Bio-inorganic Hybrid Nanomaterials

Bio-inorganic Hybrid Nanomaterials Strategies, Syntheses, Characterization and Applications Edited by Eduardo Ruiz-Hitzky, Katsuhiko Ariga and Yuri Lv...

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Bio-inorganic Hybrid Nanomaterials Strategies, Syntheses, Characterization and Applications

Edited by Eduardo Ruiz-Hitzky, Katsuhiko Ariga and Yuri Lvov

Bio-inorganic Hybrid Nanomaterials

Edited by Eduardo Ruiz-Hitzky, Katsuhiko Ariga and Yuri Lvov

Further Reading Vollath, D.

Nanomaterials An Introdution to Synthesis, Properties and Applications 2008 ISBN: 978-3-527-31531-4

Willner, I., Katz, E. (Eds.)

Bionanomaterials Synthesis and Applications for Sensors, Electronics and Medicine 2008 ISBN: 978-3-527-31454-6

Ajayan, P. M., Schadler, L. S., Braun, P. V., Keblinski, P.

Nanocomposite Science and Technology Second Completely Revised Edition 2008 ISBN: 978-3-527-31248-1

Rao, C. N. R., Müller, A., Cheetham, A. K. (Eds.)

Nanomaterials Chemistry Recent Developments and New Directions 2007 ISBN: 978-3-527-31664-9

Kickelbick, G. (Ed.)

Hybrid Materials Synthesis, Characterization, and Applications 2007 ISBN: 978-3-527-31299-3

Kumar, Challa S. S. R. (Ed.)

Nanotechnologies for the Life Sciences 10 Volume Set 2007 ISBN: 978-3-527-31301-3

Bio-inorganic Hybrid Nanomaterials Strategies, Syntheses, Characterization and Applications

Edited by Eduardo Ruiz-Hitzky, Katsuhiko Ariga and Yuri Lvov

The Editors Prof. Dr. Eduardo Ruiz-Hitzky Instituto de Ciencia de Materiales de Madrid Consejo Superior de Investigaciones Cientificas Cantoblanco 28049 Madrid Spain Dr. Katsuhiko Ariga National Institute for Material Science 1-1 Namiki 305-0044 Tsukuba, Ibaraki Japan Prof. Yuri Lvov Institute for Micromanufacturing Louisiana Technical University 911 Hergot Avenue Ruston, LA 71272 USA

All books published by Wiley-VCH are carefully produced. Nevertheless, authors, editors, and publisher do not warrant the information contained in these books, including this book, to be free of errors. Readers are advised to keep in mind that statements, data, illustrations, procedural details or other items may inadvertently be inaccurate. Library of Congress Card No.: applied for British Library Cataloguing-in-Publication Data A catalogue record for this book is available from the British Library. Bibliographic information published by the Deutsche Nationalbibliothek Die Deutsche Nationalbibliothek lists this publication in the Deutsche Nationalbibliografie; detailed bibliographic data are available on the Internet at . # 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim All rights reserved (including those of translation into other languages). No part of this book may be reproduced in any form – by photoprinting, microfilm, or any other means – nor transmitted or translated into a machine language without written permission from the publishers. Registered names, trademarks, etc. used in this book, even when not specifically marked as such, are not to be considered unprotected by law. Printed in the Federal Republic of Germany Printed on acid-free paper Cover Design WMX-Design, Heidelberg Typesetting Thomson Digital, India Printing Strauss GmbH, Mörlenbach Binding Litges & Dopf GmbH, Heppenheim ISBN: 978-3-527-31718-9

V

Contents Preface

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Contributors 1 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8

2 2.1 2.2 2.3 2.4 2.5 2.6

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An Introduction to Bio-nanohybrid Materials 1 Eduardo Ruiz-Hitzky, Margarita Darder, Pilar Aranda Introduction: The Assembly of Biological Species to Inorganic Solids 1 Bio-nanohybrids Based on Silica Particles and Siloxane Networks 4 Calcium Phosphates and Carbonates in Bioinspired and Biomimetic Materials 9 Clay Minerals and Organoclay Bio-nanocomposites 13 Bio-Nanohybrids Based on Metal and Metal Oxide Nanoparticles 20 Carbon-based Bio-nanohybrids 22 Bio-nanohybrids Based on Layered Transition Metal Solids 28 Trends and Perspectives 31 References 32 Biomimetic Nanohybrids Based on Organosiloxane Units 41 Kazuko Fujii, Jonathan P. Hill, Katsuhiko Ariga Introduction 41 Monolayer on Solid Support 45 Layered Alkylsiloxane 53 Organic–Inorganic Hybrid Vesicle “Cerasome” 59 Mesoporous Silica Prepared by the Lizard Template Method 65 Future Perspectives 69 References 71

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3

3.1 3.2 3.2.1 3.2.1.1 3.2.1.2 3.2.1.3 3.2.2 3.2.2.1 3.2.2.2 3.2.2.3 3.2.3 3.3 3.3.1 3.3.1.1 3.3.1.2 3.3.2 3.3.3 3.4 3.4.1 3.4.2 3.4.3 3.4.4 3.4.5 3.5

4

4.1 4.2 4.3 4.4 4.5 4.6 4.7

Entrapment of Biopolymers into Sol–Gel-derived Silica Nanonocomposites 75 Yury A. Shchipunov Introduction 75 Sol–Gel Processes 77 Chemistry 77 Hydrolysis 77 Condensation 78 Sol–Gel Transition 78 Silica Precursors 79 Orthosilicic Acid 80 Sodium Metasilicate 80 Alkoxides 80 Two-Stage Approach to Biopolymer Entrapment 82 Biocompatible Approaches 84 Modified Sol–Gel Processing 84 Method of Gill and Ballesteros 84 Low-Molecular and Polymeric Organic Additives 85 Organically-modified Precursors 86 Biocompatible Precursors by Brennan et al. 87 One-Stage Approach Based on a Silica Precursor with Ethylene Glycol Residues 88 Precursor 88 Role of Biopolymers in Sol–Gel Processing 89 Advantages of One-Stage Processes 96 Hybrid Biopolymer–Silica Nanocomposite Materials 98 Enzyme Immobilization 99 Perspectives 102 References 103 Immobilization of Biomolecules on Mesoporous Structured Materials 113 Ajayan Vinu, Narasimhan Gokulakrishnan, Toshiyuki Mori, Katsuhiko Ariga Introduction 113 Immobilization of Protein on Mesoporous Silica 116 Immobilization of Protein on Mesoporous Carbon and Related Materials 124 Immobilization of Other Biopolymers on Mesoporous Materials 133 Immobilization of Small Biomolecules on Mesoporous Materials 137 Advanced Functions of Nanohybrids of Biomolecules and Mesoporous Materials 141 Future Perspectives 149 References 150

Contents

5 5.1 5.2 5.3 5.3.1 5.3.1.1 5.3.1.2 5.3.1.3 5.3.2 5.3.2.1 5.3.2.2 5.3.2.3 5.3.3 5.3.4 5.4

6

6.1 6.1.1 6.1.2 6.2 6.2.1 6.2.2 6.3 6.3.1 6.3.2 6.3.3 6.4 6.4.1 6.4.2 6.5

7

7.1 7.2 7.2.1

Bio-controlled Growth of Oxides and Metallic Nanoparticles 159 Thibaud Coradin, Roberta Brayner, Fernand Fiévet, Jacques Livage Introduction 159 Biomimetic Approaches 160 In vitro Synthesis of Hybrid Nanomaterials 165 Polysaccharides 165 Alginates 165 Carrageenans 169 Chitosan 171 Proteins 174 Gelatin 174 Collagen 175 Protein Cages and Viral Capsids 177 Lipids 180 DNA Scaffolds 181 Perspectives: Towards a “Green Nanochemistry” 183 References 184 Biomineralization of Hydrogels Based on Bioinspired Assemblies for Injectable Biomaterials 193 Junji Watanabe, Mitsuru Akashi Introduction 193 Biominerals as Nanomaterials 193 Nanomaterials for Biofunctions 196 Fundamental Concept of Bioinspired Approach 197 Bioinspired Approach to Materials 197 Concrete Examples of the Bioinspired Approach 198 Alternate Soaking Process for Biomineralization and their Bio-functions 199 Nanoassembly by Polyelectrolytes 199 Alternate Soaking Process for Biomineralization 200 Biomineralization of Hydrogels for Bio-functions 201 Electrophoresis Process for Biomineralization 203 Innovative Methodology of Electrophoresis Process for Biomineralization 203 Application for Injectable Materials 204 Conclusions 206 References 206 Bioinspired Porous Hybrid Materials via Layer-by-Layer Assembly 209 Yajun Wang , Frank Caruso Introduction 209 Porous Materials 209 Microporous Materials 210

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Contents

7.2.2 7.2.3 7.3 7.4 7.4.1 7.4.2 7.4.3 7.4.4 7.5 7.5.1 7.5.2 7.5.3 7.6

Mesoporous Material 210 Macroporous Materials 211 LbL Assembly 213 LbL Assembly on MS Substrates 214 Encapsulation of Biomolecules in MS Particles 214 MS Spheres as Templates for the Preparation of Hollow Capsules 218 Preparation of Protein Particles via MS Sphere Templating 220 Template Synthesis of Nanoporous Polymeric Spheres 221 LbL Assembly on Macroporous Substrates 225 LbL Assembly on Tubular Substrates 226 LbL Assembly on 3DOM Materials 229 LbL Assembly on Naturally Occurring Porous Substrates 231 Summary and Outlook 232 References 233

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Bio-inorganic Nanohybrids Based on Organoclay Self-assembly 239 Avinash J. Patil, Stephen Mann Introduction 239 Synthesis and Characterization of Organically Functionalized 2:1 Magnesium Phyllosilicates 240 Magnesium Organophyllosilicates with Higher-order Organization 243 Intercalation of Biomolecules within Organically Modified Magnesium Phyllosilicates 246 Protein–Organoclay Lamellar Nanocomposites 247 DNA–Organoclay Lamellar Nanostructures 252 Drug–Organoclay Layered Nanocomposites 253 Hybrid Nanostructures Based on Organoclay Wrapping of Single Biomolecules 254 Organoclay-wrapped Proteins and Enzymes 254 Organoclay-wrapped DNA 258 Functional Mesolamellar Bio-inorganic Nanocomposite Films 260 Summary 262 References 262

8.1 8.2 8.3 8.4 8.4.1 8.4.2 8.4.3 8.5 8.5.1 8.5.2 8.6 8.7

9

9.1 9.2 9.2.1 9.2.2 9.2.2.1 9.2.2.2 9.2.2.3

Biodegradable Polymer-based Nanocomposites: Nanostructure Control and Nanocomposite Foaming with the Aim of Producing Nano-cellular Plastics 271 Masami Okamoto Introduction 271 Nano-structure Development 272 Melt Intercalation 272 Interlayer Structure of OMLFs and Intercalation 273 Nano-fillers 273 Molecular Dimensions and Interlayer Structure 274 Correlation of Intercalant Structure and Interlayer Opening

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9.2.2.4 9.3 9.3.1 9.3.2 9.3.3 9.4 9.4.1 9.4.2 9.5 9.5.1 9.5.2 9.5.3 9.5.4 9.5.5 9.6 9.7

Nanocomposite Structure 278 Control of Nanostructure Properties 282 Flocculation Control and Modulus Enhancement 282 Linear Viscoelastic Properties 284 Elongational Flow and Strain-induced Hardening 288 Physicochemical Phenomena 290 Biodegradability 290 Photodegradation 295 Foam Processing using Supercritical CO2 296 PLA-based Nanocomposite 296 Temperature Dependence of Cellular Structure 298 CO2 Pressure Dependence 301 TEM Observation 305 Mechanical Properties of Nanocomposite Foams 307 Porous Ceramic Materials via Nanocomposites 307 Future Prospects 309 References 310

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Biomimetic and Bioinspired Hybrid Membrane Nanomaterials 313 Mihail Barboiu Introduction 313 Molecular Recognition-based Hybrid Membranes 314 Multiple Molecular Recognition Principles 314 Self-organized Hybrid Membrane Materials 318 Ionic-conduction Pathways in Hybrid Membrane Materials 318 Ionic-conduction Pathways in Macrocyclic Hybrid Materials 319 Ionic-conduction Pathways in Peptido-mimetic Hybrid Materials 319 Self-organization in Hybrid Supramolecular Polymers 324 Self-organization by Base Pairing in Hybrid Supramolecular Polymers 325 Self-Organization of the Guanine Quadruplex in Hybrid Supramolecular Polymers 328 Dynamic Site Complexant Membranes 330 Conclusions 333 References 334

10.1 10.2 10.2.1 10.3 10.3.1 10.3.1.1 10.3.1.2 10.3.2 10.3.2.1 10.3.2.2 10.4 10.5

11

11.1 11.2 11.3 11.3.1 11.3.2 11.3.3

Design of Bioactive Nano-hybrids for Bone Tissue Regeneration 339 Masanobu Kamitakahara, Toshiki Miyazaki, Chikara Ohtsuki Introduction 339 Composite of Bioactive Ceramic Particles and Polymers 340 Bone-bonding Mechanism of Bioactive Materials 341 Interface between Bone and Bioactive Material 341 Simulated Body Fluid 342 Hydroxyapatite Formation on Bioactive Materials 343

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Contents

11.4 11.4.1 11.4.2 11.4.3 11.4.4 11.5 11.5.1 11.5.2 11.5.3 11.5.4 11.5.5 11.6 11.7

12

12.1 12.2 12.3 12.3.1 12.4 12.4.1 12.4.1.1 12.4.1.2 12.4.2 12.4.2.1 12.4.2.2 12.4.2.3 12.4.2.4 12.4.2.5 12.4.2.6 12.4.2.7 12.4.2.8 12.4.2.9 12.4.2.10 12.4.2.11 12.4.2.12 12.5

Sol–Gel-derived Bioactive Nano-hybrids 345 Silicate-based Nano-hybrids 345 Nano-hybrids Starting from Methacryloxy Compounds 347 Nano-hybrids Based on Other than Silicate 349 Nano-hybrids Combined with Calcium Phosphates 353 Nano-hybrid Consisting of Bone-like Hydroxyapatite and Polymer 354 Biomimetic Process 354 Hydroxyapatite Deposition on Polymers Modified with Silanol Groups 356 Hydroxyapatite Deposition on Natural Polymers 357 Hydroxyapatite Deposition on Synthetic Polymers 358 Control of the Structure of Hydroxyapatite 359 Nano-hybrid Consisting of Hydroxyapatite and Protein 360 Conclusion 361 References 361 Nanostructured Hybrid Materials for Bone Implants Fabrication 367 María Vallet-Regí, Daniel Arcos Introduction 367 Bone: A Biological Hybrid Nanostructured Material 369 Biomimetic Materials for Bone Repair. The Hybrid Approach 372 The Hybrid Approach 374 Synthesis and Properties of Organic–Inorganic Hybrid Materials for Bone and Dental Applications 375 Class I Hybrid Materials 375 BG–Poly(vinyl Alcohol) 375 Silica Particles–pHEMA 378 Class II Hybrid Materials 378 PMMA–SiO2 Ormosils 380 PEG–SiO2 Ormosils 380 PDMS–CaO–SiO2–TiO2 Ormosils 380 PTMO–CaO–SiO2–TiO2 Hybrid Materials 383 MPS–HEMA Ormosils 383 Gelatine–SiO2 Systems 384 Poly(e-Caprolactone)–Silica Ormosils 385 Bioactive Star Gels 387 The Synthesis of Bioactive Star Gels 388 How to Characterize Bioactive Star Gels? 389 The Bioactivity of the Star Gels 389 The Mechanical Properties of Bioactive Star Gels 391 Conclusion 392 References 393

Contents

13 13.1 13.2 13.3 13.3.1 13.3.2 13.4 13.4.1 13.4.2 13.4.3 13.4.4 13.5 13.5.1 13.5.2 13.6

14

14.1 14.2 14.2.1 14.2.2 14.2.3 14.3 14.4 14.5 14.5.1 14.5.2 14.6

15 15.1 15.2 15.2.1 15.2.2 15.3 15.3.1 15.3.2 15.3.3

Bio-inorganic Conjugates for Drug and Gene Delivery 401 Jin-Ho Choy, Jae-Min Oh, Soo-Jin Choi Introduction 401 Synthesis of Bio-inorganic Conjugates 403 Bio-inorganic Conjugate for Efficient Gene Delivery 407 Cellular Uptake Kinetics of LDH–FITC Into Cells 407 Effect of As-myc–LDH Hybrid on the Suppression of Cancer Cells 408 Bio-inorganic Conjugate for Efficient Drug Delivery 409 Cellular Uptake of MTX–LDH Hybrid 409 Effect of MTX–LDH on Cell Proliferation and Viability 409 Effect of MTX–LDH Hybrid on the Cell Cycle 410 Potential of Bio-inorganic Conjugates for Gene and Drug Delivery 411 Cellular Uptake Mechanism of LDH 412 Endocytosis of LDH 412 Endocytic Pathway of LDH 413 Conclusion 415 References 415 Halloysite Nanotubules, a Novel Substrate for the Controlled Delivery of Bioactive Molecules 419 Yuri M. Lvov, Ronald R. Price Halloysite Structural Characterization 419 Macromolecule Loading and Sustained Release 422 Nanotubule Loading Procedure 422 Drugs and Biocides 423 Globular Proteins 427 Nanoassembly on Tubules and at the Lumen Opening 428 Catalysis in a Nanoconstrained Volume of the Tubule Lumen 431 Multilayer Halloysite Assembly for Organized Nanofilms. Forming Low Density Tubule Nanoporous Materials 436 Tubule–Polycation Multilayer 436 Assembly of Tubule/Sphere Multilayer Nanocomposites 437 Applications: Current and Potential 438 References 439 Enzyme-based Bioinorganic Materials 443 Claude Forano, Vanessa Prévot Introduction 443 Enzymes versus Inorganic Host Properties 445 Enzyme Properties 445 Inorganic Host Structures 446 Immobilization Strategy 446 Adsorption Process 448 Encapsulation Processes 449 Nanostructuring of Enzyme-based Films 450

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15.3.4 15.4 15.4.1 15.4.1.1 15.4.1.2 15.4.1.3 15.4.1.4 15.4.2 15.4.2.1 15.4.2.2 15.4.2.3 15.4.2.4 15.5

Covalent Grafting 452 Bioinorganic Nanohybrids 454 Immobilization of Enzymes in 2-D Inorganic Hosts 454 Immobilization in Clay Minerals and Related Materials 454 Immobilization in Layered Double Hydroxides 457 Immobilization in Layered Metal Oxides 460 Immobilization in Layered Zirconium Phosphate and Phosphonate 461 Immobilization of Enzymes in 3-D Inorganic Hosts 464 Immobilization in SiO2 464 Immobilization on Alumina 467 Immobilization in Zeolite 469 Immobilization in Hydroxyapatite and Tricalciumphosphate 471 Enzyme–Host Structure Interactions 471 References 476 Index

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Preface Materials from living matter and inorganic materials are apparently on opposite sides in the materials' world. In this context, biological materials including polysaccharides, proteins, nucleic acids, and lipids, have soft and flexible natures, and often show incredible functions with high specificity and efficiency, which cannot be easily re-generated or replicated by combination of man-made materials. Therefore, direct use of such a class of biological materials sounds like a rational way to construct highly sophisticated functional systems. The best way to cope with the high functionality and stability of bio-related materials for practical applications is to create hybrids consisting of materials of biological origin and inorganic materials. However, simply mixing these materials together into a messy slurry is not a wise strategy. In biologically derived materials both components have their own well-organized, meaningful nanostructures, and therefore, hybridization of inorganic and biological elements in controlled structures with nanometer-scale precision is the most desirable strategy. The obtained materials can be called “bio-inorganic nanohybrids.” They are well-blessed children from both worlds, and should succeed in providing essences and advantages of both biological and inorganic materials. Research in bio-inorganic nanohybrids can be defined as an interdisciplinary field resulting from the interfaces between biotechnology, materials science, and nanotechnology. Such a new field is closely related to significant topics such as biomineralization processes, bioinspired materials and biomimetic systems. The incoming development of novel bio-nanocomposites introducing multifunctionality and taking profit from the characteristics of both types of constituents is nowadays an amazing research line taking advantage from the synergistic assembling of biopolymers with inorganic nanosized solids. Mother of pearl and marine shells, corals, teeth, bones, and microbe inclusions (such as sulfur or iron nanocrystals) are examples of bio-inorganic nanocomposites. In many cases, these composites have biopolymers and inorganic parts organized on the nanoscale, such as the regular alternation of proteins and calcium carbonate nanolayers in nacre. When struck, the layers glide over one another absorbing the shock. If cracks develop, plates simply grow back together. Such natural nanocomposite materials often combine unique mechanical properties based on the nanoscale organization of hard and soft materials and have the ability for regeneration

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Preface

and self-reproduction. However, these nanocomposites have a fatal drawback in their application. In most cases, their functions are optimized only at ambient conditions and their structural stability is maintained in a limited environment. In contrast, inorganic materials usually have incredible stability and stiffness, even in extreme conditions. In addition, they sometimes offer us the opportunity to prepare precise structures by both top-down fabrications and bottom-up assembly. Of course, superior aspects in electronic, photonic, magnetic, and mechanical properties can be expected in many inorganic materials. Nevertheless, no one can believe that the highly sophisticated functions seen in living systems may be constructed by assembly and fabrication of materials of exclusively inorganic nature. The focus of this “Bio-Inorganic Hybrid Nanomaterials” book is to cover the wide spectrum of recent developments in natural and artificial bio-hybrid materials, which is the result of the successful assembly of 15 chapters by world-wide experts in their corresponding fields. Fundamental aspects on the preparation of bioinorganic nanohybrids using various nanostructures including mesoporous materials, nanoparticles, gels, organoclays, membranes, and nanotubules with advanced methodologies such as the sol–gel process, self-assembly, intercalation, template synthesis and layer-by-layer adsorption upon the concept of supramolecular chemistry, biomimetics, and biomineralization are thoroughly described. Not limited to basic sciences, several chapters introduce practical applications of bio-inorganic nanohybrids, as exemplified in biodegradation, bone tissue re-generation, controlled delivery, and enzymatic activity. Readers can enjoy independent chapters and also feel the good harmony of the balanced assembly of the chapters. We hope that every reader can find potential possibilities of bio-inorganic nanohybrids with certain wonder, surprise, and impression, just after closing these pages. Eduardo Ruiz-Hitzky Katsuhiko Ariga Yuri Lvov

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Contributors Mitsuru Akashi Osaka University Department of Applied Chemistry Graduate School of Engineering 2-1, Yamada-oka, and The 21st Century COE Program for “Center for Integrated Cell and Tissue Regulation” 2-2, Yamada-oka, Suita Osaka 565-0871 Japan Pilar Aranda Instituto de Ciencia de Materiales de Madrid Consejo Superior de Investigaciones Científicas (CSIC) Cantoblanco 28049-Madrid Spain Daniel Arcos Universidad Complutense Departamento de Quimica Inorgánica y Bioinorganica Facultad de Farmacia Plaza Ramón y Cajal s/n 28040 Madrid Spain

Katsuhiko Ariga National Institute for Materials Science (NIMS) Supermolecules Group 1-1 Namiki Tsukuba 305-0044 Japan Mihail Barboiu Institut Européen des Membranes Adaptative Supramolecular Nanosystems Group UMR CNRS 5635 Place Eugène Bataillon CC 047 34095 Montpellier France Roberta Brayner Université Pierre et Marie Curie Université Denis Diderot Interfaces, Traitements, Organisation et Dynamique des Systèmes (ITODYS) UMR-CNRS 7086 2 Place Jussieu 75252 Paris Cedex 05 France

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Contributors

Frank Caruso The University of Melbourne Centre for Nanoscience and Nanotechnology Department of Chemical and Biomolecular Engineering Victoria 3010 Australia

Fernand Fiévet Université Denis Diderot Interfaces, Traitements, Organisation et Dynamique des Systemès (ITODYS) UMR-CNRS 7086 2 Place Jussieu 75251 Paris Cedex 05 France

Soo-Jin Choi Ewha Womans University Center for Intelligent Nano-Bio Materials Division of Nanoscience and Department of Chemistry Seoul 120-750 Korea

Claude Forano Université Blaise Pascal Laboratoire des Matériaux Inorganiques CNRS UMR 6002 63177 Aubiere Cedex France

Jin-Ho Choy Ewha Womans University Center for Intelligent Nano-Bio Materials Division of Nanoscience and Department of Chemistry Seoul 120-750 Korea Thibaud Coradin Université Pierre et Marie Curie Chimie de la Matière Coudensée de Paris (CMCP) UMR-CNRS 7574 4 Place Jussieu 75252 Paris Cedex 05 France Margarita Darder Instituto de Ciencia de Materiales de Madrid Consejo Superior de Investigaciones Científicas (CSIC) Cantoblanco 28049-Madrid Spain

Kazuko Fujii National Institute for Materials Science (NIMS) Special Subjects Group 1-1 Namiki Tsukuba 305-0044 Japan Narasimhan Gokulakrishnan National Institute for Materials Science (NIMS) Supermolecules Group 1-1 Namiki Tsukuba 305-0044 Japan Jonathan P. Hill National Institute for Materials Science (NIMS) Supermolecules Group 1-1 Namiki Tsukuba 305-0044 Japan

Contributors

Masanobu Kamitakahara Tohoku University Graduate School of Environmental Studies 6-6-20, Aoba Aramaki, Aoba-ku Sendai 980-8579 Japan

Toshiyuki Mori National Institute for Materials Science (NIMS) Nano Ionics Materials Group Fuel Cell Materials Center 1-1 Namiki Tsukuba 305-0044 Japan

Jacques Livage Université Pierre et Marie Curie Chimie de la Matière Condensée de Paris (CMCP) UMR-CNRS 7574 4 Place Jussieu 75252 Paris Cedex 05 France

Jae-Min Oh Ewha Womans University Center for Intelligent Nano-Bio Materials Division of Nanoscience and Department of Chemistry Seoul 120-750 Korea

Yuri M. Lvov Louisiana Tech University Institute for Micromanufacturing 911 Hergot Ave. Ruston, LA 71272 USA

Chikara Ohtsuki Nagoya University Department of Crystalline Materials Science Graduate School of Engineering Furo-cho, Chikusa-ku Nagoya 464-8603 Japan

Stephen Mann University of Bristol Centre for Organized Matter Chemistry School of Chemistry Bristol, BS8 1TS United Kingdom Toshiki Miyazaki Kyushu Institute of Technology Graduate School of Life Science and Systems Engineering 2-4, Hibikino Wakamatsu-ku, Kitakyushu-shi Fukuoka 808-0196 Japan

Masami Okamoto Toyota Technological Institute Advanced Polymeric Nanostructured Materials Engineering Graduate School of Engineering 2-12-1 Hisakata, Tempaku Nagoya 468 8511 Japan

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Contributors

Avinash J. Patil University of Bristol Centre for Organized Matter Chemistry School of Chemistry Bristol, BS8 1TS United Kingdom Vanessa Prévot Université Blaise Pascal Laboratoire des Matériaux Inorganiques CNRS UMR 6002 63177 Aubiere Cedex France Ronald R. Price Atlas Mining Corporation Nanoclay and Technology Division 1200 Silver City Road Eureka, UT 84628 USA Eduardo Ruiz-Hitzky Instituto de Ciencia de Materiales de Madrid Consejo Superior de Investigaciones Científicas (CSIC) Cantoblanco 28049-Madrid Spain Yury A. Shchipunov Russian Academy of Sciences Institute of Chemistry Far East Department 690022 Vladivostok Russia

María Vallet-Regí Universidad Complutense Facultad de Farmacia Departamento de Quimica Inorgánica y Bioinorganica Plaza Ramón y Cajal s/n 28040 Madrid Spain Ajayan Vinu National Institute for Materials Science (NIMS) Nano Ionics Materials Group Fuel Cell Materials Center 1-1 Namiki Tsukuba 305-0044 Japan Yajun Wang The University of Melbourne Centre for Nanoscience and Nanotechnology Department of Chemical and Biomolecular Engineering Victoria 3010 Australia Junji Watanabe Osaka University Department of Applied Chemistry Graduate School of Engineering 2-1, Yamada-oka, and The 21st Century COE Program for “Center for Integrated Cell and Tissue Regulation” 2-2, Yamada-oka, Suita Osaka 565-0871 Japan

j1

1 An Introduction to Bio-nanohybrid Materials Eduardo Ruiz-Hitzky, Margarita Darder, Pilar Aranda

1.1 Introduction: The Assembly of Biological Species to Inorganic Solids

The assembly of molecular or polymeric species of biological origin and inorganic substrates through interactions on the nanometric scale constitutes the basis for the preparation of bio-nanohybrid materials (Figure 1.1). The development of these materials represents an emerging and interdisciplinary topic at the border of Life Sciences, Material Sciences and Nanotechnology. They are of great interest due to their versatile applications in important areas as diverse as regenerative medicine and new materials with improved functional and structural properties [1–5]. It must be remarked that the development of bio-nanocomposites also represents an ecological alternative to conventional polymer nanocomposites, as the properties of the biodegradable polymers used ensure that the materials produced are environmentally friendly and renewable. Typical examples of this type of bio-nanocomposites result from the combination of polysaccharides such as starch, cellulose or polylactic acid (PLA) with microparticulated solids, which are usually called green nanocomposites or bioplastics [6,7]. Recently, special attention has been paid to strategies for synthetic approaches to bio-nanohybrids. One of these approaches is related to the preparation of bioinspired or biomimetic materials following the examples found in Nature, as for instance, bone [8], ivory [9] and nacre [10–13]. These materials show excellent structural properties due to the special arrangement at the nanometric level of their assembled components, that is biopolymers and inorganic counterparts. For instance, nacre represents a good example of a natural bio-nanocomposite, also known as native biomineral, formed by the stacking of highly oriented calcium carbonate (aragonite) platelets cemented by a fibrous protein (lustrin A). The resulting supra-architectures show exceptional mechanical properties compared to monolithic calcium carbonate [11,12]. Nowadays, bio-nanocomposites mimicking these natural materials have been prepared with the aim being to develop new biohybrids with improved mechanical properties together with biocompatibility and, in some cases, other interesting

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j 1 An Introduction to Bio-nanohybrid Materials

Fig. 1.1 Number of publications per year related to bionanohybrid materials. Data collected from the ISI Web of Knowledge [v3.0]-Web of Science. Keywords for search: (biopolymer* AND nanocomposite*) OR (natural polymer* AND nanocomposite*) OR (bio-nanohybrid*) OR (biohybrid* AND nano*).

features such as functional behavior [14–18]. In this context, the development of biohybrid systems based on biomimetic building [18,19], that is following biomineralization processes similar to those that take place in the cell wall of diatoms, where nanostructured silica nanospheres are assembled by the participation of cationic polypeptides called silaffins (SILica AFFINity) [20,21] appears to be of great significance. In relation to these natural systems, the mechanisms of interaction between colloidal silica and peptides have been particularly studied with the aim being to understand the biomineralization processes and consequently to develop, in a controlled manner, new improved synthetic bio-nanocomposites [22–24]. Inorganic solids assembled with biological species are of diverse nature with different chemical compositions, structures and textures, which determine the properties of the resulting bio-nanohybrids. In this way, single elements such as transition metals and carbon particles, metal oxides and hydroxides, silica, silicates, carbonates and phosphates, are typical inorganic components of bio-nanohybrids (Table 1.1). The affinity between the inorganic and the bio-organic counterparts, which determines the stability of the resulting bio-composites, depends on the interaction mechanisms governing the assembly processes. As indicated above, the development of bio-nanohybrids by mimicking biomineralization represents an extraordinarily useful approach. This is, for instance, the case for those bio-nanocomposites based on bone biomimetic approaches, which show excellent structural properties and biocompatibility. They are prepared by

1.1 Introduction: The Assembly of Biological Species to Inorganic Solids Tab. 1.1 Selected Examples of Bio-Nanohybrid Materials

Involving Different Types of Inorganic Solids. Inorganic moiety

Biological species

Bio-nanohybrid features

silica nanoparticles

poly-L-lysine (PLL)

biomimetic nanocomposites with controlled morphology encapsulation by sol-gel

Authors/References

Patwardhan et al. [39] siloxane networks living bacteria Fennouh et al. [62] calcium carbonate chitosan and biomimetic preparation Sugawara poly(aspartate) towards artificial nacre and Kato [88] hydroxyapatite (HAP) collagen biomimetic porous scaffolds Yokoyama for bone regeneration et al. [94] layered clay minerals chitosan functional bio-nanocomposite Darder et al. (montmorillonite) for ion-sensing applications [129] fibrous clay minerals caramel bio-nanocomposite as Gómez-Avilés (sepiolite) precursor of multifunctional et al. [153] carbon–clay nanostructured materials organoclays PLA green nanocomposites as Paul et al. [144] biodegradable bioplastics layered double deoxyribonucleic bio-nanocomposite as Choy et al. [159] hydroxides (LDHs) acid (DNA) non-viral vector for gene transfection gold nanoparticles chitosan bio-nanohybrid processable dos Santos as self-supporting films et al. [164] for biosensor applications magnetite phosphatidylcholine magnetocerasomes Burgos-Asperilla nanoparticles for targeted et al. [73] drug delivery carbon nanotubes galactose modified CNTs able to capture Gu et al. [194] (CNTs) pathogens by protein binding gelatin bio-nanocomposite thin films Ruiz et al. [220] layered perovskites with dielectric properties (CsCa2Nb3O10)

assembling hydroxyapatite (HAP), which is the main mineral constituent of bones and teeth, with biopolymers, for example collagen [25–27]. The coating of the microor nano-particulated solids with biopolymers often occurs through hydrogenbonding or metal-complexing mechanisms. In this way, the assembly of magnetic iron oxide nanoparticles (e.g., magnetite) with biopolymers (e.g., dextran) allows the preparation of magnetic bio-nanocomposites applied in NMR imaging, hyperthermia treatments or bio-carriers as drug delivery systems (DDS) [28,29]. The assembly of biopolymers with inorganic layered solids can lead to bionanocomposites in which the biopolymer becomes intercalated between the layers of the inorganic hosts [3]. The intercalation is a complex process that may simultaneously involve several mechanisms. Thus, in addition to hydrogen bonding, it has been invoked that certain biopolymers interact with the inorganic layers through

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j 1 An Introduction to Bio-nanohybrid Materials ionic bonds. This is the case for polysaccharides, proteins and nucleic acids that can act as polyelectrolytes intercalating, via ion-exchange reaction, solids provided with positively or negatively charged layers, such as layered double hydroxides (LDHs) or smectite clay minerals (see below). Microfibrous crystalline silicates such as sepiolite, similarly to amorphous silica, contain silanol groups (SiOH) covering the external mineral surface. These groups can be effectively involved in hydrogen bonding by their association to OH, NH and other polar groups belonging to the biopolymers used. Silica generated by the sol–gel method from tetraethyl orthosilicate (TEOS) in the presence of chitosan, gives biopolymer-silica nanocomposites whose morphology can be determined by the experimental conditions adopted for the preparation [30]. Chitosan and collagen can also be assembled with sepiolite to give the corresponding biopolymersepiolite nanocomposites, which exhibit good mechanical properties resulting from the combination of the fibrous inorganic substrate with the biopolymer [31–34]. The interaction mechanisms governing the formation of sepiolite-based bio-nanocomposites are mainly ascribed to hydrogen bonding, but it must be taken into account that sepiolite exhibits cationic exchange capacity (CEC 15 meq/100 g). Thus this silicate could also interact with positively charged polymers, such as chitosan, through electrostatic bonds. Although to only a minor extent, other mechanisms can be invoked, as for instance covalent bonding (grafting) between hydroxy groups on the surface of the inorganic substrates and functional groups of the biopolymers [35]. The aim of this chapter is to provide a general overview of the preparation and main characteristics of bio-nanohybrids, with emphasis on the different types of inorganic solids that can be involved in the formation of this class of materials. Special attention will be devoted to the diverse mechanisms that govern the interaction between the components of biohybrids, illustrating them with selected examples. Relevant features and potential or actual applications of recently developed bio-nanocomposites will be discussed on the basis of their structure–property relationships.

1.2 Bio-nanohybrids Based on Silica Particles and Siloxane Networks

Biominerals are produced by living organisms following a set of processes known as biomineralization, which results in a wide variety of biological materials including shells, bones, teeth, ivory and magnetic nanoparticles in magnetotactic bacteria. Biomolecules secreted by living organisms control the nucleation and growth of inorganic minerals (carbonates, phosphates, silica and iron oxide) leading to such a diversity of biological-inorganic hybrid materials, which usually exhibit a hierarchical arrangement of their components from the nanoscale to the macroscopic scale. The skeletons of diatoms and radiolarians are astonishing examples of biosilicification giving rise to amorphous hydrated SiO2 (biosilica), also formed in sponges and many higher plants [24]. As mentioned in Section 1, polycationic peptides, called silaffins, are involved in this process, controlling the assembly of silica nanoparticles to form

1.2 Bio-nanohybrids Based on Silica Particles and Siloxane Networks

Fig. 1.2 Scanning electron micrographs of (A) the silica wall of the diatom Stephanopyxis turris (reproduced from [21] by permission of WileyVCH) and (B—D) singular morphologies of silica synthesized using poly-L-lysine and pre-hydrolyzed tetramethyl orthosilicate (TMOS) under

different experimental conditions: (B) unperturbed solution, (C) flowed through a 1/800 I.D. tube and (D) stirred for 25 min. Reproduced from [39] by permission of The Royal Society of Chemistry.

these siliceous structures [20,21,36]. Similarly, silica needles in the skeleton of marine sponges involve a central filament containing silicatein, an enzyme that catalyses the synthesis of biosilica [37]. Materials scientists try to understand and reproduce these biosilicification processes taking place in nature, with the aim being to develop bioinspired or biomimetic hybrid nanostructured materials with controlled morphologies and structural properties similar to those of biosilica [24,38–42], as shown in Figure 1.2. As recently reviewed by Coradin et al. [43], proteins (collagen, gelatin, and silk) and polysaccharides (alginate, carrageenans, chitosan, as well as cellulose and its derivatives) are the main biomacromolecules involved in the synthesis of biopolymer/silica nanocomposites, while silicic acid, sodium silicate and different silicon alkoxides are employed as precursors of the silica or the polysiloxane networks assembled with the biopolymer chains. Following biomimetic processes, lysozyme and bovine serum albumin (BSA) promote the precipitation of silica particles from sodium silicate solutions, leading to entrapment of the protein [44]. Similarly, polysaccharides such as cationic and hydrophobic derivatives of cellulose also promote silica precipitation, acting as efficient templates to

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j 1 An Introduction to Bio-nanohybrid Materials develop organic–inorganic hybrid nanocomposites in combination with tetrakis(2hydroxyethyl)orthosilicate (THEOS) [45]. Chitosan is another natural polysaccharide involved in this type of silica-based hybrid material prepared by the sol–gel method. For instance, it has been assembled with siloxane networks derived from aminopropylsiloxane (APS) [46] or TEOS [30]. A similar chitosan–polysiloxane biohybrid material has been recently prepared from chitosan and 3-isocyanatopropyltriethoxysilane, where chitosan is bound to the polysiloxane network by covalent bridges. This new functional material offers photoluminiscent features and bioactive behavior, since it promotes apatite formation in simulated body fluid [47]. Chitosan–silica hybrids present as microparticulate materials showing different shapes have been prepared by the sol–gel method using TEOS or polyethoxysiloxane oligomers in the presence of the biopolymer. These materials can be used as a stationary phase in HPLC [48]. In these examples as well as in analogous materials, the interaction of the biological and the inorganic components has synergetic effects leading to hybrid materials with improved mechanical resistance, higher thermal and chemical stability and biocompatibility, and, in some cases, with functional properties. Biopolymer/silica nanocomposites are suitable for the design of membranes and coatings, drug delivery systems and also for the encapsulation of bioactive molecules such as enzymes, antibodies, yeast and plant cells or even bacteria, resulting in functional biomaterials for different biotechnological applications, including biosensors and bioreactors [43,49]. Silica-based bio-nanocomposites for drug delivery purposes have been processed as nanospheres by means of spray-drying or CO2 supercritical drying techniques. Hybrid nanoparticles based on algal polysaccharides such as alginate and carrageenan are potential carriers for the targeted delivery of drugs due to their ability to go into the intracellular space of cells and to their lack of cytotoxicity [50,51]. In other cases, silica nanoparticles serve as a support of biocide molecules and their dispersion in hydroxypropylcellulose allows the preparation of coatings and films with fungicide and pesticide activity [52]. Following a similar approach, Zhang and Dong [53] have developed functional materials based on the dispersion of Ru(bpy)32þ-doped silica nanoparticles in the biopolymer chitosan. The resulting hybrid material can be easily spread onto the surface of electrodes as a stable electroactive coating, allowing the development of chemiluminiscence sensors. Similarly to the above-mentioned entrapment of proteins by biomimetic routes, the sol–gel procedure is a useful method for the encapsulation of enzymes and other biological material due to the mild conditions required for the preparation of the silica networks [54,55]. The confinement of the enzyme in the pores of the silica matrix preserves its catalytic activity, since it prevents irreversible structural deformations in the biomolecule. The silica matrix may exert a protective effect against enzyme denaturation even under harsh conditions, as recently reported by FrenkelMullerad and Avnir [56] for physically trapped phosphatase enzymes within silica matrices (Figure 1.3). A wide number of organoalkoxy- and alkoxy-silanes have been employed for this purpose, as extensively reviewed by Gill and Ballesteros [57], and the resulting materials have been applied in the construction of optical and electrochemical biosensor devices. Optimization of the sol–gel process is required to prevent denaturation of encapsulated enzymes. Alcohol released during the

1.2 Bio-nanohybrids Based on Silica Particles and Siloxane Networks

Fig. 1.3 Schematic representation of the entrapped enzyme in a silica matrix (left side). Enzymatic activity, under extreme alkaline conditions, of acid phosphatase (A) immobilized in silica sol—gel matrices with or without CTAB, or (B) in solution. Reprinted with permission from [56]. Copyright 2005, American Chemical Society.

hydrolysis process can be harmful for the entrapped biologicals and, thus, several methods propose its removal by evaporation under vacuum [58] or the use of polyolbased silanes that generate biocompatible alcohols [59]. Catalytic activity is also preserved when silica-polysaccharide bio-nanocomposites are used as immobilization hosts. This is the case for three-dimensional hybrid matrices resulting from the combination of THEOS with xanthan, locust bean gum or a cationic derivative of hydroxyethylcellulose, which have been reported as excellent networks for the longterm immobilization of 1 ! 3-b-D-glucanase and a-D-galactosidase [60]. In addition to enzymes and antibodies, silica-based hybrid nanocomposites with a suitable porosity can successfully entrap more complex systems including yeasts, algae, lichens, plant cells and bacteria [49]. The huge volume of biological tissues, in comparison to enzymes, may hinder the polymerization processes resulting in fractures in the silica matrices. To overcome this drawback, lichen particles were embedded in a flexible network, derived from 3-(trimethoxysilyl)propyl methacrylate (MAPTS) and tetramethoxysilane (TMOS), that offers improved mechanical features (Figure 1.4A). This lichen-modified material was used to develop electrochemical sensors for the determination of heavy metal ions by anodic stripping voltammetry [61]. Similarly, algal tissue can be immobilized in sol–gel derived matrices based on TMOS and methyltrimethoxysilane (MTMOS) (Figure 1.4B).

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Fig. 1.4 Scanning electron micrographs of (A) the lichen Pseudocyphellaria hirsuta, (B) the alga Anabaena, and (D) the bacteria E. coli entrapped in sol—gel generated organopolysiloxane matrices (reprinted with permission from [65]. Copyright 2006, American Chemical Society).

(C) Transmission electron micrograph of the same bacteria, E. coli, embedded in a silica matrix containing 10 % glycerol (reproduced from [63] by permission of The Royal Society of Chemistry).

One of the first works reporting the entrapment of E. coli proposed its incorporation in a TMOS-derived silica network in which the water content was kept at about 70 wt% in order to guarantee the cells’ viability, but when silica gel was dried the bacterial activity decreased [62]. In order to overcome this drawback, the authors explored other possibilities such as the incorporation of glycerol in the silica matrix to increase bacteria viability (Figure 1.4C), leading to almost 50 % of viable bacteria after one month of ageing [63], or the addition of “quorum sensing” molecules involved in intercellular communication, which increase the cells’ viability to 100 % after one month [64]. Similar results have been achieved recently by Ferrer et al. [65], who showed that gluconolactone-bearing organopolysiloxane matrices are more efficient than pure silica in extending E. coli the cells’ viability due to their increased biocompatibility (Figure 1.4D). New materials that mimic liposomes have been recently reported as a new family of organic–inorganic hybrid compounds generated by a coupled process of sol–gel and self-assembly of long-chain containing organoalkoxysilanes [18,66–71]. These nanohybrids essentially refer to biomimetic materials derived from the assembly of a surfactant covalently bonded to a silica-based network. The name “cerasomes” was introduced by Ariga and coworkers [66] combining the terms “liposome” and “ceramic,” this last making reference to the silica network. As “ceramic” is derived

1.3 Calcium Phosphates and Carbonates in Bioinspired and Biomimetic Materials

from the Greek word keramikóz (keramikos) making reference to “inorganic nonmetallic materials whose formation is due to the action of heat” [72], the term cerasome can be confusing as they are usually formed in soft conditions. Ruiz-Hitzky suggests the use of HOILs (Hybrid Organic–Inorganic Liposomes) for this class of compounds [18]. Anyway, “cerasome” is actually the most popular term for these hybrid materials. Interestingly, the bilayers formed by the surfactant tails are able to incorporate different organophyllic species [71] making these materials potentially applicable as Drug Delivery Systems (DDS). More recently, these types of bilayers have been grafted onto magnetic nanoparticles giving rise to the so-called “magnetocerasomes” [73], which are nanohybrids simultaneously having lipophilic character and magnetic properties (see below, Section 5).

1.3 Calcium Phosphates and Carbonates in Bioinspired and Biomimetic Materials

As pointed out in Section 2, a wide number of biominerals are synthesized in nature by living organisms using organic templates. Some well-known examples include bone and ivory, where the collagen matrix controls the growth of hydroxyapatite (HAP) mineral [8,9,74], or nacre in pearls and shells, showing a brick-like structure of aragonite layers cemented by proteins [11,12]. This assortment of biological– inorganic hybrid materials, showing a hierarchical arrangement from the nano- to the macroscale, serves as a model for the development of new biomimetic and bioinspired materials. In vitro studies have demonstrated the controlled nucleation and growth of carbonates and phosphates by soluble proteins and peptides combined with insoluble polysaccharide matrices (cellulose, chitin, collagen), leading to biomimetic materials that reproduce the exceptional features of native biominerals [1,75]. Besides nacre in pearls and shells, calcium carbonate is also present in sea urchin spines, coral skeleton, eggshell, and the exoskeleton of arthropods, forming organic– inorganic hybrid structures by assembly with biomacromolecules (soluble proteins and insoluble matrices) [76–78]. Calcite and aragonite are the calcium carbonate polymorphs that constitute the biominerals found in nature, since they show a higher stability than vaterite. However, in vitro studies have confirmed that the presence of functionalised macromolecules as soluble proteins and insoluble matrices have a considerable effect on calcium carbonate crystallization, allowing the formation of the less stable polymorphs and even of amorphous CaCO3 [79,80]. Regarding soluble matrices, living organisms secrete biomacromolecules with a high content of glutamic and aspartic acids, bearing carboxyl groups that can interact with calcium ions. A similar effect has been found using polymers provided with sulfonic, hydroxy and even ether groups. Many of these studies have been carried out using the same biopolymers that act as insoluble matrices for CaCO3 crystallization in nature, such as collagen and chitin [79,81]. Calcium carbonate polymorphs are also formed on other natural and synthetic polymers including elastin that controls the formation of calcite [82], poly(ethylene glycol) that forces the selective formation of aragonite [83], poly(a-L-aspartate) that promotes vaterite formation with a helical morphology [84], or

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Fig. 1.5 SEM micrographs of (A) donut-shaped CaCO3 crystals grown on polyacrylic acid grafted chitosan, (B) CaCO3 hollow helix, fractured by micro-manipulation, formed on poly(a-L-aspartate), and (C) double layered aragonite thin films grown on a chitosan matrix in the presence of

poly(aspartate) and MgCl2 by alternate spin coating and crystallization. (A) and (B) adapted from [84] and [85] with permission from Elsevier, and (C) from [88] (reproduced by permission of The Royal Society of Chemistry).

poly(acrylate)-grafted chitosan giving rise to CaCO3 crystals of unusual morphology [85] similar to those created using poly(N-isopropylacrylamide-co-(4-vinylpyridine)) as the platform for mineralization [86]. As confirmed by these reports, materials scientists are able to produce calcium carbonate organic hybrid materials with defined morphologies (Figure 1.5) by tuning the polymers and biomacromolecules that control the nucleation and growth of calcium carbonate crystals. Many studies are currently centered on the crystallization of CaCO3 as thin films, as illustrated in Figure 1.5C, trying to mimic the nacre of shells [87–89]. The reason is that mollusk shells, where the organic matrix constitutes only 1 % of the total weight, present a fracture toughness about 3000 times higher than that of pristine calcium carbonate and offer a good example of ultra-lightweight hybrid materials with exceptional mechanical strength and interesting optical properties. Calcium phosphate minerals are present in living organisms as the most important constituents of biological hard tissues (bones, teeth, tendons, and cartilage) to provide them with stability, hardness and function [74,90]. Among the different calcium phosphates, hydroxyapatite (HAP), with a chemical composition Ca10(PO4)6(OH)2 and Ca/P ratio of 1.67, is the most widely studied due to its huge incidence in the field of regenerative medicine. Bone can be regarded as a natural nanocomposite containing HAP nanocrystallites in a collagen-rich matrix also enclosing non-collagenous proteins. Besides providing structural support, bone serves as a reservoir of calcium and phosphate ions involved in numerous metabolic functions. Due to the significant role of bone in humans, most of the synthetic hybrid materials that mimic its structure and composition are currently devoted to biomedical applications for regeneration of injured bone and this fact has led to a wide number of scientific publications on this topic in the recent years. Among synthetic materials for bone grafting, nanocomposites are replacing metals, alloys, ceramics, polymers, and composites, due to their advantageous properties: large surface area, high surface reactivity, relatively strong interfacial bonding, flexibility, and enhanced mechanical consistency. It has been proved that nanocrystalline HAP offers better results than microcrystalline HAP with respect to osteoblast cells adhesion, differentiation and proliferation, as well as