomal encapsulation, optimizationof, 7

Liposomal systems and large unilamellarvesicle (LUV), 28

Liposomal transfection efficiency, 295Liposomal vaccination system

buffers used in, 209on cytotoxic

T-cells, 216–217by fluorescence activated cell sorter

(FACS) analysis, 213Hanks balanced salt solution

used in, 209in vitro characterization and

optimization of, 213materials used in, 209–210methodology of, 208prophylactic efficiency of, 217–218rhodamine-phycoerythrin (PE)

in, 209therapeutic efficiency of, 217–218TRP-2 peptide in, 209

Liposome(s), 111, 131, 255, 322advantage of, 118and amphipathic weak base, 7–12in antitumor drugs, 52AS–SSL and AG–SSL, 8and B epitopes, 121binding and uptake of, 213BPs encapsulation in, 190

[Liposome(s)]cationic preparation of, 2and cell membranes, 370chemical structure of, 150–151complex formation of, 297concentration of, 164, 210–211CpG oligonucleotides, 212cytokine activity of, 195by dehydration–rehydration

procedure, 235, 239–240DNA entrapment within, 234–235,

239–240DOX loading into, 14effect of, 193, 196endocytosis of, 342entrapment efficiency, 136ethanol concentration in, 135–136fluorescence of, 370formulation of, 150by freeze-drying , 76hydrophilic coating on, 67and inhibitors interaction, 371internalization, multiple pathways

for, 341isotonicity agent in, 154labeling methods for, 172–181

comparison of, 181–183lipid bilayers, 12, 27, 191

matrix of, 52lipid composition of, 162lipid components of, 295lypohilization of, 180marker for, 371microscopic investigations of, 214mitochondriotropic, 322morphology of, 136and nucleic acids, 132, 295osmotic imbalances in, 163–164passive targeting of, 3

and peptides, 119peptide modification of, 295and peptides, 294pharmaceutical formulation in, 59physical properties of, 193in plasmid DNA, 131preformed vesicle (PFV) approach

for, 132

390 Index

[Liposome(s)]preparation of, 151, 323

steps in, 13properties of, 135protein vaccines into, 235, 239–240polyethylene glycol removal

from, 13RGD peptides on, 300size distribution, 17size determination, 211

cryo-TEM microscopy, 192dynamic light scattering, 192

small unilamellar, use of, 51by sucrose density gradient

centrifugation, 135suspension, 238targeting and binding of, 111,

297–299therapeutic performance of, 8unloaded, 161–162uptake of, 346–350, 362

actin, effect of, 360calcineurin inhibition, 367colchicine in, 366cytochalasins in, 360dynamin effect of, 366energy dependence on, 367factors affecting, 373considerations for, 370latrunculin A/B, 365marker for, 366microtubules, effect of, 366nocodazole for, 366preparation of, 238–241vaccine-containing

vaccine entrapmentestimation, 241

vinorelbine (VNB) in, 163zeta potential of, 324

Liposome transfection efficiency(TE), 254

calculation of, 261and configuration, 267in gene therapy, 254and hydrocarbon chains, 264methylation on, 267quantification of, 261

Liposome-encapsulated hemoglobin(LEH)

in animal models, 81biodistribution, 78–79endotoxin testing in, 72–73evaluation techniques for, 77formulation factors for, 65freeze-dry, 75–76functional capacity of circulating,

79–80gel clot method, 73homocysteine in, 70hypertonic saline and, 72lipid composition of, 65–67manufacture of, 74–75

extrusion method for, 73lipid phase homogenization, 74

methemoglobin estimation in, 77methemoglobin reduction in, 69modification of, 67osmolarity and oncotic activity

of, 72oxygen affinity of hemoglobin

in, 69particle size of, 70–71properties of, osmotic, 72source of hemoglobin for, 68stability of, 76–77sugars in, 76toxicity and immunological effects of,

80–81Liposome-labeling methods, 169

by aqueous space trapping, 172by external surface chelation, 179

Liposome-peptide constructs, 117–118application, 118targeting, 117in vaccination, 118

Liposome-polycation-DNA cationic(LPDI), 245

Liposome-polycation-DNA (LPD)aspects of, 251complexes, 245components of, 247delivery of, 246DNA in, 246and immune system, 247

Index 391

[Liposome-polycation-DNA (LPD)]limitation of, 246nanoparticles, 245transfection of, 246–247uptake of, 246vaccination studies with, 246as vaccine carrier, 249vaccines and adjuvants, 247–249

Listeria monocytogenes, 80Lurtotecan, 51LUV. See Large unilamellar vesicleLyoprotection, 76Lysosensor probes, 364Lysosome(s), 343

early or late, 363marker for, 363

Lysosome-associated membrane protein1 (LAMP 1), 364

Lysosomotropic reagents, 362Lyso tracker, 364

Macropinocytosis, 343–344Major histocompatibility complex

(MHC) molecules, 118, 249Mammalian cell membranes, 93Matrix metalloproteinases (MMPs), 189Methanol, 40, 96

concentration of, 97Methemoglobin

estimation in LEH, 77reduction, 69, 70

Methylamine, distribution of, 20Methyltriphenylphosphonium

salts, 320Micellar concentration, critical, 93Micelles, formation of, 116Micelle-transfer technique, 116Microelectrophoresis, 241Microfluidization, 238Microfluidizers, 74MilliQ1 water, 96Minimal transduction domain, 303Mitochondria (l)

bola-lipid–based, 325delivery systems, 320–322DNA delivery, 328

[Mitochondria (l)]dysfunction of, 318of eukaryotic cells, 317genome, 318inner membrane of, 317in living mammalian cells, 321, 328membrane potential of, 319molecules of, 319as pharmacological targets,

317–319outer membrane of, 317oxidative phosphorylation of, 368respiratory chain of, 317

Mitochondriotropic liposomes, 322Mitochondriotropic molecules,

chemical structure of,319–320

Mitoxantrone, 29Monocytes, 190Mononuclear phagocyte system

(MPS), 190Monophosphorylated (MP) ara-C, 54Monophosphoryl lipid A (MPL), 114Multilamellar vesicles (MLV), 15Muramyl tripeptide-PE, 119Murine melanoma cell, 209Mushroom and brushes regimes, 276Mycobacterium leprosy, 235Mycoplasma pneumoniae, 301

N 4-acyl-ara-C derivatives, 52N,N-bis(2-mercaptoethyl)-N0,N0

diethyl-ethylenediamine(BMEDA), 177

advantage of, 177–178pH gradient liposomes, 178

N-(1,2-dioleoyloxypropyl)-N,N-dimethyl-N-hydroxyethylammoniumbromide(DORI), 257

N-[1-(2,3-dioleoyloxy) propyl]-N,N,N-trimethylammoniumchloride(DOTAP), 254

N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammoniumchloride(DOTMA), 257

392 Index

N-[1-(2,3-dioleyloxy) propyl]-N,N,N-triethylammonium(DOTMA), 235

N-hydroxysuccinimidyl 3-(2-pyridyldithio) propionate, 115

Na-glyoxylyl peptide, 116Nanoprecipitation, for AmB, 97Neointima, 199Neuraminidase (NA), 221Nitrilotriacetic acid (NTA), 173N 4-octadecyl-ara-C (NOAC), 53

chemical structure of, 54duplex drugs in, 58ETC with, 54

Nuclear localization sequence, 307Nuclear pore receptor, 308Nucleic acid-based molecules

stabilized nucleic acid-lipid particle(SNALP) for, 132

stabilized plasmid-lipid particles(SPLP) for, 132

Nucleic acid therapeutics, serum-stablecarriers for, 132

Nucleus, passage of DNA into,256–257

Octasulfate, 150Oleic acid, 275Oligolysine peptides, (K)16, 294Oligonucleotides

circulation half-life of free, 141encapsulation of, 132entrapment of, 140

Oligovax, 145Oligreen assay, 134Osteolysis, tumor, 190Osteoporosis, 190Oxidative phosphorylation

(OXPHOS), 318Oxyhemoglobin, 69

oxidation and denaturation of,67–68

Paclitaxel, 51, 188Paraformaldehyde, 373Parasites, 105

Passive targetingdefinition of, 1sulfate in, 2

PEG. See Poly ethylene glycolPEG-CerC14 lipid, 139PEG-Cer oligonucleotides, 136PEG-diorthoester in SPLP, 282PEG-dipalmitoylphosphatidyl

ethanolamine (PEG-DPPE), 55PEG-DODA, incorporation of, 278PEG-linked phosphatidylethanolamines

(PEG-PE), 67aqueous solution of, 67in liposome bilayer, 67

PEG-lipids, 274acid-sensitive, 279–282advantages of, 278anionic, 278cleavable, 279degradable, 281determination of, 284–284examples of, 276incorporation of, 279and lipoplexes, 276, 279, 283lipoplexes with bi-anionic, 279in lipoplexes stabilization, 285for lipoplex incorporation, 276

PEG-lipoplexes, 283pharmacokinetics of, 283

PEG-liposomes, in humans, 180,181–182

PEGylated lipoplexes, 287–289biodistribution of, 287limitation of, 289

Peptidases, 343Peptides, 112

antigenic, 249categories of, 112conjugation of, 112construction of, 295, 304coupling of, 112endosomal escape assisted

by, 306fusogenic, 307haptotactic, 305

Percutaneous coronary interventions(PCI), 187

Index 393

[Percutaneous coronary interventions(PCI)]

procedures, 187treatment of stenotic coronary arteries

by, 187Peripheral blood mononuclear cell

(PBMC), 222Phage screening, 300

advantage of, 301peptide selection by, 301

Phagocytosis, 104Pharmacological inhibitors, 353Pharmacological therapies, categories

of, 187Phenyl-maleimide moiety, 115pH gradient techniques, 28

approach, 29columns method, 33by forming neutral HCl, 41generation

internal citrate buffer, 32–38transmembrane ammonia

gradients, 38transmembrane ion gradients,

40–41Phorbol myristate acetate

(PMA), 350Phosphatidic acid (PA), 235Phosphatidylcholine (PC), 13, 234Phosphatidylethanolamine

fluorescein, 284Phosphatidylethanolamine

(PE), 112, 235analogs of, 115

Phosphatidyl glycerol (PG),213, 235

Phosphatidylinositol-3 (PI-3) kinase, 360Phosphatidylserine (PS), 214Phospholipids (PL), 52, 155, 284

anchors, 115, 121, 122anionic, 65–66bilayer, 51concentration of, 97synthetic, 95

Photomultiplier tubes, 170Photon correlation spectroscopy

(PCS), 97

Plasmid DNA (pDNA), 131,233, 294

detergent dialysis, 141–142procedure, 134

encapsulation of, 132, 134separation of, 135

entrapment of, 235PFV approach for, 141in stabilized plasmid-lipid particles

(SPLP), 142in unilamellar liposomes, 134uptake by

APCs, 233muscle cells, 233

viral, 234Plasmodium falciparum, 328Platelet activating factor (PAF), 80Polyanetholsulfate, 138Poly (anionic) trapping agents,

chemical structuresof, 152

Polycarbonate filters, 30, 73Poly ethylene glycol (PEG), 51, 274

cholesterol, 278acid-sensitive, 280advantage of, 280

doxorubicin encapsulated, 174Polylactide capsules, 64Polymeric anions, 6–7

advantage of, 8Polymeric coated stents, 188Polyornithine reagents, 297Polyphosphate, 150Postgrafting method, 289Postinsertion techniques, 116Potassium, intracellular depletion

of, 353Preformed vesicle approach

(PFV), 132Proton gradient, transmembrane,

collapse of, 5Protons distribution, in

mitochondria, 318Pseudopeptides, 120Pseudopodia, 344Pyranine, 363

fluorescence of, 17

394 Index

Quinolinium ring system, 326Quinolone antibiotic, 38

Radiolabeled liposomal formulation,171

Radionuclide emitting photons, 170Radionuclides, 170–172Radiopharmaceuticals, 170Radiotracers, 170Rat vascular injury model, 197Red blood cells (RBCs)

enzymes, activities of, 69oxidoreductive system of, 64

Reductants, effectiveness of, 70Renca-ErbB2+ cells, 123Restenosis, 187

in hypercholesterolemic rabbitmodel, 197

inflammation and, 189inhibition of, 197

mechanism of, 199macrophages depletion in preventing,

190process of, 188rate of, 187in rat vascular injury

model, 197Reticuloendothelial cells, 171Reticuloendothelial system (RES),

64, 275Rhodamine, 319

Scintigraphic imaging technique,170–171

in nuclear medicine, 170radionuclides for, 170

Sephadex G-50 columns, 36Small unilamellar vesicles

(SUVs), 236preparation of, 236–237

Smooth muscle cells (SMCs), 188Sodium dodecyl sulphate (SDS), 234Solid ordered (SO) to liquid disordered

(LD) phase transition, 12Somatic cells, 208

Soy phosphatidylcholine(SPC), 54

Sphingosine, 353Spin columns, 36, 42SPLP. See Stabilized plasmid-lipid

nanoparticleSSL. See Sterically stabilized liposomeStabilized nucleic acid-lipid particle

(SNALP), 132administration of, 145CpG motifs in, 132half-life of, 140pharmacokinetics of, 139

Stabilized plasmid-lipid nanoparticle(SPLP), 132, 282

administration of, 143pharmacokinetics of, 143–144plasmid-containing, 145transfection of, 143tumor accumulation of, 143in tumor-bearing mice, 143

Staphylococcus aureus, 176Tc-HMPAO in rats with, 181

Stearylamine (SA), 235Stearyl triphenylphosphonium bromide,

synthesis of, 322Stearyl triphenylphosphonium (STTP)

liposomesin vitro applications of, 324and phospholipids, 325

Sterically stabilized liposome (SSL), 2ammonium sulfate gradient, 7characterization of, 7DOX, 9Dox loading, 7

condition for, 10mechanism of, 5, 8method and stability of, 9preparation of, 14

preparation of, 15Streptococcus mutans cell, 121Sulfate versus glucuronate, 8–9Surfactant protein B (SPB), 307Swainsonine, 151SYBRTM, 328Synthesized stearyl TPP (STPP)

salts, 322

Index 395

Target protein (mTOR), 188Targeted liposomal therapeutics, 160T-cell leukemia, 254T-helper (Th) peptide epitopes, 120T-lymphocytes, 20899mTc-Doxil1, 178

scintigraphic images of, 17999m Tc-HMPAO, 17599mTc-HMPAO-labeled PEG

liposomes, 17699mTc-liposomes, 175, 180

labeling method forBMEDA-glutathione of, 177hexamethylpropyleneamine

oxime-glutathione of,174–177

Tc. See Technetium99mTc-trapping methods, 174Technetium (Tc), 78-79, 169Thrombocytes, 80Tissue macrophages, 194TNF. See Tumor necrosis factorToll-like receptors

(TLRs), 118, 247Topotecan, 29, 151Transcriptional activator (TAT)

amino acids, 304with cationic liposomes, 304invasion domain of, 303liposomes, 304protein, 117

Transcytosis, 368inhibition of, 369marker for, 370stimulation of, 369

Transfection efficacy,liposomes, 274

Transmembrane ammonium iongradient, collapse of, 5

Transmembrane pH gradientcollapse by nigericin and

nonactine, 22determination

AO, 22pyranine medium, 18radioactive methylamine

method, 21

[Transmembrane pH gradient]ratiometric method, 17spectrofluorometer, 20

Transmembrane proton gradient,collapse of, 5

Transmission electron microscope(TEM), 136

Trapping agents, intraliposomal, 150anions used as, 158polyanion, 158

chemical structures of,152–153

gradient for, 154–155sulfate as, 164

Triethylammonium (TEA), 159Triphenylphosphonium

(TPP), 320Tris(2 carboxyethyl) phosphine

hydrochloride (TCEP), 329Tumor-associated antigens (TAAs),

uses of, 249Tumor cells, 208Tumor necrosis factor (TNF)-alpha,

106, 247in BMDC, 247dendritic cell, 247

Tumor osteolysis, 190Tyndall effect, 97Tyrosine-related proteins

(TRP-1, 2), 208

Ultra-Turrax T8, 211

Vaccine-containing liposomespreparation of, 241by sucrose method, 241

Vaccines, 147–148Vesicles, small unilamellar, 122Vesicular drug carriers, mitochondrial

uptake of, 321–322Vinblastine, 151Vincristine (VCR), 51, 150, 151Vinorelbine (VNB), loading capacity

of, 163Viral vectors, 293, 294

396 Index

Water-soluble nucleosides, 52chemical transformation

of, 58White blood cells (WBC), 194

Xenobiotics, 318

Yersina pseudotuberculosis, 297

Zeta potential, of PRG incorporation,278–279

Zwitterions, 38Zygospohum mansonii, 365

Index 397

Documents

Liposome Technology, Volume II Entrapment of Drugs and Other Materials Into Liposomes, Third Edition