ME T H O D S I N MO L E C U L A R B I O L O G Y

Series EditorJohn M. Walker

School of Life and Medical Sciences,University of Hertfordshire, Hatfield,

Hertfordshire AL10 9AB, UK

For further volumes:http://www.springer.com/series/7651

Nanotechnology for NucleicAcid Delivery

Methods and Protocols

Second Edition

Edited by

Manfred Ogris

Laboratory of MacroMolecular Cancer Therapeutics (MMCT), Department of Pharmaceutical Chemistry,Center of Pharmaceutical Sciences, Faculty of Life Sciences, University of Vienna, Vienna, Austria

Center for NanoScience (CeNS), Ludwig-Maximilians-University, Munich, Germany

Haider Sami

Laboratory of MacroMolecular Cancer Therapeutics (MMCT), Department of Pharmaceutical Chemistry,Center of Pharmaceutical Sciences, Faculty of Life Sciences, University of Vienna, Vienna, Austria

EditorsManfred OgrisLaboratory of MacroMolecularCancer Therapeutics (MMCT)Department of Pharmaceutical ChemistryCenter of Pharmaceutical SciencesFaculty of Life SciencesUniversity of ViennaVienna, Austria

Center for NanoScience (CeNS)Ludwig-Maximilians-UniversityMunich, Germany

Haider SamiLaboratory of MacroMolecularCancer Therapeutics (MMCT)Department of Pharmaceutical ChemistryCenter of Pharmaceutical SciencesFaculty of Life SciencesUniversity of ViennaVienna, Austria

ISSN 1064-3745 ISSN 1940-6029 (electronic)ISBN 978-1-4939-9091-7 ISBN 978-1-4939-9092-4 (eBook)https://doi.org/10.1007/978-1-4939-9092-4

Library of Congress Control Number: 2019931016

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Preface

Nanotechnology-enabled nucleic acid delivery is a rapidly evolving field and offers a multi-tude of possibilities for addressing the challenges faced by nucleic acid therapies. Since therelease of the first edition of this volume in 2013, several new technologies have emerged,and existing protocols have further improved. Within this edition, we aim at updating theexisting chapters and at the same time adding new topics to offer an up-to-date view ontechnologies used for nucleic acid delivery.

We have organized the contents of the current edition according to the sequence inwhich technologies are employed to develop formulations for nucleic acid delivery, startingwith chemical synthesis of carriers, nanoformulations, their characterization in vitro andin vivo, and their application in preclinical models. The first three chapters deal with carriersynthesis. In Chap. 1, Soren Reinhard and colleagues provide a new chapter on the synthesisof defined cationic lipid-oligomers for plasmid and siRNA transfection. David Oupickycontinues with biodegradable and disulfide-crosslinked polymers based on oligo- and poly-cations in Chap. 2. Antoine Kichler and colleagues describe synthesis and application ofcationic, histidine-rich peptides for nucleic acid delivery (Chap. 3). Direct covalent modifi-cation of oligonucleotides is described in the next two chapters. Johannes Winker andcolleagues, in Chap. 4, present a method on the generation of oligonucleotide bioconju-gates by attaching fluorophores to the 30 or 50 end via click chemistry. Maxim Antopolskiprovides detailed protocol on the covalent attachment of radionuclides for SPECT/CTin vivo imaging via end-term attached chelators in Chap. 5. Wolfgang Rodl et al. update thesynthesis protocols for peptide-polyethylenimine conjugates in Chap. 6. The new Chap. 7by Antoine Kichler and colleagues deals with a micellar delivery system for siRNA delivery,which can be stabilized by photopolymerization. Nathalie Mignet and co-workers present aprotocol for the synthesis of cationic and neutral lipids and their particle formation withnucleic acids in Chap. 8. Lipidoids are a new class of transfection reagents. In Chap. 9,Kaushik Thanki et al. describe synthesis of a lipidoid-polymer-based formulation for siRNAdelivery to cytosol. Coating of nanoparticles with successive layers of polymer and nucleicacid allows formation of a well-defined, size-controlled delivery formulation. In Chap. 10,Miriam Breunig and colleagues describe layer-by-layer technique for siRNA delivery andalso include targeting ligands. Guangzhao Mao and co-workers apply layer-by-layer tech-nique using biodegradable polymers for coating surfaces for sequential gene delivery(Chap. 11). Tatiana Segura and colleagues report a technology where polyplexes areincorporated into hydrogels for surface coating, which then allows for transfection of cellsgrown on top of the layer (Chap. 12). Analysis of nanoparticles by atomic force microscopy(AFM) gives insights not only into particle size but also surface topology. Guanzhao Maoand colleagues describe the application of AFM techniques for studying disassembly of DNAnanoparticles and nanofilms in Chap. 13. For further preclinical and clinical development,gene delivery formulations with long-term storage stability are desirable and offer ease ofusage. Wolfgang Friess and colleagues in Chap. 14 present a lyophilization protocol foroptimal freezing with cryoprotectant and freeze-drying of polyplexes. In Chap. 15, our labpresents a robust and cost-effective protocol for the measurement of luciferase reporter geneexpression for investigating nanoparticle-mediated delivery of nucleic acids. Local activationof nanoformulations and enhancement of nucleic acid delivery in vivo can be achieved by

v

several methods to achieve stimuli-based delivery. Ine Lentacker and colleagues applyultrasound-activated microbubbles to enhance local nucleic acid delivery (Chap. 16). OlgaMykhaylyk and colleagues describe in Chap. 17 the synthesis and in vitro evaluation ofmagnetic nanoparticles and microbubbles for nucleic acid delivery, which can be targetedwith magnetic force and activated by ultrasound.

Biocompatibility and toxicity are decisive parameters for nanoformulations and the finalfate of nucleic acid cargo, both on the cellular level and within an organism. Both the carriersystem and the delivered nucleic acid itself can trigger side effects, which can also influencethe therapeutic effect of nucleic acids. In three chapters (Chaps. 18–20), Moein Moghimiand colleagues provide detailed protocols for a quantitative evaluation of polycation-mediated cytotoxicity. Lactate dehydrogenase (LDH) is released after cell rupture, beingan early event in necrosis and a late event in apoptosis. Chapter 18 gives a protocol for a fastand accurate assay for LDH release, which can be applied prior to more detailed toxicologi-cal studies. Tracking onset of apoptosis gives insights into the mechanism of nanoparticle orpolymer-induced toxicity. In Chap. 19, a fluorimetric assay for detection of caspase activity isreported. For polycations, a distinct mitochondrial toxicity has been observed, which issupposed to be due to perturbation of membrane integrity. In Chap. 20, Arnaldur Hall andMoein Moghimi present a protocol on measuring mitochondrial bioenergetics by high-resolution respirometry. Both the innate and adaptive immune system can play a role in theimmunological response when an organism is exposed to exogenous nucleic acids andcarrier systems. The intrinsic effect of siRNA on cytokine induction in lung cells (afterpulmonary delivery of siRNA) is analyzed in Chap. 21 by Olivia Merkel and colleagues,presenting a protocol for detecting intracellular cytokines after sorting of immune cells usingantibody-coated magnetic beads. Although coating with polyethylene glycol (PEG) canimprove biocompatibility and blood circulation of nanoparticles, antibodies against PEGcan neutralize this effect. Tatsuro Ishida et al. in Chap. 22 provide a protocol for quantifyingthe anti-PEG induction in vivo to predict the blood clearance of PEGylated formulationsafter repeated administration. Investigation of the biodistribution of nucleic acid afterin vivo administration is imperative for desirable therapeutic interventions. Near-infraredimaging using fluorophores allows real-time tracking of nucleic acid delivery in vivo. Jean-Luc Coll and colleagues describe labeling of nanoparticles with appropriate near-infraredfluorophores and their detection in vivo (Chap. 23). The three final chapters includeprotocols for tissue-directed plasmid and siRNA delivery: In Chap. 24, Olivia Merkel andcolleagues apply siRNA by intratracheal instillation into the lung and provide a protocol onidentification of distinct cell types in the lung that take up siRNA. In Chap. 25, NathalieMignet et al. use cationic microbubbles in combination with ultrasound to allow efficienttransgene expression in the liver using optimized small plasmids. In Chap. 26, Mark Zabeland colleagues describe preparation and application of lipopeptide formulation to achievesiRNA delivery to the central nervous system.

Taken together, the second version of Nanotechnology for Nucleic Acid Delivery dealswith not only already established methods but also emerging aspects in this field. A broad setof topics is covered ranging from chemical synthesis of macromolecules and bioconjugates;novel and established nanoformulations; characterization of these nanoformulations forbiophysical, biological, and toxicological aspects; and also protocols dealing with applicationand imaging of such carrier systems in vivo.

Vienna, Austria Manfred OgrisHaider Sami

vi Preface

Contents

Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vContributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xi

1 Sequence-Defined Cationic Lipo-Oligomers Containing UnsaturatedFatty Acids for Transfection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1Soren Reinhard and Ernst Wagner

2 Synthesis of Bioreducible Polycations with Controlled Topologies . . . . . . . . . . . . 27Ye-Zi You, Jun-Jie Yan, Fei Yu, Zhi-Qiang Yu, and David Oupicky

3 Histidine-Rich Cationic Cell-Penetrating Peptides for PlasmidDNA and siRNA Delivery. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39Antoine Kichler, A. James Mason, Arnaud Marquette,and Burkhard Bechinger

4 Covalent Fluorophore Labeling of Oligonucleotides and Generationof Other Oligonucleotide Bioconjugates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61Cornelia Lorenzer and Johannes Winkler

5 Synthetic Oligonucleotides in SPECT/CT In Vivo Imaging: ChemicalModifications, In111 Complex Formation, Incorporation intoDrug Delivery Systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73Maxim Antopolsky

6 Synthesis of Polyethylenimine-Based Nanocarriers for SystemicTumor Targeting of Nucleic Acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83Wolfgang Rodl, Alexander Taschauer, David Schaffert,and Ernst Wagner, and Manfred Ogris

7 Cationic Photopolymerized Polydiacetylenic (PDA) Micelles forsiRNA Delivery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101Manon Ripoll, Patrick Neuberg, Jean-Serge Remy,and Antoine Kichler

8 Lipids for Nucleic Acid Delivery: Cationic or Neutral Lipoplexes,Synthesis, and Particle Formation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123Michel Bessodes, Helene Dhotel, and Nathalie Mignet

9 Preparation, Characterization, and In Vitro Evaluation of Lipidoid–PolymerHybrid Nanoparticles for siRNA Delivery to the Cytosol . . . . . . . . . . . . . . . . . . . . 141Kaushik Thanki, Xianghui Zeng, and Camilla Foged

10 Layer-by-Layer Assembled Nanoparticles for siRNA Delivery . . . . . . . . . . . . . . . . 153Michaela Guter and Miriam Breunig

11 Layer-By-Layer Film Engineering for Sequential Gene Delivery . . . . . . . . . . . . . . 161Lingxiao Xie, Yi Zou, Sean Carroll, Maria Muniz,and Guangzhao Mao

12 Surface- and Hydrogel-Mediated Delivery of NucleicAcid Nanoparticles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 177Angela K. Pannier, Tyler Kozisek, and Tatiana Segura

vii

13 In Situ AFM Analysis Investigating Disassembly of DNANanoparticles and Nanofilms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 199Yi Zou, Lei Wan, Jenifer Blacklock, Lingxiao Xie, Sean Carroll,David Oupicky, and Guangzhao Mao

14 Lyophilization of Synthetic Gene Carriers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 211Julia Christina Kasper, Sarah Hedtrich, and Wolfgang Friess

15 Firefly Luciferase-Based Reporter Gene Assay for InvestigatingNanoparticle-Mediated Nucleic Acid Delivery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 227Katharina Muller, Manfred Ogris, and Haider Sami

16 Enhancing Nucleic Acid Delivery with Ultrasoundand Microbubbles. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 241Heleen Dewitte, Silke Roovers, Stefaan C. De Smedt,and Ine Lentacker

17 Magnetic and Acoustically Active Microbubbles Loaded withNucleic Acids for Gene Delivery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 253Dialechti Vlaskou, Olga Mykhaylyk, and Christian Plank

18 Lactate Dehydrogenase Assay for Assessment of PolycationCytotoxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 291Ladan Parhamifar, Helene Andersen, and S. Moein Moghimi

19 Combined Fluorimetric Caspase-3/7 Assay and Bradford ProteinDetermination for Assessment of Polycation-Mediated Cytotoxicity. . . . . . . . . . . 301Anna K. Larsen, Arnaldur Hall, Henrik Lundsgart,and S. Moein Moghimi

20 Determination of Polycation-Mediated Perturbation of MitochondrialRespiration in Intact Cells by High-Resolution Respirometry(Oxygraph-2k, OROBOROS). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 313Arnaldur Hall and S. Moein Moghimi

21 Evaluating the Regulation of Cytokine Levels After siRNATreatment in Antigen-Specific Target Cell Populations viaIntracellular Staining . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 323Rima Kandil, Daniel Feldmann, Yuran Xie,and Olivia M. Merkel

22 Anti-PEG IgM Production via a PEGylated Nanocarrier Systemfor Nucleic Acid Delivery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 333Amr S. Abu Lila and Tatsuhiro Ishida

23 Near-Infrared Optical Imaging of Nucleic Acid Nanocarriers In Vivo . . . . . . . . . 347Claire Rome, Julien Gravier, Marie Morille, Gilles Divita,Anne-Laure Bolcato-Bellemin, Veronique Josserand,and Jean-Luc Coll

24 Flow Cytometry-Based Cell Type-Specific Assessment of TargetRegulation by Pulmonary siRNA Delivery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 365Olivia M. Merkel, Leigh M. Marsh, Holger Garn,and Thomas Kissel

viii Contents

25 Microbubbles for Nucleic Acid Delivery in Liver UsingMild Sonoporation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 377Nathalie Mignet, Corinne Marie, Anthony Delalande,Simona Manta, Michel-Francis Bureau, Gilles Renault,Daniel Scherman, and Chantal Pichon

26 Lipopeptide Delivery of siRNA to the Central Nervous System. . . . . . . . . . . . . . . 389Mark D. Zabel, Luke Mollnow, and Heather Bender

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 405

Contents ix

Contributors

AMR S. ABU LILA � Department of Pharmacokinetics and Biopharmaceutics, Institute ofBiomedical Sciences, Tokushima University, Tokushima, Japan; Department ofPharmaceutics and Industrial Pharmacy, Faculty of Pharmacy, Zagazig University,Zagazig, Egypt; Department of Pharmaceutics, College of Pharmacy, Hail University,Hail, Saudi Arabia

HELENE ANDERSEN � Centre for Pharmaceutical Nanotechnology and Nanotoxicology,University of Copenhagen, Copenhagen, Denmark

MAXIM ANTOPOLSKY � Teaching and Scientific Consulting, FGBPOU “Medical College”,Moscow, Russian Federation

BURKHARD BECHINGER � Institut de Chimie, CNRS, UMR7177, Universite de Strasbourg,Strasbourg, France

HEATHER BENDER � Department of Microbiology, Immunology and Pathology, Prion ResearchCenter, College of Veterinary Medicine and Biomedical Sciences, Colorado State University,Fort Collins, CO, USA

MICHEL BESSODES � Unite de Technologies Chimiques et Biologiques pour la Sante, INSERM,U 1022, Paris, France; CNRS, UMR 8258, Paris, France; Faculte de Pharmacie, SorbonneParis Cite, Universite Paris Descartes, Paris, France; Chimie ParisTech, PSL ResearchUniversity, Paris, France

JENIFER BLACKLOCK � Department of Biomedical Engineering, Wayne State University,Detroit, MI, USA

ANNE-LAURE BOLCATO-BELLEMIN � Polyplus-Transfection, Illkirch, FranceMIRIAM BREUNIG � Department of Pharmaceutical Technology, University of Regensburg,

Regensburg, GermanyMICHEL-FRANCIS BUREAU � INSERM, U1022, Paris, France; CNRS, UMR8258, Paris,

France; Faculte de Pharmacie, Sorbonne Paris Cite, Universite Paris Descartes, Paris,France; Chimie ParisTech, PSL Research University, Paris, France

SEAN CARROLL � Department of Chemical Engineering and Materials Science, Wayne StateUniversity, Detroit, MI, USA; Department of Biomedical Engineering, Wayne StateUniversity, Detroit, MI, USA

JEAN-LUC COLL � INSERM U823, Equipe 5, Institut Albert Bonniot, Grenoble, France;Universite Joseph Fourier, Grenoble, France; INSERM UGA U1209, CNRS 5309,Institute For Advanced Biosciences, La Tronche, France

STEFAAN C. DE SMEDT � Ghent Research Group on Nanomedicines, Laboratory of GeneralBiochemistry and Physical Pharmacy, Faculty of Pharmaceutical Sciences, GhentUniversity, Ghent, Belgium

ANTHONY DELALANDE � Centre de Biophysique Moleculaire and Universite d’Orleans,CNRS-UPR 4301, Orleans, France

HELEEN DEWITTE � Ghent Research Group on Nanomedicines, Laboratory of GeneralBiochemistry and Physical Pharmacy, Faculty of Pharmaceutical Sciences, GhentUniversity, Ghent, Belgium

HELENE DHOTEL � Unite de Technologies Chimiques et Biologiques pour la Sante, INSERM,U 1022, Paris, France; CNRS, UMR 8258, Paris, France; Faculte de Pharmacie, Sorbonne

xi

Paris Cite, Universite Paris Descartes, Paris, France; Chimie ParisTech, PSL ResearchUniversity, Paris, France

GILLES DIVITA � CNRS UMR5237, Montpellier, FranceDANIEL FELDMANN � Department of Pharmaceutical Sciences, Wayne State University,

Detroit, MI, USACAMILLA FOGED � Department of Pharmacy, Faculty of Health and Medical Sciences,

University of Copenhagen, Copenhagen, DenmarkWOLFGANG FRIESS � Department of Pharmacy, Pharmaceutical Technology and

Biopharmaceutics, Ludwig-Maximilians-Universit€at, Munich, GermanyHOLGER GARN � Institute of Laboratory Medicine and Pathobiochemistry—Molecular

Diagnostics, Philipps-Universit€at Marburg, Marburg, Germany; Sterna Biologicals GmbH& Co. KG, Marburg, Germany

JULIEN GRAVIER � CEA Grenoble/LETI-DTBS, Grenoble, FranceMICHAELA GUTER � Department of Pharmaceutical Technology, University of Regensburg,

Regensburg, GermanyARNALDUR HALL � Genome Integrity Unit, Danish Cancer Society Research Center,

Copenhagen, DenmarkSARAH HEDTRICH � Institute for Pharmacy, Pharmacology & Toxicology, Freie Universit€at

Berlin, Berlin, GermanyTATSUHIRO ISHIDA � Department of Pharmacokinetics and Biopharmaceutics, Institute of

Biomedical Sciences, Tokushima University, Tokushima, JapanVERONIQUE JOSSERAND � INSERM U823, Equipe 5, Institut Albert Bonniot, Grenoble,

France; Universite Joseph Fourier, Grenoble, France; INSERMUGAU1209, CNRS 5309,Institute For Advanced Biosciences, La Tronche, France

RIMA KANDIL � Department of Pharmaceutical Technology and Biopharmaceutics, Ludwig-Maximilians-Universit€at Munchen, Munich, Germany

JULIA CHRISTINA KASPER � Bioprocess and Pharmaceutical Development Biologicals,Boehringer Ingelheim Pharma GmbH and Co. KG, Biberach, Germany

ANTOINE KICHLER � Laboratoire de Conception et Application de Molecules Bioactives, LabExMedalis, Faculte de Pharmacie, CNRS, UMR7199, Universite de Strasbourg, Illkirch,France

THOMAS KISSEL � Department of Pharmaceutics and Biopharmacy, Philipps-Universit€atMarburg, Marburg, Germany

TYLER KOZISEK � Department of Biological Systems Engineering, University of Nebraska-Lincoln, Lincoln, NE, USA

ANNA K. LARSEN � Centre for Pharmaceutical Nanotechnology and Nanotoxicology,University of Copenhagen, Copenhagen, Denmark

INE LENTACKER � Ghent Research Group on Nanomedicines, Laboratory of GeneralBiochemistry and Physical Pharmacy, Faculty of Pharmaceutical Sciences, GhentUniversity, Ghent, Belgium

CORNELIA LORENZER � Department of Pharmaceutical Chemistry, University of Vienna,Vienna, Austria

HENRIK LUNDSGART � Centre for Pharmaceutical Nanotechnology and Nanotoxicology,University of Copenhagen, Copenhagen, Denmark

SIMONA MANTA � INSERM, U1022, Paris, France; CNRS, UMR8258, Paris, France;Faculte de Pharmacie, Sorbonne Paris Cite, Universite Paris Descartes, Paris, France;Chimie ParisTech, PSL Research University, Paris, France

xii Contributors

GUANGZHAO MAO � Department of Chemical Engineering and Materials Science, WayneState University, Detroit, MI, USA

CORINNE MARIE � INSERM, U1022, Paris, France; CNRS, UMR8258, Paris, France;Faculte de Pharmacie, Sorbonne Paris Cite, Universite Paris Descartes, Paris, France;Chimie ParisTech, PSL Research University, Paris, France

ARNAUD MARQUETTE � Institut de Chimie, CNRS, UMR7177, Universite de Strasbourg,Strasbourg, France

LEIGH M. MARSH � Institute of Laboratory Medicine and Pathobiochemistry—MolecularDiagnostics, Philipps-Universit€at Marburg, Marburg, Germany; Ludwig BoltzmannInstitute for Lung Vasculature Research, Graz, Austria

A. JAMES MASON � Institute of Pharmaceutical Science, King’s College London, London, UKOLIVIA M. MERKEL � Department of Pharmaceutical Technology and Biopharmaceutics,

Ludwig-Maximilians-Universit€at Munchen, Munich, Germany; Department ofPharmaceutical Sciences, Wayne State University, Detroit, MI, USA; Department ofPharmacy, Ludwig-Maximilians-Universit€at Munchen, Munich, Germany; Departmentof Pharmaceutics and Biopharmacy, Philipps-Universit€at Marburg, Marburg, Germany

NATHALIE MIGNET � Unite de Technologies Chimiques et Biologiques pour la Sante, INSERM,U 1022, Paris, France; CNRS, UMR 8258, Paris, France; Faculte de Pharmacie, SorbonneParis Cite, Universite Paris Descartes, Paris, France; Chimie ParisTech, PSL ResearchUniversity, Paris, France

S. MOEIN MOGHIMI � School of Pharmacy, Newcastle University, Newcastle upon Tyne, UK;Division of Stratified Medicine, Biomarkers and Therapeutics, Faculty of Medical Sciences,Institute of Cellular Medicine, Newcastle University, Newcastle upon Tyne, UK

LUKE MOLLNOW � Department of Microbiology, Immunology and Pathology, Prion ResearchCenter, College of Veterinary Medicine and Biomedical Sciences, Colorado State University,Fort Collins, CO, USA

MARIE MORILLE � INSERM U646, Angers, FranceKATHARINA MULLER � Laboratory of MacroMolecular Cancer Therapeutics (MMCT),

Department of Pharmaceutical Chemistry, Center of Pharmaceutical Sciences, Faculty ofLife Sciences, University of Vienna, Vienna, Austria

MARIA MUNIZ � Department of Biomedical Engineering, Wayne State University, Detroit,MI, USA

OLGA MYKHAYLYK � Institute of Molecular Immunology and Experimental Oncology,Klinikum Rechts der Isar, Technische Universit€at Munchen, Munich, Germany

PATRICK NEUBERG � Laboratoire de Conception et Application de Molecules Bioactives, LabExMedalis, Faculte de Pharmacie, CNRS, UMR7199, Universite de Strasbourg, Illkirch,France

MANFRED OGRIS � Laboratory of MacroMolecular Cancer Therapeutics (MMCT),Department of Pharmaceutical Chemistry, Center of Pharmaceutical Sciences, Faculty ofLife Sciences, University of Vienna, Vienna, Austria; Center for NanoScience (CeNS),Ludwig-Maximilians-University, Munich, Germany

DAVID OUPICKY � Department of Pharmaceutical Sciences, Wayne State University, Detroit,MI, USA; University of Nebraska Medical Center, Omaha, NE, USA

ANGELA K. PANNIER � Department of Biological Systems Engineering, University of Nebraska-Lincoln, Lincoln, NE, USA

LADAN PARHAMIFAR � Centre for Pharmaceutical Nanotechnology and Nanotoxicology,University of Copenhagen, Copenhagen, Denmark

Contributors xiii

CHANTAL PICHON � Centre de Biophysique Moleculaire and Universite d’Orleans, CNRS-UPR 4301, Orleans, France

CHRISTIAN PLANK � Institute of Molecular Immunology and Experimental Oncology,Klinikum Rechts der Isar, Technische Universit€at Munchen, Munich, Germany

SOREN REINHARD � Department of Pharmacy, Pharmaceutical Biotechnology, Center ofNanoscience (CeNS), Ludwig-Maximilians-Universit€at Butenandtstr, Munchen,Germany

JEAN-SERGE REMY � Laboratoire de Conception et Application de Molecules Bioactives, LabExMedalis, Faculte de Pharmacie, CNRS, UMR7199, Universite de Strasbourg, Illkirch,France

GILLES RENAULT � INSERM, U1016, Institut Cochin, Paris, France; CNRS, UMR8104,Paris, France; Sorbonne Paris Cite, Universite Paris Descartes, Paris, France

MANON RIPOLL � Laboratoire de Conception et Application de Molecules Bioactives, LabExMedalis, Faculte de Pharmacie, CNRS, UMR7199, Universite de Strasbourg, Illkirch,France

WOLFGANG RODL � Pharmaceutical Biotechnology, Center for System Based Drug Research,Ludwig-Maximilians-University, Munich, Germany

CLAIRE ROME � INSERM U823, Equipe 5, Institut Albert Bonniot, Grenoble, France;Universite Joseph Fourier, Grenoble, France; Grenoble Institute of Neuroscience, Grenoble,France

SILKE ROOVERS � Ghent Research Group on Nanomedicines, Laboratory of GeneralBiochemistry and Physical Pharmacy, Faculty of Pharmaceutical Sciences, GhentUniversity, Ghent, Belgium

HAIDER SAMI � Laboratory of MacroMolecular Cancer Therapeutics (MMCT), Departmentof Pharmaceutical Chemistry, Center of Pharmaceutical Sciences, Faculty of Life Sciences,University of Vienna, Vienna, Austria

DAVID SCHAFFERT � Department of Molecular Biology, Aarhus University, Aarhus, DenmarkDANIEL SCHERMAN � INSERM, U1022, Paris, France; CNRS, UMR8258, Paris, France;

Faculte de Pharmacie, Sorbonne Paris Cite, Universite Paris Descartes, Paris, France;Chimie ParisTech, PSL Research University, Paris, France

TATIANA SEGURA � Department of Biomedical Engineering, Duke University, Durham, NC,USA; Neurology and Dermatology, Duke University School of Medicine, Durham, NC,USA

ALEXANDER TASCHAUER � Laboratory of MacroMolecular Cancer Therapeutics (MMCT),Department of Pharmaceutical Chemistry, Center of Pharmaceutical Sciences, Faculty ofLife Sciences, University of Vienna, Vienna, Austria

KAUSHIK THANKI � Department of Pharmacy, Faculty of Health and Medical Sciences,University of Copenhagen, Copenhagen, Denmark

DIALECHTI VLASKOU � Institute of Molecular Immunology and Experimental Oncology,Klinikum Rechts der Isar, Technische Universit€at Munchen, Munich, Germany

ERNST WAGNER � Department of Pharmacy, Pharmaceutical Biotechnology, Center ofNanoscience (CeNS), Ludwig-Maximilians-Universit€at Butenandtstr, Munchen,Germany; Nanosystems Initiative Munich (NIM), Schellingstr, Munchen, Germany;Center for NanoScience (CeNS), Ludwig-Maximilians-University, Munich, Germany

LEI WAN � Department of Chemical Engineering and Materials Science, Wayne StateUniversity, Detroit, MI, USA

xiv Contributors

JOHANNES WINKLER � Department of Pharmaceutical Chemistry, University of Vienna,Vienna, Austria; Department of Cardiology, Medical University of Vienna, Vienna,Austria

LINGXIAO XIE � Department of Chemical Engineering and Materials Science, Wayne StateUniversity, Detroit, MI, USA

YURAN XIE � Department of Pharmaceutical Sciences, Wayne State University, Detroit, MI,USA

JUN-JIE YAN � CAS Key Lab of Soft Matter Chemistry, Department of Polymer Science andEngineering, University of Science and Technology of China, Hefei, China

YE-ZI YOU � CAS Key Lab of Soft Matter Chemistry, Department of Polymer Science andEngineering, University of Science and Technology of China, Hefei, China

FEI YU � University of Nebraska Medical Center, Omaha, NE, USAZHI-QIANG YU � CAS Key Lab of Soft Matter Chemistry, Department of Polymer Science and

Engineering, University of Science and Technology of China, Hefei, ChinaMARK D. ZABEL � Department of Microbiology, Immunology and Pathology, Prion Research

Center, College of Veterinary Medicine and Biomedical Sciences, Colorado State University,Fort Collins, CO, USA

XIANGHUI ZENG � Department of Pharmacy, Faculty of Health and Medical Sciences,University of Copenhagen, Copenhagen, Denmark

YI ZOU � Department of Chemical Engineering and Materials Science, Wayne StateUniversity, Detroit, MI, USA

Contributors xv