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Nanobiotechnology
Concepts, Applications and Perspectives
Edited byChristof M. Niemeyer and Chad A. Mirkin
InnodataFile Attachment3527605916.jpg
Nanobiotechnology
Edited by
C. M. Niemeyer andC. A. Mirkin
Further Titles of Interest
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Nanobiotechnology
Concepts, Applications and Perspectives
Edited byChristof M. Niemeyer and Chad A. Mirkin
Edited by
Prof. Dr. Christof M. NiemeyerUniversität Dortmund, Fachbereich ChemieBiologisch-Chemische MikrostrukturtechnikOtto-Hahn-Str. 644227 DortmundGermanycmn@chemie.uni-dortmund.de
Prof. Dr. Chad A. MirkinDepartment of Chemistry &Institute for NanotechnologyNorthwestern University2145 Sheridan RoadEvanston, IL 60208-3113USAcamirkin@chem.northwestern.edu
Cover illustrationMalign (top) and normal cells (bottom) on pillarinterfaces which sense cellular forces. In the middleillustration, the molecular distribution of integrin(green) and actin (red) is shown. All micrographswere kindly provided by W. Roos, J. Ulmer, andJ.P. Spatz (University of Heidelberg, Germany).
This book was carefully produced. Never-theless, editors, authors and publisher donot warrant the information containedtherein to be free of errors. Readers areadvised to keep in mind that statements,data, illustrations, procedural details orother items may inadvertently be inaccurate.
Library of Congress Card No.: applied forBritish Library Cataloguing-in-Publication DataA catalogue record for this book is availablefrom the British Library.
Bibliographic information published byDie Deutsche BibliothekDie Deutsche Bibliothek lists this publicationin the Deutsche Nationalbibliografie;detailed bibliographic data is available in theInternet at http://dnb.ddb.de.
� 2004 WILEY-VCH Verlag GmbH & Co.KGaA, Weinheim
All rights reserved (including those oftranslation in other languages). No part ofthis book may be reproduced in any form –by photoprinting, microfilm, or any othermeans – nor transmitted or translated intomachine language without written permis-sion from the publishers. Registered names,trademarks, etc. used in this book, evenwhen not specifically marked as such, arenot to be considered unprotected by law.
Printed in the Federal Republic of Germany.Printed on acid-free paper.
Typesetting Hagedorn Kommunikation,ViernheimPrinting betz-druck gmbh, DarmstadtBookbinding J. Schäffer GmbH & Co. KG,Grünstadt
ISBN 3-527-30658-7
Contents
Part I Interphase Systems
1 Biocompatible Inorganic Devices 1Thomas Sawitowski
1.1 Introduction 11.2 Implant Coatings 11.2.1 Stents 21.2.2 Seeds 71.3 Conclusion 10
2 Microfluidics Meets Nano:Lab-on-a-Chip Devices and their Potential for Nanobiotechnology 13Holger Bartos, Friedrich Götz, and Ralf-Peter Peters
2.1 Introduction 132.2 Overview 132.2.1 Definition and History 132.2.2 Advantages of Microfluidic Devices 142.2.3 Concepts for Microfluidic Devices 152.2.4 Fluid Transport 172.2.5 Stacking and Sealing 182.3 Methods 192.3.1 Materials for the Manufacture of Microfluidic Components 192.3.1.1 Silicon 192.3.1.2 Glass 192.3.1.3 Polymers 202.3.2 Fluidic Structures 212.3.3 Fabrication Methods 232.3.4 Surface Modifications 232.3.5 Spotting 252.3.6 Detection Mechanisms 262.4 Outlook 26
VContents
3 Microcontact Printing of Proteins 31Emmanuel Delamarche
3.1 Introduction 313.2 Strategies for Printing Proteins on Surfaces 333.2.1 Contact Processing with Hydrogel Stamps 333.2.2 Microcontact Printing 333.2.3 Affinity-Contact Printing 343.3 Microcontact Printing Polypeptides and Proteins 343.3.1 Printing One Type of Biomolecule 353.3.2 Substrates 363.3.3 Resolution and Contrast of the Patterns 383.4 Activity of Printed Biomolecules 403.5 Printing Multiple Types of Proteins 423.5.1 Additive and Subtractive Printing 423.5.2 Parallel Inking and Printing of Multiple Proteins 443.5.3 Affinity-Contact Printing 443.6 Methods 453.6.1 Molds and Stamps 453.6.2 Surface Chemistry of Stamps 473.6.3 Inking Methods 473.6.4 Treatments of Substrates 483.6.5 Printing 483.6.6 Characterization of the Printed Patterns 493.7 Outlook 49
4 Cell–Nanostructure Interactions 53Joachim P. Spatz
4.1 Introduction 534.2 Methods 564.3 Outlook 63
5 Defined Networks of Neuronal Cells in Vitro 66Andreas Offenhäusser and Angela K. Vogt
5.1 Introduction 665.2 Overview: Background and History 675.2.1 Physiology of Information Processing within Neuronal Networks 675.2.2 Topographical Patterning 675.2.3 Chemical Patterning 685.3 Methods 695.3.1 Topographical Patterning 695.3.2 Photolithographic Patterning 705.3.3 Photochemical Patterning 705.3.4 Microcontact Printing 715.4 Outlook 72
VI Contents
Part II Protein-based Nanostructures
6 S-Layers 77Uwe B. Sleytr, Eva-Maria Egelseer, Dietmar Pum, and Bernhard Schuster
6.1 Overview 77Abbreviations 77
6.1.1 Chemistry and Structure 786.1.2 Genetics and Secondary Cell-Wall Polymers 806.1.3 Assembly 826.1.3.1 Self-Assembly in Suspension 826.1.3.2 Recrystallization at Solid Supports 836.1.3.3 Recrystallization at the Air/Water Interface
and on Langmuir Lipid Films 836.2 Methods 846.2.1 Diagnostics 846.2.2 Lipid Chips 856.2.3 S-Layers as Templates for the Formation of
Regularly Arranged Nanoparticles 876.3 Outlook 89
7 Engineered Nanopores 93Hagan Bayley, Orit Braha, Stephen Cheley, and Li-Qun Gu
7.1 Overview 937.1.1 What is a Nanopore? 937.1.2 Engineering Nanopores 967.1.3 What Can a Nanopore Do? 977.1.4 What are the Potential Applications of Nanopores? 1007.1.5 Keeping Nanopores Happy 1037.2 Methods 1047.2.1 Protein Production 1047.2.2 Protein Engineering 1047.2.3 Electrical Recording 1057.2.4 Other Systems 1057.3 Outlook 1067.3.1 Rugged Pores 1067.3.2 Supported Bilayers 1067.3.3 Membrane Arrays 1067.3.4 Alternative Protein Pores 1077.3.5 Pores with New Attributes and Applications 1087.3.6 Theory 108
8 Genetic Approaches to Programmed Assembly 113Stanley Brown
8.1 Introduction 1138.2 Order from Chaos 113
VIIContents
8.3 Monitoring Enrichment 1168.4 Quantification of Binding and Criteria for Specificity 1198.5 Unselected Traits and Control of Crystallization/Reactivity 1198.6 Dominant Traits, Interpretation of Gain-of-Function Mutants 1208.7 Interpretation and Requirement for Consensus Sequences 1208.8 Sizes of Proteins and Peptides 1228.9 Mix and Match, Fusion Proteins, and Context-Dependence 1228.10 Mix and Match, Connecting Structures 1228.11 Outlook 123
9 Microbial Nanoparticle Production 126Murali Sastry, Absar Ahmad, M. Islam Khan, and Rajiv Kumar
9.1 Overview 1269.2 Outlook 133
10 Magnetosomes: Nanoscale Magnetic Iron Minerals in Bacteria 136Richard B. Frankel and Dennis A. Bazylinski
10.1 Introduction 13610.1.1 Magnetotactic Bacteria 13610.1.2 Magnetosomes 13710.1.3 Cellular Magnetic Dipole and Magnetotaxis 13810.1.4 Magneto-Aerotaxis 13910.1.5 Magnetite Crystals in Magnetosomes 14010.1.6 Greigite Crystals in Magnetosomes 14110.1.7 Biochemistry and Gene Expression in Magnetosome Formation 14110.1.8 Applications of Magnetosomes 14310.2 Research Methods 14310.3 Conclusion and Future Research Directions 143
11 Bacteriorhodopsin and its Potential in Technical Applications 146Norbert Hampp and Dieter Oesterhelt
11.1 Introduction 14611.2 Overview: The Molecular Properties of Bacteriorhodopsin 14711.2.1 Haloarchaea and their Retinal Proteins 14711.2.2 Structure and Function of Bacteriorhodopsin 15011.2.3 Genetic Modification of Bacteriorhodopsin 15311.2.4 Biotechnological Production of Bacteriorhodopsins 15411.3 Overview: Technical Applications of Bacteriorhodopsin 15511.3.1 Photoelectric Applications 15611.3.1.1 Preparation of Oriented PM Layers 15611.3.1.2 Interfacing the Proton-Motive Force 15811.3.1.3 Application Examples 15811.3.2 Photochromic Applications 15911.3.2.1 Photochromic Properties of Bacteriorhodopsin 15911.3.2.2 Preparation of Bacteriorhodopsin Films 161
VIII Contents
11.3.2.3 Interfacing the Photochromic Changes 16111.3.2.4 Application Examples 16111.3.3 Applications in Energy Conversion 16311.4 Methods 16511.5 Outlook 165
12 Polymer Nanocontainers 168Alexandra Graff, Samantha M. Benito, Corinne Verbert, and Wolfgang Meier
12.1 Introduction 16812.2 Overview 16812.2.1 From Liposomes in Biotechnology to
Polymer Nanocontainers in Therapy 16812.2.2 Dendrimers 16912.2.3 Layer by Layer (LbL) Deposition 17012.2.4 Block Copolymer Self-Assembly 17212.2.4.1 Shell Cross-linked Knedel’s (SCKs) 17312.2.4.2 Block Copolymer Nanocontainers 17412.3 Polymer Nanocontainers with Controlled Permeability 17512.3.1 Block Copolymer Protein Hybrid Systems 17512.3.2 Stimuli-responsive Nanocapsules 17812.4 Nanoparticle Films 17912.5 Biomaterials and Gene Therapy 18012.6 Outlook 181
13 Biomolecular Motors Operating in Engineered Environments 185Stefan Diez, Jonne H. Helenius, and Jonathon Howard
13.1 Overview 18513.2 Methods 19013.2.1 General Conditions for Motility Assays 19013.2.2 Temporal Control 19113.2.3 Spatial Control 19113.2.4 Connecting to Cargoes and Surfaces 19413.3 Outlook 195
14 Nanoparticle–Biomaterial Hybrid Systemsfor Bioelectronic Devices and Circuitry 200Eugenii Katz and Itamar Willner
14.1 Introduction 20014.2 Biomaterial–Nanoparticle Systems for Bioelectronic
and Biosensing Applications 20214.2.1 Bioelectronic Systems Based on Nanopaticle–Enzyme Hybrids 20214.2.2 Bioelectronic Systems for Sensing of
Biorecognition Events Based on Nanoparticles 205
IXContents
14.3 Biomaterial-based Nanocircuitry 21514.3.1 Protein-based Nanocircuitry 21614.3.2 DNA as Functional Template for Nanocircuitry 21814.4 Conclusions and Perspectives 221
Part III DNA-based Nanostructures
15 DNA–Protein Nanostructures 227Christof M. Niemeyer
15.1 Overview 22715.1.1 Introduction 22715.1.2 Oligonucleotide–Enzyme Conjugates 22815.1.3 DNA Conjugates of Binding Proteins 22915.1.4 Noncovalent DNA–Streptavidin Conjugates 23115.1.5 Multifunctional Protein Assemblies 23415.1.6 DNA–Protein Conjugates in Microarray Technologies 23615.2 Methods 23815.2.1 Conjugation of Nucleic Acids and Proteins 23815.2.2 Immuno-PCR 23915.2.3 Supramolecular Assembly 24015.2.4 DNA-directed Immobilization 24015.3 Outlook 241
16 DNA-templated Electronics 244Erez Braun and Uri Sivan
16.1 Introduction and Background 24416.2 DNA-templated Electronics 24616.3 Sequence-specific Molecular Lithography 24916.4 Summary and Perspectives 253
17 Biomimetic Fabrication of DNA-based Metallic Nanowires and Networks 256Michael Mertig and Wolfgang Pompe
17.1 Introduction 25617.2 Template Design 25817.2.1 DNA as a Biomolecular Template 25817.2.2 Integration of DNA into Microelectronic Contact Arrays 25817.2.3 DNA Branching for Network Formation 26117.3 Metallization 26217.3.1 Controlled Cluster Growth on DNA Templates 26317.3.2 First-Principle Molecular Dynamics Calculations
of DNA Metallization 26717.4 Conductivity Measurements on Metalized DNA Wires 27017.5 Conclusions and Outlook 272
X Contents
17.6 Methods 27417.6.1 Site-Specific DNA Attachment 27417.6.2 DNA Junctions 27417.6.3 DNA Metallization 274
18 Mineralization in Nanostructured Biocompartments:Biomimetic Ferritins For High-Density Data Storage 278Eric L. Mayes and Stephen Mann
18.1 Overview 27818.2 Biomimetic Ferritins 27918.3 High-Density Magnetic Data Storage 28018.4 Methods 28218.5 Results 28318.6 Outlook 285
19 DNA–Gold-Nanoparticle Conjugates 288C. Shad Thaxton and Chad A. Mirkin
19.1 Overview 28819.1.1 Introduction 28819.1.2 Nanoparticles 28919.1.3 DNA-functionalized Gold Nanoparticles 29119.1.4 Nanoparticle Based DNA and RNA Detection Assays 29219.1.4.1 Homogeneous DNA Detection 29219.1.4.2 Chip-based (Heterogeneous) DNA Detection Assays 29319.1.5 DNA-Nanoparticle Detection of Proteins: Biobarcodes 29919.1.6 Conclusion 30019.2 The Essentials: Methods and Protocols 30119.2.1 Nanoparticle Synthesis 30119.2.2 DNA-functionalized Au-NP Probe Synthesis 30119.2.3 Chip Functionalization with DNA Target “Capture” Strands 30319.2.4 Typical Assay Design 30419.3 Outlook 30419.3.1 Challenges Ahead 30419.3.2 Academic and Commercial Applications 305
20 DNA Nanostructures for Mechanics and Computing:Nonlinear Thinking with Life’s Central Molecule 308Nadrian C. Seeman
20.1 Overview 30820.2 Introduction 30820.3 DNA Arrays 31120.4 DNA Nanomechanical Devices 31320.5 DNA-based Computation 31520.6 Summary and Outlook 317
XIContents
21 Nanoparticles as Non-Viral Transfection Agents 319M. N. V. Ravi Kumar, Udo Bakowsky, and Claus-Michael Lehr
21.1 Introduction to Gene Delivery 31921.2 Nanoparticles for Drug and Gene Targeting 32121.3 Nonviral Nanomaterials in Development and Testing 32121.3.1 Chitosan 32121.3.2 Liposomes and Solid Lipids 32721.3.3 Poly-l-Lysine and Polyethylenimines 33221.3.4 Poly(lactide-co-glycolide) 33421.3.5 Silica 33521.3.6 Block Copolymers 33621.4 Setbacks and Strategies to Improve Specific Cell Uptake
of Nonviral Systems 33821.5 Prospects for Nonviral Nanomaterials 338
Part IV Nanoanalytics
22 Luminescent Quantum Dots for Biological Labeling 343Xiaohu Gao and Shuming Nie
22.1 Overview 34322.2 Methods 34822.3 Outlook 349
23 Nanoparticle Molecular Labels 353James F. Hainfeld, Richard D. Powell, and Gerhard W. Hacker
23.1 Introduction 35323.2 Immunogold-Silver Staining: A History 35423.3 Combined Fluorescent and Gold Probes 35623.4 Methodology 35723.4.1 Choice of Gold and AMG Type 35723.4.2 Iodinization 35923.4.3 Sensitivity 35923.5 Applications for the Microscopical Detection of Antigens 35923.6 Detection of Nucleic Acid Sequences 36023.7 Applications for Microscopical Detection of Nucleic Acids 36123.8 Technical Guidelines and Laboratory Protocols 36223.9 Gold Derivatives of Other Biomolecules 36223.9.1 Protein Labeling 36323.9.2 Gold Cluster-labeled Peptides 36423.9.3 Gold Cluster Conjugates of Other Small Molecules 36423.9.4 Gold–Lipids: Metallosomes 36523.10 Larger Covalent Particle Labels 36623.11 Gold Targeted to His Tags 36723.12 Enzyme Metallography 368
XII Contents
23.13 Gold Cluster Nanocrystals 36923.14 Gold Cluster–Oligonucleotide Conjugates:
Nanotechnology Applications 36923.14.1 DNA Nanowires 37023.14.2 3-D Nanostructured Mineralized Biomaterials 37023.14.3 Gold-quenched Molecular Beacons 37223.15 Other Metal Cluster Labels 37223.15.1 Platinum and Palladium 37323.15.2 Tungstates 37423.15.3 Iridium 375
24 Surface Biology: Analysis of Biomolecular Structureby Atomic Force Microscopy and Molecular Pulling 387Emin Oroudjev, Signe Danielsen and Helen G. Hansma
24.1 Introduction 38724.2 Recent Results 38824.2.1 DNA 38824.2.1.1 DNA Condensation 38824.2.1.2 DNA Sequences Recognized by Mica 39024.2.1.3 Drug-binding to Single ds-DNA Molecules 39024.2.2 Proteins 39024.2.2.1 Prion Proteins 39124.2.2.2 Membrane Proteins 39324.2.2.3 Spider Silk 39424.2.3 Fossils 39424.2.4 Science and Nature 39424.3 Methodology 39524.3.1 The Probe 39624.3.2 The Sample 39724.4 The Future 39824.4.1 Unity or Diversity? 39824.4.2 World-wide Research 399
25 Force Spectroscopy 404Markus Seitz
25.1 Overview 40425.1.1 Dynamic Force Spectroscopy of Specific Biomolecular Bonds 40525.1.2 Force Spectroscopy and Force Microscopy of Cell Membranes 40925.1.3 Protein (Un-)folding 40925.1.4 Elasticity of Individual Polymer Molecules 41225.1.5 DNA Mechanics 41425.1.6 DNA–Protein Interactions 41625.1.7 Molecular Motors 41725.1.8 Synthetic Functional Polymers 41825.2 Methods 419
XIIIContents
25.2.1 AFM Cantilevers 41925.2.2 Microneedles 42125.2.3 Optical Tweezers 42125.2.4 Magnetic Tweezers 42225.2.5 Biomembrane Force Probe 42325.3 Outlook 424
26 Biofunctionalized Nanoparticles for Surface-EnhancedRaman Scattering and Surface Plasmon Resonance 429Mahnaz El-Kouedi and Christine D. Keating
26.1 Overview 42926.1.1 Introduction 42926.1.2 Applications in SPR 43026.1.2.1 Nanoparticle Substrates 43026.1.2.2 Planar Substrates 43126.1.3 Applications in SERS 43426.1.3.1 Proteins 43426.1.3.2 Nucleic Acids 43726.2 Methods 43926.2.1 Planar SPR Substrate Preparation 43926.2.2 Metal Nanoparticles 43926.2.3 Bioconjugates 43926.2.4 General Comments 44026.3 Future Outlook 440
27 Bioconjugated Silica Nanoparticles for Bioanalytical Applications 444Timothy J. Drake, Xiaojun Julia Zhao, and Weihong Tan
27.1 Overview 44427.2 Methods 44527.2.1 Fabrication 44527.2.2 Particle Probes 44727.2.2.1 Dye-doped Silica Nanoparticles 44727.2.2.2 Magnetic Silica Nanoparticles 44927.2.3 Biofunctionalization of Silica Nanoparticles 44927.2.3.1 Amino-Group Cross-Linkage 45027.2.3.2 Avidin–Biotin Linking Bridge 45127.2.3.3 Disulfide-coupling Chemical Binding 45127.2.3.4 Cyanogen Bromide Modification 45127.2.4 Bioanlytical Applications for Silica Nanoparticles 45227.2.4.1 Cellular Labeling/Detection 45227.2.4.2 DNA Analysis 45327.2.4.3 Ultrasensitive DNA Detection 45327.3 Outlook 454
Index 458
XIV Contents
Preface
Nanobiotechnology is a young and rapidly evolving field of research at the cross-roads of biotechnology and nanoscience, two interdisciplinary areas each of whichcombines advances in science and engineering. Although often considered one ofthe key technologies of the 21st century, nanobiotechnology is still in a fairly embryo-nic state. Topical areas of research are still being defined, and the entire scope oftechnological applications cannot be imagined. At present, nanobiotechnology is afield that concerns the utilization of biological systems optimized through evolution,such as cells, cellular components, nucleic acids, and proteins, to fabricate func-tional nanostructured and mesoscopic architectures comprised of organic and inor-ganic materials. Nanobiotechnology also concerns the refinement and application ofinstruments, originally designed to generate and manipulate nanostructured mate-rials, to basic and applied studies of fundamental biological processes.
This book is intended to provide the first systematic and comprehensive frame-work of specific research topics in nanobiotechnology. To this end, the currentstate-of-the-art has been accumulated in 27 chapters, all of them written by expertsin their fields. Each of the chapters consists of three sections, (i) an overview whichgives a brief but comprehensive survey on the topic, (ii) a methods section whichorients the reader to the most important techniques relevant for the specific topicdiscussed, and (iii) an outlook discussing academic and commercial applications aswell as experimental challenges to be solved.
Nanobiotechnology: Concepts, Applications and Perspectives combines contributionsfrom analytical, bioorganic, and bioinorganic chemistry, physics, molecular andcell biology, and materials science in an attempt to give the reader a feel for thefull scope of current and potential future developments. The articles in this volumeclearly emphasize the high degree of interdisciplinary research that forms the back-bone of this joint-venture of biotechnology and nanoscience.
The book is divided into four main sections. The first concerns interphase sys-tems pertaining to biocompatible inorganic devices for medical implants, micro-fluidic systems for handling biological components in analytical lab-on-a-chip ap-plications, and microelectronic silicon substrates for the investigation and manip-ulation of neuronal cells. Moreover, two chapters describe methodologies regardingthe microcontact printing of proteins and the use of nanostructured substrates tostudy basic principles of cell adhesion.
XVPreface
The second section is devoted to protein-based nanostructures. Individual chap-ters concern the use of specific proteins, such as S-layers to be used as buildingblocks and templates for generating functional nanostructures, bacteriorhodopsinfor photochromic applications, protein nanopores as nanoscopic cavities for analy-tical and synthetic tasks, and biomolecular motors for the translocation of cargo insynthetic environments. The use of a variety of functional proteins as transducersand amplifiers of biomolecular recognition events is described in the chapters onnanobioelectronic devices and polymer nanocontainers. Contributions concerningthe microbial production of inorganic nanoparticles and magnetosomes as well asthe discussion of genetic approaches to generate proteins for the specific organiza-tion of particles provide insight into the body of classical biotechnology, implemen-ted in nanobiotechnology.
In the third section, DNA-based nanostructures are described, beginning withsemisynthetic conjugates of DNA and proteins, which link the advantages of nu-cleic acids to the unlimited functionality of proteins. Three contributions concernthe use of the topographic and electrostatic properties of DNA and proteins for thetemplated growth of inorganic materials. Hybrid conjugates of gold nanoparticlesand DNA oligomers are described with a focus on their applications in the highsensitivity analyses of nucleic acids. Finally, the use of pure DNA molecules for ap-plications in nanomechanics and computing is discussed.
The fourth section deals with the area of nanoanalytics, which currently includesthe majority of commercial products in nanobiotechnology. In particular, fourchapters describe the use of metal or semiconductor nanoparticles, supplementedwith nucleic acid- and protein-based recognition groups, for biolabeling, histo-chemical applications and for signal enhancement in optical detection methods.Nanoparticles are also employed as carriers for genetic material in the non-viraltransfection of cells. To exemplify the use of modern nano-instrumentation forthe study of biological systems, two chapters describe the use of the scanningprobe microscope, the key instrument in nanotechnologies, for investigating bio-molecular structure, conformation and reactivity.
The purpose of Nanobiotechnology: Concepts, Applications and Perspectives is to pro-vide both a broad survey of the field and also instruction and inspiration to all le-vels of scientists, from novices to those intimately engaged in this new and excitingfield of research. Although the collection of articles addresses numerous scientificand technical challenges ahead, the future of nanobiotechnology is bright and ap-pears to be limited, at present, only by imagination.
Dortmund, November 2003 Christof M. NiemeyerEvanston, November 2003 Chad A. Mirkin
XVI Preface
Contributors
XVIIContributors
Absar AhmadBiochemical Sciences DivisonNational Chemical Laboratory411 008 PuneIndia
Udo BakowskyDepartment of Biopharmaceutics andPharmaceutical TechnologySaarland UniversityIm Stadtwald66123 SaarbrückenGermany
Holger BartosSTEAG microParts GmbHHauert 744227 DortmundGermany
Hagan BayleyDepartment of ChemistryUniversity of OxfordMansfield RoadOxford, OX1 3TAUK
Dennis A. BazylinskiDepartment of PhysicsCalifornia Polytechnic State UniversitySan Luis Obispo, CA 93407USA
Samantha M. BenitoDepartment of ChemistryUniversity of BaselKlingelbergstrasse 804056 BaselSwitzerland
Orit BrahaDepartment of ChemistryUniversity of OxfordMansfield RoadOxford, OX1 3TAUK
Erez BraunDepartment of PhysicsSolid State InstituteTechnion-Israel Instituteof Technology32000 HaifaIsrael
Stanley BrownDepartment of MolecularCell BiologyUniversity of CopenhagenØster Farimagsgade 2A1353 Copenhagen KDenmark
XVIII Contributors
Stephen CheleyTexas A&M UniversityHealth Science CenterMedical Biochemistry and Genetics440 Reynolds Medical BuildingCollege Station, TX 77843-1114USA
Signe DanielsenNorwegian Universityof Science and TechnologyDepartment of PhysicsHøgskoleringen 57491 TrondheimNorway
Emmanuel DelamarcheIBM ResearchZürich Research LaboratorySäumerstrasse 48803 RüschlikonSwitzerland
Stefan DiezMax Planck Institute of MolecularCell Biology and GeneticsPfotenhauerstrasse 10801307 DresdenGermany
Timothy J. DrakeCenter for Research at theBio-nano InterfaceDepartment of Chemistry,McKnight Brain Institute,University of Florida,Gainesville, FL 32611USA
Eva-Maria EgelseerCenter for Ultrastructure Researchand Ludwig Boltzmann Institutefor Molecular NanotechnologyUniversity of Natural Resourcesand Applied Life SciencesGregor-Mendel-Straße 331180 WienAustria
Mahnaz El-KouediDepartment of Chemistry,The Pennsylvania State UniversityUniversity Park, PA 16802USA
Richard B. FrankelDepartment of PhysicsCalifornia Polytechnic State UniversitySan Luis Obispo, CA 93407USA
Xiaohu GaoDepartment of Biomedical EngineeringEmory University School of Medicine1639 Pierce DriveAtlanta, GA 30322USA
Friedrich GötzGelsenkirchen University of AppliedSciencesNeidenburger Str. 4345877 GelsenkirchenGermany
Alexandra GraffDepartment of ChemistryUniversity of BaselKlingelbergstrasse 804056 BaselSwitzerland
XIXContributors
Li-Qun GuTexas A&M UniversityHealth Science CenterMedical Biochemistry and Genetics440 Reynolds Medical BuildingCollege Station, TX 77843-1114USA
Gerhard W. HackerResearch Institute for FrontierQuestions of Medicine andBiotechnologySt. Johanns-HospitalLandeskliniken SalzburgMuellner Hauptstr. 485020 SalzburgAustria
James F. HainfeldBrookhaven National LaboratoryDepartment of BiologyUpton, NY 11973USA
Norbert HamppFachbereich ChemiePhilipps-Universität MarburgHans-Meerwein-Straße, Geb. H35032 MarburgGermany
Helen G. HansmaPhysics DepartmentUniversity of CaliforniaSanta Barbara, CA 93106USA
John H. HeleniusMax Planck Institute of MolecularCell Biology and GeneticsPfotenhauerstrasse 10801307 DresdenGermany
Jonathon HowardMax Planck Institute of MolecularCell Biology and GeneticsPfotenhauerstrasse 10801307 DresdenGermany
Eugenii KatzDepartment of Organic ChemistryHebrew UniversityGivat Ram91904 JerusalemIsrael
Christine D. KeatingDepartment of Chemistry,The Pennsylvania State UniversityUniversity Park, PA 16802USA
M. Islam KhanBiochemical Sciences DivisonNational Chemical Laboratory411 008 PuneIndia
Rajiv KumarCatalysis DivisonNational Chemical Laboratory411 008 PuneIndia
M. N. V. Ravi KumarDepartment of PharmaceuticsNIPERSAS Nagar, Sector 67160 062 MohaliIndia
XX Contributors
Claus-Michael LehrDepartment of Biopharmaceuticsand Pharmaceutical TechnologySaarland UniversityIm Stadtwald66123 SaarbrückenGermany
Stephen MannSchool of ChemistryUniversity of BristolBristol BS8 1TSUK
Eric MayesNanoMagnetics Ltd.108 Longmead RoadBristol BS16 7FGUK
Wolfgang MeierDepartment of ChemistryUniversity of BaselKlingelbergstrasse 804056 BaselSwitzerland
Michael MertigTechnische Universität DresdenInstitut für Werkstoffwissenschaft01062 DresdenGermany
Chad A. MirkinDepartment of Chemistry &Institute for NanotechnologyNorthwestern University2145 Sheridan RoadEvanston, IL 60208-3113USA
Shuming NieDepartment of Biomedical EngineeringEmory University School of Medicine1639 Pierce DriveAtlanta, GA 30322USA
Christof M. NiemeyerUniversität DortmundFachbereich ChemieBiologisch-ChemischeMikrostrukturtechnikOtto-Hahn-Str. 644227 DortmundGermany
Dieter OesterheltMax-Planck Institute for BiochemistryAm Klopferspitz 18A82152 Planegg-MartinsriedGermany
Andreas OffenhäusserInstitute for Thin Films and Interfaces,Bio- and ChemosensorsResearch Centre Jülich52425 JülichGermany
Emin OroudjevDepartment of PhysicsUniversity of CaliforniaSanta Barbara, CA 93106USA
Ralf-Peter PetersSTEAG microParts GmbHHauert 744227 DortmundGermany
XXIContributors
Wolfgang PompeTechnische Universität DresdenInstitut für Werkstoffwissenschaft01062 DresdenGermany
Richard D. PowellNanoprobes, Incorporated95 Horseblock RoadYaphank, NY 11980-9710USA
Dietmar PumCenter for Ultrastructure Researchand Ludwig Boltzmann Institutefor Molecular NanotechnologyUniversity of Natural Resourcesand Applied Life SciencesGregor-Mendel-Straße 331180 WienAustria
Murali SastryMaterials Chemistry DivisonNational Chemical Laboratory411 008 PuneIndia
Thomas SawitowskiInstitut für Anorganische ChemieUniversität GH EssenUniversitätsstr. 5-745117 EssenGermany
Bernhard SchusterCenter for Ultrastructure Researchand Ludwig Boltzmann Institutefor Molecular NanotechnologyUniversity of Natural Resourcesand Applied Life SciencesGregor-Mendel-Straße 331180 WienAustria
Nadrian C. SeemanDepartment of ChemistryNew York UniversityNew York, NY 10003USA
Markus SeitzDepartment of Applied PhysicsLudwig-Maximilians-UniversitätAmalienstrasse 5480799 MünchenGermany
Uri SivanDepartment of PhysicsSolid State InstituteTechnion-Israel Institute of Technology32000 HaifaIsrael
Uwe B. SleytrCenter for Ultrastructure Researchand Ludwig Boltzmann Institutefor Molecular NanotechnologyUniversity of Natural Resourcesand Applied Life SciencesGregor-Mendel-Straße 331180 WienAustria
Joachim P. SpatzInstitut für Physikalische ChemieUniversität HeidelbergINF 25369120 HeidelbergGermany
Weihong TanCenter for Research at theBio-nano InterfaceDepartment of ChemistryMcKnight Brain InstituteUniversity of FloridaGainesville, FL 32611USA
XXII Contributors
C. Shad ThaxtonNorthwestern University2145 Sheridan RoadEvanston, IL 60208USA
Corinne VerbertDepartment of ChemistryUniversity of BaselKlingelbergstrasse 804056 BaselSwitzerland
Angela K. VogtMax-Planck Institutefor Polymer ResearchAckermannweg 1055228 MainzGermany
Itamar WillnerDepartment of Organic ChemistryHebrew UniversityGivat Ram91904 JerusalemIsrael
Xiaojun Julia ZhaoCenter for Research at theBio-nano InterfaceDepartment of Chemistry,McKnight Brain Institute,University of Florida,Gainesville, FL 32611USA
1Biocompatible Inorganic Devices
Thomas Sawitowski
1.1Introduction
New technologies have always been a major driving force in medical device technology [1],and it is largely due to the high economical and social value of modern medical devicesthat new materials and processes are incorporated at a very early stage into new products.Taking this into consideration, the emergence of nanotechnology over the past few yearshas had an immediate influence on medical device technology [2]. This stems from thefact that, by changing the size of commonly known materials, new properties arise thatcan be used in many areas of today’s technologies [3–8]. For this reason, nanotechnologycan be termed an “enabling” technology. It is a highly interdisciplinary field of materialscience which involves chemists, physicist, biologists, engineers, and physicians toname just a few. At the border of those disciplines lie new problems and innovative solu-tions; for example, new implant coatings which were originally developed to improve thewear resistance of tools are now used as a biocompatible coating on stents [9]. Likewise,many other examples have been developed and currently available on a commercial basis.Some of these combine even more of the “smart” properties of nanosized material, includ-ing the enabling of drug delivery and improvement of biocompatibility [10].
1.2Implant Coatings
Implants can be classified as either permanent or temporary devices. Examples ofpermanent implants include seeds, hip joints, stents, nails, and dental implants, whilecatheters or needles are perfect examples of temporarily implanted devices. Each year,the number of implants implanted is directly related to the occurrence of the diseasestreated this way. The major diseases of the western countries relate to the cardiovascularsystem, or to cancer. In Germany during 1998, circulatory diseases (including myocardialinfarction) caused more than 500 000 deaths, or 58 % of all deaths [11]. Likewise, morethan 210 000 people died from cancer, representing 25 % of all deaths in Germany thatyear.
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Taking these facts into consideration, there is clearly a great demand for improvedtreatment of these diseases, and this involves the use of modern medical device tech-nology. This includes the concept of local rather than systemic treatment. Local treat-ment is the basis of every implant used. For example, when treating diseased vesselswith stents the implant is inserted very precisely into the stenotic area; the same holdstrue for seeds used to treat prostate cancer. Hence, implants represent the ideal carrierfor drugs to be delivered locally to the site of implantation. Different carrier systemsare currently being evaluated, including polymers, dendrimers, sol–gel-coatings, orother porous inorganic materials. In this chapter, the focus will be on nanoscale inorg-anic materials for use in local drug delivery rather than polymeric materials, amongwhich nanoporous alumina is one of the most interesting that is currently beingused in cardiology and oncology. In a more general approach, specific units such asimplantable capsules and pumps can be used as carrier technologies [12], whileMEMS devices [13] can be applied to deliver drugs locally. All of these devices must fulfila vast number of criteria before being used in humans [14]. The basic safety andefficacy requirements can be subdivided into biocompatibility, which can be furthersubdivided into tissue or hemic compatibility. For these reasons it is difficult to present acomplete and comprehensive overview of all inorganic medical devices, and sofor technical, medical, and economic reasons the focus here will be on stents andseeds.
1.2.1Stents
As mentioned previously, the current most important causes of death in modern westerncountries are vascular diseases and myocardial infarction. Various risk factors such as low-density lipoproteins, high blood pressure, nicotine abuse, and diabetes lead to changes inthe vessel and, in turn, a narrowing of the free lumen. In time, this causes angina pectorisor perhaps sudden events such as myocardial infarction. There are three invasive methodsto treat these stenoses. In cardiology, the treatment of coronary artery disease has longbeen limited to bypass surgery to circumvent the stenotic region. With this technique,veins are taken for example from the patient’s legs and used to bypass the narrowed re-gion, thus re-enabling blood flow. Such a treatment represents a significant burden for thepatient, and one of the most intriguing aspects of modern medical technologies is the op-portunity to reduce both patient burden and treatment costs by using minimally invasivemethods. A minimally invasive approach would be to use a catheter, at the end of which ismounted a small balloon. The catheter is placed into the narrowed lesion and inflated byapplying external pressure. In this way, the material which is causing the narrowing ispressed into the vessel wall, and this leads to an increase in the lumen free space. In1977, Andreas Grüntzig [15] performed the first percutaneous transluminal balloon angio-plasty (PTCA), when he opened up a narrowed vessel using a small, expandable balloon.To date, PTCA treatment remains one of the most successful applications of minimallyinvasive medical technologies. However, in addition to the many advantages of this tech-nique, inevitably there are also some drawbacks, the most important being an elastic re-
2 1 Biocompatible Inorganic Devices
coil of the vessel caused either by plaque material remaining in the blood stream or byhard plaque material which cannot be removed with the balloon [16].
In these cases an additional mechanical support is needed to improve treatment out-come, and it is for this reason that small metallic meshes – called “stents” – are implantedinto the lesion. The stents may be made from different materials, and are mounted on theballoon which, when inflated, also inflates the stent; this occurs when a stainless steelstent is used. Alternatively, the stent may self-expand when it is released from the deliverysystem; this occurs when a stent is made from Nitinol�, an alloy made from nickel andtitanium. Many different designs of stent are currently available on the market [17].Some are made of tubes which are laser-cut to build a tubular mesh, while others aremade from planar meshes that are laser-welded along the long axis to build the stent.Three typical examples of laser-cut stents are illustrated in Figure 1.1. In all of these stentsthe design consists of a rather rigid metal structure in order to ensure mechanical sta-bility.
The rigid structure guarantees a good mechanical stability of the stent on the balloonwhen finally it is implanted into the vessel. These rigid areas are interconnected by flex-ible parts that ensure stability of the stent as it is moved through the vessel towards thenarrowed lesion. In addition, in most cases the lesion to be treated is not a straight part ofthe vessel but is more often rather curved and roughened on the inside. Here, the flexibleparts ensure that the stent matches the contours of the inner lumen as well as possible.
The initial outcome of stent implantation showed a great improvement compared withPTCA. However, adverse factors such as mechanical stress and/or damage to the arterialwall, heavy metal ion dissolution (e. g., nickel, chromium, or molybdenum) from the im-plant, and disturbing turbulences in the bloodstream means that a positive outcome is notguaranteed for all patients. Thus, 30–40 % of the vessels develop a re-narrowing (resteno-sis), mainly as a result of proliferating smooth muscle cells (SMCs) resembling scar tis-sue. In clinical trials, bypass surgery was compared with PTCA and PTCA plus stent,but the problems of restenosis ultimately reduced the positive initial outcome associatedwith stent implantation [18]. Restenosis can be seen as a problem of poor biocompatibilityinvolving of course not only biochemical but also mechanical and technical aspects of theimplantation.
Bearing in mind the huge number of patients suffering from vascular diseases, there isclearly a great demand for an improved biocompatibility of stents. To achieve this, manydifferent types of coatings have been applied to stents, including silicon carbide (SiC), dia-
31.2 Implant Coatings
Figure 1.1 Different stent types : Left : Genius MEGAFlex coronary stent; Middle : Small Vessel stent(BiodivYsio SV); Right : Terumo Tsunami.
mond-like carbon (DLC), turbostratic carbon (TC), gold, iridium oxide, aluminum oxide,and many different polymeric coatings which may, or may not, be biodegradable [10]. Theinitial goal has been to improve the limited biocompatibility of stents made from stainlesssteel or Nitinol by the use of passive coatings; this prevents heavy metal ion dissolutionfrom the stents. In one study, the risk of restenosis was compared in patients with andwithout a nickel allergy [19]. The results indicated that patients who were allergic towardsnickel or molybdenum were more likely to suffer in-stent restenosis than those withoutsuch hypersensitivity. These allergic reactions may trigger the fibroproliferative or inflam-matory responses characteristic of in-stent restenosis. Almost all coatings made from in-organic materials target the dissolution of nickel, chromium, and other metals fromstents. One of the very first coatings to be applied was a thin layer of gold, though theidea of passivating the surface by chemical means generally fails. In a clinical trial invol-ving more than 700 patients, gold-coated stents caused a significant increase in neointi-mal hyperplasia compared with stainless steel stents. Thus, gold-coated stents were asso-ciated with a considerable increase in the risk of restenosis over the first year after stent-ing [20]. The precise mechanism by which gold coating causes intimal hyperplasia is stillhypothetical, though it is possible that mechanical damage of the coating leads to particleformation and hence inflammation. On the other hand, it is possible that as a stent is di-lated, due to the significant strain and stress cracks might occur in the coating. In thisway, local elements of steel and gold are formed which might even increase the dissolu-tion of certain metal ions.
Silicon carbide has also been applied to stents to cover the surface and reduce intimalhyperplasia. Initial data obtained from animals suggested that the coating shows a signif-icantly lower platelet and leukocyte adhesion at the surface of the SiC-coated tantalumstent compared with the surface of stainless-steel stents [21].
The first clinical trials conducted with silicon carbide-coated 316L stainless steel stentsshowed a better outcome after 6-month follow-up than uncoated stainless steel stents withrespect to binary restenosis rate. In a selected group of patients, the implantation of acoated coronary stent showed a high incidence of immediate success and an absence ofin-hospital cardiac events. A significant reduction of major adverse cardiac events and areduced reintervention rate compared to conventional stents was also observed by others[22].
Carbon with a mixed hybridization state between 2 and 3 has also been applied as a bar-rier layer. This type of coating has been named turbostratic carbon (TC), but it has notshown a major benefit in a clinical trial comparing bare and TC-coated stents. Anothersuggested approach is to use titanium-nitride-oxide-coated and iridium oxide-coatedstents, and initial results with these have been seen as promising [23].
Until now, it has not been clear what influences the biological response to certain sur-faces. As material, stent design, biology, chemistry, and physics represent a complex sys-tem, it is difficult to elucidate clear-cut interactions. Although passive coatings on stentshave the potential to improve biocompatibility, the next major step towards reducing in-timal hyperplasia is to bind drugs locally onto the stents themselves in order to overcomethe ultimate problems of restenosis.
Besides certain polymeric coatings, only one inorganic coating has yet proved capable oftaking-up and releasing drugs from implant surfaces, and this is nanoporous alumina.
4 1 Biocompatible Inorganic Devices
This coating, which consists of an amorphous alumina ceramic with pores in the order of5 to 500 nm diameter, can be used to store and release drugs locally [9]. The material isformed using the well-established process of anodizing aluminum in different electrolytes[24]. The oxidation is normally carried out in diluted acidic electrolytes (typically of oxalicor phosphoric acid) by applying potentials in the order of a few tens up to a few hundredsof volts, and a direct current. With this process, the aluminum surface is converted into anamorphous aluminum oxide and hydroxide surface, which can be best described as aBoehmite composition of aluminum oxide [25].
The first step of the oxidation process is the formation of a dense layer of oxide on themetal surface, the thickness of which is dependent upon the applied potential. The highelectrical field, together with some initial surface perturbation (coming from the naturalsurface roughness or from grain boundaries for example), causes the first pores to beformed. In this high-electrical field regime, the oxide crystal lattice is deformed at slightperturbations and the electrolytes dissolve the oxide more rapidly, causing pores to beformed. While the electrical field determines the oxide formation and dissolution, thepore geometry may be controlled by the electrical field and thus by the potential appliedin the process of anodizing aluminum [26]. Ultimately, a structure similar to that shownin Figure 1.2 can be obtained, with the pores ordered parallel to each other and perpen-dicular to the substrate surface [27].
At this stage, a thin oxide layer – the so-called barrier layer – remains at the bottom ofthe pores. As a rule of thumb, it can be said that for each Volt of anodic potential the porediameter increases by 1.5 nm. So, by applying 10 Volts, pores in the order of 10–15 nm areformed [28]. The pores are packed hexagonally, with an amorphous Boehmite forming thepore wall in between. Pore densities can reach values up to 1011 pores cm–2, while the por-osity always remains the same (�30 %) because small pores are packed more denselycompared with larger ones [29]. The pore length is more or less controlled by the electrical
51.2 Implant Coatings
Figure 1.2 Cross-sectional scanningelectron microscopy (SEM) imageof a nanoporous alumina membrane.The pore diameter is �120 nm.
charge, which is proportional to the time of anodic oxidation. Increasing this time leads toan increase in oxide layer thickness until an equilibrium is reached between oxide forma-tion and porous layer dissolution in the electrolyte; layer thicknesses up to �100 mm arecommon. For implants such as stents not made from aluminum, the material must firstbe coated with aluminum in a physical vapor deposition (PVD) process. During this pro-cess, the stents are mounted on a stent holder which is rotated in the aluminum plasmabeing coated at the same time. The thickness of the coating can be varied by a few hun-dred nanometers and some microns. In a second step, this layer is electrochemically con-verted into a nanoporous ceramic by using the above-described methods [30]. Dependingon the conditions, different porosities and pore sizes can be achieved (Figure 1.3).
The release kinetics for a specific drug can be varied to a certain degree by changing thelayer thickness and varying the pore sizes. Nevertheless, the solubility of the drug is also avery important aspect for the release time and the release kinetics. Until now, no hydro-philic drug has been applied to stents because there is clearly no delivery platform suitablefor the retained release of these drugs.
In order to achieve local drug delivery, there is a need for the new technologies to bind acertain amount of the substance onto a stent and assure sustained release over a few daysand up to a few weeks. For this reason, stents are typically coated with a 200–500-nm thinmetallic layer of aluminum metal, which is converted afterwards into the nanoporous alu-mina. Stents carrying 40 mg of a hydrophobic drug showed a release of this drug in phos-phate-buffered saline at 37 �C over 6 days, with good reproducibility and a narrow stan-dard deviation.
The amount of drug which is fixed onto the stent is limited by the nature of the poresystem. Assuming a 1 mm-thick coating with a porosity of 40 % on a stent having 2-cm2
surface area, it can be calculated that �80 mg of drug can be fixed. Coated stents of thistype, with and without 40 mg drug have been tested for their influence on intimal hyper-plasia in the carotid artery of rabbits. The animals were sacrificed after 4 weeks and themorphology of the vessel at the region of implantation was investigated.
6 1 Biocompatible Inorganic Devices
Figure 1.3 Surface SEM image of ananoporous alumina-coated stent.The pore diameter is �20–50 nm.
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