Materials for biomedical engineering: bioactive materials for antimicrobial, anticancer, and gene therapy 9780128184356, 0128184353

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Inorganic Micro- and Nanostructures

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Materials for Biomedical Engineering

Inorganic Micro- and Nanostructures

Edited by

Valentina Grumezescu Lasers Department, National Institute for Laser Plasma & Radiation Physics, Romania

Alexandru Mihai Grumezescu Faculty of Applied Chemistry and Materials Science, University Politehnica of Bucharest, Bucharest, Romania

Elsevier Radarweg 29, PO Box 211, 1000 AE Amsterdam, Netherlands The Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, United Kingdom 50 Hampshire Street, 5th Floor, Cambridge, MA 02139, United States Copyright © 2019 Elsevier Inc. All rights reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Details on how to seek permission, further information about the Publisher’s permissions policies and our arrangements with organizations such as the Copyright Clearance Center and the Copyright Licensing Agency, can be found at our website: www.elsevier.com/permissions. This book and the individual contributions contained in it are protected under copyright by the Publisher (other than as may be noted herein). Notices Knowledge and best practice in this field are constantly changing. As new research and experience broaden our understanding, changes in research methods, professional practices, or medical treatment may become necessary. Practitioners and researchers must always rely on their own experience and knowledge in evaluating and using any information, methods, compounds, or experiments described herein. In using such information or methods they should be mindful of their own safety and the safety of others, including parties for whom they have a professional responsibility. To the fullest extent of the law, neither the Publisher nor the authors, contributors, or editors, assume any liability for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions, or ideas contained in the material herein. British Library Cataloguing-in-Publication Data A catalogue record for this book is available from the British Library Library of Congress Cataloging-in-Publication Data A catalog record for this book is available from the Library of Congress ISBN: 978-0-08-102814-8 For Information on all Elsevier publications visit our website at https://www.elsevier.com/books-and-journals

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Contents List of Contributors .................................................................................................xv Series Preface .........................................................................................................xix Preface ....................................................................................................................xxi

CHAPTER 1 Biomedical inorganic nanoparticles: preparation, properties, and perspectives........................................ 1 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9

´ Miodrag J. Lukic, ´ Ana Stankovic, ´ Magdalena Stevanovic, ˇ ´ Maja Kuzmanovic´ and Zeljko ´ Nenad Filipovic, Janicijevi c´ Introduction ....................................................................................1 Gold Nanoparticles.........................................................................2 Silver Nanoparticles .......................................................................5 Selenium Nanoparticles................................................................10 Copper Nanoparticles...................................................................11 Iron Nanoparticles ........................................................................17 Zinc Oxide Nanoparticles ............................................................19 Hydroxyapatite Nanoparticles......................................................24 Conclusions ..................................................................................29 Acknowledgments ....................................................................... 29 References.................................................................................... 29 Further Reading ........................................................................... 45

CHAPTER 2 Inorganic composites in biomedical engineering..... 47

2.1 2.2 2.3

2.4

2.5 2.6

Murthy Chavali, Periasamy Palanisamy, Maria P. Nikolova, Ren-Jang Wu, Ravisankar Tadiboyina and P.T.S.R.K. Prasada Rao Introduction and Background.......................................................47 Categorization ..............................................................................52 Components ..................................................................................53 2.3.1 Matrices............................................................................. 53 2.3.2 Fibers................................................................................. 54 2.3.3 Particles ............................................................................. 56 2.3.4 Interface............................................................................. 57 Preparation of Composites ...........................................................57 2.4.1 Composites Based on Polymer Matrix ............................. 57 2.4.2 Composites Based on Ceramic Matrix............................. 58 Properties of Composites .............................................................59 Anomalies.....................................................................................62 2.6.1 Fracture and Fatigue Failure............................................. 62

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2.7 Biological Response.....................................................................65 2.8 Applications in Biomedical Engineering.....................................66 2.8.1 Dentistry ............................................................................ 66 2.8.2 Prosthetics and Orthotics .................................................. 68 2.8.3 Tissue Engineering............................................................ 69 2.8.4 Orthopedic......................................................................... 70 2.9 Conclusions ..................................................................................71 References.................................................................................... 72 Further Reading ........................................................................... 78

CHAPTER 3 Structural interpretation, microstructure characterization, mechanical properties, and cytocompatibility study of pure and doped carbonated nanocrystalline hydroxyapatites synthesized by mechanical alloying.......................... 81 Sushovan Lala and Swapan Kumar Pradhan 3.1 Introduction ..................................................................................81 3.1.1 Carbonation in Biological Apatites .................................. 83 3.1.2 Importance of Zn, Mn, and Mg as Trace Elements Present in Bone ................................................. 83 3.2 Materials and Methods.................................................................84 3.2.1 Mechanical Alloying......................................................... 84 3.2.2 Sample Preparation by MA .............................................. 84 3.2.3 Spark Plasma Sintering ..................................................... 85 3.2.4 Sample Characterization ................................................... 86 3.2.5 Biological Studies ............................................................. 86 3.2.6 Method of Analysis........................................................... 87 3.3 Results and Discussions ...............................................................90 3.3.1 Phase Confirmation of Unsintered HAp Samples From XRD Patterns........................................................... 90 3.3.2 Confirmation of Carbonation in HAp by FTIR Analysis ............................................................................. 90 3.3.3 Quantitative Phase Estimation of Unsintered Samples Using Rietveld’s Method ................................... 92 3.3.4 Modification in HAp Structure due to Mn/Mg/Zn Substitution........................................................................ 94 3.3.5 HRTEM Analysis............................................................ 100 3.3.6 Microstructure Characterizations of the Spark Plasma Sintered Samples ................................................ 103 3.3.7 Mechanical Properties of the Sintered HAp Samples ........................................................................... 107

Contents

3.3.8 Cytocompatibility Test.................................................... 111 3.4 Conclusions ................................................................................113 References.................................................................................. 113

CHAPTER 4 Multiparticle composites based on nanostructurized arsenic sulfides As4S4 in biomedical engineering............................................ 119 4.1 4.2

4.3

4.4

4.5

ˇ ´kova´, Peter Bala´z, ˇ Oleh Shpotyuk, Zdenka Bujna Yaroslav Shpotyuk and Adam Ingram Introduction ................................................................................119 As4S4/ZnS NC Preparation Procedure.......................................120 4.2.1 Mechanochemical Synthesis of As4S4/ZnS NCs in a Dry-Milling Mode ........................................... 121 4.2.2 Mechanochemical Synthesis of As4S4/ZnS-PX407 NSs in a Wet-Milling Mode ........................................... 121 As4S4/ZnS NC Characterization Methodology .........................121 4.3.1 Atomic-Relevant Structure ............................................. 121 4.3.2 Atomic-Deficient Structure............................................. 123 4.3.3 Biological Activity.......................................................... 126 NP-Guided Functionality in As4S4/ZnS NCs ............................127 4.4.1 Characterization of As4S4/ZnS NCs Prepared in a Dry-Milling Mode ................................................... 127 4.4.2 Atomic-Deficient Structure of As4S4/ZnS NCs ............. 130 4.4.3 Characterization of As4S4/ZnS-PX407 NSs Prepared in a Wet-Milling Mode.................................... 140 4.4.4 Biological Activity of As4S4/ZnS NPs........................... 142 Conclusions ................................................................................147 References.................................................................................. 148

CHAPTER 5 Quaternary ammonium compound derivatives for biomedical applications ..................................... 153 Mari Miura Sugii, Fa´bio Augusto de Souza Ferreira, Karina Cogo Mu¨ller, Ubirajara Pereira Rodrigues Filho and Fla´vio Henrique Baggio Aguiar 5.1 Background.................................................................................153 5.2 Biofilm Treatment and Prevention ............................................154 5.3 Quaternary Ammonium Compounds and Their Chemistry ......155 5.3.1 Cationic Acrylates and Cationic Silanes ........................ 156 5.3.2 Quaternary Ammonium Compound Disinfectants and Preservatives............................................................. 161

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5.4 5.5 5.6 5.7

5.3.3 In Situ Quaternization of Tertiary Amines to Form Quaternary Ammonium Compounds and Nanoparticle Functionalization....................................... 162 Variables Influencing the Antimicrobial Properties of Quaternary Ammonium Compound ......................................164 Cytotoxicity ................................................................................167 Antimicrobial Resistance ...........................................................168 Remarks ......................................................................................168 References.................................................................................. 169

CHAPTER 6 Block copolymer micelles as nanoreactors for the synthesis of gold nanoparticles ................... 177 Rajpreet Kaur and Poonam Khullar 6.1 Introduction ................................................................................177 6.1.1 Poloxamers and Poloxamines ......................................... 178 6.1.2 Micelle Architecture and Mixed Micelles...................... 181 6.1.3 Synthesis of Various Morphologies of Gold Nanoparticles................................................................... 183 6.1.4 Bimetallic Nanoparticles................................................. 189 6.1.5 Comparison of Poloxamers and Poloxamines................ 190 6.2 Biomedical Applications ............................................................195 6.3 Study Results..............................................................................199 6.4 Future Perspectives ....................................................................202 References.................................................................................. 203 Further Reading ......................................................................... 209

CHAPTER 7 Nanoparticles: synthesis and applications ............. 211 Nguyen Hoang Nam and Nguyen Hoang Luong 7.1 Introduction ................................................................................211 7.2 Synthesis of Nanoparticles.........................................................212 7.2.1 Chemical Reduction........................................................ 212 7.2.2 Coprecipitation ................................................................ 212 7.2.3 Seeding ............................................................................ 213 7.2.4 Microemulsion and Inverse Microemulsion................... 213 7.2.5 Hydrothermal Method..................................................... 213 7.2.6 Sonoelectrodeposition ..................................................... 214 7.3 Functionalization/Coating of Nanoparticles ..............................214 7.3.1 Functionalization of Nanoparticles................................. 214 7.3.2 Silica Coating of Magnetic Nanoparticles ..................... 215 7.3.3 Multifunctional Nanoparticles ........................................ 215 7.4 Applications................................................................................218

Contents

7.4.1 Application of Gold Nanoparticles for Breast Cancer Cell Detection..................................................... 218 7.4.2 Basal Cell Carcinoma Fingerprinted Detection ............. 219 7.4.3 Antibacterial Test Using Silver Nanoparticles............... 222 7.4.4 Magnetic Nanoparticles .................................................. 223 7.4.5 Applications of Multifunctional Nanoparticles .............. 230 7.5 Conclusion and Perspectives......................................................232 Acknowledgment ....................................................................... 233 References.................................................................................. 233

CHAPTER 8 Multimodal magnetic nanoparticles for biomedical applications: importance of characterization on biomimetic in vitro models ..... 241 8.1 8.2

8.3

8.4

ˇ Zupanci ˇ c, ˇ Jasna Lojk, Mojca Pavlin, Dasa Klemen Strojan and Mateja Erdani Kreft Introduction ................................................................................241 Characterization of Multimodal Magnetic Nanoparticles .........242 8.2.1 Properties of Magnetic Nanoparticles ............................ 242 8.2.2 Magnetic Nanoparticle Properties Change in Physiological Fluids.................................................... 244 8.2.3 Methods for Characterization of Physicochemical Properties of Magnetic Nanoparticles ............................ 245 8.2.4 Characterization of Magnetic Nanoparticle Mobility in 3D Gels and in the Artificial Extracellular Matrix ........................................................ 246 Current Biomedical Applications of Multimodal Magnetic Nanoparticles..............................................................247 8.3.1 Molecular Isolation and Magnetic Separation ............... 248 8.3.2 Magnetic Nanoparticles as Delivery Vectors................. 248 8.3.3 Cell Labeling................................................................... 249 8.3.4 Magnetic Nanoparticles as Contrast Agents for Magnetic Resonance ................................................. 249 8.3.5 Magnetofection ............................................................... 250 8.3.6 Magnetic Fluid Hyperthermia......................................... 251 8.3.7 Perspectives of Magnetic Nanoparticle Biomedical Applications................................................. 251 Endocytosis and Intracellular Fate of Multimodal Magnetic Nanoparticles..............................................................251 8.4.1 Different Endocytic Pathways ........................................ 252 8.4.2 Uptake Pathway Depends Mainly on the Properties of Nanoparticles and the Cell Type .............. 253

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8.4.3 The Intracellular Trafficking and Fate of Internalized Nanoparticles .............................................. 255 8.4.4 Endocytosis of Magnetic Nanoparticles Is an Essential Step for Most Biomedical Applications ......... 257 8.5 In Vivo and In Vitro Models (Classical Cell Cultures, Biomimetic) for Testing Nanoparticle Toxicity and Their Penetration Through Cell Plasma Membranes and Tissue Barriers .......................................................................................258 8.5.1 The Comparison of In Vivo and In Vitro Models for the Research Into Magnetic Nanoparticle Effects.... 258 8.5.2 The Routes and Model Organisms of Magnetic Nanoparticle Administration........................................... 260 8.5.3 Biomimetic In Vitro Models Represent the Bridge Between In Vitro and In Vivo Research ........................ 262 8.6 Advantages, Perspectives, and Limitations of Biomimetic In Vitro Models Versus Classical Cell Cultures .......................263 8.6.1 Skin Models .................................................................... 263 8.6.2 Lung Models ................................................................... 264 8.6.3 Gastrointestinal Tract Models......................................... 265 8.6.4 Placenta Models .............................................................. 266 8.6.5 Urothelium/Urinary Bladder Models.............................. 266 8.6.6 Perspectives of Biomimetic In Vitro Models................. 267 8.7 Conclusions ................................................................................269 Acknowledgments ..................................................................... 269 References.................................................................................. 269

CHAPTER 9 Aluminosilicate-based composites functionalized with cationic materials: possibilities for drug-delivery applications............. 285 9.1 9.2 9.3

9.4

ˇ ˇ Danina Krajisnik, Bojan Calija and Jela Milic´ Introduction ................................................................................285 Aluminosilicates as Drug Carriers—Properties, Advantages, and Limitations......................................................286 Aluminosilicate-Based Drug Carriers Functionalized With Cationic Surfactants ..........................................................288 9.3.1 Cationic Surfactants—Properties and Pharmaceutical Applications .......................................... 288 9.3.2 Preparation and Characterization of Surfactant-Modified Aluminosilicates............................ 294 9.3.3 Functionality of Surfactant-Modified Aluminosilicates as Drug Carriers.................................. 298 Chitosan-Functionalized Aluminosilicates as Drug Carriers..............................................................................303

Contents

9.4.1 Chitosan—A Versatile Biopolymer................................ 303 9.4.2 Preparation and Characterization of Chitosan-Modified Aluminosilicates .............................. 307 9.4.3 Functionality of Chitosan
Aluminosilicate Composites as Drug Carriers .......................................... 310 9.5 Conclusions ................................................................................316 Acknowledgment ....................................................................... 316 References.................................................................................. 316

CHAPTER 10 Bioactive glass nanofibers for tissue engineering ............................................................... 329

10.1

10.2 10.3

10.4 10.5

10.6

Joaquı´n Penide, Fe´lix Quintero, Jesu´s del Val, Rafael Comesan˜a, Fernando Lusquin˜os, Antonio Riveiro and Juan Pou Introduction ................................................................................329 10.1.1 Definition of Nanofiber ................................................ 329 10.1.2 Interest in Bioactive Glass Nanofibers in Tissue Engineering (Scaffolds and Composites) ..................... 330 Conventional Methods to Produce Glass Microfibers...............332 Methods to Produce Glass Nanofibers ......................................335 10.3.1 Bottom-Up Methods ..................................................... 335 10.3.2 Top-Down Methods ...................................................... 336 Bioactive Glass Fibers for Tissue Engineering and Composites .................................................................................338 Production of Glass Nanofibers by Laser Spinning Technique ...................................................................................342 10.5.1 Bioactive Glass Nanofibers for Tissue Engineering and Composites ........................................ 348 Summary and Outlook ...............................................................351 Acknowledgment ....................................................................... 351 References.................................................................................. 351

CHAPTER 11 Application of (mixed) metal oxides-based nanocomposites for biosensors ............................... 357 Ali Salehabadi and Morteza Enhessari 11.1 Introduction ................................................................................357 11.1.1 Semiconducting (Nano)Materials ................................. 358 11.1.2 Polymers........................................................................ 365 11.1.3 Nanocomposites/Particles ............................................. 365 11.2 Sensors and Biosensors ..............................................................367 11.2.1 Sensing Measurement ................................................... 367 11.3 Application of Sensors ...............................................................368

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11.3.1 Gas (Bio)Sensors........................................................... 368 11.3.2 Chemical (Bio)Sensors ................................................. 374 11.3.3 Environment Biosensors ............................................... 376 11.3.4 Biological Sensors......................................................... 379 11.3.5 Clinical Biosensors ....................................................... 382 11.4 Fabrication..................................................................................387 11.5 Selectivity, Sensitivity, and Time Factors .................................389 11.6 Summary and Recommendations for Future Work...................390 References.................................................................................. 390 Further Reading ......................................................................... 396

CHAPTER 12 Metal nanoparticles and their composites: a promising multifunctional nanomaterial for biomedical and related applications ....................... 397 12.1 12.2 12.3

12.4

12.5

Vesna V. Vodnik and Una Bogdanovic´ Introduction ................................................................................397 Some Interesting Properties of the Metals on the Nanometer Length Scale.................................................399 Nanoparticle Synthesis and Functionalization...........................402 12.3.1 Synthesis Approaches to Metal Nanoparticles............. 402 12.3.2 Functionalization of Metal Nanoparticles: Manipulation of Nanoparticles Properties.................... 403 Applications of Metal Nanoparticles and Their Polymer-Based Nanocomposites................................................406 12.4.1 Medical Applications .................................................... 407 12.4.2 Applications in Biology ................................................ 410 Conclusions and Outlook ...........................................................416 Acknowledgments ..................................................................... 418 References.................................................................................. 418

CHAPTER 13 Hybrid metal complex nanocomposites for targeted cancer diagnosis and therapeutics .............................................................. 427 13.1 13.2 13.3 13.4

Jeong-Hwan Kim, Haruki Eguchi, Masanari Umemura and Yoshihiro Ishikawa Introduction ................................................................................427 Conventional Chemotherapy......................................................429 Striving Toward Targeted Chemotherapy .................................431 Metal
Ligand Complexes as a Composite Anticancer Drug .........................................................................431 13.4.1 Iron Complexes ............................................................. 432

Contents

13.4.2 Quantitative Structure
Function Relationship of Iron-Salen Complexes .............................................. 432 13.4.3 Magnetic Nanoparticles (MNPs) as an Essential Carrier for Magnetic DDS ............................ 433 13.4.4 Molecular Magnetic Iron Complex for Magneto-DDS ......................................................... 438 13.5 Hybrid Metal Salen
Polymer Nanocomposites as Nano-DDS ..................................................................................452 13.6 Conclusion ..................................................................................454 References.................................................................................. 455

CHAPTER 14 Nanocoatings and thin films .................................... 463 Valentina Grumezescu and Irina Negut 14.1 Introduction ................................................................................463 14.2 Nanocoating Fabrication Methods .............................................464 14.2.1 Dip-Coating Method ..................................................... 464 14.2.2 Matrix-Assisted Pulsed Laser Evaporation Method...................................................... 468 14.3 Conclusion ..................................................................................473 References.................................................................................. 473 Index ......................................................................................................................479

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List of Contributors Fla´vio Henrique Baggio Aguiar University Medical Center Groningen, Biomedical Engineering Department, Groningen, Netherlands Peter Bala´zˇ ˇ Institute of Geotechnics, Slovak Academy of Sciences, Kosice, Slovakia Una Bogdanovic´ ˇ Institute of Department of Radiation Chemistry and Physics “GAMMA”, Vinca Nuclear Sciences, University of Belgrade, Belgrade, Serbia ˇ ´ kova´ Zdenka Bujna ˇ Institute of Geotechnics, Slovak Academy of Sciences, Kosice, Slovakia ˇ Bojan Calija Department of Pharmaceutical Technology and Cosmetology, University of Belgrade-Faculty of Pharmacy, Belgrade, Serbia Murthy Chavali Shree Velagapudi Ramakrishna Memorial College (SVRMC-PG StudiesAutonomous), Andhra Pradesh, India; MCETRC, Tenali, Andhra Pradesh, India Rafael Comesan˜a Materials Engineering, Applied Mechanics and Construction Department, EEI, University of Vigo, Vigo, Spain Jesu´s del Val Applied Physics Department, EEI, University of Vigo, Vigo, Spain Haruki Eguchi Advanced Applied Science Department, Research Laboratory, IHI Corporation, Yokohama, Japan Morteza Enhessari Department of Chemistry, Naragh Branch, Islamic Azad University, Naragh, I. R. Iran Fa´bio Augusto de Souza Ferreira SENAI Institute for Inovation in Surface Engineering
Campus CETEC, Belo Horizonte, MG, Brazil Ubirajara Pereira Rodrigues Filho Institute of Chemistry of Sa˜o Carlos, Sa˜o Paulo University, Department of Molecular Physics and Chemistry, Sa˜o Carlos, SP, Brazil Nenad Filipovic´ Institute of Technical Sciences of the Serbian Academy of Sciences and Arts, Belgrade, Serbia

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Valentina Grumezescu National Institute for Lasers, Plasma, and Radiation Physics, Magurele, Romania Adam Ingram Opole University of Technology, Opole, Poland Yoshihiro Ishikawa Cardiovascular Research Institute, Yokohama City University Graduate School of Medicine, Yokohama, Japan ˇ ´ Zeljko Janicijevi c´ School of Electrical Engineering, University of Belgrade, Belgrade, Serbia Rajpreet Kaur Department of Chemistry, B.B.K. D.A.V. College for Women, Amritsar, India Poonam Khullar Department of Chemistry, B.B.K. D.A.V. College for Women, Amritsar, India Jeong-Hwan Kim RadianQbio Co. Ltd., Halla Sigma Valley, Gasan Digital, Geumcheon-gu, Seoul, Republic of Korea; Cardiovascular Research Institute, Yokohama City University Graduate School of Medicine, Yokohama, Japan Danina Krajiˇsnik Department of Pharmaceutical Technology and Cosmetology, University of Belgrade-Faculty of Pharmacy, Belgrade, Serbia Mateja Erdani Kreft Faculty of Medicine, Institute of Cell Biology, Centre for Electron Microscopy, Laboratory for Cell and Tissue Cultures, University of Ljubljana, Ljubljana, Slovenia Maja Kuzmanovic´ Institute of Technical Sciences of the Serbian Academy of Sciences and Arts, Belgrade, Serbia Sushovan Lala Materials Science Division, Department of Physics, The University of Burdwan, Burdwan, India Jasna Lojk Faculty of Electrical Engineering, Group for Nano and Biotechnological Applications, University of Ljubljana, Ljubljana, Slovenia; Faculty of Medicine, Institute of Cell Biology, Centre for Electron Microscopy, Laboratory for Cell and Tissue Cultures, University of Ljubljana, Ljubljana, Slovenia Miodrag J. Lukic´ Institute of Technical Sciences of the Serbian Academy of Sciences and Arts, Belgrade, Serbia

List of Contributors

Nguyen Hoang Luong Nano and Energy Center, Hanoi University of Science, Vietnam National University, Hanoi, Hanoi, Vietnam Fernando Lusquin˜os Applied Physics Department, EEI, University of Vigo, Vigo, Spain Jela Milic´ Department of Pharmaceutical Technology and Cosmetology, University of Belgrade-Faculty of Pharmacy, Belgrade, Serbia Karina Cogo Mu¨ller Faculty of Pharmaceutical Sciences, University of Campinas, Rua Se´rgio Buarque de Holanda, Campinas, SP, Brazil Nguyen Hoang Nam Faculty of Physics, Hanoi University of Science, Vietnam National University, Hanoi, Hanoi, Vietnam; Nano and Energy Center, Hanoi University of Science, Vietnam National University, Hanoi, Hanoi, Vietnam Irina Negut National Institute for Lasers, Plasma, and Radiation Physics, Magurele, Romania; Faculty of Physics, University of Bucharest, Magurele, Romania Maria P. Nikolova Department of Material Science and Technology, University of Ruse “Angel Kanchev”, Ruse, Bulgaria Periasamy Palanisamy Department of Physics, Gnanamani College of Engineering, Namakkal, India Mojca Pavlin Faculty of Electrical Engineering, Group for Nano and Biotechnological Applications, University of Ljubljana, Ljubljana, Slovenia; Faculty of Medicine, Institute of Biophysics, University of Ljubljana, Ljubljana, Slovenia Joaquı´n Penide Applied Physics Department, EEI, University of Vigo, Vigo, Spain Juan Pou Applied Physics Department, EEI, University of Vigo, Vigo, Spain Swapan Kumar Pradhan Materials Science Division, Department of Physics, The University of Burdwan, Burdwan, India P.T.S.R.K. Prasada Rao Department of Chemistry, P B Siddhartha College of Arts & Science, Vijayawada, India Fe´lix Quintero Applied Physics Department, EEI, University of Vigo, Vigo, Spain

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Antonio Riveiro Applied Physics Department, EEI, University of Vigo, Vigo, Spain Ali Salehabadi Environmental Technology Division, School of Industrial Technology, Universiti Sains Malaysia, 11800 Minden, Penang, Malaysia Oleh Shpotyuk Vlokh Institute of Physical Optics, Lviv, Ukraine; Jan Dlugosz University in Czestochowa, Czestochowa, Poland Yaroslav Shpotyuk Faculty of Mathematics and Natural Sciences, University of Rzeszow, Rzeszow, Poland; Ivan Franko National University of Lviv, Department of Sensor and Semiconductor Electronics, Lviv, Ukraine Ana Stankovic´ Institute of Technical Sciences of the Serbian Academy of Sciences and Arts, Belgrade, Serbia Magdalena Stevanovic´ Institute of Technical Sciences of the Serbian Academy of Sciences and Arts, Belgrade, Serbia Klemen Strojan Faculty of Electrical Engineering, Group for Nano and Biotechnological Applications, University of Ljubljana, Ljubljana, Slovenia Mari Miura Sugii University Medical Center Groningen, Biomedical Engineering Department, Groningen, Netherlands; Piracicaba Dental School, University of Campinas, Department of Restorative Dentistry, Piracicaba, SP, Brazil Ravisankar Tadiboyina Aakash Educational Services Ltd. (Anna Nagar Branch), Chennai, India Masanari Umemura Cardiovascular Research Institute, Yokohama City University Graduate School of Medicine, Yokohama, Japan Vesna V. Vodnik ˇ Institute of Department of Radiation Chemistry and Physics “GAMMA”, Vinca Nuclear Sciences, University of Belgrade, Belgrade, Serbia Ren-Jang Wu Department of Applied Chemistry, College of Science, Providence University, Taichung City, Taiwan ˇ cˇ Daˇsa Zupanci Faculty of Medicine, Institute of Cell Biology, Centre for Electron Microscopy, Laboratory for Cell and Tissue Cultures, University of Ljubljana, Ljubljana, Slovenia

Series Preface In the past few decades there has been growing interest in the design and implementation of advanced materials for new biomedical applications. The development of these materials has been facilitated by multiple factors, especially the introduction of new engineering tools and technologies, emerging biomedical needs, and socioeconomic considerations. Bioengineering is an interdisciplinary field encompassing contributions from biology, medicine, chemistry, and materials science. In this context, new materials have been developed or reinvented to fulfill the need for modern and improved engineered biodevices. A multivolume series, Materials for Biomedical Engineering highlights the most relevant findings and discusses key topics in this impressive research field. Volume 1. Bioactive Materials: Properties and Applications, offers an introduction to bioactive materials, discussing the main properties, applications, and perspectives of materials with medical applications. This volume reviews recently developed materials, highlighting their impact in tissue engineering and the detection, therapy, and prophylaxis of various diseases. Volume 2. Thermoset and Thermoplastic Polymers, analyzes the main applications of advanced functional polymers in the biomedical field. In recent years there has been a revolution in thermoplastic and thermosetting polymers with medical and biological uses, which are currently being developed for medical devices, drug delivery, tailored textiles, packaging, and tissue engineering. Volume 3. Absorbable Polymers, describes the main types of polymers of different compositions with bioabsorbable and biodegradable properties. The biomedical applications of such materials are reviewed and the most innovative findings are presented in this volume. Volume 4. Biopolymer Fibers, highlights the applications of polymeric fibers of natural biological origin in biomedical engineering. Such materials are of great utility in tissue engineering and biodegradable textiles. Volume 5. Inorganic Micro- and Nanostructures and Volume 6. Organic Micro- and Nanostructures, deal, respectively, with the preparation and properties of inorganic and organic nanostructured materials with biomedical applications. Volume 7. Hydrogels and Polymer-Based Scaffolds, discusses the recent progress made in the field of polymeric materials designed as scaffolds and tools for tissue engineering. The technological challenges and advances in their production, as well as current applications in the production of scaffolds and devices for regenerative medicine are presented. Volume 8. Bioactive Materials for Antimicrobial, Anticancer, and Gene Therapy, offers an updated perspective regarding new bioactive materials with potential in the therapy of severe diseases such as infections, cancer, and genetic disorders.

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Volume 9. Nanobiomaterials in Tissue Engineering, provides valuable examples of recently designed nanomaterials with powerful applications in tissue engineering and artificial organ approaches. Volume 10. Nanomaterials-Based Drug Delivery, discusses the most investigated types of nanoparticles and nanoengineered materials with an impact in drug delivery. Applications for drug-therapy, and examples of such nanoscale systems are included in this volume. This series was motivated by the need to offer a scientifically solid basis for the new findings and approaches relevant to the biomedical engineering field. This scientific resource collects new information on the preparation and analysis tools of diverse materials with biomedical applications, while also offering innovative examples of their medical uses for diagnoses and therapies of diseases. The series will be of particular interest for material scientists, engineers, researchers working in the biomedical field, clinicians, and also innovative and established pharmaceutical companies interested in the latest progress made in the field of biomaterials. Michael R. Hamblin1 and Ioannis L. Liakos2 1

Harvard Medical School, Boston, MA, United States 2 Istituto Italiano di Tecnologia, Genoa, Italy

Preface Nanometric particles are very promising candidates for various biomedical applications. This is due to their remarkable features, which originate from their nanoscale size and unique physicochemical properties. This book aims to offer an updated collection of chapters dealing with the current situation and future trends in inorganic micro- and nanostructures. The preparation, characterization, and newest applications of such materials are discussed in great detail here. This volume is an international collection, containing 14 chapters prepared by outstanding authors, as follow. Chapter 1, Biomedical inorganic nanoparticles: preparation, properties, and perspectives, by Magdalena Stevanovi´c et al., reports on the preparation methods of different metallic and ceramic inorganic nanoparticles such as gold, silver, selenium, copper, iron, zinc oxide, and hydroxyapatite for biomedical applications. For each of these nanosystems, the main challenges regarding the currently achieved functional properties and further perspectives are also presented. Chapter 2, Inorganic composites in biomedical engineering, by Murthy Chavali et al., highlights recent developments and improvements in inorganic composite materials and their applications in biomedical engineering. Different forms and varieties of inorganic composite materials have different applications in biomedical engineering. These composite materials have high functionality, high biocompatibility, high electro-active surface area, and multiple attachment charged sites, which make them highly effective for biomedical applications in the areas of medicine and health care including for diagnostic and therapeutic purposes. Chapter 3, Structural interpretation, microstructure characterization, mechanical properties, and cytocompatibility study of pure and doped carbonated nanocrystalline hydroxyapatites synthesized by mechanical alloying, by Sushovan Lala and Swapan Kumar Pradhan, gives a comprehensive revision of recent nanocrystalline biocompatible undoped and Mn-, Mg-, and Zn-doped carbonated hydroxyapatite (HAp) powders which have been synthesized via mechanical alloying. Despite good biocompatibility, bioactivity, and osteoconductivity, the weak mechanical properties of HAp, such as high brittleness and low fracture toughness, restrict its applications in nonload-bearing or metallic implant surface coatings. Different techniques have been implemented to improve the mechanical properties of HAp including the addition of dopants, making composites, and controlling microstructures via different novel sintering methods such as hot pressing, post-hot isostatic pressing, and microwave sintering. Chapter 4, Multiparticle composites based on nanostructurized arsenic sulfides As4S4 in biomedical engineering, by Oleh Shpotyuk et al., discusses compositionally dependent nanoformulation, which is represented as common stabilization of coarse-grained As4S4 and fine-grained ZnS nanoparticles accompanied by positron-to-positronium trapping conversion. The anticancer effect on A375 and

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Bowes melanoma cell lines clearly confirms the improved biomedical efficacy of the newly developed As4S4-ZnS nanosystem. Chapter 5, Quaternary ammonium compound derivatives for biomedical applications, by Mari Miura Sugii et al., focuses on modifications promoted on the surface by organosilane compounds, containing quaternary ammonium as the functional group. This organofunctional silane was synthesized by hydrolysis and condensation reactions and could be applied to the surface of glass and resinous composites for dental applications. Chapter 6, Block copolymer micelles as nanoreactors for the synthesis of gold nanoparticles, by Rajpreet Kaur and Poonam Khullar, focuses on the applications and uses of generally nontoxic and environmentally friendly pluronics and tetronics for the synthesis, characterization, and applications of nanomaterials. Their micellar assemblies play an important role by changing the micellar environment. Their thermoresponsive nature makes them suitable for various biomedical applications. Chapter 7, Nanoparticles: synthesis and applications, by Nguyen Hoang Nam and Nguyen Hoang Luong, focuses on the synthesis, functionalization, and applications of metallic, semiconductor, magnetic, and multifunctional nanoparticles. Several applications of these nanoparticles in life sciences and the environment are discussed in this chapter. Chapter 8, Multimodal magnetic nanoparticles for biomedical applications: importance of characterization on biomimetic in vitro models, by Mojca Pavlin et al., discusses the advantages and limitations of biomimetic in vitro models versus classical cell cultures and the relevance of good in vitro models for further translation into in vivo models and finally into clinical applications. Magnetic nanoparticles (MNPs) have specific magnetic properties suitable for different biomedical applications. The most notable examples are magnetic fluid hyperthermia, visualization and tracking of cells in vitro and in vivo, and the use of MNPs as delivery platforms for innovative targeted drug-delivery systems. Chapter 9, Aluminosilicate-based composites functionalized with cationic materials: possibilities for drug-delivery applications, by Danina Krajiˇsnik et al., gives a comprehensive overview on the preparation, characterization, and properties of aluminosilicate-based composites for drug delivery, when functionalized with two distinctive groups of cationic materials: cationic surfactants and chitosan. Chapter 10, Bioactive glass nanofibers for tissue engineering, by Joaquı´n Penide et al., offers an up-to-date overview of the production techniques of glass fibers and nanofibers. The interest in this one-dimensional material in tissue engineering due to its advantageous structure and feasible compositions is discussed and achievements in its utilization as scaffolds for cellular growth are reviewed. Chapter 11, Application of (mixed) metal oxides-based nanocomposites for biosensors, by Ali Salehabadi and Morteza Enhessari, gives an up-to-date overview of the terms “sensors” and “biosensors” defined in various disciplines and applications including chemical, environment, clinical, and biology. Furthermore,

Preface

the synthesis, assembly, and applications of nanosized (mixed) metal oxides containing either covalently linked organic or inorganic compounds to form nanocomposites are explained. Chapter 12, Metal nanoparticles and their composites: a promising multifunctional nanomaterial for biomedical and related applications, by Vesna V. Vodnik and Una Bogdanovi´c, highlights the Au, Ag, and Cu nanoparticles currently being investigated in the field of health and medicine, focusing on individual and particularly polymer/organic molecules-functionalized nanoparticles—which is often employed to prevent them from agglomeration and oxidation, as well as for biofunctionalization. An overview of the synthesis, properties, and surface modifications of these nanomaterials, with particular attention given to their challenges and perspectives for relevant biomedical applications, is provided. Chapter 13, Hybrid metal complex nanocomposites for targeted cancer diagnosis and therapeutics, by Jeong-Hwan Kim et al., reviews the advances in anticancer organometallic agents based on iron complexes. In particular, chemotherapeutically active inorganic iron-complex-based advanced local magneto-drug-delivery systems composites via self-assembly of iron complexes, without the use of common magnetites and anticancer prodrugs, are highlighted. Chapter 14, Nanocoatings and thin films, by Valentina Grumezescu and Irina Negut, presents two different conventional processes for the deposition of thin films as well as nanoparticles in a form of nanocoating. Furthermore, this chapter provides the basics for both deposition techniques, dip-coating and laser deposition, in terms of processing phases to obtain ideal properties of nanocoatings and thin films in selected biomedical applications. Valentina Grumezescu1 and Alexandru Mihai Grumezescu2 1

Lasers Department, National Institute for Laser Plasma & Radiation Physics, Romania 2 Faculty of Applied Chemistry and Materials Science, University Politehnica of Bucharest, Bucharest, Romania

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CHAPTER

Biomedical inorganic nanoparticles: preparation, properties, and perspectives

1

Magdalena Stevanovic´ 1, Miodrag J. Lukic´ 1, Ana Stankovic´ 1, ˇ ´ Nenad Filipovic´ 1, Maja Kuzmanovic´ 1 and Zeljko Janicijevi c´ 2 1

Institute of Technical Sciences of the Serbian Academy of Sciences and Arts, Belgrade, Serbia 2 School of Electrical Engineering, University of Belgrade, Belgrade, Serbia

1.1 INTRODUCTION Nanotechnology enables a better understanding of the fundamental biology, physics, chemistry, and technology of nanometer-scale objects (Mitragotri et al., 2015; Stevanovi´c et al., 2008, 2013; Stevanovi´c and Uskokovi´c, 2009). It deals with the design, production, and operation of particle structures in the size range of approximately 1
100 nm. It has a wide range of applications in different areas of human activity such as in medicine, pharmacy, controlled drug delivery, optics, electronics, etc. For example, in medicine and pharmacy, a large number of studies have been focused on the use of nanoparticles as drug-delivery vehicles for therapeutics, since nanoparticles can interact with biological entities at the molecular level, and enable controlled and targeted delivery and passage through biological barriers. Moreover, in recent years, many different studies have revealed that some nanomaterials are intrinsically therapeutic. Such intense research has led to a more comprehensive understanding of cancer at the genetic, molecular, and cellular levels, providing an avenue for methods of increasing antitumor efficacy of drugs while reducing systemic side effects. It has been shown not only that nanoparticles can passively interact with cells, but also that they can actively mediate molecular processes to regulate cell functions (Kim and Hyeon, 2014). This is the case, for example, with the treatment of cancer via antiangiogenic mechanisms or the treatment of neurodegenerative diseases by effectively controlling oxidative stress (Kim and Hyeon, 2014). Among other nanomaterials, inorganic nanoparticles (Fig. 1.1) have attracted special attention since they possess unique properties such as size- and shape-reliant optical, magnetic, mechanical, and electrical properties, as well as biological responses, that is, antibacterial and antiviral properties (Stevanovi´c et al., 2015). A wide variety of techniques for the synthesis of inorganic nanoparticles (metallic and ceramic) have been reported in the literature. Inorganic nanoparticles are made by the crystallization of inorganic salts, forming a three-dimensional arrangement of linked atoms where binding is Materials for Biomedical Engineering: Inorganic Micro- and Nanostructures. DOI: https://doi.org/10.1016/B978-0-08-102814-8.00001-9 © 2019 Elsevier Inc. All rights reserved.

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FIGURE 1.1 Types of inorganic nanoparticles which are used in the biomedical field.

mainly covalent or metallic. Recently, numerous inorganic nanoparticles have been successfully produced by various different synthetic techniques. However, the obtaining of highly uniform, biocompatible inorganic nanoparticles with adequate functional properties is still a challenge. In this chapter, synthesis of different metallic and ceramic inorganic nanoparticles such as gold, silver, selenium, copper, iron, zinc oxide, and hydroxyapatite for biomedical applications will be addressed. For these nanosystems, the main challenges regarding the currently achieved functional properties and further perspectives will also be presented.

1.2 GOLD NANOPARTICLES Gold nanoparticles have occupied the attention of scientists for ages and are now heavily exploited in chemistry, biology, engineering, pharmacy, medicine, etc. (Giljohann et al., 2010; Hayat, 1989). These particles can be synthesized reproducibly, modified with apparently limitless chemical functional groups, and, in certain cases, characterized with atomic-level precision. Many examples of highly sensitive assays based upon gold nanoconjugates have been reported in the

1.2 Gold Nanoparticles

literature. Lately, among the numerous nanomaterials explored in therapeutic applications, those often found in clinical trials are gold nanoparticles (Mitragotri et al., 2015). Structures which behave as imaging-contrast agents, gene-regulating agents, drug carriers, and therapeutics have been designed and studied in the context of the diagnosis and therapy of many different diseases (Giljohann et al., 2010; Hayat, 1989). These materials have been chosen because of their unique physicochemical properties, which confer substantive advantages in cellular and medical applications. Fig. 1.2. shows different biomedical applications of gold nanoparticles. Gold nanoparticles can appear in different colors such as red, blue, or other colors, depending on their morphology, degree of agglomeration, and local environment. These visible colors reflect the underlying coherent oscillations of conductionband electrons, plasmons, upon irradiation with light of appropriate wavelengths. These plasmons underlie the intense absorption and elastic scattering of light, which in turn forms the basis for many biological sensing and imaging applications of gold nanoparticles (Murphy et al., 2008; Qian et al., 2008; Kelly et al., 2002; Kreibig and Vollmer, 1995; Link and El-Sayed, 2003; Murphy et al., 2005; Link et al., 1999; Sharma et al., 2009; Rao et al., 2000; Daniel and Astruc, 2004; Turkevich et al., 1951; El-Sayed et al., 2005; Kim et al., 2006; Chen et al., 2005).

FIGURE 1.2 Biomedical applications of gold nanoparticles. Source: Mind the graph.com

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Recently, Piella et al. described a strategy for the synthesis of highly monodisperse, biocompatible, and functionalized sub-10-nm citrate-stabilized gold nanoparticles by a kinetically controlled seeded-growth strategy (Piella et al., 2016). They found that use of traces of tannic acid, together with an excess of sodium citrate, during nucleation is essential in the formation of a high number (7 3 1013 NPs/mL) of small B3.5 nm gold seeds with a narrow size distribution. The reaction parameters such as pH, temperature, sodium citrate concentration, and gold precursor to seed ratio have been adjusted in order to produce gold nanoparticles with a precise control over their sizes between 3.5 and 10 nm. Thiolate-protected gold nanoparticles, which are uniform in size, were synthesized with no requirement for purification by Azubel and Kornberg (2016). It has been shown that the thiol-to-gold ratio controlled the size of the particles, and the choice of thiol controlled the reactivity of the particles toward thiol exchange. Lawrence et al. (2016) described a simple method for producing highly stable oligomeric clusters of gold nanoparticles via the reduction of chloroauric acid with sodium thiocyanate. The oligoclusters have a narrow size distribution and can be produced with a wide range of sizes and surface coats. By reducing dilute aqueous chloroauric acid with sodium thiocyanate under alkaline conditions, gold nanoparticles of about 2
3 nm, have been synthesized. The oligomeric clusters of these nanoparticles with narrow size distribution have been synthesized under ambient conditions via two methods. By varying the time between the addition of chloroauric acid to the alkaline solution and the subsequent addition of reducing agent (thiocyanate), in the delay-time method, the number of subunits in the oligoclusters have been controlled. The oligoclusters have been produced with sizes from B3 to B25 nm. It has been shown that this size range can be further extended by an add-on method utilizing hydroxylated gold chloride to autocatalytically increase the number of subunits in the as-synthesized oligocluster nanoparticles, providing a total range of 3
70 nm. Green synthesis of gold nanoparticles using an aqueous extract of garcinia mangostana fruit peels have been described by Lee et al. (2016). The environmentally safe synthesis of gold nanoparticles has been performed by the reduction of aqueous gold metal ions in contact with the aqueous peel extract of the plant, Garcinia mangostana. The synthesized particles were mostly spherical in shape and with sizes of about 30 nm. From the FTIR results of this research, it has been concluded that phenols, flavonoids, benzophenones, and anthocyanins all may act as the reducing agent in the synthesis of gold nanoparticles. Phenols, ketones, and carboxyls were present in the leaves of Tamarindus indica L. leaves and these leaves have been used in the research of Correa et al. Correa et al. (2016) performed biosynthesis and characterization of gold nanoparticles using extracts of T. indica L. leaves. Phenols, ketones, and carboxyls for the synthesis of gold nanoparticles were identified by gas chromatography coupled to mass spectrometry and high-pressure liquid chromatography. The synthesis was performed at

1.3 Silver Nanoparticles

room temperature during 1 hour of reaction time. The results indicated the formation of gold nanoparticles with an average size of 52 6 5 nm. Physical-vapor evaporation of metals on low vapor pressure liquids is a simple technique to synthesize nanoparticles and thin films, though only a little work has been conducted so far. Fujita et al. (2016) described obtaining gold nanoparticles by vacuum evaporation in ricinoleic acid and oleic acid. Ricinoleic acid and oleic acid are unsaturated fatty acids. The gold nanoparticles in ricinoleic acids formed aggregates and then dispersed with time, while in oleic acid large aggregates have not been observed in all timescales. The mean size of the nanoparticles was about 4 nm in both ricinoleic and oleic acids. Despite the great excitement about the synthesis and potential uses of gold nanoparticles for medical diagnostics, as tracers, and for other biological applications, researchers are increasingly aware that potential nanoparticle toxicity must be investigated before any in vivo applications of gold nanoparticles can move forward (Tong et al., 2009; Jain et al., 2008). In order to prevent bare gold nanoparticles from being agglomerated, to manipulate the gold core properties, as well as to control interfacial properties, the gold nanoparticles are generally capped by an organic layer (Zhou et al., 2009). In addition, the aggregation and dispersion of colloidal nanoparticles are one of the key issues related to their potential applications. However, the issues of how to fully achieve the colloid stability of nanoparticle dispersions as well as how to control them, have yet to be fully investigated.

1.3 SILVER NANOPARTICLES The use of silver nanoparticles as devices in biomedical applications as well as in basic research has grown enormously over the past few decades (Stevanovi´c, 2013). Not so many nanomaterials have been applied to so many diverse fields, finding their use in everything from biological sensors, chemical catalysis, drugdelivery systems, to wound healing, etc. The interest in silver nanoparticles as tools in different areas has been driven by their unique properties, which include special structural, optical, electronic, and thermal properties (Stevanovi´c, 2014; Meng et al., 2010; Stevanovi´c et al., 2012a,b). Due to these properties, silver nanoparticles are being integrated into the most diverse products. Examples include conductive inks, pastes, and fillers which utilize silver nanoparticles for their high electrical conductivity, molecular diagnostics, and photonic devices. A progressively common application is the use of silver nanoparticles for antimicrobial coatings, and many textiles, keyboards, wound dressings, and biomedical devices now contain silver nanoparticles that continuously release a low level of silver ions to provide protection against bacteria (Stevanovi´c et al., 2011, 2012a, b, 2013, 2014). In the literature, different methods for obtaining silver nanoparticles have been described. Generally, all these methods can be divided into three main groups

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which are physical, chemical, and biological methods (Iravani et al., 2014). Such methods include laser ablation, gamma irradiation, electron irradiation, microwave processing, solid-state synthesis, chemical reduction, in situ radical polymerization, photochemical methods, sonochemical synthesis, spray pyrolysis, and biological synthetic methods. It is important for the synthesis of the nanoparticles to be produced easily and at low cost. Through the optimization of experimental conditions, it is possible to synthesize nanoparticles of different sizes and morphologies. Such optimization relates to different parameters in the synthesis, such as concentrations of reactants, pH, reducing agents, different surfactants, etc. All these parameters can significantly affect the stability of the resulting particles. Silver nanoparticles have a really wide range of applications in medicine. Lin et al. (2011) reported silver nanoparticle-based surface-enhanced Raman spectroscopy (SERS) in noninvasive cancer detection. This approach is highly promising and may prove to be an indispensable tool for the future. The enhancement in the optical and photothermal properties of noble metal nanoparticles arises from the resonant oscillation of their free electrons in the presence of light, also known as localized surface plasmon resonance (LSPR) (Jain et al., 2008; Tse et al., 2011). The plasmon resonance can either radiate light (Mie scattering), a process that finds great utility in optical and imaging fields, or be rapidly converted to heat (absorption); the latter mechanism of dissipation has opened up applications in several new areas. The ability to integrate metal nanoparticles into biological systems has had the greatest impact in biology and biomedicine. In terms of therapeutics, one of the most commonly used applications of silver nanoparticles is in wound healing. Compared with other silver compounds, many studies have demonstrated the superior efficacy of silver nanoparticles in healing time, as well as achieving a better cosmetic result after healing. In the study of Kwan et al. (2011), it was shown that in wounds treated with silver nanoparticles, there was better collagen alignment after healing when compared to controls, which resulted in better mechanical strength. Regardless of the application, as already mentioned above, there are many different methods of synthesis of silver nanoparticles. Jung et al. investigated the synthesis of silver nanoparticles using a small ceramic heater which has a local heating area of about 1500 C and where source metals can be evaporated (Jung et al., 2006). The as-synthesized silver nanoparticles were spherical and nonagglomerated. The results showed that the mean diameter, the standard deviation, and the total number and concentration of nanoparticles increase with heater surface temperature. In the study of Mafune et al. (2000), silver nanoparticles were produced by laser ablation of a metal silver plate in aqueous solutions of surfactants, CnH2n11SO4Na (n 5 8, 10, 12, 16). The abundances of the nanoparticles were measured as a function of the surfactant concentration and it has been confirmed that the surfactant coverage and the charge state on the nanoparticle surface are closely related to the stability of the nanoparticles in the solutions. Tsuji et al. (2002, 2003) also have used laser ablation for the production of colloidal silver. A quantitative disagreement of the efficiency of self-absorption on both the

1.3 Silver Nanoparticles

ablation efficiency and particle size with the absorption intensity of colloids was discussed on the basis of dynamic change in the morphology of colloids. Tien et al. (2008) produced a silver nanoparticle suspension in deionized water with no added surfactants by the arc discharge method. The results have shown that a silver nanoparticle suspension, fabricated by this method, contains metallic silver nanoparticles and ionic silver. Dodecanethiol-capped silver nanoparticles have been produced by Oliveire et al. (2005) by a two-phase liquid
liquid method. Small modifications in the parameters lead to changes in the particles’ size, size distribution width, stability, and structure, as well as in their ability to selfassemble. Kim et al. (2006) synthesized spherical silver nanoparticles by the polyol process. Two different synthesis procedures were compared in order to examine the influence of reaction parameters on the resulting particle size and its distribution. In the precursor heating method, wherein a solution containing silver nitrate was heated to the reaction temperature, the ramping rate was determined to be a critical parameter affecting the particle size. However, in the precursor injection method, in which a silver nitrate aqueous solution was injected into hot ethylene glycol, the injection rate and the reaction temperature were important factors in terms of reducing the particle size and attaining monodispersity. Almost monodisperse silver nanoparticles have been prepared in a simple oleylamine 2 liquid paraffin system by Chen et al. (2007). Intensive research has established that the formation process of silver nanoparticles could be divided into three stages: growth, incubation, and Ostwald ripening stages. Both nanowires and spherical nanoparticles have been prepared in a study by Zhang et al. (2007) in which two mixed-valence polyoxometalates were shown to serve both as good reductants of silver cation and efficient capping agents for the resulting metallic nanostructures. It has been shown that polyoxometalates can also induce the synthesis of one-dimensional nanostructures in water. Colloidal silver nanoparticles have been synthesized using silver nitrate solubilized in the water core of one microemulsion as a source of silver ions, hydrazine hydrate solubilized in the water core of another microemulsion as a reducing agent, dodecane as the oil phase and sodium bis(2-ethylhexyl) sulfosuccinate as the surfactant (Zhang et al., 2007). The photocatalytic approach toward semiconductor/metal nanocomposites has been described by Cozzoli et al. (2004). In this method, metallic silver is generated upon UV illumination of deaerated TiO2 solutions containing AgNO3. By this method TiO2/Ag nanocomposite with a certain control over the metal particle size has been produced without the use of surfactants and/or additives. Synthesis of silver nanoparticles by photoreduction of metal salts has been described by Huang et al. as well as by Sato-Berru´ et al. (Huang and Yang, 2008; Sato-Berru´ et al., 2009). The photoreduction of AgNO3 in the presence of sodium citrate was carried out by irradiation with different light sources (UV, white, blue, cyan, green, and orange) at room temperature (Sato-Berru´ et al., 2009). The lightmodification process resulted in a colloid with distinctive optical properties that can be related to the size and shape of the particles. The Ag colloids, as prepared,

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were employed as active colloids in SERS. Pyridine and caffeine were used as test molecules. Huang and Yang (2008) described photoreduction of AgNO3 in layered laponite suspensions. Nadagouda et al. (2011) described the green synthesis of silver nanoparticles by the microwave chemistry method. In the literature, the application of this method has been described in the preparation of silver, gold, platinum, and gold
palladium nanostructures. Hsieh et al. (2011) also reported microwaveassisted synthesis and thermal reduction, to deposit silver nanoparticles on oxidized carbon paper electrodes. The microwave-assisted approach for depositing nanosized silver catalysts on carbon paper electrodes showed potential in the application of alkaline fuel cells because of its fast synthesis, high activity, and simplicity. In the research of Kate et al. (2011) the synthesis of silver nanoparticles by the microwave irradiation method is also described. The microwave synthesis of polypyrrole (PPy)/Ag nanocomposites does not require any oxidizing agent for polymerization. Soto et al. (2011) combined microwave-plasma and fluidized-bed technologies to decompose and vaporize silver precursors, such as silver oxide or silver nitrate. The results have shown that silver vapors condense with a homogeneous distribution of nanosize particles over the support. These particles had sizes from 3 to 50 nm, as measured using electron microscopy. Composites of polyvinylpyrrolidone and silver nanoparticles have been prepared by Yao et al. (2010) by the microwave method. The silver nanoparticles with sizes of about 25 nm were homogeneously dispersed in the polyvinylpyrrolidone matrix. Gao et al. (2011) synthesized silver nanoparticles covered by a starch layer to form spherical core
shell Ag/starch nanoparticles with a diameter ranging from 5 to 20 nm. XRD confirmed the presence of silver nanoparticles with a face-centered cubic structure. Garcı´a-Serrano et al. (2011) reported on the use of aqueous solutions of a low-molecular-weight ion-exchange polymer containing phosphonic acid groups; the poly(p-acryloylaminobenzylphosphonic acid), for the synthesis of silver particles at room temperature. The results indicated that the ion-exchange polymer is capable of protecting silver particles in the solution, leading to large-sized cubes and rectangular prisms in colloidal solutions which are stable for several months. Hebeish et al. (2011) examined and compared three different methods for obtaining silver nanoparticles: thermal, ultrasonic, or microwave. They found that thermal and ultrasonic methods are far more effective than microwave methods. A simple and inexpensive, single-step synthesis of silver nanoparticles was achieved using poly(methyl vinyl ether-co-maleic anhydride) both as a reducing and stabilizing agent (Maity et al., 2011). The synthesized silver nanoparticles were characterized and the results showed that silver nanoparticles were coated with poly(methyl vinyl ether-co-maleic anhydride)-shell with a thickness of about 5
8 nm. One of the options to synthesize metal nanoparticles is to use “natural factories,” such as biological systems. In a study by Kalishwaralal et al. (2008), the synthesis of silver nanoparticles was achieved by reduction of an aqueous solution of silver ions with the culture supernatant of Bacillus licheniformis. The process

1.3 Silver Nanoparticles

of reduction is extracellular, which makes this method one of the easier methods for the synthesis of silver nanoparticles. Venkatpurwar and Pokharkar (2011) used sulfated polysaccharide isolated from marine red algae (Porphyra vietnamensis) for the synthesis of silver nanoparticles. The obtained silver nanoparticles showed surface plasmon resonance centered at 404 nm, with average particle size measured to be about 13 nm. Biological synthesis of very small silver nanoparticles by culture supernatant of Klebsiella pneumonia has been also described by Mokhtari et al. (2009). The synthesized silver nanoparticles were uniform and with an average size of about 3 nm. The study of Korbekandi et al. (2012) demonstrated the bioreductive synthesis of silver nanoparticles using Lactobacillus casei subsp. casei at room temperature. In this research, the reaction conditions were successfully optimized to increase the yield of nanoparticle production and productivity of this biosynthetic approach. The objectives of the study by Korbekandi et al. (2013) were optimization of production of silver nanoparticles using biotransformations by Fusarium oxysporum, and a further study on the location of nanoparticle synthesis in this microorganism. The synthesized silver nanoparticles were almost spherical, single (25
50 nm) or in aggregates (100 nm), attached to the surface of biomass. Li et al. (2007) described the synthesis of silver nanoparticles by treating silver ions with a Capsicum annuum L. extract. The results indicated that the proteins, which have amine groups, played a reducing and controlling role during the formation of silver nanoparticles in the solutions, and that the secondary structure of the proteins changed after reaction with silver ions. Aqueous extract of shade dried leaves of Euphorbia hirta (L.) have been used in the study of Elumalai et al. (2010) for the synthesis of silver nanoparticles. Biosynthesis of silver nanoparticles and their activity on waterborne bacterial pathogens were investigated in the study of Krishnaraj et al. (2010). Silver nanoparticles with sizes of about 20
30 nm were synthesized using leaf extract of Acalypha indica and the formation of nanoparticles was observed within 30 min. Biosynthesis of silver nanoparticles using G. mangostana leaf extract as a reducing agent has been described by Veerasamy et al. (2011), while in the research of Singhal et al. (2011) synthesis of silver nanoparticles has been done using Tulsi (Ocimum sanctum) leaf extract. Biosynthesis of silver nanoparticles by Cacumen platycladi extract was investigated in the study of Huang et al. (2011). The objectives of the study of Iravani and Zolfaghari (2013) were the production of silver nanoparticles using Pinus eldarica bark extract and optimization of the biosynthesis process. Metallic nanoparticles are very often synthesized by wet chemical techniques, where the chemicals used are quite often toxic and flammable (Al-Marhaby and Seoudi, 2016). A simple and completely green chemical synthesis has been developed by Stevanovic et al. (2012a,b) for the synthesis of stable, bare, and capped silver nanoparticles based on the reduction of silver ions by saccharide and capping by poly(α, γ, L-glutamic acid). Poly(α, γ, L-glutamic acid) has had a dual role in the synthesis and was used as a capping agent to make the silver nanoparticles more

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biocompatible and to protect nanoparticles from agglomerating in the medium. The synthesized PGA-capped silver nanoparticles in the size range 5
45 nm were highly stable over a long period of time without sign of precipitation.

1.4 SELENIUM NANOPARTICLES Ever since its discovery, selenium has attracted the attention of many scientists due to its specific characteristics and behavior, which provided a wide range of applications for this element, from the photoelectrical industry to biology and medicine. The diverse applications are directly related to the selenium chemical form, size, and shape of its particles. When it comes to biomedical applications it is a well-known fact that selenium is an essential micronutrient for animals and humans, but with a narrow margin between beneficial and toxic effects. As a potential anticancer agent, it is found that consumption of 200 μg Se per day reduced mortality in cancer patients and decreased the incidence of lung, colorectal, and prostate cancers (Clark et al., 1996). Elemental selenium for a long time had been considered as biologically inert. Therefore, until recently, only organic forms of selenium (selenomethionine, SeMet) and some salts had been used in studying its biomedical applications. Recently, elemental selenium nanoparticles (SeNPs) have gained a lot of attention because it provides reduced risk of toxicity, but the same bioavailability and efficacy in increasing the activities of selenoenzymes compared with SeMet and selenite (Zhang et al., 2001, 2008; Wang et al., 2007). These results were a good starting point for many future types of research regarding the anticancer efficiency of SeNPs, but also opened numerous questions dealing with the mechanism and key factors responsible for the SeNP anticancer activity. When it comes to nanoparticles and their interaction with cells it is a wellknown fact that crucial parameters are the size, surface chemistry (surface charge), and morphology of particles. Therefore, in order to fully understand and summarize applications of SeNPs in biomedicine, those parameters must be strictly considered. The starting point is the synthesis procedure. Based on the literature data, numerous synthesis procedures for obtaining SeNPs have been described so far, therefore it is difficult to mention all of them. The most frequently used can be roughly divided into two groups: (1) chemical methods which include reduction of selenium from 14 oxidative state to zero, using adequate reducing agents, and (2) biosynthesis procedures, which also employ reduction of starting selenium compound, but with living organisms (bacterial strains, plants, etc.) as reducing agents. The key parameters in chemical reduction methods are the choice of the reducing agent (optimum pH) and stabilizing/dispersing agents. According to most of the papers which addressed the use of SeNPs, use of the stabilizing agents is necessary in order to hold the aggregation of Se atoms in the nanometer scale,

1.5 Copper Nanoparticles

that is, to obtain stable dispersion of nanoparticles and at the same time it could increase bioavailability and promote interaction with cells (enhanced cellular uptake, decreased cytotoxicity for normal cells, targeting, etc.). Thus, many of the researchers have been used diverse biomolecules or biocompatible surfactants to coat or functionalize the surface of the SeNPs, such as bovine serum albumin (BSA), amino acids, polysaccharides, hyaluronic acid, sodium alginate, adenosine triphosphate (ATP), etc. The choice of the right stabilizing agent should be made based on the desired application. For instance, functionalization of SeNPs with ATP provided enhanced cellular uptake and anticancer activity upon HepG2 hepatocellular carcinoma cells (Zhang et al., 2013). An interesting example can be also found in the paper published by Feng and others, where different amino acids were used to decorate SeNPs (neutral-valine, acidic-aspartic acid, and basiclysine) (Feng et al., 2014). All three types of SeNPs were successfully internalized in various human cancer cell lines through endocytosis. In another work, SeNPs were used to reduce anisomycin’s high cytotoxicity against normal cells and enhance anticancer activity (Xia et al., 2015). When compared to bare anisomycin, the conjugated system possesses better abilities to inhibit cell proliferation, arrest cell cycle, induce cell apoptosis, and block cell motility and migration. Conjugation of SeNPs with other chemotherapeutics is a promising strategy, as Liu and colleagues demonstrated using molecules of 5-fluorouracil. A stable spherical nanometer system was formed through Se
O and Se
N bonds and physical adsorption. This conjugate has shown enhanced cellular uptake and anticancer activity against five human cancer cell lines (Liu et al., 2012). Antimicrobial activity is another property of SeNPs that have been demonstrated in numerous papers. SeNPs are not considered as a strong antibacterial agent, such as silver or zinc oxide nanoparticles, so their use is limited to a certain number of bacterial strains. Bearing in mind all their properties along with their antimicrobial activity, SeNPs are still a good candidate for coating biomaterials for tissue engineering, such as bioglass scaffolds or poly(ether ether ketone) (PEEK) medical devices (Stevanovi´c et al., 2015; Wang et al., 2016).

1.5 COPPER NANOPARTICLES Copper (Cu) is a 3D transition metal. It is highly abundant in nature and presents a low-cost material compared to noble transition metals. In its bulk form, Cu has found numerous applications in the field of electronics due to its high electrical and thermal conductivity. Cu is also a physiologically important metal with numerous functions in the human body (Sengupta et al., 2014). Copper nanoparticles (Cu NPs) are heavily oriented towards biomedical applications because of their distinctive antibacterial and antifungal activity, which is coupled with unique electric, optical, and catalytic properties (Argueta-Figueroa et al., 2014). As will be illustrated later in this text, there are multiple facile synthesis methods for

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obtaining Cu NPs. However, the use of Cu NPs is still limited by their chemical instability under usual atmospheric conditions. The influence of moisture or strong Lewis bases deteriorates the oxidation resistance of Cu NPs (Benavente et al., 2013). Synthesis of Cu NPs can mainly be classified into two different groups according to: (1) the direction of nanoparticle formation and (2) the synthesis method. Nanoparticles can be obtained either through “bottom-up” (using the precursors at the atomic or molecular level to produce solid structures at the nanoscale) or “top-down” (using bulk solid material which is split into successively smaller fragments until nanoparticles are formed) approach. The bottom-up approach has gained significant popularity because it allows for better control over nanoparticle properties like size and morphology (Biswas et al., 2012; Umer et al., 2012). This approach is also easier to implement, while the top-down approach typically requires complex instruments or systems (Umer et al., 2012). Synthesis methods are commonly divided into three major categories: (1) physical, (2) chemical, and (3) biological. Some synthesis protocols use different types of methods synergistically or employ novel methods which cannot be easily placed into these categories. Physical methods utilize special equipment and experimental conditions for nanoparticle fabrication. Laser ablation is used to produce pure nanoparticles in a colloidal form dispersed in different solvents. In an interesting study, Cu NPs have been synthesized in virgin coconut oil medium (Sadrolhosseini et al., 2013). The successful synthesis of Cu NPs by laser ablation was also reported in water (Kazakevich et al., 2004; Tilaki et al., 2007), acetone (Kazakevich et al., 2004; Tilaki et al., 2007; Muniz-Miranda et al., 2011), and ethanol (Kazakevich et al., 2004). In the gas evaporation method, nanoparticles are produced by rapid condensation of the vapor produced by heating the material (Tavakoli et al., 2007). Cu NPs were synthesized using this method in the inert argon gas atmosphere (Raffi et al., 2010). Exploding wire or pulsed wire discharge technique relies on the application of high-intensity current pulses to the thin metal wire, which leads to explosion of the wire. By evaporation of the copper wire in the oleic acid vapor/mist, Murai and coworkers demonstrated that this method can be used to fabricate Cu NPs with an organic coating of a few nm thickness (Murai et al., 2007). Adaptation of the exploding wire technique was also recently utilized to obtain mixed-phase synthesis of Cu/Cu2O/CuO nanoparticles (Sahai et al., 2016). Chemical reduction methods are mainly based on the reduction of Cu(II) salts with various reducing agents [sodium borohydride (Song et al., 2004), hydrazine (Saikova et al., 2010), hypophosphite (Zhu et al., 2004a), 1,2-hexadecane-diol (Mott et al., 2007), glucose (Shenoy and Shetty, 2013), ascorbic acid (Xiong et al., 2011), carbon monoxide (Prasad and Singh, 2013) and borane compounds (Aissa et al., 2015)] in order to obtain Cu NPs. Stabilization and control of particle growth are achieved with diverse capping agents. The microemulsion method is a special adaptation of the chemical reduction methods. In this method, a thermodynamically stable isotropic solution is formed

1.5 Copper Nanoparticles

out of at least three components (polar phase, nonpolar phase, and a surfactant) (Malik et al., 2012). Cu nanocolloids with different particle sizes were synthesized using this approach (Salzemann et al., 2004; Kaminskiene et al., 2013). The sonochemical method is based on ultrasound-assisted chemical reduction. Ultrasound in the frequency range from 20 kHz to 15 MHz is utilized to promote chemical reactions mainly via acoustic cavitation (Gawande et al., 2016). It is reported that Cu NPs of variable size can be synthesized using hydrazine monohydrate as the reducing agent in the presence of ethylene glycol as the capping agent (Moghimi-Rad et al., 2010). Chemical reduction can also be modified with microwave radiation to improve the reaction rates. Microwave-assisted chemical reduction was achieved with the combination of sodium hypophosphite and ethylene glycol (Zhu et al., 2004b) as the reducing and capping agents, respectively. A similar synthesis approach can be utilized with certain plant extracts (Yallappa et al., 2013; Cheirmadurai et al., 2014). In another study, microwave heating was employed to produce nanoparticles of different sizes (Blosi et al., 2011). Specific physicochemical properties of ionic liquids have been used to synthesize metal nanoparticles (Richter et al., 2013). Cu NPs are synthesized top-down by the dissociation of Cu microparticles in ionic liquid media due to the strong interfacial interaction of Cu with ionic liquids. Several successful synthesis protocols have been reported for Cu NPs using this methodology (Kim et al., 2009; Han et al., 2011). Electrochemical synthesis of Cu NPs is performed by allowing the flow of direct current through the electrolytic cell filled with the aqueous solution of copper salt. Cu ions are reduced at the cathode and formed Cu atoms agglomerate into Cu NPs. Theivasanti and coworkers have reported the electrolytic synthesis of Cu NPs from copper sulfate precursors (Theivasanthi and Alagar, 2011). Specific morphology of Cu NPs can be obtained by employing various templates. For example, Cu nanowires were synthesized using the nanochannels of porous anodic alumina (Gao et al., 2002). Surfactants can also be used as templates and stabilizers, and Cu nanorods were successfully prepared using this approach (Yang et al., 2003). Thermal decomposition comprises heat treatment and thermal degradation of precursors for the synthesis of Cu NPs. Synthesis of Cu NPs was achieved via thermal decomposition of various precursor compounds: copper
oleate complex (Kim et al., 2006; Betancourt-Galindo et al., 2014), copper oxalate (SalavatiNiasari et al., 2008), [bis(2-hydroxy-1-naphthaldehydato)copper(II)] complex (Salavati-Niasari et al., 2010), etc. Biological methods combine different biological processes and compounds with traditional synthesis methods to fabricate nanoparticles. To date, the uses of plants and microorganisms (bacteria and fungi) were reported in the synthesis of copper nanoparticles. Using soybean extract as a chelating agent Guajardo-Pacheco and coworkers produced Cu NPs (Guajardo-Pacheco et al., 2010). A solution of CuSO4 was also

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treated with Magnolia kobus leaf extract to fabricate Cu NPs of 40
100 nm diameter (Lee et al., 2013). Rapid Cu NPs syntheses were achieved by treating the solution of CuSO4 with Zingiber officinale and Syzygium aromaticum (Subhankari and Nayak, 2013a,b). Highly stable nanoparticles were synthesized with Artabotrys odoratissimus leaf broth (Kathad and Gajera, 2014). The synthesis of Cu NPs with high yield can be attained with the treatment of CuCl2 solution with Ginko biloba L. plant extract (Nasrollahzadeh and Sajadi, 2015). Cu NPs with good antibacterial properties were fabricated with Capparis zeylanica leaf extract (Saranyaadevi et al., 2014). Varshney and coworkers utilized Pseudomonas stutzeri for the rapid biological synthesis of Cu NPs of spherical shape (Varshney et al., 2010). The same group using the same bacterial strain also prepared Cu NPs of cubic shape from electroplating wastewater (Varshney et al., 2011). Morganella morgani RP42 and Morganella psychrotolerans were employed to synthesize Cu NPs of narrow size range (15
20 nm) (Ramanathan et al., 2011). Cu NPs were also fabricated by the addition of Enterococcus faecalis supernatant to the diluted solution of CuSO4. NPs prepared with this method show strong antibacterial activity against human multidrug-resistant bacterial pathogens (Ashajyothi et al., 2014). An interesting synthesis method was reported by Majumder (2012), where F. oxysporum was used at room temperature to extract Cu from integrated circuits and fabricate NPs. Dead biomass of Trichoderma koningiopsis can be used as the reducing agent for the rapid and scalable synthesis of Cu NPs (Salvadori et al., 2014). Preparation of Cu NPs was also performed using Aspergillus species (Pavani et al., 2013). The conductive nature of synthesized Cu NPs enables the fabrication of conductive inks by adding dispersing and binding agents. There are various methods for the synthesis of conductive inks (Korada et al., 2015). Ink is commonly printed and subsequently dried and/or sintered to form a stable conducting pattern. Copper films are interesting to interconnect materials due to their high conductivity and outstanding electron-migration resistance (Vitulli et al., 2002). Patterns made from such conductive inks are of great interest in the field of flexible electronics, which has perspective applications in biosensing and implants (Korada et al., 2015). Yong et al. (2015) described a route toward improving the electrical conductivity of the copper film. This is achieved through a two-step process. Firstly, copper fine particles were synthesized using D-isoascorbic acid as a mild reductant and octylamine as a capping agent. Secondly, an oxidative preheating process in the air was used for generating convex surfaces, nanorods, or nanoparticles. Fig. 1.3 shows SEM images of these copper films oxidatively preheated at 250 C for various periods and corresponding X-ray diffraction patterns (Yong et al., 2015). Colloids of Cu NPs display a distinct absorption band in the UV-visible range arising from the plasmon resonance (Schmid and Chi, 1998; Pileni, 1998). The wavelength of the plasmon band is particle size-dependent. Plasmon resonance peak for Cu NPs in the size range of 5
20 nm is in the wavelength range 560
570 nm (Pileni, 1998). Larger particles tend to absorb at greater

1.5 Copper Nanoparticles

FIGURE 1.3 SEM images of copper films oxidatively preheated at 250 C for various periods: (A) 0 min, (B) 5 min, (C) 30 min, (D) 2 h, and (E) 4 h. (F) Corresponding X-ray diffraction patterns. (Yong, et al., 2015; This reference is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.)

wavelengths, while smaller particles and nanoclusters seem to absorb at much higher energies in the UV range (Singh et al., 2010). The optical bandgap of stabilized Cu NPs is dependent on particle size and the average reported values are around 2 eV (Mohindroo et al., 2016). Cu nanoclusters were recently assessed for applications in fluorescence imaging. It is reported that ultra-small Cu nanoclusters

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(up to several hundred atoms) exhibit discrete electronic states and size-dependent fluorescence, which could be useful in biomedical imaging (Zhou et al., 2016). The bulk form of Cu is completely diamagnetic (Anschroft and Mermin, 1976). Magnetic properties of Cu can be changed at the nanoscale due to several effects, including the interactions with the capping agents (Batsaikhan et al., 2015). Ferromagnetic behavior was reported for thiol-capped Cu NPs and superparamagnetic behavior for amine-capped Cu NPs (Garitaonandia et al., 2008). Recently, it was observed that even bare Cu NPs exhibit ferromagnetic spin polarization (Batsaikhan et al., 2015). Adjustment of the magnetic properties of Cu NPs could contribute to a plethora of biomedical applications and this area requires further research. Cu can behave as an electron donor or acceptor in certain enzymes due to its redox properties, thereby increasing the toxic effect on bacteria induced by copper ions (Karlin, 1993; Macomber and Imlay, 2009; Grass et al., 2011). The mechanism of antibacterial activity for Cu NPs relies on the interaction of enzymes and sulfhydryl groups which damages the DNA and thus generates oxidative stress (Ren et al., 2009; Santo et al., 2012; Zain et al., 2014). The main parameters determining the extent of the antibacterial effect of Cu NPs are particle size, minimum inhibitory concentration, and the degree of surface oxidation (Steindl et al., 2012). The strong antibacterial effect of Cu NPs was demonstrated for the size range from 1 to 10 nm (Cioffi et al., 2005). A decrease in cell viability was also reported for the range of NP size between 22 and 90 nm after the treatment of commonly tested bacterial species such as Clostridium difficile, Pseudomonas aeruginosa, and Escherichia coli (Yoon et al., 2007; Ruparelia et al., 2008; Mehtar et al., 2008). Gram-positive bacteria exhibit higher sensitivity to Cu NPs, while the lower viability of Gram-negative bacteria is correlated with the expressed reactive oxygen species (ROS) by Cu NPs which depend on particle size (Camacho-Flores et al., 2015). Paper sheets with Cu NPs were examined as water purifiers. The recorded bacterial inactivation was proportional to the concentration of Cu NPs (Dankovich and Smith, 2014). It was shown by Prado and coworkers that Cu surfaces are a good replacement option for stainless steel in fighting infections caused by nosocomial bacterial strains (Prado et al., 2013). Toxicity of Cu NPs was estimated by Chen et al. (2006) as moderate (class 3) with the tendency for accumulation in the kidneys, liver, and spleen. The size, morphology, and release of ions from Cu NPs were revealed as important parameters defining the toxicity of Cu NPs (Song et al., 2014). Toxic effects were observed on primary brain astrocytes and the suggested mechanism was the generation of ROS (Bulcke et al., 2014). Li et al. (2013) reported numerous signs of Cu NPs’ toxicity: oxidative stress, protein damage, DNA damage, cell membrane damage, and inhibition of cell growth. It was recently reported that Cu NPs can have adverse effects on reproductive health by crossing biological barriers (Singh et al., 2009). In vitro studies have shown that Cu-based NPs generate oxidative stress which results in reproductive

1.6 Iron Nanoparticles

toxicity (Ahamed et al., 2010). In vivo studies on a mouse population (Chen et al., 2006) found that males were more affected than females after equivalent exposure to Cu NPs. Cu NPs can have a vast potential in near future. Numerous cost-effective synthesis methods and a set of specific physicochemical properties could open up a wide array of applications. Excellent antimicrobial properties will make Cu NPs an indispensable ingredient in antimicrobial products (chemical agents, coatings, textiles, etc.) highly regarded in the biomedical field. Conductive inks and films based on Cu NPs will probably become the basis for interconnects in flexible electronic devices. This could potentially lead to the faster development of implantable electronics and biosensors. Recent findings related to the optical and magnetic properties of Cu NPs may bring up a set of perspective applications in biomedical imaging.

1.6 IRON NANOPARTICLES Iron is a metal in the first transition series. It is by mass the most common element on Earth, and the fourth most abundant element in the Earth’s crust. It is found in minerals such as hematite (Fe2O3), magnetite (Fe3O4), and siderite (FeCO3). Iron metal has been used since ancient times and it has played an immense role in the technological progress of humanity. Iron also plays an important role in biology. It is present in our bloodstream in protein hemoglobin, where it helps to transport oxygen. Iron in humans is distributed throughout the body in tissues, muscles, bone marrow, blood proteins, enzymes ferritin, hemosiderin, and transport in plasma. Nanoparticles of iron and iron oxide, in form of magnetite (Fe3O4) and maghemite (γ-Fe2O3), are widely studied for application in many fields such as magnetic fluids, catalysis, biotechnology/biomedicine, magnetic resonance imaging (MRI), data storage, and environmental remediation (Lu et al., 2007). Magnetic nanoparticles exhibit size-dependent properties, which are not observable in bulk magnetic materials. When the size of the magnetic particle is below a critical value (usually below 20 nm for iron nanoparticles), a singledomain magnetic particle will be formed and the material exhibits a unique form of magnetism called superparamagnetism. Without an external magnetic field, these single-domain magnetic particles are randomly oriented, but when an external magnetic field is applied they align in the field and produce high magnetization. A useful property of superparamagnetic particles is that for orienting of magnetic single domains relatively low magnetic fields can be used. Another useful property is that they have no coercivity. After the applied magnetic field is removed from magnetic nanoparticles, thermal energy allows them to freely reorient so that no external energy needs to be applied to demagnetize them. Below a certain temperature, called the blocking temperature, particles behave as ferromagnetic because there is not sufficient thermal energy to realign single magnetic

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domains. It should be emphasized that magnetic properties strongly depend on size. Coercivity can go from zero to the maximum value in just tens of nanometers for spherical magnetic nanoparticles (Huber, 2005). The chemical environment also strongly influences the magnetic properties of iron nanoparticles. The more reactive species that interact strongly with the iron particle surface tend to decrease magnetization more than weakly interacting species. All pure metallic nanoparticles have better magnetic performance compared to their oxide counterparts. However, pure metallic nanoparticles are highly chemically reactive, so their use is limited. Iron nanoparticles are extremely reactive with oxidizing agents, so the preparation of stable iron nanoparticles is highly challenging. The most common chemical method used for the preparation of monodisperse Fe nanoparticles is thermal decomposition of iron pentacarbonyl [Fe(Co)5] (Vitta et al., 2011). The decomposition of the iron precursors can also be accomplished by means of processes such as sonochemistry, electrochemical, and laser decomposition (Snovski et al., 2014). Controlling the oxidation of iron nanoparticles can be achieved by formation of the passive oxide layer. Iron nanoparticles can be exposed to weak oxidizer (such as low partial pressure of air, small amounts of dilute, air-free water, or carboxylic acid) so an iron oxide shell is formed on the particle surface. Iron oxide nanoparticles have been extensively investigated for their biomedical applications, such as MRI, magnetic hyperthermia (MH), magnetic targeting (MT), magnetic separation (MS), magnetofection, biosensors, and tissue engineering (Yu et al., 2017). Metallic iron nanoparticles have a much higher magnetization than iron oxide nanoparticles, however, they are still unsuitable for medical applications, which can be ascribed to their instability and iron release that can finally result in their toxicity in physiological environments. For their further application, especially in biomedicine, it is crucial to develop protecting shells that not only chemically stabilize iron nanoparticles but also that can be used for further functionalization. These strategies include coating with an inorganic layer such as precious metal, silica, or carbon, which leads to the core
shell structure. The most common way to synthesize Fe/Au core
shell nanoparticles is the reverse micelle method (Chung and Shih, 2014; Islam et al., 2013; Manjili et al., 2014). In a typical experiment, FeSO4 was added to the reverse micelle solution consisting of CTAB as a surfactant, octane as an oil phase, and 1-butanol as a cosurfactant. Then, NaBH4 was added to the solution, and the solution turned into green and then black, indicating the formation of iron nanoparticles. In the second step, HAuCl4 prepared in the same reverse micelle solution was added to the previous black solution. A micelle solution of NaBH4 was immediately added to the solution. Au (III) was reduced to Au (0) by NaBH4 and gold forms a coating on the outer surface of the iron particles. The iron-containing nanoparticles were separated by an external magnetic field. Manjili et al. (2014) investigated the effect of gold-coated iron nanoparticles in increasing the sensitivity of malignant cells to radiation and found that malignant cells treated with magnetic nanoparticles have decreased viability during

1.7 Zinc Oxide Nanoparticles

radiation in comparison with cells without nanoparticles. The study also demonstrated that cytotoxicity of gold-coated iron nanoparticles is not considerable at concentrations less than 20 μg/mL. The core
shell iron
gold nanoparticles prepared via the microemulsion process were surface-grafted with MTX (methotrexate) anticancer therapeutic and ICG (indocyanine green) fluorescent dye (Chung and Shih, 2014). The in vitro experiments verified that the nanoparticles were biocompatible; nonetheless, the Fe@Au-PSMA-ICG/MTX nanoparticles killed cancer cells via the MH mechanism and the release of MTX. Fe/SiO2 core
shell nanoparticles were prepared by a simple synthesis method in which the reaction was conducted in aqueous environments and at room temperature, resulting in Fe nanoparticles with a uniform size distribution (Yang et al., 2011). Firstly, Fe nanoparticles were synthesized at room temperature from FeCl3 using NaBH4 as a reducing agent and PdCl2 which serves as a nucleating agent. Oleic acid and citric acid were used as surface-capping agents. The Fe particle size was tailored by tuning the concentration ratio of iron ions to carboxylic acid groups. To coat the Fe nanoparticles with SiO2 layers, TEOS (tetraethyl orthosilicate) and APS (3-aminopropyl-trimethoxysilane) were added into the solution with Ar/H2 gas purging. The core
shell nanoparticles were magnetically collected from the solution. Fe/SiO2 composite covered with a thin SiO2 protection layer were synthesized as a carrier for magnetic drug targeting by using the sol
gel route and a chemical reduction method (Hsieh et al., 2015). Composite spheres prepared in this manner were made of metallic iron, and not an oxidized form, and exhibited strong magnetization. From the dye (FITC) release profile, it was confirmed that SiO2-Fe/ SiO2 composite spheres can act as a drug carrier. The results of the in vitro tests revealed that SiO2-Fe/SiO2 composite inhibited cell proliferation at a higher concentration, but was nontoxic at low concentration. The results of this study demonstrate that SiO2-Fe/SiO2 composite spheres, which are in a magnetic state and exhibit slow-release behavior, can act as a dual-functional agent in cancer therapy applications as hyperthermia and chemo agent carriers. Recently, mesoporous silica nanoparticles impregnated with zero-valent iron were investigated as nanocarrier systems for drug delivery into tumor cells (Shevtsov et al., 2016). In this case, zero-valent iron was used for labeling mesoporous silica nanoparticles for the detection of mesoporous silica nanoparticles by MR and for biodistribution studies. This study demonstrated that mesoporous silica nanoparticles can enter the blood
brain barrier and accumulate in tumor tissues.

1.7 ZINC OXIDE NANOPARTICLES Over the years, there is increasing interest in the examination of metal oxide nanoparticles because of their range of features such as: antibacterial agents

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(Sirelkhatim et al., 2015; Patrinoiu et al., 2016), biosensors (Shin et al., 2016; Tarlani et al., 2015; Hwa and Subramani, 2014), annihilation of carcinogenic cells (Tyagi et al., 2016; Souza et al., 2016), drug delivery purposes (Yadollahi et al., 2016; Chem, 2011; Bakrudeen et al., 2015), and catalytic materials for purification of waste waters (Habba et al., 2016; Taylor et al., 2013). Although the nanoparticles can be synthesized through a variety of physicochemical methods, they have their own disadvantages regarding the environment. Because of the environmental concern, most researchers focus their work in an eco-friendly way to sustain Earth. The morphological constitution of the nanomaterials presents a very important part in controlling the physical, chemical, optical, and electrical properties of the nanoparticles (Xu et al., 2007; Sirelkhatim et al., 2015; Stankovi´c et al., 2013). Among the metal oxide nanoparticles, zinc oxide (ZnO) nanoparticles are recognized as a developing field in the area of nanoscience research and development. It is known that the functional properties of zinc oxide nanomaterials depend on their physicochemical properties, such as optical properties and specific surface area, which are defined by structural characteristics, for example: the particle size and morphology, phase composition, crystallite size, crystallinity degree, as well as the crystal structure ordering, that is, the presence of lattice defects. All of these characteristics can be designed by optimizing the reaction conditions during the synthesis. ZnO nanoparticles were previously synthesized applying various chemical approaches such as: hydrothermal (Djuriˇsi´c et al., 2012), sonochemical (Khorsand Zak et al., 2013), solvothermal (Rai et al., 2013), sol
gel (Kitazawa et al., 2014), and mechanochemical methods (Stankovi´c et al., 2011). All of the above-mentioned synthesis procedures have their own drawbacks, such as instability and agglomeration of the prepared nanoparticles. To resolve these problems, most researchers have altered their research direction to the green synthesis methods of metal oxide nanoparticles (Gunalan et al., 2012; Patrinoiu et al., 2012; Jalal et al., 2010). Investigation of ZnO as an antibacterial agent started in the early 1950s. However, significant progressive examination began in 1995 (Shi et al., 2014). Sawai and colleagues stated that ZnO, MgO, and CaO powders had antibacterial activities against some bacterial strains (Sawai and Yoshikawa, 2004; Sawai et al., 1998; Sawai, 2003). Nowadays, progress in nanotechnology has supported the development of some novel antibacterial agents. A number of studies have shown that nanomaterials demonstrate enhanced antibacterial activity related to conventional materials (Raghupathi et al., 2011). ZnO nanoparticles reveal varying morphologies and show major antibacterial activity over a wide spectrum of bacterial strains discovered by many groups of researchers (Jalal et al., 2010; Seil and Webster, 2012; Stankovi´c et al., 2013). Nowadays, ZnO is investigated as a potential antibacterial agent equally in microscale and nanoscale formulations. It shows important antimicrobial activities when the particle size is reduced to the nanometer range. ZnO nanoparticles can

1.7 Zinc Oxide Nanoparticles

interact with bacterial membrane where it passes inside the cell, and afterward, exhibits different bactericidal mechanisms. The interactions among nanoparticles and bacteria cell are generally toxic, which can be exploited for antimicrobial applications, for example, in the food industry. Nevertheless, ZnO nanoparticles are testified by a number of studies as nontoxic to human cells. This aspect demanded their usage as antibacterial agents, toxic to microorganisms and biocompatible to human cells (Adamcakova-Dodd et al., 2014; Esparza-Gonza´lez et al., 2016). Different antibacterial mechanisms of ZnO nanoparticles are commonly attributed to their characteristic physicochemical properties and their high specific surface area-to-volume ratios (Seil et al., 2009). However, the precise antibacterial mechanisms are still currently under discussion, while a number of proposed ones are recommended and accepted. Bacterial infectious diseases present a continuously grown health problem that has focused the community attention worldwide as a serious human health danger, which could lead to financial and social difficulties. An increased number of epidemics and infections of pathogenic strains, bacterial antibiotic resistance, development of new bacterial mutations, the absence of an appropriate vaccine, and hospital-associated infections are a global health risk to the human population. For example, infections by Shigella flexneri cause 1.5 million deaths annually, due to contaminated food and drinks by these bacteria (Kotloff et al., 1999). Therefore, developing innovative antibacterial agents against bacteria strains, commonly important food pathogens, such as E. coli O157:H, Campylobacter jejuni, Staphylococcus aureus, P. aeruginosa, E. faecalis, Salmonella types, and Clostridium perfringens, has become an ultimate demand. The key food applications of ZnO nanoparticles are as an antibacterial agent in packaging materials (Sirelkhatim et al., 2015). In this way, the integration of ZnO nanoparticles into packing materials can reduce the quantity of antimicrobial agents directly in the food products. Additionally, ZnO nanoparticles can also play an important role in reducing pathogen contamination and extending the shelf-life of food products (Espitia et al., 2012). Nowadays, the aim of researchers is to develop novel solutions both for functional packaging (active packaging and nanocomposite materials) and low environmental influence (biodegradable materials, recyclable packaging with reduced size) (Pantani et al., 2013). The majority of materials used to conserve food are produced using synthetic polymers. Nevertheless, because of environmental pollution reasons, the attention of the researchers has recently been focused on biodegradable polymers for the preparation of food packaging materials (Bourtoom and Chinnan, 2008; Perez-Mateos et al., 2009; Findenig et al., 2012). These materials are commonly loaded with some antimicrobial agents that are in contact with food and take action on foodborne microorganisms inhibiting their growth (Gorrasi et al., 2012; Bugatti et al., 2010). Polylactic acid (PLA) and poly lactic-co-glycolic acid (PLGA) represent environmentally friendly, commercially available, and inexpensive polymers that provide huge potential as nonreusable packaging material (Madhavan Nampoothiri et al., 2010). ZnO nanoparticles are documented as environmentally friendly and

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multifunctional agents that could be considered as nanofillers for a range of polymers providing properties like antibacterial effects (Li et al., 2006). In this context, it is definitely of great interest to provide PLA or PLGA materials with antibacterial properties. With the addition of ZnO nanoparticles as fillers this could represent a significant advancement in materials research (Bussiere et al., 2012; Murariu et al., 2011; Stankovi´c et al., 2016). Unfortunately, the addition of untreated ZnO nanoparticles into PLA matrix at melt-processing temperature leads to almost total degradation of the polyester. In contrast, as reported (Murariu et al., 2011), if ZnO nanoparticles are sufficiently surface-treated with preferred ions, this can lead to polymer-based nanocomposite materials with very good maintenance of polymer fundamental molecular parameters and associated physicochemical characteristics. Diabetes is currently one of the primary causes of death and disability around the world. If the glucose concentration in the human blood is not controlled and regulated, life-threatening diseases, such as diabetic mellitus and acute diabetes, almost certainly develop (Sarangi et al., 2015). A diagnosis of diabetes means constant observing of glucose concentrations in the blood. Recently, significant efforts have been made in the improvement of highly sensitive glucose biosensors which consist of nanostructured materials (Yum et al., 2012; Wei et al., 2006; Asif et al., 2010). Along with a range of biosensor materials, ZnO represents one of the finest because of its chemical stability, nontoxicity, and major optical response, for example, the intense photoluminescence (PL). Also, ZnO has a high isoelectric point (IEP) of approximately about 9.5, which makes possible adsorption of low IEP materials, such as protein and glucose (Sarangi et al., 2015). Nowadays, there have been a number of attempts to create a significant number of glucose biosensors using ZnO nanomaterials. Most are enzymatic electrochemical sensors (Safavi et al., 2009; Kim et al., 2012; Asif et al., 2010). Immobilization of glucose results from the electrostatic interaction with ZnO molecule. Another in a series of important applications of ZnO nanoparticles is their anticancer activity. ZnO thin-film-coated chips with continual release of zinc ions are produced by accurate modification of ZnO thickness by atomic layer deposition. Their potential to release zinc ions relative to the number of deposition cycles can be estimated. ZnO chips exhibited selective cytotoxicity in human B lymphocyte Raji cells while having no effect on human peripheral blood mononuclear cells (Moon et al., 2016). The anticancer activity of ZnO nanomaterials or zinc ions might be explained by ZnO-induced cytotoxic and apoptotic activity. Zinc ions released from ZnO materials induce oxidative stress-mediated cell death (Buerki-Thurnherr et al., 2012; Petrochenko et al., 2014; Wahab et al., 2013), and the strong correlation between ZnO nanoparticle-induced cytotoxicity and free zinc ion concentration also suggests a requirement for ZnO dissolution for effective cytotoxicity (Shen et al., 2013). Consistently, extracts exhibit more cytotoxicity in suspended cells than do nanostructured ZnO chip coatings by themselves (Petrochenko et al., 2014). These findings motivate researchers to fabricate a

1.7 Zinc Oxide Nanoparticles

ZnO chip that gradually releases zinc ions and evaluate its anticancer activity. The cytotoxicity of the ZnO chip was compared to that of daunorubicin (an anticancer drug used to treat leukemia) in human B lymphocyte Raji cells (Moon et al., 2016). One of the major goals for tissue engineering today, especially in orthopedic and dental research, is to create a high-quality bone substitute in vitro, capable of repairing or replacing bone tissue, which may be used as a clinical alternative to an autograft, the present “gold standard” treatment (Grenho et al., 2015; Porter et al., 2009). Bacterial infection seriously limits the healing and regenerative capacity of tissue and remains a major limitation in the long-term utility of medical implants (Romano` et al., 2013). Therefore, it is of major importance to recognize an approach that could decrease bacterial growth without reducing mammalian cell functions. In the bone tissue engineering field, ceramic scaffolds are extensively studied as a result of their potential in regenerative medicine. Adhesion of microorganisms on biomaterials with subsequent formation of antibiotic-resistant biofilms is the most important factor in implant-related infections. Consequently, new approaches are necessary to resolve this problem. In some studies (Grenho et al., 2015), three-dimensional and interconnected porous granules of nanostructured hydroxyapatite (nano-HA) incorporated with different amounts of ZnO nanoparticles were produced using a simple polymer sponge replication method. Between the numbers of forms of calcium phosphate ceramics, hydroxyapatite (HA) has obtained significant attention due to its exceptional bioactive and osteoconductive properties as it bonds to the bone and increases bone tissue formation. Regarding ZnO nanoparticles, their high specific surface area, exceptional physiochemical properties, and improved surface reactivity (Nel, 2007) contribute to greater interactions with biological objects such as bacteria and host tissue (Seil and Webster, 2011). Additionally, ZnO particles at nanoscale dimensions with higher specific surface area have more enhanced antibacterial behavior (Raghupathi et al., 2011; Nair et al., 2009). The nanocrystals of ZnO with hexagonal (Wurtzite) structure belongs to a P63mc space group. In many cases, the application of ZnO is based on its direct wide bandgap (3.37 eV) and the large excitation binding energy (60 meV) at room temperature. However, an energy gap of 3.37 eV (368 nm) means that ZnO can only absorb UV light (Stankovi´c et al., 2012). ZnO nanoparticles have great transparency in the visible light range and also a good light doping affinity. Besides, it has several additional advantages, for example, good photocatalytic properties (Van Hoecke et al., 2011; Mary Jacob and Thomas, 2014; Mallika et al., 2014). The removal of inorganic, organic, and biological pollutants from drinking and wastewater represents one of the key steps in environmental protection. Methods such as biosorption, conventional activated sludge process, electrochemical technologies, and reverse osmosis have been applied to water treatment. In recent years a heterogeneous photocatalysis, as an efficient method for the decomposition and mineralization of pollutants from water, has been systematically studied and developed (Markovic et al., 2016). For heterogeneous

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photocatalysis, the mostly used materials to initiate the photoreaction are oxide semiconductor materials such as ZnO and TiO2. The ZnO nanoparticles can be used for degradation of toxic dyes like methylene blue, rhodamine B, malachite green, etc. (Madhumitha et al., 2016). There are some other methods which can be used to remove these toxic dyes from the environment, but they are expensive and time-consuming processes.

1.8 HYDROXYAPATITE NANOPARTICLES Contemporary materials science related to biomedical applications, and especially the fields of bone and dental tissue reconstruction, encompasses the third generation of biomaterials. Nowadays, the accent is not only on the single role of the implanting material but rather about its embedded multifunctionality and possibility to simultaneously act as a therapeutic and diagnostic agent. Moreover, understanding of the nature of interactions between implant materials and biologically important molecules, cells, and tissues, is highly appreciated to get a better insight in further perspectives. Physicochemical properties of artificial materials, chemical and phase composition, morphology and size of particles, surface charge, solubility, wettability, and water affinity, are extremely important since the foregoing processes, occurring immediately after implantation like protein adsorption, will be governed by the information transferred to the host cells. Hydroxyapatite (HAp) ceramics, with the chemical formula Ca10(PO4)6(OH)2, belonging to the family of calcium phosphate materials, were investigated for decades due to their great potential for biomedical applications. This originates from the chemical similarity with the mineral part of bone and dental tissues, itself consisting of carbonated HAp, which provides a reasonable standing point for the efforts invested toward material improvement with the final goal of synthesis a reliable and multifunctional material which can serve as an orthopedic and dental implant solution as well as a vehicle for administering appropriate therapy procedures. The possibility of preparation of HAp in the form of nanoparticles (NPs) has opened new opportunities and broadened the existing application spectra. However, the material itself has some drawbacks, especially in the mechanical performance, while its low bioresorbability in the living environment also represents an issue in specific applications. Some of these could be successfully overcome with progress in the synthesis and processing procedures, the right choice of calcium phosphate phase, as well as with appropriate composite design and functionalization of the basic material. The number of papers regarding synthesis procedures of HAp constantly increases, with small oscillations according to the most recent Scopus statistics. Among them, the major contributions were from materials science, engineering, chemical engineering, but also from medicine, pharmacy, and dentistry (Fig. 1.4).

1.8 Hydroxyapatite Nanoparticles

FIGURE 1.4 The distribution of papers related to HAp synthesis to different research areas from 1967 to 2017 according to Scopus.

The applied methods of HAp synthesis can be divided into several main groups, dry, wet, high-temperature, and biogenic procedures, as well as combinations thereof (Mehdi Sadat-Shojai, 2013). The chemical precipitation approach and its variations are probably the most often applied methods due to their simple setup, low cost, and high yield. However, this method arises as a very controldemanding approach, especially in the case of scale-up attempts, very often resulting in low reproducibility, nonstoichiometry, low crystallinity, etc. The application of simulated body fluid was found to be useful for the synthesis of “bone-like” elongated HAp NPs in a shorter time (Leena et al., 2016). Besides the chemical precipitation, hydrothermal processing is extensively used for the preparation of high-quality HAp NPs. The application of external physical fields is used to exchange the crystallization behavior of HAp nanocrystals, and it is shown to be an efficient tool to influence the crystallization of HAp NPs at its early stage and further thermal and sintering behavior of the material (Jani´cijevi´c et al., 2016). The physicochemical properties of the prepared HAp NPs are important regarding their exhibited cytotoxicity and cellular uptake (Cui et al., 2016), as well as for drug-delivery applications (Tabassum et al., 2016) and osteoblast cell response (Nathanael et al., 2016).

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The versatile functional behavior and application spectra of HAp are to a large extent influenced by its chemical nature. The hexagonal crystal structure of HAp mineral is constituted of tetrahedral phosphate groups, two structurally different types of calcium ions, and finally, hydroxyl groups positioned within the channels delimited by calcium atom triangles. The presence of hydroxyl groups in the HAp crystal structure is important for its chemical behavior and final applications: ionic substitutions in artificial and natural environments, interactions with organic compounds, NP stabilization, electrical conductivity, sintering behavior, etc. On the other hand, HAp crystals found in natural bone tissues are of nanometer size, and uniformly distributed within the organic collagen matrix. These facts clearly imply the importance of synthesis of HAp materials which possess nanostructured nature (the size of particles for powders or grains for sintered ceramics), with an increased surface to volume atomic ratio, but also the number of chemical functional groups at the surface. The process of nanostructuring of the HAp-based materials is directly reflected in the amount of surface-available hydroxyl groups, specific surface area, and consequently the higher surface energy and interaction potential. Having an apatite crystal structure, HAp can accommodate different ionic species at the positions of calcium ion, and/or phosphate and hydroxyl groups. Ionic substituents are usually mono-, di-, and trivalent ions, but also different functional groups, like SiO4, can be successfully hosted in the HAp crystal lattice, etc. Such ionic substitutions significantly influence the physicochemical properties of HAp NPs, like particle size, morphology, specific surface area, degree of crystallinity, as well as sintering behavior. The substitution in the crystal structure can also be from the incorporated carbonates from ambient conditions during the synthesis. Those changes further affect the electrical, magnetic, mechanical, and lightemitting functional properties of HAp NPs, but also the biological response reflected on osteogenesis and angiogenesis processes (Bose et al., 2013). Even the synthesis of HAp in the presence of different amounts of Zr41 ions, without doping of the HAp crystal structure, resulted in significantly changed particle size and morphology, as well as further sintering behavior (Luki´c et al., 2014). One of the targeted goals of ionic substitutions in the HAp crystal lattice is inducing the magnetic response of the reference material. Magnetic HAp NPs have found diverse applications in the field of molecular detection, magnetic imaging, drug delivery, and hyperthermia treatments (Colombo et al., 2012). The incorporation of magnetic atoms like Fe21/31 or Gd31 into the HAp crystal lattice generates a considerable magnetic response of HAp NPs. Those HAp-based materials can offer an alternative to the currently existing synthetic magnetic probes since they possess an additional advancement regarding biocompatibility, which is one of the major concerns of the magnetic probes suffering from inherent toxicity (Thomsen et al., 2006). Regarding magnetic resonant imaging, it has been shown that magnetic HAp could have even better performance compared to the commercial Gd-based contrast agents, without toxic effects (Ashokan et al., 2010). Magnetic HAp NP administration is also foreseen as a suitable approach

1.8 Hydroxyapatite Nanoparticles

for the delivery of different therapies and genetic materials, proteins, antibodies, and nucleic acids into the cells through the process of magnetic-assisted transfection (Kami et al., 2011). Furthermore, magnetic HAp NPs, successfully delivered to the targeted cancer cells, can be heated when the alternating external magnetic field is applied. This hyperthermic approach is used for precise elimination of tumor cells, which are less resistant to the temperature increase. In this context, HAp NPs with incorporated iron ions have a better response when compared to the composite material of HAp and ferrite (Inukai et al., 2011), which indicates the necessity of continual work on synthesis improvements in different HApbased chemical systems. It is important to note that effective application of magnetic HAp NPs for in vivo cancer treatment did not influence kidney function, but rather to be metabolized in the liver, with low cytotoxicity (Hou et al., 2009). Related to the other biological applications, it is worth mentioning that magnetic HAp is an efficient tool for plasmid, a small double-stranded DNA molecule, extraction from bacterial species, which is important in genetic technology as a cloning vector, but also for understanding of the bacterial resistance to antibiotics, UV irradiation, and heavy metals (Shan et al., 2012). Besides imparted magnetic behavior, HAp can serve as a host matrix for incorporation of different rare earth elements that can contribute to the light emission at different wavelengths, from visible to near infrared region (Pham et al., 2016). Moreover, within the same HAp NPs by simultaneous doping with Gd31 and Eu31, it is possible to impart several functional properties at the same time, like photoluminescence, drug delivery, and imaging modes (Chen et al., 2011). Recently, multifunctional magnetic and fluorescent HAp NPs were synthesized using Al(OH)3-stabilized MnFe2SO4 and Fe3O4 nanoparticles (Cui et al., 2015). HAp NPs are also important from the catalysis point of view since it is shown that some HAp-supported catalysts exhibit improved properties (Ho et al., 2016), and it could be envisioned as a support for in vitro study of catalysis of the biologically important processes. A recent example of the beneficial effect of a higher fraction of surface hydroxyl and carboxylic functional groups was found in aspartic acid-assisted Sr-substituted HAp NP assemblies, which exhibited strong luminescence, drug loading, and sustained drug-release properties (Park et al., 2016). The presence of light-emitting modality within the implant materials, as shown for Yb31/Ho31 codoped HAp NPs, is an extremely useful tool for longtime monitoring of the evolution of the bone reconstruction process, and tissueimplant material distribution and integration (Li and Chen, 2016). The improvement in the synthesis of HAp NPs is also important from the viewpoint of sintering, since preparation of implant materials that can supply a certain level of mechanical support requires a high-temperature consolidation process. Moreover, achieving fully dense, optically transparent HAp ceramics is suitable for making a model material for in situ and in vitro studies of biological processes that take place at the interface of implant material and the host environment. The combination of mineral and organic, collagen phase, in the natural bones, contributes to its piezoelectric properties (Fukada and Yasuda, 1957). This

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could be important for biological processes included in the constant remodeling of bone. That is the reason for the increased attention paid to the electrical properties of HAp in recent years. In the available studies, it has shown better bone healing and cell adhesion on artificial implant materials which possess an externally polarized surface (Gittings et al., 2009; Itoh et al., 2006; Nakamura et al., 2010). Although the electrical conductivity of HAp ceramics is very low, it can be enhanced by decreasing the grain size in dense ceramics (Luki´c et al., 2015), or through the preparation and processing of different composite materials with conducting phases (Dubey et al., 2013). An important area of HAp NP application is related to the controlled drugdelivery purposes. The application of HAp NPs for drug delivery requires careful synthesis and processing approaches to providing optimal materials performance. In this sense, HAp NPs can be used for the treatment of bone tissue diseases, but also for carriers of other therapeutic agents. Regarding bone disease treatments, it has been recently reviewed that appropriate combination of HAp NPs, especially porous structures, with growth factors can provide osteoconductive and osteoinductive behavior of implanted artificial bone fillers (Shi et al., 2015). Functionalization of HAp NPs with quercetin, which belongs to the group of flavonoids, for its local administration, is found to be beneficial for bone repair properties and enhancing antioxidant activity of HAp NPs (Forte et al., 2016). Also, a novel anticancer copper-based drug could be successfully encapsulated into porous HAp spheres to provide slow, pH-controlled release in a living environment (Weerasuriya et al., 2017). Insulin-loaded HAp was shown to be an effective material for controlled and long-lasting (4 days) delivery of insulin after a single intramuscular administration (Shyong et al., 2015). HAp nanorods and nanowires can be successfully used for intracellular delivery of proteins (Das and Jana, 2016). With the application of an appropriate processing method, by microwave heating, an improved release time from HAp/agarose composite in the form of nanosized powder, loaded with two different drug models, amoxicillin as antibiotic and 5-fluorouracil as an anticancer drug, can be achieved when compared to conventional processing (Kolanthai et al., 2016). Further improvement in the functional performance of HAp-based nanomaterials will be related to the understanding of the early stage of the nucleation process and growth of HAp crystals in the presence of important biological molecules, extracellular matrix, amino acids, and/or the specific polypeptide segments. In that direction, the synthesis protocols can be improved, and the possibility of functionalization of HAp NPs with therapeutic molecules in the early stages of particle growth can be achieved. This is not important only from the viewpoint of material properties, but also as a key to reveal biological calcifications and the onset of pathological processes. Furthermore, there is a large space for combining HAp NPs with versatile natural products like antioxidant compounds. Besides the direct application of HAp NPs in the therapeutic treatment of specific medical problems, this material has, maybe equally important, a role as a model material in artificial bioreactors, bone remodeling, and other kinds of research studies

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aiming to improve our current understanding of complex biological pathways. Having in mind that even some neurodegenerative diseases are presumed to be correlated with osteoporosis, and Ca21 signaling transduction, it is clear that HAp still offers a fruitful and useful research field in the area of materials for biomedical applications.

1.9 CONCLUSIONS Inorganic nanoparticles are very promising candidates for various biomedical applications. This is due to their remarkable features, which originate from their sizes in the nanoscale and unique physicochemical properties. This chapter describes various syntheses, properties, and applications of inorganic nanoparticles such as gold, silver, selenium, copper, iron, zinc oxide, and hydroxyapatite. In all synthesis procedures, there is a tendency to obtain stable, biocompatible nanoparticles by a simple, low-cost, and reproducible method. This chapter will also be a contribution to the development of commercial-scale production and further applications of inorganic nanoparticles.

ACKNOWLEDGMENTS This work was supported by a Grant Project III45004 from the Ministry of Education, Science and Technological Development of Serbia.

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2828. ˇ Stevanovi´c, M., Filipovi´c, M., Uskokovi´c, V., Skapin, S.D., Uskokovi´c, D., 2013. Composite PLGA/AgNpPGA/AscH nanospheres with combined osteoinductive, antioxidative and antimicrobial activities. ACS Appl. Mater. Interfaces 5 (18), 9034
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Further Reading

Yao, B.H., Xu, G.C., Zhang, H.Y., Han, X., 2010. Synthesis of nanosilver with polyvinylpyrrolidone (PVP) by microwave method. Chin. J. Inorg. Chem. 26, 1629
1632. Yong, Y., Yonezawa, T., Masaki, M., Hiroki, T., 2015. The mechanism of alkylaminestabilized copper fine particles towards improving the electrical conductivity of copper films at low sintering temperature. J. Mater. Chem. C 3, 5890
5895. Available from: https://doi.org/10.1039/C5TC00745C. Yoon, K.Y., et al., 2007. Susceptibility constants of Escherichia coli and Bacillus subtilis to silver and copper nanoparticles. Sci. Total Environ. 373 (2
3), 572
575. Yu, J., Chen, F., Gao, W., Ju, Y., Chu, X., Che, S., et al., 2017. Iron carbide nanoparticles: an innovative nanoplatform for biomedical applications. Nanoscale Horiz. Available from: https://doi.org/10.1039/C6NH00173D. Yum, K., et al., 2012. Boronic acid library for selective, reversible near-infrared fluorescence quenching of surfactant suspended single-walled carbon nanotubes in response to glucose. ACS Nano 6 (1), 819
830. Zain, N.M., Stapley, A.G.F., Shama, G., 2014. Green synthesis of silver and copper nanoparticles using ascorbic acid and chitosan for antimicrobial applications. Carbohydr. Polym. 112, 195
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31. Zhang, J., Gaoa, X.Y., Zhanga, L.D., Baob, Y.P., 2001. Biological effects of a nano red elemental selenium. Biofactors 15, 27
38. Zhang, G., Keita, B., Dolbecq, A., Mialane, P., Secheresse, F., Miserque, F., et al., 2007. Green chemistry-type one-step synthesis of silver nanostructures based on MoV
MoVI mixed-valence polyoxometalates. Chem. Mater. 19, 5821
5823. Zhang, Y., Xiaoling, L., Huang, Z., Zheng, W., Fan, C., Chen, T., 2013. Enhancement of cell permeabilization apoptosis-inducing activity of selenium nanoparticles by ATP surface decoration. Nanomedicine: NBM 9, 74
84. Zhou, J., Ralston, J., Sedev, R., Beattie, D.A., 2009. Functionalized gold nanoparticles: synthesis, structure and colloid stability. J. Colloid Interface Sci. 331 (2), 251
262. Zhou, M., Tian, M., Li, C., 2016. Copper-based nanomaterials for cancer imaging and therapy. Bioconjug. Chem. 27 (5), 1188
1199. Zhu, H.T., Lin, Y.S., Yin, Y.S., 2004a. A novel one-step chemical method for preparation of copper nanofluids. J. Colloid Interface Sci. 277 (1), 100
103. Zhu, H.T., Zhang, C.Y., Yin, Y.S., 2004b. Rapid synthesis of copper nanoparticles by sodium hypophosphite reduction in ethylene glycol under microwave irradiation. J. Cryst. Growth 270 (3
4), 722
728.

FURTHER READING Hsieh, C.T., Pan, C., Chen, W.Y., 2011. Synthesis of silver nanoparticles on carbon papers for electrochemical catalysts. J. Power Sourc. 196, 6055
6061.

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Kate, K., Singh, K., Khanna, P.K., 2011. Microwave formation of polypyrrole/Ag nanocomposite based on interfacial polymerization by use of AgNO3. Synth. React. Inorg. Met.
Org. Chem. 41, 199
202.

CHAPTER

Inorganic composites in biomedical engineering

2

Murthy Chavali1,2, Periasamy Palanisamy3, Maria P. Nikolova4, Ren-Jang Wu5, Ravisankar Tadiboyina6 and P.T.S.R.K. Prasada Rao7 1

Shree Velagapudi Ramakrishna Memorial College (SVRMC-PG Studies-Autonomous), Andhra Pradesh, India 2MCETRC, Tenali, Andhra Pradesh, India 3Department of Physics, Gnanamani College of Engineering, Namakkal, India 4Department of Material Science and Technology, University of Ruse “Angel Kanchev”, Ruse, Bulgaria 5Department of Applied Chemistry, College of Science, Providence University, Taichung City, Taiwan 6Aakash Educational Services Ltd. (Anna Nagar Branch), Chennai, India 7Department of Chemistry, P B Siddhartha College of Arts & Science, Vijayawada, India

2.1 INTRODUCTION AND BACKGROUND A composite material is made from two or more essential materials with knowingly different physical or chemical properties that, when combined, produce a material with characteristics different from the individual components. The argument still continues among engineers and materials scientists in explaining composite materials. More recently, the term composite has been utilized profusely by biomedical engineers for incipiently developed biomaterials, but this might not be applicable for biomaterial engineers rather than traditional engineers, who use the terms, for example, fibers, ceramics, reinforced plastics, matrices, plywood, fiberreinforced polymer or fiberglass and laminates. Composite materials are engineered from two or more fundamental materials, having different physical or chemical properties that remain fairly distinct at the bulk level. Thus, composite materials are always heterogeneous. In addition, an interface is always maintained between the phases. In composite materials, the distinct phase is dispersed through their bulk (Evans and Gregson, 1998). In composite materials, there are two main types of important materials: (1) dispersed phase and (2) matrix phase. The dispersed phases are responsible for enhancing the properties of the matrix, whereas the matrix or continuous phase is accountable for filling the volume and supports the dispersed materials. Almost all composite materials target a hasty improvement in matrix mechanical properties, such as elasticity, stiffness, strength, and flexibility; nonetheless, other properties, such as erosion stability, radiopacity, electrical properties, and thermal properties, are also of importance to scientists. This synergy creates enhanced properties within the integral product, which are not available with the discrete Materials for Biomedical Engineering: Inorganic Micro- and Nanostructures. DOI: https://doi.org/10.1016/B978-0-08-102814-8.00002-0 © 2019 Elsevier Inc. All rights reserved.

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constituents of the composite (Kickelbick, 2007; Matthew and Rawlings, 2000). Also, by regulating the volume fractions and arrangement of the dispersed phase, the properties can be altered or even customized to requirements suiting applications in relevant environments. Excluding pure elements, more or less each and everything in a composite material in one way or another contributes to its properties, for example, a piece of metal can be considered as a composite material (multicrystal) of many single crystals (grains) and become ceramic materials, alloys, steels, etc. (Schwartz, 1992). This raises a the question of how these macroconstituents were put together and their purpose? As an example, thin coatings, such as several layers of a material do not qualify as distinctive composites, and the same is true in adding resin spread fillers to various plastics, even though they are present at the macrostructure. Besides, a unit that was amassed with components made of dissimilar materials does not necessarily succeed as a composite material; on the other hand, foams and porous coatings are also not considered as composite materials. In one way, a catheter tube polymer that was reinforced with interlaced metallic wires in a polymeric sheath would be considered as a composite, but a pacemaker lead with a metallic core would not. Composite materials have two phases: 1. Continuous bulk phase: the matrix, and 2. Noncontinuous dispersed phase (may be one or more): the reinforcement. The reinforcement phase has greater mechanical or thermal properties to those of the matrix. The thin area between the phases is simply considered as an interface (third phase): an interphase. For example, facilitating adhesion of glass to a matrix polymer by a layer of coupling agent treated on glass fibers. Composites can be classified as shown in Fig. 2.1.

Composites

Structural

Laminates

Particle reinforced

Sandwich Large panels particles

Fiber reinforced

Dispersed Continuous particles (aligned)

Discontinuous (short)

Aligned

FIGURE 2.1 Classification of composites.

Random

2.1 Introduction and Background

The idea of the conception of composite materials may be ancient; based on its effectiveness the novel material is certainly unattainable by the combination of individual components or materials to produce the desired composite. A classical instance is mixing straw with fine clay for building stronger walls. In nature, examples include palm leaf, cellulose fibers/lignin matrix (wood), andcollagen fibers in the matrix (bone), etc. Recently, materials that have gained prominence include concrete with steel rods, fiberglass in resin, cement-asphalt and sand mixture, etc., which are the best-known examples. The core concept of composites is that the load is accepted by the bulk phase over larger surface areas that transfer it to the reinforcement phase. Being different from the bulk phase, the mechanical properties like stiffness, strength, toughness, or fatigue resistance change. For example, structural polymer composites have much tougher reinforcements, indicating a reduction in bulk strain on deformation and making the composite multiple orders harder than the bulk polymer. The significance is that there exist abundant matrix materials and countless reinforcement types to combine in a myriad of ways (permutations) producing materials with specific and selective properties. Most work in engineering composite materials has been carried over the past five to six decades (since the mid-1960s) most benefits being reducing weight and minimizing costs, measured in terms of ratio of strength/weight, stiffness/ weight, etc. However, tremendous progress in stiffness and strength is not always a concern in designing biomedical composites. Advanced biomedical composites already pay attention toward structural applications such as designing of dental and orthopadic implants, in combination with nanotechnology; it seems to have large-scale applications in almost every field. For example, nanotechnology in dentistry is increasing, with promising work related to nanocolloidal particles, prosthodontics, antibacterial nanofilms, etc. Biocompatibility, biological response, mimicking naturally existing structures, etc., are other central concerns, yet there are areas where composites show much promising potential to design. With diverse mechanical behaviors, physical characteristics, and processing methods and a possibility to tailor them, composite materials fascinate and challenge engineers and materials scientists, who typically work with old-fashioned materials, for example, metal-based alloys, ceramic composites, and different plastics, to design and develop novel composites (Evans and Gregson, 1998). Nanoparticles (NPs) have been particularly associtaed with various biomedical applications, for example, drug delivery systems, implants, strength-based prosthetics, diagnostic imaging, and molecular sensing (Jaspreet et al., 2005; Prasad, 2004; West and Halas, 2000, 2003; Holm et al., 2002). In this chapter, only inorganic particles used for biomedical applications are described. The characteristic dimension of NPs with a length of less than 100 nm has been shown a lot of interest for applications in the biomedical area, such as intravenous delivery, intracellular delivery, etc., where improved efficiency was be achieved using NPs (Chan, 2006; Hughes, 2005). Liposomes and polymeric drug carriers of less than 100 nm

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show increased permeability and localization of tumor spots as an application of NPs in cancer therapy, which was ascribed to the compact diffusive barrier for NPs that were estimated to be 100
600 nm (Allen, 2002; Panyam and Labhasetwar, 2003). Besides size, quantum effects and surface properties of NPs also influence their cell uptake and distribution (Moghimi et al., 2001). Cells have shown to larger interest to a more extended uptake of NPs with the more hydrophobic surface rather than hydrophilic surface both in endocytosis and phagocytosis. Surface modification of NPs is often necessary to improve stability, compatibility, transport, and functionality. Surfactants that serve as molecular linkers and improve particle stability of NPs have been engineered for the required surface characteristics (Love et al., 2005; Kossovsky et al., 1994; Caruso, 2002; Chan, 2006). Surfactants reduce the surface energy of NPs, and thus stability is enhanced. Functional groups on surfactants are responsible for enabling NP coupling with large biomolecules like antibodies (see Fig. 2.2). Surfacefunctionalized NPs also serve as drug carriers, with massive application potential for specific localization. Ceramics such as calcium phosphate [(Ca3(PO4)2], silica (SiO2), and titania (TiO2) are found to be biocompatible (Jin and Ye, 2007). Higher thermal and chemical stability than polymeric NPs is attained by the use of inorganic ceramic NPs. Ceramic particles provide a better defense of labile agents against denaturation (Hasirci et al., 2006). Bioactive ceramic has a biological affinity and also exhibits direct integration with bones in the case of bone defects. Nonetheless, their inadequate mechanical properties, such as low fracture toughness and high Young’s modulus, limit their clinical applications. Biodegradable polymers and bioactive ceramics are combined to form a variety of composite materials applied extensively in tissue engineering scaffolds. Biodegradable polyurethanes, formed of degradable polyester/polyether, contain a hydrophilic group of an ether bond, aliphatic diisocyanate, with the hydrophobic group from a chain extender (Guelcher and Gallagher, 2005; Kavlock and Pechar, 2007). Polyurethane has a controlled degradation rate due to the presence of special groups, which can be changed by changing the ratio of polyester/polyether to diisocyanate (Guelcher and Gallagher, 2005; Guelcher, 2008). The degradation byproduct must be nontoxic and not harmful to the body. To further control the degradation rate and pH of degradation products (avoiding the acid

Coating with functionalized nanoparticles

Biomolecule Inorganic particle

Inorganic particle

Inorganic particle

With functional groups and

FIGURE 2.2 Functional groups of surfactants assisting in coupling, such as nanoparticles with biomolecules, for example, antibodies.

2.1 Introduction and Background

autocatalytic effect), some researchers choose diamines (Luo and Wang, 2008), polyether urethane urea (PEUU) with PCL, and 1,4-diisocyanatobutane (BDI) and putrescine (Guan and Fujimoto, 2005). Highly pure lysine diisocyanate along with glucose reacted with synthesize polyurethane that resulted in complete degradation products, for example, lysine and glucose (LDI glucose), and entered into the human circulation system (Zhang and Beckman, 2002). Polyurethane bioactive functionalization methods are identified with three major design strategies (Bil and Ryszkowska, 2007; Chetty and Steynberg, 2008; Vitale-Brovarone and Verne, 2007), as described here. 1. Blending these polyurethanes with Ca3(PO4)2/hydroxyapatite or other inorganic ceramics (Bil and Ryszkowska, 2007; Chetty and Steynberg, 2008; Huang and Miao, 2007; Vitale-Brovarone and Verne, 2007). In this inorganic ceramic, bioactive factors further enhance the cellular compatibility and display another advantage: bone induction and conduction. 2. Providing the biomaterials [integrating soluble bioactive molecules (growth factors, plasmid DNA) with bioactivity] bioactive molecules can trigger or modulate new tissue formation (Whitaker and Quirk, 2001; Richardson and Murphy, 2001; Babensee and McIntire, 2000). 3. Incorporation of cell-binding peptides [native long chain of extracellular matrix (ECM) proteins and short peptide sequences derived from ECM proteins] into biomaterials via chemical or physical modification. These can include specific interactions with cell receptors (Suzuki and Tanihara, 2000; Buket-Basmanav and Kose, 2008). Polyhydroxyalkanoates (PHAs) are evolving as a class of biodegradable polymers forseeing applications in tissue engineering that can be combined with numerous monomers to give materials with extremely different properties as an essential component for preparing a class of composites—bioplastics. There are naturally produced by numerous microorganisms through bacterial fermentation of sugar or lipids; they also serve as a source of energy and a carbon store. These plastics can be thermoplastic or elastomeric type materials, having melting points in the range between 40 C and 180 C (Chen and Wu, 2005). The PHAs family includes an extensive range of materials, as it can be combined with numerous monomers from hard/brittle to soft and elastomeric materials. Numerous approaches have been used to understand the biomedical applications of PHAs as cardiovascular patches, dressing wounds, sutures, and tissue repair/regeneration/ engineering scaffolds (Superb et al., 2006). Innovative PHA composites were designed in combination with inorganic materials to improve the mechanical properties, for example, the rate of degradation, and also impart bioactivity. Poly(3-hydroxybutyrate), poly(3-hydroxybutyrate-co-3-hydroxyvalerate), and poly(3-hydroxybutyrate-co-3-hydroxy hexanoate) and a few polymers have been investigated thoroughly to produce different composites in combination with HA, bioactive glass, and glass 2 ceramic fillers and coatings (Chen and Luo, 2009). PHA biocomposites along with fibers of

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Posidonia oceanica (PO) were developed and their processability was assessed by extrusion, toward mechanical properties, and potential biodegradability in marine environs. PHAs were successful in compounding up to 20 wt.% with PO fibers, while, at 30 wt.% of fibers, the addition of 10 wt.% of PEG 400 was required to improve processability (Maurizia et al., 2017). Advanced nanofibers are concentrated on different types of applications, for example, water filtration, solar cells, sensors, smart textiles, tissue engineering, etc. Over the past decade, researchers have been exploring scaffolds, akin to the natural extracellular matrix, for applications in tissue engineering. With the ease of the amalgamation of drugs, natural materials, growth factors, and inorganic NPs into these nanofiber scaffolds, electrospun nanofibers have possible advantages in tissue engineering and regenerative medicine.

2.2 CATEGORIZATION The aspects contributing most to the function of the composite comprise: 1. Materials (individual components); 2. The arrangement of the components, orientation, and form; 3. The interaction between them. The reinforcement system within a composite material is responsible for determining the properties achievable by a composite, thus becoming appropriate in classifying these composites according to the reinforcement characteristics, such as shape, distribution, size, composition, orientation, etc. For the purpose of an argument on biomedical composites, this results in two broad groups, namely, fiber-reinforced and particle-reinforced composites. Another broad classification of composite materials is simply based on the matrix material used (see Fig. 2.3), which is often completed for processing rather than for performance purposes, these are: Metal matrix composites (MMC) Composite materials

Matrices

Ceramic matrix composites (CMC)

Thermoset

Polymer matrix composites (PMC)

Thermoplastic

Rubber

FIGURE 2.3 Classification of composite materials simply based on the matrix material.

2.3 Components

1. Polymer-matrix composites (PMCs); 2. Ceramic-matrix composites (CMCs); and 3. Metal-matrix composites (MMCs). The latter class (MMC) is a progressive composite, which is very unusual in biomedical applications as it is mostly used for applications with high pressure and high temperature. In addition, there is another entry for the newly developed class of composites: carbon and graphene-matrix composites (CGMCs).

2.3 COMPONENTS Composite materials have four important constituents as described elaborately below.

2.3.1 MATRICES Within a composite material, the reinforcement phase is either completely or partially engulfed by the matrix, the continuous bulk phase. Matrix assists quite a lot of important functions such as possession of fibers or particles in one place, and highly oriented composites preserve any chosen direction of fibers (Schwartz, 1992). The matrix helps in redistributing the stress by transferring the applied load to the reinforcement and also helps increase fracture toughness when used with brittle fibers, typical of a lower-stiffness material tolerating better elongation and shear forces more than the reinforcement. Here, the matrix plays an important role in determining the environmental durability of the composite through chemical resistance, hygroscopic, thermal stresses and protecting the reinforcement from these forms. With these properties, the matrix prominently affects the processing characteristics of a composite material. The matrices commonly employed in biomedical composites are listed in Table 2.1. In nonmedical uses, thermosets are the majority of matrix materials, mainly to be used in structural, automotive, automobile, aerospace applications, etc., where Table 2.1 Common Matrices in Biomedical Composites Type

Matrix

Inorganic

Glass ceramics, hydroxyapatite, calcium carbonate ceramics, carbon, steel, calcium phosphate ceramics, ceramics UHMWPE, polycarbonate, polyolefins (PP, PE), polysulfones, poly(ether ketones), polyesters Epoxy, polymethacrylates, polyesters, polyacrylates, silicon Polydioxanone, polyglycolide and their copolymers, polylactide, poly (hydroxybutyrate), alginate, collagen

Thermoplastics Thermosets Resorbable polymer

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high stiffness, anticorrosion, and temperature resistance are very significant requirements. In most medical applications, thermoplastics are still the matrix materials of choice as they are less or nonreactive in nature, flexible while processing, and have great toughness. Moreover, thermosets are nondegradable, while some biodegradable thermoplastics are a possibility. Instead of existing materials, novel matrix materials are continuously produced for numerous biomedical applications which may have desired reactivity, flexibility, strength, etc., as per the requirement. Resorbable matrices are beneficial when a permanent need of composite is questionable after implantation, this could be very exciting and challenging in designing a stiff reinforcing material that is analogous to such matrices in degradation rates. In such situations, ceramic matrices are often employed for their comprehensive properties and a wide range of bioactive possibilities which always have poor fracture toughness.

2.3.2 FIBERS Most of these materials are tougher and stiffer in their fibrous form compared to any other form, thus explaining the importance on expending fibers in designing composite materials, primarily toward structural applications, which are the chief components bearing the load. Fiber contributes to high tensile strength enhancing properties in the final part, such as strength and stiffness while minimizing weight, in a composite. The structural properties of composite materials are a result of fiber reinforcement. Fiber properties are controlled by their development process/techniques and the ingredients and coatings applied within the process. Many properties of a composite are due to its length, orientation, and volume fraction. Fibers have a high aspect ratio (length to diameter) compared to particles and whiskers; the smaller the diameter, the greater the strength of the fiber, due to a reduction in surface flaws. The vast majority of all fibers used in the composites industry are made of different types of glass. Depending upon the glass type used, filament diameter, sizing chemistry, and fiber form, a wide range of properties and performance levels can be achieved. Glass fibers are the oldest, and the most vernacular reinforcement cast in nonaerospace applications replacing heavier metal parts (as glass weighs more than carbon, and is not as stiff as carbon content), which is more impact-resistant and has higher elongation. Common fibers in biomedical composites are listed in Table 2.2. Table 2.2 Common Fibers in Biomedical Composites Type

Fibers

Inorganic Resorbable polymers Polymers

Carbon, hydroxyapatite, glass, tricalcium phosphate Silk, collagen, polylactide, and its copolymers PTFE, polyesters, UHMWPE, polyolefins

2.3 Components

Fibers are regularly developed/produced/manufactured as continuous filaments, with diameters ranging between 5 and 50 μm, prepared to form rovings, yarns, strands, mats, etc. Fabricating continuous-fiber composites is often toward large structural applications, in any case, filaments can be dissected to form short fibers as required for the applications, usually ranging in length from 3 to 50 mm to make discontinuous or short fiber composites. Such fibers are normally used for low-cost applications or smaller parts. Whiskers, for example, are singlecrystal fibers with very smaller diameters (B10 μm), but with high aspect ratios ( . 100), which also possess high strength alongside high manufacturing costs (Mallick, 1997). Compared to continuous fiber composites; short fiber composites are much less widely utilized with a preferred orientation. They have less design limitations and promising processing potential and are close to achieving their theoretical strength (Schwartz, 1992). The orientation of the short fibers (both continuous and noncontinuous) can be in 1D, 2D, or 3D, ensuing unidirectional, completely planar, and random reinforcement systems. The volume fraction of fibers strongly oriented in a chosen direction is primarily responsible for the physical properties of a composite. Anisotropy is exhibited by unidirectional planar reinforced composites, that is, their properties vary depending on the measurement axis. The third composite type has equal properties in all directions, and is isotropic. Orienting short fibers is difficult, as described above, mostly in mold-filling procedures, and the resultant composites tend to be isotropic. A type of fiberreinforced composite, a laminate composite consists of anisotropic layers or plies bonded with each other and varying both in volume fraction and relative fiber orientation, allowing high fiber-volume fractions and 3D orientation, which are not attainable in isotropic short fiber composites. There are countless naturally occurring fibers produced by plants, animals, and even geological processes (John and Thomas, 2008), such as cotton, jute, collagen, flax, wood, hemp, wool, hair, silk, etc., but these show exceptionally wide-ranging properties while presenting several processing challenges. Natural fibers show promise as biomaterials in medical applications. For example, chitin-based materials have been used in several medical applications, including bone-filling material in tissue regeneration, as a drug carrier and an excipient, and as an antitumor agent (Temenoff and Mikos, 2008). The insertion of foreign material can be either positive or negative, depending on the body’s response to the material. Naturally synthesized proteins, such as keratin, when implanted, have huge potential to be recognized as natural tissues, leading to integration and forming a superstructure. Among these are collagen fibers utilized in tissue engineering of skin and ligament; borosilicate glass fiber is omnipresent in industry but uncommon in biomedical composites, instead, adsorbable bio-glass fibers made from Ca3(PO4)2 have found a relatively good number of applications. Carbon fiber is comparable to glass fiber strength but is several times higher in stiffness owing to its fine structure with axially aligned graphite crystallites and being lighter than glass, and is extensively used to make high-

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strength lightweight prosthetics, carbon-fiber composites with fatigue resistance are very advantageous (Wise, 1996). Brittle carbon fibers are anisotropic, mostly in thermal, electrical conductivity of a composite, but can also have corrosive effects similar to metallic implants. Orthopedic applications require high resistance to impact fracture; among polymers, highly oriented aramid Kevlar fibers were suitable but their meager compressive properties make it unsuitable in bending applications and process difficulty owing to their strong cut-through resistance. Therefore, it is useful to manufacture gloves, sleeves, jackets, and other articles of clothing designed to protect users from cuts, abrasions, and heat. Flexible vascular prostheses are made from Teflon and polyester (DACRON) fibers. Polylactide and polyglycolide and their copolymers were also used to make fiber composites for applications where adsorbability is more vital than mechanical properties.

2.3.3 PARTICLES Particles are mixed into a matrix to further enhance the mechanical properties. Additional properties, such as electrical insulation, dimensional stability, and thermal conductivity can also be precisely maintained by particles, especially when added to polymer matrices. Particles can either strengthen or weaken a matrix depending on its shape, stiffness, and bond strength with the matrix. Particulate reinforcement is distributed randomly in a matrix, resulting in isotropic composites. Shape-wise, spherical particles are less effective than platelets or flakes in adding stiffness. Hard particles in a low-modulus polymer typically increase stiffness, whereas particles like silicone rubber, increase flexibility, resulting in a softer composite when added to a stiff polymer matrix. Reinforcement efficiency of bone ash and bone particles on the mechanical properties of polyester matrix composites has been examined. Cow bone ash and bone particle-reinforced tensile and flexural composite samples were developed for the purpose of biocompatibility, which is an essential requirement in biocomposites that are to be used as implants in the human body and demonstrate the best flexural properties (Isiaka, 2013). Copolymer poly(DL-lactide-co-glycolide) (PLGA)/ZnO composite materials have been investigated for various biomedical applications, such as controlled drug delivery or carriers in tissue engineering dental composites, as a constituent of creams for the treatment of a variety of skin irritations, to enhance the antibacterial activity of different medicaments (Ana et al., 2016). Particulate reinforcement support in biomedical composites is broadly utilized for dental ceramics and analog bone applications. The foremost common particle was hydroxyapatite (HA), a naturally occurring mineral component for bone, which exists in a composite structure along with collagen with poor mechanical properties and might serve more as a bioactive component than as a reinforcement component.

2.4 Preparation of Composites

2.3.4 INTERFACE The resulting composite material mechanical properties are significantly dependent on the interface between the matrix and reinforcing phases. Understanding the interfaces will improve the fabrication mechanics, fatigue, stability, and fracture behavior of fiber composites. It is presumed that the interface, as a region segregating the fiber from the matrix, will be less than one atomic layer thick (coherent interface), consisting of mechanical locking or chemical bonding between phases. Often it consists of a reaction layer and thus comprises one or more different phases. For composite containing coated fibers there can be more than one interface. A third entity, the interface, although smaller in size in almost all the composites, plays a vital role in both the fabrication and behavior of the material in service (Salkind, 1968). The interface can be studied by a number of surface analysis techniques and the interaction is being modeled. The interface aids in the transfer and dissemination of stresses from the matrix to the fibers or particles, etc. Final composite properties, retention of the property, and durability are purely dependent on the area of the interface and the interfacial bond strength. The property of wetting can be improved by different processing methods, with greater pressure (metal matrices, MMC) or lower-viscosity flow (polymer matrices). 1. Poor wetting of fiber with matrix material is due to the low interfacial area. 2. The sequence and relative magnitude (different failure mechanisms) in composites are also due to the interfacial shear strength that determines the fiber
matrix debonding process. 3. Strong interfaces that are common in PMC make ductile matrices stiff while lowering fracture toughness. 4. Weak interfaces in CMC cause brittle matrices, which become tough by encouraging matrix crack promotion while lowering strength and stiffness (Gdoutos et al., 2000). 5. When the mechanical coupling is not sufficient, coupling agents are often used to coat fibers, hence improving chemical compatibility.

2.4 PREPARATION OF COMPOSITES 2.4.1 COMPOSITES BASED ON POLYMER MATRIX Polymer matrix composite (PMC) is the material consisting of a polymer (resin) matrix combined with a fibrous reinforcing dispersed phase. PMC material uses the organic polymer as the matrix and fiber as the reinforcement. The strength and modulus of fiber are typically much higher than the matrix material. PMCs are very popular due to their low cost and simple fabrication methods and they

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possess high tensile strength, stiffness, fracture toughness, good abrasion, puncture, and corrosion resistance, while being low cost. PMCs are limited by their disadvantages such as high coefficient of thermal expansion and low thermal resistance. The properties of PMCs may be estimated by the rule of mixtures but are controlled by the properties of the fibers, like orientation, fiber concentration, and matrix properties. Two classes of polymer are used as matrix materials for fabrication of PMCs: thermosets (epoxies, phenolics) and thermoplastics [lowdensity polyethene (LDPE), high-density polyethene (HDPE), polypropylene, nylon, and acrylics], which are used in manufacturing applications such as secondary load-bearing aerospace structures, canoes, kayaks, boat bodies, automotive parts, radio-controlled vehicles, sport goods (golf clubs, skis, tennis rackets, fishing rods), bullet-proof vests and other armor parts, and brake and clutch linings. Continuous-fiber composites can be produced by simple physical laminates layup, protrusion, filament winding, and resin transfer molding. 1. Manual layup contains stacking preimpregnated tapes and sheets of parallel fiber filaments held jointly by a thermoplastic resin/partially cured thermoset resin, followed by autoclaving (neither expensive nor suitable for medical implants). 2. Filament winding, a fabrication technique mainly used for manufacturing open (cylinders) or closed-end structures (pressure vessels or tanks) mainly involves winding filaments under tension over a rotating mandrel. This is well suited for automation allowing high fiber-volume fraction, control of properties, and is limited by tubular shapes, and the fibers. 3. Pultrusion, a continuous process for the manufacture of composite materials with constant cross-section, is perfect for exceptionally firm composite rods/ beams, and can be utilized for creating orthodontic archwires. Resin transfer molding permits highly complex shapes and short spanned cycles, requiring the professional design of preforms and molds. 4. Compression molding is suitable for thermosets as well as thermoplastics but has to be preheated. In this process a plastic material is placed directly into a heated metal mold, softened by the heat, and then forced to conform to the shape of the mold as the mold closes. Short-fiber thermoplastic composites are naturally injection molded, toward quick, high volume, and production costs, and limited by length and volume fractions, expensive tooling, and very difficult to control fiber orientation and distribution.

2.4.2 COMPOSITES BASED ON CERAMIC MATRIX Fabrication of these CMCs is usually done by both pressing methods and infiltration methods. 1. In the pressing method, the reinforcement was thoroughly mixed with a powder of the matrix, further densified by hot pressing/hot isotactic pressing (HIP). Near-zero porosity is possible; the instantaneous high pressure and

2.5 Properties of Composites

temperature can degrade fibers or form a strong interfacial bond diminishing fracture toughness and durability. 2. In the infiltration method, a preformed or sintered fiber is completely filled by the matrix, such synthesis techniques include chemical vapor deposition (CVD), glass melt infiltration (GMI), and preceramic polymer or sol infiltration. Alumina-glass dental composites are prepared by this infiltration method, which is highly advantageous because of its complex shape capability, low pressure, and flexible fiber architecture. Disadvantages include that the matrix porosity is higher ( . 10%) and it involves very lengthy production cycles. Reinforcement of the particulates is done by tape-casting and slip-casting the initial preform.

2.5 PROPERTIES OF COMPOSITES Composites are increasingly produced for a multitude of tasks, for exampls, fiberreinforced composites are made to replace materials such as metals and alloys. Composite materials can be attained with a wide variety of physical, chemical, and biological properties, making it imperative to create prescient models to assist in planning with the numerous complex factors which confront engineers in composite building. Composites always offer reduced weight, stiffness, and strength, resistance against fatigue, low coefficient of expansion, ease in fabricating complex shapes, modest repair of damaged structures, resistance to corrosion, etc. In spite of the fact that these tend to be complicated by their microstructure, it is imperative to resist the temptation to treat a composite as a “black box” with wider improvements since this macroscopic approach does not have prescient control which is pivotal to avoid disappointment in the analysis. In anticipating the fundamental mechanical properties of the composite, there were three common models/approaches (Mallick et al., 2000; Gdoutos et al., 2000): 1. Mechanics; 2. Theory of elasticity; 3. Semiempirical. The mechanics of a materials model employ basic expository conditions (simple analytical equations) to reach successful shortening expectations about the stress and strain distribution of the representative component of the composite. This approach comes about where properties are related to the volume fraction of fibers and matrix, within the common rule of mixture equations for composites. Physical parameters, like density, can easily be calculated by the following equations: Vf 1 Vm 1 Vv 5 1

(2.1)

ρc 5 ρf Vf 1 ρm Vm

(2.2)

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where, Vf, Vm, and Vv are the volume fractions of the fiber, matrix, and voids, respectively, and similarly, ρc, ρf, and ρm are different component densities, fiber, and matrix. The rule of mixtures is actually advantageous; coarsely estimates an oriented tough fibrous composite, upper and lower bounds of mechanical properties, where the matrix is isotropic and the fiber is orthotropic, with coordinate 1, where the principal fiber direction and coordinate 2, transversing it. 1. For the upper bound, the Voight model was applied, where it is assumed that the fiber and matrix have the same strain. 2. For the lower bound, the Reuss model was applied, where it is assumed that the fiber and matrix have the same stress. This results in the following equations for the composite moduli: E1c 5 E1f Vf 1 Em Vm

(2.3)

1=E2c 5 Vf =E2f 1 Vm =Em

(2.4)

1=G12c 5 Vf =G12f 1 Vm =Gm

(2.5)

where, G and E are Shear modulus and Young’s modulus, respectively. The equations for transverse modulus and shear modulus are acknowledged as the inverse law of mixtures. Very few fibers, such as carbon, have diverse properties along their longitudinal and transverse axes that the preceding equations can take into account. The rule of mixtures equations also have numerous drawbacks. The iso-strain assumption within the Voight model infers strain compatibility between the phases, which is unlikely because of the dissimilar Poisson’s contractions of the phases. As fibers cannot be treated as a sheet, the iso-stress assumption in the Reuss model is also unrealistic. A fibrous composite’s ultimate tensile strength, σm  depends on whether the failure is dominated by the fiber component or the matrix component. The latter is very common when Vf is small. One result of such a treatment is σc  5 σm  =Em E1f Vf 1 σm  Vm

(2.6)



where, σm is the fracture strength of the matrix. The additional results for orthotropic composites obtained from this simple analytical approach are: α1c 5 E1f α1f Vf 1 Em αm Vm =E1f Vf 1 Em Vm
 Cc 5 1=ρc ρf Cf Vf 1 ρm Cm Vm

(2.7)

K1c 5 Kf Vf 1 Km Vm

(2.9)

(2.8)

where α is the coefficient of thermal expansion, C, the specific heat, and K, the thermal conductivity. The coefficient of hygroscopic expansion, β can be obtained by substituting α with β above. These results are for longitudinal directions only.

2.5 Properties of Composites

In an elasticity model, there were no assumptions made per unit volume around the stress and strain distributions. The difference in Poisson’s ratio between the fiber and matrix phases consider the specific fiber-packing geometry. At each point in the composite, the equations of elasticity are to be satisfied, and numerical solutions are commonly required for the complex geometries of the representative volume components. Then, estimating by the rule of mixtures, such a treatment offers tighter upper and lower bounds on the elastic properties. In semiempirical models, towards predicting experimental results, curvefitting parameters were used. The popular common model was established by Halpin and Kardos (1976; Suarez et al., 1986), and further, it has been enhanced toward aligned discontinuous fiber composites to produce such results for the longitudinal modulus, as follows: E1c =Em 5 1 1 ξηVf =1 2 ηVf     η 5 E1f =Em 2 1 = E1f =Em 1 η

(2.10) (2.11)

and the Halpin
Tsai curve-fitting parameter was assumed to be ξ 5 2L/d, where, L is the length and d, the diameter of the fiber. From this expression, E2c and G12 can be obtained using experimental values for ξ by simple substitution. For 2D randomly oriented fibers, approximating theory of elasticity equations with experimental results yielded an equation for the planar isotropic composite stiffness and shear modulus in terms of the longitudinal and transverse moduli of an identical but aligned composite system with fibers of the same aspect ratio: Ec 5 3=8 E1 1 5=8 E2

(2.12)

Gc 5 1=8 E1 1 1=4 E2

(2.13)

For a set of the 3D random orientation of fibers, a somewhat different equation was proposed for the isotropic tensile modulus: Ec 5 1=5 E1 1 4=5 E2

(2.14)

The stiffness of paniculate composites projected depending on the shape of the particles (Christensen, 1979). For rigid spherical particles in very dilute concentrations, the composite stiffness can be estimated by     Ec 5 5 Ep -Em Vp =3 1 2Ep =Em 1 Em

(2.15)

where, Ep and Vp are the stiffness and volume fraction of particles, respectively. In any model to understand composite behavior requires experimental validation, and might also prove to be relatively imprecise by not accounting for various irregularities that are representative in composite design and processing. Furthermore, these results are most typically valid for static and short-term loading.

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2.6 ANOMALIES 2.6.1 FRACTURE AND FATIGUE FAILURE An increase in different types of internal damages which gradually or tragically render a composite unsafe cause failure of fiber-reinforced composites. The damage can be induced (process or service), for example, nonuniform curing control of a dental resin related to process-induced damage and unwanted water absorption related to service-induced damage. Fiber breaking, fiber bridging, fiber pullout, matrix cracking, and interface debonding are some of the common failure mechanisms in composites, the type of loading and the properties of the constituents initiate the sequence and interaction of these mechanisms, as well as the interfacial shear strength. Energy absorption and crack deflection during fracture lead to an increase in the toughness, brittleness, and subsequent failure of the composite. Failure analysis of composites is explained by various fracture mechanics theories, between them the maimum stress theory and the maximum strain theories are predominant (Gdoutos et al., 2000). The two most vital energy-absorbing failure mechanisms in a fiber-reinforced composite are: 1. Debonding at the fiber
matrix interface, where the crack propagation is interrupted by the debonding process if the interface bonds properly; cracking, instead of moving through the fiber, is deflected to the surface, allowing the fiber to carry higher loads; 2. Fiber pull-out, where fibers do not break at the crack planes but at random locations away from this plane; pull-out occurs due to interfacial high frictional forces or shear stresses that may significantly increase fracture toughness (Mallick, 1997). In the case of particulate reinforcements, the same holds true, in which due to the smaller aspect ratio, crack deflection is more often seen than bridging and pull-out. In an Al2O3-glass composite, indentation cracks in glass, the matrix is deflected around the angular Al2O3 granules, and the cracks propagate more with the granules, indicating a stronger interface and lower toughness. A common failure mechanism in laminate composites is due to delamination loads amongst the layers, initiating at the free edge of the plate or a hole. Bone tissue has a hierarchical organization over length scales, with magnitude spanning from the macro-scale (centimeter) to the nanostructured (extracellular matrix or ECM) components and arranges itself either in a compact pattern (cortical bone) or a trabecular pattern (cancellous bone) (Ackerman and Spjut, 1976). Bone ECM comprises both predominantly type 1 collagen (a nonmineralized organic component) and 4 nm thick plate-like carbonated apatite minerality (a mineralized inorganic component) (Weiner and Wagner, 1998). Also, over 200 diverse types of noncollagenous matrix proteins (glycoproteins, proteoglycans, and sialoproteins) contribute to the abundance of signals in the immediate

2.6 Anomalies

extracellular environs. The nanocomposite structure [tough and flexible collagen fibers reinforced with hydroxyapatite (HA) crystals] is integral to the requisite compressive strength and high fracture toughness of the bone. Biomaterial-based treatments in orthopedics are growing at a swift rate. Bone and joint degenerative/inflammatory complications affect many people all over the world and will double by 2020. In addition, plentiful bone fractures, osteoporosis, spasms, low back pain, scoliosis, and other musculoskeletal problems need to be solved by different permanent, temporary, or biodegradable devices or implants. Hence, orthopedic biomaterials are destined to be implanted in the human body to perform certain biological functions by substituting or repairing different tissues such as bone, cartilage, or ligaments and tendons, and even by guiding bone repair when necessary. Previously, implantation was intended to be performed with “bio-inert” materials; scientists have now shifted deliberately to “bioactive” materials that integrate with most biological molecules or cells and regenerate tissues (Langer and Vacanti, 1993; Hench and Polak, 2002). Many bone substitute materials have been evaluated over the last two decades, and the results are bioactive ceramics, bioactive glasses, biological or synthetic polymers, and composites of these (Hench and Polak, 2002; Kretlow and Mikos, 2007; Liu and Czernuszka, 2007). It is thought that the materials will be replaced over time in tune with newly regenerated biological tissue, so there is a need for materials with mechanical properties more closely mimicking bones/tissues to be developed (Langer and Vacanti, 1993). Biodegradable and bioactive porous polymer/inorganic composite scaffolds, novel bioactive materials with different mechanical properties, and bioactive ceramics are being combined in a variety of composite materials for tissue engineering scaffolds. Bioactive inorganic materials are especially suited for use as hard-tissue scaffolds, as they can provide significant mechanical support. Bioceramics are usually created as dental implants based on alumina and zirconia, hydroxyapatite, and resorbable calcium phosphates. A varied range of bioactive inorganic materials that are similar in composition to the mineral phase of bone is of utmost clinical interest, such as tricalcium phosphate [Ca3(PO4)2], hydroxyapatite , a naturally occurring mineral form of calcium apatite—Ca5(PO4)3(OH)—bioactive glasses, a group of surface-reactive glassceramic biomaterials, and their combinations (Hench and Polak, 2002; Zijderveld et al., 2005). Bioactive glasses (Ca- and possibly P- containing SiO2 glasses), for example, when immersed in a biological fluid, can rapidly develop a bioactive hydroxycarbonate apatite layer that can readily bond to biological tissue. Additionally, these can be tailored to deliver ions, such as Si, at levels capable of activating complex gene transduction pathways leading to enhanced cell differentiation and osteogenesis (Hench and Polak, 2002; Jell and Stevens, 2006; Tsigkou et al., 2007). The resorption rate of bioactive glasses and bioceramics can be altered with crystalline HA persisting for years, while other calcium phosphates have a greater capacity to be resorbed but less strength to sustain the load. Bioactive inorganic material brittleness indicates a mismatch with the bone’s fracture toughness and is not suitable for load-bearing applications.

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Inorganic composites aim to “mimic” the composite nature of real bone with improved mechanical properties and degradation profiles by combining the toughness of a polymer phase with the strength of an inorganic generating new bioactive materials. For such composites, the alkalinity of the inorganic filler neutralizes acidic autocatalytic degradation of polymers such as PLA (Hu et al., 2003; Linhart et al., 2001). Nanosized materials have been identified as having more bioactivity than microsized ones. Tissue-engineered HA-collagen nanocomposite systems that are emerging rapidly are showing promise (Liao et al., 2004). Inorganic components at the nanoscale are combined via sol
gel processing (Pereira et al., 2005). Current composites still fall short in matching the mechanical properties of a bone. However, it is exciting to recreate the similar degree of nanoscale order of the mineral and organic components matching the body tissues. The combination of biocomposites and cell therapy research is persuaded with boundless attention. Since the early 1980s, the potential of mesenchymal stem cells in the regeneration of bone has been highlighted (Friedenstein et al., 1987). Animal models have demonstrated a number of successes using biocomposites and cells ranging from primary adult osteoblasts (bone cells) to bone marrow mesenchymal stem cells in bone tissue engineering. Nonetheless, the majority of research has come from rodents and only a handful report orthoptic applications (i.e., bone defects) in large-sized mammalian animals (Cancedda et al., 2007; Petite et al., 2000; Goshima et al., 1991; Meijer et al., 2007; Puelacher et al., 1996). Despite the clinical successes of the ectopic model in rodents, translation to human use has suffered (Meijer et al., 2007; Marcacci et al., 2007; Quarto et al., 2001; Cancedda et al., 2003; Schimming and Schmelzeisen, 2004; Morishita et al., 2006). Understandably, and arguably, the smaller size of defects in rodents, along with higher bone remodeling rates and lack of large vascular supply in larger human defects could have resulted in the significant cell death immediately after implantation of a cell-seeded biomaterial. It is also worth bearing in mind that in vivo cells in the metabolically active tissue are within 100 μm of a high oxygen source. Presenting well-controlled, interconnected, high-porosity materials into scaffolds supports successive permeability and the transport of O2 and nutrients, as well as the creation of a 3D vascular network. Extra approaches for “prevascularization” in vitro are emerging. For example, 3D systems comprising progenitor cells, differentiated mature cells, and endothelial cells in endothelial networks, engineered tissue constructs, for engineered muscle implants. Ongoing studies are revealing cell behavior in cell adhesion to modulation of the intracellular signaling pathways, which regulate transcriptional activity and gene expression (Curtis and Wilkinson, 1999). An interesting new work of relevance to bone tissue engineering explored nano-topographical features and found that the differentiation of mesenchymal stem cells produced bone mineral (Dalby et al., 2007). The relationship between nanoscale topographic features and protein adhesion and cell behavior is highly complex, remnants need to be expounded fully with respect to shape, size, in addition to protein and cell type.

2.7 Biological Response

Nanophase reinforcements [HA-collagen nanocomposites or carbon nanotube (CNT)-polymer nanocomposites] are at present producing enhancements in bioactivity and mechanical properties such as flexural and compressive moduli (Huang et al., 2005; Jell et al., 2008; Verdejo et al., 2008). Another approach for the biomimetically enhanced environment is to recreate the topographical context of native ECM through engineered 3D nanofibrous matrices. Polymer-based processing methods include electrospinning, thermally induced phase separation, and protein self-assembly generating nanofibrous matrices (Li et al., 2002, 2005). Well-ordered nanofibrous networks are created from a self-assembled peptide or peptide amphiphile-based systems (Jayawarna et al., 2006). Future approaches to progress the mechanical properties of peptide-based materials are essential if they are to be applied to load-bearing bone applications.

2.7 BIOLOGICAL RESPONSE In designing biomedical composites and predicting their performance to understand the biological response several issues must be considered. Trying to enhance the properties or have desired properties in the composite, a number of constituent materials may also increase proportionally and are responsible for variations in the response. Addressing biocompatibility is a major issue; the specific composition, arrangement, and interaction are to be examined, which influences design flexibility and regulatory approval. Previously, the biological response to nm-sized particles was not investigated thoroughly. The biological response to clinically relevant UHMWPE wear particles including nm-size and μm-size, along with polystyrene particles (FluoSpheres 20 nm
1.0 μm), and nm-sized model polyethene particles (Ceridust 3615), was estimated in terms of osteolytic cytokine release from primary human peripheral blood mononuclear cells (PBMNCs). UHMWPE nm-size particles generated from hip and knee replacements have been identified in in vitro and in vivo studies. UHMWPE particles of size range 0.1
1.0 μm have been shown to be more biologically active than larger particles, aggravating an inflammatory response concerned with late aseptic loosening of total joint replacements (Aiqin et al., 2015). The biocompatible UHMWPE was also used as an acetabular cup for a hip prosthesis. Furthermore, the performance of the composite altered by the release of enzymes can have a serious effect, varying the degradation rates/kinetics of a biodegradable composite (Ratner, 1996). The material interaction at the interface is integral to the performance of the composite material, and this can affect the tissue response in numerous ways, like filling, swelling of interfacial voids with fluid, and fibrous tissue, altering the interfacial adhesion strength between matrix and reinforcement, laminated composites, delaminate, etc. These effects are critical and may lead to failure of a composite materials’ performance because of fatigue and fracture, particularly with those in structural applications.

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Biomedical implants do not have thermosetting polymers as their materials; it is very uncommon for the said reasons, such as the presence of leftover monomer and crosslinking agents, especially in laminated composites that are made of prepreg layers. Some remaining solvents may leach out from the matrix from both thermosetting and thermoplastic polymer composites if they are not entirely eradicated during the processing period. These trace amounts are not severe and it is not considered to be an issue if it is for external applications, such as in prosthetic limbs.

2.8 APPLICATIONS IN BIOMEDICAL ENGINEERING Composite materials are used in biomedical implants/devices exemplified by the succeeding structural application examples.

2.8.1 DENTISTRY Dental biomaterials are categorized into four classes of compounds: polymers, ceramics, metals, and composites. The polymeric base material in dentistry is commonly used in the comprehensive and partial denture. In addition, resin cement, denture soft liners, pit and fissure sealants contain polymer/polymer composites. Several polymers applied for denture base include polyvinylchloride (PVC), celluloid, phenolformaldehyde (Bakelite), vulcanite, and polymethylmethacrylate (PMMA) (Alla et al., 2015; Yildiz et al., 2014). Ceramics have very good resistance against stresses, are highly vulnerable under shear and tensile stress, and are formed via a arrangement of metal elements and nonmetallic materials such as nitrides, oxides, and silicates, based on their interatomic bonding from hard, stiff, and brittle materials (McLaren and Cao, 2009; Babu et al., 2015). Composites are highly efficacious in a variety of dental applications, while meeting numerous rigorous design requirements, with difficulties in achieving homogeneous materials such as ceramics and metal alloys. Present research in biomaterial composites includes physical, chemical, and biological aspects of ceramic, metal, polymer, and composite materials. Ceramics are used as filler in the composite resins, glass ionomers, and porcelain cement (Denry, 1996). Whether it is the groundwork of crowns, repair of cavities, or even entire tooth replacement, the product needs to be harmonized esthetically in color, be translucent with other teeth, and retaining gloss. Porcelains are biocompatible dental refurbishments (such as for crowns, bridges, and veneers), and benefits include biocompatibility, solubility, and having higher toughness (Chen et al., 2011). Cavity repairs need particulate composites, which contain polymer resin matrix filled with stiff inorganics to increase their strength and impart wear resistance. The resins, typically a PMMA or a polyurethane di-methacrylate, are cured

2.8 Applications in Biomedical Engineering

by crosslinkers, UV light, or LEDs. The stiff filler particles can be ceramics, Ca2O4Si, CaF2, crystalline quartz, or Si3N4 whiskers. The filler comprises up to 50%
80% of the volume of the composite, with varying sizes (20
50 μm). In a microfilled dental resin, fused SiO2 particles (20
40 nm) merged with volume fractions up to 40% produce a high gloss translucent composite. Hybrid dental resins (particle sizes, 0.1
10 μm), allow higher filler volume, up to 80%, and higher viscosity (easy handling), and lower water absorption, compared with microfilled resins. Profitable dental composite resins have polymerization shrinkages varying from 1.6% to 2.5% (Cook et al., 1999) and water absorption of up to 1.5% (Beatty et al., 1998), causing dimensional changes. They may also have reduced adhesion to dentin (Nicholson, 1998), creating an important use for bonding agents to prevent fitting and leakage problems. All-ceramic-based dental composites were used for stress-bearing restorations of dental crowns and bridges. By including within the design, crack deflection and bridging mechanisms into the composite, which area a very important concern, fracture toughness was addressed. In-Ceram, is a common type of Al2O3 glass composite, in which a skeleton of Al2O3 particles are slip casted and sintered, followed by melt infiltration of glass into the porous core, for example, 75% by volume a-Al2O3 particles of average 3 μm size and 25% glass (Wolf et al., 1996). A mismatch in thermal expansion between Al2O3 and glass seems to have no significant effect on fracture toughness. The recent developments in aqueous tape-casting of the Al2O3 core have made it easier to confirm the composite to a tooth model (Kim et al., 1999). Such composites can be countless times harder than enamel and thus can wear it out; in order to reduce the surface hardness they must be coated with either alkali Al2O3-silicate dental porcelains or calcium phosphate composites (Francis et al., 1995). An additional application for dental composites was orthodontic archwires (Kusy 1997). One example is a unidirectional pultruded S2-glass-reinforced dimethacrylate thermoset resin (Fallis and Kusy, 2000). Dependent on the glass fiber employed, the fiber-volume fraction was wide-ranging, from 32% to 74%. The strength and modulus were analogous and very much comparable to those of titanium wires. Even orthodontic brackets can be made of these composites with a polyethene matrix reinforced with ceramic-HA particles (Wang et al., 1998), which resulted in good adhesion to enamel and enhanced isotropic properties. Nanotechnology in dentistry is increasingly focused on preventive materials and articles used in oral cavity care, such as toothbrushes incorporating nano-Ag or nano-Au colloidal particles (Raval, et al., 2016) or prosthodontic when modifying the surface of arches. Antibacterial nanofilms deposited on nanostructured electrochemically deposited TiO2 NPs on the surface of NiTi arches have been evaluated for their histopathological, genotoxic, and cytotoxic effects in Long
Evans rats (Mora´n-Martı´nez et al., 2018). ZnO NP samples exhibit less microlevel leakage in comparison with commonly used ZOE sealer, and are more or less similar to various prepared synthetic methods. Another research group (Zebarjad, 2011) synthesized more porous and spongy ZnO/MgO NPs for

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polycarboxylate dental cement used in clinical dentistry for luting agents, orthodontic attachments, cavity lining and bases, and restoration of teeth. In contrast to conventional Adhesor and Harvard zinc polycarboxylate cement, the ZnO and MgO nanostructured and ZnO/MgO nanocomposite cements have increased mechanical strength and comparable setting time for preparation of the dentin cement to those obtained by commercial samples. The bioactive particles composed of Si-O-Ca-P in an acid environment show a moderate mesoporous structure with a pore diameter around 5
30 nm. The surface of these SiO2 NPs is spherical and deposits biomimetic minerals—hydroxyapatite phase in SBF solution for 7 days, suggesting the ability for enhanced bone apposition. The ultimate goal of endodontic treatment is to eliminate bacterial infections in the root canal system, preventing microorganisms from impairing periapical healing; for this reason, Javidi et al. (2014) synthesized ZnO NPs as a sealer in endodontics. Other materials used for enhanced bone formation in endodontics are glass-ceramic materials that are nano-mimetic bioactive glasses with nano-/microstructure providing a more reactive surface than the bulk materials, it is also known that in a solution they exhibit a mild antimicrobial activity due to the release of their ionic compounds and intake of H3O1 protons, which makes them able to alter the local environment by increasing the alkalinity of the solution (Matthews et al., 2010). Huang and coworkers, in 2018, developed biominerals from different silicate bioactive glasses with and without the addition of Ca and P ions. In order to create new biomaterial for the denture base that could impede the adhesion of microorganisms to their surface and, therefore, decrease the development of denture stomatitis, Cierech et al. (2018) modified PMMA with ZnO NPs to examine the roughness, hydrophilicity, absorbability, and hardness of the surface, which demonstrated a connection with the biofilm formation. It is also observed that the antifungal properties increase with the increasing ZnO NP content in the polymer composite. The increased hydrophilicity and hardness with absorbability explain the reduced microorganism growth on the denture base.

2.8.2 PROSTHETICS AND ORTHOTICS Biomaterials which are mostly synthetic have been used in rehabilitation and regenerative medicine for more than five decades (Pilliar et al., 1976). Some of the biomaterials used in orthopedic applications include ceramics, metals, and alloys, polymeric biomaterials, and composite biomaterials. The most commonly encountered problems of biomaterials are bone cement or PMMA used in a variety of orthopedic treatments and surgeries, providing a bond between the implant and the bone tissue, which may also be reinforced by carbon fibers toward longterm stability. Biomaterials for high-performance applications like prosthetic sockets prevent ulceration with tactile clothing providing comfort have been developed.

2.8 Applications in Biomedical Engineering

The requirements of low weight, size reduction, durability, safety, and energy conservation have made fiber-reinforced plastics very attractive in replacing traditional prosthetic and orthotic materials by high-performance composites and thermoplastics (Hanak and Hoffman, 1986). For prostheses [transtibial (TT) and transfemoral (TF)], composites have been used for the socket frame component with target weight limits of 1 and 2 kg, respectively. CFR composites are ideal for such applications, with the matrix used was a blend of rigid and flexible methylmethacrylate resins for stiffness of the socket. Hybrid composites for the shank made of carbon and nylon fibers in polyester resins have improved impact resistance than carbon- or nylon-only composites. Using thermoplastics, CFR nylon 6,6, made effortlessly by injection molding, has exceptional vibrationdamping characteristics imperative for shock absorption. Nonetheless, more advanced thermoplastics like PEEK have longer fibers via injection molding for enhanced properties. The three different composites being used in the orthopedic industry are fiberglass, Kevlar (Aramid), and carbon (graphite), as these materials have completely different properties and characteristics compared from one another. Acrylic resins are a lightweight thermosetting plastic with excellent wetting properties and good inherent strength, making thin ultralight orthopedic appliances possible. Apart from being lighter, cleaner, with low forming temperatures and resistance to fatigue in bending and their combination of characteristics, materials like polypropylene and polyethene have been shown to be tremendously useful in nearly all types of orthoses, even at high unit stresses. As no material is flawless and because only a relatively small volume is needed in orthotics, materials have to be specially tailored to orthotics.

2.8.3 TISSUE ENGINEERING Biocomposites are used in the medical industry to repair and restore bone, tooth, cartilage, skin, and in tissue engineering. Synthetic bioactive and bioresorbable composite materials are showing great potential as scaffolds for tissue engineering. Next-generation biomaterials should combine bioactive and bioresorbable properties to activate in vivo mechanisms of tissue regeneration (Boccaccini and Blaker, 2005). Crosslinked hydrogel networks as scaffolds for skin regeneration were applied owing to their high water content, the properties of the barrier, and perhaps their poor mechanical properties. By reinforcing it with Spandex and gauze fibers with the hydrogel poly(2-hydroxyethyl methacrylate) (pHEMA) the tensile strength and breakpoint were enhanced. For cartilage cell growth, fibrous PGA melts and PLGA fibers were used as surfaces and scaffolds. High shear properties are wanted in cartilage repair, low-density linear polyethene was melt coated onto a woven 3D fabric of UHMWPE fibers to form a composite that closely resembles natural cartilage (Seal and Panitch, 2001). Different impermeable materials are coated on grafts forming a composite to overcome the leakage problem and to avoid lengthy preclotting times, where the

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matrix serves as a sealant, for example, vascular prosthesis is sealed by biodegradable wet DACRON fibers mixed with alginate and gelatin. For hemodialysis vascular access, PTFE fibers sandwiched between layers of porous expanded PTFE were used, thus avoiding puncture. As the needle enters, fibers are pressed aside, with needle withdrawal, the fibers move back, creating a baffle effect while reducing blood leakage (Lohr et al., 1996).

2.8.4 ORTHOPEDIC Composite materials have found widespread use in orthopedic applications, predominantly in bone fixation, such as plates, hip joint replacements, bone cement, and bone grafts. Orthopedics use many of these in their applications, as summarized by Evans and Gregson (1998), particularly in hip-joint replacements, bone fixation plates, bone cement, and bone grafts. Traditional external prosthetic and orthotic materials were aluminum, wood, and leather, which have been largely replaced by high-performance composites and thermoplastics (Hanak and Hoffman, 1986). CFR epoxy tubing replaced stainless steel in artificial arms. In total hip replacement, for the femoral stem component, common materials were replaced with 316 L stainless steel, CoCr alloys, and Ti-6Al-4V titanium alloy, which has great stiffness compared with bone. Cortical bone has a stiffness of 15 GPa and tensile strength of 90 MPa (Katz, 1966), while values for titanium (110 GPa and 800 MPa) are comparatively very high. Bone remodeling and stress shielding help in the longer term in reducing the bone mass and implant loosening, especially in the proximal side. Researchers can tailor explicit adjacent bone properties (mechanical) and also fabricated carbon-fiber composites in PEEK or polysulfone matrices with stiffness ranging between 1 and 170 GPa and tensile strength from 70 to 900 MPa (Yildiz et al., 1998). Carbon-fiber-reinforced polyetheretherketone (CFR-PEEK) has been successfully used in orthopedic implants (Chuan et al., 2015). Examples are press-fit femoral stems that are made from laminated unidirectional carbon fibers in PEEK, liquid crystalline polymer (LCP) (Kettunen et al., 1998), polysulfone, and polyetherimide (PEI) (De Santis et al., 2000). Fabricating such composites is very difficult without promising durability but has been continued for its inherent advantages of flexibility, noncorrosiveness, and radiolucency (Srinivasan et al., 2000). Polishing and coating with HA or carbon-titanium alloy, problems underlying biocompatibility attributable to paniculate carbon debris in composites can be addressed (Bacakova et al., 2001). In fracture fixation, a completely resorbable bone plate was always desired to evade the necessity for a second operation in order to eliminate the implant once healed. A tailored low-stiffness composite is a choice which also circumvent the problem of stress shielding described earlier. To maintain the mechanical properties, the rate of degradation must be controlled so that strength loss in the implant reflects a strong increase in healing. Additionally, byproducts coming out of the degradation must be nontoxic; the design of adsorbable fixation devices was

2.9 Conclusions

given by Pietrzak and his team (Pietrzak, 2000). Examples of composite bone plates include completely adsorbable laminated continuous carbon fiber in a polylactide (PLA) matrix, and totally resorbable calcium-phosphate glass fibers in PLA (Alexander 1996). Continuous poly(L-lactide) fibers in a PLA matrix form a fully resorbable composite (Dauner et al., 1998). On the other hand, not all composites had sufficient strength properties and some degrade rather rapidly. Nonresorbable carbon-epoxy bone plates with sufficient strength and fatigue properties include those available from Ortho design, Ltd. Bone cement has remained very successful in anchoring artificial joints for more than five decades. Artificial joints are secured with bone cement by filling the voids and improving adhesion amidst implants and the bone tissue that is reinforced with various fibers, preventing any loosening while enhancing its shear strength. The characteristic bone cement is PMMA, known as bone cement, and is widely used for implant fixation in various orthopedic and trauma cases. “Cement” is a misnomer as the word cement is used to designate a material that bonds two things together. PMMA powder mixed with a monomer, methacrylate type, is polymerized in situ, during fixation. Graphite, carbon, and Kevlar fibers in the low-volume fractions have been added as reinforcement to the PMMA matrix, increasing its fatigue life and reducing creep deformation (Kelly et al., 1994).

2.9 CONCLUSIONS Biomedical engineering or bioengineering oversees the application of engineering principles and design concepts to medicine and biology for healthcare purposes, closing the gap between engineering and medicine, by combining the design and problem-solving skills of engineering with medical biological sciences advancing healthcare treatment, including monitoring, diagnosis, and therapy. Biomedical engineering, with massive research activity, has focused on producing new and improved inorganics; composite materials have gained dominance due to the nonexistence of individual biomaterials satisfying the multifunctional needs. Numerous successful applications exist in bone, tissue engineering, neurological systems, etc., where the valuable properties of each individual component of composite systems act synergistically when combined, demonstrating greater bioactivity and augmented integration. Composite materials in vascular systems and networks, especially heart valve replacements, possess more limited but positive results as the deficiencies of each material were compounded, presently outweighing any additive gains. Nevertheless, these shortages may be eliminated as new biomaterials are discovered, cell sources are optimized, and delivery systems are better developed and utilized. The level of biological complexity, which needs to be recapped within a synthetic 3D environment, still remains very unclear. Additional interpretation of

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the communication between particles and the matrix will not only help researchers to focus on approaches but also enable the correct exhibition of factors chemically and in terms of their distribution. More understanding of these interactions stirring at the substrate interface is required for clinical application in structuring approaches. Vascularization of outsized tissue constructs in bone
tissue engineering remains a noteworthy challenge. Advances in microsurgical techniques can overcome a few of the current glitches in attaining quick vascularization of these implanted biomaterials. The harvestinf of pluripotent mesenchymal cells from other sources also warrants consideration, for example, from the periosteum or adipose tissue. The perspective to combine 3D printing of scaffolds and 3D printing of cells and biologics will allow the expansion of growth in innovative designer material-based hybrids. Furthermore, advanced and superior biomaterials are yet to be developed matching the biological response and complexity at the molecular level. Nevertheless, the stimulating task of designing the biomaterial surfaces for dissimilar purposes of implant integration and tissue regeneration appears to be feasible in the near future; synergistic interdisciplinary work of scientists from biology, materials science, chemistry, physics engineering, and medicine will be required to achieve this.

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FURTHER READING Benoit, D.S., Anseth, K.S., 2005. The effect on osteoblast function of colocalized RGD and PHSRN epitopes on PEG surfaces. Biomat. 26, 5209
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Further Reading

Bhattacharyya, S., Lakshmi, S., Bender, J.D., Greish, Y.E., Brown, P.W., Allcock, H.R., et al., 2004. Preparation of poly[bis(carboxylatophenoxy)phosphazene] non-woven nanofiber mats by electrospinning. Mater. Res. Soc. Symp. Proc. 842, 157
163. Bokel, C., Brown, N.H., 2002. Integrins in development: moving on, responding to, and sticking to the extracellular matrix. Dev. Cell 3, 311
321. Chavanpatil, M.D., Khdair, A., Panyam, J., 2006. Nanoparticles for cellular drug delivery: mechanisms and factors influencing delivery. J Nanosci. Nanotech. 6, 2651. Chen, G.-Q., Luo, R.-C., Chapter 8: Polyhydroxyalkanoate Blends and Composites; in Biodegradable Polymer Blends and Composites from Renewable Resources, Editor(s): Long Yu, https://doi.org/10.1002/9780470391501.ch8. Govender, S., Csimma, C., Genant, H.K., Valentin-Opran, A., Amit, Y., Arbel, R., et al., 2002. Recombinant human bone morphogenetic protein-2 for treatment of open tibial fractures a prospective, controlled, randomized study of four hundred and fifty patients. J. Bone Joint Surg. Am 84, 2123
2134. Hidaka, C., Goodrich, L.R., Chen, C.T., Warren, R.F., Crystal, R.G., Nixon, A.J., 2003. Acceleration of cartilage repair by genetically modified chondrocytes overexpressing bone morphogenetic protein-7. J. Orthop. Res 21, 573
583. Huang, C.-L., Fang, W., Chen, I.-H., Hung, T.-Yo, 2018. Manufacture and biomimetic mineral deposition of nanoscale bioactive glasses with mesoporous structures using solgel methodsCer. Intl In Press . Available from: https://doi.org/10.1016/j. ceramint.2018.06.180. Ito, H., Koefoed, M., Tiyapatanaputi, P., Gromov, K., Goater, J.J., Carmouche, J., et al., 2005. Remodelling of cortical bone allografts mediated by adherent rAAV-RANKL and VEGF gene therapy. Nat. Med 11, 291
297. Keselowsky, B.G., Collard, D.M., Garcia, A.J., 2005. Integrin binding specificity regulates biomaterial surface chemistry effects on cell differentiation. Proc. Natl. Acad. Sci. USA 26, 5953
5957. Levenberg, S., Rouwkema, J., Macdonald, M., Garfein, E.S., Kohane, D.S., Darland, D.C., et al., 2005. Engineering vascularized skeletal muscle tissue. Nat. Biotechnol 23, 879
884. Lutolf, M.P., Lauer-Fields, J.L., Schmoekel, H.G., Metters, A.T., Weber, F.E., Fields, G. B., et al., 2003. Synthetic matrix metalloproteinase-sensitive hydrogels for the conduction of tissue regeneration: engineering cell-invasion characteristics, Proc. Natl. Acad. Sci. USA, 100. pp. 5413
5418. Maheshwari, G.G., Brown, G., Lauffenburger, D.A., Wells, A., Griffith, L.G., 2000. Cell adhesion and motility depend on nanoscale RGD clustering. J. Cell Sci 113 (Pt 10), 1677
1686. Mart, R.J., Osborne, R.D., Stevens, M.M., Ulijn, R.V., 2006. Peptide-based stimuli-responsive biomaterials. Soft Matter. 2, 822
835. Murphy, W.L., Freire, E., 1992. Thermodynamics of structural stability and cooperative folding behaviour in proteins. Proteins 169, 313
361. Palecek, S.P., Loftus, J.C., Ginsberg, M.H., Lauffenburger, D.A., Horwitz, A.F., 1997. Integrin-ligand binding properties govern cell migration speed through cell-substratum adhesiveness. Nature 385, 537
540. Ryadnov, M.G., Woolfson, D.N., 2003. Engineering the morphology of a self-assembling protein fibre. Nat. Mater. 2, 329
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Shi, X., Hudson, J.L., Spicer, P.P., Tour, J.M., Krishnamoorti, R., Mikos, A.G., 2005. Rheological behaviour and mechanical characterization of injectable poly(propylene fumarate)/single-walled carbon nanotube composites for bone tissue engineering. Nanotechnol 16, S531. Shin, H., Jo, S., 2002. Modulation of marrow stromal osteoblast adhesion on biomimetic oligo[poly-thylene glycol) fumarate] hydrogels modified with Arg-Gly-Asp peptides and a poly (ethylene glycol) spacer. J. Biomed. Mat. Res. 61, 169
179. Shin, H., Jo, S., 2003. Biomimetic materials for tissue engineering. Biomat 24, 4353
4364. Shin, M., Yoshimoto, H., Vacanty, J.P., 2004. In vivo bone tissue engineering using mesenchymal stem cells on a novel electrospun nanofibrous scaffold. Tissue Eng. 10, 33
41. Silva, G.A., Czeisler, C., Niece, K.L., Beniash, E., Harrington, D.A., Kessler, J.A., et al., 2004. Selective differentiation of neural progenitor cells by high-epitope density nanofibers. Science 303, 1352
1355. Stevens, M.M., Allen, S., Davies, M.C., Roberts, C.J., Sakata, J.K., Tendler, S.J., et al., 2005. Molecular level investigations of the inter- and intramolecular interactions of pH-responsive artificial triblock proteins. Biomacromol. 6, 1266
1271. Tanahashi, M., Matsuda, T., 1997. Surface functional group dependence on apatite. J. Biomed. Mater. Res. 34, 305
315. Verma, I.M., Somia, N., 1997. Gene therapy -- promises, problems and prospects. Nature 389, 239
242. Webster, T.J., Ahn, E.S., 2007. Nanostructured biomaterials for tissue engineering bone. Adv. Biochem. Eng. Biotechnol. 103, 275
308. Woo, K.M., Chen, V.J., Ma, P.X., 2003. Nano-fibrous scaffolding architecture selectively enhances protein adsorption contributing to cell attachment. J. Biomed. Mater. Res. A 67, 531
537. Yang, F., Murugan, R., 2004. Fabrication of nanostructured porous PLLA scaffold intended for nerve tissue engineering. Biomat. 25, 1891
1900. Yang, Z.M., Liang, G., Xu, B., 2006. Supramolecular hydrogels based on β-amino acid derivatives. Chem. Commun. 7, 738
740. Yang, Z.M., Liang, G.L., Ma, M.L., Gao, Y., Xu, B., 2007. Conjugates of naphthalene and dipeptides produce molecular hydrogelators with high efficiency of hydrogelation and superhelical nanofibers. J. Mater. Chem. 17, 850
854. Zhang, S.G., 2003. Fabrication of novel biomaterials through molecular self-assembly. Nat. Biotechnol. 21, 1171
1178.

CHAPTER

Structural interpretation, microstructure characterization, mechanical properties, and cytocompatibility study of pure and doped carbonated nanocrystalline hydroxyapatites synthesized by mechanical alloying

3

Sushovan Lala and Swapan Kumar Pradhan Materials Science Division, Department of Physics, The University of Burdwan, Burdwan, India

3.1 INTRODUCTION The replacement of bone material to repair damaged bone tissues by foreign material implants within the human body first occurred more than 2000 years ago. In the mid-19th century, materials typically based on copper or bronze were frequently used to repair damaged hard tissues. However, the implantation of such materials resulted in severe inflammation, causing infections and even death of the patients (Mun˜oz, 2011). In the early 20th century, materials like glass and new alloys were used to fix these problems. In the second half of the 20th century, materials using polymers were extensively used for this purpose. The use of biomaterials in replacing human parts affected by infections, trauma, illness, or aging is now well established (Saverio Affatato and Ruggiero, 2015). A major challenge in the use of biomaterials in orthopedic applications is to devise materials which sustain their properties, such as mechanical stability, biocompatibility, and corrosion resistance, after implantation into the human body. For example, when bone joints are affected by trauma, the bone itself experiences mechanical degradation due to excessive loading or the absence of biological self-healing

Materials for Biomedical Engineering: Inorganic Micro- and Nanostructures. DOI: https://doi.org/10.1016/B978-0-08-102814-8.00004-4 © 2019 Elsevier Inc. All rights reserved.

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(Saverio Affatato and Ruggiero, 2015). Thus, the implantation of biomimetic devices made of artificial biomaterials was proposed as a way to restore or replace the function of structures under treatment (Bramowicz et al., 2016). Hydroxyapatite [HAp: Ca10(PO4)6(OH)2] is a naturally occurring mineral similar in composition to the mineral element in human bones. The name “apatite” corresponds to a family of compounds with similar hexagonal crystalline structure with space group: P63/m (Mehmel, 1930). The name “apatite” originated from the Greek word “apate” (απαταω) which means deception, due to the fact that the mineral appears in a variety of colors with different crystal habits and is often mistaken for semiprecious minerals like amethyst or aquamarine (Suetsugu and Tateishi, 2008). It is extensively used in reconstruction of damaged bone or teeth due to its excellent biocompatibility, unique bioactivity, good osteoconductivity, and stability under physiological conditions (Hench, 1991). The apatite found in native bone has a very complicated chemical composition and it differs from pure HAp in a number of ways including nonstoichiometry, rich amorphous content (63%
67%) (Boanini et al., 2010; Dorozhkin and Epple, 2002), nano-sized crystal dimensions, and calcium deficiency (Ca/P molar ratio , 1.67) due to the presence of different cations and anions such as Na1, Zn21, 2 Mg21, Mn21, CO22 3 , F , etc. (Elliott, 1994; Dorozhkin, 2011). The appearance of an amorphous phase in HAp leads to increased tendency of resorption of the material (Paluszkiewicz et al., 2010). Despite good biocompatibility, bioactivity, and osteoconductivity, the weak mechanical properties of HAp, such as high brittleness and low fracture toughness, restrict its applications in nonload-bearing or metallic implant surface coating. Different techniques have been implemented to improve the mechanical properties of HAp including addition of dopants (Bianco et al., 2010; Tan et al., 2013; Curran et al., 2011; Uysal et al., 2014), making composites (Georgiou and Knowles, 2001; Converse et al., 2009; Gergely et al., 2013; Kumar et al., 2013), and controlling microstructures via different novel sintering methods such as hot pressing (Raynaud et al., 2002), post-hot isostatic pressing (HIP) (Boilet et al., 2013), microwave sintering (Curran et al., 2010; Wang et al., 2006; Thuault et al., 2014), etc. Some advanced sintering techniques, such as HIP and spark plasma sintering (SPS), have been proven to be advantageous over conventional pressureless sintering of HAp (Kumar et al., 2013; Miao et al., 2004; Chang et al., 1997). However, HIP often results in grain coarsening or surface contamination due to sintering at high temperature for a long period of time (Miao et al., 2004). Usually, during the sintering process, HAp decomposes to β-TCP (β-tricalcium phosphate) and other phases. Anomalous grain growth of HAp in a long sintering process leads to degradation of mechanical properties. In contrast, SPS, falling in the category of field-assisted sintering technique (FAST) is a fast process of sintering and is applied to avoid grain growth and produce highly dense composites at relatively low temperature within a short time (Li and Gao, 2003; Kumar et al., 2013). In the present investigation, nanocrystalline-undoped and Mn-, Mg-, and Zn-doped HAp powders have been synthesized by mechanical alloying (MA).

3.1 Introduction

The as-milled powders have subsequently been sintered at 950 C via an SPS technique. One of our objectives is to look into the effect of the different dopants on the microstructure as well as the mechanical properties. Detailed microstructure characterizations of the synthesized samples are done by the Rietveld structure refinement method and the mechanical properties are correlated with the structural interpretation. As the synthesized nanocrystalline HAp powders have potential applications in reconstruction of damaged bone and teeth, an attempt has been made to check the biocompatibility of the synthesized powders through MTT assay by applying it to mouse cells.

3.1.1 CARBONATION IN BIOLOGICAL APATITES In biological apatites, CO3 is the main minor constituent. Bone and tooth mineral contain about 2
8 wt.% carbonate (Bigi et al., 1997). The concentration of CO3 is least in enamel (about 3.5 wt.%) and greatest in dentin (5.6 wt.%) and bone (7.4 wt.%). There exist two types of carbonated apatite in mammals: type A 2 (CO22 3 substituting OH groups) (Elliott et al., 1985) and type B carbonated apa22 32 tite (CO3 for PO4 substitution) (LeGeros, 1991). The ratio of A type to B type carbonation changes with age. Increases in positive charge due to replacement of PO32 ions by CO22 for B type substitution are balanced either by the loss of 4 3 21 Ca ions or by the introduction of Na1 or K1 ions (Suetsugu and Tateishi, 2008). The inclusion of carbonate ions into the structure of HAp results in a decrease in crystallinity and an increase in solubility of bone mineral both in vivo and in vitro (Legeros et al., 1967).

3.1.2 IMPORTANCE OF ZN, MN, AND MG AS TRACE ELEMENTS PRESENT IN BONE Zn, Mn, and Mg are the three important trace elements present in natural apatites of vertebrate bone tissue. Zn is considered to be one of the most significant elements incorporated in the HAp unit cell. It is one of the most abundant trace elements found in bone mineral having stimulatory effects to prompt bone formation both in vivo and in vitro. In addition to that, it has prohibitory effects on osteoclastic bone resorption in vivo (Matsunaga et al., 2010). Zn can support bone metabolism, bone growth, enhance bone densification, and prevent bone loss (Legeros, 2008; Kaygili and Tatar, 2012). The presence of Mn21 ions increases the ligand-binding affinity of integrin and activates cell adhesion (Gyorgy et al., 2004). Mn-doped HAp favors osteoblast proliferation, activation of their metabolism, and differentiation (Sopyan et al., 2011). Manganese helps in normal bone growth, bone metabolism, and bone remodeling. The existence of Mn21 ions in apatites helps in the synthesis of mucopolysacharides, whereas a deficiency of Mn content lowers the synthesis of organic matrix and retards endochondral osteogenesis, causing bone abnormalities

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including a decrease in the thickness or length of bones and bone deformation (Medvecky et al., 2006). Insufficiencies of Mn in bone probably cause osteoporosis (Pabbruwe et al., 2004). The most abundant minor element associated with biological apatites is Mg. Enamel, dentin, and bone contain 0.44, 1.23, 0.72 wt.% of Mg, respectively (Laurencin et al., 2011). It plays an important role in bone metabolism, stimulating osteoblast cell proliferation (Ren et al., 2010). Diminution in Mg content in bone adversely affects skeletal metabolism, causing a decrease in osteoblast activity, osteopenia, bone fragility, and bone loss (Laurencin et al., 2011; Ren et al., 2010).

3.2 MATERIALS AND METHODS 3.2.1 MECHANICAL ALLOYING With the advent of modern technology, materials scientists around the world are trying constantly to synthesize samples that can be obtained without any difficulty and the synthesized materials are expected to perform more effectively and efficiently. Bulk production of the samples is another point of interest. It has been proved that one can control the structure and properties of materials prepared through nonequilibrium methods (energizing and quenching) (Suryanarayana, 1999, 2001) and such materials show better physical and mechanical properties than those prepared by conventional routes (Suryanarayana, 2001). There are several such methods, among which mechanical alloying (MA) is one of the simplest and most inexpensive techniques. MA provides a fast and one-step synthesis of different nanocrystalline powders. MA has some unique advantages, including simple synthesis technique, low fabrication cost, and easy control of compositions. In addition, it provides a fine homogeneous nanocrystalline powder that can be consolidated according to specific requirements. By this technique, productivity of nanocrystalline powders can be increased by controlling different parameters, such as ball to powder mass ratio (BPMR), rotation speed, time of milling process, milling atmosphere, volume of the milling container, etc. MA is basically a dry (sometimes wet) milling process of the blended powder mixtures to produce scientifically interesting and commercially feasible materials and it is usually carried out by milling techniques—capable of producing highenergy compressive impact forces such as in planetary ball mills and vibrating ball mills.

3.2.2 SAMPLE PREPARATION BY MA In MA, all precursor powders (highly pure powders having a particle size of a few μm) to be ground are taken in stoichiometric ratio into vials containing grinding balls. In the present study, we used a Pulverisette-5 (P-5) (M/S Fritsch,

3.2 Materials and Methods

GmbH, Germany) high-energy planetary ball mill with chrome-steel vials (containers) of 80 mL volume containing 30 chrome steel balls of 10 mm diameter each. For pure and doped HAp preparation, calcium hydrogen phosphate, calcium carbonate, and different oxides were used as the precursor powders and dry milling was carried out in the open air. The term “planetary ball mill” arises due to the similarity of rotation of the vial mounted on the rotation disc with the kinetics of the planetary motion of our solar system. The grinding vials mounted on the rotating disc rotate about their own rotation axis in the opposite direction to that of the main rotating disc, around an axis passing through the center of the disc, analogous to the motion of a planet. Due to such a type of motion, the grinding balls do not only experience the force due to gravity (as occurs in conventional milling), but also coriolis and centrifugal forces, which further increase the kinetic energy of the system. The nanometric particles with high surface to volume ratio are obtained within a very short time due to such a high-energy mechanism. In this study, we used a Fritsch Planetary P-5 ball mill with four grinding vial fasteners. Nanocrystalline carbonated Mn-, Mg-, and Zn-doped HAp powders were synthesized with a (Ca 1 M)/P molar ratio 1.67 (M assigned for Mn/Mg/Zn) by dry milling the CaCO3 (purity 99.5%, Merck), CaHPO4. 2H2O (purity 99%, Loba Chem.) and MnO/MgO/ZnO (purity B99%, Sigma Aldrich) powder precursors taken in (4 2 x):6:x molar ratio (x 5 0.25, 0.5, 1.0 for Mn; x 5 0.25, 0.5, 1.0, 1.5 for Mg, and x 5 0.5, 1.0, 1.5, 2.0 for Zn doping), respectively. In each case, milling was carried out at room temperature for 10 hours in open air (Lala et al., 2015a,b; Lala et al., 2016a,b). The undoped HAp powder was synthesized by dry milling CaCO3 and CaHPO4. 2H2O powder mixture was taken in a 2:3 molar ratio for 10 hours under the same condition (Lala et al., 2013, 2014). The synthesis of different metal-doped carbonated HAp considering charge balance and neglecting B type carbonation (which would have been found later) can be represented by the following equation: ð4 2 xÞCaCO3 1 6 CaHPO4 U2H2 O 1 x MO 5 Ca102x Mx ðPO4 Þ6 ðOHÞ222y ðCO3 Þy 1 ð4 2 x 2 yÞ CO2 1 ð14 1 yÞ H2 O

(3.1)

where 0 , x , 4, 0 , y , 1 and M stands for Mn/Mg/Zn.

3.2.3 SPARK PLASMA SINTERING In order to obtain dense pellets of 3 mm thickness and 20 mm diameter for the mechanical studies, the as-synthesized powder compacts (undoped and 5 mol.% Mn-, Mg-, and Zn-doped HAp) were sintered at 950 C in an argon atmosphere (flow rate 5 2 L/min) using a SPS system (SPS-515S, Dr. Sinter Lab, Japan). In this study we adopted three-stage SPS for the sintering of HAp with a maximum sintering temperature of 950 C (Lala et al., 2017). A unidirectional pressure of 30 MPa was maintained throughout the entire sintering process. All the pellets

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were polished up to 0.25 μm using polycrystalline diamond suspension (cat. No. 40-6629, Buehler) followed by ultrasonic cleaning in acetone.

3.2.4 SAMPLE CHARACTERIZATION XRD patterns of unmilled and all ball-milled powder mixtures, as well as the consolidated specimens, were recorded with Ni-filtered CuKα radiation (wavelength 5 0.15418 nm) from an X-ray powder diffractometer (D8 Advance, Bruker) operated at 40 kV and 40 mA. Step-scan data (of step size 0.02 degrees 2θ and counting time 5 seconds) were recorded for the entire angular range 20
80 degrees 2θ. Microstructure characterizations of ball-milled powders were also made by HRTEM operated at 200 kV (Model HR-TEM 2100F JEOL). The FTIR spectra of as-milled HAp powders were obtained from a PerkinElmer FTIR spectrometer (Model RX1) using KBr standard in the transmission mode within the wave number range of 400
4000 cm21. For finer-scale microstructural analysis, backscattered electron (BSE) images of the consolidated specimens were obtained using a scanning electron microscope (SEM, Carl Zeiss EVO 50) at an accelerating voltage of 20 kV.

3.2.5 BIOLOGICAL STUDIES 3.2.5.1 Cell culture Chinese hamster ovarian (CHO) cells were procured from NCCS, Pune, India, and cultured in 10% FBS DMEM medium in the presence of 100 mg/L streptomycin and 100 IU/mL penicillin. Cell culture was performed in a 25-mL cell culture flask and kept at 37 C in a humidified atmosphere of 5% CO2 to about 70%
80% confluence. Subculture was done every 2
3 days. The media were changed after 48
72 hours. The adherent cells were detached from the surface of the culture flask by means of trypsinization. These cells were then used for cytotoxicity study. The materials used in the cell culture study included Dulbecco’s Modified Eagles’ Medium (DMEM) procured from Himedia, heat inactivated fetal bovine serum (FBS) obtained from Gibco, trypsin from porcine pancreas, MTT, BSA-FITC procured from Sigma Aldrich.

3.2.5.2 MTT assay Cell viability of the undoped and doped HAp samples was assessed by the microculture MTT reduction assay as previously reported (Hansen et al., 1989). In this assay the reduction of a soluble tetrazolium salt by mitochondrial dehydrogenase of the viable cells to an insoluble colored formazan product was recorded. The amount of product formed was measured spectrophotometrically after dissolution of the dye in DMSO. The enzyme activity and the amount of the formazan produced are proportional to the number of alive cells. The mammalian cells were seeded at a density of 15,000 cells per well in a 96-well microtiter plate for

3.2 Materials and Methods

18
24 hours before the assay. A stock solution of the HAp samples in dispersed state was prepared in sterile water by sonication for 30 minutes. The concentration in the microtiter plate was varied from 5 to 100 μg/mL. The cells were incubated for 24 hours at 37 C under a 5% CO2 environment. The cells were further incubated for another 4 hours in 15 μL MTT stock solution (5 mg/mL). The produced formazan was dissolved in DMSO and absorbance at 570 nm was measured using BioTek Elisa Reader. The number of surviving cells was expressed as percent viability 5 (A570(treated cells) 2 background)/A570(untreated cells) 2 background) 3 100. To study the cytocompatibility of as-synthesized HAp samples, a LIVE/ DEAD Viability/Cytotoxicity Kit for eukaryotic cells was used. The kit consists of a mixture of two nucleic acid binding stains, Calcein AM (aceto methoxy) (component A) and ethidium homodimer-1 (component B). The former has the ability to pass through the cell membrane, while the latter can only be internalized into cells with a compromised cell membrane. After the transportation of Calcein AM into the cell, the esterase enzyme present only in live cells cleaves the acetoxy group. This active form of Calcein intercalates with the DNA and results in bright green fluorescence. On the other hand, ethidium homodimer-1, upon intercalation with DNA, exhibits red fluorescence in dead cells. Just prior to assay the kit was thawed to room temperature and 4 μL of the supplied EthD-1 stock solution (2 mM) was added to 2 mL of autoclaved, tissue culture-grade PBS buffer and the mixture was vortexed. This gave an approximately 4 μM EthD-1 solution. 1 μL of the supplied 4 mM Calcein AM stock solution was then added to the 2 mL EthD-1 solution and vortexed. The resulting mixture of Calcein AM (2 μM) and EthD-1 (4 μM) was then added to HAp pretreated (up to 48 hours at 37 C) CHO cells and incubated for 30 minutes. The cells were subsequently observed under the Olympus IX51 inverted microscope using an excitation filter of BP460
495 nm and a band absorbance filter covering wavelengths below 505 nm at 10 3 magnification. The bright green color resulting from the enhanced fluorescence of oligonucleotide intercalated Calcein indicated the presence of live cells. The images of ethidium homodimer-1 intercalation were taken using the excitation filter BP530
550 and a band absorbance filter covering wavelengths below 570 nm, where negligible red fluorescence were observed.

3.2.6 METHOD OF ANALYSIS 3.2.6.1 Microstructural analysis In this study, Rietveld’s powder structure refinement analysis (Rietveld, 1967, 1969; Young and Wiles, 1982; Lutterotti, 2011) of X-ray powder diffraction patterns is performed for each sample with Rietveld software MAUD 2.26 (Lutterotti, 2011) to obtain the refined structural (lattice parameters, occupancies, atomic fractional coordinates, temperature factors, etc.) as well as microstructural parameters such as particle size and r.m.s. lattice strain of all as-milled and

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sintered undoped and doped HAp samples through the Marquardt least-squares method. Here, the peak shape is assumed to be a pseudo-Voigt function with asymmetry, as it takes individual care of both the particle size and strain broadening of the experimental profiles. The background intensity of each experimental pattern is fitted by a polynomial of degree five. For unmilled powders, the XRD pattern is simulated by CaCO3 (ICSD # 73446, rhombohedral, Sp. gr. R3c (H)), CaHPO42H2O (ICSD # 98804, monoclinic, Sp. gr. Cc), CaHPO4 (ICSD # 917, triclinic, Sp. gr. P-1), and MnO (ICSD # 9864, cubic, Sp. gr. Fm-3m)/MgO (ICSD # 52026, cubic, Sp. gr. Fm-3m)/ZnO (ICSD # 184079, hexagonal, Sp. gr. P63mc). For the as-milled samples, the initial structural parameters of HAp are taken from ICSD # 171549 (hexagonal, Sp. gr. P63/m), while for the sintered undoped, Mn- and Mg-doped HAp samples, the patterns are fitted with an additional phase of β-TCP (ICSD # 97500, trigonal, Sp. gr. R3c:H) along with HAp. TTCP (tetracalcium phosphate) (ICSD # 160461, monoclinic, Sp. gr. P21) is the third phase taken into account during refinement, in addition to HAp and β-TCP for the sintered Zn-doped HAp as the XRD pattern consists of the reflections from these phases. Initially, positions of all peaks are corrected by successive refinements of the zero-shift error. Different structural, along with microstructural, parameters such as particle size and r.m.s. lattice strain are obtained after proper fitting of the experimental XRD patterns by refining several structure and microstructure parameters of the simulated XRD patterns by a successive iterative process. Considering the integrated intensity of the peaks as a function of structural and microstructural parameters, the Marquardt least-squares procedure is implemented to reduce the intensity difference between the observed and the simulated XRD patterns and the minimization is monitored by the reliability index parameter, Rwp (weighted residual error) and Rexp (expected error) defined, respectively, as "P #1=2 2 i wi ðIo 2IC Þ Rwp 5 P 2 i wi ðIo Þ "

ðN 2PÞ Rexp 5 P 2 i wi ðIo Þ

(3.2)

#1=2 (3.3)

where Io and IC are the experimental and calculated intensities, wi (5 1/Io ) and N are the weight and number of experimental observations and P the number of fitting parameters. This leads to the value of goodness of fit (GoF) defined as, GoF 5 Rwp/Rexp (Rietveld, 1967, 1969; Young and Wiles, 1982; Lutterotti, 2011). The peak broadening, peak asymmetry, and peak shift of the experimental profiles are fitted by refining the particle size, lattice strain, and lattice parameter values (including zero-shift error). Refinements of all parameters are carried out until convergence is obtained with the value of GoF very close to 1.0 (varies between 1.1 and 1.25 in the present case), confirming the goodness of refinement. The Caglioti parameters U, V, and W, instrumental asymmetry, and Gaussianity

3.2 Materials and Methods

parameters are obtained for the instrumental setup using a specially prepared Si standard and kept fixed during refinements.

3.2.6.2 Physical and mechanical property measurement For relative density measurements of the sintered compacts, Archimedes’ method was used. Weights (Wt) of all the sintered pellets were measured in air as well as in distilled water. The density of a sample is calculated by using the following formula: Densityðg=cm3 Þ; ρo 5

WtðairÞ 3 ρðwaterÞ WtðairÞ 2 WtðwaterÞ

(3.4)

In the present study, as the samples contain more than one phase, the relative densities of the samples were calculated by a rule of mixture using the theoretical densities of pure HAp (3.16 g/cm3), β-TCP (3.11 g/cm3), and TTCP (3.05 g/cm3) from the formula below (Lala et al., 2017), Relative density; ρr 5

ρo ρA KA 1 ρB KB 1 ρC KC

(3.5)

where ρA , ρB , and ρC are theoretical densities and KA, KB, and KC are the volume fractions of phases A, B, and C, respectively. Microhardness of all consolidated samples is measured by an instrumented microhardness tester (CSM international, Rue de la Gare 4, CH-2034, Peseux, Switzerland). Both surfaces of the sintered pellets were polished with fine diamond paste (0.5 μm) and then subjected to loads varying between 200 g and 1 kg for 30 seconds holding time in each case of loading. Vicker’s microhardness (HV) of each sample was determined by measuring the length of the diagonals of the square indent shape formed after the indentation. Hardness is estimated using the formula, HV 5 0:001854 3

P d2

(3.6)

where HV is Vicker’s hardness in GPa, P is applied load in N, and d is diagonal indent length in mm. For each sample, at least 10 indentations are taken on different portions of the sample surface. Elastic modulus is determined from the discharge load versus depth curve by the Oliver and Pharr method (Oliver and Pharr, 1992) using a microhardness tester with maximum load of 2N. Fracture toughness (K1C) of the consolidated specimens is measured using the Palmqvist cracks developed by the indentions produced during the microhardness testing using Eq. 3.7 (Anstis et al., 1981).  1=2 E P K1C 5 0:016 HV C3=2

(3.7)

where K1C is the fracture toughness (MPa m1/2), E and HV are elastic modulus and Vicker’s hardness (GPa), respectively, P is the applied load (N), with C being the Palmqvist crack length (mm).

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In addition, the brittleness index (B) has been determined by the following equation (Lawn and Marshall, 1979): B5

HV K1C

(3.8)

3.3 RESULTS AND DISCUSSIONS 3.3.1 PHASE CONFIRMATION OF UNSINTERED HAp SAMPLES FROM XRD PATTERNS Fig. 3.1A and B depicts the growth of nanocrystalline undoped and doped HAp powders by mechanical alloying the CaCO3 and CaHPO42H2O powder mixture taken in a 2:3 molar ratio and CaCO3, CaHPO42H2O, and MnO/MgO/ZnO taken in (4 2 x):6:x molar ratio (x 5 0.25, 0.5, 1.0 for Mn; x 5 0.25, 0.5, 1.0, 1.5 for Mg and x 5 0.5, 1.0, 1.5, 2.0 for Zn doping) respectively. In the unmilled undoped powder mixture only reflections of CaCO3 and CaHPO42H2O are present in accordance with their stoichiometric ratios, whereas, in the unmilled mixtures with metal oxides, traces of CaCO3, CaHPO42H2O, CaHPO4, and MnO/ MgO/ZnO can clearly be found. In Fig. 3.1A, all possible reflections of the precursor phases in the unmilled mixtures have been identified and marked with different peak markers. It may be noted that in the unmilled mixture with metal oxides there is a significant presence of triclinic CaHPO4 phase, though it is absent in the unmilled powder mixture without metal oxides (undoped). It seems that in the presence of metal oxides, water of crystallization (2H2O) of monoclinic CaHPO42H2O has been released and the compound transformed partially to triclinic CaHPO4 phase. After 10 hours of milling all powder mixtures result in complete formation of single-phase HAp without any contamination either from the milling media or from the initial precursors. In Fig. 3.1B, all possible HAp reflections are indexed properly. It is also evident that due to doping an amorphous-like background intensity increases significantly.

3.3.2 CONFIRMATION OF CARBONATION IN HAp BY FTIR ANALYSIS FTIR spectra of as-milled undoped and 5 mol.% Mn-, Mg-, and Zn-doped HAp powders are shown in Fig. 3.1C, which are typical spectra obtained for carbonated HAp. The FTIR spectra exhibit a significant amount of structural carbonate in all as-milled apatite samples. Two regions of the spectra are characteristic of carbonate vibrations in the as-milled samples (Fig. 3.1C) (Li et al., 2012): 1. 850
890 cm21 assigned to ν2 vibrations of carbonate groups; 2. 1400
1600 cm21 assigned to ν3 vibrations of carbonate groups.

3.3 Results and Discussions

FIGURE 3.1 (A) XRD patterns of initial unmilled precursor powder mixture and (B) milled mixtures of undoped and 5 mol.% Mn-, Mg-, and Zn-doped powders, each milled in open air at room temperature for 10 h. The major peaks of HAp are indexed according to ICSD #171549, (C) FTIR spectra of as-milled undoped and 5 mol.% Mn-, Mg-, and Zn-doped samples, (D) Rietveld output plot of 10 mol.% Mn-doped A-cHAp fitted with (i) both crystalline and amorphous A-cHAp phases and (ii) with only crystalline A-cHAp phase.

In the first region, the sharp band at 878 cm21 in the FTIR spectra corresponds to ν2 bending vibration of C
O bonds in the carbonate group, which is 2 the characteristic of A type carbonation in HAp (substitution of CO22 3 for OH channel ions) (Anwar et al., 2016; Sroka-Bartnicka et al., 2017; Li et al., 2012). In the other region there appears a relatively broad band within 1400
1580 cm21 due to ν3 stretching vibration of CO22 3 group in all the as-milled samples (Liu et al., 2015; Kannan et al., 2011; Guo et al., 2011, 2013). This broad band comprises of different overlapping frequencies of ν3,1450, 1470, 1540, and 1550 cm21 due to A type (substitution of CO22 for OH2 ions) and 1419 and 3 21 1465 cm due to B type carbonation (substitution of CO22 for PO32 ions) 3 4 (Anwar et al., 2016; Sroka-Bartnicka et al., 2017). However, in the entire spectra

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we did not observe any prominent bands resulting from vibration of the OH2 group, which usually appears at 630 (libration mode) and 3570 cm21 (stretching vibration) (Shaltout et al., 2011; Wu et al., 2010; Gasga et al., 2013) These obser2 vations confirm the complete A type substitution of CO22 3 for OH in all ballmilled HAp samples. FTIR spectra of all the as-milled samples show characteristic bands due to PO32 ions appeared at 472, 561, 598, 957, 1038, and 1106 cm21 (Zou et al., 4 2012; Li et al., 2012; Gamelas and Martins, 2015; Wu et al., 2010; Gasga et al., 2013). Among the phosphate-derived bands, the weak vibration band at 472 cm21 in the spectra corresponds to phosphate asymmetric stretching (ν2) vibration. The sharp doublet peaks at 561 and 598 cm21 are assigned to O
P
O bending mode (ν4) vibration. The band at 957 cm21 results from symmetric stretching (ν1) vibration of P
O bonds, whereas the strong band between 1038 and 1106 cm21 is due to P
O asymmetric stretching (ν3) vibration. The broad band at 3400 cm21 (stretching vibration) and another band at 1640 cm21 (flexural vibration) are attributed to the presence of adsorbed water from the air by the HAp molecule (Zhang et al., 2009). The absorption band present at 2350 cm21 in all the samples corresponds to CO2 absorption by the sample. We have also fitted the entire FTIR spectrum of each sample with multiple Gaussian functions to find out the phosphate to carbonate (P:C) content ratio in the as-milled samples. The P:C ratios are almost the same (varying between 3.98 and 4.04:1) in all the as-milled samples. Considering the charge balance, the structural formula of carbonated undoped HAp in the present work may be written as: Ca10 [(PO4)6
2y/3 (CO3)y][(OH)2
2x(CO3)x]. Considering the P:C ratio as B4:1 and complete substitution of OH2with CO22 (complete A type substitu3 tion), that is, when x 5 1, y comes out to be B0.43, which is equivalent to B 5.7 P atoms per unit cell instead of the ideal 6.0. Though the absence of OH vibrations confirms the complete A type substitution, this result reveals that there may be a small amount of B type carbonation in HAp. Hence, no impurities within the synthesized as-milled samples are detected by FTIR analysis.

3.3.3 QUANTITATIVE PHASE ESTIMATION OF UNSINTERED SAMPLES USING RIETVELD’S METHOD The Rietveld method is well accomplished in quantifying the relative phase abundance in a multiphase material. Experimental XRD patterns of all 10 hours milled undoped and doped HAp samples are fitted very well by refining the structural and microstructural parameters of the respective simulated patterns. Though in the as-milled samples the simulated pattern consists of CaCO3, CaHPO42H2O, CaHPO4, metal oxides, and HAp phases, apart from the HAp phase no trace of other phases is found in any of the samples. This confirms that all milled samples contain only a single HAp phase. The “goodness of fittings” (GoFs) in all cases lie between 1.1 and 1.25, signifying the fitting qualities are good enough for all experimental patterns.

3.3 Results and Discussions

From Rietveld’s analysis it is found that a part of the crystalline HAp phase is transformed to amorphous phase with very low particle size and high lattice strain and due to MA in undoped sample and cumulative effect of MA and cationic substitution (Mn21, Mg21, Zn21) in the doped samples milled for 10 hours. We have fitted the XRD patterns of all the as-milled samples without and with an additional amorphous HAp phase with very low particle size and high strain, along with crystalline HAp phase and such a plot for a 10 mol.% Mn-doped sample is shown in Fig. 3.1D. It can be noted that when the XRD pattern is fitted only with the crystalline phase (Fig. 3.1D-i) the quality of fitting was very poor and the unmatched intensity pattern at around 30 and 38 degrees 2θ indicates the presence of an amorphous-like phase, similar to the distorted crystalline HAp phase. Inclusion of the amorphous phase during refinement results in perfect fitting of the XRD pattern (Fig. 3.1D-ii) with a significant reduction in GOF value from 1.8 to 1.15. The residual of fittings (IO 2 IC) between observed (IO) and calculated (IC) intensities of fitting is plotted under the respective XRD patterns. The relative phase abundance of crystalline and amorphous HAp in all the as-milled samples as obtained from Rietveld’s analysis is shown in Fig. 3.2A-i, ii, iii. In undoped HAp, almost 50% amorphization is obtained due to MA and in addition to this inclusion of dopant ions into the HAp structure leads to an increase in this amorphization. For Mn-doped HAp B 60%, amorphization is obtained for 10 mol.% doping (Fig. 3.2A-i). Almost 70% amorphization is found for 15 mol.% Mg doping (Fig. 3.2A-ii), whereas B 80% amorphization is obtained for 20 mol.% Zn doping (Fig. 3.2A-iii). From the perspective of amorphous content in native biological apatites (B63%
67%) (Boanini et al., 2010; Dorozhkin and Epple, 2002), the prepared HAp can be considered as excellent biomimetic product for bone transplantation. Such amorphization should also improve the mechanical properties of HAp, along with homogeneous bone resorption (Lala et al., 2015a,b). Variation of lattice parameters, a and c of hexagonal HAp phase with increasing doping percentage of Mn/Mg/Zn are shown in Fig. 3.2B-i, ii and iii, respectively. In Mn-doped samples the lattice parameter a decreases slowly, whereas c decreases at a relatively faster rate with increasing Mn doping concentrations (Lala et al., 2015a,b), while for Mg-doped samples both these parameters decrease continuously with increasing Mg concentrations (Lala et al., 2016a,b). In the case of Zn-doped samples, the lattice parameter a remains almost invarient, whereas c decreases nonlinearly with increasing Zn substitution and finally tends to saturate after 15 mol.% of Zn substitution (Lala et al., 2015a,b). Such a decrease in lattice parameters can be attributed to the continuous substitution of ˚ ) by the smaller Mn21 (ionic radius 0.83 A ˚ )/Mg21 Ca21 ions (ionic radius 0.99 A 21 ˚ ˚ (ionic radius 0.72 A)/Zn (ionic radius 0.74 A) ions during solid solution formation. In Mn and Mg-HAp samples, the consequence of an almost linear decrease in both the lattice parameters with an increase in solute (Mn/Mg) concentrations into solvent (HAp) matrix follows Vegards’s law for substitutional alloys. This result is consistent with the results obtained by Ren et al. (2010).

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FIGURE 3.2 (Ai, ii, iii) Variation of volume fractions of different phases present in the as-milled samples with different mol.% of Mn, Mg, and Zn doping, respectively. (Bi, ii, iii) Variation of lattice parameters in the as-milled samples with different mol.% of Mn, Mg, and Zn doping, respectively.

3.3.4 MODIFICATION IN HAP STRUCTURE DUE TO MN/MG/ZN SUBSTITUTION In the HAp unit cell there are two nonequivalent Ca sites, designated as Ca1 and Ca2, which can be replaced by the substitutional cations. Local atomic arrangements of Ca1 coordinated by oxygen atoms are illustrated in Fig. 3.3A. The Ca1 site is surrounded by six PO32 tetrahedral, where six oxygen ions 4

3.3 Results and Discussions

FIGURE 3.3 (A) Oxygen coordination of columnar Ca1 sites in HAp and linking of two Ca1 columns via PO4 tetrahedral, (B) 3D view of partially substituted A type carbonated HAp unit cell structure, (C) distribution of PO4 tetrahedrons in and around the HAp unit cell, (D) local environment of carbonate hexagonal channel along with Ca2
O coordination.

(designated as O1 and O2) at the vertices of PO32 4 tetrahedral are located at the first nearest neighboring sites and another three oxygen ions (designated as O3) are present at the second nearest neighboring sites, forming the Ca1O9 substructure, as shown in Fig. 3.3A. Four of the 10 Ca21 ions in the unit cell are located at the first site (Ca1), and are usually designated as columnar calcium sites as they form single atomic columns perpendicular to the basal plane/ab plane (Fig. 3.3A). The remaining six Ca21 ions located at the second site (Ca2) form hexagonal channels along the c-direction of the unit cell. Among these six Ca221 ions, one group, consisting of three Ca2, forms a triangle located at 1/4th

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of the c-axis and the other group of three forms another triangle at 3/4th of the c-axis of the unit cell (Fig. 3.3B). The Ca221 site is exposed onto the crystal surface, consequently playing an important role in the physicochemical properties of HAp, such as surface charge distribution and interactions with organic compounds. In pure HAp lattice, there are partially occupied OH21 ions along the crystallographic c-axis forming the OH channel. The oxygen atoms in this channel are identified as O4 sites. In the CO channel of A type carbonated HAp (A-cHAp) lattice, the C atom is situated at the 2b Wyckoff site [fractional coordinate (0,0,0)]. Each of them is surrounded by six O5 atoms (the bottom part of the CO channel as shown in Fig. 3.3B) in which two triangular shaped C1
(O5)3 layers are lying parallel to each other containing three alternative O5 atoms in each plane (the top part of the CO channel in Fig. 3.3B). Six of the Ca221 ions surrounding the C
O channel of the A-cHAp lattice forms a hexagonal channel along the c-axis. In Fig. 3.3C the arrangement of PO4 polyhedrons in and around the HAp unit cell is shown. Of these 10 polyhedrons, six lie at the interior of the unit cell along the opposite diagonal of the unit cell and the remaining four remain at the ac or bc plane of the unit cell (Fig. 3.3C). In each of these PO4 polyhedra the central P atom is surrounded by four O atoms lying at the vertices (one O1, one O2, and two O3 atoms, which are shared by Ca1O9 and Ca2O7 substructures). These PO4 polyhedra are the main building block of the HAp unit cell. The local environment of the carbonate hexagonal channel along with Ca2
O coordination is depicted in Fig. 3.3D. Bivalent cations are thought to replace Ca21 ions in the HAp lattice. There exist two crystallographically different calcium sites in the HAp unit cell: Ca1 and Ca2. Although the Ca2 (triangular Ca site about the OH arrangement along the c-axis) site is expected to be energetically more favorable than the Ca1 (columnar Ca atoms parallel to c-axis) site for such substitution (Matsunaga et al., 2010), still other sites for such occupation (substitution or insertion mechanism) have also been reported (Gomes et al., 2011, 2012, 2014). The dopants may occupy interstitial sites in HAp (Wyckoff site 2b). However, this particular site is occupied by C atoms in the A-cHAp structure and hence is excluded. To find out the exact location of cationic substitution in the HAp lattice we have considered the following three different models of the HAp lattice structure exposed to Rietveld structure refinements: (1) cations replace Ca1 site only (Model 1), (2) cations substitute both the Ca1 and Ca2 sites (Model 2), and (3) cations replace Ca2 site only (Model 3). During refinements one additional crystallographic site has been introduced in the HAp lattice structure for metal atoms. For Model 1, the Ca1 site occupancy has been set bound to an additional site referred as M1 (M designates Mn/Mg/Zn) having fractional coordinates the same as the Ca1 site. In Model 2, two additional sites, M1 and M2, with fractional coordinates the same as Ca1 and Ca2, respectively, were set bound to the respective Ca1 and Ca2 sites. Finally, in Model 3 the Ca2 site was set bound to an additional M2 site

3.3 Results and Discussions

having coordinates the same as the Ca2 site. Occupancies of both Ca1 and Ca2 sites in the HAp structure are refined carefully. In both Mn- and Mg-doped HAp samples the occupancy of the Ca2 site is found to reduce by a significant amount, whereas that of the Ca1 site remained almost invariant. Consequently, the occupancy of the Mn2/Mg2 site takes a significant value. The numbers of Ca2 and Mn2/Mg2 atoms per unit cell with increasing dopant concentration are also evaluated. The number of Ca2 atoms in undoped HAp unit cells is six and it decreases continuously to nearly five with an increase in Mn/Mg substitution into HAp from 0.00 to B 1.00 for 10 mol.% substitution for each. Such a plot for Mn-doped HAp is shown in Fig. 3.4. The occupancy of Ca2 decreases accordingly with a decrease in the quantity of Ca2. Therefore, Rietveld’s analysis confirms the substitution of Ca2 atoms with Mn/ Mg atoms and such substitution along with local environment around CO channel in A-cHAp lattice is depicted in Fig. 3.3D.

FIGURE 3.4 Variation of quantity of Ca2/Mn2 atoms per unit cell in all as-milled samples with increasing Mn content. Inset of the figure shows the variation of occupancy of Ca2/Mn2 atoms in all as-milled samples. Reprinted from Lala, S., Ghosh, M., Das, P.K., Kar, T., Pradhan, S.K., Mechanical preparation of nanocrystalline biocompatible single-phase Mn-doped A-type carbonated hydroxyapatite (A-cHAp): effect of Mn doping on microstructure. Dalton Trans. 44, 20087
20097. copyright (2015), with permission from Dalton Trans.

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For Zn-doped HAp samples in all three structure models, Ca2 site occupancy is found to be nearly unity, even in Model 2 and Model 3, where there is a possibility of Zn substitution in the Ca2 site. In both Model 2 and Model 3, occupancy of the Zn2 site remains at almost zero, indicating no substitution of Zn in the Ca2 site. However, in Model 1 and Model 2, where Zn substitution for Ca1 is possible, it shows the nearly identical nature of variation of Ca1 and Zn1 occupancy. In both of these models we have found a significant amount of occupancy of the Zn1 site, which increases with mol.% of Zn doping. Analysis of all these results reveals that the Zn atoms substitute the Ca1 atoms in the HAp structure. Substitution of the Ca2 site by Mn/Mg is manifested by significant changes in bond lengths between Ca2/M and O sites in undoped and Mn/Mg-doped HAp with different percentages of dopant concentration and these changes have been measured using ATOMS 6.2 software and are depicted in Fig. 3.5A and B. It is evident that in the undoped sample, Ca2
O bonds are of different lengths, similar to John
Teller distortions in HAp lattice. In both Mn- and Mg-doped samples all Ca2/M (Mn/Mg)
O bond lengths decrease, in general with different rates, from the undoped one, and these contractions increased further with an increase in dopant mol.%. It may be noted that in undoped HAp one of the Ca2
O3 bonds with ˚ bond length is almost parallel to the c-axis and the other two Ca2
O3 2.34 A ˚ bond lengths make an B45 degrees bond angle with the cbonds with 2.53 A axis. The Ca2
O1 and Ca2
O2 bond lengths are also unequal and parallel to the b- and a -axes, respectively. After Mn/Mg doping, maximum contraction is noticed for Ca2
O3 bonds in comparison to both Ca2/M
O1 and Ca2/M
O2 bond lengths and the degree of contraction increases with increasing doping concentrations. This may be due to the fact that the decrease in lattice parameter c is more than a in doped samples. It is also noticed that the Ca2
O1:Ca2
O2 bond length ratio 1.153 in undoped samples increases gradually to 1.162 for 10 mol.% Mn doping and 1.174 for 15 mol.% Mg doping. The Ca2
O3 (making 45 degrees with c axis):Ca2
O3 (parallel to c axis) ratio also increases from 1.081 in undoped HAp to 1.086 and 1.096 with an increase in Mn and Mg doping concentrations to 10 mol.% and 15 mol.%, repectively. This indicates that the lattice distortion increases with increasing doping concentrations. It may lead to increased disorder in the lattice and results in transformation of crystalline to amorphouslike HAp phase. However, the change in Ca2/M
O5 bonds is small as O5 sites are connected to C atoms residing at 2b Wyckoff position with fixed (0,0,0) coordinates and did not respond to lattice distortion significantly. It appears that the O5 site approaches slowly the Mn/Mg atom during substitution of Ca221 position by smaller Mn21/Mg21. Therefore, the overall results confirm the substitution of Mn21/Mg21 in the Ca221 site only. We also have compared the changes in different bond lengths in Ca1O9 polyhedron with increasing Zn mol.%. All possible bond lengths are marked and shown in Fig. 3.5C. It is evident that with increasing Zn mol.%, all Ca1/Zn
Ca1/ Zn, Ca1/Zn
O1, Ca1/Zn
O2, and Ca1/Zn
O3 bond lengths reduce gradually due to substitution of larger Ca121 ions by smaller Zn21 ions. The Ca1/Zn
Ca1/Zn

3.3 Results and Discussions

FIGURE 3.5 (A) and (B) Changes in Ca2/M (Mn/Mg)
O bond lengths (A˚) in different Mn/Mg-doped samples in comparison to undoped sample, (C) position and shape of Ca1O9 polyhedron in 10 h milled Zn-doped A-cHAp lattice with different mol.% of Zn doping. (D) Atomic structure of PO4 tetrahedron in 10 h milled Zn-doped A-cHAp lattice with different mol.% of Zn doping.

˚ , Ca1/Zn
O1 bond length 2.36 A ˚ , and Ca1/Zn
O3 bond bond length 3.33 A ˚ ˚ , respectively, in a length 2.81 A in 5 mol.% Zn reduce to 3.27, 2.02, and 2.59 A 20 mol.% Zn-doped sample. In comparison to 5 mol.% Zn-doped sample, O1(Ca1/Zn)-O1 bond angle as It resembles the angle subtended by O1-(Ca/Zn) and (Ca/Zn)-O1 bonds angle in a 20 mol.% Zn-doped sample is decreased due to very high strain values for the planes parallel to c-axis (hk0). Therefore, an increase in

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Zn substitution leads to generating deformation in the HAp structure as well as length contraction of the lattice along the c-axis. However, Ca1/Zn
O2 bond lengths in all doped samples remain almost invariant. This indicates that the base of the Ca1O9 polyhedron remains almost unchanged but the upper and central part reduces gradually with increasing Zn doping and results in a change in the shape and size of the polyhedrons. This result is consistent with the changes in lattice parameters a and c with variation of concentration of Zn doping into HAp structure. As the Zn atoms substitute the ninefold coordinated Ca1 sites lying along the c-axis, lattice parameter a remains almost invariant when c decreases. All these results confirm substitution of Ca1 sites by Zn21 ions in the Zn-HAp sample. The HAp unit cell contains six PO4 tetrahedrons. An ncrease in cationic substitution into the HAp lattice increases the appreciable distortion in PO4 tetrahedrons. Such distortion manifested in tilting of PO4 polyhedrons is shown in Fig. 3.5D for Zn substitution. This tilting is recognized as a decrease in O3-P-O2 bond angle as it resembles angle subtended by O3-P and P-O2 bonds angle with an increase in Zn doping as shown in Fig. 3.5D. Furthermore, with an increase in Zn content, the central P atom gradually shifts toward the surface of the tetrahedron and finally comes out partially from the polyhedron and forms an incomplete tetrahedron due to high strain concentrations especially at higher Zn mol.% of doping. The Rietveld analysis is a superior technique to explore the shape and size of crystallite in a powder sample. It reveals that crystallites of all doped samples are isotropic in nature. Variations of crystallite size with different mol.% of metal doping obtained from Rietveld analysis are shown in Fig. 3.6. The crystallite size of a 2.5 mol.% doped sample is found to be B 18 nm, which reduces slowly to B 15 nm for 10 mol.% Mn doping. The particle size of an Mg-doped sample is found to be minimum (11 nm for 15 mol.% doping), whereas for a Zn-doped sample the particle size reduces from 18 nm (5 mol.% doping) to 14 nm (20 mol.% doping). Therefore, in all cases, crystallite size decreases with an increase in doping concentrations. This is due to the fact that with an increase in cationic substitution, the lattice distortion increases and long range order of periodicity of atomic sites decreases resulting in a decrease in particle size. The r.m.s. lattice strain generated in doped HAp lattice during milling as well as metal ion substitution has also been obtained from the Rietveld analysis and is shown in Fig. 3.6. As crystallite size decreases, r.m.s. lattice strain generated within the lattice gradually increases. Consequently, we notice that the r.m.s lattice strain increases significantly with increasing doping concentration. These results signify the dominance of strain broadening over particle size broadening in the XRD patterns.

3.3.5 HRTEM ANALYSIS The selected area electron diffraction (SAED) pattern of a 10 mol.% Mn-doped HAp sample is shown in Fig. 3.7A. It clearly reveals the formation of the

3.3 Results and Discussions

FIGURE 3.6 Variation of particle size and lattice strain in the as-milled samples with different mol.% of (A) Mn, (B) Mg, and (C) Zn doping.

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FIGURE 3.7 (A) Selected area electron diffraction (SAED) pattern of 10 h milled 10 mol.% Mn-doped HAp powder, (B) HRTEM image of crystalline zone and amorphous zone in 20 mol.% Zndoped sample, (C) HRTEM micrograph of an isolated well-grown nano-sized HAp single crystal, (D) magnified fringe pattern showing some multiorder reflections and the indexed FFT pattern (inset) of the region of 15 mol.% Mg-doped HAp.

nanocrystalline HAp phase. These reflections are properly indexed as per the ICSD # 171549 (Sp. gr. P63/m) and the intensity distributions of each individual reflection are in accordance with the XRD patterns of the milled sample shown in Fig. 3.1B. Fig. 3.7B gives direct evidence of the presence of both, the crystalline and the amorphous HAp phases in the 20 mol.% Zn-doped sample. In fact, the abundance of amorphous phase in the sample is obvious from the HRTEM image—consistent with the Rietveld analysis. From the Rietveld analysis, particles are found to be isotropic in nature and the image of such an individual spherical particle is shown in Fig. 3.7C. It consists of (2 2 0) lattice planes only. Such a particle may be considered as a tiny single crystal of extremely small dimension (B13.14 nm) and the perfectly parallel lattice

3.3 Results and Discussions

fringes confirm that the particle is free from lattice imperfections. Fig. 3.7D shows the lattice fringe pattern and the FFT pattern (inset) of the HRTEM image of a 15 mol.% Mg-doped sample along the [1 0 0] direction. The interplanar spacings are measured as 0.31, 0.47, and 0.27 nm, which correspond to the (1 0 2), (1 1 0), and (1 1 2) planes, respectively. The FFT pattern is properly indexed using the same ICSD standard. The halo around the center of the FFT pattern arises due to the presence of the amorphous phase in the sample, which is consistent with the XRD analysis result.

3.3.6 MICROSTRUCTURE CHARACTERIZATIONS OF THE SPARK PLASMA SINTERED SAMPLES Fig. 3.8A illustrates the XRD patterns of the undoped and all 5 mol.% doped samples sintered at 950 C. SPS of undoped and doped HAp as-synthesized samples leads to partial decomposition to other phases. In undoped, 5 mol.% Mn- and Mgdoped samples, the HAp phase partially transforms to the β-TCP phase, and in the 5 mol.% Zn-doped sample, the HAp phase partially decomposes to both β-TCP and TTCP phases (Lala et al., 2017). The outputs of Rietveld’s fitted patterns of a selected portion (2θ 5 30
37 degrees) of the sintered samples are shown in Fig. 3.8B. From the figure it is evident that when the as-synthesized undoped HAp sample is sintered at 950 C, the most intense (0 2 10) reflection of β-TCP begins to grow from the HAp lattice. It is to be noted that HAp and β-TCP phases have similar crystal structure (the HAp unit cell has a hexagonal structure with Sp. gr. P63/m and the β-TCP unit cell is trigonal with a hexagonal base, Sp. gr. R3c:H) and interplanar spacings (d-values) of the most intense reflection of HAp (d1 2 1 5 0.282 nm) and that of β-TCP (d0 2 10 5 0.286 nm) are very close (Lala et al., 2016a,b). Thus, it seems that the (0 2 10) plane of β-TCP lattice grows coherently on the (1 2 1) plane of HAp lattice. In undoped sintered HAp, these two planes are overlapped, while the presence of the isolated second highest intense peak (2 2 0) of β-TCP phase has been found. In 5 mol.% Mn-doped HAp sintered samples, (0 2 10) and (1 2 1) peaks of β-TCP and HAp phases are completely isolated and the intensities of (0 2 10) and (2 2 0) peaks are much higher than those in the undoped sintered HAp. This indicates that a significant amount of Mn-doped HAp phase has been transformed to the β-TCP phase at the same sintering temperature. The rate of decomposition further increases in Mg-doped sintered HAp. In a 5 mol.% Zn-doped sintered HAp sample, the intensity of the (0 2 10) peak of β-TCP exceeds the intensity of the (1 2 1) peak of HAp phase; indicating the amount of decomposed β-TCP phase surpasses the amount of the parent HAp phase. The presence of some other reflections of β-TCP along with (1 2 3) reflection of TTCP is also noticed in the XRD pattern and they have been accordingly indexed. Thus, it is clear that although the sintering temperature (950 C) and concentration of dopant (5 mol.%) are the same, different kinds of dopant have an enormous influence on the

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FIGURE 3.8 (A) XRD patterns of the undoped and 5 mol.% Mn-, Mg-, and Zn-doped HAp sintered at 950 C via SPS. (B) The output of Rietveld’s fitted patterns of (2θ 5 30
37 degrees) the undoped, and 5 mol.% Mn-, Mg-, and Zn-doped sintered HAp samples. Reprinted from Lala, S., Maity, T.N., Singha, M., Biswas, K., Pradhan, S.K., Effect of doping (Mg, Mn, Zn) on the microstructure and mechanical properties of spark plasma sintered hydroxyapatites synthesized by mechanical alloying. Ceram. Int. 43, 2389
2397, copyright (2017), with permission from Elsevier.

3.3 Results and Discussions

decomposition and sintering behavior of HAp. After sintering, all undoped and doped as-synthesized nanocrystalline single-phased HAp samples turn into composites containing a mixture of HAp and mainly β-TCP phases with different compositions. These sintered undoped and doped HAp composites are expected to evolve with different mechanical properties. However, the origin of different sintering behaviors may be due to the following reasons: (1) the size mismatch of ˚ ) and the doping ions (Mn21 5 0.83 A ˚ , Mg21 5 the Ca21 (ionic radius 0. 99 A 21 ˚ ˚ 0.72 A, Zn 5 0.74 A) and (2) the difference in melting temperature of different dopants (1250 C, 650 C, and 419 C for Mn, Mg, and Zn, respectively). Among the sintered samples, the maximum decomposition of HAp is obtained for the Zndoped sample. Along with phase quantification of different phases present in the sintered samples, the structural and microstructural parameters as obtained from Rietveld’s analysis are tabulated in Table 3.1. In the undoped sintered sample, decomposition of HAp into β-TCP is found to be B6%, which increases for Mn, Mg, and Zn doping in ascending order up to B49%. For a Zn-doped sample along with β-TCP, partial decomposition of HAp into TTCP (B12%) also occurred (Lala et al., 2017). However, the phase of α-TCP is absent in all these sintered samples. The lattice parameters and lattice volume values of HAp along with other phases of all the sintered samples are also given in Table 3.1. Due to replacement of Ca21 by other smaller cations, the lattice volume of HAp for all doped samples decreases from that of sintered undoped HAp. Among the doped sintered samples, the lattice volume is found to be maximum for Mn-HAp and minimum for MgHAp, while Zn-HAp possesses an intermediate value due to the decreasing order of ionic radii of Mn21, Zn21, and Mg21 ions. The particle size and lattice strain values of different phases of all sintered samples are also tabulated in Table 3.1. It is interesting to note that for doped samples, the particle size of the HAp phase increases with the atomic number of the dopant, whereas the grain size of β-TCP increases with a decrease in the melting temperatures of doping elements. This implies that grain growth during SPS is dominated by the elements having lower melting temperature with relatively weak binding energy. The grain growth is relatively higher in the β-TCP phase, which indicates that this phase contains a significant part of the dopants during its coherent growth from the HAp phase. Subsequently, the volume percentage of the β-TCP phase increases with a decrease in the melting temperature of the dopant elements. For the undoped sintered sample, the maximum lattice strain of the β-TCP is found and it gradually decreases with an increase in the phase content of β-TCP in the doped samples. A free growth of β-TCP results in a decrease of the lattice strain. However, the r.m.s. lattice strain of HAp does not change significantly. It is thus evident from the above findings that nanocrystalline undoped and doped HAp composites with restricted grain growth have been achieved by SPS with significant lattice strain inside the compacted samples.

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Table 3.1 Percentage Phase Quantity, Structural and Microstructure Parameters of Different Phases Present in the Sintered Undoped and Doped HAp Samples Sample (Sintered at 950 C)

Phase % (Error , 1%)

Undoped HAp

HAp (94.1%) β-TCP (5.9%) HAp (78.3%) β-TCP (21.7%) HAp (61.5%) β-TCP (38.5%) HAp (38.44%) β-TCP (48.89%) TTCP (12.66%)

5 mol.% Mn-HAp 5 mol.% Mg-HAp 5 mol.% Zn-HAp

Lattice Parameters (Å) (Error: 6 0.0003 to 6 0.0006 Å) a a a a a a a a a c

5 9.465, c 5 6.883 5 10.360, c 5 37.292 5 9.476, c 5 6.861 5 10.329, c 5 37.275 5 9.459, c 5 6.875 5 10.291, c 5 37.253 5 9.469, c 5 6.868 5 10.330, c 5 37.251 5 7.102, b 5 10.960, 5 10.075

Lattice Volume (Å3)

Particle Size (nm) (Error: 6 4 to 6 8 nm)

R.m.s. Lattice Strain (Error: 6 0.03 3 1023 to 6 0.07 3 1023)

534.1 3466.4 533.4 3444.1 532.7 3416.3 533.3 3442.4 784.2

82 87 95 98 88 130 153 262 167

2.13 3 1023 3.47 3 1023 2.2 3 1023 1.92 3 1023 2.4 3 1023 1.8 3 1023 1.8 3 1023 1.2 3 1023 1.1 3 1023

Reprinted from Lala, S., Maity, T.N., Singha, M., Biswas, K., Pradhan, S.K., Effect of doping (Mg, Mn, Zn) on the microstructure and mechanical properties of spark plasma sintered hydroxyapatites synthesized by mechanical alloying. Ceram. Int. 43, 2389–2397, copyright (2017), with permission from Elsevier.

3.3 Results and Discussions

Fine-scale microstructural characterization of the sintered compacts has been performed using SEM and localized EDX analysis. Fig. 3.9 shows the backscattered (BSE) SEM images of the undoped and doped samples with a magnified micrograph shown in the inset. Fig. 3.9A shows an SEM image of undoped HAp indicating the presence of major HAp phase (gray phase) with a very small amount of β-TCP (white phase). Fig. 3.9B represents an SEM image of the Mndoped sample, showing the predominant presence of the HAp phase (light gray phase) with little β-TCP as the relatively dark gray phase along with few pores. The Mg-doped sample consists of HAp phase (light gray phase) with a relatively higher amount of β-TCP phase (dark gray phase) (Fig. 3.9C). However, sintering of Zn-doped sample leads to transformation of HAp to both β-TCP (white phase) as well as TTCP phases (dark gray phase) (Fig. 3.9D). The dark black regions are pores. The elemental analysis of different phases present in the samples is also done. The EDX spectra obtained from different-colored regions corresponding to different phases for each sample are also presented in the respective micrographs in Fig. 3.9A
D. From the EDX spectra it is clear that in the doped HAp samples, along with the parent HAp phase, the doping elements are also present in the decomposed secondary phases. In the undoped sample the Ca/P ration in the HAp phase is 1.65 and in β-TCP it is 1.53, whereas the (Ca 1 Mn)/P ratio in the HAp phase is 1.65 and in β-TCP it is 1.52 in the Mn-doped sample. In the Mg-doped sample, the (Ca 1 Mg)/P ratio in the HAp phase is 1.68 while that in the β-TCP phase is 1.53 and in the Zn-doped sample the (Ca 1 Zn)/P ratio in HAp, β-TCP, and TTCP phases are 1.65, 1.53, 1.92, respectively (Lala et al., 2017).

3.3.7 MECHANICAL PROPERTIES OF THE SINTERED HAp SAMPLES The sintered compacts (pellets) of undoped and doped HAp composites prepared in the present investigation exhibit higher relative density and finer grain size at a relatively low sintering temperature. As the sample has potential applications in orthopedics, it is very important to measure the mechanical properties of the sintered specimens. The mechanical properties of all sintered pellets are given in Table 3.2. In the present investigation, one of our prime objectives is to achieve the dense nanocrystalline HAp with restricted grain growth. For all the samples, almost 97% relative density is obtained after sintering at 950 C. Maximum density is obtained for undoped HAp, where the phase content of β-TCP is the lowest. The theoretical density of the secondary phases, like β-TCP (3.11 g/cm3) and TTCP (3.05 g/cm3), are less than that of pure HAp (3.16 g/cm3). That is why an increase in the content of the secondary phases results in relatively lower densification of the doped HAp composites. In addition, finer particle size at this temperature would have its influence on the mechanical properties. The elastic modulus of each sample is determined from the load
displacement curves, with a maximum load of 2N. The maximum value of elastic modulus is found for sintered undoped HAp, whereas the minimum value is obtained for the Zn-doped sample. However, the value of elastic

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FIGURE 3.9 SEM backscattered images with EDX spectra of different-colored regions in (A) undoped, (B) Mn-doped, (C) Mg-doped, and (D) Zn-doped HAp samples sintered at 950 C by SPS. Reprinted from Lala, S., Maity, T.N., Singha, M., Biswas, K., Pradhan, S.K., Effect of doping (Mg, Mn, Zn) on the microstructure and mechanical properties of spark plasma sintered hydroxyapatites synthesized by mechanical alloying. Ceram. Int. 43, 2389
2397, copyright (2017), with permission from Elsevier.

Table 3.2 Mechanical Properties of the Spark Plasma Sintered Undoped and Doped HAp Samples Sample (intered at 950 C)

Relative Density ρr ð%Þ

Elastic Modulus (E) (GPa)

Reduced Modulus (Er) (GPa)

Hardness (HV) (MPa)

Fracture Toughness (K1c in MPa m1/2)

Brittleness Index, B (μm21/2)

Undoped HAp 5 mol.% Mn-HAp 5 mol.% Mg-HAp 5 mol.% Zn-HAp

97.2 96.8 96.3 96.1

6 6 6 6

173.37 6 17.34 63.12 6 11.5 89.11 6 6.5 58.38 6 2.5

162.41 6 14.1 61.31 6 4.1 90.23 6 6.1 60.76 6 2.5

13613.51 6 2289 3446.3 6 621 5142.6 6 1089 2058 6 210

0.53 6 0.14 1.69 6 0.35 1.0 6 0.31 1.54 6 0.50

25.68 2.039 5.142 1.336

0.4 0.4 0.3 0.5

6 6 6 6

4.1 0.06 0.82 0.43

Reprinted from Lala, S., Maity, T.N., Singha, M., Biswas, K., Pradhan, S.K., Effect of doping (Mg, Mn, Zn) on the microstructure and mechanical properties of spark plasma sintered hydroxyapatites synthesized by mechanical alloying. Ceram. Int. 43, 2389
2397, copyright (2017), with permission from Elsevier.

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modulus of undoped HAp is relatively very high compared to the doped samples (Table 3.2). From the load
displacement plots we found that the slope of the unloading curve of the undoped HAp sample is less than the doped samples and the slopes of the Mn-, Mg-, and Zn-doped samples are nearly identical, resulting in high elastic modulus for the undoped HAp. Similar to elastic modulus, the hardness of the undoped HAp is exceedingly high compared to the doped HAp samples. The minimum hardness value is obtained for the Zn-doped sample. This result can be interpreted by the dependence of hardness on the grain size according to Eq. 3.9 derived from the Hall
Petch equation (Furukawa et al., 1996) given by, HV 5 Ho 1 KH d2ð1=2Þ

(3.9)

where Ho and KH are material constants related to the hardness measurements, and d is the average grain size. For undoped HAp, the grain size of the primary HAp phase is minimum (B82 nm) and the phase abundance of secondary β-TCP phase is very less with particle size B87 nm; resulting in significantly high hardness of the sample. In Mg- and Mn-doped HAp samples, the grain sizes of both HAp and β-TCP phases are relatively higher than that of the undoped HAp and consequently the hardness of Mg- and Mn-doped HAp samples is less than that of undoped HAp. In the Zn-doped HAp sample, after decomposition, the β-TCP becomes major phase, along with the minor TTCP phase. It may be noted that there is a significant grain growth in all three phases and, as a consequence, microhardness is found to be minimum in the Zn-doped sample among all other samples. The most remarkable results are found in the variation of fracture toughness of the doped samples. The minimum fracture toughness (K1C) is found for the undoped sample and it increases more than threefold with only 5 mol.% Mn doping in the HAp sample (Table 3.2). The enhancement in the value of K1C in the doped samples may be correlated with the microstructure and phase quantity of the secondary phases present in the samples. The β-TCP phase is reported to be almost 1.3 times tougher than HAp (Lopez et al., 1999). Hence, abundance of β-TCP in the composites increases the toughness of the HAp samples. The transformation of HAp into β-TCP is also accompanied by the expansion of the lattice volume of the HAp phase, leading to the accumulation of the residual stress around the crack tip and the excess energy is required to propagate the crack through the sample (Lopez et al., 1999; Yazdanpanahn et al., 2015). In other words, the abundance in β-TCP causes crack deflection during the application of load and increases the toughness of the sample. In undoped HAp, the content of β-TCP is very much less and, consequently, the cracks can easily propagate through the grains, resulting in a minimum value of K1C. In addition, the larger grain size of the phases results in easy crack propagation, leading to lowering of the toughness. In the Mn-doped sample, there is a significant amount (B22%) of β-TCP and also particle sizes of both HAp and β-TCP phases are relatively low, resulting in a maximum value of the toughness among all the samples. In the Zn-doped sample, β-TCP and TTCP phases have

3.3 Results and Discussions

larger lattice volume than HAp and cumulatively they constitute the major volume percentage in the composite. As a result, the K1C of the Zn-doped HAp composite is significantly higher. However, as the crystallite size of the sample is relatively larger than the Mn-doped sample, the toughness of the sample is less than for the Mn-doped HAp composite. Similarly, the brittleness index of all the sintered specimens are calculated using Eq. 3.8. The brittleness index of the undoped HAp is exceedingly higher than the doped samples. Therefore, addition of the doping elements to HAp significantly reduces the brittleness index of the sample (Table 3.2). Especially, the values of brittleness index of Zn- and Mn-doped HAp are less as compared to that of undoped HAp. Incorporation of doping elements mainly leads to a considerable increase of fracture toughness of the samples along with a consequent decrease in the brittleness index. This behavior suggests that the addition of explicit ions can play a significant role in crack propagation mode and fracture toughness of substituted HAp and thus improves the mechanical properties.

3.3.8 CYTOCOMPATIBILITY TEST The cytocompatibility of the as-synthesized undoped and doped HAp samples is analyzed using MTT assay. Initially, a stock solution of 1000 μg/mL is prepared by sonication for 30 minutes. The samples are incubated with CHO cells for 12, 24, and 48 hours. Ninety percent or more of the cells are alive in all the samples up to 20 μg/mL, and with a further increase in the doping concentration of HAp, the viability of the cells marginally decreases to around 85% up to a concentration of 100 μg/mL for an incubation time of 24 hours (shown in Fig. 3.10A). Interestingly, even after 48 hours of incubation, the cytocompatibility of the cells is (B80%) at a maximum concentration of 100 μg/mL for each sample. Thus, from the time-dependent cytocompatibility study it can be concluded that all the samples demonstrate a sufficiently high percentage of cell viability (B80%) even after 48 hours of incubation, confirming the cytocompatibility of the mechanically synthesized HAp samples. The cytocompatibility of all undoped and doped as-synthesized HAp samples is further confirmed by incubating CHO cells with 100 μg/mL of all the assynthesized HAp samples for 48 hours followed by treating it with the LIVE/ DEAD viability kit and then observed under fluorescent microscope. This is presented in Fig. 3.10B for Mg-doped samples. Fluorescence images showed predominant presence of green cells and absence of red cells, which clearly indicates the presence of live cells and sufficient cytocompatibility of the as-synthesized HAp samples (Fig. 3.10B-ii, iv, vi, viii). Moreover, in each case the bright field images (Fig. 3.10B-i, iii, v, vii) show cells having the same morphology in all the samples with different dopant concentrations. This also verifies the samples as being biofriendly.

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FIGURE 3.10 (A) Viability of CHO cells treated with varying concentrations of as-milled undoped and doped HAp samples for 24 h incubation. Percent errors are within 6 5% in triplicate experiments, (Bi, iii, v, vii): bright field images of CHO cells incubated with 100 μg/mL each of 2.5, 5, 10, and 15 mol.% Mg-doped HAp samples, respectively, for 48 h. (Bii, iv, vi, viii): LIVE/DEAD microscopic fluorescence images of the cells incubated for 48 h with 2.5, 5, 10, and 15 mol.% Mg-doped HAp samples, respectively.

References

3.4 CONCLUSIONS Nanocrystalline single-phase undoped and Mn-, Mg-, and Zn-doped carbonated HAp powders are synthesized at room temperature by mechanical alloying of the CaCO3, CaHPO42H2O, and respective metal oxide powder mixture. FTIR spectra 2 analysis reveals A type carbonation (substitution of CO22 3 for OH ) in the HAp lattice. Rietveld refinement of XRD patterns reveals detailed microstructure characterization in terms of lattice imperfections, measures the relative abundance of crystalline and amorphous, and confirms the substitution of Ca221 by Mn21/ Mg21 in Mn/Mg-doped HAp lattice, while Zn21 ions substitute into the ninefold coordinated Ca21 sites, that is, the Ca1 sites in the HAp unit cell. HRTEM image analyses confirm the presence of an amorphous phase originated due to the cumulative effect of mechanical alloying and ionic substitution in the HAp lattice. MTT assay, along with bright field and fluorescent images of the cells incubated with the stock solution of the samples, reveal a high percentage of cell viability and hence confirms the cytocompatibility of all the HAp samples. In order to obtain dense pellets of the samples for mechanical characterizations, the assynthesized powders are sintered at 950 C using SPS. Subsequent sintering of the samples causes the partial decomposition of HAp phase to β-TCP in undoped, Mn- and Mg-doped HAp, while Zn-doped HAp further decomposes to TTCP phase. The sintering also results in the formation of highly dense compact without significant grain growth. The sintered undoped HAp composite shows the highest hardness of B13 GPa, the lowest fracture toughness of 0.53 MPa m1/2, and the highest brittleness index of B25μm21/2 in comparison to the doped HAp composites. The minimum hardness of B2 GPa and the lowest brittleness index of 1.3 μm21/2 are obtained for Zn-doped HAp composites. The maximum fracture toughness (B1.7 MPa m1/2) is obtained for the sintered Mn-doped HAp composite. Hence, the mechanical properties of the sintered nanocrystalline HAp composites can be adjusted as per the requirement by addition of a small amount of impurities into the HAp lattice through SPS.

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CHAPTER

Multiparticle composites based on nanostructurized arsenic sulfides As4S4 in biomedical engineering

4

ˇ ´ kova´3, Peter Bala´zˇ 3, Yaroslav Shpotyuk4,5 Oleh Shpotyuk1,2, Zdenka Bujna and Adam Ingram6 1

Vlokh Institute of Physical Optics, Lviv, Ukraine 2Jan Dlugosz University in Czestochowa, Czestochowa, Poland 3Institute of Geotechnics, Slovak Academy of Sciences, Koˇsice, Slovakia 4 Faculty of Mathematics and Natural Sciences, University of Rzeszow, Rzeszow, Poland 5Ivan Franko National University of Lviv, Department of Sensor and Semiconductor Electronics, Lviv, Ukraine 6Opole University of Technology, Opole, Poland

4.1 INTRODUCTION Since prehistorical times, the chemical compounds of arsenic, often also called arsenicals in the medical literature (Dilda and Hogg, 2007; Liu et al., 2008), have been known as promising and versatile drugs in medicine, owing to their angiogenesis-inhibiting effect on a number of human malignancies (Dilda and Hogg, 2007; Liu et al., 2008; Gibaut and Jaouen, 2010; Wang, 2001). Thus, pronounced anticancer functionality of known tetra-arsenic tetrasulfide As4S4 polymorphs has been of great interest in biomedicine because of its promising antileukemic activity (Gibaut and Jaouen, 2010; Wang, 2001). To overcome negative feedback due to poor bioavailability as a consequence of limited water solubility, these arsenicals are often reduced to a nanosize, covering the characteristic nano- and subnanometer interatomic length scales (Tian et al., 2014; Deng et al., 2001; Bala´zˇ et al., 2007; Bujnakova et al., 2015a; Pastorek et al., 2014; Wang et al., 2009). Being subjected to high-energy ball milling in solutions with some biocompatible polymers, they form nanocomposites (NCs), which possess excellent medicinal efficacy (Bujnakova et al., 2015a; Pastorek et al., 2014; Wang et al., 2009; Bala´zˇ et al., 2003; Bala´zˇ et al., 2016). Thus, the polymerassembled suspensions of As4S4 nanoparticles (NPs) exhibit improved bioavailability and cytotoxicity as compared with bulk arsenicals, and enhanced pharmacological in vitro anticancer activity, producing DNA damage and apoptosis-induced cellular effects on human cancer cell lines MCF7, HepG2, and

Materials for Biomedical Engineering: Inorganic Micro- and Nanostructures. DOI: https://doi.org/10.1016/B978-0-08-102814-8.00005-6 © 2019 Elsevier Inc. All rights reserved.

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A549 (Tian et al., 2014), ECV-304 (Deng et al., 2001), RPMI-LR5 and OPMI (Bala´zˇ et al., 2007), H460 (Bujnakova et al., 2015a), BOWES and A375 (Pastorek et al., 2014), HL-60 (Wang et al., 2009), etc. Generally, in different medicinal applications, the NP subsystem is supplemented by many components to facilitate the needed functions, such as fluorescent emission (ZnS, ZnSe, CdS, CdSe), magnetically addressable drug delivery (Fe3O4), etc. (Jean et al., 2003; Shpotyuk and Filipecki, 2003). Therefore, in the case of NP-based composites, we deal with their higher structural diversity, resulting in a more complicated nature of anticancer functionality. In all the above-mentioned cases, the comprehensive information on atomic arrangement of these systems plays a pivotal role in understanding their anticancer behavior and prepare NCs with guided anticancer functionality. Positive consequences of nanostructurization in such substances are governed not only by the chemistry of the NPs themselves (their type, content, and phase composition), but also the interconnection between different space-filling fragments forming free-volume elements (FVEs), such as atomic vacancies, vacancy-type clusters, inner voids, pores, NP interfaces, cracks, etc. (Ou et al., 2010; Cheng and Zhang, 2013; Balazs et al., 2006). To control such tiny atomic-deficient entities, positron annihilation lifetime (PAL) spectroscopy, with the probe grounded on experimental space
time continuum determination for electron interaction with its antiparticle (positron e1) seems to be one of the most promising (Krause-Rehberg and Leipner, 1999; Jean et al., 2003; Shpotyuk and Filipecki, 2003). Within this method, the electron
positron (e2
e1) interaction is employed to study the atomic- and subatomic imperfections (such as free-volume defects, vacancies, vacancy-like clusters and their complexes, interfacial voids and pores, intergranual boundaries, etc.) in nanomaterials whatever their chemistry. This chapter gives a comprehensive analysis of nanostructurization effects in multiparticle composite systems, exploring possibilities of the PAL method supported by traditional atomic-relevant microstructure probes, such as X-ray diffraction (XRD), Raman scattering (RS), and transmission and scanning electron microscopy (TEM and SEM) with energy-dispersive X-ray spectroscopy (EDS) as an example of NP-based As4S4-ZnS composites prepared by high-energy ball milling.

4.2 AS4S4/ZNS NC PREPARATION PROCEDURE Mechanochemical treatment of arsenic sulfide (As4S4) polymorphs is known to be an effective thermodynamically nonequilibrium approach, which can be realized in so-called dry- or wet-milling modes using high-energy milling in various mills (Bala´zˇ , 2008; Bala´zˇ et al., 2009, 2013, 2017; Shpotyuk et al., 2017).

4.3 As4S4/ZnS NC Characterization Methodology

4.2.1 MECHANOCHEMICAL SYNTHESIS OF AS4S4/ZNS NCS IN A DRY-MILLING MODE The As4S4/ZnS NCs were prepared in different molar ratios between As4S4 and ZnS (5:0, 4:1, 1:1, 1:4, 0:5) by comilling of commercial As4S4 (98%, Sigma Aldrich) and precursors for ZnS [zinc acetate, (CH3COO)2Zn.2H2O (99%, Ites) and sodium sulfide Na2S (98% Acros Organics)] according to the procedure described in detail previously (Shpotyuk et al., 2017). Simply, comilling was performed in a planetary mill Pulverisette 6 (Fritsch) using a tungsten carbide milling chamber and milling balls (10 mm in diameter) in argon atmosphere for 20 minutes. After synthesis, the mixture was washed several times and after drying the final powder samples of As4S4/ZnS were produced.

4.2.2 MECHANOCHEMICAL SYNTHESIS OF AS4S4/ZNS-PX407 NSS IN A WET-MILLING MODE The nanosuspensions (NSs) were prepared in a laboratory circulation mill MiniCer (Netzsch). As4S4/ZnS samples (in different molar ratios) were subjected to milling in the presence of 300 mL Poloxamer 407 (PX407) water solution (0.5 wt.%) for 120 minutes at a milling speed of 4000 rpm. The mill was loaded with yttrium-stabilized ZrO2 milling balls (0.6 mm in diameter). The resulting As4S4/ZnS-PX407 NSs were centrifugated at 5500 rpm and filtered through a 0.22-μm sterile filter. The overall technological flowsheet for the preparation of the samples of As4S4/ZnS NCs and As4S4/ZnS-PX407 NSs is given in Fig. 4.1.

4.3 AS4S4/ZNS NC CHARACTERIZATION METHODOLOGY 4.3.1 ATOMIC-RELEVANT STRUCTURE The XRD measurements were carried out using a D8 Advance diffractometer (Bruker) equipped with a Θ/Θ goniometer, Cu Kα radiation (40 kV, 40 mA), and scintillation detector. The diffraction data were collected over 10 , 2Θ , 80 degrees angular range with 0.03 degree steps and 20 s/step counting time. Commercial Bruker software, Topas, was used for Rietveld fitting, and Diffracplus Eva software was applied on the ICDD-PDF2 database for phase analysis. The Fourier-transform infrared (FTIR) spectra were recorded using a Tensor 29 (Bruker) spectrometer in a transmission mode using KBr pelletization method. For TEM studies, the powder was added to ethanol to create a suspension sonicated for 10 minutes. After sonication, this suspension was applied to the TEM Cu grid with lacey carbon film covering it. Before TEM examination, the sample was dried at ambient temperature. High-resolution TEM (HRTEM) characterization was carried out with the help of two instruments, a 300 kV FEI

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FIGURE 4.1 Flowsheet of As4S4/ZnS NCs and As4S4/ZnS-PX407 NSs preparation.

CM300 and a 200 kV JEM ARM200F. A JED 2300 EDS analyzer with Silicon Drift Detector was used for elemental analysis. Low-magnification images were taken at 160 kV with a JEM 2000FX microscope. Field emission SEM (FESEM) in secondary electrons with JEOL 7600F was used for morphology studies. The specific surface areas, adsorption isotherms, and pore size distribution were obtained with the low-temperature nitrogen adsorption method using a NOVA 1200e analyzer (Quantachrome Instruments). The NP size distribution was detected by photon cross-correlation spectroscopy with a Nanophox analyzer (Sympatec). A portion of each NS was diluted with stabilizer-containing solution to achieve a suitable concentration for measurement. This analysis was averaged for three separate measurements for each sample using dispersant with a refractive index of 1.33. The zeta potential (ZP) was determined using a Zetasizer Nano ZS (Malvern) device, which measures the electrophoretic mobility of NPs, converting it into ZP through the Helmholtz
Smoluchowski equation built into the Malvern zetasizer software. The ZP was measured in the original dispersion medium, the results being averaged for three measurements.

4.3 As4S4/ZnS NC Characterization Methodology

The dissolution experiments were performed stirring 100 mg of solid phase into 100 mL of simulated gastric fluid (SGF) composed of 0.2% NaCl in 0.7% HCl (pH 5 1.3) and into 100 mL of simulated intestinal fluid (SIF) composed of 0.042% NaOH, 0.4% NaH2PO49H2O, and 0.6% NaCl (pH 5 6.5) at temperature 36.5 C. Leaching was allowed to occur for 120 minutes. Next, aliquots of solution were withdrawn at appropriate time intervals to determine the dissolved As content using atomic absorption spectroscopy (Spectr AA-30, Varian). These measuring conditions were acceptable with respect to previous research (Amidon et al., 1995).

4.3.2 ATOMIC-DEFICIENT STRUCTURE The void structure of the pelletized As4S4/ZnS NCs was studied using PAL spectroscopy. The PAL spectra were recorded with fast coincidence system (ORTEC) of 230 ps resolution (the full width at half maximum of single Gaussian determined for control 60Co isotope) at a temperature of 22 C and relative humidity of 35% (Shpotyuk and Filipecki, 2003). To ensure precise lifetime measurement, each spectrum was recorded in normal-measurement statistics reaching B106 elemental annihilation events. The channel width of 6.15 ps allows the total number of channels to be 8000. The 22Na isotope of low B50 kBq activity prepared from aqueous 22NaCl solution (wrapped in Kapton foil of 12 μm thickness and sealed) was used as a positron source sandwiched between two identical NC pellets. The best fitting of the detected PAL spectra was achieved under their decomposition into three single exponents (three-term 3 3-decomposition route under normalized component intensities I1 1 I2 1 I3 5 1), covering channels caused by positrons e2 annihilating in defect-free bulk, trapped in spatially extended free-volume defects and forming bound positron
electron (e1
e2) state (the positronium Ps atom). This procedure was performed by processing the raw PAL spectra with LT 9.0 program (Bujˇna´kova´ et al., 2017). The resulting accuracies in positron lifetimes τi and intensities Ii were not worse than 6 0.005 ns and 0.5%, respectively. Under such conditions, the error bar in the e2 trapping rate κd does not exceed 6 0.01 ns21. Positron e1 trapping formalism allows an adequate description of PAL spectra in terms of a two-state model ( 3 2-term decomposition) with one kind of e1-trapping defect (Krause-Rehberg and Leipner, 1999; Jean et al., 2003; Shpotyuk and Filipecki, 2003; Kansy, 1996; Tuomisto and Makkonen, 2013; Seeger, 1974). Corresponding e1 trapping modes, for example, mean τ av and defect-free bulk τ b positron lifetimes along with trapping rate in defects κd are defined as: τav 5

τ1 I1 1 τ2 I2 ; I1 1 I2

(4.1)

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CHAPTER 4 Multiparticle composites

τb 5 κd 5

I1 I1 τ1

1 I2 ; 1 τI22

 I2 I I 2 τ2 I1 τb

(4.2)

(4.3)

In addition, the difference between defect-related and bulk positron lifetimes (τ 2
τ b) is calculated within a 3 2-model as the size signature of free-volume e1-trapping defects is defined as the equivalent number of vacancies, whereas the τ 2/τ b ratio is indicative of the nature of these defects (Krause-Rehberg and Leipner, 1999). Ps trapping formalism describes positrons which annihilate in porous substances as free particles or pick-up an electron from environment forming a bound positron
electron (e1
e2) state (Jean et al., 2003). The Ps atom exists as a singlet para-positronium p-Ps, which decays intrinsically emitting two γ-quanta (the character lifetime in a vacuum approaches 0.125 ns), and triplet ortho-positronium o-Ps, which decays with three γ-quanta (the character lifetime reaches 142 ns). Because of the overlapping of the positron wave function with that of the electron from the surroundings, annihilation with such an electron (having an antiparallel spin) decreases the positron lifetime to 0.5
10 ns (“pick-off” annihilation), resulting in two γ-rays (Jean et al., 2003). To stabilize Ps, the following conditions should be satisfied, the first being sufficiently high size of void-captured Ps and the second being low electron density preventing direct e1
e2 annihilation (Jean et al., 2003). The Ps localized in free volumes give an indication on their mean radii R in terms of the longest τ 3 lifetime due to the semiempirical Tao
Eldrup equation (Shpotyuk et al., 2015a, 2016a):

τ3 5 0:5U 12


2πR 21 R 1 1 Usin ; R1ΔR 2π R1ΔR

(4.4)

where ΔR 5 0.166 nm is the fitted empirical electron layer thickness (Jean et al., 2003). The relative intensity of this component I3 correlates well with the density of the Ps trapping sites, giving a fractional free volume fv (in %) as f v 5 CUVf UI3 ;

(4.5)

˚ 3) is the void volume in spherical approximation (4/3πR3) and C is where Vf (in A an empirically determined constant (0.0018 for epoxy and 0.0014 for polystyrene polymers) (Jean et al., 2003). The 3 3- 3 2-coupling decomposition algorithm ( 3 3- 3 2-CDA) can be applied to parameterize the mixed e1-Ps trapping channels in the case of highly inhomogeneous substances, such as polymers or composites (Eldrup et al., 1981; Chakraverty et al., 2005). These channels are mutually interconnected in nanostructurized composites so that interplay between them results in significant

4.3 As4S4/ZnS NC Characterization Methodology

complication in meaningful interpretation of the detected PAL spectra. The formalism of simply separated e1 and Ps trapping events cannot be further explored as a realistic signature of the annihilation phenomenon because of uncompensated admixture in the first component arising from p-Ps (Shpotyuk et al., 2016a; Chakraverty et al., 2005). In such a case, greater understanding on the eventual behavior of mixed e1-Ps trapping channels is needed to distinguish their contributions unambiguously and, thereby, to develop a meaningful methodological algorithm of their description in dependence on structure evolution processes. In the model (Eldrup et al., 1981; Shpotyuk et al., 2015a), the nanostructurization caused by incorporation of guest NPs in host matrix was described as substitution e1-Ps trapping, for example, a process which occurs as conversion of o-Ps traps in pure host matrix into e1 trapping sites in NP-modified host
guest matrix. By accepting the tight interconnection between these traps, this approach has been defined as 3 3- 3 2-CDA, to distinguish it from the conventional 3 3-decomposition procedure dealing with PAL spectra originated from unresolved e1-Ps trapping. Hence, in respect to this approach, an additive two-state e1 trapping model, describing conversion from Ps to e1 trapping sites in host matrix due to embedded guest NPs, can be validated by a mathematical treatment procedure allowing consideration of the measured 3 3-term PAL spectra in a generalized 3 2-term form. host host This procedure is applied for both host matrix (pure PVP with τ host 1 ; τ2 ; τ3 host host host lifetimes and I1 ; I2 ; I3 intensities) and NP-modified guest
host composite       (As4S4-PVP with τ 1 ; τ 2 ; τ 3 lifetimes and I1 ; I2 ; I3 intensities), as in Shpotyuk et al. (2016a) and Chakraverty et al. (2005). The second component in this generalized 3 2-decomposition involves contributions from all possible trapping channels, including e1 trapping, input from o-Ps decaying (formerly being in the third com ponent with I3host or I3 intensities) and p-Ps self-decaying (formerly being in the first component with 0.125 ns lifetime and 1/3I3 yield) (Jean et al., 2003). Thus, we can easily separate contributions to the first channel, which are different from p-Ps   input, these being denoted as (τ a,Ia) and (τ a ; Ia ) for host and host
guest matrix, respectively. Under this condition, additional e1 trapping input with lifetime τ int and intensity Iint can be resolved in the second component of the generalized 3 2-term PAL spectrum of the host
guest matrix (with the remainder of the o-Ps trapping sites taken as in the host matrix) so that 



I2 5 Iint 1 I3 UðI2host =I3host Þ;      τ 2 UI2 5 τ int UIint 1 τ host 2 U I2 2 Iint :

(4.6) (4.7)

The compensating (τ n,In) input in the first channel of NP-embedded composite arising from this additional e1 trapping can be found under requirement of full equilibrium between channels: 







τ n UIn =τ a UIa 5 τ int UIint =τ 2 UI2 :

(4.8)

Thereby, the physical parameterization of NP-related trapping sites can be finally performed by accepting (τ n,In) and (τ int,Iint) as corresponding first and second components of the 3 2-term PAL spectrum, where defect-related τ int lifetime

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CHAPTER 4 Multiparticle composites

reflects e1 trapping which appeared due to the embedded guest NP. Thus, by converting 3 3-term PAL spectra of host matrix and host
guest NC in the generalized 3 2-term form in respect to 3 3- 3 2-CDA (Eldrup et al., 1981; Shpotyuk et al., 2015a), we extract just this additive part, which corresponds entirely to NPrelated traps. Under accepted prerequisites, the e1 trapping defects can be associated with pseudogap holes at the interface between the outer surface layer of agglomerated guest NPs and the innermost layer of surrounding host matrix. The bulk positron lifetime τ b calculated, respectively, to (τ n, In) and (τ int, Iint) components using Eq. (4.2) can be attributed to the defect-free positron lifetime of distinct NP. In the case of highly monolith NPs, this τ b value tends toward the bulk positron lifetime of the corresponding substance, while in more loose aggregates of many NPs, it is higher, reflecting their inner compactness. In general, the value of τ int lifetime itself, as well as its difference and ratio with bulk lifetime, are attributed to the geometrical size and nature of e1 trapping interfacial holes, their trapping rate being estimated via two-state trapping formalism (Kansy, 1996; Tuomisto and Makkonen, 2013; Seeger, 1974).

4.3.3 BIOLOGICAL ACTIVITY Two human melanoma cancer cell lines, A375 and BOWES, were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated FCS, 2 mM L-glutamine, 100 μg/mL penicillin, and 50 μg/mL streptomycin, in 5% CO2 humidified atmosphere, at 37 C. Approximately 2.5105 cells/well were seeded in 12-well plates, cultivated for 1 day, and then treated with As4S4/ZnS NCs for 24 hours. The cells were detached using EDTA:trypsin, collected by centrifugation at 1000 g for 3 minutes and washed twice with cold PBS. For cytotoxic assay using FDA/PI staining, cells were exposed to As4S4/ZnS samples with various concentrations of As (0.156; 0.312; 0.625; 1.25; 12.5 μg/mL) for 24 hours. Trypsinized and washed cells were resuspended at 1105 cells in 300 μL of PBS/0.2% BSA containing 10 nM FDA (from a 5 mM stock in DMSO) and left for 30 minutes at room temperature. The cells were then cooled and 3 μL of PI (1 mg/mL) were added. Finally, after 15 minutes incubation, the fluorescence intensities of at least 104 cells were measured using a Canto II flow cytometer. The data were analyzed with FCS Express 4 software (De Novo Software, LA, USA). The values of 50% inhibition concentration (IC50) were determined for each sample from dose
effect dependence calculated by CalcuSyn 1.1 software. The cell cycle analysis was based on the measurement of the DNA content of nuclei labeled with propidium iodide (PI). The treated cells were resuspended in 300 μL of 0.05% Triton X-100 and 15 μL of RNA-se A (10 mg/mL) in PBS for 20 minutes at 37 C. The cells were then placed on ice and incubated for at least 10 minutes before PI (50 μg/mL) was added. Finally, after 15 minutes of incubation, data for 104 cells were acquired using Canto II flow cytometers. DNA cell cycle was analyzed with Multi-Cycle AV plug-in to FCS Express 3 software (De Novo Software, LA, USA).

4.4 NP-Guided Functionality in As4S4/ZnS NCs

4.4 NP-GUIDED FUNCTIONALITY IN AS4S4/ZNS NCS 4.4.1 CHARACTERIZATION OF AS4S4/ZNS NCS PREPARED IN A DRY-MILLING MODE The XRD patterns are shown in Fig. 4.2, confirming that the 5:0 sample is pure arsenic sulfide (As4S4) (JCPDS 01-072-0686) with most intensive reflexes at 15.3, 17.57, 22.38, 29.5, and 30.95 2θ corresponding to ð111Þ, (111), ð112Þ, (221), and ð222Þ diffraction planes in a monoclinic C2/c structure. It is noteworthy that the XRD pattern is a superposition of crystalline and significant amorphous contribution. The mean crystallite size was calculated as 25 nm. On the other hand, the XRD pattern of 0:5 confirms pure zinc sulfide (ZnS) with sphalerite structure (JCPDS 01-0792). Considerable broadening of peaks indicates that ZnS has fine nanocrystalline structure, composed of crystallites of 3.4 nm size. Three diffraction peaks at 28.75, 48.2, and 56.5 2θ correspond, respectively, to (111), (220), and (311) planes in cubic F 43 m structure. The changing ratio between As4S4 and ZnS is found from XRD patterns, the estimated weight fractions being satisfactory with respect to nominal compositions (see Table 4.1). The sizes of As4S4 crystallites vary within the 25
40 nm domain, while ZnS crystallites are approximately one-order finer (2.4
3.4 nm). The deeper insight into structure, crystallinity, morphology, and size of NPs was provided with the techniques of SEM and TEM; with sample 1:4 being used for this research. The representative FESEM and low-magnification TEM images (Fig. 4.3A and B) illustrate that this NC consists of densely packed irregular

FIGURE 4.2 XRD patterns of As4S4/ZnS NCs with different molar ratio of components.

127

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Table 4.1 Estimation of Crystallite Size From XRD Analysis for As4S4/ZnS NCs As4S4:ZnS

Crystallite Size (nm)

Weight Ratio Molar Ratio

Nominal

Experimental From XRD

As4S4 (nm)

ZnS (nm)

5:0 4:1 1:1 1:4 0:5

100:0 94.5:5.4 81.4:18.6 52.4:47.6 0:100

100:0 94.8:5.2 70.0:30.0 68.0:32.0 0:100

2.6 1 25 2.2 1 27 1.3 1 40 1.2 1 40



2.4 2.9 3.1 3.4

FIGURE 4.3 FESEM (A) and low-magnification TEM (B) images of agglomerated crystallites in 1:4 As4S4/ZnS NC. The SAED pattern in the inset of (B) is indexed to ZnS (rings 2, 3, 4) and As4S4 (ring 1).

agglomerates of fine crystallites. As was determined, both the As4S4 and ZnS crystallites are distributed within individual NPs. Structural information on NCc can be obtained from the SAED pattern (inset of Fig. 4.3B), which consists of broader concentric diffraction rings typical for nanocrystalline samples. The interplane distances of 0.316, 0.193, and 0.165 nm determined from rings marked as (2), (3), and (4) are assigned to (111), (220), and (311) planes of cubic ZnS phase (a 5 0.54 nm, F 43 m space group, JCPDS 01-0792). The interplane spacing of 0.572 nm determined from the SAED ring labeled as “(1)” correlates well with the most intense 111 reflection of the As4S4 phase (JCPDS 72-0686). It can be anticipated that other, less intense reflections of this As4S4 phase are overlapped with more intense reflections of ZnS phase. The HRTEM confirms that NPs consist of randomly oriented crystallites of size 3
8 nm. They are mostly oriented along the {111} plane parallel to a primary electron beam, which follows from the most intense 111 reflection

4.4 NP-Guided Functionality in As4S4/ZnS NCs

exhibiting an interplanar spacing of 0.316 nm in the relevant FFT pattern (inset of Fig. 4.4A). Some of the crystallites are defect-free (Fig. 4.4B), but many of them are defective (Fig. 4.4C and D). A single crystalline spherical-like ZnS NP with a size around 7.3 nm is shown in Fig. 4.4B. The straight zinc-blende ZnS 111 fringes exhibiting interplanar spacing of 0.316 nm are seen running through the NP. It is generally known that stacking faults or twins in FCC crystals can be revealed only for NPs oriented along the [110] zone axis. Therefore, it is supposed that these seemingly defect-free NPs can also contain some crystal defects. Further HRTEM studies show that a large percentage of ZnS NPs exhibits single or multiple twinning (Fig. 4.4C and D). Multiple stacking defects, seen in the circled grain in Fig. 4.4D, are revealed in many NPs. The values of specific surface area (SA) of As4S4/ZnS NC defined by the BET method are gathered in Table 4.2. The smallest SA 5 0.3 m2/g was estimated for

FIGURE 4.4 HRTEM imaging of 1:4 As4S4/ZnS NCs: (A) micrograph with relevant FFT pattern in the inset; (B) micrograph of tightly agglomerated NPs; (C) details of zinc-blende ZnS NP showing lattice fringes with 0.316 nm interlayer distance [NP exhibits twinning across the (111) plane; B region is a mirror image of parent A crystal]; (D) micrograph of multiple stacking faults in the packing of ZnS NPs (the steps at surfaces of NPs are arrowed).

129

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Table 4.2 Surface Properties of As4S4/ZnS NCs Molar Ratio

SA, m2/g

Total Pore Volume, cm3/g

Average Pore Diameter, nm

5:0 4:1 1:1 1:4 0:5

0.3 11 11 68 126

0.0034 0.0837 0.0417 0.1039 0.1544

52.78 29.74 14.93 6.09 4.90

sample 5:0 (sample As4S4). By analyzing the adsorption
desorption isotherms, this sample was determined to be nonporous. By introducing fine ZnS crystallites into NC, the SA value is obviously increased. Only a small amount of ZnS (as in the case of 4:1 NCs) causes more than a 30-fold enhancement of this parameter, and SA reached as high as 126 m2/g in the 0:5 sample (pure ZnS). Such an obvious increase in SA values is assisted by the nature of ZnS as a mesophore material.

4.4.2 ATOMIC-DEFICIENT STRUCTURE OF AS4S4/ZNS NCS 4.4.2.1 Expected channels of mixed positron-Ps trapping in NP-based composites Positron annihilation in NP-based systems is known to be defined by NPs themselves (their chemical nature and geometrical specification), the interfacial freevolume defects, or triple junctions (TJs) with the volume of a few missing atoms at the intersection of three or more grain boundaries (Nambissan, 2011; Shpotyuk et al., 2015b, 2016b; Keeble et al., 2012). These TJs are highly diverse, even for NP-uniform composites, being revealed within intra- and inter-NP agglomerates (Weibel et al., 2005). In NP-based composite systems, the above diversity of positron-Ps traps can be substantially enhanced due to mixing and segregation of different NPs (Boldyreva, 2013). Let us consider possible positron-Ps trapping defects in an NP-based composite system assuming a homogeneous physical mixture of two principally defferent NPs, these being coarse-grained A (25
40 nm, as for As4S4 crystallites after milling from bulk precursors, Table 4.1) and fine-grained B (2.4
3.4 nm, as for ZnS crystallites after milling from chemically synthesized precursors, Table 4.1). The bottommost level of VFE in NP-based composites comprising intercrystalline interactions is composed of multivacancies in A and B components and intercrystallite TJ in view of the rather nonspherical approximation for such crystallites. The agglomerated homogeneous (A and/or B) or inhomogeneous A-B close-packed crystallites serve as precursors for NPs in the composite system. At the uppermost level of FVEs in the composite system, the distinct NPs are formed by interactions between agglomerated loosely packed crystallites (the spherical approximation to NPs is assumed). For mixed A-B composite, we

4.4 NP-Guided Functionality in As4S4/ZnS NCs

assume constituent segregation under competitive A and B content (1:1 composition) or ordered unit segregation approaching 5:0 or 0:5 composition (Boldyreva, 2013; Yip and Hersey, 1977). These prerequisites are valid for dry-milled As4S4/ ZnS NCs (Shpotyuk et al., 2016b; Bala´zˇ et al., 2003). The boundary 5:0 and 0:5 NCs forming interfacial TJs in homochemical A- and B-environments are depicted in Fig. 4.5A and B, respectively. Within the approach of three hard contacting spheres of R radius, such interfacial TJs can be imagined as equilateral triangles with BR side. For mixed A-B NCs, these TJs attain an A- or B-preferential heterochemical environment as is shown in Fig. 4.6. With going from coarse-grained A (5:0) to

FIGURE 4.5 FVE in coarse
fine-grained A-B NC showing interfacial TJs in homochemical A 1 A 1 A (A) and B 1 B 1 B (B) environments, and vacancy-type voids in A- (C) and B-subsystems (D
F).

FIGURE 4.6 FVE in coarse
fine-grained A-B NC showing interfacial TJs in heterochemical Apreferential A 1 A 1 B (A), B-preferential A 1 B 1 B (B), A 1 B 1 B 1 B (C), and grainboundary A 1 nB (D) environments.

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FIGURE 4.7 Amplified cartoon view showing FVE in interfacial TJs of coarse-grained A subsystem (red triangles) due to occupancy with fine B NPs (green).

fine-grained B (0:5) NCs, the homochemical A-type TJs (Fig. 4.5A) are gradually replaced by heterochemical A- and B-preferential TJs, as shown, respectively, in Fig. 4.6A
D, so the B-rich NCs demonstrate higher diversity of expected TJs. As to their own vacancy-type defects, they are not important for positron-Ps trapping in the A-subsystem (Fig. 4.5C) in view of overestimated open volumes (a few thousands of nm3), which are far beyond measuring limits of PAL spectroscopy (Krause-Rehberg and Leipner, 1999; Jean et al., 2003; Shpotyuk and Filipecki, 2003). In contrast, the FVEs in a form of vacancies in the B-subsystem (Fig. 4.5D, E, and F) are more PAL-sensitive, enhancing the trapping rate in B-rich NCs. By accepting the irregularity and closer packing arrangement of B NPs, the volumes of positron traps are expected to be less than for geometrically regular packing shown in Fig. 4.5. The third kind of FVEs meaningful for mixed positron-Ps trapping is realized in A-B composites having TJs in coarse-grained A subsystem filled with fine B NPs (see Fig. 4.7). This channel can be validated provided an essential difference in the NP sizes, especially when Ps-trapping TJs in the A subsystem (distinguished by large red triangles in Fig. 4.7) are reduced in volume due to embedded B NPs, thus producing effective positron traps. A spherical-like approach to these FVEs allows their separation on components contributing to different trapping channels, while their volumes can be essentially disturbed in realistic composites, owing to more irregular NP shape. Shape irregularity causes denser NP packing and underestimated volumes of voids. This concerns interfacial TJs between coarse-grained crystallites in the A subsystem, which possess gradually lower volumes than those assuming hard contacting spheres. The expected volumes of these TJs are depressed due to the amorphous phase which appeared after milling. This means that all estimated free volumes should be accepted only as upper limits in a mixture of hard A-B spheres forming a realistic NP-based composite system.

4.4.2.2 Compositional evolution of FVEs in As4S4/ZnS NCs The above model proposed for an NP-based coarse
fine-grained A-B system gives a key to understanding the compositional evolution of FVEs in As4S4/ZnS NCs detected with PAL spectroscopy.

4.4 NP-Guided Functionality in As4S4/ZnS NCs

The PAL spectra registered under a 50 ns channel width for pelletized 5:0 and 0:5 samples of As4S4/ZnS NCs ( 3 3-term fitting) are shown in Fig. 4.8, the bestfit positron and Ps-trapping modes are gathered in Table 4.3. Similar spectra were registered for all intermediate As4S4/ZnS NCs (4:1, 1:1, 1:4). The PAL data are well described by this fitting (as it follows from narrow statistical scattering of variance around 0-axis), except the 0:5 sample composed of ZnS NPs. In the latter, it was possible to decompose the PAL spectrum (Fig. 4.8B) on four or five components under a channel width of 500 ns without a

FIGURE 4.8 PAL spectra of 5:0 (A) and 0:5 (B) representatives of pelletized As4S4/ZnS NCs reconstructed from 3 3-fitting at the background of source contribution (bottom inset shows statistical scatter of variance; channel width of PAL measurements is 50 ns).

133

Table 4.3 Fitting Parameters and PAL Trapping Modes Describing Positron Annihilation in Pelletized As4S4/ZnS Nanocomposites (The Channel Width of PAL Measurements Is 50 ns) PAL Spectra Fitting Parameters τ1

τ2

τ3

I2

I3

Positron Trapping Modes τb

κd 21

Ps Trapping Modes

τ 2
τ b

τ 2/τ b

R

fv

Composite As4S4:ZnS

ns

ns

ns

a.u.

a.u.

ns

ns

ns

a.u.

nm

%

5:0 4:1 1:1 1:4 0:5

0.209 0.202 0.202 0.194 0.185

0.433 0.399 0.387 0.378 0.375

2.089 1.856 1.705 1.804 1.955

0.212 0.250 0.288 0.286 0.341

0.010 0.011 0.015 0.013 0.008

0.235 0.231 0.235 0.226 0.224

0.53 0.61 0.69 0.73 0.94

0.20 0.17 0.15 0.15 0.15

1.84 1.73 1.65 1.68 1.67

0.296 0.275 0.259 0.269 0.284

0.19 0.17 0.20 0.19 0.14

4.4 NP-Guided Functionality in As4S4/ZnS NCs

FIGURE 4.9 PAL spectra of 0:5 NC reconstructed from 3 5-fitting at the background of source contribution (bottom inset shows statistical scatter of variance, channel width of PAL measurements is 500 ns).

decrease in the goodness of fitting (Fig. 4.9), the results of this procedure are presented in Table 4.4. This testifies in favor of many Ps trapping channels in small-sized ZnS NCs due to input from FVEs at the bottommost level (vacancies and intercrystalline TJs) and multivacancy voids in the packing of ZnS crystallites (as shown in Figs. 4.5F, 4.6C and D). It should be noted that the sizes of o-Ps traps estimated in a spherical approximation using Eq. (4.4) are well fitted to R D 0.27
0.30 nm with free volume fraction fv D 0.14%
0.20% (Table 4.4). In monoparticle ZnS-based NCs (0:5 As4S4/ZnS), the contribution of larger o-Ps traps with R D 12
14 nm and fv D 43%
44% (Table 4.4) is essential. Before analysis of the PAL data in As4S4/ZnS NCs, it is necessary to recognize the possible annihilation paths. Arsenic sulfide (As4S4), as a coarse-grained component, exists in three crystalline polymorphs, these being low-temperature α-As4S4 structurally identical to mineral realgar, high-temperature β-As4S4, and pararealgar as an alteration product from both the α- and β-phases (Shpotyuk et al., 2015b; Bonazzi and Bindi, 2008). All polymorphs are built of cage-like As4S4 molecules filling a ˚ 3 per molecule in realgar α-As4S4) or looser space to form a denser (B14.8 A ˚ structural arrangement (B15.7 A3 per molecule in pararealgar) (Bonazzi and Bindi, 2008). Reliable PAL measurements for realgar testify to defect-free bulk positron lifetime τ b  0.223 ns, and defect-specific positron lifetimeτ d 5 τ 2 D ˚ 3 volume characteristics for tri- and 0.346 ns due to positron traps with B80 A tetra-atomic vacancies (such traps are overlapped low electron-density spaces

135

Table 4.4 PAL SPECTRA Parameterization of Pelletized 0:5 Nanocomposites (Formed of Pure ZnS) Under Channel Width of 500 ns Employing unconstrained 3 4- and 3 5-Fitting Procedures PAL Spectra Fitting Parameters Lifetimes, ns

Ps Trapping Modes

Intensities (%)

R3

f3

R4

f4

R5

f5

τ1

τ2

τ3

τ4

τ5

I2

I3

I4

I5

nm

%

nm

%

nm

%

0.192 0.183

0.400 0.360

2.092 1.473

37.06 15.41


42.34

0.41 0.51

0.008 0.010

0.029 0.004


0.023

0.296 0.233

0.16 0.10

1.262 0.827

44.2 1.86


1.354


43.0

4.4 NP-Guided Functionality in As4S4/ZnS NCs

around S atoms forming As4S4 molecules) (Shpotyuk et al., 2015b). Because of similar covalent bonding and space-filling efficiency in all As4S4 polymorphs, there seems to be reasonably close proximity between the respective positron traps. Zinc sulfide (ZnS), as a fine-grained component, belonging to II
VI group wide band-gap semiconductors, exists in the form of hexagonal wurtzite and cubic zinc blende (Biswas et al., 2006). Whichever technology is used, this material demonstrates bulk lifetimes τ b within the 0.215
0.230 ns domain (Pareja et al., 1992; Adams et al., 1995; Krause-Rehberg et al., 1998) in good agreement with known calculations (Plazaola et al., 1994), vacancy-type components (0.266 ns for monovacancy and 0.286 ns for divacancy) (Pareja et al., 1992), and 0.430 ns lifetime attributed to voids or grain boundaries (Adams et al., 1995). As can be seen from Table 4.4, the bulk lifetimes τb for 5:0 and 0:5 NCs are close to those for realgar α-As4S4 (0.223 ns) and ZnS polycrystals (0.230 ns), testifying that FVE exists in the chemical environment of these crystalline species. The defect-related lifetimes τ d 5 τ 2 are higher than those for vacancy-type defects in these crystals [0.342 ns for α-As4S4 (Shpotyuk et al., 2015b) and 0.266
0.286 ns for ZnS (Pareja et al., 1992)], meaning that other types of FVEs are essential in both subsystems. These defects are interfacial TJs between nanocrystallites as their character is similar to nanostructurized substances (Nambissan, 2011; Shpotyuk et al., 2015b, 2016b). When going from 5:0 (As4S4) to 0:5 (ZnS) NCs, there is a strong growing tendency observed in the trapping rate κd due to an increase in the content of these defects (due to growing trend in I2 intensity; see Table 4.4). In contrast, the o-Ps trapping modes are in an opposite dependence, demonstrating a decrease in I3 accompanied by an increase in τ3 toward both boundary compositions in respect to 1As4S4:1ZnS NC. The main void-evolution process governing the behavior of the third component in 3 3-term decomposed PAL spectra can be imagined as a contribution from interfacial TJs caused in the coarse-grained As4S4-subsystem due to occupancy with fine-grained ZnS NPs (Fig. 4.7). Additional input to Ps-trapping channel in As4S4/ZnS NCs is expected for high content of agglomerated ZnS NPs due to multivacancy voids (Fig. 4.5F). Hence, nanostructurization in the As4S4/ZnS NC system can be imagined as a conversion from o-Ps-trapping sites to positron traps, thus allowing 3 3- 3 2-CDA formalism (Shpotyuk et al., 2015b, 2016b) to analyze the PAL spectra. The corresponding NP-related PAL trapping modes resulting from such treatment are given in Table 4.5. As follows from the 3 3- 3 2-CDA modes for boundary 5:0 and 0:5 NCs, unique As4S4-related trapping sites are rather o-Ps traps with τ int 5 0.875 ns, which can be ascribed (due to τ b  0.271 ns, increased in respect to τ b  0.223 ns proper for realgar α-As4S4) (Shpotyuk et al., 2015b) to interfacial TJs in a random network of loosely packed As4S4 NPs (Fig. 4.5A). In contrast, the ZnS-related trapping sites are positron traps with τ int 5 0.365 ns, which can be attributed (due to proximity in τ b  0.209 ns to bulk positron lifetimes of ZnS) (Pareja et al., 1992; Adams et al., 1995; Krause-Rehberg et al., 1998) to multivacancy clusters in a network of more closely packed ZnS NPs (Fig. 4.5D, E, and F) and free-volume

137

Table 4.5 NP-Related PAL Trapping Modes in Pelletized As4S4/ZnS Nanocomposites Treated Within 3 3- 3 2-CDA in Respect to 1As4S4:1ZnS Composite I Component

II Component

PAL Trapping Modes

τn

τ int

τ av

In

Iint

τb

κd 21

τ 2
τ b

τ 2/τ b

Composite Samples As4S4:ZnO

ns

a.u.

ns

a.u.

ns

ns

ns

ns

a.u.

5:0 4:1 1:4 0:5

0.244 0.202 0.145 0.172

0.127 0.133 0.099 0.373

0.875 0.464 0.316 0.365

0.020 0.039 0.036 0.187

0.330 0.261 0.191 0.237

0.271 0.231 0.170 0.209

0.40 0.63 1.00 1.02

0.604 0.233 0.146 0.156

3.23 2.01 1.86 1.74

4.4 NP-Guided Functionality in As4S4/ZnS NCs

voids in preferential ZnS environment (Fig. 4.6C and D). The latter defects are dominant in ZnS-rich NCs along with defect-related monovacancy (τ d 5 0.266 ns) and divacancy (τ d 5 0.286 ns) traps. In As4S4-rich 4:1 NCs, the preferential traps are interfacial Ps-trapping TJs filled with fine-grained ZnS NPs (Fig. 4.7). The whole hierarchical model illustrating compositional diversity of interchangeable positron-Ps trapping sites in NP-based As4S4/ZnS NCs is depicted in Fig. 4.10.

FIGURE 4.10 Microstructure-hierarchical model showing compositional diversity of interchangeable positronPs trapping sites in coarse
fine-grained As4S4/ZnS NCs (bottom row represents FVEs in interfacial TJ of coarse-grained As4S4-system due to occupancy with fine-grained ZnS NPs).

139

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CHAPTER 4 Multiparticle composites

The interfacial TJs in the homochemical As4S4 and ZnS environment along with multivacancy defects in fine-grained ZnS subsystem are shown to be governing FVEs in boundary NCs. The greatest variety of positron-Ps trapping paths owing to interfacial TJs in a mixed heterochemical As4S4-ZnS environment is expected just for 1:1 As4S4/ZnS NC.

4.4.3 CHARACTERIZATION OF AS4S4/ZNS-PX407 NSS PREPARED IN A WET-MILLING MODE For biomedical applications, it is necessary to provide the As4S4/ZnS NCs in the form of stable NSs. This was achieved by further milling (in a circulation mill) using 0.5 wt.% water solution of PX407 as stabilizer. As an example, the particle size distribution of 1:4 As4S4/ZnS-PX407 NS after 120 minutes of milling is depicted in Fig. 4.11. The main size fraction of NPs in this NC is within the 60
300 nm range. Some minor fractions in the areas with larger NP sizes are visible, documenting that the sample still contains coarse particles in the micrometer range. Due to centrifugation at 5500 rpm, it was possible to remove these particles, and to obtain the distribution in a range from 60 to 190 nm (lower part of Fig. 4.11). Stability of the obtained NSs was confirmed by ZP measurements in the original dispersion media, which, in fact, are measurements of diffuse layer thickness (results with appropriate pH values are given in Table 4.6). With the exception of 0:5 As4S4/ZnS, all samples dispersed in distilled water (i.e., As4S4/ZnS-H2O)

FIGURE 4.11 Particle size distribution in 1:4 As4S4/ZnS NC after 120 min of milling in PX407 (top) and additional centrifugation at 5500 rpm (bottom).

4.4 NP-Guided Functionality in As4S4/ZnS NCs

Table 4.6 Zeta Potential ζ and pH Values of As4S4/ZnS NSs in Water and PX407 Solution Molar Ratio

d50 (nm)

As4S4/ZnS-H2O; ζ (mV)

As4S4/ZnS-PX407; ζ (mV)

pH

5:0 4:1 1:1 1:4 0:5

112 133 126 112 870

2 40.6 2 46.3 2 42.6 2 42.4 2 23.5

2 21.4 2 27.7 2 26.1 2 26.0 2 5.48

5.88 6.13 6.68 6.90 7.39

exhibit relatively high ZP values averaged as
43 mV. In contrast, after milling in PX407, these As4S4/ZnS-PX407 NSs show a reduction in the averaged ZP to
25 mV. If PX407 with its high molecular weight (12,500) is successfully introduced in a system, it shifts the plane of shear to a further distance from the NP surface, reducing ZP values, thus indicating a thick adsorbed layer and good stabilization (Groen et al., 2003). On the other hand, the milling of pure ZnS in PX407 was unsuccessful, because of the high average NP size (d50 5 870 nm) and low ZP value (
5.48 mV), this sample was excluded from further experiments. To confirm the chemical interaction between As4S4/ZnS and PX407, the FTIR spectra were recorded. As an example, the FTIR spectrum of the 1:4 As4S4/ZnSPX407 sample containing peaks originated from both ZnS NPs and PX407 is depicted in Fig. 4.12. When comparing with the spectrum of 1:4 As4S4/ZnS NC, the most visible peaks ascribed to ZnS at 637 and 1467 cm21 (Mishra et al., 2009; Ganguly et al., 2013a; Ummartyotin et al., 2012) are well distinguished. Other peaks belong to PX407, for instance, the intensive peak at 1093 cm21 is attributed to
C
O stretching mode and absorption in the wide B2800
3000 cm21 region is due to
C
H stretching mode. This spectral region can be conveniently employed to monitor changes after interaction (Mosquera and Carvajal, 2014). When comparing the spectrum of this sample with one of pure PX407 presented in Guo et al. (1999), significant changes in this region should be noted, speaking in favor of a more hydrophobic environment of polyoxypropylene center block of PX407, resulting from PX407 molecule adsorption on the surface of As4S4/ZnS NPs. Long-term stability of As4S4/ZnS-PX407 NSs was also studied by measuring NP size distribution using the photon cross-correlation technique. It was determined that the higher amount of ZnS in NC causes the smaller aggregation effect between NPs in NSs. Such prepared suspensions were stable for more than 1 year. This phenomenon can be explain due to the enlarged specific surface areas SA in ZnS-contained NCs (see Table 4.2) causing more free-volume sites suitable for copolymer adsorption and subsequent prevention of Ostwald ripening (the nature of open pores in NCs is also important for copolymer adsorption) (Guo et al., 1999).

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CHAPTER 4 Multiparticle composites

FIGURE 4.12 FTIR spectra of 1:4 As4S4/ZnS NC and 1:4 As4S4/ZnS-PX407 NS.

4.4.4 BIOLOGICAL ACTIVITY OF AS4S4/ZNS NPS 4.4.4.1 Dissolution of As from mixed As4S4/ZnS NPs It is well known that water solubility of arsenic sulfides is extremely low. Thus, for example, the solubility of realgar α-As4S4 in water reaches only 7.1 μg/g (Bujnakova et al., 2015b), which may essentially hamper its application for oral administration. The poor solubility is generally related to slow dissolution, thereby poorly soluble drugs typically exhibit insufficient bioavailability responses (Groen et al., 2003). Moreover, it can be found in the literature that there is a direct correlation between in vitro dissolution and in vivo bioavailability (Zhang et al., 2011). The results of dissolution testing of As from As4S4/ZnS NPs leached in SGF and SIF are given in Fig. 4.13. It is obvious that the highest As dissolution is character for 1:4 As4S4/ZnS NC, which also exhibits the largest specific surface areas (SA) (see Table 4.2). The dissolution is expressed more by SIF (pH 5 6.5) than by SGF (pH 5 1.3), where As releases are 9% and 2.25%, respectively. On the other hand, the 5:0 As4S4/ZnS NC with smallest SA 5 0.3 m2/g exhibits no As release after 120 minutes of leaching in both fluids. The importance of improved surface properties of NCs by increasing SA values is also confirmed in this case.

FIGURE 4.13 Dissolution of As from As4S4/ZnS NCs in SGF (A) and SIF (B) at 36.5 C over 120 min.

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4.4.4.2 In vitro anticancer functionality of As4S4/ZnS-PX407 NSs FDA/PI staining was used to determine the effect of As4S4/ZnS-PX407 NSs on the viability and proportion of apoptotic and necrotic cells. The similar proportion of apoptotic and necrotic cells in A375 and BOWES lines was achieved with increasing As concentration in As4S4/ZnS-PX407 NSs up to 1.25 μg/mL, with the exception of Bowes cells treated with 1:1 As4S4/ZnS-PX407 NS (Fig. 4.14). Apoptosis is obviously not a dominant mode of death in cells exposed to As4S4/ ZnS-PX407 NSs. One can speculate that ZnS additions do not preclude DNA damage, induction of γH2AX, and autophagy activation (Ho¨rter and Dressman, 2001). The viability of cancer cell lines (human melanoma A375 and BOWES) treated with various concentrations of As from As4S4/ZnS-PX407 NSs is shown in Fig. 4.15. All samples clearly exhibit concentration-dependent toxicity with increased cytotoxic effect. The half-maximal inhibitory concentrations IC50 (i.e., concentration of As at which 50% of cell population is inhibited) after 24-h treatment by As4S4/ZnS-PX407 NSs are summarized in Table 4.7. The highest toxicity (defined by minimal IC50) is reached for both A375 and BOWES cell lines treated with 1:1 As4S4/ZnS-PX407 NS. This effect can be remarkably depressed with a further increase in the concentration of each component (As4S4 or ZnS, especially to 4:1 or 1:4 compositions), thus revealing a way toward purposefully guided anticancer functionality in a compositional row of these NSs. The cell cycle analysis of both A375 and BOWES lines reveals a concentration-dependent decrease in the proportion of G1 cells (Fig. 4.16A). The G1 decrease is accompanied by accumulation of cells in the G2/M phase of the cell cycle. Although a proportionate decrease of G1 cells in the BOWES line is also detected, it does not induce such significant concentration-dependent changes as in the case of A375 cells (Fig. 4.16B). There is no increased proportion of G2/M cells for both cell lines for 1:1 As4S4/ZnS-PX407 NS. This phenomenon is probably caused by the high toxicity of this sample (see IC50 values in Table 4.7). Interestingly, this NS shows some anomalies also in the specific surface area (SA) (see Table 4.2). Similarly, the highest concentration (12.5 μg/mL of arsenic in NSs) does not follow a concentration-dependent decrease of G1 proportion due to high toxicity. The detected anticancer effects on human melanoma cell lines A375 and BOWES confirm the high toxicity of the studied arsenic sulfides (As4S4) with improved biological activity as in the case of NCs containing admixture of zinc sulfide (ZnS). This result is in harmony with Dash et al. (2014) and Ganguly et al. (2013b), where selectively induced cytotoxic and genotoxic effects on leukemic cells and antimicrobial effects of ZnS NPs were observed. Based on a comparison of viability data and the proportion of cell cycle phases, one can conclude that the cell cycle is more readily modulated in Bowes line than in A375, and the cell cycle as a whole is an earlier, more sensitive parameter.

4.4 NP-Guided Functionality in As4S4/ZnS NCs

A375 cell line, 24 h

(A) 100

% of cells

80 60 40 20

Apoptotic

Necrotic

0.312 0.625 1.25 12.5

0.156 0.312 0.625 1.25 12.5

0.156 0.312 0.625 1.25 12.5

As4S4/ZnS 5:0

0.156

1.25 12.5

0.156 0.312 0.625

0

0

As4S4/ZnS 4:1

As4S4/ZnS 1:1

As4S4/ZnS 1:4

Viable

Concentration of As (µ µg/mL)

Bowes cell line, 24 h

(B) 100

% of cells

80 60 40

Apoptotic

Necrotic

0.156 0.312 0.625 1.25 12.5

As4S4/ZnS 4:1

As4S4/ZnS 1:1

Viable

0.156 0.312 0.625 1.25 12.5

0.312 0.625 1.25 12.5

As4S4/ZnS 5:0

0.156

1.25 12.5

0.156 0.312 0.625

0

0

20

As4S4/ZnS 1:4

Concentration of As (µg/mL)

FIGURE 4.14 FDA/PI staining of A375 (A) and BOWES (B) cell lines after 24 h of treatment with various As concentrations of As4S4/ZnS-PX407 NSs.

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CHAPTER 4 Multiparticle composites

FIGURE 4.15 Viability of A375 and BOWES cancer cell lines after 24 hours of treatment with various As concentrations of As4S4/ZnS-PX407 NSs.

Table 4.7 IC50 of A375 and BOWES Cell Lines After 24 h Treatment With Various As Concentrations of As4S4/ZnS-PX407 NSs IC50 (μg/mL of As) As4S4/ZnS-PX407

A375

BOWES

5:0 4:1 1:1 1:4

7.86 4.34 0.87 3.04

6.21 6.36 0.42 7.03

4.5 Conclusions

FIGURE 4.16 Cell cycle analysis of A375 and BOWES cell lines after 24 hours of treatment with various As concentrations of As4S4/ZnS-PX407 NSs.

4.5 CONCLUSIONS Arsenic sulfide (As4S4) NPs combined with zinc sulfide (ZnS) ones taken in different molar ratios were prepared by a high-energy mechanochemical route in a dry mode. Afterward, these As4S4/ZnS samples were milled in a water solution of biocompatible copolymer Poloxamer 407 to obtain stable NS using a wetmilling approach. The multiexperimental characterization approach evolving atomic-sensitive methods of XRD, RS, Fourier-transform infrared spectroscopy, as well as atomicdeficient methods employing annihilating positrons supported by structuremorphology study with SEM and TEM is systematically and comprehensively applied to characterize the developed NCs.

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The ZnS NPs consisting of fine-grained 2.4
3.4 nm crystallites are shown to be stabilized preferentially at the surface of coarse-grained As4S4 ones composed of larger coarse-grained 25
40 nm crystallites, increasing the specific surface area of the NCs from 0.3 to 68 m2/g. The presence of ZnS in NCs appears to be very beneficial in many aspects. As a consequence of the higher specific surface areas of these NPs, the NSs are more stable (their stability is prolonged from 16 weeks for As4S4 to more than 1 year for As4S4/ZnS). The positron lifetime spectra for As4S4/ZnS NCs are reconstructed from 3 3term fitting and subjected to parameterization using original 3 3- 3 2-coupling decomposition algorithm. To separate contributions in positron-Ps trapping channels, the hierarchical model considering FVEs in composites at the level of interacting crystallites (nonspherical approximation) and their agglomerates (spherical approximation) is developed. Assuming packing of hard contacting spheres for both coarse-grained As4S4 and fine-grained ZnS NPs in different chemical environments, the void-evolution process governing the behavior of the third component in the 3 3-term decomposed PAL spectra is identified as a contribution from interfacial TJs in coarse-grained As4S4-subsystem due to occupancy by fine-grained ZnS NPs. The defect-formation processes in coarse
fine-grained As4S4/ZnS composites occur in a homochemical environment of more compacted fine-grained ZnS particles inserted in a looser coarse-grained As4S4 environment. The calculated trapping parameters are shown to characterize adequately nanospace filling in As4S4/ZnS composites. The anticancer effects were tested on two melanoma cell lines, A375 and Bowes, giving promising results, which confirm the increased biomedical efficacy of the prepared As4S4/ZnS NCs. The evolution of an atomic-deficient structure tested within modified algorithms of PAL spectroscopy is shown to play a decisive role in anticancer functionality of the NCs. According to the presented results, the combination of coarse-grained arsenic sulfide (As4S4) as an efficient anticancer drug with fine-grained zinc sulfide ZnS can usefully contribute to drug delivery.

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CHAPTER

Quaternary ammonium compound derivatives for biomedical applications

5

Mari Miura Sugii1,2, Fa´bio Augusto de Souza Ferreira3, Karina Cogo Mu¨ller4, Ubirajara Pereira Rodrigues Filho5 and Fla´vio Henrique Baggio Aguiar1 1

University Medical Center Groningen, Biomedical Engineering Department, Groningen, Netherlands 2Piracicaba Dental School, University of Campinas, Department of Restorative Dentistry, Piracicaba, SP, Brazil 3SENAI Institute for Inovation in Surface Engineering
Campus CETEC, Belo Horizonte, MG, Brazil 4Faculty of Pharmaceutical Sciences, University of Campinas, Rua Se´rgio Buarque de Holanda, Campinas, SP, Brazil 5 Institute of Chemistry of Sa˜o Carlos, Sa˜o Paulo University, Department of Molecular Physics and Chemistry, Sa˜o Carlos, SP, Brazil

5.1 BACKGROUND Although medical devices, implants, prostheses, and equipment are sterilized by autoclave or radiation, exposure to air can infect these surfaces (Subbiahdoss et al., 2013). Given acceptable growth conditions, they can multiply from one organism to more than one billion in just 18 hours. Contamination and colonization by microorganisms on surfaces can result in problems as insignificant as bad odor up to serious human infections (Moriarty et al., 2013). Bacteria can settle and build biofilms. Biofilms are cohesive and protective communities sheltering microorganisms in a three-dimensional extracellular polysaccharide matrix. Bacteria embedded in biofilms are much more resistant to antibiotics and the host immune system than planktonic ones, making their eradication more difficult. Thus, biofilms are usually related to chronic and persistent infections. Chronic sinusitis and otitis, dental caries, periodontal diseases, pulmonary or urinary infections, and chronic wounds are among the complications that biofilms unleash (Høiby et al., 2015; Wilkins et al., 2014). Not surprisingly, bacterial contamination is still the most common cause of prosthesis losses (Moriarty et al., 2013). We are facing what some call the “postantibiotic era,” in which infections that were easily treatable are now a threaten to life, therefore alternative strategies must be found (WHO). Materials with antimicrobial properties for biomedical purposes are a promising field to be explored (Kenawy et al., 2007; Riga et al., 2017; Wilkins et al., 2014; Xiao et al., 2012). The term “antimicrobial agent” refers to a broad range of substances able to kill pathogenic microorganisms, Materials for Biomedical Engineering: Inorganic Micro- and Nanostructures. DOI: https://doi.org/10.1016/B978-0-08-102814-8.00006-8 © 2019 Elsevier Inc. All rights reserved.

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providing varying degrees of protection (Kenawy, 2001). In this context, a group of antimicrobial agents called quaternary ammonium compounds (QAC) has met dental and medical requirements for fighting bacteria. The aim of this chapter is to cover biofilm-arising problems, current antimicrobial materials containing QAC for dental and biomedical purposes, means of obtaining these materials, proposed mechanisms of action, and variables influencing the antimicrobial activity.

5.2 BIOFILM TREATMENT AND PREVENTION The first step in biofilm formation is bacterial adherence to a conditioning layer comprised of proteins, at this stage bacterial adhesion is still reversible. Bacteria will start dividing and others will adhere, initiating the irreversible attachment. Extracellular polymeric substance (EPS) secretion and quorum-sensing mechanism will start and three-dimensional structuring will begin. After this stage, biofilm is considered mature. With a mature biofilm, a dispersal step will take place in which small segments of the biofilm will be detached, releasing bacteria to colonize other surfaces (Riga et al., 2017; Wilkins et al., 2014). Fig. 5.1 depicts the biofilm formation steps. Antibiotic therapy is the main approach against infections nowadays. However, diffusion and penetration of antimicrobials is hindered due to the EPS surrounding

FIGURE 5.1 Biofilm formation steps: (1) attachment; (2) irreversible attachment; (3) EPS and quorum sensing start triggering three-dimensional structuring; (4) mature biofilm with water channels; and (5) detachment of biofilm segments releasing planktonic bacteria. EPS, Extracellular polymeric substance.

5.3 Quaternary Ammonium Compounds and Their Chemistry

bacteria. Bacteria sheltered in biofilms can be 1000 times more resistant to antibiotics than when in planktonic form. Additionally, the widespread production and extensive use of antibiotics have contributed to the emergence of multiple drugresistant infectious organisms, the so-called superbugs (e.g., meticillin-resistant Staphylococcus aureus) (Munhoz-Bonilla and Ferna´ndez-Garcı´a, 2015). Materials with antimicrobial capability come across as a reasonable alternative to antibiotics. Antimicrobial materials can be classified into two main types: leaching materials and contact-active materials. Leaching materials are biocide carriers and their mechanism of action relies on release of the biocide, usually low-molecular-weight compounds, in the environment and microorganisms’ chemical eradication. Contact-active materials have a modified surface that will prevent bacterial adhesion or kill bacteria upon contact (Riga et al., 2017). Leaching materials are mostly preferable in cases in which high initial doses are advantageous, for instance for preventing contamination over newly placed implants. Some biocides have been cited for leaching purposes such as silver ions, antimicrobial peptides, and even some low-molecular-weight QACs (Riga et al., 2017). When the biocide is released into the body, it does not exert a specific and localized action (Chen et al., 2000; He et al., 2016; Kenawy et al., 2007; Moriarty et al., 2013). In this regard, some thermo- or pH-responsive leaching material could amend this issue (Dadsetan et al., 2013). Concerns over leaching materials are related to the toxicity of released doses and to loss of the antimicrobial potential over time (Chen et al., 2000; Li and Shen, 2000). In contact-active materials, biocides are not leached but presented in the bulk of the material or as a coating on the surface, thus diminishing the possibility of toxicity, enhancing selectivity and effectiveness, and preventing loss of antimicrobial activity in the long term (Kenawy et al., 2007). Contact-active materials can perform antimicrobial activity by preventing protein and bacterial adhesion or by damaging the bacterial membrane. With respect to the former mechanism, proteins or organism-specific interactions with the surface are minimized and therefore the adhesion is nonexistent or easily reverted. Examples are poly(ethylene glycol), Teflon, or poly(dimethylsiloxane)-based materials (Riga et al., 2017). Regarding the second mechanism, a cationic charged surface will interact with the bacterial membrane to kill bacteria, as is the case with the QAC mechanism of action.

5.3 QUATERNARY AMMONIUM COMPOUNDS AND THEIR CHEMISTRY Antimicrobial agents, including antibiotics, disinfectants, and antiseptics have been substantially developed (Siedenbiedel and Tiller, 2012; Xiong et al., 2012; Zhang and Serpe, 2014). Antimicrobials can vary in their chemical nature, mechanism of action, impact on the human body and environment, half-life

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CHAPTER 5 Quaternary ammonium compound derivatives

characteristics, endurance on various substrates, synthesis, and costs. The ideal antimicrobial polymer would exhibit: antimicrobial activity against a broad range of microorganisms; long-term properties; chemical stability (should not leach out toxic subproducts); environmentally friendly synthesis; low-cost; and insolubility in body fluids (Arora et al., 2013). Quaternary ammonium polymers have matched a great part of these requirements as antimicrobial materials. QACs are conceived as antimicrobial agents extensively studied since Domagk discovered the antimicrobial property of benzalkonium chlorides in 1935. QACs constitute a group of cationic antimicrobial agents that contain functional groups covalently bonded to a central nitrogen atom (R4N1), with at least one of the R groups consisting of an alkyl group (Tezel and Pavlostathis, 2011). The classical Menschutkin reaction is one of the most common routes to obtain quaternary ammonium cations. The reaction is based on the addition reaction between tertiary amines and organohalides, thus representing a facile approach to produce a wide variety of potentially antibacterial monomers, oligomers, and polymers. This technique was adapted to synthesize free radical, photocurable, dimethacrylate monomers containing quaternary ammonium functionalities, miscible with common dental resinous composite (Antonucci et al., 2012; Jaeger et al., 2010; Wi´sniewski et al., 2011). When QAC is obtained it can be included or attached to different sorts of material as this chapter will explore. The QAC mechanism of action relies on strong electrostatic interactions between the positively charged nitrogen and the negatively charged bacterial membrane, resulting in its disruption and loss of cytoplasmic content. Generations of QAC with various structures have been explored as disinfectants (Xue et al., 2015) in many fields, such as water treatment, agriculture, medicine and healthcare products, food, and the textile industry (He et al., 2011; Imazato et al., 2014). The use of a quaternary ammonium biocide can provide durable antimicrobial protection against a wide variety of microorganisms without the side effect of leaching heavy metals, phenolic compounds, or other toxic compounds. The wellestablished fungicidal and bactericidal properties make QACs promising candidates for modifying a great variety of surfaces (Chru´sciel and Le´sniak, 2015). Stable binding and immobilizing of quaternary ammonium moieties into biomaterials are commonly achieved by means of covalent bonds. Routes for obtaining a polymeric material containing stable quaternary ammonium biocides attached are: polymerization of quaternary ammonium-bearing monomers; hydrolysis and condensation of silanized quaternary ammonium groups; binding to a previously prepared material (Gong et al., 2013); and functionalizing nanoparticles (NPs) (Farrugia and Camilleri, 2015).

5.3.1 CATIONIC ACRYLATES AND CATIONIC SILANES Recent scientific reports evidence the need for materials with long-lasting antimicrobial properties to overcome biofilm in dentistry. The most common and costly biofilm-dependent oral disease worldwide is dental caries (Baelum et al., 2007;

5.3 Quaternary Ammonium Compounds and Their Chemistry

Dye et al., 2007). Biofilm plays an important role in dental caries development. Oral biofilms also follow the steps of biofilm formation formerly described. The initial colonization occurs over acquired pellicle (glycoproteins, mucins, statherins, α-amylase, agglutinins) microorganisms like Streptococcus sanguinis, Streptococcus gordonii, Streptococcus oralis, Streptococcus mitis (Horiuchi et al., 2009; Shimazu et al., 2015; Struzycka, 2014), pathogenic Streptococcus mutans, and Actinomyces spp. will interact with each other (Bowen et al., 2018). If the individual diet is rich in fermentable sugars, acidogenic species will prevail and fermentation of these dietary carbohydrates will bring the environmental pH down (Conrads et al., 2014; Hamada and Slade, 1980; de Soet et al., 2000). A pH lower than 5.5 initiates demineralization of tooth structure, which will lead to white spot lesions (initial dental caries lesions) (Sundararaj et al., 2015). Once a cavitation is originated and bacteria infiltrate in dentin tubules it is hard to obtain aseptic dentin for restoration placement (de Almeida Neves et al., 2011; Imazato et al., 2006). In this setting, studies about bonding systems containing antimicrobial properties have been launched. A series of studies were dedicated to evaluating the antimicrobial activity of quaternary ammonium monomers inserted into primers or bonding agents. Methacrylate monomers containing quaternary ammonium groups were developed to this end: methacryloyloxydodecyl pyridinium bromide (MDPB), dimethylaminododecyl methacrylates (DMADDM), and methacryloxyethyl cetyl dimethyl ammonium chloride (DMAE-CB) are the most common (Ge et al., 2015). MDPB monomers were the first successfully included in commercial bonding systems which exhibited antimicrobial activity against seven oral streptococci, some lactobacilli, and anaerobic and endodontic pathogens like Enterococcus faecalis, Fusobacterium nucleatum, and Prevotella nigrescens. Due to this large spectrum of action some have tried incorporation of MDPB into restorative composite resins but reported diminished inhibitory effects after polymerization of the monomers (Imazato et al., 2006, 2012). DMAE-CB is another effective monomer against cariogenic S. mutans (Antonucci et al., 2012), S. sanguinis, and Streptococcus sobrinus when incorporated in commercialized bonding systems (Ge et al., 2015). DMADDM exhibited antimicrobial properties against S. mutans when inserted in primers (Chen et al., 2016; Li et al., 2014; Zhou et al., 2016) and adhesives from bonding systems, in nanocomposites for tooth restoration (Zhou et al., 2013), and in composite resins for orthodontic cementation (Melo et al., 2014). Biofilm accumulation around orthodontic devices is a common situation in dental practice. All cemented appliances hinder hygiene because of their complex geometry and also block muscle and saliva clearance activity, leading to biofilm accumulation (Gorelick et al., 1982; Makvandi et al., 2015; Melo et al., 2014; Rosenbloom and Tinanoff, 1991; Wang et al., 2014; Zhou et al., 2016). The development of oral biofilms in orthodontic composite resins, as well as on other accessories, such as brackets, metal ligatures, wires, and elastomeric rings, may compromise patients’ oral health, jeopardizing the efficiency of orthodontic treatment (Gong et al., 2013). White spot lesions take 6 months to develop in normal

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conditions and in patients with fixed orthodontic appliances they can occur much faster, in up to 1 month after cementation (O’Reilly and Featherstone, 1987; Øgaard et al., 1988). To address this problem, this group has developed a new silane-based material containing QAC [iodide quaternary ammonium methacryloxy silicate (IQAMS)]. IQAMS was inserted in a commercial composite resin for bracket cementation (Transbond XT Light Cure Adhesive) or applied as a coating over this composite surface. IQAMS exhibited S. mutans biofilm inhibition effect when applied as a coating but not when inserted into the composite resin. As hypothesized by others, the diminished antimicrobial activity of IQAMS inserted into the composite could be due to unavailability of the compound on the surface after photoactivation. Therefore application as a coating was advocated for enhanced antimicrobial properties (Sugii et al., 2017). Considerable interest has emerged over the application of functional quaternary ammonium-containing silanes and polysiloxanes. These materials differ from the methacrylate-based ones because the anchoring unit is an organofunctional trialkoxy or tetralkoxysilane and quaternary ammonium groups are linked by siloxane bonds (Fig. 5.2). These materials are commonly synthesized via the sol
gel process and present the possibility of adjusting the properties of the final product at a molecular level. Different end-functional macromonomers may be synthesized with nonleaching QAC distributed into the bulk of the material (Yoshino et al., 2011). Such materials are extremely versatile in terms of application and kinetically and thermodynamically stable in both strongly acidic and slightly basic media (Perrin, 2011). Organosilane matrices offer tailored hydrophilic, hydrophobic, ionic, and hydrogen-bonding capacities, as well as electrochemical properties and adjustable porosity (Tripathi et al., 2006). They assemble inert, nonbiodegradable materials and promising antimicrobial results have been reported considering functionalization with quaternary ammonium groups. In the mid-1960s, researchers discovered that antimicrobial functionalized silanes could be strongly bonded to reactive substrates by siloxane (Si
O) linkages (Isquith et al., 1972). Hydrolyzable groups (halogens or alkoxy groups) on the silicon atom enable

FIGURE 5.2 (A) Quaternary ammonium methacrylate-based monomer with reactive double bond ready for free radical polymerization. (B) Anchoring units of silane after hydrolysis ready for further condensation reaction.

5.3 Quaternary Ammonium Compounds and Their Chemistry

silanes carrying specific functions to be bonded to the substrate. Thereafter, the antimicrobial activity of the [3-(trimethoxysilyl)propyldimethyloctadecyl] ammonium chloride (SiQAC) has been studied extensively on a variety of treated surfaces. Because of the presence of reactive silanol groups generated during hydrolysis, quaternary ammonium silanes (QASs) can attach covalently to substrate surfaces via Si
O linkages to exert nonleachable antimicrobial functions (Isquith et al., 1972). The surfaces on which they can be used include metal, plastic, glass, rubber, ceramic, porcelain, marble, cement, granite, tile, silica, sand, appliances that are melamine or phenolic, siliceous, polycarbonate, and wood. The bridge-building of organofunctional silanes is particularly important in three fields of application: adhesion promotion, surface modification, and polymer crosslinking (Kregiel, 2014). The sol
gel process is based on a sol, generated from alkoxy metal or metalloid. Such compounds readily react with water via hydrolysis, generating products with hydroxyl groups bonded to the metal or metalloid atom. The hydrolyzed molecules will bond via condensation reactions, from which smaller molecules, such as water and ethanol, are formed and the result is a colloidal suspension of solid particles or polymers in a liquid. The process continues forming larger molecules until the sol turns into a gel, colloidal, or polymeric network nonfluidic containing crosslinked covalent bonds. The solvent evaporation from the gel can lead to a xerogel or an aerogel, if the solvent is removed under supercritical conditions (Brinker and Scherer, 1990). Fig. 5.3 is a schematic representation of the reactions involved in the sol
gel process. The sol
gel process is influenced by temperature, synthesis duration, presence of catalysts, concentration of reagents, etc. All these factors will determine the

FIGURE 5.3 Representation of the reactions that take place during the sol
gel process.

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characteristics of the final material. Regarding the catalysts, an acid environment is usually applied when the aim is film development, while bases are mainly used for synthesis of particles that can be incorporated into different matrices (Brinker and Scherer, 1990). The disadvantage of the sol
gel process versus controlled polymerization techniques is the lack of control over polymer polydispersity and architecture. Nevertheless, polymers prepared from quaternized ammonium silane macromonomers may have improved toughness and damping properties, due to the flexibility of the siloxane backbone as compared with rigid C
C bonds. Additionally, the incorporation of more flexible siloxane linkages can demonstrate enhanced polymerization characteristics and the ability to self-repair damage caused by water sorption and mechanical stress relief over time (Gong et al., 2012b). Important studies in dentistry brought together QAS-functionalized methacrylate (QAMS) groups to be inserted in an experimental photocurable composite resin (Gong et al., 2012b) and in a commercial autopolymerizing acrylic resin for removable orthodontic devices (Gong et al., 2012a). In both cases, materials containing QAMS demonstrated contact-killing effect for S. mutans, Actinomyces naeslundii, and Candida albicans. The authors hypothesized that the presence of siloxane crosslinking could delay cracking propagation in the restorative composite resin since the silicate network could deflect and dissipate energy (Gong et al., 2012b). For the orthodontic acrylic resin, QAMS insertion improved toughness without affecting the flexural strength and modulus (Gong et al., 2012a). In biomedical devices, QASs were used to coat silicon rubber tracheoesophageal shunt prostheses. Biofilms readily settle and impair the functioning of these prostheses leading to a short useful lifetime, ranging from 3 to 6 months (Hilgers and Balm, 1993). The silane-containing quaternary ammonium groups formed a positively charged surface with great efficacy in preventing mixed biofilms (Candida tropicalis, C. albicans, S. aureus, Staphylococcus epidermidis, and Streptococcus salivarius). Besides the inhibitory effect, the QAS was noncytotoxic to mammalian cells and stable even in a moist environment. QAS coating could increase tracheoesophageal prostheses’ lifetimes and could also be useful in other biomedical devices (Oosterhof et al., 2006). Infections starting in catheters are also a concern. Hospitalized patients can remain with catheters for extended periods of time and bacterial biofilms can easily form, leading to infections. Zanini et al. (2015) modified the surface of a commercialized catheter with a silane-based QAC and found that the polyurethane catheters could display antimicrobial activity against Escherichia coli for 4 up to 24 hours. Surgical sutures and wound dressings are also a concern in the biomedical field as surgical sites are easily infected by bacteria, regardless of the spot in the human body and represent a risk for bacteremia (Meghil et al., 2015), and increased morbidity and hospital stay (Iovino et al., 2017). Preliminary studies with chromic gut, nylon, and polyester sutures impregnated with silane-based QAC-QACK21 demonstrated antimicrobial action against Porphyromonas

5.3 Quaternary Ammonium Compounds and Their Chemistry

gingivalis and E. faecalis (Meghil et al., 2015). Wound dressings and textile fibers impregnated with silane-based QAC can also help to prevent wound contamination and hospital crossinfections. Silane-based QAC can be covalently bound to these fibers and therefore resistant to laundering. Functionalized coatings were effective against E. coli, Pseudomonas aeruginosa, S. aureus, S. epidermidis, and fungi Saccharomyces cerevisiae and C. albicans (El-Ola, 2008; Lin et al., 2003; Varesano et al., 2011). Bone cements are used to immobilize prostheses or fragments of fractured bone, as other medical devices are prone to biomaterial-associated infections. Quaternized chitosan derivatives inserted into bone cement prevented biofilm formation over this surface better than gentamicin-loaded bone cement. This effect was observed for S. epidermidis, S. aureus, and meticillin-resistant strains. Besides the robust antimicrobial property, quaternized chitosan-loaded bone cements were also biocompatible with osteogenic cells (Tan et al., 2012). From an industrial point of view bacteria, fungi, algae, and other organisms can consume and degrade surfaces during shipment, storage, and use, causing loss of product as well as exposing the consumer to contamination. Once the material is anchored on the substrate it can protect it from microbial contamination and guarantee product quality (Monticello, 2010).

5.3.2 QUATERNARY AMMONIUM COMPOUND DISINFECTANTS AND PRESERVATIVES Chlorhexidine digluconate is one of the most notorious quaternary ammoniumbased disinfectants and undoubtedly efficient for a wide spectrum of pathogenic bacteria. In dentistry it is the gold standard chemotherapy, being recommended for treating gingivitis, periodontal therapy, adjuvant treatment for patients with high risk of caries, disinfecting prostheses, pre- and postoperative rinsing, and root canal irrigation (Ankola et al., 2009). The prescription should be precautious though as extensive use of chlorhexidine can induce tooth staining, calculus formation, and changes in taste perception. Other applications of chlorhexidine include antimicrobial soaps, hand antisepsis, and skin disinfection in hospitalized patients (Karki and Cheng, 2012; Pittet et al., 2009). Alkyl trimethyl ammonium bromide and chloride, cetyltrimethyl ammonium bromide and chloride, lauryltrimethyl ammonium bromide, behentrimonium chloride, and stearyltrimethylammonium chloride have been added to hair products and face cosmetics (Halla et al., 2018). Chitosan and its derivatives and quaternary ammonium cellulose derivatives have been reportedly used in hair conditioners, shampoos, and skin products (Kenawy et al., 2007). QAC is also present in the food processing industry (Kenawy et al., 2007). Concerns over foodborne poisoning and demand over increased shelf-life for food products led to the use of QAC in food packaging and processing. QAC could be used as disinfectants or edible films against food spoilers such as E. coli, S. aureus, P. aeruginosa, Campylobacter jejuni (Gunther et al., 2018), Listeria monocytogenes, and Salmonella (Bastarrachea et al., 2015). Benzalkonium chloride, benzethonium

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chloride, cetyl pyridinium chloride, and didecyldimethylammonium chloride are some of the already-commercialized disinfectants (Gunther et al., 2018) and quaternized chitosan derivatives have been studied for edible films (Arora et al., 2013). As QAC can bind to surfaces in a stable manner it has also caught attention as an application in water treatment filters because of the diminished risk of toxicity for consumers. Quaternized chitosan derivatives in combination with graphene oxide were able to reduce 99.99% of E. coli from contaminated water without leaching of QAC (Wang et al., 2015). Other chitosan quaternary ammonium salts (Jin et al., 2017) and quaternary ammonium cationic polymers such as poly(diallyldimethylammonium chloride) and epichlorohydrin-dimethylamine have been used as flocculating agents (Zeng et al., 2016).

5.3.3 IN SITU QUATERNIZATION OF TERTIARY AMINES TO FORM QUATERNARY AMMONIUM COMPOUNDS AND NANOPARTICLE FUNCTIONALIZATION Cationic poly(ethylene imines) (PEI) are an example of QAC obtained by means of in situ quaternization of tertiary amines. For this kind of approach the surface or polymeric structure should present reactive alkyl groups (Fig. 5.4). If the substrate originally does not present these groups it is possible to functionalize via plasma and akylation treatments prior to the quaternization. Once the substrate or polymer has alkyl groups it is possible to react them with tertiary amines existing in PEI (Elena and Miri, 2018). In situ quaternization has a drawback of limited functionalization due to steric hindrance (Zheng and Du, 2013). A minimum inhibitory concentration (MIC) test was performed for a series of cationic PEI solutions with different molecular structures for: S. aureus, E. coli, and Bacillus subtilis. It was concluded that polymeric solutions of cationic PEI are effective against the three bacterial strains and the best molecular arrangement for enhanced antimicrobial activity was a cationic group directly connected to an alkyl chain resulting in an amphiphilic molecule which then could be linked to PEI without spacers or intermediate molecules (He et al., 2012). For application in dentistry purposes, quaternary ammonium functionalized PEI NPs were synthesized and inserted in three different commercial products: a restorative composite resin (Filtek Z 250, 3M ESPE); a low-viscosity composite resin (Filtek Flow, 3M ESPE), and a bonding agent (Adper Single Bond, 3M ESPE). Materials incorporated with 1% (w/w) quaternary ammonium functionalized PEI NPs exhibited antimicrobial properties against S. mutans. Flexural strength was however decreased for the low-viscosity composite resin after insertion of quaternary ammonium functionalized PEI NPs (Beyth et al., 2006). Quaternary ammonium functionalized PEI NPs were also tested in root canal sealers with a broader spectrum of action: besides S. mutans also A. naeslundii, E. faecalis, and C. albicans (Jiao et al., 2017). Silica NPs can deliver biocides entrapped in their structure or immobilized in their surface. Because of their high surface area, NPs are capable of carrying and releasing great amounts of biocides (Fig. 5.5). Incorporating antimicrobial

FIGURE 5.4 Schematic representation of the in situ quaternization process.

FIGURE 5.5 Schematic representation of nanoparticle quaternization.

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compounds by modifying the NP surface and entangling these particles into a polymeric matrix can also enhance the mechanical features. Dental materials profited from inclusion of QAC-functionalized NPs (Jiao et al., 2017). QACs were attached to the surface of nanosilica particles and the results revealed that composite filled with these quaternary ammonium methacrylatemodified nanosilica (QMSNs) inhibited growth of Gram-positive S. mutans, S. aureus, B. subtilis, Gram-negative E. coli, P. aeruginosa, and fungi C. albicans. These modified NPs functioned as reinforcement particles and composites showed improved mechanical properties with higher flexural strength values (Makvandi et al., 2015). There are disadvantageous aspects of functionalizing silica NPs in the sense that the particles could be lost by wear if they are not strongly chemically bound to the matrix or if they have to transpose thick mature biofilms when used as drug carriers. NPs can bind to EPS but their variability in size, charge, and shape can also change this interaction. Therefore, researchers have been trying to understand how NPs’ characteristics play a role in the attachment and diffusion through the biofilm. Evidence has pointed out that for some Pseudomonas and E. coli biofilms, positively charged NPs could attach and diffuse more easily into the biofilm compared to negatively charged or neutral NPs.

5.4 VARIABLES INFLUENCING THE ANTIMICROBIAL PROPERTIES OF QUATERNARY AMMONIUM COMPOUND There are some factors to be considered when analyzing the antimicrobial properties of polymeric materials containing QAC, including surface charge density (Kamal et al., 1991), effect of molecular weight (Moriarty et al., 2013), counterion effect (Xiao et al., 2012), effect of alkyl chain length (Kenawy et al., 2007) and bacterial singularities (Kenawy, 2001). The influence of the surface charge was tested between different compounds containing QAC. It was noticed that positively charged surfaces, even when attracting more bacteria at first, prevented biofilm growth for Gram-negative bacteria. A threshold of 1014 charges per cm2 has been reported for a surface to exert antimicrobial activity (van de Lagemaat et al., 2017). The authors inferred that a strong attachment between positive surface charge and negative bacterial membrane impeded the elongation necessary for cell division. Gram-positive bacteria were not affected as much because of their thicker and more rigid peptidoglycan layer. Negatively charged surfaces, in contrast, promoted exponential Grampositive and Gram-negative growth, even though the initial adhesion was lower (Gottenbos et al., 2001). The molecular weight was also correlated with the antibacterial potential. It was demonstrated through synthesis of polymeric biocides with different molecular weights that antibacterial activity increases as the molecular weight rises, as does

5.4 Variables Influencing the Antimicrobial Properties

the cationic charge density. As the bacterial cell surface is negatively charged, the higher the cationic charge density of a surface, the easier is the adsorption of the polymeric biocide to the cell membrane and the consequent process of membrane lyses, cytoplasmic material leakage, and cell death (Ikeda et al., 1986). It has been discussed that the counterion also plays a role in the antimicrobial activity. Some have stated that there was an increase or decrease in the antimicrobial efficacy against E. coli and S. epidermidis depending on the counterion. The exact mechanism for these changes, however, could not be elucidated (Ingalsbe et al., 2009). Counterions with weak ionic bonds, that could easily dissociate into free ions, exhibited higher antimicrobial activity than tight ion pairs (Kanazawa et al., 1993). Concerning quaternary ammonium groups, it was found that bromide counterions were more efficient than chloride. On the other hand, some authors found no difference between chloride, bromide, and iodide counterions (Panarin et al., 1985). Some have found that chloride-containing quaternized amine polyurethanes were less bactericidal than iodide-containing quaternized amine polyurethanes. Although the antimicrobial activity of iodine has already been established, the influence of iodide counterions was not conclusive in this study (Flemming et al., 2000). Polymer final conformation and charge density vary according to spacer length. This characteristic could also play a role in the way that the polymer interacts with the cytoplasmic membrane (He et al., 2012; Ikeda et al., 1986). When considering quaternary ammonium chlorides, the hydrophilic
lipophilic balance affected its antimicrobial potential. It was found that even low concentrations of QACs were capable of hindering osmoregulatory activity and leakage of K1 and H1 (Lambert and Hammond, 1973) when a long carbon chain is attached to the N1. This additional antimicrobial mechanism was explained by chain intake into the bacteria cell enhancing the process of membrane physical disruption (Simoncic and Tomsic, 2010). At this point, it is important to mention that the carbon chain length has also been reported as an important factor influencing the antimicrobial activity. Longer chains seem to demonstrate greater antibacterial activity (Li et al., 2013b; Simoncic and Tomsic, 2010; Zhou et al., 2013). An optimum chain length has been cited as between 16 and 18 carbon atoms (Lindstedt et al., 1990). To prove the influence of the carbon chain in the antimicrobial activity of compounds a series of polymeric iodine QAC with different alkyl chain lengths obtained by reacting dimethylaminoethyl with different alkyl iodides were synthesized. MIC determination showed that all chain lengths between C10 up to C18 showed significant antibacterial activity. The antibacterial activity increased with increasing alkyl chain length from 5 to 16. Increasing the chain length to more than 16 carbons did not reflect on effectiveness (He et al., 2011). Another comparative analysis was made between methacrylate containing quaternary ammonium groups with different carbon chain lengths inserted on a dental bonding agent. Bacterial early attachment and biofilm CFU decreased by 4 log

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when increasing the chain size up to 16 carbons. Also, with longer chains the MIC and minimum bactericidal concentration decreased by five orders of magnitude. However, when the chain size was extended to 18, the antibacterial efficacy decreased. This was attributed to chain bending and consequent prevention of electrostatic interactions between the quaternary ammonium group and the bacterial membrane. The improvements in antimicrobial activity did not compromise cytotoxicity or the bond strength to dental structure of the bonding agent (Li et al., 2013b). It has been stated that a maximum antimicrobial efficiency is granted for Gram-positive bacteria when the chain length is between 12 and 14 carbons. As for Gram-negative bacteria, the ideal chain length would be between 14 and 16 (Daoud et al., 1983; Gilbert and Al-taae, 1985). It is important to emphasize that differences in bacterial structure also influence antimicrobial activity. Gram-negative bacteria present besides the cell wall an outer membrane that enhances the barrier against antimicrobial substances. Studies with S. aureus (Gram-positive) showed that molecules weighing in a range of 5 3 104 to 9 3 104 Da could diffuse across the cell wall without difficulty (Kenawy et al., 2007). On the other hand, it is worth remembering that Grampositive strains have a thicker and more rigid peptidoglycan layer (Gottenbos et al., 2001) that can work as a barrier against molecules with high-molecularweight (Costerton and Cheng, 1975). Differences between bacteria and fungi must be cited. Both, Gram-positive and Gram-negative bacteria are prokaryotic cells, whereas fungi are eukaryotic cells. Because of the more complex structure of eukaryotic cells, higher resistance to antimicrobial activity for these is expected (Zhang and Srinivasan, 2004). The most common methods described in microbiology to evaluate the antimicrobial activity of materials are the agar or disk diffusion test, and the quantitative methods that include MIC and broth macro/microdilution due to their simplified methodology and cost
benefit. Unfortunately, as a lot of adaptations in methodologies have been done to test the antibacterial activity of antimicrobial materials (different bacterial strains or growth media) it may be difficult to compare the results of different studies, suggesting a need for a standardized method as reported by other researchers. Another issue is the duration of the antibacterial effect. Most studies for dental material science only document the short-term effects on antibacterial activity of the material. With a maximum aging period of 6 months in in vitro studies, while the maximum aging in in vivo studies was 12 months, suggesting the need for further long-term randomized controlled trials for assessing dental materials with antimicrobial agents (Farrugia and Camilleri, 2015). Based on the data survey, there is a multitude of factors to be considered when developing polymeric materials containing antimicrobial moieties. It is necessary to take into count the characteristics of the polymer to which the immobilization will be carried out, such as thermal and chemical stability, mechanical properties, and affinity to the antimicrobial moiety chosen. Also, the antimicrobial

5.5 Cytotoxicity

moiety peculiarities concerning potential toxicity (depending on the molecular weight), the hydrophobicity promoted by the carbon chain, which in turn will directly impact the antimicrobial activity, and the immobilization of the group on the surface of the substrate should be considered. The characteristics of the microorganisms to which the antimicrobial effect is aimed will also guide the features of the molecule.

5.5 CYTOTOXICITY In general, low-molecular-weight antimicrobial agents tend to be more toxic than high-molecular-weight ones but given the mechanism of bacterial membranetargeting of QAC, cytotoxicity is of obvious concern and still under debate. Important studies compared the toxicity of MDPB monomer to others already in use in dental materials like triethylene glycol dimethacrylate (TEGDMA), bisphenol A-glycidyl methacrylate (Bis-GMA), and methacryloyloxydecyl phosphate (MDP) over mouse fibroblasts L929, odontoblast-like cells, and human pulpal cells. Toxic concentrations of MDPB for fibroblasts L929 were in the same range as for TEGDMA (Imazato et al., 1999). Bis-GMA and MDP caused higher mineralization inhibition from odontoblast-like cells compared to MDPB (Nishida et al., 2010). After these evidences the first antibacterial adhesive system containing MDPB started to be commercialized by Kuraray Medical (Clearfil SE Protect in USA and Clearfil Mega Bond FA in Japan). Subsequently studies followed comparing Clearfil SE Protect to other commercial dental adhesives. Clearfil SE Protect was less cytotoxic than Adper Scotchbond 1, Excite, Tyrian SPE, and One Step plus (Koulaouzidou et al., 2008). Also, Clearfil SE Protect was as cytotoxic as Clearfil SE Bond which does not contain antimicrobial monomer (Kusdemir et al., 2011). Gong and collaborators also tested cytotoxicity by means of MTT assay and flow cytometry. Quaternary ammonium-containing acrylic resins were compared to quaternary ammonium-free acrylic resins on murine dental papilla-derived odontoblast-like cells (MPDC-23). The authors concluded that acrylic resins containing QAC were as biocompatible as those free of QAC and that cytotoxicity observed could be related to leaching of residual methacrylate monomers from the acrylic resin (Gong et al., 2013). Composite resins containing QPEI showed a similar effect on cell viability of MBT cell to composites that did not contain QPEI. In vivo studies in rats were also carried out and revealed no inflammatory signals after implantation of the restorative composite resin containing QPEI 2% (w/w). DMADDM also had much lower cytotoxicity than Bis-DMA on human gingival fibroblasts (Li et al., 2013a). Rat tooth models evidenced that DMADDM promoted little pulpal inflammation when compared to a commercial adhesive and a glass-filled composite resin (Li et al., 2013b).

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5.6 ANTIMICROBIAL RESISTANCE Antimicrobial resistance is an alarming issue worldwide, and is the target of discussions not only in academic spheres but also among public health leaderships. QAC emerged in this scenario as a good approach to counteract antimicrobial resistance. Antimicrobial resistance to QAC has already been reported related to the presence of subinhibitory concentrations. Environments such as sewage, sediments, and water treatment stations can contribute to the selection of QAC-resistant bacteria (Jiao et al., 2017). Therefore special care should be taken concerning discarding of QAC. Soil and sediments are naturally negatively charged, which makes contamination by QAC much easier (Tezel and Pavlostathis, 2011). Resistance mechanisms arose from both kinds: intrinsic and acquired means. Intrinsic resistance is related to components naturally found in bacteria (structural, physiological, or biochemical) that contribute to decreased susceptibility to biocides, for instance, the outer membrane of Gram-negative bacteria. Acquired mechanisms are related to modifications on the hyperexpression of efflux pump genes due to oxidative stresses, stress-induced mutagenesis, and acquisition of efflux pump genes through integrons, plasmids, or transposon (Jennings et al., 2016; Jiao et al., 2017; Tezel and Pavlostathis, 2011). Genes associated with acquired resistance to QAC are qacA, qacB, qacC-H, qacJ, and qacZ, which are responsible for efflux pump expression (Jennings et al., 2016). An up-to-date list of QAC-resistant bacteria points to Salmonella typhimurium, Acinetobacter baumannii, L. monocytogenes, E. coli, S. aureus, E. faecalis, Klebsiella pneumoniae, and P. aeruginosa. Further developments have been directed to inactivation of efflux pumps as a measure to fight resistant bacteria (Jiao et al., 2017).

5.7 REMARKS Bacterial contamination is an everlasting human problem. Combating diseases caused by bacterial infection is not as simple in this postantibiotic era. Therefore there is an urgency for alternative strategies to tackle bacterial-associated infections. In view of this worrying ambience, grafting antimicrobial activity to materials used for dentistry and biomedical purposes is meaningful. QACs have been for decades included in daily-life products and more recently also in biomaterials with undeniable efficiency against pathogens. Given the variety of QACs and means for achieving a QAC-containing material it is of major importance for investigators to know the variables affecting antimicrobial activity, how to better benefit from the mechanism of action, and what is the aimed-for final application. Queries about QACs still have to be addressed, such as cytotoxicity to the human body and QAC-resistant bacteria.

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Block copolymer micelles as nanoreactors for the synthesis of gold nanoparticles

6

Rajpreet Kaur and Poonam Khullar Department of Chemistry, B.B.K. D.A.V. College for Women, Amritsar, India

6.1 INTRODUCTION Nanoscience deals with nanoscale materials with a range in the nanometer scale (1
100 nm). The word “nano” is derived from the Greek word “nano,” meaning “dwarf.” Metal nanoparticles (NPs) have long been considered to exhibit unique physical and chemical properties differing from those of the bulk state or atoms, due to the quantum size effect, resulting in specific electronic structures (Rotello, 2004; Schmid, 2006; Caruso, 2004; Liz-Marzan and Kamat, 2003; Burda et al., 2005; Chen and Mao, 2007; Xia et al., 2009). The first scientific description of the properties of NPs was provided in 1857 by Michael Faraday in his famous paper “Experimental Relations of Gold to Light” (Faraday, 1857) In 1959 Richard Feynman gave a talk describing a molecular machine, built with atomic precision. This was considered the first talk on nanotechnology. This was entitled “There’s plenty of room at the bottom.” Various metal NPs have been synthesized, including AuNPs, AgNPs, PdNPs, and SeNPs. Among all the nanostructured materials, gold nanoparticles (AuNPs) have attracted considerable interest and have versatile applications in various fields, such as drug delivery, catalysis, bioimaging, sensing, photothermal therapy, nanoelectronics, and in the fabrication of photonic and plasmonic devices (Eustis and El-Sayed, 2006; Jain et al., 2008; Murphy et al., 2008a,b; Hu et al., 2006; Cobley et al., 2011; Sardar et al., 2009; Ghosh and Pal, 2007; Hashmi and Hutchings, 2006; Sperling et al., 2008; Boisselier and Astruc, 2009; Giljohann et al., 2010). AuNPs can be synthesized using two approaches: “top down” and “bottom up.” In the top-down approach bulk material is broken down to generate NPs of desired dimensions. However, this approach suffers from the limitations of controlling the size and shape of the particles as well as further functionalization

Materials for Biomedical Engineering: Inorganic Micro- and Nanostructures. DOI: https://doi.org/10.1016/B978-0-08-102814-8.00007-X © 2019 Elsevier Inc. All rights reserved.

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(Nguyen et al., 2011). The alternative “bottom up” approach involves the formation of AuNPs from individual molecules using either a chemical or a biological reduction (Parab et al., 2011). A further two steps are involved, that is, nucleation and successive growth. However, when both these steps are completed in the same process, it is termed in situ synthesis, while the other one is called the seed growth method. Nonionic amphiphilic copolymer micelles composed of polypropylene oxide (PPO) and polyethylene oxide (PEO) units and surfactants are extensively used in the synthesis of nanostructured materials. Pluronics performs multiple functions, that is, it acts as a reducing agent, shape-directing agent, as well as stabilizing the NPs obtained. This chapter accounts for the recent advance in the synthesis of AuNPs using environmentally friendly, nontoxic, linear-chained poloxamers, and branched poloxamines and the further scope of block copolymer-coated gold NPs in various biomedical fields.

6.1.1 POLOXAMERS AND POLOXAMINES Poloxamers are also known as Pluronics (BASF), Synperonics (ICI), or Genapol and are water-soluble polymers. Amphiphilic block copolymers, composed of two units, the PEO and PPO, are available in a variety of molecular weights and PEO/ PPO ratios commercially. It is because of the hydrophilic nature of PEO blocks and the hydrophobic nature of PPO blocks that these block copolymers are amphiphilic in nature. The linear block copolymers with the structure PEO-bPPO-b-PEO are known as poloxamers (Fig. 6.1) and tetra-branched copolymers with four PEO
PPO blocks in a central ethylenediamine bridge are called poloxamines (Fig. 6.2). These surfactants were first introduced in the 1950s by BASF, NJ, United States. Recently, these water-soluble block copolymers have attracted significant interest due to environmental concerns (Krishna et al., 2006; Hobbs et al., 2012) and their water-based food and pharmaceutical formulations (Naohika and Atsushi, 2013; Yusa et al., 2005). Pluronics with different molecular compositions can be categorized using an easy description of the structure and properties of a given copolymer. In all the trade names, that is, Pluronics, Lutrol, or Synperonics, the prefix letters describe the physical appearance of the pure copolymer, that is, “L” refers to liquid, “P” to paste, and “F” to flakes. In addition, the name contains

FIGURE 6.1 Structure of poloxamers composed of two PEO blocks and a central PPO block.

6.1 Introduction

FIGURE 6.2 (A) Structural formula of star-shaped T904 and its schematic representation. H (white), C (gray), O (red), and N (blue). (B) A graphical model of the structure and (C) its relaxed structure. (D) A graphical model of an aggregate of three molecules and (E) their relaxed structure.

information about the block length ratio in the respective polymer. The approximate molecular weight of PPO can be obtained by multiplying the first one or two digits by 300 and that of PEO can be obtained by multiplying the last digit by 10. It is also possible to convert the nomenclature for Pluronics to the generic name for poloxamers by multiplying the first one or two digits by ´ ez et al., 2014). a factor of 3 (Torcello-Gom The working process of these copolymers is micellization with metal. The micellization process of these polymers in aqueous solutions is governed by the interplay between the hydrophobicity and hydrophilicity of the building blocks and their interactions with the solvent. In block copolymer, each segment shows a particular function, such as the reducing and anchoring function. An appropriate variation in the number of PEO and PPO repeating units causes a shift in the overall nature from predominant hydrophilic to predominant hydrophobic. Such a versatile nature also makes them widely useful in various applications such as dispersion stabilizer (Lin and Alexandridis, 2002; Barnes and Prestidge, 2000), pharmaceutical ingredients (Yang and Alexandridis, 2000; Ivanovo et al., 2002;

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FIGURE 6.3 A TBP micelle with the core occupied by PPO units and the corona constituted by PEO units. Red dotted circle shows a possible surface cavity whose size is related to the number of PEO and PPO units. A larger cavity can easily accommodate a guest molecule in comparison to a smaller cavity.

Kabanov et al., 2002), biomedical materials (Ahmed et al., 2001; Cohn et al., 2003), and templates for the synthesis of mesoporous materials and NPs (Solee-Illia et al., 2003; Zhao et al., 1998; Han et al., 2000; Karanikolos et al., 2004; Kim et al., 2004; Wang et al., 2004). In order to understand the synthesis of AuNPs using the micelles of block copolymers, we need to ensure the presence of the micellar phase along with the gold salt. It is to be noted that the monomeric form of block copolymer does not effectively involve the reduction reaction due to the lack of surface cavities and, hence, reduction is caused by the micelles only (Fig. 6.3). The reducing ability of the cavity which is formed by the PPO and PEO block is directly connected to its size. Since one polymer is contributing only one surface cavity because it accepts only one guest ion, that is, an oxidizing agent as accepted by the partially hydrated or dehydrated cavities with high aggregation number at high temperature (Khullar et al., 2011). Such reducing agents are called structured reducing agents where structural factors are the deciding factors for reduction. Reduction of gold salt is carried out by the surface cavities produced by the compact arrangement of the Triblock polymer (TBP) monomers in the corona layer of TBP micelles (Fig. 6.4). The extent of hydration of these surface cavities is the deciding factor as greater hydration reduces the nucleation process because it hinders the approach of gold ions. But, with the increase of dehydration, compact micelles are formed that bring the nucleation center in close proximity, to produce larger AuNPs. Since soft micelles are unable to hold the larger NPs, such NPs find their way into the bulk phase. In contrast, larger micelles are fully capable of holding these NPs. The overall shape, structure, and the temperature are the key parameters for the production of AuNPs.

6.1 Introduction

FIGURE 6.4 Demonstration of the overall redox process taking place in the surface cavities at the micelle
solution interface of TBP micelles.

6.1.2 MICELLE ARCHITECTURE AND MIXED MICELLES TBPs with a greater number of PEO units rather than PPO units form compound micelles but predominantly hydrophobic TBPs usually produce well-defined micelles. Micelles undergo several structure transitions (i.e., micelles to threadlike micelles to vessels, etc.) with concentration and temperature variations. It is observed that whenever a transition in the structure of the micelle occurs, it alters the interfacial arrangement of the surface cavities and hence it affects the overall mechanism that is actually occurring through the ligand and metal charge transfer (LMCT) complex. TBPs with a larger number of cavities with sufficient size are able to produce ordered morphologies of gold NPs, whereas a TBP with less surface cavities and small micelle will lead to an unstable LMCT complex and hence the nucleation depends on the extent of intermicelle collisions due to diffusion. In order to achieve well-defined morphologies of AuNPs, a predominantly hydrophilic TBP with a greater number of PEO units is desired because of the presence of more PEO units, the instant reduction can be achieved. The other deciding parameter is the PPO/PEO ratio, TBPs with a high PPO/PEO ratio produce stable micelles, with respect to temperature (Fig. 6.5).

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FIGURE 6.5 Schematic representation of the proposed mechanism for the synthesis of AuNPs by Using Micelles of (A) L31, (B) L64, and (C) P123, respectively (see details in the text). (D) Plot of the average size of NPs estimated from TEM images versus cavity size for L31, L64, and P123.

6.1 Introduction

TBPs which are predominantly hydrophobic, for example, L121, are used as templates for the synthesis of AuNPs. However, the presence of convectional surfactant is a must to solubilize L121 in the aqueous phase. By comparing the different zwitterionic surfactants, such as dimethyl- dodecylammoniopropanesulfonate (DPS), Tetrapropylenebenzene sulfonate (TPS), N-hexadecyl- N-N0 dimethyl-3-ammonio-1-propane-sulfonate (HPS), it is observed that the order of hydrophobicity of these surfactants follows DPS , TPS , HPS (C12 , C14 , C16). The less hydrophobic nature of DPS produces welldefined micelles, whereas the more hydrophobic TPS and HPS dismantle the micelle template because of their enhanced solubilizing effects. The low concentration and low hydrophobicity of neutral surfactant cause minimal disturbance to the arrangement and hence produce well-defined micelles loaded with tiny NPs. One of the remarkable features of these copolymers is their thermoresponsive behavior. The micellization process is extremely temperature-dependent. With an increase in temperature, Critical Micelle Concentration (CMC) values decrease dramatically. Due to this property, such polymers are called “smart and intelligent polymers” and find applications in controlled drug delivery, thermal printing, biomedical processing, and sensor development (Parmar et al., 2013; Zou et al., 2012; Huynh et al., 2012).

6.1.3 SYNTHESIS OF VARIOUS MORPHOLOGIES OF GOLD NANOPARTICLES Over the past decade, various methods, such as seed-mediated growth processes (Iqbal et al., 2007), template-directed patterning (Liang et al., 2005; Johnson et al., 2002), biomineralization (Xie et al., 2007), two-phase reactions (Daniel and Astruc, 2004), and inverse micelles (Pileni, 2003), have been used to synthesize nanostructures including rods (Keul et al., 2007; Niidome et al., 2007), plates (Xie et al., 2007), spheres (Liang et al., 2005), and cages (Chen et al., 2005). The template-based approach (either hard or soft) is the most commonly used approach to prepare uniform gold nanorods. The hard templates usually are alumina or polycarbonate membranes for 1D gold nanostructures, whereas the soft templates are usually surfactants that form rod-shaped micelles. The block copolymer-mediated synthesis method offers many advantages and needs only an environmental and economic method, but it is also a very simple procedure that simply requires the mixing of metal salt with a block copolymer ratio. This type of methodology offers many possibilities, like simply varying the block copolymer type, amphiphilic character, concentration, temperature, and solvent. It is observed that use of a weak reducing agent modifies the thermodynamic controlled reaction pathway to kinetic controlled pathway as a result of slowing down of precursor decomposition or using a weak reducing agent or by Ostwald ripening (Xia et al., 2009).

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The seed growth method can be employed for anisotropic NP synthesis such as nanoprisms (Millstone et al., 2005, 2006). Another mechanism that is normally employed to describe the growth of NPs is the Lamer mechanism (LaMer and Dinegar, 1950; Mer, 1952). In this mechanism, after initial nucleation, the nuclei grow into particles by the molecular addition of nutrient species on the surface of the particles known as Ostwald ripening. In this method, the particles will be mostly monodisperse in nature. The “aggregative nanocrystal model” is used to describe Au nanocrystal growth (Njoki et al., 2010). This model explains that initial nucleation and growth result from a number of critically sized aggregates of smaller nanocrystallites in a nonclassical aggregative nucleation step. The seed growth method is a soft template approach and is one of the most popular techniques for the synthesis of AuNPs. Initially, gold seeds of B3
4 nm size are prepared by the borohydride reduction of the gold salt in the presence of citrate or CTAB as the capping agent. Then, these preformed seeds are added into the growth solution which contains a reducing agent such as ascorbic acid. The addition of silver nitrate in the growth stage helps improve and increase the yield of nanorods. The nanostructures thus obtained have potential applications in medicines due to tunable plasmonic properties but they are restrained due to the toxicity of CTAB (Murphy et al., 2008a,b). Also, CTAB binds strongly to the surface and restricts subsequent functionalization (Fig. 6.6). This limitation can be improved by using a mixture of CTAB and Pluronic (F-127) in the seed growth method (Fig. 6.7). The nanorods thus prepared in the presence of Pluronic (F-127) are quite stable and the presence of pluronic also results in a higher yield of nanorods. The reducing nature of PEO blocks of pluronics is responsible for the enhanced yield of nanorods (Fig. 6.8). The formation of a stable complex between CTAB and block copolymer due to hydrophobic interactions between the PPO blocks of pluronics and the hydrophobic surfactant tail is the cause of stabilization of gold nanorods. The presence of ascorbic acid is required for the synthesis of gold nanorods as the pluronic alone is not sufficient to reduce the metal salt in the presence of CTAB. However, at high concentration, PEO can reduce the gold ions but the PPO blocks get adsorbed onto the gold cluster and lead to the stabilization of AuNPs, which hinder the growth of NPs into nanorods. Generally, the size and shape of the metal NPs depend on the competition between the nucleation (metal ion reduction in bulk) and the growth process (metal ion reduction on nuclei). If the metal ion reduction on nuclei is more dominant than in a bulk solution, then particle growth is more significant. However, if the metal ion reduction in bulk is more dominant than on nuclei, then new particle formation is more significant than particle growth.

6.1.3.1 Icosahedral gold nanoparticles F88, P85, P104, and P105 Pluronics have also been used to prepare icosahedral AuNPs. However, regular icosahedron shapes are formed only when a

6.1 Introduction

FIGURE 6.6 Schematic representation of the mixed micelle formation between L121 and zwitterionic surfactants and their subsequent use as micelle templates for the self-assembled AuNPs. The top reaction shows the formation of spherical L121 1 DPS mixed micelles in the L121-rich region of the mixture by incorporating the hydrocarbon chains of DPS molecules in the L121 micelles. In the DPS-rich region, predominantly hydrophobic L121 is solubilized by the hydrocarbon tails of DPS in the form of a typical compound micelle, where DPS molecules occupy the shell, while L121 resides in the core, resulting in the formation of flower-like morphologies with AuNPs mainly accommodated in the core due to the presence of L121 surface cavities. The lower reaction shows that using TPS or HPS instead of DPS induces their longer hydrocarbon tails in the L121 micelles, thus causing structure transitions with the formation of oval or elongated morphologies that are visualized by the self-assembled AuNPs.

high-molecular-weight copolymer, that is, P85, is used. This is due to the fact that higher molecular weight copolymer is more flexible and can adsorb on the surface of gold cluster more efficiently, which is required for the formation of regular icosahedral AuNPs. Also, the size of these NPs can be controlled in the range of 100 nm to 1 mm by simply varying the experimental conditions such as temperature, concentration etc. (Figs. 6.9 and 6.10).

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FIGURE 6.7 SEM images of gold nanorods prepared from 25 mL of seed in the presence of AgNO3 (a) without Pluronic F-127 and (b) with Pluronic F-127 (4.75 3 10 exponent-4 M). Scale bars: 500 nm.

6.1 Introduction

FIGURE 6.8 TEM images of gold nanorods prepared from 25 mL of seed (5.5 3 10 exponent-7M): (a) in the presence of AgNO3 (without Pluronic F-127), after storage for 1 week in an aqueous environment; (b) in the presence of AgNO3 and Pluronic F-127 (c) in the absence of Pluronic F-127 and AgNO3; (d) in the presence of Pluronic F-127 and in the absence of AgNO3. Scale bars: 5 nm.

6.1.3.2 Nanoplates Pluronics, like F-127, has also been used in single-step seed-mediated synthesis to prepare biocompatible IR-responsive gold nanoplates. F-127 is approved by the FDA (Food and Drug Administration of the United States) for in vivo biomedical applications (Alexandridis and Hatton, 1995). The PPO blocks of F-127 bind to the surface of NPs through hydrophobic interactions and control the growth of AuNPs by specific crystallographic directions. In this case, the growth rate of NPs is kinetically controlled rather than thermodynamically, by

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FIGURE 6.9 Characterization of gold icosahedrons synthesized at 40 C with a reaction time of 1 day, and the concentrations of F88 and HAuCl4 are 0.84 and 5.8 mM, respectively: (a) SEM image; (b) higher magnification SEM image; (c) and (d) SEM images of a single gold particle observed from different angles of view; (e) geometrical model of the obtained icosahedron particles; and (f) representative TEM image.

FIGURE 6.10 SEM images of the as-synthesized particles when using different pluronic copolymers at 40 C, the concentrations of HAuCl4 and copolymers were 0.84 and 5.8 mM, respectively. (a) P85, (b) P104, (c) P105. The concentration of the pluronics was 8.4 mM. The insets of (b) and (c) show the magnified SEM images of the icosahedrons.

different sticking probabilities by atoms on a particular crystallographic face (Nam et al., 2002). The order of sticking probabilities of different faces is ɣ(110) . ɣ(100) . ɣ(111). Therefore FCC metals tend to grow and nucleate along the [111] facets. Because of this the [111] facet of FCC metals has the lowest surface energy as compared to other facets, that is, [110] . [100] . [111]. The FCC metal confers its tendency to nucleate and grow into NPs with its surface bounded by [111] facets (Xu et al., 2008). At a particular F-127/HAuCl4 molar ratio, the PPO blocks of the pluronics get adsorbed on the [111] planes of FCC Au nuclei, therefore inhibiting the growth on [111] crystallographic planes and promoting anisotropic growth along [110] facets (Khan et al., 2012;

6.1 Introduction

FIGURE 6.11 Schematic illustration of proposed growth mechanism of AuNPs: (A) crystallographic facets with basal {111} plane and h110i side facet, (B) the formation of a hexagonal plate, triangular plate, and truncated triangular plate like shapes along the different h110i directions, (C) the expected particles shapes, and (D) representative TEM images of hexagonal NPs, triangular NPs, and truncated triangular NPs.

Xia et al., 2009; Jin et al., 2001; Smith et al., 1986; Jana et al., 2001; Sau et al., 2001; Sau and Murphy, 2004; Zou et al., 2006; Nam et al., 2002; Zhang et al., 2004; Wiley et al., 2005; Heinz et al., 2009; Feng et al., 2011; Kan et al., 2006) (Fig. 6.11). Under ideal conditions, the nuclei grow uniformly along six [110] directions if the growth results in the formation of a hexagonal plate. But deviation of the crystal growth forms uniform growth rate along [110] directions due to the local fluctuations, resulting in the formation of a triangular and truncated triangular plate. Such nanostructures are found to be quite stable when preserved in Pluronic F-127 (Kaur and Chudasama, 2014) and hence they have the potential to be used for in vivo applications of cancer diagnosis and therapy.

6.1.4 BIMETALLIC NANOPARTICLES Pluronic F-127 has also been employed for the synthesis of Ag
Au bimetallic NPs in which maltose-coated AgNPs are added to the aqueous solution of F-127

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followed by the addition of HAuCL4. These bimetallic NPs have enhanced catalytic activities for the reduction of 4-nitrophenol by NaBH4 as compared to individual Ag and Au NPs. The enhanced efficiency is attributed to the greater surface areas containing higher energy facets and Ag/Au interfacial regions, where homogeneous surface aggregation (alloying) occurs, and that have excess electron density compared to monometallic NPs.

6.1.5 COMPARISON OF POLOXAMERS AND POLOXAMINES In comparison to the linear pluronic copolymers, tetronics consist of an X-shaped structure which is made up of an ethylenediamine central group bonded to four chains of PPO
PEO. It is because of this unique structure that tetronics are widely used in various biomedical and pharmaceutical applications (Dong et al., 2004). The presence of a tertiary amine group plays a very important role in the response to pH variation as well as in conferring thermodynamic stability (Alvarez-Lorenzo et al., 2007; Gonzalez-Lopez et al., 2008; Longenberger and Mills, 1995) that distinguishes their properties from poloxamers. The amphiphilic nature of the tetronics is due to the presence of both PPO and PEO blocks, whereas the PEO block is responsible for the reduction of the gold salt, the PPO block causes the adsorption of the copolymer on the NP surface. This creates a competition between the reduction (in bulk) and growth (on the surface). The Au(III) ion binds both to the diamine groups and PEO groups present in the copolymer via ion
dipole interactions, as a result of which the metal ion Au(III) gets entrapped and reduced in the micelle to Au(s). The reduction process is directly proportional to the number of micelles. The higher the number of micelles with such a type of loosely packed structure, the more easily the metal ion gets penetrated inside the micelle and hence the more NPs that will be produced. Tetronics which gives rise to micelles with loose structure act as a better stabilizer for the synthesis of AuNPs than their linear counterpart pluronics because of better coverage of the NP surface. A comparison of Tetronic T904 and Pluronic P105, both having the same percentage of PEO and PPO units, demonstrates the good reducing power of T904, even at as low a concentration as 0.07 M with 0.1 mM HAuCl4. This is due to the presence of a peculiar X-shaped structure (Fig. 6.12) that eases the formation of pseudocrown cavities, and secondly the presence of reducing amino groups in the copolymer molecule (Sakai and Alexandridis, 2005a,b; Newman and Blanchard, 2006). Both these factors favor reduction. Effect of pH: pH plays a very crucial role during the synthesis of AuNPs using block copolymers. At low pH, in acidic medium, a stronger hydrogen bond between block copolymers and water molecules is formed. This is due to the attachment of protonated water molecules with the PEO block and hence PEO corona holds more positive charge. However, when AuNPs are produced, H1 are

6.1 Introduction

FIGURE 6.12 (a) TEM image of gold nanoplates formed by reduction of HAuCl4 in the presence of T904 at a copolymer/metal salt molar ratio (MR) of 1.5 (0.5 mM HAuCl4) at 25 C. (b) Absorption spectrum of the nanoplates. (c) SAED pattern taken from an individual nanoplate and its assigned reflection indexes. (d) TEM image of gold nanoplates displaying bending contours.

also produced and the presence of H1 ions interrupts the binding of metal ions to the PEO block and the reduction is slowed down, increasing the size of the micelle (Yang et al., 2006) and hence a lower number of AuNPs will be produced. Also, at low pH, because of hydration of the micelle, the AuNPs cannot be entrapped by the micelle and hence they find their way into the bulk. However, as the pH is increased, according to Le Chatelier’s principle, more and more number H1 ions will be neutralized and so the reduction of the gold salt is increased, resulting in a large number of AuNPs. Low pH results in poor yield of AuNPs and high pH, that is, basic conditions result in a good yield of stabilized AuNPs. This stability at high pH can be attributed to the hydrophobic interactions because of the coating of AuNPs with the block copolymer. Therefore the coating makes the NPs hydrophilic and easily dispersible in water without further surface modification, which is desirable for

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biomedical applications. Increasing the pH value enhances both the reaction rate (Piao et al., 2007) and the coordination ability of the block copolymers as well as resulting in a change in the morphology of AuNPs that results in an Ostwald ripening process. Pluronics get adsorbed on the gold surface to form a core
shell structure via hydrophobic interactions. Poloxamine, that is, Tetronic T904, contains two nitrogen atoms and its titration shows two inflection points, pKa1 and pKa2, at 4.0 and 8.8, respectively. At pH , 4, T904 exists in diprotonated form, which results in Coulombic repulsions among the positively charged amine groups and hence prevents self-aggregation. Tetronics are highly pH-sensitive, and the size and shape of AuNPs can be controlled by varying the pH of the reaction mixture. At a low pH of 3.0, triangular NPs are formed and as the pH is increased more spherical NPs are formed. With the further increase of pH, the size of the spherical particles further decreases. It is also observed that acid used to decrease the pH itself exerts a strong effect on the shape and size of NPs because of the specific adsorption of Cl2 ions (HCl/NaCl) on the NP surface. As the pH decreases, the micellization becomes more difficult, which causes an increase in the CMC and a decrease in micellar size. At low pH, that is, under acidic conditions, the H1 ion disturbs the binding of metal ions to the PEO block and, therefore, the reduction rate decreases. Also, an increased population of plates is observed at low pH (Fig. 6.13) due to the adsorption of Cl2 ions on the specific planes, whereas high pH, spherical NPs are produced due to accumulation of T904 on different crystallographic planes, that promotes the growth in all directions and hence produces spherical particles. Spherical particles are also produced at high temperature, which indicates that reduction rate is under thermodynamic control (Fig. 6.14). In tetronics, the presence of amine groups raises the pH of the solution up to 8.9
9.3. It is easier to predict the morphology of micelles in pluronics rather than in tetronics, as in pluronics, the hydrophobic PPO blocks form the core and hydrophilic PEO blocks form the corona/shell. Significant steric constrains in the four arms of tetronics lead to the formation of highly hydrated micelles. In pluronics, PEO blocks act as a mild reducing agent and the redox reaction is mainly carried out in the micelle surface cavities that act as an active site for the entrapment of gold ions (Khullar et al., 2010, 2011, 2013). It is also observed that tetronic-coated NPs, produced at pHB2, can prove to be excellent models for pH-responsive drug-delivery vehicles (Mishra et al., 2012; Cuestas et al., 2011) in the colon at pHB10, where they change from ionic to the nonionic form for controlled drug delivery. In the case of tetronics, the presence of a micellar phase is a must for the synthesis of AuNPs as it is also true for the pluronics (Khullar et al., 2010, 2011, 2013; Sakai and Alexandridis, 2005a,b). The formation of a surface cavity by PPO and PEO blocks in the aggregated state is the most important requirement for the reduction process.

6.1 Introduction

FIGURE 6.13 TEM images of gold particles formed by reduction of HAuCl4 at molar ratio (MR) 4 (0.5 mM HAuCl4) and pH (a) 3.0, (b) 4.0, (c) 5.5, (d) 6.5, and (e) 7.5 at 25 C and at MR 16 and pH (f) 3.0, and (g) 7.5 at 25 C.

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FIGURE 6.14 TEM images of gold particles formed by reduction of HAuCl4 at molar ratio (MR) 1.5 (0.5 mM HAuCl4) and pH (a) 3.5, (b) 4.5, (c) 6.0, and (d) 7.5 at 75 C for (e) MR 4 and (f) MR.

In the case of Tetronic T904, it is observed that at low pH, diamine groups get protonated, which results in the formation of highly hydrated vesicles/micelles that electrostatically attract negatively charged AuCl42 ions. This results in the formation of an LMCT (ligand and metal charge transfer) complex that requires high temperature for the formation of compact micelles and facilitates the reduction reaction. On the other hand, at high pH, no protonation of the amine group

6.2 Biomedical Applications

FIGURE 6.15 Schematic representation of a T904 monomer and its vesicles at low and high pH. A hydrated vesicle with core
shell type morphology is formed at low pH, while a compact compound micelle with no clear core
shell regions is produced at high pH.

occurs and hence the reduction of Au31 to Au(0) occurs instantaneously at low temperature. Further growth process occurs at the surface of the micelles, which is why AuNPs are decorated at the micellar assemblies. Tetronics are less surface-active than cationic or nonionic surfactants and hence they do not show any shape control effects and result in the formation of only spherical or polyhedral morphology (Fig. 6.15). However, the presence of any other surfactant, for example, C14E8, which is a nonionic surfactant, leads to the formation of well-defined geometries of AuNPs (Fig. 6.16). This is because of the active participation of the surfactants in the shape control effects on the crystal growth of NPs. The micelles of tetronics, due to their pH and thermoresponsive behavior, act as excellent nanoreactors for the nonmaterial synthesis.

6.2 BIOMEDICAL APPLICATIONS Two important factors that control targeting the attachment of cell-specific ligands required for the increased selectivity are the size and shape of the particles. In the medical field, it is desired to achieve the selective delivery of drugs to specific areas in the body to increase the efficiency of drugs and decrease their side effects. Each drug, in addition to benefits, also offers some side effects. This kind of problem is commonly encountered in drugs used for the treatment of

195

FIGURE 6.16 (a) TEM image shows the presence of fine thin square Au NPs synthesized in the presence of 20 mM C14E8. (b) Shows a high resolution image of Au NPs lying one above the other bound with {100} lattice planes. (c) and (d) Show the TEM images of low and high magnifications of the liquid crystalline phase of T904 in the presence of 20 mM SDS. (e) Shows the high resolution image of the lattice planes of 2
3 nm Au NPs embedded in the liquid crystalline phase. (f) Show the TEM images of several pentagonal/hexagonal and triangular shaped NPs which are produced in the presence of 20 mM HTAB.

6.2 Biomedical Applications

cancer chemotherapy. Similarly, cytotoxic compounds can kill not only target cells but also normal cells in the body. Stimuli-responsive polymers or smart or intelligent polymers are those that respond sharply to small changes in physical or chemical conditions with relatively large phase. Below Cp, cloud point, the copolymer dissolves in water. These polymeric delivery systems respond to even small changes in temperature due to their tendency to undergo sol
gel transitions near body temperature and, therefore, controlling the release rate of loaded drugs while maintaining their physiochemical stability and biological activity. Poloxamers and poloxamine nonionic surfactants are approved by the FDA to be used as drug carriers in parenteral systems, food additives, and pharmaceutical ingredients, and have diverse applications in various biomedical fields ranging from drug delivery and medical imaging to management of vesicular diseases and disorders. The PPO block of the copolymers undergoes hydrophobic interactions with the hydrophobic surfaces of nanospheres. This kind of adsorption results in free movement of PEO side arms, which also causes steric repulsions. The extent of adsorption depends on both the size of PEO and PPO blocks, as well as the type of interactions present, such as NP surface charge, hydrogen bond between PEO unit, and the constituent groups on the particle surface. These kinds of engineered NPs exhibit reduced adsorption of proteins and blood as compared to the uncoated NPs and hence resist ingestion by phagocytic scavenger cells (Moghimi et al., 1993; Li and Caldwell, 1996). Poloxamer micelles are composed of a PPO core and PEO corona. Hence, they permits encapsulation of a large number of hydrophobic compounds, as a result of which they have full potential to be used as drug- and gene-delivery systems (Kabanov et al., 2002). Because of their self-assembled nature, emulsifying properties, as well as their biocompatible nature, pluronics have been used for biomedical and pharmaceutical applications and also in the development of drugdelivery systems (Fusco et al., 2006; Kabanov et al., 2002). Pluronic formulations have shown enhancement in cytotoxic activity of chemotherapeutic drugs like dexorubin toward multidrug-resistant (MDR) cancer cells (Batrakova et al., 1999a,b; Alakhova and Kabanov, 2014; Alakhov et al., 1996). Also, these formulations have the potential to modify the sensitivity of MDR cancer cells and lead to an increase in drug transport across the cell membrane. This tendency is directly proportional to the number of PEO and PPO units. It is observed that these block copolymers have 40 PPO units and an adjacent PEO segment consisting of at least 70 PEO units (e.g., Poloxamine-908, Poloxamine-1508, Poloxamer-407). Such engineered NPs are observed to remain in the systemic circulation for prolonged periods when injected intravenously into mice, rats, and rabbits. These nanovehicles are promising candidates for various applications such as medical imaging and drug delivery for controlled release of therapeutic materials. Because of the tendency of some poloxamers, for example, Poloxamer-407, to be converted to gel form, they have potential to be used for the slow release of peptides and therapeutic proteins, which include interleukin-2, urease, and human

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growth hormone (Morikawa et al., 1987; Fults and Johnson, 1990; Katakana et al., 1997). When injected into the body, the gel slowly dissolves and slowly releases the entrapped protein molecules over a period of 1
2 days and a substantial fraction is removed from the body in the form of renal excretion. Poloxamer-407 has also been used as an artificial skin for the treatment of third-degree burns because of its bactericidal properties (Nalbandian et al., 1987). Owing to its surfactant nature, it also cleans the wound of tissue detritus. Another major problem in chemotherapy is the development of drug resistance (Van Even and Konings, 1997; Germann, 1993). Block copolymers can be used for the treatment of such problems. However, this activity depends on hydrophilic
lipophilic balance (HLB) properties and the size of the poloxamer molecules. It is suggested that the greater the size of the PPO segment, the more effective will be the block copolymer. This is due to the interaction of the PPO block with the cell membrane (Batrakova et al., 1999a,b). Therefore the use of these block copolymers in pharmaceutical formulations results in increased drug bioavailability as well as drug accumulation in selected organs (e.g., the brain) and might overcome the problem of drug resistance, which limits the effectiveness of many therapeutic reagents. Nearly 70% of the drug molecules coming from the synthesis have solubility problems, while nearly 40% of newly developed active pharmaceutical ingredients (API) are rejected in early-phase development. The surface-active nature, low toxicity, and minimal immune response make pluronics a promising candidate for controlled drug delivery. Due to the improved bioavailability, low drug degradation makes them the topic of research. Due to the presence of PPO blocks in the micellar core, these micelles serve as the reservoirs for hydrophobic drugs. Due to low CMC values, these micelles have greater thermodynamic and kinetic stability. Two types of routes, that is, physical and chemical, can be used to keep the drugs in the hydrophobic core of the micelle. Nanocrystallization is the process to enhance the bioavailability of poorly soluble drugs and drug association rate. Drugs are available in the submicron size range with a crystalline API core covered by a stabilizer layer. It offers the following advantages: 1. improved bioavailability, 2. high drug loading, and 3. potential for targeted drug delivery. The selection of the stabilizer is a key factor. A limited number of stabilizers in the nano range are being used, that is, polymeric stabilizers (Lindfors et al., 2008; Lee et al., 2008), pluronics (Lai et al., 2009; Xiong et al., 2008), surfactant stabilizers, etc. Due to the difference in the physiochemical properties, each API needs a specific stabilizer. Indometacin is a water-insoluble hydrophobic drug. It is used as a model drug to study the interactions of poloxamers (L64, F-68), reverse poloxamers (17R4), and poloxamines (T908, T1107). Surface plasmon resonance and contact angle techniques are used to study the interaction of polymers with the drug using the wet ball milling technique.

6.3 Study Results

6.3 STUDY RESULTS These studies concluded that a good stabilizer must fulfill the following requirements: 1. It should firmly attach to the surface. 2. The polymer should properly coat the particle. Compared to their linear counterparts, that is, pluronics, poloxamines have been neglected for a long time. However, these are the potential candidates for drug delivery and tissue engineering. Tetronics can stabilize DNA (Pitard metal) and hence negatively charged DNA-poloxamine supramolecular assemblies were used in a gene-delivery system for the therapy of skeletal and heart musclerelated diseases. Poloxamine has also been employed in the form of coating over the hydrophobic NPs in order to prolong the particles’ blood circulation time and render them potential drug-delivery systems for medical and pharmaceutical purpose. The molecular weight and composition of the PEO
PPO are the deciding factors for the coating efficiency. It is observed that with an increase in the hydrophilicity and molecular weight, the shielding effect becomes more important. Tetronic T304 is a promising drug carrier and an effective transport inhibitor for the treatment of MDR tumors. Similarly, aqueous micellar solutions of Tetronic T904 micelles have been employed to solubilize quercetin (QN), a hypolipidemic drug, at different salt concentrations, pHs, and temperatures. It is the special architecture of tetronics that the pH, temperature, and ionic strength of the medium strongly influence the solution behavior (Nivaggioli et al., 1995). The solubility of drug in the micellar system was examined by a UV
visible spectrophotometer. The micellar size was determined using DLS (dynamic light scattering) and the possible locus of the drug molecule in the micellar aggregate was estimated from two-dimensional nuclear overhauser effect spectroscopy. Theoretical studies suggest that, as the molecular weight of the copolymer increases, micellar size also increases. As the concentration of T904 increases, the number of micelles also increases and also the solubility of QN increases. Most of the drugs are usually salts for pH buffering and ionic strength balance, QN tends to influence the behavior of T904 in aqueous medium. It is due to the presence of the 2-degree amine group in tetronics that they show pH and thermoresponsive behavior. When tetronics T904 is compared with P84, which have the same percentage of PEO block, that is, 40%, it is observed that QN is much less soluble in T904 at low pH but as the pH is increased up to 6, due to its ionization, a drastic increase in solubility is observed, while in P84, up to 3 mM of QN is soluble at low pH but with an increase in pH, no drastic increase in solubility is observed. It is due to the absence of pH-sensitive moieties in pluronics. That is why Pluronics P85 shows considerable solubility even at low pH and with an increase in pH, solubility increases due to dehydration. However, this increase is not as drastic compared to that in T904.

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The enhanced solubility of QN in T904 at high pH is due to both hydrophobic and electrostatic interactions. At low pH, T904 gets protonated, which results in Coulombic repulsions and hence micellization does not occur. At low pH, QN molecules interact with T904 unimers. But at high pH, micellization occurs. The QN gets solubilized in these micelles. Above pH 10, the phenolic
OH group of QN gets dissociated (at position 17,19) and results in the formation of a mixture of neutral, anionic species, which indicates the existence of both hydrophobic and electrostatic interactions that result in a dramatic increase in the QN solubility. However, the drug-loaded micelles suffer from thermodynamic stability, that is, with dilution, these drug carrier micellar systems become unstable in nature. This situation holds for very hydrophilic block copolymers having high CMC/ CMT values. Several strategies have been employed to overcome this shortcoming, for example, formation of an interpenetrating network via light-initiated crosslinking of a tetra-functional acrylate monomer. Another approach involves conversion of the
OH group into aldehyde and introducing mine linkages via the addition of diamine (Yang et al., 2007). Pluronics are also used for the surface coating of the drug-loaded hydrophobic NPs to enhance the blood circulation time of the drug carriers. The size and properties of the coated surfaces are deciding factors for the site of deposition within the body is observed. The size and properties of the coated surfaces are very important deciding factors for the effective absorption of drug within the body. Effective enhancement of serum life time has been achieved for polymeric NPs in a size ranging from 70 to 200 nm. Several investigations have been done on the solubilization and delivery of hydrophobic drugs using block copolymer micelles (Kadam et al., 2009, 2011; Parekh et al., 2011; Barreiro-Iglesias et al., 2005; Bae et al., 2007; Oh et al., 2005; Bhattacharya et al., 2010), for example, carbamazepine and hydrochlorothiazide have been solubilized using plutonic micelles (Kadam et al., 2009, 2011). Another study involved the solubilization of nimesulide using star block four-armed PEO
PPO
BCP (block copolymer) and concluded that the solubility increases with temperature and pH, while it decreased in the presence of added salt. Pluronics such as P103, P104, and P105 have also been used for the solubilization of the nimesulide drug. It is observed that CMC is the major thermodynamic parameter for deciding the micellar stability. Micelles below the CMC values in the body fluids disintegrate and the drug is released into the external media (Jones and Leroux, 1999). CMC and CMT are vital parameters for the application of block copolymer micelles in a controlled drug-delivery system. The CMC and CMT values of pluronics can be determined by UV
visible measurement and they indicate that they form stable micelles that remain intact on dilution. It is observed that the entrapment of drugs in these nano-sized core
shell micelles results in increased bioavailability as well as improvement of membrane transportation. The greater the hydrophobicity of the pluronics, the greater will be the solubility of the drug.

6.3 Study Results

Due to the biodegradable and biocompatible nature of NPs from pluronics, they are employed in various biomedical applications. Also, they undergo phase separation in the concentration above CMC, resulting in micelles with cargo space in the core for lyophilic drugs and hydrated exterior for stabilizing the micelles. There are several advantages to employing drug-incorporated micellar systems compared with traditional drug formulations, such as reduction of multidrug resistance, increase of bioavailability, and targeted drug delivery. There are several factors that control the pharmaceutical properties of the NPs: 1. 2. 3. 4. 5. 6.

the miscibility of drug and core-forming block; the physical state of the micellar core; the amount of the loaded drug; the molecular volume of the drug; the length of the core-forming block; and localization of the drug within the micelle (Allen et al., 1999).

To improve the low encapsulation capacity, high PDI, and large size of pure F-68 NPs, mixed micelles of F-68 with polycaprolactone (PCL) derivative have been synthesized. The PCL derivatives, including PCL homopolymers, triblock, pentablock, and spherical NPs, are synthesized. Of all the given PCL derivatives, the pentablock-based particles in both pure ratio (2:1 molar ratio of PCL to PPO in the core) and mixed ratio (1:1 molar ratio of PCL to PPO in the core) forms displaying minimum crystallinity, gave the best results. They were found to have the best drug encapsulation, most optimized particle size, better PDIs, and finally faster mono-mechanism releases. These investigations also indicated the role of the core-compatibilizer molecule for the miscibilization of the PCL and PPO in the hydrophobic part of the mixed micelles. Currently, micelles of Pluronics L61 and F-127 loaded with dexorubin are tested for treatment of cancer cells. Similar investigations have been done using paclitaxel as an anticancer agent. These formulations have shown improved pharmokinetic properties and enhanced blood circulation time. In addition to being a drug carrier system, they are also known to penetrate the blood
brain barrier (Kabanov et al., 2003) as well as a potential candidate for gene therapy to increase the efficiency of the gene transfer technologies. Compared to pluronics, at fixed PEO/PPO ratio, tetronics show unique properties, such as better penetrability power, more soluble in water, and higher interfacial activity. The aggregation behavior of tetronics can be altered by changing the block sequence or temperature. In case of Tetronics T1107, the hydrophilic PEO blocks are present at both ends and result in a “brush”-like adsorption model, whereas with that for T904, we get an “umbrella.” With the increase in temperature, the PPO blocks progressively lose their hydration layer and hence results in the compact packing arrangement. Such studies help in the development of pharmaceutical formulations as well as for controlled drug delivery.

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Tetronics are nowadays also used as the matrix to produce nanocomposite hydrogels. Such materials are emerging as an attractive concept to craft materials with tailored properties such as optical, electronic, mechanical, as well as promoting a specific biological function. Tetronic T1107 has been used to study the phase behavior using various techniques such as SANS (small-angle neutron scattering), DLS, and FTIR (Fourier-transform infrared spectroscopy)-IR (infrared) spectrum. At low concentration of T1107, spherical micelles are formed, but at a high concentration, it forms gels. As this process initiates, the shell of the micelles gets dehydrated and long range bcc order is revealed. However, in the presence of BT NPs (barium titanate), that are modified with CD (cyclodextrin), the sol
gel transition (temperature decrease from 25 C to 12 C) as well as broadening of the gel phase region is observed. The presence of NPs does not disturb the bcc arrangement of the micelles in the gel. The low pH hinders the formation of gel, hence the gel phase can be readily used for biomedical applications. Polymeric micelles are appealing Trojan horses to use for water-soluble hydrophobic drugs formulated for oral administration (Sosnik et al., 2008). Mixed micelles formed by the comicellization of two amphiphiles displaying different HLB have become an effective approach to optimize the encapsulation performance of poorly water-soluble drugs and the physical stability of the system (Oh et al., 2004; Wei et al., 2009). Nowadays, developing novel poorly water-soluble anti-HIV drugs and preparing highly concentrated aqueous formulations of EFV is the main goal to improve the pharmacotherapy of the pediatric population (Sosnik, 2010). It is observed that the comicellization of poloxamine/poloxamer polymeric micelles, that is, T904/F-127, enhances the encapsulation capacity as well as the physical stability. Such drug-loaded nanocarriers are sufficient in size to ensure the appropriate absorption in the intestine after oral administration. In such systems, F-127 micelles play the role of micellar templates and then the incorporation of T904 makes the system more hydrophobic. These mixed micelles show synergistic behavior. It was observed that the mixed micelles are more stable than pure poloxamer/poloxamine micelles. Such mixed micelle behavior offers the following advantages: 1. The greater encapsulation capacity of Tetronic T904. 2. The greater physical stability of poloxamer F-127, which helps to prepare a more concentrated and stable aqueous solution. Such systems help to prepare scalable and cost-effective PEO
PPO polymeric micelles to finely tune not only the encapsulation performance but also the size of the drug-loaded aggregate.

6.4 FUTURE PERSPECTIVES This chapter introduces the applications and uses of generally nontoxic and environmentally friendly pluronics and tetronics for the synthesis,

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7777. Sardar, R., Funston, A.M., Mulvaney, P., Murray, R.W., 2009. Gold nanoparticles: past, present, and future. Langmuir 25, 13840. Sau, T.K., Murphy, C.J., 2004. Room temperature, high-yield synthesis of multiple shapes of gold nanoparticles in aqueous solution. J. Am. Chem. Soc 126, 8648
8649. Sau, T.K., Pal, A., Jana, N.R., Wang, Z.L., Pal, T., 2001. Size controlled synthesis of gold nanoparticles using photochemically prepared seed particles. J. Nanopart. Res 3, 257
261. Schmid, G. (Ed.), 2006. Nanoparticles: From Theory to Application. Wiley-VCH. Smith, D.J., Long, A.K.P., Wallenberg, L.R., Bovin, J.O., 1986. Dynamic atomic-level rearrangements in small gold particles. Science 233, 872
875. Solee-Illia, G.J., de, A.A., Crepaldi, E.L., Grosso, D., Sanchez, C., 2003. Block copolymertemplated mesoporous oxides. Curt. Opin. Cooled Interface Sci. 8 (1), 109
126. Sosnik, A., 2010. Nanotechnology contributions to the pharmacotherapy of paediatric HIV: a dual scientific and ethical challenge and a still pending agenda. Nano Med. 5, 833
837. Sosnik, A., Carcaboso, A.M., Chiappetta, D.A., 2008. Polymeric nano carriers: new endeavors for the optimisation of the technological aspects of drugs. Recent Pat. Biomed. Eng. 1, 43
59. Sperling, R.A., Gil, P.R., Zhang, F., Zanella, M., Parak, W.J., 2008. Biological applications of gold nanoparticles. Chem. Soc. Rev. 37, 1896. ´ ez, A., Wulff-Pe´rez, M., Ga´lvez-Ruiz, M.J., Martı´n-Rodrı´guez, A., Torcello-Gom Cabrerizo-Vı´lchez, M., Maldonado-Valderrama, J., 2014. Block copolymers at interfaces: interactions with physiological media. Adv. Colloid Interface Sci 206, 414
427. Van Even, H.W., Konings, W.N., 1997. Multi drug transport from bacteria to man: similarities in structure and function. Semin. Cancer Biol. 8, 183
191. Wang, L., Chen, X., Zhan, J., Sui, Z., Zhao, J., Sun, Z., 2004. Controllable morphology formation of gold nano- and micro-plates in amphiphilic block copolymer-based liquid crystalline phase. Chem. Lett. 33 (6), 720
721. Wei, Z., Hao, J., Yuan, S., Li, Y., Juan, W., Sha, X., et al., 2009. Paclitaxel-loaded Pluronic P123/F127 mixed polymeric micelles: formulation, optimization and in vitro characterisation. Int. J. Pharm. 376, 176
185.

Further Reading

Wiley, B., Sun, Y., Chen, J., Cang, H., Li, Z.Y., Li, X., et al., 2005. Silver and gold nanostructures with well-controlled shapes. MRS Bull. 30, 356
361. Xia, Y., Xiong, Y., Lim, B., Skrabalak, S.E., 2009. Shape-controlled synthesis of metal nanocrystals: simple chemistry meets complex physics? Angew. Chem. Int. Ed. 48, 60
103. Xie, J.P., Lee, J.Y., Wang, D.I.C., 2007. Synthesis of single-crystalline gold nanoplates in aqueous solutions through biomineralization by serum albumin protein. J. Phys. Chem. C. 111, 10226
10232. Xiong, R., Lu, W., Li, J., Wang, P., Xu, R., Chen, T., 2008. Preparation and characterisation of intravenously injectable nimodipine nanosuspension. Int. J. Pharm. 350 (1
2), 338
343. Xu, J., Li, S., Weng, J., Wang, X., Zhou, Z., Yang, K., et al., 2008. Adv. Funct. Mater. 18, 277
284. Yang, L., Alexandridis, P., 2000. Physicochemical aspects of drug delivery and release from polymer-based colloids. Curt. Opin. Colloid Interface Sci 5, 132
143. Yang, B., Guo, C., Chen, S., Ma, J.H., Wang, J., Liang, X.F., et al., 2006. Effect of acid on the aggregation of poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) block copolymers. J. Phys. Chem. B 110, 23068. Yang, T.F., Chen, C.N., Chen, M.C., Lai, C.-H., Liang, H.F., Sung, H.W., 2007. Shellcrosslinked pluronic L121 micelles as a drug delivery vehicle. Biomaterials 28, 725
734. Yusa, S.I., Fukuda, K., Yamamoto, T., Ishihara, K., Morishima, Y., 2005. Synthesis of well-defined amphiphilic block copolymers having phospholipid polymer sequences as a novel biocompatible polymer micelle reagent. Biomacromolecules 6, 663
670. Zhang, J.M., Ma, F., Xu, K.W., 2004. Calculation of the surface energy of FCC metals with modified embedded-atom method. Appl. Surf. Sci. 229, 34
42. Zhao, D., Feng, J., Huo, Q., Melosh, N., Fredrickson, G.H., Chmelka, B.F., et al., 1998. Triblock copolymer synthesis of mesoporous silica with periodic 50 to 300 angstrom pores. Science 279 (23), 548
552. Zou, X., Ying, E., Dong, S., 2006. Seed-mediated synthesis of branched gold nanoparticles with the assistance of citrate and their surface-enhanced Raman scattering properties. Nanotechnology 17, 4758
4764. Zou, P., Suo, J.P., Nie, L., Feng, S.B., 2012. Temperature-sensitive biodegradable mixed star-shaped block copolymers hydrogels for an injection application. Polymer 53, 1245
1257.

FURTHER READING Alkilany, A.M., Nagaria, P.K., Hexel, C.R., Shaw, T.J., Murphy, C.J., Wyatt, M.D., 2009. Cellular uptake and cytotoxicity of gold nanorods: molecular origin of cytotoxicity and surface effects. Small 5 (6), 701
708. Bromberg, L., 2005. Intelligent hydrogels for the oral delivery of chemotherapeutics. Expert Opin. Drug Delivery. 2, 1003
1013. Goy-Lopez, S., Taboada, P., Cambon, A., Juarez, J., Lorenzo, A.C., Concheiro, A., et al., 2010. Modulation of size and shape of Au nanoparticles using amino-X-shaped poly(ethylene oxide)-poly(propylene oxide) block copolymers. J. Phys. Chem. B 114, 66
76.

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Khi, S., Hassanzadeh, S., Goliaie, B., 2007. Effects of hydrophobic drug-polyesteric core interactions on drug loading and release properties of poly(ethylene glycol)-polyesterpoly(ethylene glycol) trip block core-shell nanoparticles. Nanotechnology 18 (17). Available from: https://doi.org/10.1088/0957-4484/18/17/175602. Lim, B., Jiang, M., Tao, J., Camargo, P.H.C., Zhu, Y., Xia, Y., 2009. Shape-controlled synthesis of Pd nanocrystals in aqueous solutions. Adv. Funct. Mater. 19, 189
200. Moschwitzer, J.P., 2013. Drug nanocrystals in the commercial pharmaceutical development process. Int. J. Pharm. 453 (1), 142
156. Muller, R.H., Gohla, S., Keek, C.M., 2011. State of the art of nanocrystals—special features, production, nanotoxicology aspects and intracellular delivery. Eur. J. Pharm. Biopharm. 78 (1), 1
9. Rabinow, B.E., 2004. Nanosuspensions in drug delivery. Nat. Rev. Drug Discov. 3, 785
796. Weinheim, Shields, S.P., Richards, V.N., Buhro, W.E., 2010. Nucleation control of size and dispersity in aggregative nanoparticle growth. a study of the coarsening kinetics of thiolate-capped gold nanocrystals, Chem. Mater., 22. p. 3212. Zhang, C., Zhang, J., Han, B., Zhao, Y., Li, W., 2008. Synthesis of icosahedral gold particles by a simple and mild route. Green Chem. 10, 1094
1098. Zia, Y., Xiong, Y., Lim, B., Skrabalak, S.E., 2009. Shape-controlled synthesis of metal nanocrystals: simple chemistry meets complex physics? Angew. Chem. Int. Ed. 48, 60.

CHAPTER

Nanoparticles: synthesis and applications

7

Nguyen Hoang Nam1,2 and Nguyen Hoang Luong2 1

Faculty of Physics, Hanoi University of Science, Vietnam National University, Hanoi, Hanoi, Vietnam 2Nano and Energy Center, Hanoi University of Science, Vietnam National University, Hanoi, Hanoi, Vietnam

7.1 INTRODUCTION Nanoparticles are defined by the worldwide federation of national standards bodies, the International Organization for Standardization (ISO), as nanoobjects with all external dimensions in the nanoscale, where the lengths of the longest and shortest axes of nanoobjects do not differ significantly (ISO/TS 80004-2:2015). Though nanoscale is basically ranged from 1 to 100 nm, nanoparticles can be categorized by three size ranges: larger than 500 nm, between 100 and 500 nm, and between 1 and 100 nm (European Commission, 2010). With respect to the size and the size distribution, nanoparticles may exhibit size-related intensive properties. If they are small enough to confine their electrons, they produce quantum effects and exhibit unexpected properties, for example, gold nanoparticles appear red in solution (see, for instance, Eustis and El-Sayed, 2006), and melt at much lower temperatures than that in slab form (Buffat and Borel, 1976). The high surface-area-to-volume ratio of nanoparticles provides the significant changes in properties related to contact/surface area, such as catalytic (Astruc, 2008), surface-enhanced plasmon resonance (Melaine et al., 2015), etc. Depending on the composition and structure, nanoparticles can be of single properties such as metallic, dielectric, semiconductor, magnetic, or multifunctional which include more than one feature from single-property nanoparticles. Their applications, or potential applications, are in many different fields (Salata, 2004; Mody et al., 2010; Lu et al., 2007; Zhang et al., 2008; Nguyen et al., 2015; and references therein). Among those, the advantages of nanoparticles in applications in life sciences and the environment are due to the fact that their size is comparable with the dimensions of objects such as viruses (about 10
100 nm) or cells (about 1
10 μm). This gives nanoparticles an ability to attach to biological entities without changing their functions, while the high surface-area-to-volume ratio of

Materials for Biomedical Engineering: Inorganic Micro- and Nanostructures. DOI: https://doi.org/10.1016/B978-0-08-102814-8.00008-1 © 2019 Elsevier Inc. All rights reserved.

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nanoparticles permits strong bonds with surfactant molecules. In environmental applications, the specific features (small size, large surface area) of nanoparticles can provide a tool for very sensitive detection of a specific contaminant from the presence of which pollution often arises. The engineering of nanoparticles can also offer opportunities to treat environmental contamination. In this chapter we focus on the synthesis, functionalization, and applications of metallic, semiconductor, magnetic, and multifunctional nanoparticles. Compiling all the literature would greatly exceed the scope of this work, instead, we present typical and representative examples for discussion on the synthesis, functionalization, and applications of those nanoparticles.

7.2 SYNTHESIS OF NANOPARTICLES 7.2.1 CHEMICAL REDUCTION Chemical reduction is an effective wet-chemical method for making zero-valent nanoparticles based on chemical-reducing aqueous salts of metals, such as silver nitrate (AgNO3) in the case of synthesis of silver nanoparticles, for instance. To reduce the precursor metal salt, at least one reducing agent is used to produce electrons for metal ions that reduce them to become zero-valent. Commonly used reductants are borohydride, citrate, and ascorbate. Reduced nanoparticles are stabilized by a stabilizing agent. An example of a stabilizing agent is cetyltrimethylammonium bromide [(C16H33)N(CH3)3Br; CTAB], which is widely used in gold nanoparticle synthesis. The stabilizing agents can be reducing agents themselves, such as citrate of sodium in making silver nanoparticles (Shenava Aashritha, 2013). For more details the reader is referred to the review paper by Alaqad and Saleh (2016) and references therein.

7.2.2 COPRECIPITATION Precipitation is the carrying down by a precipitate of soluble substances under certain conditions (Patnaik, 2004). Generally, when the concentration of substances reaches supersaturation, a nucleation suddenly appears in solution. The nucleation will be grown by the diffusion on to its surface which then becomes nanoparticles. During the growth, the nucleation needs to be slowed down in order to get uniform nanoparticles. Several methods can be listed as precipitation: coprecipitation, microemulsion/inverse microemulsion, polyol, etc. Coprecipitation is a convenient way to synthesize Fe3O4 nanoparticles (Lu et al., 2007; Quy et al., 2013; Dung et al., 2016; Khalil, 2015; Mascolo et al., 2013). The mixture of two chloride salts of FeCl2 and FeCl3 with 1:2 molar ratios of Fe21/Fe31 was vigorously stirred and kept at 70 C before NH4OH was added resulting in the black color precipitation. The Fe3O4 nanoparticles were collected after purifying through magnetic separation with ethanol and distilled water

7.2 Synthesis of Nanoparticles

several times to decontaminate the residual chemicals. By modifying the pH and ion concentration in solution, the size of nanoparticles can be controlled.

7.2.3 SEEDING The seeding method was discovered by Frens (1972, 1973), where nanoparticles are grown by the reduction of salt in aqueous solution which contains seed nanoparticles. In this method, the stabilizers are used to control the size and shape of growing nanoparticles. This method was developed over the years using various types of seeding, reductant agents, and stabilizers (Xu et al., 2007; Han et al., 2009; Perrault and Chan, 2009; Ziegler and Eychmuller, 2011; Rioux and Meunier, 2015). For example, the method which was developed by Perrault and Chan in 2009, reduces HAuCl4 in gold nanoparticles seed-contained aqueous solution using hydroquinone. The gold nanoparticle seed can act in conjunction with hydroquinone to catalyze the reduction of gold ions into their surface. If the stabilizer is citrate, typically the seed nanoparticles were prepared by the citrate method. Using this method, the size of nanoparticles can be grown at least 30
300 nm.

7.2.4 MICROEMULSION AND INVERSE MICROEMULSION Microemulsion is a popular method to synthesize nanoparticles, where microemulsions are an isotropic and thermodynamically stable mixture of “oil,” water, and surfactant, or in combination with a cosurfactant. The basic types of microemulsions are direct (oil dispersed in water) and reverse (water dispersed in oil). The small drops of aqueous phase (micelles) may contain salts and/or other ingredients, and the “oil” may actually be the mixture of the surfactants. The reaction to form nanoparticles also can be realized when the micelles mix with each other and the growth of the nanoparticles is controlled by the surfactants in “oil” (Lopez-Quintela and Rivas, 1993). Using this method, the Fe3O4 nanoparticles can be synthesized (Feltin and Pileni, 1997) and also can be functionalized with a silica layer. This method can also be used to prepare Fe3O4/Au core
shell nanoparticles to prevent the oxidation of magnetic nanoparticles (MNPs) as well as form the biocompatibility (Boutonnet et al., 1982). Furthermore, the inverse microemulsion method is the simplest way to produce multifunctional nanoparticles by creating the silica matrix in aqueous phase, which will fix the single particles inside (Dung et al., 2016).

7.2.5 HYDROTHERMAL METHOD In the hydrothermal method, the crystals of nanoparticles are grown by heterogenous reaction under conditions of high temperature and high pressure from substances which are insoluble at normal temperature and pressure (Byrappa and Adschiri, 2007). The crystal growth is carried out in an apparatus consisting of a

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steel pressure vessel called an autoclave, in which nutrients are supplied along with water. Hydrothermal synthesis is usually carried out below 300 C. The critical condition gives a favorable reaction field for formation of nanoparticles, owing to the enhancement of the reaction rate and large supersaturation based on the nucleation theory. This method has been used to synthesize metal oxide nanoparticles in supercritical water (Hayashi and Hakuta, 2010), metal nanoparticles (Kim et al., 2014), and semiconductor nanoparticles (Bui et al., 2014; Hoa et al., 2011; Williams et al., 2007).

7.2.6 SONOELECTRODEPOSITION Sonoelectrodeposition is a useful synthesis method for nanoparticles and has been successfully applied to prepare metallic nanoparticles such as FePt and CoPt (Luong et al., 2011; Nam et al., 2012; and references therein). Sonoelectrodeposition is a technique combining the advantages of electrodeposition and mechanical waves of ultrasound to produce metallic nanoparticles (Zhu et al., 2000). In Section 7.4 we discuss silver nanoparticles. One of main disadvantages of the conventional synthesis methods for silver nanoparticles, including chemical reduction, is the presence of unexpected toxic ions in the final products. The toxic ions in the product are mostly the ions of the silver precursor, such as nitrate and thiolsulfate. A good silver precursor such as silver acetate can be used (Irzh et al., 2007), however, this chemical is expensive and manipulation is difficult under ambient conditions. Tuan et al. (2011) reported a modified sonoelectrodeposition technique to obtain silver nanoparticles in a nontoxic solution. The modification is that a silver plate was used as the cathode instead of silver salts thus allowing the avoidance of unexpected ions from the salts.

7.3 FUNCTIONALIZATION/COATING OF NANOPARTICLES 7.3.1 FUNCTIONALIZATION OF NANOPARTICLES Functionalization of nanoparticles can be defined as the addition of a chemical functional group on their surface in order to achieve surface modification that enables their self-organization and renders them compatible (Subbiah et al., 2010). The most widely used functional groups are amino, biotin, steptavidin, carboxyl, and thiol groups (Bruce and Sen, 2005). The main purpose of functionalizing nanoparticles is to cover their surface with a molecule that possesses the appropriate functionality needed for the designed application. For many biomedicine applications, nanoparticles need to be functionalized in order to conjugate with biological entities such as DNA, antibodies, and enzymes. For more details on the functionalization of nanoparticles, its methods, and class, as well as its implications in biomedical sciences, the reader may be referred to, for instance, the review by Subbiah et al. (2010).

7.3 Functionalization/Coating of Nanoparticles

We focus here on the functionalization of gold nanoparticles and MNPs discussed in Section 7.4. For application in detecting breast cancer cells, gold nanoparticles synthesized by a chemical reduction were functionalized with 4-aminothiolphenol (4-ATP, sometimes called p-aminothiolphenol [PATP]). For basal cell carcinoma (BCC) detection, different amounts of 4-ATP solutions were added to gold nanoparticles coated by CTAB. CTAB on the surface of gold nanoparticles was replaced by 4-ATP to form gold nanoparticles functionalized with 4-ATP (Au-4ATP). Fe3O4 nanoparticles were functionalized using 3-aminopropyl triethoxysilane (APTS). APTS is a bifunctional molecule, an anchor group by which the molecule can attach to free
OH surface groups. The head group functionality
NH2 is for conjugating with biological objects. The amino-NP is ready to conjugate with the DNA of the herpes virus and with the antiCD4 antibody.

7.3.2 SILICA COATING OF MAGNETIC NANOPARTICLES Maintaining the stability of MNPs for a long time without agglomeration or precipitation is an important issue (see, for instance, Lu et al., 2007). The protection of MNPs against oxidation by oxygen, or erosion by acid or base, is necessary. The common method is protection by a layer which is impenetrable, so that oxygen, for example, cannot reach the surface of the particles. It is noted that the stabilization and protection of particles are often closely linked with each other. One of the ways to protect MNPs is coating them with silica. A silica shell not only protects the magnetic cores, but can also prevent direct contact of the magnetic core with additional agents linked to the silica surface that can cause unwanted interactions. The coating thickness can be controlled by varying the concentration of ammonium and the ratio of tetraethylorthosilicate (TEOS) to H2O. The surfaces of silica-coated MNPs are hydrophilic, and are readily modified with other functional groups (Ulman, 1996). Quy et al. (2013) and Hieu et al. (2017) have prepared Fe3O4/SiO2 nanoparticles by coating MNPs with silica using TEOS.

7.3.3 MULTIFUNCTIONAL NANOPARTICLES Recently, multifunctional nanoparticles have gained wide attention due to their advantages in the goal of applications. Potentially, multifunctional nanoparticles which include individual physicochemical properties of nanoparticles, such as plasmonic metallic nanoparticles, photoluminesable semiconductor nanoparticles or quantum dots, and MNPs, can complement some of the limitations of conventional applications using single nanoparticles, particularly in biomedicine. For example, bifunctional nanoparticles which are composed of MNPs and metallic nanoparticles not only can be used as optical labels in bioimaging, diagnosis and therapy, but also allow some biomolecules to be tagged and separated, together with targeted drug delivery and magnetic resonance imaging under the induction of an external magnetic field (Cai et al., 2014; Sotiriou et al., 2011; Ilovitsh et al., 2015; Giani et al., 2012; Sun et al., 2006). Multinanoparticles can be in

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core
shell structures or in the complex structures which are a combination of at least two types of single nanoparticles. Core
shell structured nanoparticles can be classified into inorganic/inorganic, inorganic/organic, organic/inorganic, organic/organic, core/multishell, and movable core/hollow shell nanoparticles (Chaudhuri and Paria, 2012). The synthesis approaches to nanoparticles can be divided into top-down and bottomup methods. The top-down approaches often use externally controlled tools to cut, mill, and shape materials into the designed nanoscale structures, for example, lithography methods, laser beams, mechanical techniques. The bottom-up approaches exploit the chemical properties of the molecules to cause them to self-assemble to become nanoparticles, such as chemical synthesis discussed above. The bottom-up methods can produce much smaller nanoparticles and are cost-effective, compared to the top-down methods. Both methods are used in the synthesis of core
shell structured nanoparticles. However, since ultimate control is needed for achieving a uniform coating of the shell, the bottom-up approach has proven more suitable. A combination of the two methods can also be utilized, for example, core particles synthesized by the top-down method but then coated by the bottom-up approach in order to maintain precise shell thickness. In general, various methods were used to prepare core
shell nanoparticles. For example, to produce iron oxide@Ag core
shell nanoparticles, several methods were used including impregnation (Liu et al., 2012), surface functionalization followed by deposition (Liu et al., 2010), solvo-thermal reduction (Liu and Li, 2009), and chemical reduction (Hu et al., 2010; Sun et al., 2012). Reducing agents such as glucose and sodium borohydride are used for the reduction of silver salts, and the surface functionalization of iron oxide nanoparticles by different surface-modifying agents is required. Hu et al. (2010) used glucose for the reduction of Ag(NH3)21 to Ag, which is adsorbed onto the surface of silicacoated iron oxide which is prepared by the coprecipitation method. Liu et al. (2010) have reported the surface functionalization of Fe3O4 surface by APTS followed by reduction of AgNO3 using sodium citrate and sodium borohydride. Sun et al. (2012) have used sodium borohydride as the reducing agent for the reduction of Ag(NH3)21 to obtain Fe3O4@Ag core
shell nanoparticles. Dung et al. (2016) used ultrasound to assist in the reduction of silver ions following this strategy. Liu and Li (2009) have used dimethylformamide as the reducing agent during solvo-thermal synthesis of γ-Fe2O3@Ag microspheres. In parallel, one-step synthesis using thermal decomposition of silver acetate in the presence of iron oxide microspheres is also applicable and does not require the addition of any external reducing agent or surface modification of iron oxide (Sharma and Jeevanandam, 2013). To control the overall size and the shell thickness, a microemulsion method, where water droplets act as a template or nanoreactor, is preferable to a bulk medium. Other types of multifunctional nanoparticles are the complex structures of materials, which is the combination of at least two types of single nanoparticles. Similar to a core
shell structure, the nanomaterials used in complex structures

7.3 Functionalization/Coating of Nanoparticles

can be categorized by two types of nanoparticles: organic, which includes micelles, liposomes, nanogels, dendrimes, and inorganic, which includes magnetic, semiconductor, lanthanide, and metallic nanoparticles. The combination can be achieved in many ways, however it is needed to fulfill the requirements of the applications. For example, the multifunctional nanoparticles should have superparamagnetic properties in order to be applied in drug delivery and DNA separation, and they should have plasmonic properties in order to be applied as biolabeling agents, and they should also be biocompatible. The simplest combination which fulfills these requirements is the complex of Fe3O4 nanoparticles with superparamagnetic properties and Ag nanoparticles with plasmonic properties in a matrix of SiO2 which provide the biocompatibility and also improve the stability of Ag and Fe3O4 nanoparticles. Surface activator polyvinylpyrrolidone (PVP) was used to control the size of silver nanoparticles, which were synthesized by a wet-chemical reduction method with NaBH4 as reductant. The synthesized nanoparticles were coated with 4-ATP to form functionalized Ag4ATP nanoparticles. These functionalized nanoparticles were combined with the above-prepared Fe3O4 nanoparticles by an inverse microemulsion method to form multifunctional nanoparticles (Dung et al., 2014). In this method, the microemulsion was created by mixing the hydrophilic phase of the mixture of Ag-4ATP and Fe3O4 solution and the hydrophobic phase of toluene. The mixture of Ag-4ATP/Fe3O4 with different mass rates was moderated under a sonic bath for 2 hours, then TEOS was added to react with water in solution as in reaction (7.1). The formed SiO2 coating layer in amorphous conformation covers both initial particles. SiðOC2 H5 Þ4 1 2H2 O-SiO2 1 4C2 H5 OH

(7.1)

The multifunctional composites were also successfully prepared in a complex form using an ultrasound-assisted chemical method (Dung et al., 2017a). MNPs were firstly prepared by the coprecipitation method, then coated by a silica layer. The silica layer, after that, was modified by APTS. Silver ions were then absorbed on the surface of APTS-functionalized silica-coated MNPs. Under the ultrasonic wave of 200 W acting for 60 minutes these silver ions were reduced by sodium borohydride. In XRD characterization after synthesis, the relative intensity of diffraction peaks of silver crystals increases when the atomic ratio of silver to iron increases from 0.208 to 0.455. In parallel, all nanoparticles showed superparamagnetic properties with the saturation magnetization decreased from 44.68 to 34.74 emu/g with increasing silver:ion atomic ratio. The coexistence of strong surface plasmon absorption at 420 nm and these superparamagnetic properties make these particles promising for biomedical applications. In another way, MNPs can be directly functionalized with an amino group without coating by silica layer (Dung et al., 2017b). In this way, Fe3O4-ZnO multifunctional nanoparticles were successfully synthesized in aqueous solution by ultrasound-assisted thermolysis. The as-prepared Fe3O4 MNPs were modified by APTS to have free amine (2NH2) groups on their surface. Zn21 ions then were

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added and stirred to adsorb onto the surface of Fe3O4-NH2 nanoparticles in alkaline solution at pH 11. The solution was decomposed through thermolysis in an ultrasound bath. The characterization shows that photoluminescence of Fe3O4ZnO multifunctional nanoparticles was enhanced in visible light at a wavelength of 565 nm to allow detection, labeling, diagnosis, and therapy in biomedicine. Furthermore, they exhibit superparamagnetic properties of Fe3O4 with high saturation magnetization, which can be used for separation applications in biomedicine under an external magnetic field.

7.4 APPLICATIONS 7.4.1 APPLICATION OF GOLD NANOPARTICLES FOR BREAST CANCER CELL DETECTION Gold nanoparticles are promising candidates for cell imaging and tumor-targeted drug delivery (Sokolov et al., 2003; Paciotti et al., 2004; Jain, 2005), breast cancer diagnosis, and targeted therapy (Yezhelyev et al., 2006). Being a member of the epidermal growth factor receptor tyrosine kinase family, HER2 is found to be overexpressed in 20%
30% of human breast cancers (Harries and Smith, 2002; and references therein). Therefore, HER2 is an interesting target for breast cancer therapies. Anti-HER2 (trastuzumab, trade name Herceptin) is a humanized monoclonal antibody (mAb) designed specifically for antagonizing the HER2 function. Quynh et al. (2011) and Nguyen et al. (2015) have synthesized gold nanoparticles by a chemical reduction then applied them for imaging KPL4 breast cancer cells after conjugating them with trastuzumab. Fig. 7.1 shows the bright-field and dark-field microscopy images of breast cancer cells after being incubated with gold nanoparticles nonconjugated with trastuzumab as well as conjugated with trastuzumab (Nguyen et al., 2015). As can be seen from Fig. 7.1, when the gold nanoparticles were not conjugated with trastuzumab, the dark-field image showed no signal of the gold nanoparticles (A2). When the gold nanoparticles were directly conjugated with trastuzumab, the gold nanoparticles were bound onto cancer cells and these cancer cells were clearly observed in the dark-field image (A4) through the scattering light from the gold nanoparticles. When the 4-ATP functionalized gold nanoparticles (amino-gold nanoparticles) were covalently conjugated with trastuzumab through l-ethyl-3-(3dimethylaminopropyl) ethylcarbodiimide (EDC) connection, the gold nanoparticles also concentrated on the cancer cells, but these cancer cells were observed with slightly lower intensity in the dark-field image (A6) compared to those in image A4. Nguyen et al. (2015) pointed out, however, that the gold nanoparticles directly conjugated with trastuzumab could be stored in a freezer for only about 2 weeks before they lost their activity, while the gold nanoparticles covalently conjugated with trastuzumab were stable with storage for about two months.

7.4 Applications

FIGURE 7.1 Bright-field (A1, A3, A5) and dark-field (A2, A4, A6) microscopy images of breast cancer cells after being incubated with gold nanoparticles nonconjugated with trastuzumab (A1, A2), the gold nanoparticles conjugated with trastuzumab (A3, A4) and the amino-gold nanoparticles covalently conjugated with trastuzumab through EDC connection (A5, A6). After Nguyen, H.L., Nguyen, H.N., Nguyen, H.H., Luu, M.Q., Nguyen, M.H., 2015. Nanoparticles: synthesis and applications in life science and environmental technology. Adv. Nat. Sci.: Nanosci. Nanotechnol. 6, 015008.

7.4.2 BASAL CELL CARCINOMA FINGERPRINTED DETECTION Skin cancer is the most common cancer in humans and its incidence is increasing (Baxter et al., 2012). Worldwide, BCCs constitute about 74% of skin cancer cases (NCIN, 2013). Among the treatments for “high-risk” BCCs (i.e., BCCs on the

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face and neck, or recurrent BCCs), Mohs micrographic surgery (MMS) is the most efficient (Mohan and Chang, 2014). This procedure helps to maximize the removal of tumor cells, while spares as much healthy tissue as possible. However, the need for a pathologist or specialized surgeons to diagnose frozen sections during surgery has limited the wider use of MMS, which leads to cases of inappropriate inferior treatment. Frozen-section histopathology also requires arduous and time-consuming procedures, increasing the costs compared to standard methods of BCC excision. For skin cancer diagnosis, Raman spectroscopic imaging is a promising technique, because of its high sensitivity to molecular and structural changes associated with cancer. However, raster scanning Raman mapping requires long times for data acquisition, typically days for tissue specimens of 1 cm 3 1 cm. Recently, multimodal spectral imaging based on Raman spectroscopy and tissue autofluorescence was used to reduce the BCC diagnosis time to only 30
60 minutes, which becomes suitable for use during MMS (Kong et al., 2013; Takamori et al., 2015). An alternative method that could allow to reduce data acquisition and BCC diagnosis times during MMS is surface-enhanced Raman spectroscopy (SERS). It was discovered that strongly increased Raman scattering signals can be obtained in the very close vicinity of metal nanostructures, which are mainly due to resonances between optical fields and the collective oscillations of the free electrons in a metal. Thus SERS has attracted great interest in the biolabeling field because significant enhancement of the labeling signals of molecular vibrations on the metallic nanoparticles surface can be obtained. Quynh et al. (2016) studied surface-enhanced Raman (SER) signal of 4-ATP that was linked to the surface of gold nanoparticles conjugated with skin carcinoma cell antibody BerEP4. Gold nanoparticles with sizes ranged from 2 to 5 nm were prepared by a wet-chemical method using CTAB. The Au-4ATP-antibody solutions were dropped on the surface of the tissue sample and the SER scattering signals were collected and analyzed. Fig. 7.2 shows the fingerprinted landscape of SER signals of Au-4ATPantibody on a BCC tissue. Fig. 7.2A shows the colored image of a Gram-stained tissue, where the cancer cell area may be the dark-colored regions, for example, region A1, A2. However, the result of diagnosis essentially depends on the subjective decision of the pathologists because this nonspecific method may lead to misinterpretation of noncancer regions as cancer ones. Fig. 7.2B shows a brightfield microscopy image of the tissue, where B1 region corresponds to a hair follicle position, and B2 does not, although B1 and B2 have the same position on the tissue as regions A1 and A2 in Fig. 7.2A. Fig. 7.2C shows the result of an SER signal obtained by the principal component analysis (Quynh et al., 2016). Fig. 7.2D shows the result of the SER signal analyzed using only the intensity of SER peaks at 1075 cm21. The Au-antibody colloids are oriented close to the BCC surface by the antigen
antibody coupling. The carcinoma sections act as a dock where a high concentration of Au-4ATP-antibody particles is distributed, then the SER peak intensity at 1075 cm21 will be higher in these areas. In

7.4 Applications

FIGURE 7.2 Fingerprinted landscape of SER signals of Au-4ATP-antibody on BCC tissue. (A) Image of Gram-stained BCC tissue where A1 and A2 are the areas of suspected BCC; (B) bright-field microscopy image of tissue, where regions B1 and B2 are in the same position on the tissue as regions A1 and A2, respectively; (C) SER signal landscape obtained by principal component analysis, where regions C1 and C2 are in the same position on the tissue as regions A1 and A2, respectively; (D) the fingerprinted landscape of intensity of SER peaks at 1075 cm21, where regions D1 and D2 are in the same position on the tissue as regions A1 and A2, respectively. The difference between D1 and D2 shows that only red-colored D2 (and the similar-colored area) are the infected area, while D1 is not. After Quynh, L.M., Nam, N.H., Kong, K., Nhung, N.T., Notinger, I., Henini, M., et al., 2016. Surfaceenhanced Raman spectroscopy study of 4-ATP on gold nanoparticles for basal cell carcinoma fingerprint detection. J. Electron. Mater. 45, 2563
2568.

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Fig. 7.2C, the colored areas such as C1 and C2 can be considered as cancer regions. However, in Fig. 7.2D the area D1 does not show the high intensity of the peak at 1075 cm21, while the others, such as the D2 area, indicate very high intensity of the peak at 1075 cm21. From Fig. 7.2, only A2, B2, C2, and D2 regions can be definitely considered as the cancer areas, while A1, B1, C1, and D1 may assigned as the position of hair follicles where the cell concentration is higher than in other parts. Quynh et al. (2016) pointed out that, while the whole SER map collecting time should be longer than 2 hours (the collecting time of each spectrum was nearly 5 seconds), the fingerprinted image using peak height at 1075 cm21 can be observed in around 5 minutes. Hence, this method may represent a solution for quick diagnosis, even during operation.

7.4.3 ANTIBACTERIAL TEST USING SILVER NANOPARTICLES Silver nanoparticles (AgNPs) are commonly utilized nanomaterials due to their antibacterial properties, high electrical conductivity, and unique optical properties that can be used in various applications (Sondi and Salopek-Sondi, 2004). It is believed that the high affinity of Ag toward sulfur or phosphorus is the key element of its antibacterial property. As sulfur and phosphorus are found in abundance throughout cell membranes, AgNPs react with sulfur-containing proteins inside or outside the cell membrane, which in turn affects cell viability (Pal et al., 2007; Elechiguerra et al., 2005). Another theory proposed that Ag1 ions released from AgNPs can interact with phosphorus moieties in DNA, resulting in inactivation of DNA replication, or can react with sulfur-containing proteins to inhibit enzyme functions (Sharma et al., 2009). These properties allow the incorporation of AgNP into various matrices such as activated carbon (AC), polymer networks, textiles, and wound dressing materials (Sedaghat and Nasseri, 2011). Many approaches have been developed to obtain AgNP of various shapes and sizes, including chemical reduction, laser ablation, gamma irradiation, electron irradiation, chemical reduction by inorganic and organic reducing agents, photochemical method, microwave processing, thermal decomposition of Ag oxalate in water and in ethylene glycol, and sonoelectrochemical method (see references in Tuan et al., 2011). As pointed out in Section 7.2.6, one of main disadvantages of those methods is the presence of unexpected toxic ions in the final products. Tuan et al. (2011) report a modified sonoelectrochemical technique to obtain AgNP in a nontoxic solution. The silver particles are then directly loaded on AC produced by thermal activation of coconut husk. Here we concentrate to their work on the antibacterial properties of AgAC examined by inhibition growth of Escherichia coli. Fig. 7.3 shows the quantitatively antibacterial study of AgNP in LuriaBertani (LB) broth. It presents the dynamics of E. coli growth in only LB broth (negative control), TSC control [LB broth supplemented with 120 μL trisodium citrate (TSC) solution], and AgNP antibacterial tests (LB broth supplemented with AgNP of concentration from 2 to 200 μg/mL). In this figure, OD595 represents optical density at 595 nm (1 optical density at 595 nm, OD595, equals the

7.4 Applications

FIGURE 7.3 Bar chart of optical density at 595 nm, OD595, presenting Escherichia coli concentration in LB in the presence of different concentrations of AgNP (μg/mL) as a function of time (h). Each test was conducted after 4, 8, 24, and 30 h. It is clear that, with the concentration of AgNP $ 16 μg/mL, E. coli growth was inhibited. After Tuan, T.Q., Son, N.V., Dung, H.T.K., Luong, N.H., Thuy, B.T., Anh, N.T.V., et al., 2011. Preparation and properties of silver nanoparticles loaded in activated carbon for biological and environmental applications. J. Hazard. Mater. 192, 1321
1329.

concentration of 1.7 3 109 cells/mL). The initial number of E. coli inoculated into 2 mL LB medium of the tested tube was 1.7 3 106 cells, giving the final bacterial concentration of 8.5 3 105 cells/mL. It is observed that E. coli grew normally in the negative control and the TSC control. After 30 hours in the TSC control, the concentration of E. coli (OD595 5 2.5) is higher than that in the negative control (OD595 5 1.5) which suggests that TSC was not toxic to E. coli and may be even enabled for the growth of the bacteria. With the presence of AgNP, the situation is different because of the well-known antibacterial property of AgNP (Kim et al., 2007). When AgNP concentration was 2 μg/mL, the result is similar to that of the negative control because the low value of AgNP could not inhibit bacterial growth. With a higher AgNP concentration, the inhibitory effect appeared within 8 hours even at a low AgNP concentration of 4 μg/mL. Fig. 7.3 clearly shows that, with the concentration of AgNP . 16 μg/mL, the E. coli growth was inhibited.

7.4.4 MAGNETIC NANOPARTICLES MNPs are of great interest in biomedicine applications. In the applications described below these nanoparticles were synthesized by the coprecipitation method.

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7.4.4.1 Arsenic removal from water MNPs Fe3O4 were reported to adsorb arsenic ions from contaminated water (Leslie-Pelecky et al., 2005). In this environmental application, compared to other techniques currently used to remove arsenic from contaminated water, such as centrifuges and filtration systems, this method using MNPs has the advantage of being simple, and, most importantly, not requiring electricity. This is very important, because arsenic-contaminated sites are often found in remote areas with limited access to power (Filipponi and Sutherland, 2010). The arsenic adsorption abilities of Fe3O4, Fe12xCoxFe2O3 (Co-ferrites) and Fe12yNiyOFe2O3 (Ni-ferrites) with x 5 0, 0.05, 0.1, 0.2, 0.5 and y 5 0.2, 0.4 were studied with different conditions of stirring time, concentration of nanoparticles, and pH (Hai et al., 2008). The starting arsenic concentration of 0.1 mg/L was reduced about 10 times down to the maximum permissible concentration (MPC) of 0.01 mg/L after a few minutes of stirring. The removal process seemed not to depend considerably on the concentration of x in the Co-ferrites. Similar results were found for the Ni-ferrites, where the arsenic concentration was reduced to the MPC value after a stirring time of a few minutes and the removal did not change considerably with y. Studying also the effects of the weight of the nanoparticles on the removal process, Hai et al. (2008) showed that, after 3 minutes of stirring, the optimal weight to reduce arsenic concentration down to a value lower than the MPC was 0.25 g/L for Fe3O4 and 0.5 g/L for Co- and Ni-ferrites. Studying the desorption process, Hai et al. (2008) showed that 90% of the arsenic ions was desorbed from nanoparticles. After desorption, the nanoparticles did not show any difference in arsenic readsorption ability. Repeating the adsorption
desorption process four times, Hai et al. (2008) proved that the nanoparticles could be reused for arsenic removal.

7.4.4.2 Herpes DNA separation Herpes simplex virus, or herpes, causes extremely painful infections in humans (Ryan and Ray, 2004). Thus, the determination of the presence of herpes is important. A simple and fast way to recognize the presence of the DNA of the virus is to use an electrochemical sensor. However, electrochemical sensors exhibit a sensitivity limit, so they cannot measure concentrations lower than a few tens of nM/L (Tuan et al., 2005). Therefore, a virus DNA separation before the measurement by using the electrochemical sensor is needed in order to increase the concentration of the DNA. Hai et al. (2008) used a DNA sequence, which is representative of the herpes, as a probe to hybridize with the target DNA in the sample. After being activated with EDC and 1-methylimidazole (MIA), the probe DNA was mixed with the amino-NP to have nanoparticles with the probe DNA on the surface (DNA-NP). The herpes DNA separation was carried out as follows: 1 mL of the solution containing 2 wt.% of DNA-NP was mixed with 2
20 mL of a solution with 0.1 nM/L of the herpes DNA. The hybridization of the probe DNA and the target DNA appeared at 37 C for 1 hour. Then, the

7.4 Applications

nanoparticles with hybridized DNA were collected and redispersed in 0.1 mL of water using magnetic decantation. The dehybridization of the nanoparticles with the probe and target DNA was obtained at 98 C. Hai et al. (2008) obtained a solution with a high concentration of the DNA of the herpes virus after removing the DNA-NP from the solution by using magnetic decantation. When all the target DNA were separated, the DNA concentration had increased from 20 to 200 times. Fig. 7.4 shows the dependence of the output signal on the initial volume of the solution containing 0.1 nM/L of the herpes DNA before and after the magnetic separation (Hai et al., 2008). The initial solution contained 0.1 nM/L of the DNA, which was much smaller than the sensor sensitivity. Therefore, the output signals before magnetic enrichment were almost zero (Fig. 7.4, open squares). After magnetic enrichment, the output signals linearly increased with increasing initial solution volume, depending on the initial volume of the solution. The higher the volume, the higher the concentration. The result is higher output signals were obtained. This means that the concentration of the herpes DNA was much higher after the enrichment. With the highest initial volume that Hai et al. (2008) used in their studies, the concentration after magnetic enrichment was 200 times higher than the initial concentration.

10

V (mV)

8

6 After magnetic separation

4

2 Before magnetic separation 0 0

5 10 15 Initial volume (mL), concentration 0.,1 nM/L

20

FIGURE 7.4 Dependence of the output signal on the initial volume of solution containing herpes DNA before and after magnetic separation. After Hai, N.H., Chau, N., Luong, N.H., Anh, N.T.V., Nghia, P.T., 2008. Application of magnetite nanoparticles for water treatment and for DNA and cell separation. J. Korean Phys. Soc. 53, 1601
1606.

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7.4.4.3 CD41 cell separation In human immunodeficiency disease, such as HIV/AIDS, helper T cells (CD41 T cells) are considerably destroyed by HIV. For mechanisms of CD41 T cell depletion in HIV infection we refer the reader to the review of, for instance, Okoye and Picker (2013). A dropping number of CD41 T cells (which are often referred to as CD4 cells) in blood is an indicator of immunodeficiency as in the case of HIV/AIDS. The number of CD41 T cells in the blood of HIV-infected patients is often reduced to less than 500 cells/μL. According to the World Health Organization (WHO) classification, a person is diagnosed with AIDS if the CD4 count is less than 200 cells/μL (World Health Organization, 2007). Thus, the CD4 count is very important for doctors to adjust treatment strategies. The principle of counting the number of CD41 T cells in blood is based on the specific linker between the antiCD4 monoclonal antibody and CD41 T receptor on the lympho T surface (Casset et al., 2003; and references therein). Fluorescent-labeled antiCD4 antibody has been commonly used to count CD41 T cells of HIV/AIDS patients due to its binding specificity to the cells and fluorescent emission signals. However, the fluorescent signals of labeled CD41 T cells are sometimes interfered with by autofluorescence of other dead white cells, such as killers (CD81) T cells, B cells, macrophages, or neutrophils, which contribute to the background in detection. To minimize this background interference, CD41 T cells can be magnetically sorted from other cells in the blood, followed by fluorescent signal detection. Hai et al. (2008) and Khuat et al. (2008) used Fe3O4 MNPs coated with fluorescent-labeled antiCD4 antibody (antiCD4-MNPs) to count the CD41 T cells. The antiCD4-MNPs were prepared through covalent linking between the carboxyl group of the antiCD4 antibody and the amino group of amino-modified MNPs. The antiCD4-MNPs were then used as a material to conjugate with CD41 T cells for magnetic separation. These authors observed a number of cells bound with magnetic clusters and particles. Fig. 7.5 shows the conventional microscope visualization of the blood cells after being coupled with the antiCD4 antibody and antiCD4-MNPs and separated using a magnet (Hai et al., 2008). For observing the CD41 T cells, using fluorescence isothiocyanate labeled antiCD4-MNPs, the fluorescent intensity was improved by about two times compared to when cells were only labeled with the antiCD4 antibody. This result indicates the role of the MNPs and can be used for the treatment of an HIV-infected patient with a simple fluorescent microscope.

7.4.4.4 Detection of pathogenic viruses Purification of nucleic acids (DNA and RNA) from clinical samples is an important step in diagnostics, such as detection of pathogenic viruses and bacteria using the polymerase chain reaction (PCR), paternity testing, genetic research, DNA fingerprinting, and DNA sequencing. The nucleic acid purification method based on interaction with silica, developed by Boom et al. (1990), is commonly used. It

7.4 Applications

FIGURE 7.5 Microscope visualization of the blood cells under white light (A, C) and under blue light excitation (B, D), after being coupled with the antiCD4 antibody and antiCD4-MNPs and separated by using a magnet. After Hai, N.H., Chau, N., Luong, N.H., Anh, N.T.V., Nghia, P.T., 2008. Application of magnetite nanoparticles for water treatment and for DNA and cell separation. J. Korean Phys. Soc. 53, 1601
1606.

is known that DNA binds to silica, even though both DNA and the silica surface are negatively charged. Thus, to provide insight into important issues such as the mechanism behind DNA binding to silica is of great interest. Using molecular dynamics simulations, Shi et al. (2015) showed that the two major mechanisms for binding of DNA to silica are attractive interactions between DNA phosphate and surface silanol groups and hydrophobic bonding between DNA base and hydrophobic region of silica surface. These short-range attractions can be sufficiently strong to overcome the electrostatic repulsion between negatively charged DNA and negatively charged silica surface.

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Micrometer-size silica-coated magnetic beads have been developed by different groups (see, for instance, Akutsu et al., 2004) and biotech companies such as Roche Diagnostics, Life Technologies, Promega, and Beckman Coulter to improve the efficiency of purification. Berensmeier (2006) described methods based on magnetic microparticles for nucleic acid purification. Recently, the investigation and application of silica-coated MNPs for separation and purification of nucleic acids has become an emerging area. Ashtari et al. (2005) reported a method for recovery of target ssDNA using amino-modified silica-coated MNPs and used them to recover trace concentrations of target ssDNA fragments of severe acute respiratory syndrome virus with high efficiency and good selectivity. Quy et al. (2013) presented a method for synthesis of the silica-coated Fe3O4 MNPs and their application for isolation and enrichment of DNA of Epstein
Barr virus (EBV), which is associated with particular cancers and lymphoma, and hepatitis virus type B (HBV) which causes hepatitis. Quy et al. (2013) have shown that the purification efficiency of DNA of both EBV and HBV using synthesized silica-coated Fe3O4 MNPs was superior to that obtained with commercialized silica-coated Fe3O4 magnetic microparticles. Quy et al. (2013) reported also on time saving in detection of EBV and HBV, namely the time required for DNA purification using silica-coated Fe3O4 nanoparticles was significantly reduced as the particles were attracted to magnets more quickly (15
20 seconds) than the commercialized silica-coated Fe3O4 microparticles (about 2
3 minutes). These results were attributed to the fact that silica-coated Fe3O4 nanoparticles have a larger total surface area compared to that of the commercialized silica-coated Fe3O4 microparticles.

7.4.4.5 Specific and rapid tuberculosis detection The worldwide effort to eradicate tuberculosis (TB), the highly infectious disease caused by Mycobacterium tuberculosis (MTB), has thus far led to significant decreases in the number of incidents and mortality rates. TB, however, remains the second leading cause of death from an infectious disease and a major global health problem (World Health Organization, 2011). Unfortunately, in the absence of an effective screening method, there are many cases of TB and multidrugresistant-TB which are not opportunely detected or treated. In the early 1990s, a diagnostic procedure based on the amplification of the insert sequence (IS) 6110 was developed and soon became prevalent. This method is displaying advantages regarding detection limit and specificity through the amplification of this signature sequence using the PCR technique (Kolk et al., 1992, 1998; Kox et al., 1994; Sankar et al., 2011; Shukla et al., 2011). However, this procedure requires the time-consuming extraction of DNA from each sample, including a cell lysis step which is usually inefficient on account of the highly complex bacterial cell wall (Noordhoek et al., 1994; Ellis and Zabrowarny, 1993; Ogbaini-Emovon, 2009). Recently, the collaboration between biologists and physicists has allowed the development of nanomaterials in DNA extraction from different organisms. More importantly, using MNPs, multiple samples could be processed simultaneously on

7.4 Applications

a microtiter plate, which would enhance the testing rate and reduce the contamination risk for testing personnel, especially in the case of dangerous pathogens (e.g., TB). Furthermore, this material could be constructed to form bioconjugates containing specific antibodies which would enhance the specificity of the detection method (Arruebo et al., 2009). Recently, Pham et al. (2015) reported for the first time the development of a specific and rapid TB detection using MNPs. The MNPs were functionalized with amino groups to facilitate coupling with anti-TB antibodies. The coupled nanoparticles were used to enrich Mycobacterium. In addition, preliminary assessment of this method in testing clinical samples (sputum and throat wash specimens) was also noted. The results of this study indicated potential for the establishment of a high-throughput semiautomated TB diagnostic procedure, which is currently being studied. Specificity, or the capability of improving signal-to-noise ratio, is a critical criterion in any diagnostic procedure. Samples collected from patients (sputum in most cases) normally contain other microorganisms which might be the contamination source. By pretreatment with N-acetyl L-cysteine-sodium hydroxide (NALC-NaOH), the decontamination could be done for sputum samples. This technique, however, could not eliminate nonspecific signal entirely. Besides the specifically designed primers for the amplification of the signature sequence IS6110, the coupled anti-MTB antibody served as a sieve which captured only the MTB antigens. The whole procedure was done in approximately one hour, which was half of the total time required for the traditional DNA extraction method.

7.4.4.6 Biological treatment targeting Mycobacterium tuberculosis in contaminated wastewater Wastewater from hospitals and facilities receiving patients infected with contagious microorganisms has dense concentrations of these pathogens, which may represent a danger to public health. Therefore, proper wastewater treatment to remove these contaminants before discharging to the sewage system is a great societal concern. Most common wastewater treatment methods are divided into physical (heating, ultraviolet radiation, etc.), chemical (e.g., hypochlorite/chlorination, ozonation), and biological categories. The main disadvantages of the first two categories are complex implementation and high maintenance cost (e.g., due to corrosion of the pipe systems). With the additional advantage of being environmentally friendly, biological methods have become valuable alternatives. Most of these methods utilize live microorganisms in either fermentation processes to remove toxic chemicals or filtration applications through the formation of biofilm (Sewage water treatment vat., 2016) and, thus, are without specific targets. Nguyen et al. (2016) reported biological treatment targeting MTB in contaminated wastewater using lysing enzymes coupled to Fe3O4 MNPs. This study was a further development from previous results obtained with a complex comprised of magnetic amino nanoparticles and anti-TB antibody molecules (Pham et al., 2015). The complex, referred to as NP-NH2-anti-TB, was shown to be capable of specifically capturing MTB (Pham et al., 2015), which would be killed with the

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addition of lysing enzymes. The major role of the nanoparticles is to bring these molecules in close proximity so that the lysing enzymes can work on the bacteria captured through the conjugated antibody on the same surface area. This, ideally, would solve the limit of diffusion, thus enhancing the reaction rate. Nguyen et al. (2016) also reported an initial assessment of the developed method in a wastewater model by spiking the wastewater samples collected from a hospital and a facility receiving TB patients with Mycobacterium bovis from bacillus CalmetteGue´rin vaccine. The results of this study indicated potential applications of the NP-NH2-anti-TB complex combined with enzymes to efficiently treat MTB-contaminated wastewater.

7.4.5 APPLICATIONS OF MULTIFUNCTIONAL NANOPARTICLES Multifunctional nanoparticles are gradually attracting more and more attention because of their ability to combine numerous properties, such as electronic, magnetic, optical, and catalytic. They are highly functional materials with modified properties which can be quite different from those of the individual materials. The properties of core
shell nanoparticles can be modified by changing either the constituting materials or the core-to-shell ratio (Oldenburg et al., 1998). Multifunctional nanoparticles show distinctive properties of the different materials employed together to meet the diverse application requirements. The purpose of the functionalization is manyfold, such as surface modification, the ability to increase the functionality, dispersibility, and stability, controlled release of the single nanoparticles, reduction in consumption of materials, etc. Applications of different multifunctional nanoparticles are summarized in review articles by Karele et al. (2006), Seleci et al. (2016), Jia et al. (2013), and Bao et al. (2013). The multifunctional nanoparticles are widely used in various applications such as pharmaceutical applications (Caruso, 2001), biomedical (Balakrishnan et al., 2009; Salgueirino-Maceira and Correa-Duarte, 2007), catalysis (Daniel and Astruc, 2004; Phadtare et al., 2003), electronics (Kortan et al., 1990; Qi et al., 1996), enhancing photoluminescence (Mews et al., 1994; Kamat and Shanghavi, 1997), creating photonic crystals (Scodeller et al., 2008), etc. Especially in the biomedical field, these nanoparticles are used for bioimaging (Laurent et al., 2008; Babes et al., 1999; Dresco et al., 1999), controlled drug release (Dresco et al., 1999), targeted drug delivery (Laurent et al., 2008; Gupta and Gupta, 2005; Dresco et al., 1999; Yan et al., 2009), cell labeling (Laurent et al., 2008; Jaiswal et al., 2003; Michalet et al., 2005), and tissue engineering applications (De et al., 2008). For instance, core
shell nanoparticles have attracted considerable attention in clinical and therapeutic applications (Hirsch et al., 2003; Loo et al., 2004). Core
shell nanoparticles, which are strong absorbers, can be used in photothermal therapy, while those which are efficient scatterers can be used in imaging applications. Silica core
gold shell nanoshells, a novel core
shell nanostructure, which either absorb or scatter light effectively, can be designed by varying their core-to-shell ratio (Loo et al., 2004). In imaging applications of core
shell

7.4 Applications

nanoparticles, they can be conjugated with specific antibodies for diseased tissues or tumors. When conjugated nanoparticles are inserted in the body, they get attached to diseased cells and can be imaged. In parallel, when the tumor has been located, resonance wavelength absorption of the core
shell nanoparticles will lead to localized heating of the tumor and it is destroyed. In other words, the imaging and photothermal therapy can be carried out together with core
shell or multifunctional nanoparticles. In drug-delivery systems using core
shell nanoparticles, the drug can be encapsulated or adsorbed onto the nanoparticle surface (Sparnacci et al., 2002) via a specific functional group or by an electrostatic stabilization technique. The nanoparticles will come into contact with the biological medium, and then direct the drug. For instance, the enzyme- and antibodyconjugated core
shell nanoparticles, which are strong absorbers with gold shells, can be embedded in a matrix of the polymer, such as nisopropylacrylamide (NIPAAm), and acrylamide (AAm) (West and Halas, 2000). These polymers exhibit a melting temperature which is slightly above body temperature. If the core
shell nanoparticles absorb heat from the illumination with resonant wavelength, the heat will transfer to the local environment, then cause collapse of the polymer matrix and release of the drug. Furthermore, when the core is MNPs, the core
shell nanoparticles with the bifunction of magnetic and gold nanoparticles can drive the drug and kill the tumor under the external magnetic field as well as by the irradiation of resonance wavelength. The usual methods of tumor treatment, such as chemotherapy or radiotherapy, have various side effects such as substantial loss of hair, lack of appetite, diarrhea, etc. The process of attacking the tumor also leads to the loss of many healthy cells. Core
shell or multifunctional nanoparticles offer an effective and relatively safer strategy to cure these ailments by significantly reducing the amount of chemicals or radiation using local treatments. Another strategy of using multifunctional nanoparticles is using multiple types of nanoparticles in one application, such as in fast DNA diagnosis (Quynh et al., 2014). This fast DNA kit created from NH2-modified SiO2-coated Fe3O4 nanoparticles and highly fluorescent Mn-doped ZnS nanoparticles in a sandwich structure, which can be seen in Fig. 7.6. The sandwich configuration attached the fluorescent particles to the docking matrix of MNPs with the complementary hybridization of the detector probe
target
capture probe structure. In one side of this sandwich structure, SiO2-coated Fe3O4 nanoparticles, which were modifed by the NH2 group, were employed as a docking matrix. The docking matrix was linked with a capture probe oligonucleotide chain, which specifically identifies the target DNA. In the other side of the sandwich, the detector probe formed by the signaling semiconductor nanoparticles contacted with another oligonucleotide chain. This sandwich configuration was applied to detect DNA of EBV using separation function of MNPs and the signaling function of semmiconductor nanoparticles. The kit firstly was stored as three separated solutions containing magnetic probe nanoparticles, semiconductor detector nanoparticles, and target DNA molecules, respectively. In order to measure the target DNA concentration, those solutions

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FIGURE 7.6 Schematic sandwich structure of DNA detection Fast kit using multifunctional magnetic
semiconductor nanoparticles. After Quynh, L.M., Hieu, N.M., Nam, N.H., 2014. Fast DNA diagnostic using Fe3O4 magnetic nanoparticles and light emitting ZnS/Mn nanoparticles, VNU J. Sci.: Math.
Phys. 30(3), 1
11.

were mixed to assemble the magnetic donor
target DNA
semiconductor detector complexes via the specific hybridization of catcher probe and detector probe with the target DNA. The complexes, then, are easily collected by an outside magnetic field. The other components, which do not contain MNPs will be washed out. The collected complexes were redistributed in solution and measured by photoluminescence. The luminescent intensity at 586 nm of Mn-doped ZnS nanoparticles changes with changing the initially added DNA target concentration. The detection limit of target DNA is around 2 3 106 copies/mL (B0.3 fM), showing the ability of using the named multifunctional magnetic
semiconductor sandwich structure for fast KIT DNA detection during scene investigation and viral DNA detection. Finally, in addition to the improved material properties, multifunctional materials are also important from an economic point of view. Multifunctional nanoparticle-based drug-delivery systems have been developed to improve the efficiency and reduce the systemic toxicity of a wide range of drugs, where additional capabilities like targeting and image contrast enhancement added to the nanoparticles. However, additional functionality means additional synthetic steps and costs, more convoluted behavior and effects in vivo, and also greater regulatory hurdles. The tradeoff between additional functionality and complexity is discussed by Cheng et al. (2012).

7.5 CONCLUSION AND PERSPECTIVES This chapter reviews various methods of synthesis and functionalization of metallic, semiconductor, magnetic, and multifunctional nanoparticles. Some applications of the fabricated nanoparticles in life sciences and the environment are discussed.

References

In the synthesis domain, it is expected that new preparation approaches will be introduced allowing the use of less energy and less toxic materials (“green manufacturing”). In order to meet requirements for different applications, the functionality of nanoparticles becomes more complex. Thus the major trend in the further development of nanoparticles is to make them multifunctional, with the potential to integrate various functionalities. Smart multifunctional nanoparticles will be very promising for a variety of applications.

ACKNOWLEDGMENT The authors would like to thank Prof. N.H. Hai, Prof. N.T.V. Anh, Prof. P.T. Nghia, Dr. P. Yen, Mr. L.M. Quynh, Dr. I. Notingher, and Prof. M. Henini for close collaboration.

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671. Alaqad, K., Saleh, T.A., 2016. Gold and silver nanoparticles: synthesis, methods, characterization routes and applications towards drugs. J. Environ. Anal. Toxicol. 6, 384. Arruebo, M., Valladares, M., Gonza´lez-Ferna´ndez, A., 2009. Antibody-conjugated nanoparticles for biomedical applications. J. Nanomater. 2009, 439389. Ashtari, P., He, X., Wang, K., Gong, P., 2005. An efficient method for recovery of target ssDNA based on amino-modified silica-coated magnetic nanoparticles. Talanta 67, 548
554. Astruc, D. (Ed.), 2008. Nanoparticles and Catalysis. Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim. Babes, L., Denizot, B., Tanguy, G., Le Jeune, J.J., Jallet, P., 1999. Synthesis of iron oxide nanoparticles used as MRI contrast agents: a parametric study. J. Colloid Interface Sci. 212, 474
482. Balakrishnan, S., Bonder, M.J., Hadjipanayis, G.C., 2009. Particle size effect on phase and magnetic properties of polymer-coated magnetic nanoparticles. J. Magn. Magn. Mater. 321, 117
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CHAPTER

Multimodal magnetic nanoparticles for biomedical applications: importance of characterization on biomimetic in vitro models

8

ˇ cˇ 3, , Jasna Lojk1,3, Klemen Strojan1 and Mateja Mojca Pavlin1,2, , Daˇsa Zupanci Erdani Kreft3 1

Faculty of Electrical Engineering, Group for Nano and Biotechnological Applications, University of Ljubljana, Ljubljana, Slovenia 2Faculty of Medicine, Institute of Biophysics, University of Ljubljana, Ljubljana, Slovenia 3Faculty of Medicine, Institute of Cell Biology, Centre for Electron Microscopy, Laboratory for Cell and Tissue Cultures, University of Ljubljana, Ljubljana, Slovenia

8.1 INTRODUCTION Magnetic nanoparticles (MNPs) are, due to their electron-dense core and specific magnetic properties, suitable for different biomedical applications and could be used for enormous benefit in the medicine and veterinary fields. Nevertheless, over the last two decades, humans and animals have been exposed to numerous nanoscale materials, and the explosive spread of nanotechnology has become another threat to the environment and our health (Bahadar et al., 2016; Oberdo¨rster et al., 2005). Thus, the toxicology of MNPs is being extensively investigated, including their genotoxicity, immunotoxicity, as well as the teratogenic and carcinogenic potential of MNPs. Since safety is an issue of constant concern, the emphases on the importance of investigating and developing credible in vivo and in vitro models should represent one of the priorities of nanotechnology research. In this chapter we overview the multimodal MNPs, their characterization, main biomedical applications, MNP
cell interactions, and discuss the importance of appropriate biomimetic in vitro and in vivo models. We first introduce the main properties of MNPs, the importance of analysis in physiologically relevant conditions and standard and innovative physicochemical characterization methods



Mojca Pavlin and Daˇsa Zupanˇciˇc are cofirst authors, since they contributed equally to this chapter.

Materials for Biomedical Engineering: Inorganic Micro- and Nanostructures. DOI: https://doi.org/10.1016/B978-0-08-102814-8.00009-3 © 2019 Elsevier Inc. All rights reserved.

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for the analysis of MNPs. The review of the most important and promising biomedical applications is followed by a description of the key mechanism of MNP uptake and the importance of understanding the route of internalization and intracellular fate of MNPs is discussed. Next, the most commonly used in vitro and in vivo models are described with an emphasis on innovative biomimetic in vitro models. Finally, we give an opinion on the advantages and disadvantages of biomimetic in vitro models, which enable us to overcome the gap between classical in vitro research (immortalized cell lines, primary and secondary cell cultures) and in vivo research followed by clinical trials and biomedical applications introduced into routine clinical use in human and veterinary medicine.

8.2 CHARACTERIZATION OF MULTIMODAL MAGNETIC NANOPARTICLES Adequate characterization of MNPs is crucial for the development of new MNPbased applications and for successful translation into clinical use. Furthermore, it is also essential for the assessment of the potential toxicity and immunogenicity of biomedically relevant MNPs. In general, MNPs can be characterized with a variety of methods available for characterization of nanoparticles (NPs). We will describe the most important methods and specifically mention those that are specific to the magnetic nature of MNPs.

8.2.1 PROPERTIES OF MAGNETIC NANOPARTICLES Typically MNPs are composed of a solid, electron-dense magnetic core composed of iron oxide or other magnetic materials like cobalt-ferrite (Bregar et al., 2013; Gupta and Gupta, 2005). Due to their superparamagnetic properties they are also referred to as SPIONs. The magnetic core has to have high magnetization saturation in order to enable magnetic field-based applications like hyperthermia or cell separation. For specific applications, MNPs are further functionalized in order to: (1) stabilize the MNPs in a physiological suspension with a pH around 7.4, for example, 0.9% NaCl (Fig. 8.1), (2) provide functional groups at the surface for further derivatization, and finally (3) to avoid immediate uptake by the reticuloendothelial system (Neuberger et al., 2005). The surfaces of MNPs could be modified through the addition of layers of organic polymer (e.g., PLGA, polyethylene glycol (PEG), dextran, chitosan, etc.), inorganic metals (e.g., gold), or oxide surfaces (e.g., silica), suitable for further functionalization through the attachment of various bioactive molecules such as DNA, antibodies, proteins, or ligands (Gupta and Gupta, 2005; Laurent et al., 2008). This functionalization changes the properties of the nanoparticles (NPs) and affects their bioactivity.

8.2 Characterization of Multimodal Magnetic Nanoparticles

FIGURE 8.1 (A) Hydrodynamic diameter for naked cobalt-ferrite (CoFe2O4) NPs and poly-acrylic acidcoated NPs (CoFe2O4-PAA) for different molar concentrations of NaCl. (B) Measured average hydrodynamic diameter (circles) and zeta potential (triangles) of cobalt-ferrite NP suspension in water. Suspension destabilization is observed at zeta potential . 2 20 mV.

The most important parameters of MNPs for drug-delivery or cell-labeling applications are physicochemical properties such as size, shape, crystallinity, surface charge, surface chemistry, and magnetization saturation. These properties greatly affect the stability of MNPs, their binding capacity and release properties, blood circulation time, mobility, and the bioavailability of the NPs within the body (Berry and Curtis, 2003; Fro¨hlich, 2012; Hajipour et al., 2014; Xu et al., 2006).

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Surface charge and size are the key parameters responsible for MNP behavior in biological systems. For example, larger NPs with diameters greater than 200 nm are sequestered mainly by the spleen when administered systemically and are removed from the blood by the phagocyte system. On the other hand, very small NPs with diameters less than 10 nm are rapidly removed from the body through extravasation and renal clearance. Therefore, particles in the range from 10 to 100 nm are optimal for systemic administration in magnetic drug targeting, since they have prolonged circulation times and are small enough to penetrate small capillaries and thus enable effective distribution in tissue (Berry and Curtis, 2003; Sun et al., 2008).

8.2.2 MAGNETIC NANOPARTICLE PROPERTIES CHANGE IN PHYSIOLOGICAL FLUIDS The first contact of MNPs with biological systems usually occurs with the physiological media, such as growth media or blood, which have a distinctive composition of ions, proteins, and other molecules (Pavlin and Bregar, 2012). Therefore, the most important and challenging aspect of MNP characterization is measurement of their properties under physiological conditions that closely resemble in vivo and/or in vitro conditions. For instance, noncoated MNPs tend to aggregate in physiological fluids (Pavlin and Bregar, 2012) as shown in Fig. 8.1, and many NPs adsorb proteins and aggregate during in vitro assays and after intravenous administration in the blood. Therefore, it is crucial to perform thorough physicochemical characterization of MNPs also in physiological fluids, such as culture media or blood (Fig. 8.2). The most important measurements for such characterization are MNP size measurement by dynamic light scattering (DLS) or complex magnetic susceptibility, because it can provide a measure of MNP hydrodynamic size and zeta potential

FIGURE 8.2 DLS measurements of the hydrodynamic diameter for very stable PAA-coated CoFe2O4 NPs and for the less stable polyethyleneimine (PEI)-coated NPs. The shift of the solid curve for PEI NPs is due to aggregation of NPs in the culture media with FBS.

8.2 Characterization of Multimodal Magnetic Nanoparticles

(Bhattacharjee, 2016; Pavlin and Bregar, 2012). Many NPs, which are stable at low pH or in deionized water are not stable and aggregate in physiological fluids (Bregar et al., 2013; Lojk et al., 2015; Pavlin and Bregar, 2012; Strojan et al., 2017a,b), where the abundance of proteins and ions destabilizes the colloid suspension of NPs. Furthermore, based on the physicochemical properties of NPs, different molecules adhere to the NP surface and form a so-called corona. Corona is composed mainly by proteins and consequently the term protein corona is usually used (Treuel et al., 2015). Since the outermost layer of the NPs determines the effect of NPs on the cells and on the whole body, it is therefore of key importance to analyze the protein corona structure (Aggarwal et al., 2009; Walczyk et al., 2010) in order to understand NP uptake, toxicity, and potential immunogenicity. Several studies had already stressed the importance of analyzing the protein corona that is formed on the NP surface in different media a decade ago. Clearly, even though this is not part of intrinsic NP properties like size or zeta potential, the protein corona crucially determines the behavior of NPs in biological environments. Thus, in recent decades the proteomic analysis of protein corona has become one of the standard methods that help to understand the interactions of NPs with cells in vitro and their behavior in animal or human bodies. Moreover, it was shown that protein corona composition very importantly determines the potential immune response (Hajipour et al., 2014; Strojan et al., 2017b).

8.2.3 METHODS FOR CHARACTERIZATION OF PHYSICOCHEMICAL PROPERTIES OF MAGNETIC NANOPARTICLES Most widely used methods for characterization of physicochemical properties of MNPs are transmission electron microscopy (TEM), X-ray diffraction, nuclear magnetic resonance (NMR), Fourier-transform infrared spectroscopy, and different thermal methods (e.g., thermogravimetric analysis, differential scanning calorimetry, size-exclusion chromatography, surface-enhanced Raman spectroscopy, small-angle neutron scattering, and others) (Barrera et al., 2009; Fee and Van Alstine, 2004; Li et al., 2008). However, most of these methods are either destructive or require special sample preparation. In addition, there is also a problem to characterize the organic layer (Bhattacharjee, 2016; Fro¨hlich, 2012). For characterization of hydrodynamic size and surface charge of MNPs, the DLS—also known as photon correlation spectroscopy—and zeta potential measurements are available (Fischer et al., 2005; Petersson et al., 2006; Petri-Fink et al., 2005; Zhao et al., 2008). In fact, both DLS and zeta potential have evolved into standard methods for characterization of NPs. Both techniques are noninvasive, require minimal sample preparation, and no preexperimental calibration, however they require optically transparent samples.

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Specifically, for measurements of the hydrodynamic size of the MNPs (Connolly and St Pierre, 2001) a method called AC magnetic susceptibility for detection of Brownian relaxation was developed (Fannin et al., 2000). This method allows dynamic analysis of NP functionalization in real time and in physiological fluids, such as culture media and blood, which together with the nondestructive nature of the method can give an advantage with respect to the other methods. Also, the method is compatible with the usual biomedical protocols since the samples can be prepared in physiological, isotonic solutions and similar systems. This eliminates additional preparation of samples in “artificial” nonphysiological environments. Furthermore, due to the electron-dense core of MNPs, TEM and scanning electron microscopy analysis (Fig. 8.3) proved to be invaluable techniques that enable direct visualization and characterization of MNPs. This can significantly add to direct observation of MNP size, aggregation, and shape in the native physiological environment in vitro, in vivo, or on ex vivo tissue samples.

8.2.4 CHARACTERIZATION OF MAGNETIC NANOPARTICLE MOBILITY IN 3D GELS AND IN THE ARTIFICIAL EXTRACELLULAR MATRIX Gel electrophoresis and magnetophoresis represent useful methods for analysis of MNPs in artificial in vitro [gels, the extracellular matrix (ECM)] and in ex vivo

FIGURE 8.3 Direct visualization of internalization of CoFe2O4-PAA NPs into CHO cells by scanning electron microscopy. NPs (arrows) on the plasma membrane of the cell. Note that some NPs are in a plasma membrane invagination. Individual NP crystallites can be observed. Scale bar: 100 nm.

8.3 Current Biomedical Applications of Multimodal

(tissues) environments. These methods complement standard DLS and zeta potential in the characterization of MNPs and additionally enable analysis of mobility properties of MNPs. Gel electrophoresis is a well-established method for separation and characterization of macromolecules. It was also shown that it can be effectively used for characterization of charged NPs (Arias et al., 2008; Hanauer et al., 2007; Parak et al., 2003). Furthermore, gel magnetophoresis was developed specifically to analyze MNPs (Holligan et al., 2003). Some in vitro studies analyzed particle velocities and forces in agarose gels (Holligan et al., 2003), while only a few studies (Kuhn et al., 2006) analyzed particle mobility and interaction with the environment in a 3D ECM, which represents a controllable in vitro model of tissue. It was shown that there is a critical size of MNP entrapment in ECM pores for given particle magnetization that advance surface coating like PEG coating minimizes particle adhesion to ECM and aggregation. Several in vitro studies also showed that the theoretical analysis of particle diffusion in complex 3D systems is important for understanding of NP mobility and behavior within tissues (Kuhn et al., 2006; Pluen et al., 2001; Ramanujan et al., 2002; Zborowski et al., 1995). Multimodality and other unique properties of MNPs represent their main advantage over other types of NPs, which led to the huge advancements in the development of new MNPs in the last few decades. These new MNPs represent promising tools for various biomedical applications that are described in the text that follows.

8.3 CURRENT BIOMEDICAL APPLICATIONS OF MULTIMODAL MAGNETIC NANOPARTICLES In the last decade, advancements in the field of nanotechnology led to the development of new MNPs that enable several promising biomedical applications, such as cellular targeting, labeling of the cells’ separation, tissue repair, drug delivery, new contrast agents for magnetic resonance imaging (MRI), hyperthermia, and magnetofection (Gupta and Gupta, 2005; Laurent et al., 2008; Pankhurst et al., 2003). For these applications, the particles must have combined properties of high magnetic saturation, monodisperse size, biocompatibility, and appropriate surface properties. One of the main reasons for their applicability lies in their multimodality. Most importantly, electron-dense cores of MNPs and their magnetic properties facilitate retention of MNPs in desired locations using an external target-directed magnetic gradient field, the technique called magnetic targeting (Prijic et al., 2012). Moreover, MNPs can be efficiently detected and visualized in vitro and in vivo by using TEM (Bregar et al., 2013; Kocbek et al., 2013). The surface of MNPs can be tailored to bind fluorescent labels, antibodies, enzymes, receptor ligands, or other small molecules (Gupta and Gupta, 2005). These properties are the main driving force behind applications of MNPs that will be addressed in the following text.

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8.3.1 MOLECULAR ISOLATION AND MAGNETIC SEPARATION Specific high-affinity interactions between biological entities, such as antibody
antigen interactions, are used in several biomedical and biotechnological applications. When one or more such molecules are immobilized on MNPs, we can achieve specific high-affinity binding of its counterpart on MNPs. Importantly, the resulting complexes can be manipulated with magnetic fields, providing completely new ways of applicability. Several applications of functionalized MNPs, such as DNA, protein and cell separation, molecular biosensing, and pathogen detection, are based on these concepts. The basic idea is simple: MNPs modified with antibodies, antigens, or aptamers (oligonucleotide or peptide molecules that bind to a specific target molecule) are added to a mixture of molecules (cell lysate, whole blood) where binding between molecules with affinity occurs. Afterwards, MNPs with bound specific molecules can be isolated using a magnetic field. The most common method for protein isolation is based on the reaction between His-tagged proteins and nitrilotriacetic acid coating on MNPs (Li et al., 2007). The process is highly selective and effective in isolation of proteins present in low concentrations (Li et al., 2007). Another advantage is its costefficiency and scalability. Similarly, specific DNA or RNA can be isolated from complex biological fluids, including saliva (Yi et al., 2013), by grafting specifically designed oligonucleotides to the surface of MNPs (Berensmeier, 2006). Unfortunately, the efficiency of such nucleic acid isolation is generally modest. Innovative, more efficient isolation of total DNA from the blood of patients is possible using MNPs coated with dimercaptosuccinic acid (Min et al., 2014).

8.3.2 MAGNETIC NANOPARTICLES AS DELIVERY VECTORS The highest goal of biomedical nanotechnology has always been the targeted delivery of effector molecules to their targets (e.g., chemotherapeutic molecules to cancer cells in tumors and in metastasis). While the delivery itself has already been proved in numerous FDA-approved NP-formulations (e.g., liposomes), efficient targeted delivery remains elusive (Bobo et al., 2016). At this point we must emphasize that MNPs themselves are not necessarily the best candidates for such a task, but their properties can be efficiently used in some more complex MNPbased systems. Such systems may, for example, consist of MNPs and liposomes (i.e., magnetic liposomes) (Santhosh et al., 2015), which are capable of carrying drugs (Di Corato et al., 2015). MNPs are used in such systems for two main reasons: (1) visualization of such MNP-based systems (in vitro and in vivo) and (2) magnetically assisted targeting by a precisely applied magnetic field (Wahajuddin and Arora, 2012). The first reason is of great importance, especially at the development stages of such a system, while the latter can be understood in the means of active targeting.

8.3 Current Biomedical Applications of Multimodal

8.3.3 CELL LABELING Multimodality of MNPs enables extremely efficient cell-labeling. Due to the electron-dense core of MNPs they can be directly visualized by TEM after preparation of the samples from different in vitro cell cultures or tissue samples obtained from in vivo studies. Moreover, when functionalized with fluorescent label MNPs could be used for cell labeling and monitoring labeled cells in vitro by fluorescent microscopy (Lojk et al., 2015). Those cells can be analyzed when they are still alive by live cell imaging or can be first fixed and additionally labeled with immunofluorescence. Furthermore, labeled cells can be also tracked in vivo using MRI. MNPs can be used to label specific subpopulations of cells already present in the tissue by affinity reactions (specific cell targeting) or cell type specific uptake of MNPs. Gallo et al. showed that protein G-IgG antibody labeled MNPs can be used to tag peripheral blood mononuclear cells in human blood (Gallo et al., 2013). Perhaps more useful is the cell type unspecific ex vivo loading of MNPs into cells by the means of unspecific endocytosis, prior to cell therapy and tracking of labeled cells (injection of living labeled cells into a patient). In such a way, MNPs can be, for example, used for high-resolution spatial and temporal tracking of stem cells post implantation. Recent progress in the field is described by Li et al. (2013). Moreover, an external magnetic field can be used to enhance labeling efficiency, which is a valuable technique for cells that are hard to label (Adams et al., 2015). Commercially available MNP formulations for cell labeling are described by Wang et al. (2013b).

8.3.4 MAGNETIC NANOPARTICLES AS CONTRAST AGENTS FOR MAGNETIC RESONANCE In clinical practice, one of the most frequently performed MNP application is the use of MNPs as contrast agents for MRI. MRI is a powerful imaging technique based on the principles of NMR (Shin et al., 2015). Specifically, hydrogen protons are aligned in a magnetic field, alignment is followed by energy relaxation which forms a voltage in a radio antenna surrounding the patient. Tissues with large amounts of freely mobile water can be clearly distinguished in the MRI images. Magnetic contrast agents, when in proximity to hydrogen protons, modify relaxation mechanism, enhancing the natural contrast (Lo´pez-Cebral et al., 2014). Surface modification of such MNP-based contrast agents is an absolute necessity, because noncoated MNPs tend to aggregate in physiological fluids (Pavlin and Bregar, 2012). Due to this instability, there is a considerable amount of effort being invested into this specific subfield of contrast agent development. Another important aspect is the specificity of contrasting. Current formulations are very good for contrasting “detoxifying” organs, such as liver, because those formulations are passive in regards to targeting abilities and, as a result, a majority of such contrast agents are accumulated in the liver (Gallo et al., 2013), as discussed in detail below. Another form of passive targeting in MRI is targeting of

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some tumors, which utilizes the so-called enhanced permeability and retention (EPR) effect. In those cases, NPs are accumulated in tumors due to poorly developed vasculature and insufficient lymphatic drainage. Despite the EPR effect, passive accumulation of NPs in such tumors is not efficient enough (Sykes et al., 2014). To overcome this problem, MNPs can be functionalized to target specific organs, tissues, or even cells. Such approach was already used for active targeting of orthotopic ovarian tumors in nude mice with MNPs decorated with HER-2 antibodies (Satpathy et al., 2014). A similar approach can be used also in targeted drug delivery, as described above.

8.3.5 MAGNETOFECTION The responsiveness of MNPs to magnetic fields led researchers to develop MNPbased gene vectors, which could be guided to specific tissue using a magnet and resulting in transfection of cells in the selected tissue, which is referred to as magnetofection (Mykhaylyk et al., 2007; Scherer et al., 2002). The technique is based on iron oxide MNPs coated with polyethyleneimine (PEI), a positively charged polymer capable of electrostatic interactions with DNA. A magnetic device with neodymium-iron-boron permanent magnets was designed for the first in vitro experiments, to sediment MNP
DNA complexes on the cell surface. Transfection kinetics were greatly improved and strongly increased concentration of vectors at the cell surface improved dose
response profiles already in the early studies. Important in vitro features of magnetofection are drastically lowered vector dose, reduced incubation time, and the possibility of gene delivery to otherwise nonpermissive cells (Scherer et al., 2002). Magnetofection increases the efficiency of nucleic acid delivery by enhancing the concentration of MNPs on the cell surface, while the mechanism enabling internalization of MNPs remains endocytosis (Scherer et al., 2002). Detailed protocols for this approach were discussed in Mykhaylyk et al. (2007). The mechanisms behind magnetofection are broadly discussed in Plank et al. (2011). Despite numerous studies, the transport of nucleic acids from vesicles such as endosomes and lysosomes to ribosomes or cell nucleus remains elusive. The fact is that nucleic acids leave the intracellular vesicles, as confirmed by the successfully achieved transfection or silencing. The ultimate aim of magnetofection is to produce therapeutic benefit in patients by delivering nucleic acid sequence of interest (gene therapy) to the targeted cells by using a magnetic field (Ortiz et al., 2012). While there are numerous magnetofection studies that have been performed in vitro (Govindarajan et al., 2013; Prosen et al., 2013), there is also an ever-growing field of research regarding magnetofection in vivo (Bhattarai et al., 2008; Dames et al., 2007; Galuppo et al., 2006). Specifically, Dames et al. achieved MNP-aerosol delivery to the lungs of mice in combination with target-directed magnetic gradient field (Dames et al., 2007).

8.4 Endocytosis and Intracellular Fate of Multimodal

8.3.6 MAGNETIC FLUID HYPERTHERMIA Magnetic fluid hyperthermia is a therapeutic procedure which involves exposure of MNP-loaded cells to an alternating external magnetic field, which causes vibration of MNPs and generation of heat (Jordan et al., 2001). This rises the temperature in the targeted body region, such as tumors. The rationale is based on a direct cancer cell-killing effect by elevated temperatures of the tumor (Wust et al., 2002). Magnetically induced interstitial hyperthermia offers an attractive approach for treating cancer, because it is a local therapy and can be combined with other treatments. In general, MNPs are injected into the tumor tissue where they are stimulated by an alternating magnetic field (Jordan et al., 2001). As a consequence of Brownian and Ne´el relaxation processes, heat is produced. The first human trials with magnetic fluid hyperthermia were started in 2003 on patients suffering from glioblastoma and recurrent prostate carcinoma (Thiesen and Jordan, 2008). Although most research has been evaluated in preclinical studies, the first clinical trials are very promising. For now it is too early to claim a therapeutic advantage because survival and disease progression were not the endpoints of the clinical studies (Laurent et al., 2011). Nevertheless, it should be noted that the company MagForce is in preparation of an FDA application for magnetic fluid hyperthermia treatment of glioblastoma.

8.3.7 PERSPECTIVES OF MAGNETIC NANOPARTICLE BIOMEDICAL APPLICATIONS The applications described above represent the most notable areas of biomedical applicative research regarding MNPs. Some are already fully operational on in vitro models (magnetic separation, magnetofection) and others are already being tested in clinical trials (hyperthermia). Despite the immense potential for complex applications, nowadays the majority of FDA-approved MNP-based formulations are used for iron deficiency treatment in chronic kidney disease (Bobo et al., 2016). Nonetheless, multimodal MNPs are on the verge of wide practical use. One of the main obstacles to the use of MNPs in an even wider range of biomedical applications is in insufficient understanding of their endocytosis and fate within cells, tissues, and whole animal or human bodies.

8.4 ENDOCYTOSIS AND INTRACELLULAR FATE OF MULTIMODAL MAGNETIC NANOPARTICLES Understanding the mechanism of MNP uptake by cells and their intracellular fate is crucial for the development and optimization of each specific MNP-based biomedical application. The majority of NPs have been shown to enter the cells through an energy-dependent process of endocytosis. Endocytosis is cells’

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intrinsic mechanism for internalization of various extracellular substances, regulation of plasma membrane composition, and a requirement for signaling of certain membrane receptors (Doherty and McMahon, 2009). Several endocytic pathways are known, from phagocytosis (Freeman and Grinstein, 2014) to different pinocytotic pathways, namely clathrin-dependent endocytosis (McMahon and Boucrot, 2011), caveolin-dependent endocytosis (Parton and del Pozo, 2013), macropinocytosis (Lim and Gleeson, 2011), and several clathrin- and caveolin-independent pathways, distinguished by different proteins involved in the process (e.g., flotillin, Arf6, RhoA) (Mayor and Pagano, 2007).

8.4.1 DIFFERENT ENDOCYTIC PATHWAYS Phagocytosis is primarily used in the uptake of infectious agents, apoptotic, and senescent cells by phagocytic immune cells such as macrophages and dendritic cells. As such, phagocytosis enables the uptake of particles as large as several micrometers. The phagocytosis of NPs is driven by interactions with specific receptors (e.g., mannose receptors, integrins, scavenger receptors, receptors for antibodies, opsonins) that recognize complementary ligands present in the protein corona of NPs. NPs are taken up by progressive formation of invaginations around the cargo that ultimately fuse to form a phagosome (Freeman and Grinstein, 2014). Similarly, clathrin- and caveolin-dependent endocytic pathways are also receptor-mediated, but the volume of formed vesicles is much smaller. NPs bind to the receptors (e.g., low-density lipoprotein receptor or transferrin receptor) in the clathrin-coated pits, which invaginate and are pinched off the plasma membrane to form a clathrin-coated vesicle with a diameter of 85
120 nm. Thus with this pathway, which is employed by the majority of cell types, only small NPs or small NP aggregates can be internalized (McMahon and Boucrot, 2011). In caveolin-dependent endocytic pathways small flask-shaped invaginations named caveolae are formed, which measure 50
100 nm in diameter. Caveolae, which are present mainly in fibroblasts, smooth muscle cells, adipocytes, and endothelial cells, are relatively static structures which internalize only after binding of specific ligands, such as albumin or αvβ3 integrins. The forming caveolae might elongate into a tubular invagination or detach from the plasma membrane and fuse into caveosomes. These caveosomes are usually transported along microtubules towards the center of the cell or through the cell to its opposite site in the process called transcytosis (Kafshgari et al., 2015; Parton and del Pozo, 2013). Receptor-mediated internalization pathways are specific, since the cargo is selected based on the plasma membrane receptors. NPs internalized through these pathways thus must contain ligands for such receptors in their protein corona (Banerjee et al., 2014; Kafshgari et al., 2015). On the other hand, macropinocytosis enables nonspecific internalization of larger volumes of fluid phase with intensive plasma membrane evaginations in the form of blebs, ruffles, and lamellar evaginations (Fig. 8.4A), forming a vesicle called a macropinosome with 0.5
10 μm diameter (Fig. 8.4B) (Lim and Gleeson, 2011). This pathway is

8.4 Endocytosis and Intracellular Fate of Multimodal

FIGURE 8.4 Endocytosis and intracellular fate of MNPs in immortalized cell lines. (A) Pinocytosis of three-layer cobalt-ferrite MNP coated with PAA-PEI-PAA (thick arrow). Cytoplasmic protrusion (asterisk) is elongated from the apical plasma membrane (arrowhead). (B) Endosome (thick arrow) with endocytosed MNP. (C, D) Multivesicular body or late endosome with cobalt-ferrite MNPs coated with PAA (thin arrows). (E) Massive perinuclear accumulation of cobalt-ferrite MNPs coated with PAA (thin arrows); apical plasma membrane (arrowhead); cell nucleus (N). (F) Partially degraded three-layer cobaltferrite MNP coated with PAA-PEI-PAA (thick arrow) within the late endosome. Scale bars: A, B, F 5 500 nm; C, D 5 250 nm; E 5 2 μm.

present in all cell types, including nonphagocytic cells, and enables internalization of larger NPs or NP aggregates.

8.4.2 UPTAKE PATHWAY DEPENDS MAINLY ON THE PROPERTIES OF NANOPARTICLES AND THE CELL TYPE The utilized endocytic pathway for NP internalization depends on the physicochemical properties of NPs (Fadeel et al., 2015), extracellular environment (e.g., pH, ion, and protein composition) (Petters et al., 2014), on the composition of protein corona (Treuel et al., 2015), and on the presence or activity of certain endocytic pathways in the observed cell type (Joris et al., 2013). Positively

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charged NPs are generally more readily endocytosed due to the electrostatic binding to the negatively charged plasma membrane (Barrow et al., 2015; Calatayud et al., 2014; Kralj et al., 2012), however the opposite was also demonstrated (Matahum et al., 2016). The lowest internalization rate was observed for neutral NPs, such as PEG-coated NPs, which also show lower protein absorption, decreased recognition by immune cells, and longer circulation time in vivo (Liu et al., 2011; Ni et al., 2012). The size and shape of NPs have an important effect on the internalization rate as well, but the optimal size/shape parameters vary between different NPs and cell types examined (Albanese et al., 2012). For example, 50-nm gold NPs showed the most efficient uptake into human cervical cells compared to other sizes (Chithrani et al., 2006; Chithrani and Chan, 2007). On the other hand, 100nm polystyrene NPs were most readily internalized in the cells of a colon adenocarcinoma cell line, while 50-nm polystyrene NPs showed the lowest efficiency for internalization among the tested sizes (Yin Win and Feng, 2005). In general, the optimally internalized size usually varies around 50
100 nm. Interestingly, the maximal phagocytosis was obtained with 2
3-μm polymeric microspheres (Champion et al., 2008), which coincides with the general size of bacteria normally internalized with this pathway, but much smaller NPs were also reported to enter via phagocytosis (Franc¸a et al., 2011). Protein adsorption on the NP surface and the formation of protein corona in physiological fluids is a very important parameter, since it can change the properties of NPs such as the hydrodynamic diameter and surface charge, and consequently influence cellular uptake (Banerjee et al., 2014; Treuel et al., 2015). Moreover, the absorbed proteins can interact with the receptors on the cell surface and stimulate NP uptake through receptor-mediated pathways (Kafshgari et al., 2015). Adsorbed serum proteins, such as complement components or immunoglobulins, can act as opsonins and trigger immune recognition (Chen et al., 2016), which can have profound consequences in vivo. Unfortunately, most studies do not analyze the protein corona composition or changes in physicochemical properties of NPs after they are added to culture medium or administered to the living organism. This might be the reason for the lack of consensus on the effect of NP properties on the selection of internalization pathway. Besides relying on the physicochemical properties of NPs to target a specific internalization pathway, targeting can also be improved by functionalizing NPs with specific ligands or antibodies, which bind to a certain receptor, specific for the desired endocytic pathway. Such ligands are, for example: (1) manose-6phosphate, transferrin, or nicotinic acid to target receptors in clathrin-coated vesicles; (2) folic acid, albumin, or cholesterol to target caveolae (Bareford and Swaan, 2007); (3) opsonins such as antibodies or components of the complement system and bacterial components to induce phagocytosis (Freeman and Grinstein, 2014), and many others. It is also possible to target a specific cell type, for example, cancer cells, by targeting receptors that are expressed or overexpressed only on cancer cells. Cancer cells are frequently targeted with antibodies against

8.4 Endocytosis and Intracellular Fate of Multimodal

specific cancer markers or with ligands such as epidermal growth factor to target epidermal growth factor receptor or RGB tripeptides (arginine-glycine-aspartic acid) to target integrins, which are amply expressed in certain cancer cells (Hadjipanayis et al., 2010). Nevertheless, this approach still has several problems when applied in vivo. Moreover, the presence of NPs in the vesicles of intracellular trafficking routes can alter the normal pathways within the cell. Even if the NPs were receptor-targeted, their intracellular trafficking might not resemble the trafficking of a free ligand (Tekle et al., 2008), so it should be analyzed for each new MNP formulation. Several studies have shown that processing of internalized NPs and their intracellular fate is cell type specific, although this area is still poorly explored. The array of active endocytic pathways depends on the cell type (Doherty and McMahon, 2009) and on the stage of cell differentiation (e.g., with differentiation of urothelial cells the rate of endocytosis diminishes) (Kreft et al., 2009). Moreover, cancer cells, which escape the control system of the human body might have different endocytic pathways than normal cells, which was already suggested as a method for selective targeting of NPs in tumors (Sahay et al., 2010). To gain a better understanding of potential NP
cell interactions, every new biomedical MNP formulation should thus be separately evaluated on several cell types.

8.4.3 THE INTRACELLULAR TRAFFICKING AND FATE OF INTERNALIZED NANOPARTICLES Once internalized, NPs undergo intracellular trafficking and processing. Most endocytic pathways lead to degradation in lysosomes. The phagosome directly fuses with lysosomes, while macropinosomes first undergo membrane recycling to concentrate the cargo before degradation (Doherty and McMahon, 2009). Following clathrin-mediated endocytosis, the cargo undergoes the endolysosomal intracellular trafficking route; formed primary vesicles undergo fusion to form the early endosomes, where the cargo is released into the lumen of the vesicle due to acidification (pH 6.0). In early endosomes, the receptor could be recycled back to the cell surface or sorted for degradation by budding into the lumen of the endosome (Maxfield and McGraw, 2004). The formation of these intraluminal vesicles marks the maturation of the endosome to the multivesicular body and thereafter to the late endosome (Fig. 8.4C), which then fuses with lysosomes to form the hybrid organelle (Fig. 8.4D), where degradation occurs in the presence of low pH (pH 4.5) and lysosomal enzymes (Luzio et al., 2007). MNPs are enzymatically nondegradable, so such NPs tend to accumulate in the perinuclear region of the cells, still enclosed in vesicles (Fig. 8.4E) (Bregar et al., 2013; Lojk et al., 2015; NDong et al., 2015). Accumulation is desirable for biomedical applications such as hyperthermia, cell labeling, and tracking or magnetic separations (Gupta and Gupta, 2005; Laurent et al., 2008; Pankhurst et al., 2003). However, prolonged retention of MNPs can also have negative

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consequences on the cells. For example, carboxydextran-coated iron oxide MNPs following clathrin-dependent internalization (Lunov et al., 2011) undergo endolysosomal intracellular trafficking route and accumulate in the macrophages in vitro. Although these MNPs did not affect cell viability 24 hours after incubation, they induced caspase 3-dependent apoptosis, reactive oxygen species (ROS), and secretion of tumor necrosis factor α 3 days after incubation (Lunov et al., 2010a). Prolonged NP presence might also induce autophagy and lysosomal dysfunction, defects in intracellular trafficking and cell signaling, or could prevent vesicle fusion. All these processes are important for cellular homeostasis, removal of damaged cellular components, and stress response (Stern et al., 2012). Iron oxide MNPs and EGFR-targeted plasmonic MNPs have been shown to induce autophagy in lung epithelial cancer cells (Khan et al., 2012; Yokoyama et al., 2011), sodium citrate-coated iron oxide MNPs caused induction of autophagy in HeLa cells (Huang et al., 2015), and PLGA-coated iron oxide MNP induced accumulation of autophagosomes in human breast cancer cells (Zhang et al., 2016), to mention just a few. High intracellular loading of NPs might alter cell functions, as in the case of NP-labeled macrophages, where a decrease in uptake capacity was observed in vitro (Hsiao et al., 2008; Lunov et al., 2010a), which might impede pathogen removal and immune response. However, even if MNPs are considered nondegradable, lysosomal enzymes might still damage the MNP coating. The retention of fluorescently labeled carboxydextran-coated iron oxide MNPs in lysosomes gradually decreased the intracellular fluorescence due to degradation of the NP coating (Fig. 8.4F) (Lunov et al., 2010b). Damage to the coating layer might also lead to acid etching of the MNPs, generation of free metal ions (Sabella et al., 2014), and a gradual decrease in the MNP core diameter (Gutie´rrez et al., 2015). More and more evidence indicates that these free ions might be one of the mediators of toxicity of these MNPs (Sabella et al., 2014). Degradation of iron oxide MNPs could increase ROS formation (Hohnholt and Dringen, 2011; Wu et al., 2014), apoptosis, and inflammation (Lunov et al., 2010b). Ion release is usually also accompanied by changes in MNP morphology, loss of magnetic properties and labeling function (Gutie´rrez et al., 2015). On the other hand, degradation can also be positive, since it could be a mechanism of activation of MNP-delivered prodrugs or a process of MNP removal from the organism after they are no longer needed. An acidic environment in lysosomes can also be exploited for lysosomal damage and release of endocytosed cargo into the cytosol through the so-called “proton-sponge effect.” Lysosomal release can be achieved with polycationic coatings, which prevent acidification of lysosomes by a buffering effect and at the same time increase osmotic pressure and induce swelling. This can result in damage to the lysosomal membrane, leakage, or even bursting of the lysosome (Guo and Huang, 2011). Such NPs are coated, for example, with PEI (Prijic et al., 2012; Strojan et al., 2017a; Wang et al., 2014), chitosan, or poly-L-lysine and are mainly used for drug or nucleic acid delivery into the cytosol (Guo and Huang, 2011). However, disruption of lysosomes not only releases NP-delivered cargo,

8.4 Endocytosis and Intracellular Fate of Multimodal

but also lysosomal enzymes, which can have toxic effects on cells. For example, iron oxide MNPs can induce release of lysosomal enzyme cathepsin D (Cengelli et al., 2010), which acts as a signal for apoptosis (Serrano-Puebla and Boya, 2016). Moreover, NPs can nonspecifically associate and damage other organelles in the cell, such as mitochondria, induce formation of ROS and mitochondrialmediated induction of apoptosis (Parhamifar et al., 2010; Park et al., 2014; Zhang et al., 2016). A possible NP fate is their excretion back into the ECM. The mechanisms of NP excretion are not well understood, but it is believed that some vesicles containing NPs can undergo exocytosis either as a regular mechanism of membrane recycling or during cell stress as a part of membrane repair (Sakhtianchi et al., 2013). Exocytosis and intracellular retention time are dependent especially on cell type, but also on the size and surface properties of NPs. For example, exocytosis of 15-nm superparamagnetic iron oxide NPs trapped in porous silicon carriers from murine macrophages in vitro is greater than exocytosis of 30-nm NPs (Serda et al., 2010). NPs can also be exocytosed by directly passing through the cell in the process of transcytosis, which is typical for caveolin-dependent uptake (Kafshgari et al., 2015; Parton and del Pozo, 2013). In this case, the cargo normally bypasses lysosomes and is secreted intact on the other side of the cell. For this reason, this pathway is a useful target for various NPs, which aim to deliver drugs and active agents that should not be degraded in the cells. This route could be employed for delivery across endothelial barriers to the underlying target tissues (Wang, 2014; Wang et al., 2011). In most other cases, exocytosis is not desirable, since it removes NPs from the cells, reducing the efficacy of the application.

8.4.4 ENDOCYTOSIS OF MAGNETIC NANOPARTICLES IS AN ESSENTIAL STEP FOR MOST BIOMEDICAL APPLICATIONS MNPs have been designed to improve drug delivery (1) by increasing selective delivery and accumulation of MNPs in certain cell types, (2) by delivering prodrugs, which require proteolytic degradation in lysosomes for drug activation, or (3) for delivery into cytosol through lysosomal escape. These approaches improve drug delivery to selected targets and decrease systemic toxic effects. Endocytosis of NPs also provides a mechanism for delivery of membrane-impermeable drugs or DNA molecules, which require internalization to achieve their therapeutic action. The employed endocytic pathway and consequent intracellular fate of MNPs not only defines the efficiency of the biomedical application, but also the toxicity mechanisms of the selected MNP type. Understanding the intracellular fate is therefore important to design more efficient MNPs through precisely determined size, shape, and surface properties to better fit the desired application, as well as to more precisely evaluate their biosafety.

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Nowadays, most biosafety studies of MNPs are performed using in vitro cell lines, which are far from conditions in vivo as discussed in Section 8.5. Moreover, in vitro systems used for toxicity assays should be carefully selected based on the likelihood of which cell types the NPs could come into contact within the animal or human body. Therefore, in vivo studies on animal models are of great importance as well.

8.5 IN VIVO AND IN VITRO MODELS (CLASSICAL CELL CULTURES, BIOMIMETIC) FOR TESTING NANOPARTICLE TOXICITY AND THEIR PENETRATION THROUGH CELL PLASMA MEMBRANES AND TISSUE BARRIERS MNPs’ small size enables them to easily enter the human or animal body and cross the various biological barriers (Pourmand and Abdollahi, 2012). Once in the body, they come in contact with various tissues and cell types, depending on the route of administration, where they can damage the tissues and cause cell toxicity, immunotoxicity, immunogenicity, and genotoxicity. The severity of damage is often dependent on the time of exposure, therefore rapid action followed by quick elimination from the body is often preferable. Nevertheless, long-term exposures are often imminent. The majority of nanotoxicity studies have been conducted on various short-term in vitro cell models (Mahmoudi et al., 2012), but only a limited number of long-term biomimetic in vitro studies and especially long-term in vivo studies have been done, and there is an urgent need for more research in this area.

8.5.1 THE COMPARISON OF IN VIVO AND IN VITRO MODELS FOR THE RESEARCH INTO MAGNETIC NANOPARTICLE EFFECTS In vivo studies are performed first on animal models (especially mice and rats, but also cats and dogs) and later in clinical trials on humans. Such animal and human studies have shown that in vivo interaction of MNPs and biological systems is quite complicated and very dynamic (Malindretos et al., 2007; Schlachter et al., 2011; Shen et al., 2011). When MNPs enter into the body, they can be distributed into various cells, tissues, and organs, where they may accumulate for a prolonged time in the same type of nanostructure as administered or may become metabolized and therefore change their nanostructure. Small MNPs (,10 nm) are usually rapidly removed from the body through extravasation and renal clearance, while large MNPs ( . 200 nm) are sequestered by the spleen via mechanical filtration. Besides the size of the MNPs, coating material and surface engineering is the main factor that influences pharmacokinetics (i.e., absorption, distribution, metabolism, and excretion) of MNPs in the body (Gupta and Gupta, 2005). On average, the typical final distribution of MNPs after intravenous administration is

8.5 In Vivo and In Vitro Models (Classical Cell Cultures, Biomimetic)

approximately 80%
90% in liver, 5%
8% in spleen, and 1%
2% in bone marrow (Duguet et al., 2006). After inhalation, MNPs were found to be distributed in the brain, liver, spleen, and lungs, showing their ability to cross the blood
brain barrier (Kwon et al., 2008). Nevertheless, the main goal of MNP applications is the use of magnetic targeting, which could enhance the delivery of MNPs to the target organ in the selected organism. Therefore, one of the major issues to test is MNP distribution with and without magnetic targeting. Since within the living organism it is inevitable that MNPs are distributed as mentioned previously, it is imperative to check hepatotoxicity, spleen and pulmonary toxicity, nephrotoxicity, and also immune system activation, hematological toxicity, and oxidative stress among others (Kumar et al., 2012). In vivo studies have shown that MNPs exert their most notable toxic effects in the form of cell death, inflammation, and disturbing the blood coagulation system (Zhu et al., 2008). Moreover, in in vitro studies decreased cell viability has been demonstrated as the most common toxic effect of MNPs, depending on the MNP coating (Delcroix et al., 2009; Ge et al., 2009; Naqvi et al., 2010). All the abovementioned toxic effects of MNPs are mainly due to the production of excess ROS, including free radicals such as the superoxide anion, hydroxyl radicals, and the nonradical hydrogen peroxide (Liu et al., 2013). These generated ROS further damage cells mainly by disrupting DNA, peroxidizing lipids, and altering gene transcription and protein structure (Sharifi et al., 2012; Soenen and De Cuyper, 2010). On the other hand, studies where magnetic targeting is employed after intravenous injection or aerosol delivery of the MNPs showed no toxicity observations in rats and mice, respectively (Chertok et al., 2008; Dames et al., 2007). Taken together, toxicity data generated by employing various in vitro and in vivo models is often conflicting, inconsistent, and inadequate, partially due to the differences in the models used and consequent experimental settings. One of the possibilities to bridge the gap between in vitro and in vivo research is the development of biomimetic in vitro systems, which could, at least to some extent, deepen our knowledge of MNP toxicity and their potential usefulness for biomedical applications. Information on the pharmacokinetics of the MNPs in the human or animal body is necessary for risk assessment purposes, because of their tendency to accumulate. Besides previously mentioned intravenous injection and inhalation, oral, skin (dermal), and urinary bladder exposure could also be addressed as possible route of MNP administration. Therefore, in vivo studies on animal models must be performed in a way to assess the ability of MNPs to cross the lung, gut, skin, placental, blood
brain, and blood
urine barriers. Whereas these in vivo studies offer irreplaceable and comprehensive data on the biodistribution of the MNPs in a living organism, the number of animal studies should be decreased as much as possible for several reasons. The main two reasons are: (1) the use of animals is ethically debatable and (2) animal models do not fully simulate the physiology of humans. Thus, various in vitro models, especially primary and secondary cell cultures, and immortalized cell lines have been developed to study cytotoxic,

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teratogenic, and cancerous effects of NPs, while biomimetic in vitro models enable us to analyze their ability to penetrate through different body barriers. The results of research on such models can help us understand the precise mechanisms of MNP toxicity and thereafter appropriately modify MNPs to decrease toxicity. Moreover, in vitro research enables us to predict in vivo internal exposure and minimize undesirable toxicity of MNPs within the living organism. This kind of approach would enable us to plan in vivo studies with more knowledge about possible effects and complications that different kinds of MNPs could cause. Moreover, in this way we can easily follow the 3Rs (replacement, reduction, and refinement) concept of Russell and Burch regarding animal experiments (Russell and Burch, 1959). Nowadays, the main two focuses are: (1) the development of the appropriate in vitro models that mimic the barriers of the human body, socalled biomimetic in vitro models, and (2) the use of ex vivo models, where small pieces of human tissue are transferred into specially designed chambers to maintain the complexity of the physiological barriers (Saunders, 2009). Another emerging possibility is the concept of organ-on-a-chip models, which are designed as hybrid devices joining cells and microfabricated structures aiming to reconstruct the dynamic physical, cellular, and functional characteristics of human tissues (Bhatia and Ingber, 2014). After appropriate in vitro assays are performed and before the clinical studies, in vivo research aims to estimate the effects of MNPs on the whole organism. Such knowledge is crucial to allow us to design specific, focused, and safe clinical trials aiming to develop new strategies for the applied use of MNPs (Fig. 8.5).

8.5.2 THE ROUTES AND MODEL ORGANISMS OF MAGNETIC NANOPARTICLE ADMINISTRATION One of the most important aspects to consider before starting the in vivo study is the route of MNP administration, especially in higher vertebrates and

FIGURE 8.5 Schematic highlighting the route of testing MNPs from in vitro immortalized cell lines to clinical application.

8.5 In Vivo and In Vitro Models (Classical Cell Cultures, Biomimetic)

mammalians. The most widely used routes of various drugs administration are also applicable to MNP administration. Indeed, the main researched routes of NP administration are: (1) skin or dermal administration, where NPs are incorporated into appropriate gel or cream and applied onto the skin; (2) inhalation, where NPs have to be prepared in the form of an aerosol, which is then taken by the animal by breathing; (3) intravenous or intraperitoneal injection, where NPs in appropriate physiological fluid are administered directly into the blood or into the peritoneal cavity, respectively; (4) oral administration through consummation of the NPs with fluids or food. For skin administration, also referred to as dermal exposure or percutaneous absorption, one of the most suitable model organisms is zebrafish, which are genetically well-defined vertebrates that have several important advantages. The housing and maintenance is not expensive, which makes zebrafish an attractive model for preliminary research of toxicity and/or aquatic environmental contamination. Another advantage is easy extrapolation of the results to other vertebrates and humans. Moreover, the short life cycle of this aquatic vertebrate makes it very suitable for the study of embryo viability and genetic malformation during exposure to MNPs (Griffitt et al., 2009; Hu et al., 2011). Another useful option to test skin administration is the use of nude mice (Lin et al., 2013; Wang et al., 2013a). Although their skin is much thinner than human skin the data about penetration of NPs through the stratum corneum barrier and subsequent localization within the epidermal and dermal layers of the skin could be useful to predict NP distribution within the skin of a larger animal (e.g., pig) or human. For administration via inhalation, most commonly the models of subchronic inhalation in rats and mice are used. These studies performed on different groups of female and male rats attempt to establish a complete data of toxicity and therefore measure the absorption, distribution, metabolism, and excretion of the MNPs (Sung et al., 2011a). For inhalation research different types of protocols are used. One option is the use of inhalation chambers, where animals are housed for different periods of time (Sung et al., 2011b). Another option is an intratracheal instillation of MNPs dispersed in sterile saline (0.9% NaCl) when animals are under anesthesia. For example, recent a in vivo study by Smulders et al. (2016) showed that 84 days after intratracheal instillation of SiO2-Fe3O4 core
shell NPs there is almost complete MNP clearance from the lung, with some distribution to the spleen and kidney. Nevertheless, after intravenous administration of the same MNPs, these MNPs mainly accumulate in the liver, and are retained there for over 84 days (Smulders et al., 2016). A very useful option is to prepare MNPs in a form of inhalable dry powders through spray-drying a feed consisting of MNPs (Stocke et al., 2015). This formulation could be easily used with inhalers, but was not jet-tested on animal models. Intravenous and intraperitoneal injections of MNPs into mice are commonly used routes for systemic administration. These two pathways allow the study of MNP organ distribution after different time points and hematological and biochemical studies to test the toxicity of MNPs in tissues and blood cells.

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Additionally, the evaluation of mutagenic potential, chromosomal aberrations, and genotoxicity for germinal cells could be done (De Jong et al., 2008; Xie et al., 2011). Furthermore, intravenous administration enables us to follow renal excretion and accumulation in the urinary bladder (Shibu et al., 2013). During renal excretion, MNPs come in contact with the epithelium, named the urothelium, which lines the luminal side of the urinary bladder, and could potentially target urothelial cancer cells or urothelial cells infected by uropathogenic Escherichia coli. Moreover, the urinary bladder is an easily reachable organ by catheter, through which MNPs could be applied, and represents great advantages, since systemic application is avoided. Recently it was shown that incorporation of iron oxide MNPs improved MRI contrast of the urinary bladder scaffolds implanted operatively (Sun et al., 2014). Oral administration is usually done by oral gavage to a mice or other animal model or by supplementing the basic diet of the animals with MNPs. Through this administration route, MNPs first come in contact with the esophagus and stomach, before they can be potentially absorbed in the intestine. The stomach acid could damage the NPs and induce dissolution of toxic ions. Moreover, the interaction of MNPs with protons in the stomach could promote an increase in the release of HCO32, which results in an increase of the pH of the stomach, causing damage and electrolyte metabolic alterations (Chen et al., 2006). Nevertheless, several studies employed different NPs for improved oral delivery of insulin and heparin (He et al., 2015: 12; Paliwal et al., 2011).

8.5.3 BIOMIMETIC IN VITRO MODELS REPRESENT THE BRIDGE BETWEEN IN VITRO AND IN VIVO RESEARCH Studies of MNP cytotoxicity and tissue barrier penetration are most commonly investigated by employing biomimetic in vitro models by culturing different types of cells on porous membrane inserts, which are composed of a permeable membrane separating an apical and a basolateral compartment. Cells are seeded and cultured on the inserts to form a barrier (after cells reach confluence) between the two compartments. Depending on the cell type selected, such models can be used to study lung, gastrointestinal, urinary, or placental penetration of MNPs (Braakhuis et al., 2015). They are also used to measure not only uptake of MNPs into the cells from the apical compartment but also efflux from the cells to the basolateral compartment as an extent of permeability and/or translocation (Braakhuis et al., 2015). The majority of MNP toxicity research was until recently performed on different kinds of cell lines such as cancer cell lines (Kocbek et al., 2013; Lojk et al., 2015; Prijic et al., 2012), lung cells, liver cells, fibroblasts, mesenchymal stem cells, kidney cells, macrophages, nerve cells, endothelial cells, mesothelioma cells, keratinocytes, smooth muscle cells, and lymphocytes (Braakhuis et al., 2015; Liu et al., 2013; Mahmoudi et al., 2012). Besides different kinds of cell

8.6 Advantages, Perspectives, and Limitations of Biomimetic

lines used in these studies, another important variable is various kinds of MNPs and various coating materials and coating methods used. This is quite an impressive portfolio of research and the results show that the degree of toxicity varies with cell type, MNP type, and the exposure protocol. The cells of immortalized cell lines often do not possess all phenotypical characteristics of the cells within the organisms. In this respect, the use of primary and secondary cell cultures seems more appropriate. Due to the problems associated with such cell cultures (discussed in detail in Section 8.6), MNPs toxicity tests are less frequently performed on these kinds of cell cultures than on cell lines. Data revealing marginal or no toxicity via in vitro tests led onto in vivo studies, but it was often revealed that the in vitro and in vivo results were contradictory. Therefore, the development of biomimetic in vitro culture systems and ex vivo models was necessary. Indeed, some research on those kind of models was performed with MNPs. Baroli developed a vertical diffusion cell for establishment of full-thickness human skin (Baroli, 2010). Placental BeWo b30 Transwell model was widely used for various nanoparticles (Braakhuis et al., 2015), including MNPs (Correia Carreira et al., 2015). Recently, well-established biomimetic in vitro models of porcine urothelial cells (Viˇsnjar et al., 2012; Viˇsnjar and Kreft, 2013, 2015) were used for long-term assessment of MNP toxicity and endocytosis (Lojk et al., 2017; Skoˇcaj et al., submitted).

8.6 ADVANTAGES, PERSPECTIVES, AND LIMITATIONS OF BIOMIMETIC IN VITRO MODELS VERSUS CLASSICAL CELL CULTURES Generally, over the last 10 years a lot of more or less biomimetic in vitro models have been developed for the investigation of translocations of different kinds of NPs through plasma membranes of the cells and through tissue barriers. The developed biomimetic in vitro models correspond to the most exposed organs and tissues, to which NPs could come in contact within the living organisms, such as skin, lung, gastrointestinal tract, urothelium of the urinary bladder, and even placenta. Although only a few of them were used for MNP testing, the added relevance of obtained results, their recent improvements, validations, and simplified maintenance will undoubtedly encourage researchers to use them instead of classical monolayer cell cultures.

8.6.1 SKIN MODELS Biomimetic in vitro skin models are important especially for the study of negative consequences of dermal exposure of NPs or NP products, such as cosmetics, clothes, or medical gauzes containing NPs. An excellent example of a validated biomimetic in vitro model is that of EpiDerm (Cotovio et al., 2005), which

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FIGURE 8.6 Comparison of normal porcine in vivo (A) and human in vitro (B) skin model. Scale bar for A and B: 50 μm.

represents a normal human 3D model of epidermal tissue as we confirmed also with ultrastructural analysis using electron microscopy (our unpublished data) and on histological paraffin sections shown in Fig. 8.6. This model is permitted by the EU for full replacement of animal models in skin irritation assays. Although it was first designed for testing the dermal absorption of chemicals, it has already been used also for the analysis of transdermal NP penetration (Murthy et al., 2012). Unfortunately, specifically for NP toxicity evaluation, there are no wellestablished guidelines prescribing the type of skin, species, and experimental protocol. Moreover, critical evaluation of current models is missing. Skin biomimetic in vitro models vary in the type of skin membrane, which could be full-thickness skin or dermatomed skin. Human versus animal skin is another important issue. While human skin is considered as the gold standard, a large number of studies on percutaneous penetration of NPs use diverse animal models including mice, rat, and porcine skin (Labouta and Schneider, 2013). The structural and morphological differences between human and animal skin, particularly the density of the hair follicles, thickness of the skin layer, and skin lipid composition, could significantly affect the percutaneous penetration of NPs (Labouta and Schneider, 2013). All these limitations currently hinder the possibility for the comparison and interpretation of the results obtained by the above-mentioned different models (Braakhuis et al., 2015).

8.6.2 LUNG MODELS Classical in vitro models of lungs most frequently use lung epithelial cell lines such as A549, Calu-3, and H441, although those immortalized cells do not have phenotypical characteristics of lung epithelial cells in vivo. For example, cells of the cell line A549 do not form tight junctions (Chowdhury et al., 2010), which is

8.6 Advantages, Perspectives, and Limitations of Biomimetic

the major feature of all epithelial cells regardless of which epithelium they compose. Despite that, the A549 cell line is often referred to as a lung epithelial cell model. In general, one of the greatest advantages of testing on all kinds of cell lines is their low cost, easy establishment, and maintenance. Nevertheless, one must be aware that immortalized cell lines could not be addressed as a model for cells and barriers which employ complexity and tissue architecture within the body of the whole organism. Therefore, for a more credible lung in vitro model, primary rat alveolar cells have been frequently used for testing of different NPs (Fazlollahi et al., 2011; Yacobi et al., 2008). In addition to the differences in cell types, another important issue of in vitro lung barrier models is whether cells are submerged into culture medium or cultured at the air
liquid interface. Submerged models have the advantage of being technically simpler. Nevertheless, the culture medium can alter the properties of the MNPs, and subsequently their penetration and effects. Moreover, air
liquid models mimic more realistically the inhalation administration of NPs in the aerosol phase and thus many air
liquid models have been established recently (Blank et al., 2006; Fro¨hlich et al., 2013; Kreft et al., 2015a,b; Lenz et al., 2009, 2013). Limitations represent the complexity of the system needed to maintain constant temperature and humidification, and the high cost in comparison to submerged models. In recent years, coculture models, which are composed of more than one cell type, are used to biomimic the lung barrier more precisely than monocultures (Klein et al., 2011). In all of those models the lung epithelial cells (primary cultures or immortalized cell lines) are used as a basis, while different cell types such as endothelial cells, dendritic cells, macrophages, type II alveolar cells, fibroblasts, and different combinations of these cells are added to the basic model (Brandenberger et al., 2010; Hermanns et al., 2010; Papritz et al., 2010; Rothen-Rutishauser et al., 2008). Disadvantages of coculture models are their higher cost in comparison with monocultures and their higher technical complexity. Besides that, in cocultures some unexpected interactions between different cell types could emerge and have an influence on the results, which are therefore difficult to interpret. On the other hand, those interactions between different cell types often mimic actual interactions within the living organism and are therefore considered as beneficial.

8.6.3 GASTROINTESTINAL TRACT MODELS Orally administered NPs should first be dispersed in appropriate dietary fluids or gels or encapsulated. After consummation they slowly trespass into the gastrointestinal tract and are therefore exposed to continuously changing conditions, which have an effect on their nature and characteristics (Bellmann et al., 2015). Moreover, orally administered NPs come in contact with different kinds of tissue barriers, mainly with oral mucosa, esophagus, stomach, small and large intestine epithelia, and corresponding proteoglycan layers covering those epithelia. Thus, monoculture of the most widely used gut epithelial cell line Caco-2 is not a reliable model. Usually, coculture and three-culture models are used with the

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addition of human colon adenocarcinoma mucus-secreting cells (HT29-MTX) and/or human intestine microfold (M) cells to the Caco-2 basis (Mahler et al., 2012). Different biomimetic in vitro gastrointestinal models attempt to mimic the gastrointestinal environment as faithfully as possible, to generate physiologically relevant results. In order to do so, these models must focus on one of two main issues. They can focus on mimicking the penetration of NPs through plasma membrane or through whole-tissue/cell layers of either oral, gastric, small intestine, and large intestine and are therefore called static models (Lefebvre et al., 2015). More complex static models often include most of the relevant gastrointestinal conditions and could be to some extent compared to the in vivo situation (Van de Wiele et al., 2007). On the other hand, they can focus on the dynamically altering conditions within the gastrointestinal tract during digestion by simulation of the passing of NPs along the gastrointestinal tract and are therefore named dynamic digestion models. A better understanding of changes in the physicochemical properties of NPs due to their possible digestion in the gastrointestinal tract and consequent changes in the interactions between these NPs and cells is very important.

8.6.4 PLACENTA MODELS Several in vivo animal studies have shown that NPs can penetrate the placenta and cause defects in developing embryos (Cela´ et al., 2014), but the ability and mechanisms of NP penetration are still not well understood. The most relevant models for investigating transplacental penetration of nanoparticles are based on the isolated human placenta used in ex vivo models. This is due to the maintenance of the complexity of the intact placenta, which is ensured in such models (Saunders, 2009). However, penetration studies in the ex vivo intact placenta models are technically difficult to perform properly and require large volumes of NP suspensions to be analyzed. Consequently, biomimetic in vitro models employing representative placental cell lines on porous membrane systems are being developed as an alternative. Human placental cell lines that are usually used are the BeWo, Jar, and JEG-3 cell lines (Buerki-Thurnherr et al., 2012). The furthermost widespread model contains the BeWo cell line, which represents a choriocarcinoma-derived placental cell line that is similar to cytotrophoblastic cells. The BeWo cells are usually grown on Transwell inserts until reaching the confluence. This enables the quantification of both endocytosis into the cells from the apical compartment (maternal side) and exocytosis from the cells to the basolateral compartment (fetal side) (Buerki-Thurnherr et al., 2012).

8.6.5 UROTHELIUM/URINARY BLADDER MODELS Urothelium is the lining of the luminal side of the urinary bladder and establishes the blood
urine barrier, which is the tightest barrier in mammalian organism (Lasiˇc et al., 2015). The cell line that is usually addressed as a urothelium in vitro

8.6 Advantages, Perspectives, and Limitations of Biomimetic

model system is RT4, however these cells are derived from human urinary bladder papillary urothelial neoplasm and do not mimic highly differentiated, functional urothelium. Therefore, it was necessary to develop a biomimetic in vitro model composed of well-differentiated urothelial cells, which exhibited high transepithelial resistance characteristics for the in vivo situation (Viˇsnjar et al., 2012). Although normal human differentiated urothelial cells could be obtained during transurethral resection of the urinary bladder, practice has shown that these cells are usually poorly differentiated and do not grow successfully in vitro. The major reason for this is the fact that they derive from patients with some sort of urinary bladder disorder, most frequently low-grade or high-grade papillary urothelial carcinoma, and therefore even the portions of urothelium that cystoscopically look normal do not contain highly differentiated urothelial cells (Zupancic and Romih, 2013). Another possibility is to obtain highly differentiated urothelial cells from human ureters (Cross et al., 2005), from heart-beating brain-stem dead donors (Garthwaite et al., 2014) or healthy pigs. The pig is a very suitable animal model, since normal porcine urothelial cells have many biological properties equivalent to normal human urothelial cells (Fo¨llmann et al., 2000; Turner et al., 2008). Moreover, as pigs are domestic animals, which are raised for food, their bladders are readily available at low cost in nearly unlimited amounts from local farms, which represents a huge advantage. Finally, recent advances in the field of pig-to-human xenotransplantations (Tonsho et al., 2014) point toward the possibility of using porcine urothelial cells in human tissue engineering applications. Another advantage of such a model is its durability, since normal porcine urothelial cells can easily be grown in cultures for more than 30 days, enabling us to conduct long-term studies (Viˇsnjar and Kreft, 2015). Moreover, by adjusting the concentration of Ca21 in the growth medium one can mimic either normoplastic or hyperplastic urothelium (Viˇsnjar et al., 2012). By culturing of normal porcine urothelial cells on amniotic membrane scaffolds we developed a tissue-engineered urothelium with molecular and ultrastructural properties comparable to those of native urothelium (Jerman et al., 2014). In Fig. 8.7, normal porcine urinary bladder urothelium in vivo in comparison to our established tissue-engineered urothelium in vitro is shown. Even coculturing of porcine normal urothelial cells with porcine urinary bladder fibroblasts and/or smooth muscle cells was successful (Zupanˇciˇc et al., 2018), enabling testing of penetration of NPs not only into the urothelial cells, but also through a biomimetic in vitro model of multilayered bladder wall.

8.6.6 PERSPECTIVES OF BIOMIMETIC IN VITRO MODELS Due to the rapid expansion in the development, research, and use of MNPs in recent years, it is vital that we improve our understanding of MNP interactions with biological systems. Only in this way will we be able to establish safety standards and improve the design of MNPs. Such mechanisms can of course be studied on in vivo models, but the request and necessity for reliable in vitro

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FIGURE 8.7 Comparison of normal porcine in vivo (A) and in vitro (B) urothelial model. Scale bar: for A and B: 50 μm.

experimental models is growing. With the introduction of new regulations intended to limit the use of animals in research, investigators are being forced to design and validate novel in vitro models to perform the experiments (Jerman and Kreft, 2018). Some described in vitro models use primary and secondary cultures, while others use immortalized cell lines. On one hand, in comparison with cell lines, cells of primary and secondary cultures have a more differentiated phenotype closely resembling cells in vivo. On the other hand, the isolation of primary cells and maintenance of highly differentiated and viable secondary cell cultures is often experimentally difficult, since the cells can dedifferentiate after isolation, differ from subculture to subculture, and proliferate to a limited extent and are therefore often of a short duration. Cell lines are easy and low cost to work with, well characterized, and usually more homogeneous, yet they exhibit only few or none of the features of differentiated cells. Overall they very poorly represent the situation in vivo. Therefore, many innovative approaches have been used to develop new biomimetic in vitro models and improve existing ones. When compared to in vivo studies, such models are usually quicker to perform, more cost-effective, and they permit well-controlled experiments. These characteristics make them suitable for performing large-scale screening research, which is of particular value for the field of nanotechnology bearing in mind the huge number of new and improved NPs, including MNPs. Moreover, the usage of human cells in biomimetic in vitro models can make them more representative of the human response to the MNPs when compared to animal models. Obviously, there are also disadvantages and limitations to the employment of biomimetic in vitro models. The major one is the inability to imitate the systemic complex interactions between cells, tissues, and organs, which is constantly occurring in vivo and has a huge influence on the response of the cells and the whole organism to the administration of MNPs. This is due to the fact, that these models are limited to only one or a few different cell types. Although biomimetic in vitro models can be beneficial for studying whether MNPs can cross plasma membranes and to some extent tissue barriers, studies of overall MNP toxicological, pharmacological, cancerous, and teratogenic effects are restricted to animal in vivo models. The significance of in vivo studies

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nevertheless depends on the investigated biological system and the species used. To sum up, biomimetic in vitro models own numerous benefits making them to some degree more valuable than in vivo models and by using them in combination with in vivo models, they can provide an invaluable link between preclinical animal studies and human clinical trials.

8.7 CONCLUSIONS MNPs are one of the most promising NP types, not only in diagnostic, visualization, and tracking applications such as MRI and cell labeling, but also in therapies such as magnetic fluid hyperthermia for innovative targeted drug delivery. Their unique magnetic properties enable targeting by precisely applied magnetic field or enables retention of MNPs in desired locations within the tissues and organs. Nevertheless, systemic applications of MNPs can cause undesired effects, including accumulation in certain organs or too rapid clearance from the body, hindering translation into clinical settings. Besides the standard physicochemical characterization and different mobility assays, it is therefore of great importance to test MNPs in living cells, tissues, and organisms, including humans. The most widely used models to test MNPs are in vitro immortalized cell lines, which enable us to test MNP mechanisms of uptake, their intracellular fate, and potential cytotoxicity and genotoxicity. Although such classical in vitro analysis is a first step in development of new MNP formulations, an additional evaluation on advanced biomimetic in vitro models is crucial before planning studies on animal models and clinical trials. However, due to the complexity of those models, their further improvements and standardizations are needed to explore promising new biomedical applications of MNPs and to efficiently design new MNP formulations.

ACKNOWLEDGMENTS The authors thank V.B. Bregar who developed PAA-coated cobalt-ferrite nanoparticles. This work was supported by the Slovenian Research Agency within projects J3-7494, J26758, J3-6794, J7-7424, P3-0108, P1-0055, Z4-8229, Young Researchers Program, and ˇ MRIC UL IP-0510 Infrastructure Program. The authors acknowledge to Sanja Cabraja, ˇ ˇ Linda Strus, Nada Pavlica Dubariˇc, and Sabina Zeleznik for technical assistance.

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Aluminosilicate-based composites functionalized with cationic materials: possibilities for drugdelivery applications

9

ˇ Danina Krajiˇsnik, Bojan Calija and Jela Milic´ Department of Pharmaceutical Technology and Cosmetology, University of Belgrade-Faculty of Pharmacy, Belgrade, Serbia

9.1 INTRODUCTION Composites are materials consisting of at least two materials combined to obtain a material with characteristics different from its constituents, whereas the constituents remain separate and distinct within a composite structure (Zafar et al., 2016). One of the starting materials is usually designated as continuous phase or matrix, and the other, used to improve the properties of the continuous phase is referred to as the reinforcement agent. This approach could be used to improve the drug-delivery properties of various materials of both natural and synthetic origin. These properties include prolonged/delayed drug release, drug loading, stability, biocompatibility, selectivity for target tissues, etc. Aluminosilicates have been traditionally used as excipients in pharmaceutical preparations due to their biocompatibility, high surface area, ion-exchange ability, and excellent thermal and mechanical stability (Lopes et al., 2014). Modification/ functionalization of aluminosilicates with various materials can be employed to obtain composite materials with improved functional characteristics, such as drug-loading capacity, swelling and drug-release properties, sensitivity to biological stimuli, and mechanical and rheological characteristics. This chapter summarizes the most important properties of aluminosilicates as drug carriers and discusses the preparation, characterization, and functional properties of aluminosilicate-based composite materials intended for drug delivery functionalized with two distinctive groups of cationic materials: cationic surfactants and chitosan. Additionally, the structure, physicochemical characteristics, and safety of these diverse cationic materials are given for a better understanding.

Materials for Biomedical Engineering: Inorganic Micro- and Nanostructures. DOI: https://doi.org/10.1016/B978-0-08-102814-8.00010-X © 2019 Elsevier Inc. All rights reserved.

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9.2 ALUMINOSILICATES AS DRUG CARRIERS—PROPERTIES, ADVANTAGES, AND LIMITATIONS Clays belong to a category of silica layered ingredients that are commonly used in the pharmaceutical industry, either as ingredients or in combination, in composites and hybrids (Jafarbeglou et al., 2016). There is increasing interest in the use of clay minerals as agents on which drugs can be adsorbed and then delivered to target sites in the body in a manner which allows for controlled release of the drug (Zhou and Keeling, 2013). According to the literature, clays commonly used in pharmaceutical products are kaolinite, talc, smectites [montmorillonite (MMT), saponite, and hectorite], palygorskite, sepiolite, etc. (Carretero et al., 2013; Rodrigues et al., 2013; Rowe et al., 2009; Viseras et al., 2010; Viseras and Lopez-Galindo, 1999). The interest in clays as drug carriers amongst the scientific community has increased significantly in recent years due to their composition, which can be easily modified to serve different purposes (Aguzzi et al., 2007; Chrzanowski et al., 2013; Park et al., 2016; Rodrigues et al., 2013; Viseras et al., 2010; Yu et al., 2013; Zhou and Keeling, 2013). The European Pharmacopoeia 9.0 (Ph. Eur. 9.0; EDQM, 2017) describes bentonite as a natural clay containing a high proportion of MMT (Al2O34SiO2H2O), a native hydrated aluminum silicate in which some aluminum and silicon atoms may be replaced by other atoms such as magnesium and iron. Hectorite is a naturally occurring 2:1 phyllosilicate clay of the smectite (MMT) group and is a principal component of bentonite clay. The USP32
NF27 describes magnesium aluminum silicate as a blend of colloidal MMT and saponite that has been processed to remove grit and nonswellable ore components (Rowe et al., 2009). A similar definition for aluminum magnesium silicate is given in Ph. Eur. 9.0. Based on the favorable physicochemical properties, such as high specific surface area, adsorption capacity, ion exchange capacity, colloid and thixotropy, swelling property, chemical inertness, and low toxicity for oral administration, these naturally occurring substances have been used in various pharmaceutical dosage forms as adsorbents, stabilizing agents, suspending agents, viscosity-increasing agents, tablet and capsule disintegrants, or tablet binders (Rowe et al., 2009). In addition to aluminosilicates of pharmacopoeial quality, other representatives of tubular clays (halloysite) and tectosilicates (zeolites) have been investigated as potential drug carriers. Halloysite (Al2Si2O5(OH)4nH2O) is an abundant and inexpensive aluminosilicate clay chemically similar to kaolin, with hollow tubular geometry and length from 500 to 1500 nm, outer diameter between 40 and 60 nm, and inner diameter between 10 and 15 nm (Lvov et al., 2008, 2016). Its nanotubular geometry, biocompatibility, negatively charged inner surface, and positively charged outer surface under physiological conditions and high surface to volume ratio, make this clay material particularly interesting as a carrier for both positively and negatively charged drug molecules, with relatively high drug loading (10
30 wt.%) and prolonged release (from a few hours up to few months) (Lvov et al., 2016).

9.2 Aluminosilicates as Drug Carriers—Properties

A zeolite is a crystalline, hydrated aluminosilicate of alkali and alkaline earth cations having an infinite, open, three-dimensional structure, which is further able to lose and gain water reversibly and to exchange extraframework cations, both without change of crystal structure (Mumpton, 1999). Depending on the structure, mineralogical and petrographical features, and chemistry, natural zeolites exhibit a series of properties such as: (1) alkaline nature, (2) cation exchange, (3) physical sorption, and (4) external surface activity, which are directly or indirectly involved in reactions and/or processes of biochemical relevance (Colella, 2011). Among a wide variety of zeolites which have been identified in sedimentary deposits, clinoptilolite [a mineral from the heulandite group of zeolites, (Na, K)6(Al6Si3O)O72nH2O], is the most widespread and marketed worldwide, and up to date, it has been the most investigated for pharmaceutical applications (Cerri et al., 2016; Colella, 2011; Krajiˇsnik et al., 2017; Mili´c et al., 2014). The use of clinoptilolite has been demonstrated for purposes of both an active ingredient (in the external skin treatment, as an antidiarrheal, or as an antacid drug) and functional drug carrier enabling modified (prolonged) drug release. In addition to previously mentioned properties involved in reactions and/or processes relevant for its biomedical applications, the possibility of external surface property alterations via modification with organic molecules, that is, long-chain cationic surfactants, is the focus of researches relevant for their applications as drug carriers (Cerri et al., 2016; Colella, 2011; Krajiˇsnik et al., 2017; Mili´c et al., 2014). The way and degree to which an excipient meets the intended function in the final formulation is referred to as excipient functionality (Mili´c et al., 2017). Controllable physical or chemical characteristics affecting excipient functionality are known as functionality-related characteristics (Ph. Eur. 9.0; EDQM, 2017). Since the 5th edition of the European Pharmacopoeia (Ph. Eur. 5.0; EDQM, 2005), a number of excipient monographs have contained a nonmandatory section on functionality-related characteristics (FRCs). The aim of this section is to provide users with a list of physical and physicochemical characteristics that are critical to the typical uses of the concerned excipient, and to provide the general methods required to assess these characteristics. For diverse mineral excipients listed in Ph. Eur. 9.0, characteristics such as specific surface area, particle-size distribution, swelling power with water, or sedimentation volume, are recognized as being relevant control parameters for one or more functions of the substance when used as an excipient. Additionally, numerous investigations of aluminosilicates have revealed that ion exchange and external surface sorption ability, thermal properties, X-ray powder characteristics, bulk and tapped density, as well as powder flow properties, could be also considered as functional characteristics of these materials as drug carriers in various dosage forms. The application of natural aluminosilicates as biocompatible and environmentally friendly drug carriers is an advantageous feature, but with certain limitations, such as low encapsulation efficiency and drug-loading capacity, and sometimes even unwanted drug release kinetics. Aiming to overcome these limitations, aluminosilicate-based drug carriers can be functionalized by various materials, such as polymers and surfactants.

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9.3 ALUMINOSILICATE-BASED DRUG CARRIERS FUNCTIONALIZED WITH CATIONIC SURFACTANTS 9.3.1 CATIONIC SURFACTANTS—PROPERTIES AND PHARMACEUTICAL APPLICATIONS Certain compounds, because of their chemical structure, have a tendency to accumulate at the boundary between two immiscible phases. Such compounds are termed amphiphiles, surface-active agents, or surfactants. Surfactants have a characteristic structure, possessing both polar (hydrophilic, i.e., water-liking) and nonpolar (hydrophobic, i.e., water-hating) regions in the same molecule. The existence of two such regions in a molecule is referred to as amphipathy and the molecules are consequently often referred to as amphipathic molecules (from the Greek word “αμϕις, amphis” meaning “both”). The hydrophobic portions (typically referred as to tails) are usually saturated or unsaturated hydrocarbon chains, although fluorocarbon and siloxane chains can be used or, less commonly, heterocyclic or aromatic ring systems. The hydrophilic portions (referred as to head groups) can be nonionic, ionic, or zwitterionic, accompanied by counter ions in the last two cases (Attwood, 2013; Corrigan and Healy, 2006; dos Santos et al., 2013; Tadros, 2014). Surfactants are generally classified according to the nature of the hydrophilic group within the molecule. Four main classes of surfactants may be distinguished, namely anionic, cationic, amphoteric (ampholytic or zwitterionic), and nonionic (Attwood, 2013; Corrigan and Healy, 2006; Myers, 2006). Additionally, a fifth class of surfactants, usually referred to as polymeric surfactants, has been used for many years for the preparation of emulsions and suspensions and their stabilization (Jain et al., 2013; Tadros, 2014). Structures of some typical surfactants are shown in Fig. 9.1. Due to their amphiphilic nature, surfactant molecules display two very interesting and useful properties: reduction of the surface tension when adsorbing at a specific interface (i.e., air
water or oil
water) and the ability to self-associate and self-organize (dos Santos et al., 2013). The driving force for surfactant adsorption is the lowering of the free energy of the phase boundary (at the air
liquid, the oil
water, or the solid
liquid interface), while the degree of surfactant adsorption at the interface depends on the surfactant structure and the nature of the two phases that meet the interface (Tadros, 2014). Micellization is an alternative to interfacial adsorption for removing hydrophobic groups from contact with the aqueous environment, thereby reducing the free energy of the system (Corrigan and Healy, 2006). The concentration at which micelles (aggregates, usually spherical, of colloidal dimensions) are formed in solution is termed the critical micelle concentration or CMC (Attwood, 2013; Jain et al., 2013). Micellar shape can be affected by changes in temperature, concentration, and the presence of added electrolyte to the liquid phase. Changes in any of these factors may affect micellar size, shape (from small spherical over elongated cylindrical to the large vesicular), and aggregation number (number of surfactant monomers in the micelle) (Corrigan and Healy, 2006; dos Santos et al., 2013; Nagarajan, 2014).

9.3 Aluminosilicate-Based Drug Carriers Functionalized

FIGURE 9.1 Typical surfactant representatives.

Surfactants find wide application in science and almost every chemical industry from primary production processes, such as the recovery and purification of raw materials in the mining and petroleum industries, to a wide range of

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commercial and consumer uses in paints, detergents, antistatic, wetting and softening agents, fibers, plastics, etc., alongside cosmetic, pharmaceutical and agrochemical applications (Jadhav et al., 2017; Maddan and Tyagi, 2008; Myers, 2006; Tadros, 2014). The adsorption of surfactants at the various interfaces between solids, liquids, and gases results in changes in the nature of the interface, which are of considerable importance in pharmacy (Attwood, 2013). Because of their unique functional properties, surfactants find a wide range of uses in various pharmaceutical preparations (Corrigan and Healy, 2006; Jain et al., 2013).

9.3.1.1 Physicochemical properties of cationic surfactants In the molecule of cationic surfactant, the hydrophilic part consists of a positively charged group which is, almost invariably, centered on one or more nitrogen atoms (Cross, 1994). There are two important categories of cationic surfactants that differ mainly in the nature of the nitrogen-containing group (Myers, 2006). The first consists of the alkyl nitrogen compounds, such as simple ammonium salts containing at least one long-chain alkyl group and one or more amine hydrogen atoms: 2 Cn H2n11 NHR1 2X

and quaternary ammonium compounds (quats for short) in which all amine hydrogen have been replaced by organic substituents: 2 Cn H2n11 NR1 3X

The amine substituents may be long-chain or short-chain alkyl, alkylaryl, or aryl groups (Fig. 9.1B1 and B2). The counterion may be a halide, sulfate, acetate, or similar compound. The second category contains heterocyclic materials typified by the pyridinium, morpholinium, and imidazolinium derivatives (Fig. 9.1B3). Other cationic functionalities are also possible, but are much less common than these two major groups. Cationic surfactants are generally water-soluble when there is only one long alkyl group, but the product becomes dispersible in water and soluble in organic solvents if there are two or more long-chain hydrophobes in their structure. They are generally compatible with most inorganic ions and hard water, stable to pH changes, both acid and alkaline (quats retain their cationic character at any pH, unless molecular breakdown occurs) (Cross, 1994) and compatible with nonionic surfactants. Cationic surfactants are incompatible with metasilicates and highly condensed phosphates, protein-like materials and with most anionic surfactants, but they are generally chemically stable and can tolerate electrolytes. The CMC of cationic surfactant is close to that of anionics with the same alkyl chain length (e.g., the CMC of benzalkonium chloride is 0.17%) (Tadros, 2014).

9.3 Aluminosilicate-Based Drug Carriers Functionalized

One of the most important applications of cationic surfactants is based on their tendency to adsorb a variety of negatively charged solid surfaces. Therefore, the representatives of this class of surfactants are used as anticorrosive agents for steel, flotation collectors for mineral ores, dispersants for organic pigments, antistatic agents for plastics, antistatic agents and fabric softeners, hair conditioners, anticaking agents for fertilizers, bactericides, etc. (Cross, 1994; Maddan and Tyagi, 2008; Myers, 2006; Parida et al., 2006; Tadros, 2014). The majority of minerals and a high proportion of organic substances present surfaces that have high energy and are hydrophilic and polar in nature. For example, minerals with high silica content possess surface 2 OH groups that engage readily in ion exchange with cationic surfactants leaving the solid with a hydrophobic coating (Cross, 1994): 2Si 2 O 2 H 1 R4 N1 5 2 Si 2 O 2 R4 N 1 H1

The displacement of inorganic cations, such as sodium and magnesium, from the surface of clay particles by quaternary ammonium anions, results in the formation of organophilic clays (Cross, 1994). The most suitable clays for this modification are the bentonite (clays) type with a high cation-exchange capacity (Cross, 1994; Sarkar et al., 2012; Sun et al., 2013). The end product of clay modification with cationic surfactants already has various industrial and environmental applications (Park et al., 2013; Khenifi et al., 2009; Xi et al., 2005; Yariv et al., 2011).

9.3.1.2 Pharmaceutical application of cationic surfactants Cationic surfactants first became important when the commercial potential of their bacteriostatic properties was recognized in the 1930s (Jungermann, 1970). Since then, these materials have been introduced for application in pharmaceutical and cosmetic products, and environmental as well as biological fields, which led to an increase in their economic importance due to their unique properties (Begum et al., 2016; Brayfield, 2014; Cross, 1994; De Guertechin, 2009; Jadhav et al., 2017; Myers, 2006; Rowe et al., 2009). The quaternary ammonium and pyridinium cationic surfactants have bactericidal activity against a wide range of Gram-positive and, at higher concentration, against some Gram-negative bacteria. They are ineffective against bacterial spores, having variable antifungal activity, and are effective against some viruses (Brayfield, 2014). The bactericidal effect of positively charged quaternary ammonium compounds could be attributed to their electrostatic interaction with negatively charged biological lipid membranes of bacteria, fungi, and other microorganisms (Jadhav et al., 2017). Moreover, it has been proposed that the cationic agents hypothetically react with the phospholipid components in the cytoplasmic membrane, thereby producing membrane distortion and protoplast lysis under osmotic stress (Jadhav et al., 2017; Vieira and Carmona-Ribeiro, 2006). Quaternary ammonium compounds are most effective in neutral and slightly alkaline solution, and their bactericidal activity is appreciably reduced in acid

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media; their activity is enhanced by alcohols (Brayfield, 2014). Due to their bactericidal action, quaternary ammonium compounds have been used as antiseptics or disinfecting agents, preservatives, and for sterilizing contaminated surfaces or surgical instrument disinfection. In addition, they also have emulsifying and detergent properties. They may be used on the skin, especially in the cleaning of wounds (Brayfield, 2014; Jadhav et al., 2017; Rowe et al., 2009). To date, a significant number of quaternary ammonium compounds, such as benzalkonium chloride, benzethonium chloride, benzododecinium bromide, benzododecinium chloride, benzoxonium bromide, cetalkonium chloride, cetrimide, cetrimonium bromide, cetrimonium chloride, and cetylpyridinium chloride have been used as constituents of various pharmaceutical preparations (Anderson et al., 2006; Brayfield, 2014; Fahr, 2018; Rowe et al., 2009). The most important representatives, their typical properties, and applications in pharmaceutical formulation or technology are presented in Table 9.1. Table 9.1 Cationic Surfactants: Definitions, Functional Categories, and Applications in Pharmaceutical Formulations and Technology (Brayfield, 2014; Rowe et al., 2009)

Surfactant

Functional Category

Benzalkonium chloride (Fig. 9.1B2) C19H42BrN, H2O [CAS—800-54-5] Definition (Ph. Eur. 9.0): Mixture of alkylbenzyldimethylammonium chlorides, the alkyl groups mainly having chain lengths of C12, C14, and C16

Antimicrobial preservative; antiseptic; disinfectant; solubilizing agent; wetting agent

Cetrimide C19H42BrN, H2O (Mr 364.5) [CAS—1119-97-7 (trimethyltetradecylammonium bromide); 1119-94-4 (dodecyltrimethylammonium bromide); 8044-71-1 (cetrimide)]. Definition (Ph. Eur. 9.0):

Antimicrobial preservative; antiseptic; cationic surfactant; disinfectant

Applications in Pharmaceutical Formulations or Technology Antimicrobial preservative in applications similar to other cationic surfactants, such as cetrimide; in ophthalmic preparations, it is one of the most widely used preservatives at a concentration of 0.01%
0.02% w/v; it is also used as preservative in nasal, otic, and small-volume parenteral preparations Antimicrobial preservative in pharmaceutical formulations; cationic surfactant; preservative (in eye drops) at a concentration of 0.005% w/v; topical antiseptic, generally as 0.1
1.0% w/v aqueous solutions cream or spray for skin, burns, and wounds; solutions containing up to 10% w/v cetrimide are used as (Continued)

9.3 Aluminosilicate-Based Drug Carriers Functionalized Table 9.1 Cationic Surfactants: Definitions, Functional Categories, and Applications in Pharmaceutical Formulations and Technology (Brayfield, 2014; Rowe et al., 2009) Continued

Surfactant

Functional Category

Cetrimide consists of trimethyltetradecylammonium bromide and may contain smaller amounts of dodecyland hexadecyltrimethylammonium bromide

Cetylpyridinium chloride (Fig. 9.1B3) C21H38ClN, H2O (Mr 358.0) [CAS—7773-52-6 (cetylpyridinium); 123-03-5 (anhydrous cetylpyridinium chloride); 6004-24-6 (cetylpyridinium chloride, monohydrate)]. Definition (Ph. Eur. 9.0): Cetylpyridinium chloride contains not less than 96.0% and not more than the equivalent of 101.0% of 1hexadecylpyridinium chloride, calculated with the reference to the anhydrous substance Cetrimonium bromide (Fig. 9.1B1) C19H42BrN, H2O (Mr 364.5) [CAS—6899-10-1 (cetrimonium); 57-09-0 (cetrimonium bromide, sin. Hexadecyltrimethylammonium bromide)]. Definition (USP): Cetrimonium bromide contains not less than 96.0% and not more than 101.0% of hexadecyltrimethylammonium bromide, calculated as C19H42BrN, on dried substance.

Applications in Pharmaceutical Formulations or Technology shampoos to remove the scales in seborrheic dermatitis; cleanser and disinfectant for hard contact lenses, although it should not be used on soft lenses; ingredient of cetrimide emulsifying wax, and in o/w creams (e.g., cetrimide cream)

Antimicrobial preservative; antiseptic; cationic surfactant; disinfectant; solubilizing agent; wetting agent

Cetylpyridinium chloride is a quaternary pyridinium antiseptic with actions and uses similar to those of other cationic surfactants (see Cetrimide). It is used chiefly as lozenges or solutions for the treatment of minor infections of the mouth and throat. It is also used topically for the treatment of skin and eye infections, and as antimicrobial preservative in pharmaceutical formulations

Cetrimonium bromide is a quaternary ammonium antiseptic with actions and uses similar to those of other cationic surfactants (see Cetrimide)

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As listed in Table 9.1, cationic surfactants are included in various pharmaceutical preparations (inhalations, IM injections, nasal, ophthalmic, otic, and topical), and some of the representatives (such as benzalkonium chloride, benzethonium chloride, cetylpyridinium chloride, cetrimonium chloride) are included in the FDA database of inactive ingredients (FDA Inactive Ingredient Database, 2018c) for parenteral and nonparenteral routes of applications. Although their usage within recommended concentrations is usually well tolerated, prolonged and repeated contact with the skin and mucosal membranes can occasionally cause irritation and hypersensitivity (Brayfield, 2014). If ingested orally, cetrimide and other quaternary ammonium compounds can cause nausea, vomiting, muscle paralysis, CNS depression, and hypotension; concentrated solutions may cause esophageal damage and necrosis (Rowe et al., 2009). Besides its common use for antiseptic purposes in various pharmaceutical formulations, cationic surfactants have also been employed as solubilizers in drug release testing (Abrahamsson et al., 1994; Huang et al., 2011; Park and Choi, 2006; Ullah et al., 2014), to control the release rate of drugs from various matrices (Nokhodchi et al., 2008; Nokhodchi et al., 2002), as well as for the modification of micro- and solid lipid nanoparticles (Briones et al., 2001; Singh et al., 2003; Wischke et al., 2006; Yu et al., 2009). The strong electrostatic interaction of quaternary ammonium compounds with the bacterial cell wall is advantageous, especially when used as surfactants in nanoformulations for targeted and sitespecific drug delivery (Jadhav et al., 2017). Therefore, these surfactants have been used for multiple purposes, such as bactericides and steric stabilizers for nanoparticle formulation, and as components of the vesicles for antibiotics, peptide, or gene delivery (Botequim et al., 2012; Castillo et al., 2004; Cui et al., 2010; Jadhav et al., 2017; Ma et al., 2010; Zakharova et al., 2016). Additionally, in the last two decades, several cationic surfactants have been intensively investigated by means of surface modification of natural aluminosilicates (mostly zeolites) in order to obtain functionalized materials for their potential biomedical application.

9.3.2 PREPARATION AND CHARACTERIZATION OF SURFACTANT-MODIFIED ALUMINOSILICATES As previously mentioned, the chemical modification of aluminosilicate external surfaces with cationic surfactants has been intensively studied in the last two decades, as a promising approach for designing materials with novel surface properties, such as providing a high affinity for organic, that is, drug molecules. According to the literature, the most investigated minerals for this purpose were representatives of tectosilicates (zeolites) (Colella, 2011; Mili´c et al., 2014; Krajiˇsnik et al., 2017; Paveli´c and Hadˇzija, 2003). The preparation of surfactant-modified aluminosilicates is based on the interaction of cationic surfactant molecules with a porous three-dimensional

9.3 Aluminosilicate-Based Drug Carriers Functionalized

aluminosilicate framework which is negatively charged (due to the isomorphic substitution of some of the quadrivalent Si with trivalent Al), and normally neutralized by extraframework mono (mainly Na1 and K1) and/or divalent (mainly Ca21 and Mg21) cations that are commonly exchangeable. Since surfactant mole˚) cules are too large to enter zeolite channels (molecular dimensions of 3
10 A (Wang and Nguyen, 2016) or to access internal cation-exchange positions, their sorption is limited to the external surfaces of the zeolite particles. The external cation exchange capacity (ECEC) characterizes the exchange capacity of the mineral surfaces for surfactants and in addition to surface area (ESA), and the external surface charge density (ESCD 5 ECEC/ESA) is one the key parameters in the evaluation of the external surface activity of zeolites. These parameters depend, besides the zeolite nature and structural features, on several other variables, such as grain size, framework charge density, that is, Si/Al ratio of the zeolite, extraframework cation composition and, in the case of zeolite-rich rocks, on zeolite content, mineral composition, etc. (Colella, 2011). The adsorption of cationic surfactants such as hexadecyltrimethylammonium bromide or cetylpyridinium chloride onto a negatively charged zeolitic surface involves both cation exchange and hydrophobic binding. Namely, during this process, two different products could be obtained (Colella, 2011; Haggerty and Bowman, 1994; Sullivan et al., 1998): 1. Formation, via cation exchange, of a monolayer or vhemimicellev at the solid 2 aqueous interface when the amount of surfactant is below ECEC of zeolite. The zeolite retains most of its total CEC, and simultaneously it becomes enriched in organic carbon and acquires additional capacity to adsorb or dissolve nonpolar organic molecules; 2. Formation of a bilayer vadmicellev on the external surface of zeolites at surfactant concentrations above ECEC of zeolite. The bilayer formation results in charge reversal on the external surface, providing sites where anions will be retained, cations repelled, while neutral species can partition into the hydrophobic core. The possible arrangement of surfactant molecule (hexadecyltrimethylammonium bromide) at the zeolitic surface, in accordance with the above concept, is presented in Fig. 9.2. Conversion of the negative zeolite surface charge by modification with cationic surfactants enabled their application in environmental remediation for removal of hydrophobic organic pollutants (such as benzene, toluene, and xylene) and inorganic anions (e.g., chromate, selenate, sulfate, arsenate, antimonate, and nitrate) from contaminated water (Bowman et al., 1995; de Gennaro et al., 2014; Luo et al., 2017; Seifi et al., 2011; Sullivan et al., 2003; Torabian et al., 2010; Wingenfelder et al., 2006). Surfactant-modified zeolites have also been investigated as adsorbents for mycotoxins, metabolites, and pollutants (Benkli et al., 2005; Dakovi´c et al., 2010; Hrenovic et al., 2008; Markovi´c et al., 2017; Roˇzi´c et al., 2009). Furthermore, interactions of cationic surfactants with natural zeolites were extensively studied, since surfactant-modified zeolites proved to be excellent adsorbents for various drug molecules, contributing to the possibility of their

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FIGURE 9.2 Concept of surfactant adsorption at the zeolitic surface (below and above ECEC). ˇ ´ J., Dakovic, ´ A., Krajisnik, Reprinted with permission from Milic, D., Rottinghaus, G.E., 2014. Modified natural zeolites
functional characterization and biomedical application. In: Tiwari, A. (Ed.), Advanced Healthcare Materials. John Wiley & Sons, Inc., and Scrivener Publishing LLC, Hoboken, pp. 361
403. Copyright (2014) Wiley.

application in drug delivery (Colella, 2011; Krajiˇsnik et al., 2017; Mili´c et al., 2014; Serri et al., 2017). Preparation of the drug
surfactant-modified zeolite composites was, in most cases, carried out by batch adsorption method (also denoted as liquid phase adsorption), which included two steps: in the first one, the surfactant-modified zeolites (SMZs) were prepared, followed by drug adsorption from a solution (generally aqueous) in the second one. It is important to emphasize that adsorption from aqueous solutions is a useful and safe alternative to already reported organic media such as methanol, n-hexane, dimethyl sulfoxide, dimethylformamide, and diethylether, employed for drug adsorption on both natural and synthetic aluminosilicates (Charnay et al., 2004; Horcajada et al., 2006; Khodaverdi et al. 2014; Rimoli et al., 2007). To date, the most commonly used zeolite for preparation of SMZs is mineral clinoptilolite (de Gennaro et al., 2015; Farı´as et al., 2011; Krajiˇsnik et al., 2010, 2011, 2013a,b, 2015; Nezamzadeh-Ejhieh and Tavakoli-Ghinani, 2014; Rivera and Farı´as, 2005; Serri et al., 2017), while other natural zeolites, such as phillipsite and chabazite have been also investigated recently (Cappelletti et al., 2017; de Gennaro et al., 2016; Markovi´c et al., 2017; Serri et al., 2016). The cationic surfactants used in these investigations were hexadecyltrimethylammonium bromide (HB), benzalkonium chloride (BC), and/or cetylpyridinium chloride (CPC), while sulfamethoxazole, metronidazole, diclofenac sodium, diclofenac diethylamine, ibuprofen, and/or cephalexin were utilized as drug molecules. In brief, during modification procedures the surfactant concentrations were below, equal to, or above ECEC (e.g., ranging from 25% to 400%) of the starting zeolite, at a

9.3 Aluminosilicate-Based Drug Carriers Functionalized

constant solid/liquid ratio. Suspensions were shaken, usually at room temperature, taking into account sufficient time to reach equilibrium, which was typically from 24 to 48 hours. Additionally, modification procedures were also performed at much higher speeds (5000
18,000 rpm) within a much shorter activation time (usually 10
15 minutes). Afterwards, the solid phase was separated (by filtration or centrifugation) and dried, resulting in the preparation of SMZs. Preparation of drug
SMZ composites was also investigated in a one-step procedure (i.e., direct method) from drug (diclofenac sodium or aceclofenac)/cationic surfactant solutions, containing the surfactants (HB) and in an amount equivalent to ECEC of the starting zeolite (clinoptilolite) (Krajiˇsnik et al., 2012, 2013c). Numerous techniques are involved in the characterization of both SMZs and drug
SMZ complexes/composites. Theoretical investigations of surfactants and drugs with natural zeolite (clinoptilolite) were performed using semiempirical calculations to study the interactions of the clinoptilolite channel model with two surfactants (surfactant
zeolite system) and three drugs (drug
zeolite system) (Lam and Rivera, 2006). Atomic emission spectroscopy with inductively coupled plasma (ICP-AES) was performed to determine the silicon-to-aluminum ratio and the composition of the original natural zeolite and the surfactant-modified sample, while X-ray diffraction (XRD) analysis was used for the evaluation of structural changes in SMZs compared to the starting material (Farı´as et al., 2010). Zeta potential measurements of SMZs and drug
SMZ composites compared to untreated starting material were valuable for the determination of the organic phase organization at the composite surface, that is, transition from a monolayer to a patchy bilayer (Krajiˇsnik et al., 2013a, 2015; Markovi´c et al., 2017), while additional structural information was obtained using Fourier transform infrared (FTIR) spectroscopy (Krajiˇsnik et al., 2013a, 2015; Markovi´c et al., 2017; Nezamzadeh-Ejhieh and Tavakoli-Ghinani, 2014). Thermal properties of the composites were evaluated using thermogravimetric (TG), differential thermogravimetric (DTG), and differential thermal analysis (DTA) (Farı´as et al., 2011; Krajiˇsnik et al., 2010, 2013b; Markovi´c et al., 2017; Nezamzadeh-Ejhieh and Tavakoli-Ghinani, 2014; Rivera and Farı´as, 2005). To study the probable changes of morphology for ground zeolite powders after ball milling, scanning electronic microscopy (SEM) was used (Nezamzadeh-Ejhieh and Tavakoli-Ghinani, 2014). Laser light scattering (LLS) of SMZs enabled determination of the mean volume diameter and size distribution of the particles (de Gennaro et al., 2015). Confocal laser scanning microscopy (CLSM) observations were used for the discovery of the localization of different molecules in/on SMZs according to their chemical nature (de Gennaro et al., 2015; Serri et al., 2016). Pharmaceutical/technical characterization of SMZs and drug-SMZ composites was performed related to their application in the formulation of an oral dosage form. Powder flowability, Carr’s index, and Hausner ratio were determined according to standard pharmacopoeial procedures (de Gennaro et al., 2015; Krajiˇsnik et al., 2010; Serri et al., 2016). Dissolution tests for drug
SMZ composites were conducted according to pharmacopoeial requirements for solid dosage

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forms using a paddle apparatus (Krajiˇsnik et al., 2013a,b, 2015; Serri et al., 2017) and other nonpharmacopoeial equipment that ensured sink conditions during the testing period (de Gennaro et al., 2015; Farı´as et al., 2011; Rivera and Farı´as, 2005; Serri et al., 2016). Complete biopharmaceutical evaluation of the tested composites was accompanied with analysis of drug release profiles (Farı´as et al., 2011; Krajiˇsnik et al., 2013a, 2015) and biocompatibility (via acute toxicity, cytotoxicity, and cytokine production) (Krajiˇsnik et al., 2014; Serri et al., 2017). Besides SMZs, other organoclays combining the properties of catanionic surfactants and clay minerals as new materials with potential uses in medicine, wastewater treatment, and antibacterial applications have been investigated recently (Yapar et al., 2015). The aggregates formed in an aqueous solution by simultaneously dissolved single-tailed anionic and cationic surfactants are described as catanionic systems (Bramer et al., 2007; Caillet et al., 2000; Ghosh et al., 2006; Yapar et al., 2015). The surfactants chosen to make the catanionic surfactant were cetylpyridinium (CP) and lauroyl sarcosinate (SR), which interact strongly in aqueous media and cause specific aggregations, such as ion-pair amphiphiles and needle- and leaflike structures. New ternary systems through the addition of MMT were formed. The surface and interlayer structures of the different MMT-CP-SR samples using CP and SR in amounts equal to various ratios of cationic exchange capacity of the clay mineral were studied. TG and XRD analyses in addition to zeta-potential measurements were used to elucidate the interlayer- and external-surface structures. It was determined that the external and interlayer surface properties of the new material formed are rather different from those of the organoclays prepared with a single cationic surfactant. The strong interaction between the cationic and anionic surfactants and MMT in the interlayer surface allows the possibility of the complex to capture and retain various organic materials (Yapar et al., 2015).

9.3.3 FUNCTIONALITY OF SURFACTANT-MODIFIED ALUMINOSILICATES AS DRUG CARRIERS Based on the previously presented literature data, the main advantage of potential pharmaceutical application of SMZs as drug carriers is their ability to achieve modified (prolonged) drug release. The initial investigations related to properties of SMZs as prospective excipient for pharmaceutical application were mainly based on interactions within drug
SMZ composites and mechanism of subsequent in vitro drug release. Nevertheless, since SMZs would represent a constituent [besides active substance(s)] of a final dosage form (up to date, mainly for oral administration), further investigations of its overall composition and possible interactions of SMZs with other auxiliary substances are necessary. Additionally, the investigation of excipient functionality in terms of improved flow properties, compressibility, content uniformity, dilution potential, or performance, such as disintegration and dissolution profile (EDQM, 2017; Gupta et al., 2006), is also important for evaluation of its overall potential as a drug carrier.

9.3 Aluminosilicate-Based Drug Carriers Functionalized

Functionality of surfactant-modified aluminosilicates as drug carriers started almost 15 years ago, when the first paper on preliminary characterization of drug support systems based on natural zeolite clinoptilolite was published by Rivera and Farı´as (2005). The authors investigated the influence of different treatments (at pH 1.2 and 5.5) and modifications involving a surfactant (BZ) and three drugs (sulfamethoxazole, acetylsalicylic acid, and metronidazole) aiming at the future preparation of slow-release systems. It was determined that the presence of surfactant and drugs on the zeolite does not produce structural changes, and resulted in a strong decrease of the specific surface area. Based on these findings, it was reported that the zeolitic materials are able to support drugs of a very different nature. These results were the basis for further theoretical (Lam and Rivera, 2006) and practical investigations of clinoptilolite
surfactant composites as drug support by the same research team (Farı´as et al., 2010, 2011; Rivera and Farı´as, 2005). Within these researches it was verified that the zeolite structure remained unchanged after the modification with the surfactant, additionally the amount of the drug adsorbed was related to its structure and surfactant amount. Finally, it was determined that the drug desorption from drug
SMZ composites was prolonged, which was of great significance for the practical application of these functionalized drug carriers. The drugs most commonly used in the reported and following papers were representatives of nonsteroidal antiinflammatory drugs (NSAIDs): acetylsalicylic acid, diclofenac diethylamine (DDEA), diclofenac sodium (DS), and ibuprofen (IBU), of which the two last were the most investigated. Their short elimination half-lives (t1/2) (1
2 hours for DS and 2
4 hours for IBU, respectively), in addition to the side effects (the most frequent being gastrointestinal and cardiovascular) make them good candidates for modified-release dosage forms, where the rate and/or place of release of the active substance(s) is different from that of a conventional-release dosage form administered by the same route. Therefore, the use of oral-modified NSAID preparations is well established and is especially important for NSAIDs with short half-lives (i.e., diclofenac, IBU) (Tomi´c et al., 2017). Besides BC, modification of clinoptilolite with HB in amounts equal to and twice the ECEC of the starting zeolitic tuff was investigated (Krajiˇsnik et al., 2010). The prepared SMZs were used for the additional investigation of three model drugs: DDEA, DS, and IBU, by means of liquid drug adsorption. It was revealed that variations between drug adsorption levels were influenced by the surfactant type and the amount present at the surface of the composites. Furthermore, determination of flow properties showed that modification of the zeolitic surface reflected on powder flow characteristics by improving it at the higher surfactant level, that is, a more hydrophobic surface. In vitro drug release profiles of the drug
SMZ composites revealed sustained drug release over a period of 8 hours, which was particularly important for DS and IBU and their use in oral dosage forms. Results of the adsorption of DS by modified natural zeolite composites at three levels of HB (10, 20, and 30 mmol/100 g, that is, 100%, 200% and 300% of

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zeolitic ECEC), also showed a proportional increase of drug adsorption by increasing the amount of surfactant used for the modification (Krajiˇsnik et al., 2013b). In vitro release data from the drug-modified zeolite composites (denoted as DHB 10
30C) and physical mixtures of ZHBs and DS (denoted as DHB 10
30P) at the appropriate molar (mmol HB/mmol DS) ratio showed that the prolonged DS release for both groups of samples over a period of 8 hours was achieved (Fig. 9.3). The presented results indicated that the investigated materials could be successfully used as functional drug formulation excipients, which was confirmed in vivo as DS-modified zeolite mixtures produced a significant dose-dependent reduction of the rat paw edema induced by the proinflammatory agent carrageenan (Krajiˇsnik et al., 2014). In the same study, the DS antiedematous effect was intensified and significantly prolonged by modified zeolite, suggesting a potential improvement in the treatment of inflammation by DS-modified zeolite mixtures. Functionalization with another cationic surfactant cetylpyridinium chloride (CPC) at three levels (corresponding to 100%, 200%, and 300% of zeolitic ECEC) enabled preparation of DS-SMZ composites for prolonged drug release (Krajiˇsnik et al., 2013a). The results of drug release testing showed that the prolonged release of DS from all three composites, as well as from a physical mixture containing SMZ [modified with the lowest CP amount (DS/ZCPC-10)] and DS was achieved over a period of 8 hours. The drug release from both DS/ZCPC10 (max. 55%) and corresponding physical mixture (max. 38%) was remarkably lower than that from the physical mixture of starting zeolite and DS (max. 85%). The authors proposed the possible mechanism of DS release from the ZCPC 10
30 composites, taking into account the different CPC amounts and their organization on the zeolitic surface (monolayer vs. bilayer). The kinetic analysis of the DS release data for the tested samples indicated a combination of drug diffusion and ion exchange as the predominant release mechanisms in the dissolution medium. Adsorption of DS by zeolite
clinoptilolite modified with the same surfactant
CPC in an amount above the ECEC of clinoptilolite (150% of ECEC) was studied by de Gennaro et al. (2015). The adsorption kinetics showed a very fast loading (within 20 minutes) of the DS at the surface of CPC-modified zeolite. Powder flowability of the starting material, estimated by means of Carr’s index [CI (%)] revealed that it was cohesive with scarce flow properties, while the addition of surfactant and drug slightly enhanced the flow properties of the powders, probably by discouraging van der Waals interparticle interactions. These findings were in accordance with the literature data related to the investigation of SMZs modified with CPC in an amount equal to and above the zeolitic ECEC (Mili´c et al., 2014), where it was shown that even flowability of the sample with the smallest amount of CPC was significantly improved (more than 50%) compared to the starting zeolite. A wide distribution of volume diameters of SMZ particles, along with their irregular shape (determined by LS), suggested that a granulation process is most likely required during production procedure of a dosage form for oral administration.

9.3 Aluminosilicate-Based Drug Carriers Functionalized

FIGURE 9.3 In vitro dissolution profiles of DS from (A) drug-modified zeolite composites (closed symbols) and drug-modified zeolite physical mixtures (open symbols); (B) physical mixtures with different (mmol HB/mmol DS) molar ratio. ˇ ´ A., Malenovic, ´ A., Milojevic-Raki ´ ´ M., Dondur, V., Reprinted with permission from Krajisnik, D., Dakovic, c, ˇ et al., 2013b. Investigation of adsorption and release of diclofenac sodium by modified ´ Z., Radulovic, zeolites composites. Appl. Clay Sci. 83
84, 322
326. Copyright (2013) Elsevier.

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SMZs were also found to be a very versatile drug carrier since molecules with different chemical properties can be potentially loaded in/on SMZs. CLSM observations revealed that cationic and polar probes prevalently localize in the SMZ bulk, anionic probes preferentially arrange themselves on the SMZ surface, while the loading of a nonpolar molecule of in/on SMZs is discouraged. Drug release from DS-SMZs (denoted as SMNZ) in simulated intestinal fluid was prolonged over 5 hours through a mechanism prevalently governed by anionic exchange with a rapid final phase probably ascribed to the release of DS fraction within the patchy bilayer of SMZs (Gennaro et al., 2015). Versatility of SMZs toward adsorption of drug molecules with different polarity was also demonstrated for SMZs modified with BC and CPC (in amounts equivalent to 100%
300% of the zeolitic ECEC value) (Krajiˇsnik et al., 2015). Sorption investigations of IBU (practically insoluble in water in contrast to DS, which is sparingly soluble in water) from aqueous buffer solutions onto the SMZs revealed that variations between adsorption levels were influenced by the type of surfactant and the amount present at the surface of the composites. The drug uptake by the surfactant/zeolite composites confirms that the sorption of IBU is closely related to the overall hydrophilic/hydrophobic interactions of the ionizable drug molecule with the modified zeolitic surfaces and that it can be described by the adsorption/partition model. Prolonged drug release from the IBU-SMZ composites over an 8-hour period was achieved and differences in dissolution rates were in agreement with drug-sorption findings and are explained by combined hydrophobic and partial electrostatic interactions in the drug/surfactant/zeolite samples. The sustained drug delivery, as one of the most important characteristics of SMZs, was also investigated for SMZ granules (Serri et al., 2017). A granulate for the oral controlled delivery of DS has been realized by wet granulation, using an SMZ (clinoptilolite modified with CPC) as an excipient. DS-loaded granules have been prepared by a wet granulation method (utilizing HPMC as a binding/controlled-release excipient carrier) resulting in product with suitable technological features. The granules possessed satisfactory dosage uniformity and flowability suitable for oral dosage form manufacturing, along with sustained drug release of up to 9 hours, driven by both ion exchange and transport kinetics. In addition to the aforementioned functional advantages of SMNs as prospective drug carriers, various tests related to their biocompatibility have been carried out as a part of complete biopharmaceutical characterization. So far, many studies have demonstrated that zeolite (mainly clinoptilolite) administration to animals and humans, even for several months, caused no change that could be considered a toxic effect of treatment (Adamis et al., 2000; Alexopoulos et al., 2007; Colella, 2011; Paveli´c and Hadˇzija, 2003). The safety of both natural and SMZs as a potential excipient was evaluated during an acute toxicity testing. A dose of 2000 mg/kg of clinoptilolite and the sample with the highest cationic surfactant (HB) content (equivalent to 300% of zeolitic ECEC) was administered to NMRI HAN mice via oral route according to FDA guidance for Potential excipient intended

9.4 Chitosan-Functionalized Aluminosilicates as Drug Carriers

for short-term use (CDER, 2005) for acute toxicity testing. During the observation period (72 hours), treated animals were observed for symptoms of toxicity. The results demonstrated that both natural clinoptilolite and the tested SMZ did not cause death or any kind of toxicological reaction during the period of observation. Additionally, in other studies related to stability of SMZs in vivo (Krajiˇsnik et al., 2013b; Mili´c et al., 2014) desorption of surfactants in water and phosphate buffer at pH 6.8 was investigated. A minimum of surfactant desorption (#5%) from the SMZs alongside the results of nontoxic properties enabled application of this functionalized mineral material as an excipient in the following in vivo assessment of antiedematous activity (Krajiˇsnik et al., 2014). The cytotoxicity of free DS, placebo SMZ granulate, and DS-loaded granulate against mouse macrophages (RAW264.7 cell line) was screened by measuring the activity of mitochondrial dehydrogenase through a modified MTT assay (Serri et al., 2017). The results indicated the biological safety of the formulations for concentrations up to 5 mg/mL, since no significant toxicity was observed. Moreover, cytotoxicity profiles have shown that DS formulated in the granulate is less cytotoxic than the free drug, probably due to the slower release of the drug from the dosage form in the culture medium. In order to investigate the pharmacological activity of the granulate, cytokine analysis was performed. The results obtained by in vitro cell experiments have shown the ability of the DS-loaded granulate to exert an antiinflammatory activity on macrophages. Namely, compared to the treatment of cells with free DS, the drug-loaded granulate showed a different pharmacological profile, with a prolonged antiinflammatory action by virtue of the granulate’s ability to sustain DS release. Although the presented results encourage application of SMZs as potential pharmaceutical excipient, according to recommended strategies to support marketing of new excipients in drug products, all pivotal toxicology studies should be performed in accordance with state-of-the art protocols and good laboratory practice regulations (CDER, 2005), and such studies are expected in the near future.

9.4 CHITOSAN-FUNCTIONALIZED ALUMINOSILICATES AS DRUG CARRIERS 9.4.1 CHITOSAN—A VERSATILE BIOPOLYMER Chemically, chitosan is a binary copolymer comprised of randomly distributed glucosamine and N-acetyl glucosamine monomer units linked by β-(1,4) glycosidic bonds (Fig. 9.4) (Wu et al., 2014). It is naturally present in some fungi (Muzzarelli et al., 2012), but is commonly obtained by partial deacetylation from chitin (2-acetamido-2-deoxy-β-D-glucan), the second most abundant polymer in nature after cellulose (Hajji et al., 2014). Partial removal of acetate moieties from chitin can be achieved by its treatment with sodium or potassium hydroxide at high temperatures (de Moura et al., 2011) or by enzymatic deacetylation in the

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FIGURE 9.4 Chemical structure of chitin and chitosan.

presence of chitin deacetylase (Jaworska, 2012). It was discovered in 1859 by C. Rouget and named by F. Hoppe Seyler in 1894 (Yeul and Rayalu, 2013). One hundred years later, owing to its unique cationic nature, viscosity-enhancing and gel-forming ability, biocompatibility, and versatility, this aminopolysaccharide has become one of the most intensively studied biopolymers in the field of drug delivery.

9.4.1.1 Physical and chemical properties of chitosan In its dry form, chitosan usually appears as off-white powder or flakes. In contrast to the majority of natural polysaccharides, it is a weak base due to the presence of amino groups on the C2 position of the glucosamine monomer units. Depending on the glucosamine/N-acetyl glucosamine monomer ratio, its pKa value ranges between 6.4 and 6.7 (Sorlier et al., 2001). It is highly hydrophilic with a rigid crystalline structure due to inter- and intramolecular hydrogen bonding (Dash et al., 2011; Thakur and Thakur, 2014). The most important structural properties of chitosan are molecular weight, deacetylation degree, and distribution of free amino groups along the polymer backbone. The molecular weight of chitosans usually ranges between 10 and 1000 kDa (Zargar et al., 2015). Although there are no generally accepted limits, these polymers can be categorized as low- (,150 kDa), medium- (150
300 kDa), and high- ( . 300 kDa) molecular-weight chitosans. Chitosans having a molecular weight below 10 kDa are commonly known as oligochitosans or chitoˇ oligosaccharides (Calija et al., 2013; Xia et al., 2011). These oligomers can be obtained by physical, chemical, or enzymatic depolymerization from their highmolecular-weight precursors (Kittur et al., 2005; Popa-Nita et al, 2009; The United States Pharmacopeia, 2011). Molecular weight affects several critical chitosan properties such as solubility, crystallinity, tensile properties, and rheological behavior of its aqueous solutions, as well as its biodistribution, biodegradation, and elimination (Dash et al., 2011; Kean and Thanou, 2010; Nunthanid et al., 2001).

9.4 Chitosan-Functionalized Aluminosilicates as Drug Carriers

Deacetylation degree is a measure of free amino groups within a chitosan structure expressed as the ratio of glucosamine to N-acetyl-glucosamine monomer units. It affects chitosan reactivity, solubility, rheological properties, and biodegradability. The deacetylation degree of commercially available chitosans is usually about 85% (Yeul and Rayalu, 2013). Chitosan is insoluble in water and common organic solvents and soluble in dilute aqueous acidic solutions (e.g., acetic acid, formic acid, nitric acid) (Thakur and Thakur, 2014; Yeul and Rayalu, 2013). The solubility of chitosan largely depends on its structural characteristics, primarily its molecular weight and deacetylation degree. That is, the aqueous solubility of chitosans increases with decreasing molecular weight and increasing deacetylation degree (Kubota et al., 2000; Thakur and Thakur, 2014). Therefore, oligochitosans are easily soluble in water, unlike their high-molecular-weight precursors (Yin et al., 2009). The distribution pattern of acetyl moieties also affects the solubility of chitosan, since they are involved in interchain interactions and aggregation (Philippova et al., 2012; Rinaudo, 2006). The rheological behavior of chitosan solutions is another feature that is important for various biomedical applications of this polymer. Chitosan is frequently used as a viscosity-enhancing agent, therefore, its viscosity-enhancing ability is highly desirable, but it also can be a limiting factor due to handling and processing difficulties. Chitosan aqueous solutions exhibit pseudoplastic behavior, with more pronounced influence of shear rate on viscosity at higher chitosan concentrations (Bansal et al., 2011; Hwang and Shin, 2000). The viscosity of chitosan solutions is increased by increasing the chitosan molecular weight, deacetylation degree, and concentration, and is decreased with temperature increase and addiˇ tion of salts (Anitha et al., 2014; Calija et al., 2011; Martinez-Ruvalcaba et al., 2004). In contrast to their precursors, oligochitosans form nonviscous aqueous ˇ solutions, even at high concentrations (Calija et al., 2015). Chitosan functionality can be improved via chemical modification or complexation with various chemical entities. There are three functional groups in chitosan structure available for interaction with other molecules and/or (poly)anions: primary amino group at the C-2 position, and primary and secondary hydroxyl groups at the C-6 and C-3 positions, respectively. The quaternization of the primary amino group is commonly used to improve solubility under physiological conditions, binding capacity for polyanions, such as genes, and stability of the resulting complexes (Xiao et al., 2012). This modification also improves chitosan mucoadhesivity, antibacterial activity, and enhances their permeability (Wu et al., 2006). In reaction with monohalocarboyilic acids, chitosan can form N- or O-carboxyalkil derivatives, such as N-carboxymethyl chitosan. This water-soluble chitosan derivative forms viscous solutions and possesses excellent film- and gelforming properties (Mourya and Inamdar, 2008). Hydroxyalkylation of amino and hydroxyl groups can also be used to improve both the solubility and some biological properties of chitosan (Peng et al., 2005). Chitosan derivatization by grafting is widely used for improvement of its various functional properties including

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solubility, chelating and complexation ability, mucoadhesivity, antioxidant, and antibacterial activity (Jayakumar et al., 2005). These derivatives are also used for the preparation of “smart drug carriers” sensitive to biological stimuli, such as pH and temperature (Li et al., 2013; Mukhopadhyay et al., 2014). Furthermore, being a polycation chitosan can electrostatically interact with anionic polymers, of both natural (e.g., pectin, alginate, xanthan gum, carboxymethyl cellulose, and carrageenan) and synthetic origin (e.g., polymethacrylate copolymers and carbomers) forming complex structures known as (inter)polyelectrolyte complexes (Araujo ˇ ˇ et al., 2014; Cerchiara et al., 2016; Calija et al., 2015; Calija et al., 2013; Mustafin et al., 2015; Tsai et al., 2014). In contrast to covalently modified chitosans, these complexes are reversible structures that can be easily degraded in vivo and prepared in various forms suitable for various routes of administration. The cationic nature of chitosan is also responsible for electrostatic interaction with negatively charged surfaces of some aluminosilicate clays, such as halloysite and MMT, and subsequent formation of polymer/clay nanocomposites with improved drug-delivery properties, which is discussed in detail in this chapter (Liu et al., 2012; Salcedo et al., 2014).

9.4.1.2 Safety and regulatory status of chitosan Despite the fact that chitosan has become one of the most extensively studied biopolymers in the field of drug delivery, there are no approved drug products containing this polymer as an inactive ingredient (FDA Inactive Ingredient Database, 2018c). Still, chitosan has been approved in various dietary supplements as it stimulates the maintenance of normal blood LDL-cholesterol concentrations (EFSA, 2011), and also in wound dressings due to its excellent wound healing properties (FDA, 510(k) Premarket Notification Database, 2018a). The compendial status of chitosan was changed in 2002, when a monograph for chitosan hydrochloride was included in The European Pharmacopoeia (EDQM, 2002). In 2011, a chitosan monograph was introduced in the 2nd Second Supplement to USP 34-NF 29 (USP 34-NF 29, 2011). Chitosan is commonly regarded as a biocompatible and safe biopolymer, however, up to now, it has not been included in the FDA database of Generally Recognized As Safe (GRAS) food substances (FDA GRAS Substances (SCOGS) Database, 2018b). This could be explained by its biological activity, structural versatility, and variability in its biodegradation and biodistribution pathways. Namely, numerous studies have shown that the biodistribution, biodegradation, and overall safety of chitosan largely depend on its structural properties, primarily its molecular weight and degree of deacetylation (Huang et al., 2004; Kean and Thanou, 2010; Ren et al., 2005). The main degradation mechanism is depolymerization by lysozyme, an enzyme present in human body fluids, and by bacterial enzymes in the colon (Kean and Thanou, 2010; Va˚rum et al., 1997). In vitro studies have shown that the enzymatic degradation rate decreases with increasing both deacetylation degree and molecular weight (Yang et al., 2007; Zhang and Neau, 2001). The in vivo degradation of chitosan has not been sufficiently

9.4 Chitosan-Functionalized Aluminosilicates as Drug Carriers

investigated, but some reports suggest that the deacetylation degree might affect the in vivo degradation rate (Yang et al., 2007). Toxicological studies have shown that the toxicity of chitosan largely depends on the route of administration. A total daily dose of 0.7
0.8 mg/kg/day and up to 6.75 g showed no or slight evidence of toxicity upon oral administration in rabbits and human volunteers, respectively (Hirano et al., 1988; Tappola et al., 2008). On the other hand, subcutaneous administration of doses above 50 mg/kg caused anorexia, while doses above 150 mg/kg had a lethal outcome in dogs, respectively (Minami et al., 1996). Intravenous administration of 4.5 mg/kg/day in rabbits showed no abnormal changes, while a dose of 50 mg/kg/day caused death (Hirano et al., 1991; Hirano et al., 1988).

9.4.2 PREPARATION AND CHARACTERIZATION OF CHITOSANMODIFIED ALUMINOSILICATES In the last decade, increasing interest has been focused on the functionalization of aluminosilicates with chitosan in order to improve their drug-delivery properties. Generally, there are three types of polymer/aluminosilicate composites: phaseseparated, intercalated, and exfoliated composites. In the phase-separated composites, polymer surrounds aluminosilicate agglomerates. On the other hand, in the intercalated composites, polymer penetrates between the aluminosilicate layers, whereas, in the exfoliated composites, aluminosilicate is exfoliated into single layers surrounded by a polymer (Jafarbeglou et al., 2016). Aluminosilicate/chitosan composites for drug delivery are usually obtained by mixing acidic chitosan solution with aluminosilicate dispersion or dispersion of aluminosilicate dry powder in acidic chitosan solution under vigorous stirring. This leads to the cationic exchange of protonated chitosan with exchangeable aluminosilicate cations and subsequent chitosan intercalation between the aluminosilicate layers. The mechanism of biopolymer aluminosilicate interaction and selection of a suitable preparation procedure depend on the properties of both constituents. Having negative surface charge, aluminosilicates such as MMT and halloysite nanotubes (HNT) can interact with protonated amino groups of chitosan under appropriate conditions (Ambrogi et al., 2017; Liu et al., 2012; Salcedo et al., 2014). MMT/chitosan composites are among the most intensively studied chitosan/aluminosilicate composites for drug delivery. In 2003, Darder and coworkers investigated MMT
chitosan composites as potential anion exchangers in bulk-modified electrodes for the potentiometric determination of anions (Darder et al., 2003). They prepared MMT
chitosan nanocomposites by slow addition of acidic chitosan solution in an MMT suspension under constant stirring. Intercalation of chitosan was driven mainly by an electrostatic interaction between protonated amino groups of the polymer and negative moieties of MMT. When the amount of chitosan exceeded the cationic exchange capacity of MMT, chitosan bilayers were formed due to hydrogen bonding between the amino and hydroxyl groups (Fig. 9.5).

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CHAPTER 9 Aluminosilicate-based composites functionalized

FIGURE 9.5 Schematic representation of chitosan intercalation into Na 1 MMT. Reprinted with permission from Darder, M., Colilla, M., Ruiz-Hitzky, E., 2003. Biopolymer 2 clay nanocomposites based on chitosan intercalated in montmorillonite. Chem. Mater. 15, 3774
3780. Copyright (2003) American Chemical Society.

9.4 Chitosan-Functionalized Aluminosilicates as Drug Carriers

In 2010, Aguzzi and coworkers prepared MMT
chitosan nanocomposite intended for modified delivery of 5-aminosalicylic acid, a nonsteroidal antiinflammatory drug for the treatment of Crohn’s disease and ulcerative colitis (Aguzzi et al., 2010). The nanocomposite was prepared by a solid
liquid interaction between the polymer and the clay mineral. In brief, MMT was dispersed in chitosan acidic solution under vigorous stirring to allow cationic exchange between protonated chitosan and the cationic species from the interlayer space of the clay mineral. The pH value of chitosan solution was adjusted to 5.0 to achieve protonation of chitosan amino groups and avoid any changes in MMT structure. The resulting nanocomposite was loaded with 5-aminosalicylic acid by passive diffusion of the drug molecules through cellulose membranes into a compartment filled with nanocomposite dispersion. Based on the available literature data, there are two approaches for the preparation of MMT/chitosan nanocomposites: (1) the addition of acidic chitosan solution into MMT dispersion under vigorous stirring or vice versa (Darder et al., 2003; Hsu et al., 2012; Lertsutthiwong et al., 2012) and (2) the addition of MMT powder into acidic chitosan solution (Aguzzi et al., 2010; Sandri et al., 2014). More rarely, chitosan powder is added into a previously formed MMT dispersion (Ambrogi et al., 2017; Hua et al., 2010). A model drug can be dissolved in an MMT dispersion or chitosan solution (Azhar and Olad, 2014; Hua et al., 2010) or upon nanocomposite formation (Aguzzi et al., 2010; Salcedo et al., 2014). Further treatment of the resulting MMT/chitosan nanocomposite dispersion depends on the desired final form of the composite. The dispersion can be cast into glass dishes and oven-dried to obtain nanocomposite films (Hsu et al., 2012), or the composite can be separated by filtration or centrifugation, washed with distilled water, and oven-dried or freeze-dried (Aguzzi et al., 2010; Sandri et al., 2014). Another possibility is to encapsulate the nanocomposites by using ionotropic gelation (Hou et al., 2015; Hua et al., 2010). This approach is based on the ability of chitosan to form hydrogels in contact with some anions, such as tripolyphosphate. Briefly, MMT/chitosan nanocomposite dispersion is dropwise added into a tripolyphosphate solution under gentle stirring. Once chitosan from the dispersion reaches tripolyphosphate ions, a rapid reaction of ionotropic gelation starts transforming the droplets into hydrogel particles. The most common HNT/chitosan nanocomposite preparation procedure is similar to the above-described solid
liquid preparation procedure proposed for MMT/chitosan nanocomposite. In this procedure, HNTs are added into acidic chitosan solution under vigorous stirring, usually followed by sonication to obtain homogeneous dispersion of the resulting nanocomposite (Kelly et al., 2004; Liu et al., 2012; Peng et al., 2015). The nanocomposite formation is based on electrostatic interactions between protonated amino groups of chitosan and negatively charged HNT surfaces arising from substitution of Al31 for Si41 in its tetrahedral sheet (Bailey, 1990; Liu et al., 2012). The composite is additionally stabilized by hydrogen bonding between chitosan amine and hydroxyl groups and HNT Si
O bonds (Liu et al., 2012). The nanocomposite dispersion can be further cast into

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CHAPTER 9 Aluminosilicate-based composites functionalized

Petri dishes or molds and dried to obtain HNT/chitosan nanocomposite films and scaffolds, respectively (Liu et al., 2012, 2013). The nanocomposite dispersion can be easily encapsulated by a simple one-stage microencapsulation procedure without or with crosslinkers. Namely, dropwise addition of the nanocomposite dispersion into an alkaline solution results in chitosan precipitation and formation of HNT/chitosan or composite hydrogel beads (Peng et al., 2015). In the latter case, the nanocomposite dispersion is added dropwise into sodium tripolyphosphate solution and rapid reaction of gelation between chitosan and tripolyphosphate ions results in the formation of HNT/chitosan hydrogel beads crosslinked with ˇ TPP (Calija et al., 2017). Various techniques can be used for characterization of aluminosilicate/chitosan composites. FTIR spectroscopy and zeta potential measurements are commonly used to scrutinize interactions between chitosan and aluminosilicates ˇ (Azhar and Olad, 2014; Calija et al., 2017; Huang et al., 2017; Lertsutthiwong et al., 2012; Li et al., 2013; Liu et al., 2013; Paluszkiewicz et al., 2011; Silva et al., 2012). SEM, transmission electron microscopy (TEM), energy-dispersive X-ray (XEDS) analysis, and XRD are powerful tools for the analysis of morphology and microstructure of nanocomposite beads, films, hydrogels, and scaffolds (Aguzzi et al., 2014; Azhar and Olad, 2014; Huang et al., 2017; Li et al., 2016; Liu et al., 2013; Onnainty et al., 2016; Peng et al., 2015; Rao et al., 2018). Atomic force microscopy (AFM) is useful for investigation of nanotopography of the chitosan/HNT nanocomposite films (Liu et al., 2012). TG, DTG, DTA analysis, and differential scanning calorimetry (DSC) are valuable methods for the ˇ analysis of thermal properties of the composites (Aguzzi et al., 2010; Calija et al., 2017; Darder et al., 2003; Liu et al., 2013; Peng et al., 2015). Mechanical properties of the nanocomposite films and hydrogels can be examined by dynamic mechanical analysis (DMA) and different tensile properties tests (Liu et al., 2012).

9.4.3 FUNCTIONALITY OF CHITOSAN
ALUMINOSILICATE COMPOSITES AS DRUG CARRIERS Chitosan possesses several properties which make it a good candidate for the functionalization of aluminosilicates as drug carriers: cationic nature, pHsensitive behavior, biocompatibility, and mucoadhesivity. Its cationic nature is essential, since the composite formation is based on ionic exchange, as discussed in the previous section. On the other hand, aluminosilicates possess a high surface to volume ratio and excellent chemical and mechanical stability. Therefore, combining these two distinctive materials under appropriate conditions may result in the formation of composite materials with significant drug-delivery potential. A brief overview of literature data on aluminosilicate functionalization with chitosan for drug-delivery and wound-healing applications is presented in Table 9.2.

Table 9.2 An Overview of the Literature Data on Aluminosilicate Functionalization With Chitosan for Drug-Delivery and Wound-Healing Applications

Aluminosilicate Montmorillonite

Montmorillonite

Montmorillonite

Montmorillonite

Montmorillonite

Montmorillonite

Functional Characteristics of Chitosan Average molecular weight: 251 kDa Deacetylation degree: 98% Average molecular weight: 8.4 kDa Deacetylation degree: 80% Average molecular weight: 71, 220 and 583 kDa Deacetylation degree: 85
90% Average molecular weight: n/a Deacetylation degree: 98% Average molecular weight: 251 kDa Deacetylation degree: 98% Average molecular weight: n/a Deacetylation degree: 95%

Final Carrier Type

Model Drug

Improved Properties

References

Composite powder

5-Amino salicylic acid

Sustained drug release in acidic medium

Aguzzi et al. (2010)

Composite powder

5-Fluorouracil

Decreased drug toxicity

Kevadiya et al. (2012)

Composite powder




Antibacterial activity

Lertsutthiwong et al. (2012)

Composite powder




Mucoadhesivity Wound-healing properties

Salcedo et al. (2014)

Composite powder

Silver sulfadiazine

Bacteriostatic and bactericidal properties

Sandri et al. (2014)

Composite nanoparticles

Betaxolol hydrochloride

Enhancement of precorneal retention Sustained release

Hou et al. (2015)

(Continued)

Table 9.2 An Overview of the Literature Data on Aluminosilicate Functionalization With Chitosan for Drug-Delivery and Wound-Healing Applications Continued

Aluminosilicate Montmorillonite

Montmorillonite

Halloysite

Halloysite

Halloysite

Functional Characteristics of Chitosan Average molecular weight: 50
190 kDa Deacetylation degree: 85% Average molecular weight: n/a Deacetylation degree: n/ a Average molecular weight: 1000 Da (oligochitosan) Deacetylation degree: 75.4% Average molecular weight: mediuma Deacetylation degree: 75
80% Average molecular weight: 600 kDa Deacetylation degree: 95%

Final Carrier Type

Model Drug

Improved Properties

References

Composite nanoparticles

Chlorhexidine digluconate

Sustained release Mucoadhesivity

Onnainty et al. (2016)

Composite films

Chlorhexidine diacetate

Antimicrobial and antibiofilm activity

Ambrogi et al. (2017)

Composite powder




Wound-healing properties (improved re-epithelialization effect)

Sandri et al. (2017)

Hydrogel containing halloysite
chitosan composite

Tetracycline hydrochloride

Sustained drug release

Kelly et al. (2004)

Composite hydrogel

Doxorubicin hydrochloride

Drug entrapment efficiency Mechanical properties

Huang et al. (2017)

Halloysite

Halloysite

Halloysite

a

Average molecular weight: n/a Deacetylation degree: 90% Average molecular weight: 310
370 kDa Deacetylation degree: .75% Average molecular weight: mediuma Deacetylation degree: n/ a

According to the supplier’s data.

Composite microspheres

Acetylsalicylic acid

Drug loading Sustained drug release in acidic medium

Composite microspheres

Verapamilhydrochloride

Morphology Drug entrapment efficiency Sustained drug release

Composite nanoparticles

Curcumin

pH-sensitive drug release

Li et al. (2013)

Ï Calija et al. (2017)

Rao et al. (2018)

CHAPTER 9 Aluminosilicate-based composites functionalized

50 300 40 200

30 20

100

0

0

20

40

60 Time (min)

80

5-ASA/VHS 10 5-ASA/CS 5-ASA/CS/VHS 0 100 120

Drug released (mg/cm2) from 5-ASA/CS and 5-ASA/CS/VHS

One of the first studies related to aluminosilicate functionalization with chitosan for drug-delivery purposes was published in 2004. Namely, Kelly and coworkers functionalized tetracycline-loaded HNTs with medium-molecular-weight chitosan to achieve sustained release and prolonged local action in treatment of periodontitis (Kelly et al., 2004). HNT/chitosan composite had somewhat lower, but still relatively high, encapsulation efficiencies (32.5% and 41% for tetracycline base and tetracycline HCl, respectively) in comparison to the HNTs (39.05% and 49.3% for tetracycline base and tetracycline HCl, respectively). On the other hand, in vitro drug release tests in phosphate buffer pH 6.8 showed significant reduction of the drug release rate from chitosan-treated HNTs in comparison to the untreated HNTs, with 78% of the drug released after 9 days. This difference was particularly visible in the first-burst release stage and can be ascribed to chitosan insolubility in pH-neutral dissolution medium. More importantly, in vivo test in dogs confirmed slow but sufficient drug release to achieve prolonged microbiological activity of the composites in the final formulation over 6 weeks. Similar effects of chitosan functionalization on MMT were reported by Aguzzi and coworkers (Aguzzi et al., 2010). They investigated and compared release profiles of 5-aminosalicylic acid from MMT/chitosan composite and from both constituents, and confirmed their synergistic effect on drug release. As can be seen in Fig. 9.6, MMT/chitosan nanocomposite exhibited sustained release of 5-aminosalicylic acid, with lower drug release rates in comparison to chitosan and MMT alone. Furthermore, encapsulation of the drug was more effective and the

Drug released (mg/cm2) from 5-ASA/VHS

314

FIGURE 9.6 5-Aminosalicylic acid in vitro release profiles from the composite, pure chitosan, and MMT in 0.1 M HCl. Reprinted with permission from Aguzzi, C., Capra, P., Bonferoni, C., Cerezo, P., Salcedo, I., Sa´nchez, R., et al., 2010. Chitosan
silicate biocomposites to be used in modified drug release of 5-aminosalicylic acid (5-ASA). Appl. Clay Sci. 50, 106
111. Copyright (2010) Elsevier.

9.4 Chitosan-Functionalized Aluminosilicates as Drug Carriers

higher values of encapsulation efficiencies were achieved for the nanocomposite than for its individual components. This approach also proved to be effective for microparticulate drug carriers. It was shown that the mixing of chitosan with drug-loaded MMT dispersion prior to encapsulation by ionotropic gelation results in increased drug loading, improved swelling behavior, and decreased release rates of encapsulated drug (Hua et al., 2010). Chitosan microparticles crosslinked with tripolyphosphate are prone to swelling and degradation in acidic conditions, similar to those in upper parts of the gastrointestinal tract (Fan et al., 2012). This leads to the rapid release of encapsulated drug from the particles. MMT/chitosan composite microparticles are stabilized by an interaction between MMT and chitosan, and, thus, are able to provide slower drug release in an acidic environment. By increasing MMT content, drug release from the particles decreased in both acidic and slightly alkaline conditions (Hua et al., 2010). MMT/chitosan nanocomposites also have potential for drug delivery via various mucosal routes of administration, such as buccal and nasal mucosa, due to their good mucoadhesive properties (Onnainty et al., 2016). Mucoadhesivity allows prolonged retention at the site of administration, resulting in improved local and/or systemic bioavailability of the administered drug (Sosnik et al., 2014). This feature of MMT/chitosan nanocomposites originates from chitosan and its ability to interact with mucus electrostatically and via hydrogen bonding (Onnainty et al., 2016; Sogias et al., 2008). Mucoadhesivity of MMT/chitosan composites is lower than that of pure chitosan but still higher than that of pure MMT (Salcedo et al., 2014). Lower mucoadhesivity of the composite could be ascribed to the reduced mobility of chitosan chains as a consequence of interaction with MMT. This approach can also be used to reduce unwanted drug effects. Kevadiya et al. performed intercalation of 5-fluorouracil in MMT/chitosan nanocomposites with the aim of reducing drug toxicity and improving its bioavailability (Kevadiya et al., 2012). In vitro genotoxicity and cell viability assay showed significant DNA damage reduction and unchanged efficacy toward cancer cells upon drug intercalation in the composites. On the other hand, in vivo pharmacokinetic tests in Wistar rats revealed increased residence time of the drug in plasma when drug was intercalated in the composites prior to administration. In addition, hepatotoxicity was significantly reduced in comparison to the pure drug. The biocompatibility of aluminosilicate/chitosan nanocomposites is another important issue that has to be considered prior to potential application. Liu et al. investigated in vitro fibroblast response on HNT/chitosan nanocomposite films and compared it with the response on pure chitosan (Liu et al., 2012). Both the composite and chitosan showed acceptable and comparable biocompatibility, and no reduction of mouse NIH3T3 cell viability was observed. SEM analysis revealed a minor difference in cell morphology between cells grown on chitosan and the composite, most likely due to surface roughness of the composite. Acceptable biocompatibility was also confirmed for MMT/chitosan composites. Salcedo and

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CHAPTER 9 Aluminosilicate-based composites functionalized

coworkers performed an in vitro cytotoxicity test for chitosan and MMT/chitosan composites on colorectal adenocarcinoma cell lines (Caco-2) and no significant reduction of cell viability was observed in comparison with the blank (Salcedo et al., 2014).

9.5 CONCLUSIONS Natural aluminosilicates (typically clays) have been traditionally used as excipients in pharmaceutical preparations/products due to their favorable physicochemical and functionality-related characteristics. Over the last two decades, halloysite (as a representative of tubular clays) and zeolites (as representatives of tectosilicates) have emerged as materials for prospective pharmaceutical applications. Surface modification of these materials is aimed at improving their technological features, drug-loading capacity, and achieving modified drug release. Based on the above discussion, functionalization of aluminosilicates using organic longchain species, such as cationic surfactants and aminopolysaccharide chitosan, can be used as a valuable approach for improvement of their drug-delivery potential in terms of sustained drug release, encapsulation efficiency, and mucoadhesivity. Still, there is a need for further studies on drug
modified aluminosilicates interactions within the composites, their pharmaceutical and biopharmaceutical properties, followed by safety and biocompatibility assessment.

ACKNOWLEDGMENT This work was realized within the framework of the projects TR 34031 and ON 172018 supported by the Ministry of Education, Science and Technological Development of the Republic of Serbia.

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CHAPTER

Bioactive glass nanofibers for tissue engineering

10

Joaquı´n Penide1, Fe´lix Quintero1, Jesu´s del Val1, Rafael Comesan˜a2, Fernando Lusquin˜os1, Antonio Riveiro1 and Juan Pou1 1

Applied Physics Department, EEI, University of Vigo, Vigo, Spain 2Materials Engineering, Applied Mechanics and Construction Department, EEI, University of Vigo, Vigo, Spain

10.1 INTRODUCTION 10.1.1 DEFINITION OF NANOFIBER The most common manifestation of nanotechnology is science, engineering, and technology conducted at the nanoscale, which is about 1
100 nm. This definition was popularized by the National Nanotechnology Initiative (NNI) of the United States federal government program and this range of dimensions for the nanoscale was specified in an International Standard (ISO/TS 80004-1:2015). According to its definition, a nanofiber is a fiber with two external dimensions in the range of 1
100 nm and the third dimension significantly larger (ISO/TS 80004-2:2015). However, there does not seem to be a consensus about how much “significantly larger” it must be. If we consider the diverse definitions of fibers, this dimension should vary from more than 100 times the diameter (Wallenberger et al., 2000), to greater than 1000 times (Tanioka and Takahashi, 2016). Alternatively, there are some definitions of nanomaterials, and specifically of nanofibers, which describe them in terms of their properties and functionality rather than their dimensions. In fact, the very same standard which specifies the range of dimensions for the nanoscale, states that nanotechnology manipulates matter, predominantly in the nanoscale, to make use of size- and structure-dependent properties and phenomena distinct from extrapolation from larger sizes of the same material (ISO/TS 80004-2:2015). In this sense, the extension of the range of diameters proposed by some authors in the definition of nanofiber from 1 to 1000 nm is justified, because some significant phenomena are observed between 100 and 1000 nm (Tanioka and Takahashi, 2016). Specifically, in the field of bioactive materials, different noticeable “nanoeffects” were demonstrated in several studies for nanofibers with diameters in the range of 0.1
1 μm: Woo et al. (2003) produced nanofibrous architectures of poly(L-lactic acid) with diameters of between 50 and 500 nm by thermally induced phase separation, and they demonstrated that the nanofibrous scaffolds adsorbed larger amounts of fibronectin and Materials for Biomedical Engineering: Inorganic Micro- and Nanostructures. DOI: https://doi.org/10.1016/B978-0-08-102814-8.00012-3 © 2019 Elsevier Inc. All rights reserved.

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vitronectin than solid-walled scaffolds. Kim et al. (2006) produced bioactive silicate glass nanofibers with average diameters between 84 and 630 nm by electrospinning of a precursor sol
gel. They selected the fibers with average diameters of 220 nm as representative of other-sized nanofibers, and demonstrated their potential in the enhancement of bone bioactivity and in vitro osteogenic differentiation of mesenchymal stem cells (MSCs). More recently, the same technique was employed to produce bioactive glass fibers with the same composition and diameters of hundreds of nanometers: the addition of these nanofibers to a poly (lactic acid) (PLA) biopolymer formed a composite which increased cell proliferation (Kim et al., 2012). Finally, Bioglass 45S5 fibers with diameters in the range 70 nm
1 μm, produced using the laser spinning technique, showed a distinct way of degradation and hydroxyapatite (HA) precipitation than microfibers, since they transformed into hollow fibers of HA (Quintero et al., 2009b).

10.1.2 INTEREST IN BIOACTIVE GLASS NANOFIBERS IN TISSUE ENGINEERING (SCAFFOLDS AND COMPOSITES) The discovery and development of new advanced materials has rapidly transformed the biomedical engineering, particularly the medical implants field. This field requires materials with singular properties and features, which were not fulfilled until recent times. Specifically, materials are required to promote osseointegration or to gradually release active compounds (drug delivery) inside a host. Despite the high-performance capabilities of many of the novel materials that can be produced nowadays, there is still a need to develop special materials for tissue engineering since their requirements are extremely restrictive. Indeed, a material suitable in this field must be biocompatible, but is also requested to fulfill some really special mechanical properties (Hollister, 2005; Kim and Mooney, 1998; Reis and Roma´n, 2004; Yang et al., 2001). Therefore, the development of 3D substrata, which are specifically called scaffolds, suitable for efficient in vitro and in vivo cell culture (Kim and Mooney, 1998) is crucial and a vital step for this aim. Evidently, the first and most important requirement for a scaffold is favoring cell growth and cellular differentiation, as well as showing a bioresorbable behavior, that is, they should be naturally dissolved (biodegradable) inside the host while promoting a favorable biological response and tissue growth. Moreover, materials suitable for scaffolding do not only need to be biocompatible and bioresorbable, but also need to show proper mechanical and structural properties. Particularly, the chosen biomaterial must be able to act as a supporting structure for the new tissues, scaffold, while these tissues are regenerated. For this aim, they are required to present certain mechanical strength, combined with flexibility similar to that of the tissue that is finally going to be substituted. Of course, this includes the capacity of evenly distributing the corresponding loads throughout the scaffold so as to avoid any stress concentration. This necessity is even more important and demanding when dealing with hard tissues, since the stresses

10.1 Introduction

(loads) that they must withstand are generally higher than when dealing with soft tissues. Finally, another important condition for an appropriate material for tissue engineering lies in its morphology. It must be such that it favors the adhesion of new cells and the creation of connection channels in the entire volume for the transportation of nutrients. Specifically, they must show a foam-like structure with proper spatial distribution and size of the pores. As the reader can see, all these requirements together turn the search for a suitable material into a real challenge. Most materials employed as scaffolds for the regeneration of soft tissues are bioresorbable polymers or biological materials, such as collagen (a component that is plentiful in skin and bones), since they are easily shaped into the required geometries. On the other side, when the aim is the regeneration of mineralized tissues (hard tissues, such as those of the teeth), bioactive ceramics are the most appropriate. The final election of the proper material is mainly based on the kind of cells whose growth must be promoted, the inflammatory response, absorption speed, etc. Several types of structures and manufacturing techniques have been proposed with the aim of fulfilling the structural requirements of scaffolds. Noticeably, these solutions are closely related to the target material considering that the fabrication techniques for a polymeric scaffold are quite different from those for a ceramic one. The structures that show the best results acting as a scaffold are high-porosity foams and mesh of fibers (Stevens, 2008). Certainly, they present an ideal configuration which combines a macroporous network (larger pores than 100 μm) with a nanoporous one (smaller than 50 nm). The first network promotes tissue growth inside it and the nutrient supply. The nanoporous network favors cell adhesion and intensifies the bioactivity of the scaffolds (Jones and Hench, 2004) for two reasons: these nanostructures truly increase the surface to volume ratio of these scaffolds which clearly improves their behavior as biomaterials (Stevens and George, 2005); on the other hand, the components of the biological tissues as well as the interactions between cells and biomaterials are in the nanoscale (Hajiali et al., 2010; Fisher et al., 2007). Despite the high-performance capabilities exhibited by high-porosity foams, nanofibers demonstrated higher efficiency in the promotion of tissue regeneration. Specifically, scaffolds formed by nanofibers present an excellent protein absorption and intensify the cell adhesion and growth with regard to those made of foams (Woo et al., 2003). Therefore, thanks to their extremely high specific surface area and their natural solubility and reactivity, bioactive glass nanofibers are one of the most appropriate choices to serve as scaffolds (Erol-Taygun et al., 2013). This material shows all these attributes which, together, lead to a more efficient stimulus to the cell receptors. Consequently, a mesh of nanofibers presents superior potential to be employed as scaffolds for cell culture and the regeneration of tissues. Bioactive glass nanofibers arouse enormous interest for many reasons, in the previous paragraphs we have described their bioactive benefits, but they have also remarkable advantages from their mechanical properties. In the case of

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fiber-reinforced polymeric composites, it is well-known that the mechanical properties of the composite are optimized in the axial direction of the fibers provided their length is greater than the critical length (Sergent, 2001). This critical length is proportional to the fiber diameter; therefore, decreasing the diameter would improve the reinforcing capabilities of fibers. This is one of the reasons why nanofibers can improve the mechanical properties of microfiber composites, but the advantages with decreasing diameter have many aspects (Al-Saleh and Sundararaj, 2011): the tensile strength of fibers may increase due to a decrease in the number of defects; the contact area between the filler and polymer matrix increases due to an increase in fiber surface area/volume ratio; and the fiber flexibility increases.

10.2 CONVENTIONAL METHODS TO PRODUCE GLASS MICROFIBERS Accordingly, nanofibers are striking structures for tissue engineering; however, they are as useful as they are difficult to produce. It should be noted that, in most materials, the nanosize (in the case of nanofibers, the diameter) means that there are just a few hundreds of molecules straight, one after the other. Therefore, a process aiming to produce a material as thin as a nanofiber must be very sophisticated, accurate, and probably complex. Generally, glass fiber and microfiber (and some nanofibers) production methods are based on heating the precursor material up to a fiber-forming temperature, defined as a temperature at which the melt is stable as a liquid and the viscosity is within the proper range for the fiber-forming process. Therefore, the fiber-forming temperature must be above the liquidus temperature in order to prevent crystallization of the melt during fiber formation (Seward and Varshneya, 2001), where the liquidus temperature is defined as the temperature at which crystals first begin to appear on cooling the melt under equilibrium conditions. Crystal development in the melt during glass forming is called devitrification, being an undesired effect in glass processes aiming to produce a completely amorphous solid. Particularly, in fiber-forming processes devitrification frequently leads to failure. At the same time, the temperature of the melt must correspond to a viscosity within the proper range for fiber formation. This is the range of viscosity where the process is able to stretch the molten material to form a filament but avoiding any breaking down. Particularly, if the viscosity is too high, the material will not flow under the stretching force applied to form the fiber; on the contrary, if the viscosity is too low, the molten material breaks down due to capillary forces and forms little spheres (drops) instead of fibers. Conventionally, viscosities in the range of 100
1000 Pas are accepted as proper for stretching the molten material (Seward and Varshneya, 2001; Wallenberger and Bingham, 2010).

10.2 Conventional Methods to Produce Glass Microfibers

These conditions for the fiber-forming temperature may not be satisfied for every glass composition. The liquidus temperature for some glasses is above the range of viscosities for fiber formation, therefore these glasses are very likely to devitrify. On the other hand, the influence of temperature on viscosity may be too restrictive in order to make stable the fiber formation process. When a liquid is cooled, the transition from the liquid state to glass does not occur at a single, sharp value of the temperature. On the contrary, there is a range of temperature, termed the glass transformation range, where the viscosity rises from a value in the order of 108 Pas to a value higher than 1015 Pas to qualify for appearance as a solid (Varshneya, 1994). Frequently, a fictive temperature called the glass transition temperature (Tg) is defined for easy reference to this transformation. Ceramics whose viscosity fits with the Arrhenius equation in a wide range of temperatures are termed strong melts. Particularly, in these materials such as silica or germania, viscosities vary slightly when the temperature is close to the glass transition temperature (Tg). On the other hand, there are some ceramics whose viscosity only fits the Arrhenius equation in a narrow temperature range. Specifically, at temperatures higher than Tg, their viscosity sharply changes, inasmuch as the activation energy of fragile liquids significantly varies with temperature. That is why they are called fragile melts (Ojovan, 2009; Wallenberger and Bingham, 2010). Typical examples of these are certain aluminates, boron oxides, or fluorides. Finally, there are some ceramics in which transition from solid to liquid is relatively fast. It can be said that, when the temperature rises at a certain point (above the liquidus line), their viscosity suddenly becomes almost negligible (i.e., ,1 poise) (Wallenberger and Bingham, 2010). These ceramics are usually called inviscid melts and it is quite difficult to produce fibers from this kind of material by conventional methods. This is because the viscosity is only appropriate for the stretching process in a quite restricted range of temperatures. In summary, it is critical to guarantee good control over the viscosity of the precursor material through its temperature in order to endorse a correct formation of fibers. Strong melts do not show sharp changes in their viscosity as a function of the temperature, therefore it is relatively easy to obtain fibers from them. However, in fragile melts and especially in inviscid melts, viscosity is quite sensible to temperature changes, so there must be a more accurate control on that temperature to succeed in the production of fibers. There are five major methods that may be called “traditional,” aiming to produce fibers with minimum diameters as small as tens of microns from different ceramics and glasses (Fig. 10.1) (Wallenberger and Bingham, 2010). Following, there is a brief description of each one: A. Drawing the precursor material by a spinning drum which is bellow a crucible that has a small hole where the molten material falls. B. Employing an inert gas to extrude the precursor molten material so it passes through a small gap and bring it to a chamber with controlled reactive atmosphere.

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FIGURE 10.1 Schematic representation of the techniques for the production of micrometric glass fibers from the glass melt.

C. Updrawing the molten material from the pool by a spinning drum while cooling it down. D. Heating and melting the precursor material by a laser beam in inert atmosphere and pulling it by a spinning drum to stretch it. E. Starting from a cylindrical preform, heating it in a particular point by electric resistors and pulling it in a controlled way so as it is stretched to produce a fiber. Most silicates with relatively high viscosities that can be stretched at temperatures usually below 1500 C, are processed by method A in order to produce fibers. Some aluminosilicates can be processed by method A too, but a special alloy crucible may be required due to the necessity of higher fiber-forming temperatures. Glass compositions which give fragile melts, such as some aluminosilicates and quaternary calcium aluminate, can be formed using method C to updraw the filament for the supercooled melt. Optic fibers and structural fibers, which show elevated viscosities too, are often transformed into fibers by method E at temperatures exceeding 2000 C. With respect to method B, it is useful to produce aluminate or metallic fibers since they are inviscid melts so they need to be chemically stabilized by a controlled atmosphere. Other fragile melts, such as YAG (yttrium aluminum garnet), must be shaped by method D when the aim is the production of amorphous fibers. In the particular case of YAG, there is the need to use an argon atmosphere (Weber et al., 1998).

10.3 Methods to Produce Glass Nanofibers

10.3 METHODS TO PRODUCE GLASS NANOFIBERS Glass nanofibers are tremendously useful, not only in the biomedical area as explained above, but also in other different fields such as the energy sector, textiles, microelectronics, or cosmetics. Due to the high number of distinct applications of this particular material, there are several techniques aiming to produce nanofibers (Kuchibhatla et al., 2007). Particularly, this section is focused on the techniques for the production of glass nanofibers because of their usefulness in tissue engineering. The techniques aiming to produce fibers in the nanoscale, nanofibers, are classified in two main groups: top-down and bottom-up methods. This classification defines basically the size of the precursor materials employed in each case (Zhang, 2003). In the first one, the process starts with a material whose size is above the nanoscale and then, it reduces the size of the material somehow down to the nanoscale. On the other hand, in the bottom-up methods the precursor material presents a size that is below the nanoscale, such as aggregates of molecules, molecules themselves, or even single atoms, which are eventually joined to build larger structures such as nanofibers.

10.3.1 BOTTOM-UP METHODS These methods are generally characterized by a low production rate due to the nature of the mechanisms involved in the process. They essentially consist of growing the nanomaterial by addition of material from a different state of matter, in the case of nanofibers it is mainly from the vapor phase. These methods are typically classified by the phase of the precursor material: liquid, vapor, or solid. However, the only methods capable of producing glass nanofibers start with precursor materials that are either in the vapor or solid phase. In this latter case, the solid material is evaporated as a first step, so virtually all the glass nanofibers produced by bottom-up methods are grown from the vapor phase. The most important method for the production of glass nanofibers is the vapor
liquid
solid method (VLS), which was proposed by Wagner (Wagner and Ellis, 1964) to produce silicon nanoneedles (nanofibers joined to a substratum). It typically employs a metallic drop as catalytic, which promotes the growth of a fiber by deposition of silicon from the vapor phase. Several authors reported the production of amorphous silicon oxide nanowires by VLS. Some examples are those made with catalysts such as platinum (Gurylev et al., 2015) or vanadium oxide (Banis et al., 2011). The technique is the same as for the silicon nanowires, but in this case the silicon is oxidized in the atmosphere as soon as the nanowires are formed. The obtained nanowires show very small diameters (tens of nanometers), but are relatively short (maximum lengths of several hundreds of micrometers). By this method, it is also possible to produce crystalline nanoneedles of different oxides such as In2O3, Ga2O3, or TiO2.

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Chemical vapor deposition mainly consists of introducing the substratum where the nanowires are going to be formed (and attached) in a recipient where the precursor material is in vapor phase. Then, through a chemical process, the deposition is forced so that the precursor material aggregates and forms the nanofibers. This method was successfully employed for the production of amorphous silicon oxide nanofibers with diameters of a few hundreds of nanometers (Cao et al., 2014). In sum, some of these bottom-up methods are able to produce amorphous silicon oxide nanowires with extremely small diameters and lengths up to several hundreds of micrometers. However, they show some disadvantages that make their use as bioactive glass nanofibers quite difficult: first of all, their low production rate is an important drawback if a macroscopic mass of nanofibers is needed. On the other hand, using these techniques it is not easy to tailor the composition of the produced nanofibers. Instead, they are basically composed by silicon oxide [some of them even present an unknown proportion between silicon and oxygen, SiOx (Cao et al., 2014; Liu et al., 2011; Nie et al., 2011)]. Therefore, in order to obtain bioactive glass nanofibers, a postprocessing step would be needed so as to add components such as calcium oxide or phosphate which enhance their bioactivity.

10.3.2 TOP-DOWN METHODS Usually, these methods involve melting of the precursor material and stretching it to form the fibers. It can be said that these methods are the result of a natural evolution of the conventional techniques which were explained in Section 10.2. There are different possibilities for both melting the material and stretching it. The most commonly employed methods are rotary jet spinning and electrospinning, which are described here.

10.3.2.1 Rotary jet spinning This system is basically formed by a rotor with a deposit in the inner part which contains the pool of the molten precursor material. This material flows toward the external part of the rotor and, thanks to the centrifugal force, the material is ejected through some holes strategically made in the outer part of the rotor. Finally, fibers are formed and cooled down before they reach the collector (Fig. 10.2). By this method, it the production of microfibers of polymer and ceramic materials is feasible and, in some specific cases, production of nanofibers can be achievable.

10.3.2.2 Electrospinning Electrospinning is currently the most commonly employed technique for the production of nanofibers from several different materials at the laboratory scale. It is based on employing an electric tension to induce an attraction force between the precursor material and the collector. This force is the one that creates the

10.3 Methods to Produce Glass Nanofibers

FIGURE 10.2 Scheme of the rotary jet spinning technique.

stretching effect on the liquid material. Electrospinning is able to produce microand nanofibers from several different materials, mainly polymers but some ceramics as well. The great advantage is that it permits the production of continuous fibers with very small diameters (Bhardwaj and Kundu, 2010). Though the origin of the electrospinning technique can be dated back to 1900 (Thenmozhi et al., 2017), the research activity on the application of this method for the production of nanofibers was notably boosted after the pioneer work of Doshi and Reneker (1995). The working principle of this technique can be found clearly described in many publications, such as those previously cited in this paragraph. The main advantage of this method is its relatively low cost if compared with the bottom-up methods and its higher production rate (Dzenis, 2004). Fibers obtained by this technique present diameters from 50 nm to 5 μm, depending on the precursor material, and they are remarkably long (Doshi and Reneker, 1995). This method allows the production of fibers from several different materials: mainly these materials are natural compounds like spider silk, polymers, and collagens. Some ceramics are also valid as precursor materials for the production of fibers, but they are not as easy to obtain as the natural compounds mentioned before. Generally, postprocessing is needed for that kind of material, and only some specific ceramics are valid for being electrospun (processed by electrospinning). The reason for this limitation lies in the most important disadvantage of this technique: the precursor material must show a convenient viscous to temperature relationship, that is, there must be a wide range of temperatures at which the viscosity is suitable for electrospinning. This means that the viscosity must be in

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such a range of values that it allows the force produced by the electric field to stretch this particular material. The problem is mainly with ceramic materials, particularly those that present an inviscid melt. As mentioned before, they abruptly change from a high viscosity to an almost negligible one in a very small range of temperatures, so that the control of the viscosity becomes extremely troublesome. In summary, there is a wide variety of methods and technologies that aim to produce different kind of nanofibers: crystalline and amorphous, continuous or with limited length, cylindrical or not, and with a wide variety of materials. Notwithstanding, every explained technique has its own advantages and disadvantages. Generally, those techniques that allow to obtain nanofibers with a wide variety of materials show some necessities that increase the costs of production: high vacuum chamber, accurate control of the atmosphere, etc; moreover, they cannot produce high quantities of nanofibers. On the other hand, those techniques that do not have this disadvantage only allow the production of nanofibers with some particular materials since they must satisfy certain conditions to be appropriate for processing. If we aim to obtain glass nanofibers with a great length, electrospinning is the only capable method. However, the problem comes when there is the need to obtain nanofibers from some fragile melts. In the next section, a new method called laser spinning is presented. It is able to produce fibers from materials in which electrospinning fails. Moreover, this method show a high production rate, allows to obtain nanofibers with a relatively simple experimental set-up, and the process does not require atmospheric control.

10.4 BIOACTIVE GLASS FIBERS FOR TISSUE ENGINEERING AND COMPOSITES The most common polymers used in bioactive glass composites are PLA, polyglycolic acid, and their copolymers (PLGA), which have been used clinically for many years (Jones, 2013). These polymers were employed to produce different kinds of composites reinforced with bioactive glasses, most of them in the form of particles. Examples of composites using these polymers were prepared with different techniques and compositions of bioactive glass particles. Poly(DL-lactide) (PDLLA) and PLGA matrix composites including Bioglass 45S5 (composition 46.1 SiO226.9 CaO24.4 Na2O2.6 P2O5 in mol %) powder with mean particle size ,5 μm were prepared using the thermally induced solid
liquid phase separation method (Maquet et al., 2004). PDLLA composites with 13-93 bioactive glass particles (50
125 μm) were produced by extrusion (Niemela¨ et al., 2008). The main drawback of these composites is that the phases degrade at different rates (Blaker et al., 2010, 2011). This problem can be overcome by tailoring the compositions of the polymer and the bioactive glass so that they interact during

10.4 Bioactive Glass Fibers for Tissue Engineering and Composites

bioresorption, autoregulating their dissolution rate. An alternative is to employ different polymers which can react positively with the best-known compositions of bioactive glasses: such as poly(glycerol sebacate) (PGS) with Bioglass 45S5 (Liang et al., 2010); polymethyl methacrylate (PMMA) with bioactive glass S53P4 granules (size of 0.5
0.8 mm) (Peltola et al., 2012); or natural polymers which degrade by enzyme action as a collagen
chitosan mixture with sol
gelderived bioactive glass particles (Peter et al., 2010). The other possibility is to design specifically the composition of the glass and to process it to obtain the optimum morphology. However, this is not always easy due to the necessity of combining the proper composition to react with the polymer together with the adequate viscosity
temperature dependence in order to process it and avoid devitrification. Another concern is that the mechanical properties of polymer composites reinforced with glass particles do not always improve significantly those of the single polymer. One reason is that the interfacial bonding between the glass particles and the polymeric matrix must be increased in order to optimize the mechanical properties of the composite. Conversely, the dispersed bioactive glass phase can be incorporated in the form of fibers instead of particles and the mechanical properties are indeed upgraded, at least in the direction of the axis of the fibers. This may yield to uniaxial enhancement in the case of unique orientation of fibers, bidirectional improvement in the case of mats, or isotropic properties if short and disordered fibers are dispersed in the matrix. Additionally, we have already mentioned above the benefits of bioactive glass fibers for tissue engineering. For these reasons, several groups early explored the production of bioactive glass fibers for the production of fibrous scaffolds and composites. The conventional method of melt fusion and drawing through a spinneret was firstly employed to produce microfibers of different compositions, mainly silicates. However, despite the potential and widely extended clinical use of Bioglass 45S5, this inverted glass composition is more difficult to form into fibers due to devitrification of the melt and its fragility that may lead to break-up during stretching. Consequently, many authors studied different compositions with a higher silica content than Bioglass 45S5 in order to avoid the inverted glass effect that makes it prone to crystallization during processing, but simultaneously giving up its higher bioactivity: such as the earlier work by Marcolongo et al. (1997); the S520 bioactive glass (Clupper et al., 2003) with composition 52.0 SiO2, 20.9 Na2O, 7.1 K2O, 18.0 CaO, 2.0 P2O5 in mol % and diameters of 20 μm; and the 13-93 bioactive glass (Brown et al., 2008; Pirhonen et al., 2006) with composition 54.6 SiO2, 6.0 Na2O, 7.9 K2O, 22.1 CaO, 7.7 MgO, 1.7 P2O5 in mol %. A different strategy explored by other researchers was to design a silicate glass composition with extended processing window, which still conserved the high bioactivity of the 45S5. This strategy essentially consists of conserving the high molar content of the silica network modifiers to attain the same network connectivity of 45S5 but partially substituting potassium and calcium for sodium so as to take advantage of the mixed alkali effect, which reduces the onset of

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crystallization. In this sense, it is worth mentioning the composition ICIE16 (49.5 SiO2, 6.6 Na2O, 6.6 K2O, 36.3 CaO, 1.1 P2O5 in mol %) developed to avoid devitrification during sintering (Wu et al., 2011), which also improved its capability for fiber drawing, microfibers with diameters between 40 and 200 μm were produced (Do¨hler et al., 2016). Other compositions exploring the same approach were also proved to form into fibers with diameters around 40 μm (GabbaiArmelin et al., 2015, 2017). Also, the addition of small amounts of fluoride inhibits crystallization (Do¨hler et al., 2016). Notwithstanding the difficulties mentioned, a couple of laboratories succeeded to produce fibers of the Bioglass 45S5 by melt drawing: firstly a work led by the inventor of Bioglass, Larry Hench, produced thick fibers with diameters between 165 and 310 μm (De et al., 2000), while the laboratories of MO-SCI Corporation, USA, produced fibers with diameters in the range of 20
40 μm (Clupper et al., 2004) and later thinner fibers with a mean diameter of 2.3 μm (Liu et al., 2013). Alternatively, other works explored the properties of borate-based glasses formed into fibers using the same melt drawing technique (Liu et al., 2013, 2014) with promising results in their bioactive properties. The 13-93B3 borate microfibers (composition in wt.% 56.6 B2O3, 5.5 Na2O, 11.1 K2O, 4.6 MgO, 18.5 CaO, 3.7 P2O5, and diameters between 0.2 and 3.0 μm) degrade faster than 45S5 fibers with similar diameters, however, the amorphous calcium phosphate layer formed on the borate fibers crystallized to hydroxyapatite more slowly than the silicate fibers. This result is proposed to explain the high capacity of borate fibers to heal soft-tissue wounds (Liu et al., 2013). A different line of research explored the capabilities of phosphate glass fibers for soft-tissue regeneration. These glass compositions were proposed with the aim of preventing long-term toxicity of silicon (Abou et al., 2009), and their fibrous morphology was intended to mimic the nature of muscle and ligament tissue. The initial formulations formed into fibers were explored in the system CaONa2OP2O5 with amounts of phosphate of 45, 50, and 55 mol %. However, only the compositions with 50 and 55 mol % P2O5 (metaphosphate and ultraphosphate glass structure respectively) could be conventionally drawn into fibers with diameters between 8 and 40 μm (Ahmed et al., 2004b). Following this work a series of different compositions was studied. Several glasses were obtained by substituting small amounts of Fe2O3 (1
5 mol %) for the Na2O from the formulations of the former work with 50 mol % P2O5. They were successfully drawn into fibers with diameters between 10 and 40 μm (Abou et al., 2005; Ahmed et al., 2004a). The biocompatibility results showed an increase of solubility with decreasing diameter (related to an increase of surface area to volume ratio), whereas the degradation rate is decreased with the incorporation of Fe2O3 (Abou et al., 2005). Therefore, the addition of this quaternary component could facilitate control of the solubility of the fibers without changing the metaphosphate structure of formulations with 50 mol % P2O5 which gives long phosphate chains. This structure would facilitate good melt-forming capability, because long phosphate chains exist and they are strong enough to withstand the stresses used in

10.4 Bioactive Glass Fibers for Tissue Engineering and Composites

pulling the fluid filament during fiber drawing, and the viscosity is increased by the crosslinks between chains (Ahmed et al., 2004b). Additionally, they observed the formation of myotubes along the axis of the fibers, and that varying the iron content of the glass can influence the ability of muscle precursor cells to attach and subsequently proliferate and differentiate (Ahmed et al., 2004a). Another formulation containing a major quantity of phosphate (62.9 P2O521.9 Al2O315.2 ZnO mol %) was also formed into fibers with diameters of 6.5 μm (Shah et al., 2005). This work demonstrated that these phosphate fibers can support the proliferation and differentiation of human masseter muscle-derived cells. An alternative strategy incorporated TiO2 to different glass formulations with a fixed amount of 50 mol % P2O5 as the network former in order to keep the metaphosphate structure which ensures good fiber drawability (Vitale-Brovarone et al., 2011). At the same time, the varying amount of TiO2 acts as a selector of the glass dissolution rate and limits the pH decrease in the solution caused by the acidic degradation products. They produced fibers of compositions with fixed amounts of 50 P2O5, 30 CaO, 9 Na2O, 3 SiO2, 3 MgO, and varying amounts of TiO2 and K2O between 0 and 5 mol %. The fibers had diameters between 35 and 180 μm. In a different work (Vitale-Brovarone et al., 2012) these fibers showed biocompatibility and suggested that they can be proper substrates for glial cell adhesion and alignment, as well as for axonal growth. Phosphate glass fibers proved also promising results as a reinforcing phase in glass fiber
polymer composites. In a more recent work, Joo et al. (2012) produced a composite made of collagen and glass microfibers, having the composition of one of the metaphosphates containing 5 mol % of Fe2O3 proposed in a previous work (Ahmed et al., 2004a). This composite showed a beneficial effect in spinal cord regeneration in rats, providing physical guidance cues to regenerating axons. In contrast, the same composites did not promote a directional growth of axons in the case of peripheral nerves (Kim et al., 2015). On the other hand, the techniques based in forming a sol
gel precursor may elude the problems of devitrification. These include some unusual methods to produce microfibers: spraying the sol
gel (Hatcher et al., 2003; Ore´fice et al., 2001) or dry-spinning of pure silica fibers (Jokinen et al., 2000; Peltola et al., 2012). However, these methods were only employed to produce glasses with very high content of silica (60
80 mol % SiO2 in the case of spraying and pure silica for dry-spinning) probably due to the need of extruding a high viscosity gel, and their highly porous structure endangers their mechanical properties. In contrast, the more usual method of electrospinning of a sol
gel precursor was also used to produce bioactive glass microfibers with compositions, in mol %, of 60
80 SiO215
35 CaO5 P2O5 (Yi et al., 2008). In a different work, the 70 SiO225 CaO5 P2O5 microfibers (diameters between 0.5 and 2.0 μm) showed good cell attachment and spread (Poologasundarampillai et al., 2014). This technique has nevertheless shown its outstanding potential for the production of nanofibers. In fact, most of the works which succeed in the production of bioactive glass nanofibers employed the technique of electrospinning of a sol
gel. The first successful

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work reported the production of nanofibers with composition 70 SiO225 CaO5 P2O5 (mol %) and diameters between 84 and 630 nm (Kim et al., 2006). Many works followed this: fibers with composition 70 SiO230 CaO and diameters 50
800 nm (Lu et al., 2009) showed good mechanical properties to form scaffolds; 70 SiO225 CaO5 P2O5 nanoporous solid fibers with diameters of 500 nm (Hong et al., 2010b) and hollow fibers with diameters of 600 nm (Hong et al., 2010a), were loaded with drugs that were released in a controlled manner. More recently, nanofibers with the composition of Bioglass 45S5 and diameters between 300 and 400 nm were produced by electrospinning of a glass sol mixed with aqueous solution of polyvinyl alcohol (Deliormanlı, 2015). However, these fibers crystallized forming different phases during calcination, so they strictly did not preserve the 45S5 composition. In the same way, several works explored the potential of bioactive glass nanofibers obtained by electrospinning to produce a polymer composite: a nanocomposite made of PLA reinforced with bioactive glass fibers (composition 58 SiO238 CaO4 P2O5, average diameters of 320 nm) demonstrated high bioactivity, osteoblastic cells showed favorable cell attachment and proliferation behavior on the nanocomposite, and the incorporation of the bioactive nanofiber significantly enhanced further differentiation and mineralization behaviors of the cells (Kim et al., 2008). Additionally, a similar nanocomposite with 70 SiO225 CaO5 P2O5 nanofibers reinforcing PLA demonstrated improved cell proliferation and osteogenic induction of MSCs (Kim et al., 2012). Furthermore, scaffolds made of fibers with composition 60 SiO236 CaO4 P2O5 and average diameters of 450 nm reinforcing poly(ε-caprolactone) were implanted in rats, revealing good bone regeneration capability (Jo et al., 2009). However, the main drawbacks of the glass or ceramic fibers obtained from a sol
gel precursor are that they used to be all stuck together due to drying or calcination of the gel; therefore they form films or mats and are difficult to post-process to form different structures. Also, the sol
gel fibers present high porosity, consequently they tend to be very fragile (Dai et al., 2011).

10.5 PRODUCTION OF GLASS NANOFIBERS BY LASER SPINNING TECHNIQUE Laser spinning is a novel technique capable of producing very long glass nanofibers from ceramic materials with a great variety of compositions (Quintero et al., 2007a,b, 2009a). Fig. 10.3 shows an illustration schematizing the laser spinning process. The precursor material is in solid phase, usually in the form of a plate with a thickness of several millimeters. The technique is based on heating and melting a small volume of this material by a high-power laser beam, while a supersonic gas jet rapidly stretches and cools it down to form nanofibers. All the process takes some microseconds to produce the nanofibers because the laser beam provides enough power density to heat a little volume of material from

10.5 Production of Glass Nanofibers by Laser Spinning Technique

FIGURE 10.3 Schematic representation of the laser spinning process.

ambient temperature to melting temperature (which in some cases is beyond 2000 C) in very short time intervals. Therefore, an extremely high temperature gradient (over 1000 C/mm) is produced. The supersonic gas jet is capable of stretching and cooling the fibers down very fast so they keep their characteristically cylindrical filament shape. In practice, this process is quite similar to laser cutting. Indeed, laser spinning produces a cut in the precursor material plate, where the material removed to produce this cut is transformed into nanofibers. Therefore, a high quantity of glass nanofibers can be produced in only a matter of seconds by laser spinning. So far, nanofibers with different compositions have been obtained from several types of precursors: inorganic glasses, ceramics, and natural rocks. Moreover, the composition of the obtained nanofibers is practically identical to that of the precursor material. The temperature of the molten material and the elongation velocity of the fluid filament must be controlled for effective production of nanofibers. In particular, the viscosity of the molten material must be low enough that the dragging force exerted by the supersonic air jet can stretch the fluid filament. However, it must be high enough to avoid its premature breaking down, which would form drops or particles instead of fibers. The temperature (and viscosity) of the melt is mainly controlled by the density of radiant power of the laser beam and by the relative velocity of the precursor material with regard to the laser beam (Quintero et al., 2014). The gas employed to stretch the molten material is usually compressed air, since no chemical reaction is involved in the production of this kind of nanofiber (like oxidation), and hence there is no need for using any different gas. The kind

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of laser chosen in each case mainly depends on the material from which we aim to obtain nanofibers. In the case of ceramic materials, for example, CO2 laser is a good choice considering that its wavelength (10.6 μm) is efficiently absorbed by most ceramics. This is a method that goes from macro- to nanoscale (top-down) so, similar to most of them, it demonstrates a high production rate. The maximum production rate of laser spinning is close to 60 mg/s of fibers, which means that 1 g of fibers can be produced in approximately 16 seconds (Dieste et al., 2011). This result clearly denotes the high production rate that can be achieved by laser spinning if compared with other methods or techniques. In practice, laser spinning produces a disordered mesh of very long fibers, which can even reach some decimeters. Macroscopically, their appearance is quite similar to cotton wool, as can be seen in Fig. 10.4. However, these fibers are totally independent and detached from each other, so they can be separately taken in a single fiber whenever necessary. Scanning electron microscope (SEM) and transmission electron microscope (TEM) images reveal that the fibers have a well-defined cylindrical shape, with diameters that are almost constant along their entire length. The typical appearance of the fibers is represented by the SEM images in Fig. 10.5. The micrograph presented in Fig. 10.5A shows the morphology of nanofibers produced by laser spinning with composition 58 SiO214 Na2O24 CaO4 P2O5 (mol %) and diameters between 300 and 800 nm. The image in Fig. 10.5B exhibits an example of the nanofibers of Bioglass 45S5 with diameters between 400 nm and 1.2 μm. Fibers are flexible and, as mentioned before, they are detached, so they can be aligned, ordered, separated in individual fibers, and even woven.

FIGURE 10.4 Image of a mat of glass nanofibers produced by laser spinning, which are compared with a 1 euro coin.

10.5 Production of Glass Nanofibers by Laser Spinning Technique

FIGURE 10.5 Micrographs obtained by SEM showing the typical appearance of the nanofibers produced by laser spinning. The image presented in (A) shows nanofibers with composition 58 SiO214 Na2O24 CaO4 P2O5 (mol %). The image in (B) exhibits an example of the nanofibers of Bioglass 45S5.

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Statistical analysis of the diameters measured using SEM images revealed that the distribution of diameters fits quite precisely a log-normal distribution, ranging from tens of nanometers to several microns. The characteristic parameters of the distribution (median, mode, standard deviation) can be varied depending on the processing parameters: laser power, relative speed between the precursor material and the laser beam, etc. (Dieste et al., 2011). It has been demonstrated that, if the processing conditions are correctly chosen, the statistical mode of the diameters can be as small as 180 nm. In particular, the relative speed of the precursor material with regard to the laser beam is the most influential processing parameter in the resulting distribution of diameters. The TEM micrograph in Fig. 10.6 shows a detail of some Bioglass 45S5 nanofibers produced by laser spinning (Quintero et al., 2009a); the inset presents a representative electron diffraction pattern of those nanofibers, demonstrating its amorphous structure. In fact, the electron diffraction analyses made from multiple samples demonstrate that every fiber is amorphous, independently of the nature of the precursor material from which these fibers were obtained, even though this precursor material is originally crystalline. This is produced because of the high cooling speed of the molten material.

FIGURE 10.6 TEM micrograph of some Bioglass 45S5 nanofibers produced by laser spinning; the inset presents a representative electron diffraction pattern of these nanofibers revealing their amorphous structure.

10.5 Production of Glass Nanofibers by Laser Spinning Technique

Another important advantage of laser spinning lies in the composition of the fibers produced. They show almost the same chemical composition as the precursor material, despite the high temperatures reached during the process. In fact, by this technique a great quantity of glass fibers from different materials were produced (Quintero et al., 2007a,b, 2009a). Table 10.1 shows an example of the analysis by X-ray fluorescence of the composition of several soda-lime silicate glass plates specifically melted for the experiment and the fibers obtained from them (Quintero et al., 2007b). It confirms that they both show very similar compositions, with only small differences. The highest differences are in the volatile components, such as Na2O and P2O5. In fact, the increase of the relative percentage of the other components is due to a decrease in the proportion of sodium and phosphorus. This important property of laser spinning is again due to the high speed of the process. The material is melted and solidified in milliseconds; therefore the evaporation of the volatile components is restricted. The analysis of the process by an ultrahigh-speed camera revealed the formation mechanism of fibers (Quintero et al., 2007a). The greater limitation to producing nanofibers is the breaking down due to capillary forces. This occurs when the viscosity is too low, so the capillary forces due to the surface tension of the molten material are stronger than the cohesion forces in the molten material and it breaks to form small drops. This effect is similar to that when a faucet is slightly turned on. The volume of flow would be very low, so the water jet would be really thin. If it is thin enough, the capillary forces produced by the surface tension overcome the cohesion forces of the liquid, so it is consequently divided in drops. On the other hand, a theoretical analysis on the production rate of glass nanofibers was carried out by a mathematical model developed specifically for laser spinning (Quintero et al., 2009b). These analyses pointed out to formation times of nanofibers on the order of microseconds, which allow an understanding of why this technique always produces amorphous fibers, and why it is suitable to Table 10.1 Chemical Composition in Weight Percent of Four Different Soda-Lime Silicate Glass Samples and the Fibers Obtained From Them by Laser Spinning Sample 1 2 3 4

Precursor Fibers Precursor Fibers Precursor Fibers Precursor Fibers

SiO2

Na2O

CaO

P2O5

49.86 51.71 54.10 56.80 60.83 62.37 65.00 65.97

17.87 15.81 16.04 13.49 12.71 10.65 11.98 8.92

25.42 26.42 23.35 24.50 20.72 22.06 16.17 19.76

5.78 4.79 5.36 4.17 4.50 3.60 5.68 3.98

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produce nanofibers from low-viscosity melts: the elongating process is much faster than the breaking flow, preventing filament rupture. Another interesting result obtained from the mathematical model was to make an estimate of the viscosity range suitable for the formation of a fiber. Specifically, the maximum viscosity which allows stretching of the fluid filament is in the range of 100
300 Pas. With melt viscosity close to this limit the process will yield the thickest fibers with diameters in the order of several micrometers, higher viscosity would impede fiber formation. Conversely, the lower viscosity limit in the order of 1 Pas corresponds to the processing conditions which will produce the thinnest nanofibers, with diameters of tens of nanometers (Quintero et al., 2014). If the viscosity of the filament is less than this limit, it will break down due to the flow driven by capillary forces. Actually, this lower limit is two orders of magnitude smaller than the conventional methods of fiber drawing (100
1000 Pas), and consequently broadens the range of compositions that can be processed by laser spinning in comparison with other techniques.

10.5.1 BIOACTIVE GLASS NANOFIBERS FOR TISSUE ENGINEERING AND COMPOSITES As we mentioned in Section 10.5, laser spinning is able to produce nanofibers from a wide range of different ceramic materials. In particular, laser spinning was the first technique that was able to produce Bioglass 45S4 and 52S4.6 nanofibers (Quintero et al., 2009a) among other different compositions. Plates of precursor materials with the desired composition and suitable width were prepared by conventional melting techniques in a common furnace. These plates were then directly processed by laser spinning and a dense net of glass micro- and nanofibers was obtained. These fibers are totally detached and independent from each other, and they show lengths of up to several centimeters. Moreover, there is no need for postprocessing, as they can be directly employed as bioactive glass. Table 10.2 presents a comparison between the composition of these plates of precursor material and the nanofibers obtained from them. Both compositions are very similar and, again, this is because of the high speed of the melting and solidification processes, restricting the evaporation of the volatile components. Table 10.2 Chemical Composition in Weight Percent of the Plates of Bioactive Glasses Employed as Precursor Materials and the Fibers Obtained From Them by Laser Spinning Sample 45S5 52S4.6

Precursor Fibers Precursor Fibers

SiO2

Na2O

CaO

P2O5

45.54 46.00 51.47 52.27

23.87 23.19 21.09 20.00

27.42 27.68 24.13 24.71

2.55 2.43 2.53 2.30

10.5 Production of Glass Nanofibers by Laser Spinning Technique

Bioactivity tests show the potential of these bioactive nanofibers produced by laser spinning. After 12 hours of immersion in simulated body fluid the nanofibers are completely covered with amorphous calcium phosphate. Then, after 1 or 2 incubation days, many of the fibers are hollow. This is a distinct behavior which has not been observed in thicker fibers. In this case, the fast dissolution of the silicate network of the bioactive glass occurs at a similar rate as the external layer of calcium phosphate is being formed, and, since the fibers are so thin they can completely dissolve through its full thickness. As a result, the original nanofibers completely disappear giving way to a hollow calcium phosphate tube. This is an important result, which means that at the end of the process, the only remaining material at the body would be the calcium phosphate and the tissue produced by the body itself, while the fibers (material that comes from outside of the body) totally disappeared. Finally, after 5 incubation days, the calcium phosphate layer crystallizes in the form of hydroxyapatite (Quintero et al., 2009a). This means that bioactive laserspun glass nanofibers were capable of promoting the generation of biologic tissue over them and then disappear; therefore, they present high bioactivity and are completely bioresorbable. The antibacterial properties of some different compositions of laserspun glass nanofibers were also analyzed. In a first work, the antibacterial effect of ICIE16 bioactive glass nanofibers was tested and compared with nanofibers of a bioinert glass (Echezarreta-Lo´pez et al., 2014). As described above, the ICIE16 glass is a formulation derived from Bioglass 45S5, where potassium and calcium were partially substituted for sodium, therefore the ICIE 16 glass has a significantly higher content of Ca than 45S5, but conserving the same network connectivity. Fibers with diameters from tens of nanometers up to 5 μm were produced using the laser spinning technique. The ICIE16 nanofibers showed biocompatibility since they are not cytotoxic and developed an amorphous CaP layer after 4 days of incubation in culture medium. At the same time, they present a bactericidal effect on Staphylococcus aureus, which is significantly higher than the bacteriostatic result of the bioinert fibers. This effect was related to the release of alkaline ions and the increase of the pH of the medium. In a more recent work, the antibacterial activity against S. aureus of 45S5 Bioglass nanofibers was compared to those with composition ICIE 16 M, a modification of 45S5 where sodium was partially substituted for potassium, magnesium, zinc, and strontium (Echezarreta-Lo´pez et al., 2017). The 45S5 dissolved and formed a crystalline layer of calcium phosphate faster than the ICIE 16 M nanofibers. Both showed biocompatibility under the conditions of a standard in vitro cytotoxicity test performed with fibroblast (B 323 ALB/3T3) cells. Notwithstanding, they presented a different bactericidal effect: the 45S5 nanofibers shown a bateriostatic effect, while the ICIE 16 M demonstrated higher bactericidal results for longer times, which can be justified by the progressive release of Zn and Sr ions. Consequently, these fibers demonstrated their potential to be used as filler for composites, keeping their disinfectant properties. Two new compositions of bioactive glasses were also processed by laser spinning to produce nanofibers which were tested against Escherichia coli,

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Streptococcus oralis, Streptococcus mutans, S. aureus, and Candida krusei (Esteban-Tejeda et al., 2016). The compositions were a soda-lime silicate glass with high CaO content (43.7 SiO2, 6.3 B2O3, 22.2 CaO, 20.8 Na2O, 6.5 Al2O3, 0.4 K2O in mol %) and a ZnO glass (23.3 SiO2, 35.7 B2O3, 6.5 Na2O, 3.6 Al2O3, 30.9 ZnO in mol %). The laserspun nanofibers showed the capability of decreasing the presence of E. coli, S. oralis, S. mutans, and C. krusei up to a no detectable quantity in less than 24 hours. The results against S. aureus were different: the CaO nanofibers induced a 4-log reduction, while the ZnO nanofibers produced a 6-log reduction. Both compositions demonstrated biocompatibility in the standard cell viability tests performed with mouse embryonic fibroblast cells. Moreover, the zinc oxide laserspun fibers were incorporated as fillers for composites destined for dental materials and similar antimicrobial results were obtained. They were tested in the presence of human saliva in order to simulate the real working conditions and their antibacterial properties remained after immersion for 8 days and their structure was not degraded. Additionally, to the best of our knowledge, nanofibers of phosphate glasses have been produced for the first and only time using the laser spinning technique. A series of phosphate invert glasses (less than 40% mol P2O5) were previously developed with the objective of obtaining compositions that dissolve completely in aqueous solutions, degrade more slowly than the glasses with metaphosphate structure, and with the ability of forming an apatite layer in in vitro tests (Brauer et al., 2010). The metaphosphate and ultraphosphate formulations described in Section 10.4 were not suitable for bone tissue engineering since they do not form an hydroxyapatite layer in the in vitro or in vivo tests and they dissolve too fast to support cell adhesion (Abou et al., 2009). Furthermore, the addition of TiO2 to the phosphate glass reduces its degradation rate, enhances cell proliferation and gene expression (Abou et al., 2009), and improves their processing capabilities (Brauer et al., 2010). The compositions of three of these formulations developed by Brauer et al. (2010) are presented in Table 10.3. They were all successfully transformed into nanofibers using the laser spinning technique (Quintero et al., 2013). The in vitro analyses with preosteoblast cells (MC3T3-E1) demonstrated no cytotoxicity, and showed good cell adhesion and alignment along the fibers (unpublished results). Finally, although the following example does not correspond to a bioactive glass, it is worth mentioning that the laser spinning technique also demonstrated its capacity to directly produce nanofibers with silver nanoparticles embedded Table 10.3 Composition in Mol Percent of the Invert Phosphate Glasses Processed by Laser Spinning to Form Nanofibers Glass

P2O5

CaO

Na2O

MgO

TiO2

Mg5NT5 Mg5NT10 Mg5NT15

37 37 37

26.7 24.4 22.1

22.1 20.2 18.3

9.2 8.4 7.6

5 10 15

References

(Cabal et al., 2013). Therefore, this technique is capable of producing composites with the shape of nanofibers directly without any postprocessing. These nanofibers also showed significant antibacterial properties due to the effect of silver nanoparticles.

10.6 SUMMARY AND OUTLOOK A review of the production of different compositions of bioactive glass fibers— spanning silicates, borates and phosphates—with special emphasis in their methods of production, properties, dimensions, and applications has been presented. This survey aims to demonstrate the potential of this kind of bioactive materials and structures in the fields of tissue regeneration and tissue engineering directly as scaffolds forming a porous net, as well as a reinforcing phase of a bioactive composite. In this context, nanofibers are singular structures that present high potential to be used in tissue engineering. This is mainly due to their high surface area to volume ratio, and the 3D structure that forms a mesh of nanofibers mimicking the morphology of the extracellular matrix at the nanoscale, which elicits stimuli that promote cell adhesion, proliferation, and differentiation. There are several techniques aiming to produce nanofibers, and electrospinning is currently the most commonly employed one. Laser spinning is a novel technique that can be employed for the production of nanofibers with a wide variety of compositions. The high velocity of melting and solidification of the precursor material allows to produce glass fibers even from compositions of fragile melts and a high tendency for devitrification. On the other hand, it is easy to vary the composition of the nanofibers with the aim of improving their properties in any way. So far, laserspun glass nanofibers with different compositions have been successfully tested as bioactive, bioresorbable, and antibacterial applications.

ACKNOWLEDGMENT This work was partially supported by the EU research project Bluehuman (EAPA_151/ 2016 Interreg Atlantic Area), Government of Spain (MAT2015-71459-C2-P), and Xunta de Galicia (ED431B 2016/042, ED481B 2016/047-0, ED481D 2017/010).

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246. Kuchibhatla, S.V.N.T., Karakoti, A.S., Bera, D., Seal, S., 2007. One dimensional nanostructured materials. Prog. Mater. Sci. 52, 699
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27.

CHAPTER

Application of (mixed) metal oxides-based nanocomposites for biosensors

11

Ali Salehabadi1 and Morteza Enhessari2 1

Environmental Technology Division, School of Industrial Technology, Universiti Sains Malaysia, 11800 Minden, Penang, Malaysia 2Department of Chemistry, Naragh Branch, Islamic Azad University, Naragh, I. R. Iran

11.1 INTRODUCTION Smart materials are very difficult to define in explicitly. Inspiration for the search for new solutions of advanced material-based sensors is common throughout nature (Cichosz et al., 2018). Changing the daily optical-based glucose check for diabetes patients to electrochemical sensors, with millions of sensors sold annually, the method of production, cost, and easiness of use would have a huge impact on the market. Interdigitated transducers in biosensor technology are important to fulfill the requirements of the diverse criteria in the market, such as their combination with biomolecules to develop high-selectivity sensors. Their electrical excitation makes it easy to develop biosensors to perform automatically measurement and fluidics manipulation. Using advanced materials (plastic, nanomaterials, etc.) as a substrate in sensors technology helps to further reduce the costs of the sensors. Quality, cost, suitability, and automatization are important factors which make sensors excellent candidates as potential equipment for diverse applications. Biosensors were first fabricated in the 1960s. These were macroscopic membranes consisting of transducers and gluing wires. For smallscale production, as is required for clinical analyzers, conventional biosensors are not useful. In this chapter present-day applications of biosensors to clinical chemistry are reviewed, including basic and applied research, commercial applications, and fabrication techniques. Recognition elements include enzymes as biocatalytic recognition elements and immunoagents and DNA segments as affinity ligand recognition elements, coupled to electrochemical and optical modes of transduction. The future will include biosensors based on synthetic recognition elements

Materials for Biomedical Engineering: Inorganic Micro- and Nanostructures. DOI: https://doi.org/10.1016/B978-0-08-102814-8.00013-5 © 2019 Elsevier Inc. All rights reserved.

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to allow broad applicability to different classes of analytes and modes of transduction extending the lower limits of sensitivity. Microfabrication will permit biosensors to be constructed as arrays and incorporated into lab-on-a-chip devices. The learning objectives of this chapter are: • • • • • •

To understand the various (bio)sensor devices and the principle of measurement; To be able to know the mechanism of detection of various analytes; To illustrate the criteria of the sensing mechanism; To find the materials (metal oxides, mixed metal oxides, nanocomposites, etc.) appropriate for sensing devices; To select the best sensing materials (composition) based on performance characteristics; To understand the range, linear range and detection limits, response times, recovery times, and lifetimes.

11.1.1 SEMICONDUCTING (NANO)MATERIALS Semiconducting materials are widely used in miscellaneous applications, such as energy-storing devices (Salehabadi et al., 2018a,b,c,d), sensing devices (Enhessari and Salehabadi, 2016; Hulanicki et al., 1991; Korotcenkov et al., 2009), etc. They consist mostly of metal oxides, some metals, or polymers. Titanium oxides, zirconia, silicon dioxide, etc. are semiconductors used as effective immobilization scaffolds in biosensor technology. These nanomaterials are able to promote direct electron transfer of the entrapped biomolecules and maintain the long-term bioactivity. Various synthesized nanostructural forms of semiconductors, including nanoparticles (NPs), nanotubes (NTs), nanowires, nanorods, nanobelts, nanosheets, nanotips, quantum dots (QDs), hollow spheres, etc. have been synthesized. Sol
gel synthesis, hydrothermal or solvothermal growth, physical or chemical vapor deposition, low-temperature aqueous growth, chemical bath deposition, or electrochemical depositions are methods used for nanoscale material formation. Electronic materials include insulators, semiconductors, conductors, and superconductors. A solid insulator is a substance with a very low electrical conductivity and there is a considerable energy gap before an empty orbital becomes available (Fig. 11.1A). A semiconductor is a substance which has electrical conductivity that increases with increasing temperature, and at room temperature, the conductivities are typically intermediate between conductors and insulators (Enhessari, 2013; Khanahmadzadeh et al., 2015; Zare et al., 2009; Enhessari et al., 2010, 2012a,b, 2013, 2016a,b; Nouri et al., 2016). Semiconductors are classified as intrinsic or extrinsic semiconductors; the former band gap is very small (Fig. 11.1B), therefore, the energy of thermal motion results in jumping of some electrons from the valence band into the empty upper

11.1 Introduction

band, and the former is a semiconductor in the presence of impurities; p-type doping atoms remove electrons from the valence band and n-type doped atoms supply electrons to the conduction band (Fig. 11.1C). A conductor is a substance with an electric conductivity that decreases with increasing temperature, and has zero band gaps (Fig. 11.1D). A superconductor (classified into three types) is a class of materials with zero electrical resistance. The origin of this class of materials is related to electron
phonon coupling and the resultant pairing of conduction electrons (Askeland and Phule, 2006). A metallic conductor, a semiconductor, and a superconductor can be distinguished based on the temperature dependence of the electrical conductivity. This variation can be observed in Fig. 11.2.

FIGURE 11.1 The structure of a typical (A) insulator, (B) intrinsic semiconductor, (C) extrinsic semiconductor, (D) conductor. Adapted from Atkins, P., Overton, T., Rourke, J., Weller, M., Armstrong, F., 2006. Inorganic Chemistry, fourth ed. Oxford, USA.

FIGURE 11.2 Variation of the electrical conductivity of a substance with temperature (Atkins et al., 2006).

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Semiconductors have many applications because their properties can be easily modified by the addition of impurities; these applications include computer chips, diodes, transistors, lasers, and LEDs. Furthermore, the electrical conductivities of semiconductors can be controlled by exposure to an electric field, light, pressure, and heat, hence, they can be used in many sensor devices (Atkins et al., 2006). An electron sea can be formed when elements lose electrons. This governs the chemical properties of the metallic elements and accounts for metallic bonding. The terms “metals” and “nonmetals” elements are directed toward the ionization energies. This definition can be observed in from 13 to 16 groups. The elements in these groups start with nonmetals and end with metals. There are allotropic variations in the sense that some elements exist as metals and nonmetals, like group 15; N and P are nonmetals, As is a nonmetal, a metalloid, and a metallic allotrope, and Sb and Bi are metals. Among all the elements, transition metalbased nanocomposites have been most widely investigated. Moreover, metalloids like Si are also used in biosensor technology. The most well known elements reported in biosensing technology are cobalt, copper, iron, manganese, nickel, osmium, titanium, zinc, and zirconium. Materials with high ionic conductivity have important applications in sensors and fuel cells of various kinds. Metal-oxide semiconductors can be used as sensors for monitoring environmental pollution, fire, and vehicle emissions. The fundamental sensing mechanism relies upon the change in electrical conductivity due to the interaction between the gases in the environment and oxygen in the grain boundaries (Hunter et al., 2006). Metal oxides nanostructures (MONs) have received enormous attention for their promising sensing applications. Unique features of MONs, such as controllable size, functional biocompatibility, biosafety, chemical stability, and catalytic properties make them suitable for fabrication of (bio)sensors. Moreover, the physicochemical properties of MONs, such as enhanced electron-transfer kinetics and strong adsorption capability, and the possibility of chemical modification of their surfaces, make them more advantageous than other materials in order to enhance chemical and biological sensor performances. MONs are good candidates for optical emitters, electronic conductors, catalysts, carriers for amplified detection signals, and biosensing interfaces. The term “nanocomposite” has been widely distributed in biosensor technology. Almost all elements can play a role in biosensor technology and its respective nanocomposites. The amalgamation of conducting and semiconducting NPs like gold, silver, platinum, carbon nanotubes, graphene, etc. has been reported. The optical, electrical, and magnetic properties of MONs can be enhanced as an amalgamate with NPs. As a result, the final materials, called “nanocomposites,” have improved selectivity, stability, and sensing performances. The selection, design, and application of MONs have an important role in the generation of new sensing devices with novel functionality, enhanced signal amplification, and coding strategies.

11.1 Introduction

The elemental distribution of transition metals is mostly focused on these elements. In transition metals, iron (Fe) have stimulated a great deal of interest. For example, facile electrochemical biosensors for hydrogen peroxide using efficient catalysis of hemoglobin on the porous Pd@Fe3O4-MWCNT nanocomposites are reported (Baghayeri and Veisi, 2015). A hydrogen peroxide biosensor based on electromagnetic poly(p-phenylenediamine) @ Fe3O4 nanocomposite was fabricated by Baghayeri and his coworkers (Baghayeri et al., 2010). They found that a pair of well-defined redox peaks of Hb at the Hb
PpPDA@Fe3O4 modified glassy carbon electrode with reproducibility and high sensitivity to H2O2. PFu@Fe3O4 conductive nanocomposites have been examined as a host of hemoglobin (Baghayeri et al., 2014). In this system, the Hb immobilizes on PFu@Fe3O4 nanocomposites. Bionanocomposite of antihuman IgG/COOH
multiwalled carbon nanotubes/Fe3O4 was made for electrochemical immunoassay of human tetanus IgG (hIgG) as a model antigen (Zarei et al., 2012). Fe3O4/polydopamine/Au nanocomposites were fabricated for detection of human leptin in serum (He et al., 2015). This immunosensor exhibited high sensitivity, good specificity, and a wide linear range for human leptin sensing from 1.0 to 8.0 3 102 pg/mL. Fig. 11.3 shows the fabrication process of this immunosensor. Xanthine molecules can serve as an indicator of meat spoilage, determined using a REGO/Fe3O4 bionanocomposite sensor (Dervisevic et al., 2015). Here, the current response of the linear range was in the range of 2
36 μM with a

FIGURE 11.3 Fabrication of a biosensor for human leptin (He et al., 2015).

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sensitivity of 0.17 μA/M, a response time of B3 seconds, and a detection limit of 0.17 μM. The basic principle of the electrochemical reaction with amperometric xanthine biosensors are as illustrated in Eq. (11.1): Xanthine+O 2 +H 2 O

Uric acid+ H 2 O 2 2H + +O2 + 2e–

(11.1)

Working electrode

Immobilization of a lipase (Candida rugosa) on functionalized Fe3O4@SiO2, which covalently bound to the nanoparticles and was entrapped in the membrane, was reported by Aghababaie et al. (2016). They reported that the relative activity and loading capacity of ENCM is higher than lipase immobilized on a UF membrane. Fe3O4/g-C3N4/HKUST-1 composites as a platform for ochratoxin A (OTA) are another class of biosensor recently fabricated (Hu et al., 2007). This is a fluorescence biosensor with strong adsorption capacity for dye-labeled aptamer and capable of completely quenching the fluorescence of the dye. The fluorescence intensity of the biosensor has a linear relationship with the OTA concentration. A stable colloid solution of core
shell Fe3O4/polyaniline nanoparticles in chitosan (CHT)/H2PtCl6 over the surface of a carbon paste electrode (CPE) was synthesized for determination of xanthine (Sadeghi et al., 2014). Copper is the second most applicable element in biosensor technology. Copper oxide nanowires/single-walled carbon nanotubes (Chen et al., 2016), CuO/grapheme (Hsu et al., 2012), Cu/Cu2O nanocrystals and reduced graphene oxide (Wang et al., 2015), copper oxide/polypyrrole/reduced graphene oxide (Moozarm et al., 2015), Cu2O/MWCNTs (Zhang et al., 2009a) nanocomposites are widely used in clinical and biological sensing applications. Zinc (Zn) and zirconium (Zr) are also used in the fabrication of biosensors. Graphene/zinc oxide nanocomposites were synthesized via liquid-phase exfoliation and solvothermal growth for DNA sensing (Shin et al., 2015). The results indicate that the graphene/zinc oxide nanocomposite can enhance the sensitivity and efficiency of electrochemical DNA biosensors. Fig. 11.4 shows a schematic fabrication of grapheme/ZnO biosensor-based nanocomposites. ZnO/CH3NH3PbI3/NCQD nanocomposites were used for detection of fibroblast-like synoviocyte (FLS) cells (Pang et al., 2015). A wide linear range from 1.0 3 104 to 10 cells/mL and a low detection limit of 2 cells/mL was obtained from current sensors. The authors proposed that this kind of bisosensor would provide an enhanced strategy for FLS cell detection. Indian researcher Mogha and his coworkers (Kumar et al., 2016) reported a biosensor made from acetylcholinestrase (AChE), and reduced graphene oxide (RGO)-supported zirconium pxide (ZrO2/RGO) nanoparticles for chlorpyrifos (pesticide) detection. They schematically illustrated the significance of this biosensor (Fig. 11.5).

11.1 Introduction

FIGURE 11.4 Schematic representation of graphene/ZnO biosensor-based nanocomposites (Shin et al., 2015).

FIGURE 11.5 Process of nanocomposite pesticide biosensing and detection from nature to the laboratory.

The concept of huge molecules is a globally accepted subject by many researchers. Natural, elastomer, and synthetic polymers are three main groups of polymers. Natural polymers have very complex structures as compared to synthetic polymers. Elastomers, include rubbery materials, have found to use widely

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in various bio-applications owing to their structural and mechanical properties. Elastomers can be prepared either synthetically or naturally. The term “conductivity” can be used for both, inorganic and organic materials (Fig. 11.6). The structural requirement for conducting polymer (CP) is a conjugated π-electron system. The CPs are also possible when the π-electron is a side chain. In this chapter, we attempt to answer two fundamental questions that may be encountered by researchers in developing biosensor performance, including: 1. The type of analytes that nanosized metal oxides can measure in an appropriate condition. Metals or metalloids

Analyte

Complement (CNT, graphene, clay, polymer)

Oxygen

(glucose, cholestrol,...)

Method (Electrochemical,...)

FIGURE 11.6 Conductivity of advanced materials.

Measurement

11.1 Introduction

2. The best criteria for selection of metal oxides capable for analyte measurement. Analyte Metal oxide

Method measurment

Analyte biosensor

11.1.2 POLYMERS Polymers are inherently low in electrical conductivity. However, there are several synthetic polymers with electrical conductivity, such as polypyrrole, polythiophene, etc. (Table 11.1). These polymers are not thermoplastics. Admixing conducting fillers into nonconducting polymers is an effective and economical way to produce electrically CP components with better processability, flexibility, and mechanical properties. Various elements can be used as a sensing element: DNA, antibodies, cells, molecularly imprinted polymers (MIPs), and conducting and semiconducting polymers (CPs and SCPs). The high application potential of CPs and SCPs in chemical and biological sensors is one of the main reasons for their intensive investigation and development. Conductive polymers or electroactive polymers are a famous subgroup of synthetic polymers, bearing at least one conjugated π-electron system. Polymers with conjugated π-electron systems display unusual electronic properties, including high electron affinities and low ionization potentials, hence, they are easily oxidized or reduced by charge transfer agents. The as-mentioned phenomena in poly (radicalcation) (p-type) or poly(radical-anion) (n-type)-doped conducting materials are close in relation to metallic conductivity. Fig. 11.6 illustrates the possible conductivity range of various materials.

11.1.3 NANOCOMPOSITES/PARTICLES Nano-ordered composite materials consisting of inorganic/inorganic or organic/ inorganic materials have been attracting attention for the purpose of creating high-performance functional materials (Salehabadi et al., 2014; Salehabadi, 2014; Tan et al., 2016). The advent of NPs in the 21st century with their many advantages, such as large surface-to-volume ratio, high surface reaction activity, and strong adsorption ability to immobilize desired biomolecules, especially enzymes, has changed the technology of making high-performance materials. NPs may not always be very useful in biomedical and technological applications. They have a large ratio of surface area to volume and strong dipole
dipole

365

Table 11.1 Conductive Polymers The Main Chain Contains

Heteroatoms Present No Heteroatom

Nitrogen-Containing

Sulfur-Containing

Aromatic cycles

Poly(fluorene)s Polyphenylenes Polypyrenes Polyazulenes Polynaphthalenes

The N is in the aromatic cycle: Poly(pyrrole)s (PPY) Polycarbazoles Polyindoles Polyazepines The N is outside the aromatic cycle: Polyanilines (PANI)

The S is in the aromatic cycle: Poly(thiophene)s (PT) Poly(3,4ethylenedioxythiophene) (PEDOT) The S is outside the aromatic cycle: Poly(p-phenylene sulfide) (PPS)

Double bonds Aromatic cycles and double bonds

Poly(acetylene)s (PAC) Poly(p-phenylene vinylene) (PPV)

11.2 Sensors and Biosensors

attractions; hence, they can easily aggregate. Moreover, the number of functional groups usually limits selective binding (Chen et al., 2011).

11.2 SENSORS AND BIOSENSORS Sensor technology is a basic enabling technology in many instances. In intelligent manufacturing processing, sensors have been considered in a range of applications, from assessing aircraft integrity to monitoring chemicals in the environment. The structural principle of sensor technology is instructed by ceramic materials. The oxygen-deficient crystal structure in semiconducting oxide materials can change significantly the resistance of an oxide sensor. A biosensor or a bioreceptor or transducer, is an integrated receptor-transducer device, which converts the biological-recognition events into a measurable physicochemical signal that is proportional to the target concentration (Lamabam and Thangjam, 2016). Electrochemical, optical, piezoelectric, and electromechanical sensors are four types of biosensors (Fig. 11.7). We will discuss the various types of biosensors in the coming sections.

11.2.1 SENSING MEASUREMENT The sensors, in general, are also classified by various criteria, including primary input quantity (measurand), transduction principles (using physical and chemical effects), material and technology, property and application. Certain features have to be considered in sensor technology. Accuracy, environmental condition (temperature/humidity), range measurement (limit of sensor),

FIGURE 11.7 Classification of biosensors.

367

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CHAPTER 11 Application of (mixed) metal

and calibration are essential criteria for selecting sensing devices. Moreover, resolution, linearity range, cost, and finally repeatability are the most important factors in sensor technology. In sensor measurement, in fact, the sensor performance measures from the Vout of RL that cascades RS (the resistance of sensor) using Eqs. (11.2) and (11.3). RS 5 RL 3 S5

ðVc 2 Vout Þ Vout Ra Rg

(11.2) (11.3)

where RS is the resistance of the sensor, S is the sensor response, Ra and Rg are the resistance in air and in the air mixed with detected gases, respectively (Enhessari and Salehabadi, 2016).

11.3 APPLICATION OF SENSORS Sensor applications can be expressed based on their classification into the following groups: •

•

•

•

Accelerometers: These are instruments for measuring the acceleration of a moving or vibrating body (pace makers and vehicle dynamic). They are based on microelectromechanical (MEM) sensors. Biosensors: As mentioned above, biosensors are based on electrochemical technology and are used for all bioactive sites like food testing, medical care device, water testing, and biological warfare agent detection. Image sensors: These are based on the complementary metal-oxide semiconductor technology and are used in consumer electronics, biometrics, traffic and security surveillance and PC imaging. Motion detectors: These are based on infrared, ultrasonic, and microwave/radar technology and are used in video games and simulations, light activation, and security detection.

In this section, the general observations of the sensors will be discussed in five categories: gas, chemical, environment, biology, and clinical.

11.3.1 GAS (BIO)SENSORS The advent of high-performance solid-state gas sensors has motivated several scientists in searching for new materials and investigating their gas-sensing properties. Spinels, illuminites, and perovskites are three famous groups in gas sensor

11.3 Application of Sensors

technology. The mixed metal oxides are oxidation catalysts or oxygen-activated catalysts. The stability of the mixed metal oxide structure allows synthesis of new compounds with a high extent of oxygen deficiency. In our previous chapter (Enhessari and Salehabadi, 2016), we summarized the as-reported perovskite nanomaterials in gas-sensing devices. We mentioned that the high catalytic activity of perovskite oxides depends on the high surface activity to oxygen reduction ratio or a large number of oxygen vacancies in the particular structure. Among the various reactions studied, automobile exhaust gas, various pollutant gases such as H2S (Shandiz et al., 2013) and NH3 (Song et al., 2011), NOx decomposition reaction gas (Zhou et al., 2014), hydrogen gas (Mukherjee and Majumder, 2014), methanol (Sen et al., 2015), and LP gas (Ranjith Kumar et al., 2014) have attracted particular attention. The perovskite materials can be used as a thin film (nanocomposites) or nanopowders. Table 11.2 represents the most important perovskite materials in gas-sensing devices. Lanthanum iron oxide nanocrystals (Bhargav et al., 2014, 2015), NiFe2O4 (spinel)-La0.8Pb0.2Fe0.8Co0.2O3 (perovskite) (Maity et al., 2015), SmFe0.7Co0.3O3 perovskite oxide (Zhang et al., 2010), lanthanum cuprate (La2CuO4) (Dharmadhikari et al., 2014), nanocrystalline La12xCaxFeO3 (Shi et al., 2014), barium stannate and aluminum oxide-based gas sensor (Kocemba et al., 2007), LaCoO3 (Velciu et al., 2015; Ding et al., 2016), NdCoO3 (Malavasi et al., 2005), Mg0.5Zn0.5Fe2O4 (Mukherjee and Majumder, 2013), NdCoO3 perovskite (Malavasi et al., 2005) are some examples of mixedmetal oxides used as promising materials for CO/CO2-sensing devices. The mechanism of CO sensors is as depicted in Eq. (11.4). 2 O2 ads 1 CO.CO2 1 e 1 COg .COaq: .COads 1 e2 2 COads 1 O2 2 .CO2 1 e COg 1 Oxo .CO2 1 e2 1 Vxo CO 1 1=2 O2 1 Vxo 1 e2 .CO2 1 Oxo

(11.4)

11.3.1.1 NOx Lanthanum-based mixed metal oxides are considered for NOx gases detection. (La0.8Sr0.2)2FeNiO62δ (Zhou et al., 2014), LaFeO3, and LaMnO31δ (Armstrong et al., 2011) are some examples of this sensing device. According to mixed potential theory, in a potentiometric sensor, the redox reactions always govern the potential difference between a sensing electrode and a reference electrode, according to Eq. (11.5), NO2 1 2e2 3NO 1 O22 2O22 3O2 1 4e2

(11.5)

Here, the reactions for NO2 exposure or reverse reactions for NO exposure occur at a sensing electrode where the reference electrode is exposed to air (Armstrong et al., 2011). NO2 acts as an oxidizing gas, hence, oxygen is produced at the electrode while NO is a reducing gas and consumes the oxygen. The former

369

Table 11.2 Gas-Sensing Materials Based on Mixed Metal Oxides (Enhessari and Salehabadi, 2016) Sensing Materials

O2

H 2O

CO/CO2

C2H5OH

Titanate

Sr(Ti0.65Fe0.35)O3 Pb(Zr0.2Ti0.8)O3

BaTiO3 CdTiO3 Na2Ti3O7

Li0.35La0.55TiO3

CoTiO3, Cr1.7Ti0.3O3, Zn2TiO4,

Ferrite

SrTi0.6Fe0.4O3aδ BaFeO3

La0.8Sr0.2Fe1axCuxO3 Cu0.5Zn0.5W0.3Fe1.7O4

Co1axNixFe2O4 Mg0.5Zn0.5Fe2O4

Co1axNixFe2O4 ZnFe2O4 NixZn1axFe2O4 GaFeO3 Ni1axCoxFe2O4

NOx

Ref.

Perovskite

LaFeO3, Co1axMnxFe2O4 CoFe2O4

[Argirusis et al., 2011; Belle et al., 2014; Du et al., 2010; Imran et al., 2013; Isarakorn et al., 2011; Santhaveesuk et al., 2015; Wang et al., 2011; Yoon et al., 2013; Zhang et al., 2008] [Armstrong et al., 2011; Bagade and Rajpure, 2016, 2015; Cavalieri et al., 2012; Chapelle et al., 2011; Iio et al., 2014; Joshi et al., 2016; Karthick Kannan and Saraswathi, 2014; Kazin et al., 2011; Meuffels, 2007; Mukherjee and Majumder, 2014, 2013; Sen et al., 2015; Sutka et al., 2013; Tudorache et al., 2013]

Cobaltite

Bi10Co16O38 Ln1axSrxCoO3

NdCoO3 Nd0.8Sr0.2CoO3 Bi12(Bi0.55Co0.45)O19.6 LnBaCo2O51δ Bi10Co16O38 Ba0.5Sr0.5Co0.8Fe0.2O32δ

Cobaltate

Mangenate

YCoO3 LaCoO3 La12xCexCoO3 NdCoO3

La0.8Sr0.2Al0.9Mn0.1O3 Ln1axCa(Sr)xMnO3

La0.6Ca0.4Mn1axNixO3

Cerate

BaCe0.90Gd0.1O3aδ

Niobate

BaNbO3

Nickelate Stanate

YCoO3

Ba12xNixSnO3 Ba12xLaxSnO3 ZnSnO3

AlNbO4 CrNbO4 InNbO4 LaNi03 BaSnO3

CaSnO3, Zn2SnO4,

LaNi03 Zn2SnO4

[Addabbo et al., 2015; CasasCabanas et al., 2011; Gómez et al., 2015; Malavasi et al., 2005; Michel et al., 2007; Shuk et al., 1993; Tealdi et al., 2007; X. Wei et al., 2010a] [Addabbo et al., 2015; Ding et al., 2015; Malavasi et al., 2005; Shi et al., 2014; Tealdi et al., 2007] [Armstrong et al., 2011; Franke et al., 2016; Mullen et al., 2014; Shuk et al., 1993] [Luyten, 1991; Schutter et al., 1992; Wei et al., 2011; Zhou et al., 2015] [Gnanasekar et al., 1999; Zhang et al., 2009b] [Xuchen, 2000] [Ganbavle et al., 2014; Huang et al., 2012; Jiang et al., 2011; Kocemba et al., 2007; Song et al., 2011; Tharsika et al., 2015; Upadhyay and Kavitha, 2007] (Continued)

Table 11.2 Gas-Sensing Materials Based on Mixed Metal Oxides (Enhessari and Salehabadi, 2016) Continued Sensing Materials

O2

H 2O

CO/CO2

C2H5OH

NOx

Ref.

Perovskite Zirconate

CaZrO3

Chromate

MgCr2O4

Molybdate

Tungstate

Bi3FeMo2O12

ZnMoO4 Bi3FeO4(MoO4)2 NiMoO4 CuMoO4 PbMoO4 ZnWO4 MnWO4

[Andre et al., 2014; Deng et al., 2001; Zhou and Ahmad, 2008] LaCr1axTixO3

Bi3FeMo2O12

CoWO4 SnxWO31x SnW04

[Pingale et al., 1996; Pokhrel et al., 2007; Saha et al., 2005] [Edwin Suresh Raj et al., 2002; Sears, 1989; Sundaram and Nagaraja, 2004]

Bi3FeMo2O12

CuWO4 SnW04 MgWO3 ZnWO4 BaWO4

[Gonzalez et al., 2012; Kärkkänen et al., 2010; Solis and Lantto, 1995; Solis et al., 2000; Suresh Raj et al., 2002; Tamaki et al., 1995; You et al., 2012]

11.3 Application of Sensors

Table 11.3 Examples of Alcohol Sensors and Their Respective Linearity Range Materials

Linearity

Ref.

Ni/nafion/graphene CNT/Ni Pd-Ni/SiNWs Ni
CF La0.8Pb0.2Fe12xMgxO3 LaCoxFe12xO3 In22xNixO3 Zn2TiO4

0.43
88.15 mM 500
600 μM 0
20.4 mM 0.25
87.5 mM 500 ppm 137
500 ppm 1
500 ppm 50
200 ppm

[Jia and Wang, 2013] [Chen and Huang, 2010] [Tao et al., 2009] [Zhang et al., 2015] [Azizi et al., 2015] [Feng et al., 2011] [Feng et al., 2012] [Santhaveesuk et al., 2015]

increases the potential at the sensing electrode (positive response) and the latter decreases this potential (negative response).

11.3.1.2 Ethanol Various compositions of materials, either mixed metal oxides or composites are used for alcohol sensing. The sensitivity, selectivity, and stability are three important factors in alcohol sensors. In Table 11.3, the famous materials and the respective properties are summarized. In the case of perovskite-based sensors, O2 adsorbs on the surface substrate, trap electrons and the negative-charged chemisorbed oxygen species in the form of O22 and O2. As sensing materials react with reducing gases, the gas molecules primarily react with oxygen, the carrier density is depressed (due to the electron-donating nature of gases), and finally increasing the resistance (Eq. 11.6). 2 C2 H5 OH 1 6On2 ðads:Þ -2CO2 1 3H2 O 1 6ne

(11.6)

For example, Ni/nafion/graphene nanocomposites are fabricated for alcohol sensing (Fig. 11.8). Here, nanoscale Ni is electrochemically deposited on the surface of nafion/graphene film to fabricate a nonenzymatic ethanol sensor (Jia and Wang, 2013). The high electrocatalytic activity of this sensor toward oxidation of ethanol in alkaline media is reported with linearity range of 0.43
88.15 mM.

11.3.1.3 Oxygen Monitoring oxygen as dissolved in medical, food processing, and waste management industries is important. The sensors for such detection including clark electrodes, aqueous electrochemical cells, paramagnetic gas sensors, and optical sensors (Ramamoorthy et al., 2003). In oxide semiconducting oxygen sensors the defects, that is, oxygen vacancies and free charge carriers, create oxygen adsorbates on the surface of the oxide semiconductor at elevated temperatures as illustrated in Eq. (11.7),

373

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CHAPTER 11 Application of (mixed) metal

FIGURE 11.8 Preparation of Ni/nafion/graphene nanocomposites (Jia and Wang, 2013).

O2ðgasÞ 2O2ðadsÞ O2ðadsÞ 1 e2 -O2 2ðadsÞ 2 2 O2 2ðadsÞ 1 e -2OðadsÞ

(11.7)

For example, the effect of Ce-doped BaFeO3 perovskite materials as oxygen sensors was fabricated by Penwell and Giorgi (2014). At 800 C, the conductivity of Ba0.95Ce0.05FeO32δ reaches 3.3 S/cm. Moreover, linear and reproducible responses of the sensor at 500 C and 700 C were observed.

11.3.1.4 Water (humidity) In many industries, monitoring of the water vapor concentration is very important. Industrial drying plant, ovens, and other processing activities, effluent gases from power plants, incinerators, metal refining furnaces, skin humidity, mineral processing kilns and chemical plants require measurement of humidity at elevated temperatures. Different ceramic sensors for humidity detection are investigated including ionic, electronic, solid-electrolyte, and rectifying-junction types. The polymeric resistive humidity sensors are also reported based on polyelectrolytes and conjugated polymers (Chen and Lu, 2005). Humidity sensors detect the relative humidity (%RH 5 (PV/PS) 3 100) of the moisture and temperature in the air. It is defined as a ratio of moisture to the maximum amount that can be held in the environment at the present temperature and pressure. Absolute humidity (AB 5 mw/v) is a ratio of the mass of water vapor in air to the volume of air. Fig. 11.9 shows the mechanism of humidity sensing.

11.3.2 CHEMICAL (BIO)SENSORS A chemical (bio)sensor is a device that transforms chemical information into an analytically useful signal. The chemical information may originate from a

FIGURE 11.9 Schematic representation of a typical humidity sensor.

Table 11.4 Classification of Chemical Sensors (Enhessari and Salehabadi, 2016) Type of Chemical Sensor

Source

Example

Optical (optodes)

Optical phenomena

Electrochemical

Electrochemical process

Electrical

Electrical properties

Mass sensitive

Mass change at a specially modified surface Change of paramagnetic properties Heat effects of a specific chemical reaction or adsorption

Absorbance, reflectance Luminescence Fluorescence Opto-thermal effect Light scattering Voltammetric sensors Potentiometric sensors Chemically sensitized field effect transistor potentiometric solid electrolyte gas sensors Metal oxide semiconductor Organic semiconductor Electrolytic conductivity Electric permittivity Piezoelectric devices Surface acoustic wave devices

Magnetic

Thermometric

Radiation

Change of physical properties

Oxygen monitors

Combustion reaction Enzymatic reaction Optothermal device X-, p- or r-radiation Chemical composition

chemical reaction of the analyte or from a physical property of the investigated system. The chemical biosensors can be used in diverse applications such as medicine, home safety, environmental pollution, and many others. Chemical sensors are classified according to the operating principle of the transducer (Table 11.4).

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CHAPTER 11 Application of (mixed) metal

11.3.2.1 Drugs As discussed in previous sections, a biosensor is a sensing device comprising a biological component which can be connected to a physical transducer. The interaction of a drug compound and immobilized biomatter allows quantitative investigation on the basis of this dual configuration (Yu et al., 2005). Enzyme-based biosensors can be applied in the pharmaceutical industry for monitoring chemical parameters in bioreactors (Wang et al., 2013; Sin et al., 2014; Olaru et al., 2015; Bachan Upadhyay and Verma, 2013; Ahmed et al., 2014). The determination of salbutamol by adsorptive stripping voltammetry on a CPE modified by iron titanate nanopowders has been reported by Attaran et al. (2012). They expressed that the resulting electrode-based nanocomposites have a linear response in the range of 0.2
25 nM with a detection limit of 90 pM. In an electrochemical study of nickel titanate nanoceramic modified electrode for salicylic acid (SA), Ghoreishi et al. (2015) observed that under optimized conditions, two linear calibration ranges of 3.0
40.0 μM and 40.0
1000.0 μM for SA were obtained with a detection limit of 68.0 nM (S/N 5 3). A study on captopril in the presence of para-aminobenzoic acid (Mehdi et al., 2015) showed that the MnTiO3/CPE nanocomposite is a promising metal oxide for catalytic oxidation of drug. The sensing device showed two linear dynamic concentration ranges of 1.0 3 1028 to 1.0 3 1027 and 1.0 3 1027 to 1.0 3 1026, with a detection limit of 1.6 nM. Co3O4/SnO2 nanocomposites are also used for diltiazem detection in tablets and biological fluids (Attaran et al., 2016). Anodic stripping voltammeter setup of chemically modified carbon paste electrode containing Co3O4/SnO2 nanopowders has been used with a linear response to a drug concentration range of 50
650 nM, with a lowest detection limit value of 15 nM.

11.3.3 ENVIRONMENT BIOSENSORS The constant increase in the number of harmful pollutants in the environment calls for fast and cost-effective detection analytical techniques. The requirement for disposable systems or tools for environmental applications in place of traditional analytical systems, in particular for environmental monitoring, is a driver for the development of new technologies for quantitative detection. The biosensor is an appropriate alternative or complementary tool, a subgroup of chemical sensors, in which a biological mechanism is used for analyte detection. Food, heavy metals, and pesticides, and dust are three important pollution areas that need to be detected/controlled. In this section the heavy metals and pesticides will discussed.

11.3.3.1 Heavy metals Heavy metals are essential nutrients (Fe, Co, Zn, etc), harmless (Au, Ag, In, etc), however, they can be toxic in larger amounts, and poisonous (Cd, Hg, Pb, etc). Cadmium, mercury, and lead are heavy metal environmental contaminants.

11.3 Application of Sensors

Heavy metal biosensors

Protein based

Non enzymes

Enzymes

Inhibition

Activation

Whole-cell based

Fusion

Antibodies

Regular

Natural

Genetically engineered

Monoclonal

FIGURE 11.10 Classification of heavy metal biosensors (Verma and Singh, 2005).

They are bioaccumulative and can impose serious organ damage. Efficient, economical, and deployable techniques are still required. Sensing devices based on CPs are reported while the metal-oxide-based nanocomposites are not widely reviewed. The enhancement of analytical performance for trace analysis using sensors is still in progress. Heavy metal ion detection using biosensors can be monitored using protein-based and whole-cell-based approaches (Fig. 11.10). An optical biosensor for the detection of trace heavy metals was fabricated by Shtenberg and his coworkers (2015). In this system, an unknown aqueous solution is incubated with enzyme functionalized P-SiO2, using a simple CCD spectrometer setup. The optical monitoring correlates to the trace elements in the sample. The results indicate the high specificity and sensitivity of the system towards three metal ions (Ag1 . Pb21 . Cu21), with a detection limit range of 60
120 ppb.

11.3.3.2 Pesticide and dust In the 21st century, new classes of pollutants like pesticides and dust directly affect human health. Agriculture is a main source of pesticide consumption. On the other hand, the dust in the atmosphere comes from soil, dust lifted by weather, volcanic eruptions, and pollution. Generally, for detection of these types of pollutions, chromatographic separation techniques such as gas and liquid chromatography are traditionally used. Nowadays, some sensors have been invented for quantitative sensing of pesticides and dust. Enzyme-based amperometric biosensors, such as acetylcholinesterase (AChE), are reported as important sensing devices for the detection of pesticides in healthcare measurement, the food industry, and environmental analysis. The use of (mixed) metal oxide nanoparticles and their respective nanocomposites were reported as promising materials with improved response time, linear range, detection limit, reproducibility, and long-term stability of biosensors

377

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CHAPTER 11 Application of (mixed) metal

(Kumar et al., 2016; Zheng et al., 2016; Li et al., 2014; Zhao et al., 2013; Won et al., 2010; Zhang et al., 2012; Gupta et al., 2010; Prabhakar et al., 2016). A biosensor based on graphene/Co3O4 nanocomposites was designed for organophosphate (OP) pesticide detection, electrochemically (Zheng et al., 2016). The synergic effect of nanocomposites, including high surface area, good biocompatibility, and excellent conductivity for this biosensor, were observed toward OP detection in the low detection limit, good repeatability, wide linearity, and short response inhibition time. Carboxylic silica nanosheet (SNS)/platinum nanoparticle modified glass carbon electrodes for pesticide detection were also fabricated by Li et al. (2014). They prepared a Pt NPs
CSNS
NF nanocomposite as shown in Fig. 11.11. SNSs were prepared from montmorillonite, modified using APTES, and functionalized by succinic anhydride in DMF to form CSNS followed by suspension of CSNS in H2PtCl6 and NaBH4 to get Pt NPs
CSNS. Finally, the as-prepared nanocomposites were mixed with NF to get Pt NPs
CSNS
NF. A GCE electrode were covered by Pt NPs
CSNS
NF and coated by AChE-CS. The above electrodes were immersed in PBS containing various concentrations of pesticide and transferred into an electrochemical cell (pH 7.4 PBS containing 0.5 mM ATCl) to study the amperometric response. The inhibition of pesticide can be measured using Eq. (11.8).  Inhibition % 5

ip;control 2 ip;exp 3 100 ip;control

(11.8)

where ip,control and ip,exp are the peak currents of ATCl on the biosensor in the absence or presence of pesticide inhibition, respectively.

FIGURE 11.11 Preparation of pesticide biosensor-based Pt NP
CSNS
NF/GCE/AChE-CS nanocomposites (Li et al., 2014).

11.3 Application of Sensors

11.3.4 BIOLOGICAL SENSORS In bioscience, the sensors which can detect analytes are called biosensors. They are based on biological components, such as cells, protein, nucleic acid, or biomimetic polymers. On the other hand, nonbiological sensors are also used for biological sensing, called nanosensors. Biological agents like viruses, bacteria, pathogenic organisms, and the toxins they produce are difficult to detect. The sensing method in biological agents should be specific, that is, capable of discriminating between closely related pathogenic and nonpathogenic organisms or toxins, sensitive enough to detect trace targets, high affinity for maintaining binding even through repeated washing steps; and finally stable to long-term use (Sapsford et al., 2008). Biochemical compounds can be detected by sensors. Compared with the traditional analytical systems, sensors are relevant tools for bioanalyte detection, quantitatively. Sensors are composed of active sensing materials coupled with a signal transducer. These devices transmit the signal selectively and sensitivity electrically, thermally, or optically. Thus, the development of potential active materials plays a key role in designing efficient, reliable, and innovative sensing devices. Various metal oxide-based nanocomposites are shown in Table 11.5.

11.3.4.1 DNA Cancer is today’s most pressing health concern. Hence, very sensitive monitoring of cancer cells must be provided for an easy and effective way to monitor progression of the disease, DNA, and cells (Huang and Jie, 2013). Various sensors are biologically fabricated for DNA detection, such as MIPs, conducting and SCPs, and nanomaterials, either pristine or nanocomposites. In CPs, there is almost no conductivity in the neutral state. The charge carriers upon oxidizing (p-doping) or reducing (n-doping) their conjugated backbone are responsible for conductivity. Oxidation followed by relaxation processes causes the formation of localized electronic states (polaron) (Billmeyer, 2007). Complexity, timeconsuming procedures, and narrow targets are some of the drawbacks of traditional DNA detection. Recently, electrochemical DNA biosensors have been used for detection of bacteria, because of their fast response time, convenience, and high sensitivity (Li et al., 2013). A schematic diagram of the preparation, sensor fabrication, and detection process of GOx
Thi
Au@SiO2 (Fig. 11.12) was reported by Li et al. (2013). The nanocomposite-based (mixed) metal oxides for DNA detection are tabulated in Table 11.5.

11.3.4.2 Protein Rapid and low-cost determination of proteins is needed immediately. The most widely used tests are based on colorimetric procedures. In this technique, the proteins react to produce colored complexes. Various factors govern the detection, such as absolute protein quantity, amino acid composition, protein purity, and association state. Biosensors have achieved great interest due to their operational

379

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CHAPTER 11 Application of (mixed) metal

Table 11.5 Examples of Biosensors-Based Mixed Metal Oxides Nanocomposites Analyte

Composite

DNA

CuO/SWCNT [Chen et al., 2017, 2016, 2015; Deng et al., 2014; Gupta et al., 2010; Hushiarian et al., 2015; Li et al., 2013; Shukla et al., 2012; Tak et al., 2016; Wang et al., 2015; Yang et al., 2015]

TiO2/GO/ PPy Protein

Glucose

Cholesterol

Ru-silica/gold Chitosan/Fe2O3 CNT/MnO2 Co3O4/polyaniline Fe3O4/polydopamine/ Au Chitosan/Au ZnO/Au CNT-gold-titania ZnO-silicon ZnO-CuO CuO-graphene CuNiO-graphene CuO/polypyrrole/ graphene oxide RGO/ZnO Fe3O4/chitosan ZnO/chitosan-g-PVA Graphite/SrPdO3 Indium tin oxide (ITO)/ MMT Graphene/CNT/ZnO/ Au CeO2/chitosan/ITO CHIT/CeO2
GR CNT/Fe3O4 Au/TiO2 NiFe2O4/CuO/FeOchitosan

Linearity Fe3O4@3D-GO 3D NG/Fe3O4 Poly(3,4-ethylenedioxythiophene)/Au GOx-Thi/Au@SiO2 SiO2
CeO2 Zn/chitosan-PVA CuO/GO Sm/CeO2 ZnO/CNT 0.007
3.5 nM 0.2
200 pM
0.2
100 ng/mL 12
1280 μM 1.0
8.0 3 102 pg/mL 0.6
110 ng/mL 0.1
33.0 μM 0.1
8 mM

0.001
8 mM 0.05
16 mM 1
100 mM 5
400 μM 1
26 mM
0.1
6 mM 1
20 mM
10
400 mg/dL 0.02
1.3 mM
7
100 μM 50
5000 mg/L

Ref. 1.0 3 10214
1.0 3 1028 M 0.01
100 nM 1.0 3 10214
1.0 3 1026 M
0.02
50.0 nM

0.001
8 mM 1 3 10213
1 3 1026 M 5
180 ng/μL

[Afsharan et al., 2016; Cai et al., 2015; He et al., 2015; Masoomi et al., 2013; Prabhakar et al., 2016; Wang et al., 2014]

[Alizadeh and Mirzagholipur, 2015; Elads et al., 2015; Karuppiah et al., 2015; Kaushik et al., 2008; Miao et al., 2015; Moozarm et al., 2015; Shukla et al., 2012; Wei et al., 2010; Wu et al., 2013; Zhang et al., 2014; Zhang et al., 2015]

[Du et al., 2015; Joshi et al., 2015; Sandeep Kumar et al., 2015; Malhotra and Kaushik, 2009; Singh et al., 2012; Tang et al., 2016; Zarei et al., 2012]

(Continued)

11.3 Application of Sensors Table 11.5 Examples of Biosensors-Based Mixed Metal Oxides Nanocomposites Continued Analyte

Composite

Linearity

Ref.

Urea

ZrO2/PPI ZnO/CNT TiO2/ZrO2 CoFe2O4-HSA/CT Fe3O4/polydopamine/ Au ZnO/LiTaO3 GCE/chitosan/Au Fe3O4/Au/3-((2mercaptoethylimino) methyl) benzene-1,2diol CNT/Fe3O4 CNT
MnO2 Graphene oxide/ITO ZnO/LiTaO3 TiO2/Au ZrO2
RGO Polyaniline
TiO2

0.01
2.99 mM 10
100 mg/dL 5
100 mg/dL 1.99
19.23 μM 0.3
8.0 3 102 pg/mL

[Charan and Shahi, 2016; Shukla et al., 2014; Srivastava et al., 2013; Tak et al., 2013]

Immunology

100 μg/mL 0.6
110 ng/mL 0.6
110 ng/mL

[An, 2016; He et al., 2015; Jamil et al., 2015; Suveen Kumar et al., 2015; Masoomi et al., 2013; Tu et al., 2015; Zarei et al., 2012; Zhou et al., 2010; Zhu et al., 2015]

30
1000 ng/mL 0.2
100 ng/mL 10
450 ng/mL 100 μg/mL 2
22 ng/mL 10
2500 μM 0.01
2.5 mM

FIGURE 11.12 Schematic representation of typical biosensors for DNA detection (Li et al., 2013).

381

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CHAPTER 11 Application of (mixed) metal

FIGURE 11.13 Schematic representation of a protein biosensor. Adapted from Leca-Bouvier, B., Blum, L.J., 2005. Anal. Lett. 38, 1419.

advantages over standard photometric methods. These advantages are rapidity, ease-of-use, mass manufacture, cost, simplicity, and portability. By appropriate recognition element selection, it is possible to detect either a particular target protein or a broad range of proteins (Fig. 11.13) (Leca-Bouvier and Blum, 2005).

11.3.5 CLINICAL BIOSENSORS The applications of biosensors in clinical chemistry have been reviewed by many researchers, including for both commercial applications and fabrication techniques. Electrochemical, optical, and piezo-optical modes of transduction are used for recognition elements including enzymes (biocatalytic recognition elements) and immune agents and DNA-affinity ligand recognition elements. Microfabrication will allow biosensors to be constructed as arrays and incorporated into lab-on-a-chip devices (D’Orazio, 2003). In clinical chemistry laboratories, clinical analyses are no longer used for clinical measurements. Biological fluids generally need to measure in hospital point-of-care settings, by caregivers in nonhospital settings, and by patients at home. Biosensors are a good candidate for measurement of analytes in clinical chemistry and are also suitable for new biological applications. Of all the clinical biosensors fabricated since 1962, glucose, cholesterol, urea, and immune biosensors have been commercially fabricated and used in various clinical aspects.

11.3.5.1 Glucose The advent of glucose biosensors has been concentrated on recently by many researchers due to their extensive applications in clinical diagnosis. Two types of

11.3 Application of Sensors

FIGURE 11.14 Working reaction of an amperometric glucose biosensor.

glucose biosensor have been used, based on enzymatic catalysts and nonenzymatic catalysts. The sensing capabilities of biosensors like sensitivity, stability, biocompatibility, reproducibility, and selectivity are the most important objectives. Amperometric biosensors use enzymes as the recognition element used for the first time as a sensor for glucose in blood and involved immobilization of the enzyme glucose oxidase (D’Orazio, 2003). A solution of glucose oxidase was physically entrapped between two membranes; a gas-permeable membrane and a dialysis membrane. The reaction is illustrated in Fig. 11.14. Various metal oxide-based nanocomposites with different linearity ranges have been reported by many researchers for glucose detection (Table 11.5). The detection device is mostly based on amperometric transducers. For example, the amperometric responses of various modified GC electrodes to glucose based on CuO/graphene nanocomposites were reported by Hsu and his coworkers (2012). In this setup, an appropriate amount of glucose is mixed with NaOH solution in three modified electrodes system. After adding various concentrations of glucose into a potassium hydroxide solution, the oxidation currents of a working electrode are monitored at a fixed potential. From the amperometric curve, the linear relationship between the oxidation current and glucose concentration can be detected. Fig. 11.15 shows the amperometric responses of graphene, CuO nanoparticles, and CuO/grapheme-modified GC electrodes as expressed by Hsu et al. (2012).

11.3.5.2 Cholesterol Cholesterol is a crucial biobased material for human body as it is a structural component of biological membranes, nerve, and brain cells. Cholesterol is synthesized in the liver and supplied from food, and takes part in the production of hormones, bile acids, vitamin D, and other vital molecules (Saxena and Das, 2015). Above 200 mg/dL can damage the blood vessels, causing several diseases such as hypertension, arteriosclerosis, coronary heart disease, lipid metabolism dysfunction, brain thrombosis, etc. Typical (mixed) metal oxide-based nanocomposites for cholesterol detection are listed in Table 11.5. An interesting nanocomposite for cholesterol detection

383

CHAPTER 11 Application of (mixed) metal

600 500

Current (µA)

384

Graphene

400

CuO CuOG

300 200 100 0 0

300

600

900

1200

1500

1800

Time (s)

FIGURE 11.15 Typical representation of amperometric responses of graphene, CuO nanoparticles, and CuO/grapheme-modified GC electrodes (Hsu et al., 2012).

2FeOOH Ni(NO3)2.6H2O + Cu(NO3)2.3H2O + Fe(NO3)3.9H2O NH4OH

Ni(OH)2 + Cu(OH)2 + Fe(OH)2+ NH4NO3+H2O

NiFe2O4 + CuO+FeO + H2O Chitosan

FIGURE 11.16 Schematic representation of NiFe2O4/CuO/FeO/H2O/chitosan film nanocomposites.

has been reported by Singh et al. (2012). They fabricated an electrochemical bioelectrode of ChOx/NiFe2O4/CuO/FeO-chitosan/ITO nanocomposite. The bioelectrode detects as a function of cholesterol concentration. A multistep synthesis technique is used for preparation of magnetic parts of nanocomposites (Fig. 11.16)

11.3 Application of Sensors

11.3.5.3 Urea Urea is toxic above certain concentrations. It is produced during nitrogen metabolism, therefore, it has great significance in clinical chemistry (blood urea), food chemistry, and environmental monitoring. Hence, continuous real-time monitoring in clinical, environmental, and food-related environments is important. The conventional techniques are time-consuming and mostly laboratory-bound. Biosensors can play important roles due to their ease of use, portability, and the ability to furnish real-time signals. The biocomponent of a urea biosensor is urease. Urease can catalyze the hydrolysis of urea to ammonium and hydrogen carbonate ions (Eq. 11.9). O H +

O H2N

NH2

+3 H

Urease H

N 2H

H H

–

–

+ HO + HCO3

(11.9)

A setup of an amperometric urea biosensor is fabricated based on urease and glutamate dehydrogenase. The immobilization is done on the nylon nets coupled with a Pt electrode. In this system, glutamate dehydrogenase in the presence of sodium hydroxide enhanced the reproducibility and stability of the sensor (Singh et al., 2008). ZnO
MWCNT/ITO is one of the novel urea biosensor-based nanocomposites (Tak et al., 2013). The nanocomposite exhibits good linearity over a wide range of urea concentrations (10
100 mg/dL), high sensitivity, low value of Km (0.85 mM), and a low detection limit of about 0.23 mM. The process of electron transfer is shown in Fig. 11.17. In this process, the analyte (urea) is added into the PBS solution containing [Fe(CN)6]32/42 as the redox mediator. As oxidation occurs, chemical products such as NH3 and carbon dioxide (CO2) are formed and subsequently the urease is reduced. The Fe31 ions present in the PBS solution capture the released electrons and reduce to Fe21 ions. The reverse reaction releases electrons which are transferred to the ITO layer of the efficient matrix of bioelectrode.

11.3.5.4 Immunology Electrochemical immunosensors have recently received great clinical and academic attention. However, some deficiencies, like poor sensitivity and slow electron transfer kinetics, have enhanced the use of electrochemically active tags such as enzymes, noble metals, and transition metal oxides for signal amplification (Tu et al., 2015). The impedimetric immunosensor is a sort of sensing device based on singlefrequency impedance measurements, and is a robust and label-free device. The bioreagents develop as the immunoassay is performed on the electrode surface.

385

386

CHAPTER 11 Application of (mixed) metal

Urea

NH3

+

CO2

Matrix (ZnO/ZnO-MWCNTs)

Ureaseoxi

Ureasered

ITO

Urease Glass Fe2+

Fe3+

e–

FIGURE 11.17 The process of electron transfer (Tak et al., 2013).

FIGURE 11.18 Immunosensor reaction.

As shown in Fig. 11.18, an immunochemical reaction between the pesticide residues and the immobilized antigen is generated and the limited amount of antibody (Ab) on the surface is determined (Rodrı´guez and Valera, 2013).

11.4 Fabrication

11.4 FABRICATION As mentioned above, biosensors are fascinating analytical tools appropriate for revolutionizing the market in the near future. However, due to technical and production problems, the biosensors are not distributed widely. The fabrication of a multibiosensor system is needed immediately for various bioapplications. Traditionally, biosensors were fabricated in the form of membranes and transducers. It must be noted that with the long historical background of biosensors, very few of them are able to perform measurements in undiluted biological fluids for long-term applications (Urban, 2000). A biosensor system consists of sample handling, biorecognition, transduction, and signal interpretation. Fig. 11.19 shows the relationship between different parts of a sensing system. For bioactive target monitoring, small, integrated and reliable sensing elements are necessary. In principle, the “(bio)sensors” in general are elements receiving information either physically or chemically or physicochemically and transforming it into an electric response (Fig. 11.20). Biosensor devices are generally classified into four groups: biosensors based on acoustic transducers, optical microtransducers, calorimetric transducers, and electrochemical. The classification of biosensors and their respective working devices is tabulated in Table 11.6. External

External Biorecognition

Transduction Data processing

Sample

Observer Wet

Dry

FIGURE 11.19 Junction between main compartments of a biosensing system.

FIGURE 11.20 A typical biosensor.

387

388

CHAPTER 11 Application of (mixed) metal

Table 11.6 Classification of Biosensors Based on Transducers Acoustic transducers

[Urban, 2000]

Optical microtransducers

[Dey and Goswami, 2011]

Calorimetric transducers

[Urban, 2000]

Electrochemical

Conductometric biosensors

[Isildak et al., 2012]

Potentiometric transducers

[Wang et al., 2012]

Amperometric biosensors

[Shimomura et al., 2013]

11.5 Selectivity, Sensitivity, and Time Factors

11.5 SELECTIVITY, SENSITIVITY, AND TIME FACTORS Selectivity: This factor is the essence of sensors. It is rare to find a sensor which will respond to only one analyte. It is more usual to find a sensor that will respond mainly to one analyte, with a limited response to other similar analytes. The extent of interference based on Nicolskii
Eisenman’s equation (Eq. (11.10)) can be calculated in terms of the electrode potential and a selectivity coefficient, ki,j, as follows, n=z

E 5 K 1 Slogðai 1 ki;j aj Þ

(11.10)

where ai and aj are the activity of the primary analyte of charge n and the activity of the interfering analyte of charge z, respectively. Sensitivity: The concentration range covered by any analytical technique from the calibration point of view, and the linearity response over the section of this range, in general, is called the sensitivity. At the lower level is the “detection limit.” It is the concentration of analyte at which the extrapolated linear portion of the calibration graph intersects the baseline. The linear ranges are generally much larger for potentiometric sensors covering 12 powers of 10 of the hydrogen ion concentration. Amperometric sensors and biosensors generally do not have ranges of much more than two or three powers of 10 (Eggins, 2002). Response times: The time required for a system to reach equilibrium is called the “response time.” The response time for chemical or biochemical nature-based sensors is offset by the simplicity of the measurement and the minimal sample preparation time. The response times in biosensors can be from a few seconds to a few minutes. Recovery times: The time that elapses before a sensor is ready to be used for another sample measurement is called the “recovery time.” A sensing device should rest immediately or after one measurement to resume its base equilibrium. Lifetimes: The response during continuous use of the sensor when it is in contact. The time after which the response has declined by a given percentage is called the “lifetime.” On the other hand, it is the time over which the assembled sensor is stored. To compare all the above factors in experimental results, three glucose biosensors are compared in Table 11.7. From the table, it can be seen that CuO/ grapheme has an excellent response time and detection limit. Table 11.7 Some Examples of Important Factors in Sensing Measurements Sensing Materials

Analyte

Detection Limit (mM)

Sensitivity

Linear Range (mM)

Response Time (s)

ZnO/CHIT-g-PVAL Au
Fe3O4@SiO2 CuO/graphene

Glucose Glucose Glucose

0.2 0.01 0.001

40.04 V/mM
1065 μA/mmol/L cm2

2
1.2 0.05
8.0 0.001
8

3 5 1

389

390

CHAPTER 11 Application of (mixed) metal

11.6 SUMMARY AND RECOMMENDATIONS FOR FUTURE WORK It is interesting to note that even though biosensors have a long history, very few are capable of performing commercially in undiluted biological fluids for longterm applications. Nanotechnology can facilitate the synthesis of novel materials with desired applications. The mechanism of analyte detection varies based on the nature of the measurand, biosensors, etc. It can be concluded that the most appropriate biosensors are applied based on analyte conditions and optimum linearity range, detection limits, response times, recovery times, and lifetimes. From the view point of sensing materials (MONs, NPs, polymers, and their respective nanocomposites) and the fabrication process, the selected biosensors have unique characteristics. Therefore, preparation of novel materials for fabrication of “easy to use biosensors” and “multidisciplinary biosensors” are still required for everyday life. The “global health communities” would have urgently reacted for development of clinical biosensors.

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FURTHER READING Dey, D., Goswami, T., 2011. J. Biomed. Biotechnol. 2011, 348218. Ghasdi, M., Alamdari, H., 2010. Sens. Actuators B Chem. 148, 478. Ghasdi, M., Alamdari, H., Royer, S., Adnot, A., 2011. Sens. Actuators B Chem. 156, 147. Isildak, I., Cubuk, O., Altikatoglu, M., Yolcu, M., Erci, V., Tinkilic, N., 2012. Biochem. Eng. J. 62, 34. Kumar, S., Ahlawat, W., Kumar, R., Dilbaghi, N., 2015a. Biosens. Bioelectron. 70, 498. Kumar, S., Sharma, J.G., Maji, S., Dhar, B., 2015b. Biosens. Bioelectron. 78, 497. Lee, I., Jung, B., Park, J., Lee, C., Hwang, J., Park, C.O., 2013. Sens. Actuators B Chem. 176, 966. Shimomura, T., Sumiya, T., Ono, M., Ito, T., Hanaoka, T., 2013. Anal. Bioanal. Chem. 405, 297. Wang, J., Wu, C., Hu, N., Zhou, J., Du, L., Wang, P., 2012. Biosensors 2, 127.

CHAPTER

12

Metal nanoparticles and their composites: a promising multifunctional nanomaterial for biomedical and related applications

Vesna V. Vodnik and Una Bogdanovic´ ˇ Institute of Nuclear Department of Radiation Chemistry and Physics “GAMMA”, Vinca Sciences, University of Belgrade, Belgrade, Serbia

12.1 INTRODUCTION The dynamics of development technology in all science aspects moves forward very rapidly. Among the more transformative technologies, nanotechnology has the opportunity to offer the most, as it tends to create new materials with new properties and functionalities, such as nanomaterials. Their investigation is interesting from a technological standpoint since the transition from individual atoms to nanoscale material and the bulk state of matter have a great fundamental importance in science (Edelstein and Cammarata, 1996; Kreibig and Vollmer, 1995). A dramatic increase in the ratio of surface atoms to interior atoms with particle size decrease, accompanied by a great change of their physicochemical properties, is still the goal of today’s studies. Metal nanoparticles (NPs) are in the limelight of modern nanotechnology, especially colloidal Au, Ag, and Cu, which in transparent media provide a wide range of colors. They have been fascinating scientists for a long time due to very intense coloration, since their absorption and re-emission of light are dependent on the wavelength at which conduction electrons oscillate. For other metals (Pb, In, Co, Sn, and Cd) the plasma frequency lies in the UV region and NPs do not display strong color effects. Unlike them, noble metal NPs, as their size is reduced to tens of nanometers, exhibit a strong absorption band in the visible region, absent in the individual atom and in the bulk. The imaginary part of the dielectric constant at the plasmon frequency is very small and the near-field effect is so high that it makes the plasmon excitation of these NPs very interesting. Most commonly, surface plasmon resonance (SPR) frequencies are carried out with AuNPs, AgNPs, and CuNPs, and strongly depend

Materials for Biomedical Engineering: Inorganic Micro- and Nanostructures. DOI: https://doi.org/10.1016/B978-0-08-102814-8.00014-7 © 2019 Elsevier Inc. All rights reserved.

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on their size, shape, interparticle distance, electron density, dielectric properties, and local environment (Mulvaney, 1996). Some of these interesting metal NPs properties, how they influence and interact with their surrounding, and how we can make these properties useful for different applications will be discussed in greater detail in this chapter. The first reason for studying metal NPs from a technological standpoint is their applications as optical systems. Their optical response appears when their surface plasmon excitations strongly couple with external light, which induces localization of SPR and consequently the electromagnetic field (EM) enhancement of plasmons. In particular, this optical signal enhancement allows the detection of Raman signals from a single molecule. The study of optical phenomena related to the EM response of metal NPs led to the development of the research field called plasmonics (Ozbay, 2006). From a plasma model, where the free electrons of a metal can be treated as an electron liquid of high density, it follows that electron density waves (called plasma oscillations or Langmuir waves) with an energy of the order of 10 eV will propagate along the interface of a metal. Interacting plasmons give rise to a wide variety of important properties of nanosystems, such as surface-enhanced Raman excitation (SERS), as one of the more important exploitations of metal NPs in biology, medicine, and engineering (Thomas et al., 2013; Hoppmann et al., 2013). Also, optical data communication and data storage need new materials with nonlinear optical phenomena. Tuning the optical properties with size and shape, together with the extremely high extinction coefficient of SPR compared with dye molecules have made them very interesting materials (Du et al., 1998). Plasmonics also allow new possibilities to treat cancer or the ability of using metal NPs as multimodal, intracellular optical sensors based on near infrared absorption or Raman scattering for biological and medical applications (Li et al., 2010; Gupta and Verma, 2014; Ravalli and Marrazza, 2015). Many sensitive and selective NPs have special physicochemical properties that offer a suitable platform for quantitation of metal ions, anions, proteins, and DNA, based on analyte-induced changes in their absorption, fluorescence, and scattering intensity, which is important in medical, environmental, material, pharmaceutical, and food science (Song et al., 2011). The successful applications of metal NPs in these fields require them to enter cells across the cell membrane with embedded or peripherally attached proteins (Rasch et al., 2010). These processes may be carried out through endocytosis, direct microinjection of NPs dispersions, by electroporation or a mediated uptake process using known biological interactions or promoters with surfacefunctionalized NPs (Medintz et al., 2005). As the NPs should be compatible with biological systems, their surface functionalization offers a convenient possibility for interaction with the cells. In addition, this process maintains good water solubility of NPs, their electrostatic/steric stabilization through the formation of a double electrical layer and binding organic molecules (biomolecules) to the particle surface. Also, the metal NPs could be embedded in polymer matrices in order

12.2 Some Interesting Properties of the Metals

to avoid their oxidation, aggregation/agglomeration, and the formation of the thermodynamically favored bulk material (Vodnik et al., 2013; Bogdanovi´c et al., 2015a,b; Stamenovic et al., 2018). The interparticle interaction in these nanocomposites enables them to act as molecular bridges in the polymer matrix. The correct choice of polymer, NPs type, and polymer
NPs interaction, influence nanocomposite use in vastly differing applications. This chapter also discusses current efforts and research challenges in their emerging usage for potential biomedical and related applications. According to different applications of these nanostructures, their synthesis, size and shape control, surface modification, and characterization have attracted considerable attention from a fundamental and practical point of view. While it would be worth discussing the wide range of preparation methods more thoroughly, some of the synthetic procedures will be reviewed here, only to serve as a general introduction to the appropriate technique for preparation of nanomaterials with desired properties and property combinations. This chapter focuses on the characteristic properties of the metal NPs and their polymer-based nanocomposites which show pronounced features for biomedical and related applications, without going into biological or chemical detail. In addition, conjugation of metal NPs to biological molecules before their use for biological or medical applications will be also considered. The discussion will be illustrated with examples from the literature and from our experimental results. However, we have tried to summarize the most recent developments in the field of applied NPs, but due to the overwhelming nature and size of this research field, we only provided a cursory overview of some materials and thus apologize in advance for all omissions.

12.2 SOME INTERESTING PROPERTIES OF THE METALS ON THE NANOMETER LENGTH SCALE With characteristic physicochemical properties and promising applications in diverse fields, from the medical and pharmaceutical to the high-tech (Ruparelia et al., 2008; Grumezescu, 2016; Aiken and Finke, 1999), metal NPs are one of the most extensively studied colloidal systems in the nanoscience domain. Progress in this area depends on the possibility to synthesize stable metal NPs with different sizes and shapes since their electronic, optical, and catalytic properties are closely related to their morphology. With particles size decreasing to the nanometer scale, the motion of electrons is reduced, and surface effects become very important (Link and El-Sayed, 2003). The main feature of NPs significant for their properties and consequently various applications is their great surface-to-volume ratio. With decreasing particles size from macroscopic crystal to nanometer magnitude, the percentage of surface atoms increases—3.0 nm particles 35% of the surface atoms, while decreasing particle size to 0.7 nm increases this percentage to 92% (Aiken and Finke, 1999).

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Since large fractions of uncoordinated edge- and corner-atoms are located on the surface area, particles have high surface energy, they are highly reactive, and unstable, and should be stabilized/functionalized by different agents, which will be explained later. Additionally, the chemical reactivity of NPs is also affected by their morphology since their surface structure changes as a function of size and particles shape. Among the metal NPs, noble metals AuNPs, AgNPs, and CuNPs stand out, since they show specific behavior when exposed to light. Metal NPs have the ability to absorb/interact with electromagnetic radiation in a narrow wavelength range, which leads to collective oscillations of free conduction electrons in their surface. When these oscillations become resonant with the incident light, the absorption band called SPR appears in the absorption spectrum of metal colloid (Kreibig and Vollmer, 1995). These effects are more pronounced for noble metal NPs than for other metals, considering d
d band transitions that push their plasmon frequencies into the visible part of the spectrum (Kreibig and Vollmer, 1995). The SPR band is directly influenced by particle size and shape (Fig. 12.1A) (Vodnik et al., 2008). With decreasing particle size, charge density increases, inducing changes to their electronic structure and optical properties. For small metal nanospheres, incident radiation induces a dipole mode that oscillates in phase with the electric field of the incoming light. This interaction is expressed as a narrow, intensive peak in the spectrum (Fig. 12.1A, Au nanospheres), that becomes wider and shifts toward higher wavelengths when the

FIGURE 12.1 (A) Size- and shape-dependent absorption spectra of AgNPs and AuNPs; (B) TEM images of metal NPs: AgNPs—nanospheres (a), truncated triangular nanoplates (b), dendrites (c), different shapes (d), nanoplates, discs, and rods (e); CuNPs— nanospheres (a), nanocubes (b); AuNPs—nanospheres with 17 nm (a) and 40 nm (b) diameters, and nanorods with different lengths (c,d).

12.2 Some Interesting Properties of the Metals

particles size is increased, due to light’s inability to homogeneously polarize larger NPs. In this case, higher-ordered modes are excited (Kreibig and Vollmer, 1995). With the introduction of the second, longitudinal dimension, particles shape becomes important for their optical properties. For nanorods, the resonance wavelength, and therefore absorption spectrum, depend on the electron oscillations across the transversal and/or longitudinal direction of rods (Momi´c et al., 2016). High-energy, transversal peaks originate from the electrons oscillating in the direction perpendicular to the major (long) rod axis, located at the same wavelength as the nanosphere of the same size, while the electron oscillations along the long axis are manifested as low-energy, longitudinal plasmon on greater wavelengths. In comparison to the transverse SPR wavelength, the longitudinal SPR peak is more sensitive to the changes in the dielectric properties of the surroundings and can be tuned by varying the aspect ratio of nanorods (Fig. 12.1A, Au nanorods). Further reduction of symmetry causes the appearance of new absorption bands. Due to dipole resonance in plane and quadrupole in and out of the plane, nanoprisms are indicated in the absorption spectrum by three peaks (Fig. 12.1A, AgNPs), and similarly, nanocubes by four (Vukoje et al., 2014a; Im et al., 2005). In concentrated colloid dispersions, when NPs are close to each other, their mutual interaction shifts SPR toward smaller energies together with the appearance of the additional band (Kreibig et al., 1981). The surrounding medium, and surfactant molecules/stabilizing agents on their surface have an influence on the nanoparticle optical properties as well (Vukovi´c and Nedeljkovi´c, 1993; Vukoje et al., 2012). Research within our group is partly related to the chemical synthesis of the metal NPs with various sizes and shapes. Their TEM micrographs are presented in the Fig. 12.1B. The electronic structure of metal NPs is also closely related to their catalytic properties. With great selectivity, efficiency, and recyclability, these particles have a great future as possible catalysts for various types of reactions, from ammonia detection to biosensing—detection of biomolecules (Gupta and Verma, 2014; Ravalli and Marrazza, 2015). The catalytic properties are influenced by their geometric structure, that is, size and shape, and crystallographic planes on their surface (Wang et al., 2011). Crystallographic planes have different surface energies that increase with charge density increase, when the fraction of edgeand corner-atoms is large. These uncoordinated atoms represent active places that could easily interact with surrounding molecules, enhancing the effectiveness of the catalytic reaction. Among metals, PtNPs, with certain characteristics that distinguish them from Au, Ag, and Cu, are the most commonly used as catalytic material (Nguyen and Minteer, 2015), but cheaper alternatives are noble metals (Okitsu and Mizukoshi, 2016). Knowing the overall properties of the metal NPs, their mutual interactions and interaction with surrounding molecules, whether they are capping or stabilizing agents, biomolecules, or electrolytes, allows one to design NPs with tailored properties that can be exploited for various purposes.

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12.3 NANOPARTICLE SYNTHESIS AND FUNCTIONALIZATION 12.3.1 SYNTHESIS APPROACHES TO METAL NANOPARTICLES The successful utilization of metal NPs in various applications depends on their size, shape, composition, and the surface functionalities. In order to extend the array of their properties, a large number of synthetic procedures is developed. Whether these methods are chemical or physical, the selection of the synthetic route depends on the desired characteristics. Physical synthetic procedures (evaporation/condensation, laser ablation) have some advantages over chemical methods in the form of easier control of experimental conditions, larger uniformity of metal NPs distribution, and the absence of solvent contamination (Oseguera-Galindo et al., 2016). On the other hand, since the equipment used in these methods is expensive and large, chemical methods are more affordable and accessible. They are based on chemical reduction of metal ions to metal NPs by an appropriate reducing agent, for example, sodium borohydride, sodium citrate dihydrate, hydrazine hydrate, etc. (Vodnik et al., 2008; Vukovi´c and Nedeljkovi´c, 1993; Turkewich et al., 1951; Bogdanovi´c et al., 2014b; Laban et al., 2016). Metal NPs characteristics, including the possibility and kinetics of particle formation, their size and shape, crystallinity, and stability, are affected by the characteristics of the reducing agent (primarily strength) and solvent (type, polarity). Lately, there has been a growing need for the development of eco-friendly or so-called “green” synthesis metal NPs, in order to reduce the use of toxic chemical agents. There is great potential in prokaryotic and eukaryotic microorganisms, marine organisms, extracts of biomolecules and plants, for use as reduction agents in metal NPs formation (Singhal et al., 2011; Jeevan et al., 2012). In addition, the literature offers many different types of metal NPs synthetic routes, such as radiolytic reduction of metal ions (Spasojevi´c et al., 2017), electrochemical and sonochemical reduction (Surudˇzi´c et al., 2013; Kumar et al., 2014), etc. A special class of nanomaterials with usually improved characteristics compared to metal NPs is represented by polymer-based nanocomposites. Besides the increased stability of metal NPs incorporated in polymer matrix, their electrical, optical and catalytic properties, antimicrobial activity, and sensing of biomolecules or metabolite products are more pronounced (Bogdanovi´c et al., 2015a,b; Stamenovic et al., 2018). On the other hand, metal NPs also have a positive effect on the polymer by increasing its thermal stability, mechanical, and electrical properties (Vodnik et al., 2011, 2013; Bogdanovi´c et al., 2014a, 2015b). Similarly to metal NPs, nanocomposites can be synthesized by physical (Feng et al., 2006) and chemical methods. The lack of physical synthetic routes (mechanical mixing of nanocomposite constituents) creates the great possibility of NPs aggregation and decomposition of the polymer matrix, whereby their main characteristics are lost. Much stronger binding between metal NPs and polymer matrix than hydrogen and van der Waals bonds characteristic for physically synthesized nanocomposites, is achieved by chemical and electrochemical reactions (Vodnik et al., 2011, 2013;

12.3 Nanoparticle Synthesis and Functionalization

Bogdanovi´c et al., 2014a, 2015b; Jovanovi´c et al., 2014). Depending on whether the metal NPs are already synthesized when the polymerization process is initiated (Bogdanovi´c et al., 2014a) or are formed together with the polymer (Bogdanovi´c et al., 2015a,b; Vukoje et al., 2014b), nanocomposite synthetic procedures can be divided into ex situ and in situ methods. In our research experiments, we have used both of these procedures for synthesis of metal NPs (Cu and Au)
polyaniline (PANI) nanocomposites (Bogdanovi´c et al., 2014a, 2015a,b). The main advantage of in situ over ex situ methods is a decreased number of reactants that participate in the reaction—there is no need for additional capping/stabilizing agents for NPs protection nor for an oxidizing agent for the monomer polymerization, since metal ions have the role of oxidizing agent that will, for example, polymerize aniline to PANI, while simultaneously aniline monomers will reduce metal ions to NPs. Additionally, the formed polymer matrix stabilizes metal NPs and protects them from oxidation. One of today’s challenges in the nanotechnology field is designing new hybrid and advanced biomaterials that could be exploited for controlled drug delivery purposes, tissue engineering, bone and dental repairing/reconstructing, imaging, etc. Bionanocomposites may be able to fulfill the requirements needed for possible biomedical applications. In order to be used in medicine, bionanocomposites have to be biocompatible and nontoxic, biodegradable, and easily absorbed or eliminated from the body. The choice of an appropriate polymer matrix, nanofiller type, and their mutual interaction are important for the characteristics of the bionanocomposite as the final product with the desired characteristics, and for its further biomedical application. Natural polymers, such as polysaccharides, starch, and alginate (Boˇzani´c et al., 2011; Ghasemzadeh and Ghanaat, 2014), as well as synthetic ones that are water-soluble [poly(vinyl alcohol) (PVA), poly(ethylene glycol) (PEG), and guar gum] (Gupta and Verma, 2014; Vodnik et al., 2011, 2013; Ghasemzadeh and Ghanaat, 2014; Liu et al., 2012) or electrically conductive polymers (PANI, polyvinylpyrrolidone, and polypyrrole) (Bogdanovi´c et al., 2014a, 2015a,b; Stamenovic et al., 2014, 2018; Jiang et al., 2013) are used as polymer matrices in bionanocomposites, together with metal NPs as fillers. Nanocomposites based on metal NPs and the various aforementioned polymers can inherit characteristics of their constituent materials, including optical, electrical, or catalytic properties of metal NPs and thermal, mechanical, electrical, or chemical properties of polymers. However, due to the mutual interaction between metal NPs and polymer matrix, these characteristics can be modified, more or less pronounced, or express some new features.

12.3.2 FUNCTIONALIZATION OF METAL NANOPARTICLES: MANIPULATION OF NANOPARTICLES PROPERTIES In order to stabilize metal NPs and manipulate their properties at the same time, before their use for the desired applications, surface functionalization with organic

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molecules has been utilized as a powerful tool. This determines their physicochemical properties and could lead to processability, surface chemistry, and biocompatibility enhancement. The creation of specific sites on their surface is selective for molecular attachment and their interaction with the environment, and may yield a controlled assembly or the delivery of NPs to a target. Simple structures of NPs surrounded by functional molecules can be easily produced, and/or interparticle crosslinking can occur with other molecules. The core particle is often protected by a layer/several layers of molecules adsorbed or chemisorbed on their surface. The same layer might act as a biocompatible material, but more often an additional layer of linker molecules with reactive groups at both ends is required to allow further functionalization, depending on the application (Fig. 12.2). As the biological processes are typically performed in an aqueous environment, a hydrophilic NPs surface is desired for reactions with biological molecules. Functional molecules on NPs should prevent them from aggregation, maintain their good water solubility, retain their functionalities, and ensure biocompatibility before they interact with targeted subjects. Ligands containing amino or carboxy groups have been used to functionalize water-soluble NPs by electrostatic repulsion, and can be exploited for the conjugation of other molecules to the particles (Dojˇcilovi´c et al., 2016; Pajovi´c et al., 2015). In addition, metal NPs are not stable in micromolar concentrations in electrolytic solutions, which induce their aggregation. It can be avoided by adsorption of negatively charged phosphine molecules or nonthiolated ssDNA on the AuNPs surfaces (Liu, 2012; Koo et al., 2015). The functionalization of NPs can be accomplished during their synthesis by a suitable agent. Among the surface coatings there is an increasing interest in using polysaccharides as linear or branched polymeric carbohydrate structures, for tailoring NPs surface functionality (Habibi and Dufresne, 2008). In this case, carbohydrates as biomimetic functional molecules on the NPs surface can be used for the diagnosis and treatment, as a carrier for anti-HIV prodrugs (Chiodo et al., 2014), or for detecting carbohydrate-binding proteins (Adak et al., 2014). Various organic molecules with different features can be exploited for NPs functionalization or spatial assembly, bringing the unique properties and functionality of both materials, NPs and biomolecules. Proteins, enzymes, DNA, RNA, lipids, vitamins, peptides, and water-soluble polymers are able to stabilize and functionalize NPs without undesirable consequences on the environment and biosystems. This functionalization could be performed through chemisorption of thiol groups, by electrostatic adsorption of positively charged biomolecules on the negatively charged NPs surfaces or vice versa, and covalent/noncovalent binding between biomolecule functional groups and particles. DNA and RNA can be employed as generic polymeric molecules to functionalize AuNPs in an aqueous solution by a thiol
metal bond, induce AuNPs assembly into aggregates, or organize them into spatially defined

12.3 Nanoparticle Synthesis and Functionalization

FIGURE 12.2 Schematic representation of metal NPs conjugation with functional molecules and polymers and their applications.

structures (dimers, trimers) (Herne and Tarlov, 1997; Mirkin et al., 1996; Alivisatos et al., 1996). Selective sensitivity of the AgNPs can be improved by their functionalization with protein cytochrome c, while the specific uptake of AuNPs by cells is optimized by their conjugation with the corresponding

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peptide (Sivanesan et al., 2011; Nativo et al., 2008). Efficient stabilization/ functionalization of AuNPs could be performed by enzymes, α-amylase, or lysozyme (Rangnekar et al., 2007; Eby et al., 2009). Biomacromolecules and fluorescent dyes have also been used to functionalize nonfluorescent metal NPs to form fluorescence for detection proteins, DNAs, cells, and bacteria or to determine the activity/concentration of enzymes (Laban et al., 2016; Dojˇcilovi´c et al., 2016; Pajovi´c et al., 2015; Tseng et al., 2014; Laban et al., 2014). Metal NPs functionalization includes ampicillin and tetracycline molecules (Brown et al., 2012; Buszewski et al., 2016), when these systems become potent bactericidal agents with unique properties that subvert antibiotic resistance mechanisms of multiple-drug-resistant bacteria. Besides biological molecules, various polymer molecules can also be used to prevent their aggregation and functionalize them for a great variety of purposes. This combination results in the development of new functions that are noncharacteristic for individual components and represents a simple way to use their advantages. For example, NPs could be stabilized by surface-active polymers adsorbed strongly onto their surface due to van der Waals attractive forces, since there is a large decrease in their surface energy in comparison with native NPs (Rozenberg and Tenne, 2008). The interaction between NPs in these nanocomposites enables them to act as molecular bridges in the polymer matrix. Of the polyesters that show promise in biomedical fields, PEG is the most prevalent. Due to its amphiphilic properties, PEG-functionalized metal NPs can be soluble in a number of solvents, with intermediate polarity, and can be used for biological, chemical, and biomedical applications (Liu et al., 2012). Another hydrophilic and nontoxic polymer discussed in the literature is chitosan, which is able to stabilize metal NPs and provide them with antibacterial activity (Vukoje et al., 2014a; Gu et al., 2014).

12.4 APPLICATIONS OF METAL NANOPARTICLES AND THEIR POLYMER-BASED NANOCOMPOSITES In the previous sections we noted that successful utilization of metal NPs in biology and biomedicine, and interdisciplinary fields, critically depends on their structural features and surface chemistry. Desirable NPs characteristics can be achieved before their use, and different environmentally friendly methods have been developed. Particularly, great progress in the manipulation of NPs in the last decades has allowed us to encroach into the fascinating world at the length scale of molecules and DNA strands. Moreover, the possibility to concentrate, amplify, and manipulate light at the nanoscale level gave rise to the idea to use metal NPs in cells. With the biological size, comparable to proteins (B5 nm) and DNA chains, but smaller than cells, bacteria, and viruses, metal NPs have found applications in various fields (Fig. 12.2).

12.4 Applications of Metal Nanoparticles

In the following sections, we give selected examples of different types of these nanosystem applications that have been reported recently, discussing their challenges and perspectives.

12.4.1 MEDICAL APPLICATIONS As mentioned above, NPs have a biological size and their successful applications in medicine sometimes require them to enter cells across the cell membrane. Surface functionalization of NPs offers mediated/targeted uptake by the cells using known biological interactions. These reactions can take place over biomolecules such as antibodies, collagen, glutathione, fluorophores, etc., depending on the function required by the application.

12.4.1.1 Cancer immunotherapy/drug delivery In the last few years, significant progress has been made in the field of cancer immunotherapy. The goal is targeting immune-suppressive populations and stimulating immune effector cells against cancer. Recent investigations have shown that the delivery and efficacy of immunotherapeutic agents and molecular therapies can be enhanced, together with reduced adverse outcomes, through the use of NPs. They have been explored as immunotherapy carriers due to their preferential accumulation within tissues and cells of the immune system. AuNPs have been applied as a promising carrier for immune therapies, including cancer antigen and immune adjuvant delivery (Lin et al., 2013a,b). Their size- and shape-dependent optical properties can be exploited in photothermal ablation and light-triggered drug delivery (Arvizo et al., 2010). Exactly these characteristics affect blood clearance and organ accumulation of AuNPs in vivo, that is, the smaller NPs circulate in the blood longer and can be distributed more widely than larger NPs (Lin et al., 2013a,b; Arvizo et al., 2010). Their simple surface coating with PEG can reduce opsonization and uptake by the reticuloendothelial system. However, as the PEG molecules can be displaced with cystine present in the blood (causing protein absorption and macrophage uptake), this problem was solved by adding an alkyl linker between the PEG and the thiol group bound to the AuNPs surface (Larson et al., 2012). On the other hand, AuNPs blood half-life increases with decreasing NPs size and increasing PEG molecular weight (Perrault et al., 2009), while a higher percentage of smaller PEGylated AuNPs reaches the targeted tumor site, but accumulate in the liver and spleen to a lesser extent than larger ones (Zhang et al., 2009). In addition, positively charged NPs were taken up to a much higher extent by nonphagocytic cells than negatively charged ones (Liu et al., 2013). Varying the hydrophobicity of 2 nm AuNPs, Moyano et al. determined that more hydrophobic NPs would induce higher expression of inflammatory cytokines by mouse splenocytes (Moyano et al., 2012). Furthermore, NPs can be used for photothermal therapy (PTT) by varying their size, shape, and shell thickness, to absorb light in the near-infrared (NIR) range. Among the large variety of NPs shapes, nanorods, nanocubes, and

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nanocages would have large absorption cross-sections and display a large photothermal effect with absorbed photons, which can be converted into phonons— lattice vibrations to produce a localized temperature jump. Within the NIR range (where blood and tissue are relatively transparent), the NPs show strong SPR absorption of light which has penetrated through the healthy tissue, then they heat up and destroy the nearby cancer cells (Kennedy et al., 2011). Based on this effect, conjugate Au nanocages and tumor-targeting antibody (anti-HER2) were used to destroy breast cancer cells in vitro owing to the large absorption crosssection of particles that facilitates the conversion of NIR irradiation into heat (Chen et al., 2007). Bear et al. exploited Au nanoshells in the elimination of metastatic melanoma by PTT, which can promote a tumor-specific immune response against a distant, subcutaneous B16-ovalbumin (B16-OVA) tumor (Bear et al., 2013). They found that a combination of PTT with adoptive T-cell therapy could inhibit metastatic tumor growth to a greater extent than either treatment alone. Another potential combination PTT with immune modulatory agents such as drugs, oligonucleotides, or proteins together with optically and thermally responsive metal NPs in delivering such agents, could be exploited. For instance, macrolide-coated, NIR tuned Au nanorods target tumors in vivo as a combination PTT and immunotherapy (Dreaden et al., 2012), while doxorubicin-loaded hollow Au nanoshells apply a combination PTT and chemotherapy treatment in vivo (You et al., 2012). This system could potentially be applied in a metastatic disease model, when drug and the thermal treatment could induce a systemic immune response against distant, untreated sites. A number of combination treatment possibilities of thermally responsive metal NPs, such as a combination of optical contrast enhancement and the photothermal effect, or thermal ablation or light-triggered release which can be combined with delivery of nucleic acid immune adjuvants, have been previously reviewed (Almeida et al., 2014; Zeng et al., 2014). Thus, metal NPs, especially AuNPs, may act as a new class of anticancer nanomedicine agent, combining therapy and diagnosis. Moreover, as mentioned above, metal NPs are attractive candidates for adjuvant delivery and safety of immunotherapy agents. For example, several groups have investigated AuNPs-mediated CpG oligonucleotide delivery as short DNA sequences that mimic bacterial DNA and thus stimulate immune cells via interaction with toll-like receptor (TLR9) (Lin et al., 2013b) or they have investigated AuNPs vaccines for their ability to deliver a large payload which recently have been used in an HIV model or as a cancer vaccine platform (Safari et al., 2012).

12.4.1.2 Imaging of tissues and cells/nanoparticles in diagnostics Metal NPs have been in active use in cell imaging owing to their size-dependent efficiency of absorption or scattering light. The larger NPs exhibit more scattering, while smaller ones exhibit higher absorption cross-sections. Thus, their size and absorption/scattering cross-sections should be carefully considered when designing a cell imaging system. The larger metal NPs ($30 nm) are ideal for cellular labeling using dark-field microscopy, while small NPs (,10 nm) can be

12.4 Applications of Metal Nanoparticles

used in photothermal techniques. Cellular labeling using NPs offers more possibilities for quantitative imaging of biological molecules (i.e., DNA, proteins, viruses, etc.) in the cell’s endogenous environment or detection of biospecific interactions. They can be used as in vitro and in vivo imaging contrast probes. Their bioimaging relies on intense Rayleigh/Mie light scattering from their surface, providing very bright and stable light-scattering signals which are important for long-term imaging of living cells, the plasmonic field near their surface or between coupled NPs, which enhance the Raman signals of molecules near this fields, allowing monitoring of the changes in the molecular environment around the NPs, the enhancement or quenching of the fluorescence of the molecules in the vicinity of the NPs, and on the SPR mechanism that produces multiple effects, that is, photothermal, photoacoustic, etc., for multimodal single-cell imaging. Numerous reports have appeared describing the use of Au particles of different sizes and shapes for real-time detection of their penetration into living cells, which permits extensive visualization of the cells under continuous illumination to achieve multicolor imaging (Austin et al., 2014). The light-scattering effect of metal NPs can be used to differentiate diseased cells (i.e., cancerous) from the healthy cell population, for molecular recognition applications, and in the identification of cell-surface receptors (Austin et al., 2015). El-Sayed’s group employed plasmonically enhanced Raman and Rayleigh scattering from AuNPs to identify human oral cancer cells and to selectively destroy them with AuNPs-assisted photothermal therapy (El-Sayed et al., 2006). Other groups have used various antibody-labeled NPs in conjunction with SERS, spectral and fluorescence imaging to selective label and visualize cancerous cells, both in vitro and in vivo (Seekell et al., 2011; Lee et al., 2014). Moreover, Kneipp et al. used SERS and surface-enhanced hyper-Raman scattering demonstrated to probe and image the pH in live cells by nonspecific intracellular AuNPs (Kneipp et al., 2007). These NPs can target mitochondria (Wang et al., 2011; Ju et al., 2014a) or they have been used to probe intracellular events involved in cell growth and death (Kang et al., 2012). This platform further allows to determine the drug efficacy of chemotherapeutics and monitor apoptotic molecular events in real-time, when AuNPs have been coupled with fluorescence to monitor the induction of apoptosis and caspase biomarkers due to energy transfer between NPs and target acceptor molecule (Chen et al., 2014; Wen et al., 2014). This AuNPs fluorescence imaging platform was also used for the real-time tracking of oxidative stress levels in both in vitro and in vivo models (Ju et al., 2014b), to detect two forms of tumor mRNA in breast cancer cells (Qiao et al., 2011) or to monitor enzymatic activity and cellular metabolism (Wu et al., 2013; Han et al., 2014). Future efforts in using plasmonic NPs, especially AuNPs, toward real-time single-molecule mapping within single cells should involve higher imaging resolution, by using smaller NPs and detecting the light absorption instead of scattering using photothermal imaging, together with novel bioconjugation strategies to target specific molecules in single cells and to provide complex information from the cell sample (El-Sayed et al., 2006; Leduc et al., 2013).

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12.4.2 APPLICATIONS IN BIOLOGY Due to their properties, especially small size and great surface area, metal NPs and their composites with various natural and synthetic polymers have great potential for biological applications, either for detection and imaging of biomolecules, or as an antimicrobial agent. Functionalization of metal NPs or their incorporation in polymer matrix enable their easy interaction and bonding with different functional groups present in the biomolecules or on the surfaces of cells, which is the basis for their biological application—labeling and detection of the specific molecules.

12.4.2.1 Fluorescent biological labeling Imaging of living systems, specific cells/tissues, or biomolecules, based on fluorescence, is very sensitive and selective toward certain chemical and/or biological molecules that fluoresce initially or are labeled by fluorescent species, NPs, or sensors, and is not harmful toward living organisms. Incorporation of fluorescent NPs into cells or tissues enables their easy visualization, while NPs functionalized by proper oligomers/polymers, ligands, or antibodies can be used for targeted imaging of biomolecules. The possibility of noble metal NPs functionalization by fluorescent molecules, together with their characteristic features, make them applicable to cellular imaging. Unlike fluorescent probes (organic fluorophores), metal NPs have good photostability, strong photoluminescence, high emission rate, and sizedependent tunable fluorescence, that make them very attractive and suitable for biological labeling. In addition, varying surface species is a simple way to optimize the intensity of fluorescence and to design metal NPs with specific spectral properties. Fluorescent metal NPs could be used to illustrate the dynamics of the intercellular network. Fluorescent AuNPs showed great potential as markers for in vitro and in vivo cell targeting (Shang and Nienhaus, 2012) and they could be used as a biocompatible fluorescent probe. Furthermore, in order to explore and clarify metal NPs uptake by human cells, prior to their biomedical use, live HeLa cells were exposed to lipoic acid-protected AuNPs (Yang et al., 2013). Knowing the uptake kinetics, intercellular localization, and the uptake path is crucial for metal NPs utilization for drug delivery and cell diagnosis in a controllable and biocompatible manner. For example, Duan et al. synthesized biocompatible Au nanoclusters coated with chitosan-N-acetyl-L cysteine which showed low cytotoxicity, allowing their usage for living cell imaging (Duan et al., 2018). Also, AuNPs showed great potential for diagnostic purposes, as a fluorescence probe for the selective determination of glutathione, in living cells and human blood (Tian et al., 2012), or to label hematopoietic cells (Huang et al., 2011). The folic-acid-conjugated fluorescent AuNPs could be used for folate receptortargeted imaging of oral squamous cell carcinoma and breast adenocarcinoma cell MCF-7 (Retnakumari et al., 2010). Silica-coated AuNPs with incorporated

12.4 Applications of Metal Nanoparticles

thrombin-activatable fluorescent peptide showed great potential for in situ detection of a thrombotic lesion in a mouse model, as well as for possible therapy in clinical applications (Kwon et al., 2018), while a nanohybrid of Au nanorods and carbon dots with silica as a bridge between them were successfully used for in vitro detection of synthetic macrophages, enabling their possible application as a theranostic contrast agent for atherosclerosis (Liu et al., 2018). Our previous investigations in this field showed that AuNPs and AgNPs, functionalized by tryptophan and riboflavin, could be used for deep UV fluorescence labeling study of microbial cells (Dojˇcilovi´c et al., 2016; Pajovi´c et al., 2015). Nanocomposites of metal nanoparticles with polymers also could find their role in cell imaging. PVA-borax hydrogel functionalized by Ag dots was used as the sensing probe to detect insulin in human blood, while the three-layer core
shell nanostructure of AgNPs, silica, and fluorescent dye, is highly sensitive to prostatespecific antigen in the human serum (Pourreza and Ghomi, 2017; Xu et al., 2017). Besides their biocompatibility, metal NPs need to target specific tissues with high selectivity in vivo, and to be efficiently released from the living organism by the metabolic system, while their long-term influence on the cells and their cytotoxicity, should be clarified.

12.4.2.2 Biodetection of proteins Interesting optical properties of metal NPs enable their possible application as sensors of various molecules, including biomolecules. Great chemical reactivity of metal NPs, based on functional groups present on their surface, makes them very useful for detection of different proteins, nucleic acids, and metabolites. These sensors work on the key
lock principle, whereby properly functionalized metal NPs can selectively conjugate with the desired analyte (biomolecule). For example, AuNPs could be used for malaria antigen detection, sensing of aflatoxin, and determination of the content of hCG hormone from human blood (Guirgis et al., 2012; Wang et al., 2014; Schneider et al., 2000). The designation of DNA sequences is of great concern in modern diagnostic methods. Among noble metal NPs, AuNPs have potential to be used as a biochip for precise DNA sequence determination. Because of their high stability, resistance to agglomeration, and easy preparation, AuNPs functionalized with oligonucleotides are usually used for DNA detection (Ma et al., 2018). Several methods have employed AuNPs for DNA detection—colorimetric, electrical, SERS, etc. Colorimetric DNA detection is based on NPs’ color change, that is, SPR shifting. Targeted DNA is complementary to the DNA chains from the particle surface, which in turn results in their interaction (bonding), and finally, in the formation of NPs aggregates (Nourisaeid et al., 2016). The lower sensitivity of the colorimetric DNA detection method is overcome by the electrical method, whereby the interaction between targeted DNA and corresponding metal NPs is manifested by the change of the electrical signal (Song et al., 2011). Furthermore, based on the strong electric field generated on the metal NPs surface, they have great potential for the SERS technique which provides molecular fingerprints. There is a signal

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enhancing when NPs aggregate upon bonding to targeted DNA or they are functionalized with oligonucleotides that contain Raman-active dyes for identification (Lin et al., 2015; Xu et al., 2015). Also, nanocomposites based on metal NPs and polymers can be used for DNA detection (Zhu et al., 2015; Jin et al., 2018). AuNPs and AgNPs, of different sizes, were used for the detection of different proteins (Li et al., 2017; Zong et al., 2017). As bare and unmodified NPs, they easily can interact with proteins, at the same time as receptors and indicators (Saptarshi et al., 2013), which make them a simple sensing material for colorimetric detection of proteins. With the change of NPs species and their size, as well as the protein and their concentration, particular absorbance responses were created, together with a visual color change. This enables simple protein differentiation and potential applications in medical diagnosis (Sriram et al., 2015). Biodetection based on noble metal NPs as a novel diagnostic method for detection of various biological molecules can provide vital information in considering many diseases. Besides demands for higher selectivity and sensitivity toward key biomolecules, with minimal cost, there is a need for real-time and simple detection.

12.4.2.3 Biosensing applications Metal NPs represent receptors for various biological species—bacteria and viruses, DNA, or proteins from antibodies and antigens, to more simple molecules, like glucose, acting as a connection between nanotechnology and biotechnology. To be considered as a reliable biosensor of a certain analyte, it needs to effectively display (optical/electrical signal) its interaction with required molecules, even when they are in traces. Using NPs as biosensors enables increasing the sensitivity toward biomolecules, as well as lowering the detection limit, even to an individual molecule. Among noble metal NPs, AuNPs have been commonly used as biosensors. Since their optical properties are environment-dependent, the changes to particle surfaces through interaction with the surrounding molecules induces a change in colloid color that can be observed by the naked eye, together with changes in the SPR position, shape, intensity, etc. This feature is the basis for immunosensors (Lesniewski et al., 2014; Liu et al., 2015) or for efficient colorimetric biosensing of nucleic acids (Zaher et al., 2018). For example, AuNPs modified with covalently bonded anti-T7 antibodies serve as a simple and selective colorimetric immunosensor for T7 bacteriophages, as a model organism for adenoviruses (Lesniewski et al., 2014). T7 bacteriophages form a complex with modified AuNPs, causing their agglomeration, and consequently a change in their color and SPR. Also, this investigation pointed out that AuNPs as immunosensors can detect all T7 bacteriophages present in the sample, compared to biological tests that label only biologically active ones. Similarly, modified AuNPs could be used as colorimetric immunosensors for influenza A virus (Draz and Shafiee, 2018). Besides AuNPs, AgNPs and CuNPs also have the possibility to be used as colorimetric immunosensors for respiratory syncytial virus (Valdez et al., 2016). This

12.4 Applications of Metal Nanoparticles

type of virus detection has an advantage over classic immunoassays, since it is a simple and one-step sensing technique, and does not require additional amplification. Similarly, pathogen microorganisms, such as Escherichia coli, of which the enterohemorrhagic serotype is responsible for the production of shiga-like toxin that causes bloody diarrhea in humans, or Lactobacillus species and botulinum neurotoxin, could be detected by a colorimetric biosensor based on AuNPs (Jyoti et al., 2010; Verdoodt et al., 2017; Liu et al., 2014). AuNPs immobilized by appropriate bioreceptor units are used for the detection of electroactive biological species, viruses, hormones, and cancer biomarkers, whereby modified AuNPs transfer electrons between the biomolecule and electrode, catalyzing their oxidation or reduction (Ravalli and Marrazza, 2015; Chandra et al., 2013; Alipour et al., 2013; Mazloum-Ardakani et al., 2015; Arya et al., 2011). As a response to this process, the electrical signal appears as amperometric, potentiometric, or impedimetric. Besides AuNPs, several studies have pointed out the significance of other metal NPs for the detection of various biomolecules (Xu et al., 2006; Wang et al., 2016).

12.4.2.4 Antimicrobial testing With microbes evolving, and their rising resistance to commonly used antimicrobial agents (antibiotics and antimycotics), there is a need for the development of new antimicrobial materials. Hence, science today is also aiming to find new, useful, and efficient antimicrobial agents (Bogdanovi´c et al., 2014b; Theron et al., 2008; Giannossa et al., 2013). With the emergence of nanotechnology and the possibilities that it offers, metal NPs have found their place as antimicrobial agents in various fields, from medicine to food industry and wastewater treatment (Theron et al., 2008; Giannossa et al., 2013). Their great characteristics in this area were recognized even in ancient times, although they were not known as NPs (Lemire et al., 2013). Ag and Cu have proved to be excellent antimicrobial materials, and could be used for this purpose in the form of NPs, either alone or incorporated in the polymer matrix, in the form of ions or hybrid structures. The exact mechanism of their antimicrobial activity is not completely known yet. It is assumed that, when in contact with microbial cells, AgNPs are incorporated in the cell membrane, or they penetrate, causing damages and leaking of cellular content, and finally cell death (Cho et al., 2005). Similarly, when CuNPs are in a medium with microbes, there is a release of copper ions (Cu21 and Cu1) from the particle surface, together with cyclic redox reactions between them on the microbial cell surface, which cause its destruction, and finally penetration of copper into the cell. Microbes’ cell function is disrupted in multiple ways, such as permeability, respiratory and biochemical functions together with DNA damages (Ruparelia et al., 2008). The most important property of these NPs is their ability to participate in redox reactions, representing catalytic cofactors for microbial cell enzymes that can generate or catalyze reactive oxygen species, responsible for cell oxidation and damage (Lemire et al., 2013). Also, a term called “ionic/molecular mimicry”

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is described as the displacement of original elements (sulfur, nitrogen, oxygen) from microbe biomolecules by Cu ions, for example, that form organic complexes with these elements, causing changes to osmotic balance and deformations to proteins and nucleic acid structure (Lemire et al., 2013). Metal NPs are suitable as an antimicrobial material, since their small size fits the size of biomolecules, which makes their mutual interaction possible and easy. In addition to their size, shape and surface structure, characteristics of microbes— type, structure, as well as the conditions of their exposure to the antimicrobial agent (time, temperature, pH, etc.)—are also important for examination of material antimicrobial activity (Raza et al., 2016). Based on literature data, AuNPs and PtNPs did not show any antibacterial activity as bare NPs, but in the form of bimetal NPs, functionalized or in composites, their antimicrobial efficiency becomes evident (Boomi et al., 2014; Zhang et al., 2015). Our researches are related to the examination of antimicrobial activity of AgNPs and CuNPs, and their composites (Bogdanovi´c et al., 2014b, 2015a; Bogdanovi´c et al., 2018; Ili´c et al., 2010; Lazi´c et al., 2013). An interesting and very intensive antimicrobial property was observed for CuNPs (B6 nm) whether bare or incorporated in conducting polymer PANI (Bogdanovi´c et al., 2014b, 2015a; Bogdanovi´c et al., 2018). Some of these results are presented in Fig. 12.3,

100 90 80 Cell reduction (%)

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70 60 50 40 30 20 10 0 E. coli

S. aureus 8 ppm

16 ppm

C. albicans 32 ppm

FIGURE 12.3 CuNPs concentration-dependent reduction ability on E. coli, S. aureus, and C. albicans over 2 h incubation time.

12.4 Applications of Metal Nanoparticles

as quantitative (chart) measurements for bare CuNP antimicrobial activity, and qualitative, AFM measurements of the microbial morphology deformations after their exposure to Cu/PANI nanocomposites (Figs. 12.4
12.6). This system could find practical applications in the rapid control of microbial infections in contaminated water before further processing. In addition to the previous study, many other nanocomposites based on AgNPs and polymers with excellent antibacterial behavior against some representative bacteria, fungi, and foodborne pathogens have also been reviewed (Link and El-Sayed, 2003; Zare and Shabani, 2016; Solairaj and Rameshthangam, 2017). Another attractive research area in progressive applications of metal NPs is in the production of antimicrobial textiles (medical textiles, sportswear, and everyday clothing). A major work on the functionalization of these materials has been done with AgNPs due to their extraordinary efficiency against microbes applied to the surface or incorporated into textile fibers, providing a product which kills/ inhibits microbial growth. Our recent studies reported the intrinsic antimicrobial efficiency of AgNPs deposited on different textile materials (Ili´c et al., 2009, 2010; Lazi´c et al., 2012). Overall, the multiple functionalities of metal NPs make them attractive for various approaches in this field, such as introducing NPs onto textile and different surfaces, or using them in water treatment. However, in order to be safely used, metal NPs need to be minutely characterized. The mechanisms of their

FIGURE 12.4 AFM images of E. coli (A) before and after incubation with Cu/PANI (20 ppm) for (B) 1 h and (C and D) 2 h.

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FIGURE 12.5 AFM images of S. aureus (A) before and after incubation with Cu/PANI (20 ppm) for (B) 1 h and (C and D) 2 h. ˇ Reprinted with permission from Bogdanovic´ U., Vodnik V., Mitric M., Dimitrijevic S., Skapin S.D., ˇZunicˇ V., et al., Nanomaterial with high antimicrobial efficacy-copper/polyaniline nanocomposite, ACS Appl. Mater. Interfaces 7, 2015a, 1955
1966. Copyright (2015) American Chemical Society.

toxicity and safe exploitation of their antimicrobial properties without negatively impacting human health and the environment need to be well examined and understood.

12.5 CONCLUSIONS AND OUTLOOK Exceptional accomplishments are performed in designing nanomaterials with different functionalities that can be used for various medical and biological applications. Considering unique size- and shape-dependent physicochemical properties, metal NPs are at the forefront of scaffolds for designing such bioactive materials of the novel opportunities as diagnostic, delivery, and disease-treated systems. Thus, in vitro applications of metal NPs are well established, while in vivo ones

12.5 Conclusions and Outlook

FIGURE 12.6 AFM images of C. albicans (A) before and after incubation with Cu/PANI (20 ppm) for (B) 1 h and (C and D) 2 h. ˇ Reprinted with permission from Bogdanovic´ U., Vodnik V., Mitric M., Dimitrijevic S., Skapin S.D., ˇZunicˇ V., et al., Nanomaterial with high antimicrobial efficacy-copper/polyaniline nanocomposite, ACS Appl. Mater. Interfaces 7, 2015a, 1955
1966. Copyright (2015) American Chemical Society.

show promising results. However, their application requires a directed design, providing actuation and stability in complex environments, such as living organisms. Among these processes, their surface functionalization by known biological interactions or promoters holds great promise and offers a compatible surface (in addition to the required good water solubility) to interact with cells before realizing their own functionalities. By appropriate modification, specific binding to target surfaces, cells or organelles can be tuned, for the controlled targeting of metal NPs. Up to now, highly attractive features such as biosensing capabilities in connection with SERS imaging, novel therapies based on local drug delivery, and photothermal therapy, are being explored. Their future applications should be considered for actual biological problems and the molecular precision of the interaction between them and intracellular subjects, which should lead to novel routes in biomedical practice.

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ACKNOWLEDGMENTS This work was supported by the Ministry of Education, Science, and Technological Development of the Republic of Serbia (Grants 172056 and 45020).

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886.

CHAPTER

Hybrid metal complex nanocomposites for targeted cancer diagnosis and therapeutics

13

Jeong-Hwan Kim1,2, Haruki Eguchi3, Masanari Umemura2 and Yoshihiro Ishikawa2 1

RadianQbio Co. Ltd., Halla Sigma Valley, Gasan Digital, Geumcheon-gu, Seoul, Republic of Korea 2Cardiovascular Research Institute, Yokohama City University Graduate School of Medicine, Yokohama, Japan 3Advanced Applied Science Department, Research Laboratory, IHI Corporation, Yokohama, Japan

13.1 INTRODUCTION Cancer is a fatal illness which also significantly and adversely impacts the social and economic status of patients and hence there is an urgent need for its timely elimination. In the past decades, tremendous fascinating progresses in medicinal and pharmaceutical technology have been accomplished in treating cancer (Balderas-Renteria et al., 2012). Specifically, a significant proportion of research investment is dedicated to improving chemotherapeutic efficacy, which is often the only hope in healing a cancer patient. However, anticancer chemotherapeutic agents in the available market are not only unaffordable to many because of their high prices, but they also lack the capability to cure cancer due to the higher death cases over survival rates, deadly effects on normal tissue, inadequate targeting, and impaired transport to the tumor. The long-term use of the available anticancer drugs also develops resistance by tumor cells, which critically restricts their applications (Pereira et al., 2012). Determination of suitable drug dosage, optimal drug concentration, and drug release kinetics at the tumor site, can also be challenging. Hence, there is an urgent need to develop medications with greater anticancer activity and less toxicity than present treatments. Recently, the arena of nanoparticle (NP)-based DDS has reached beyond the confines of traditional structures (e.g., geometries, sizes, interfaces) and chemical compositions so as to rationally design entities specifically tasked with overcoming sequential biological barriers (Alexiou et al., 2000; Jain and Stylianopoulos, 2010; Sun et al., 2014; Lee et al., 2015; Sao et al., 2015; Carregal-Romero et al., 2015;

Materials for Biomedical Engineering: Inorganic Micro- and Nanostructures. DOI: https://doi.org/10.1016/B978-0-08-102814-8.00015-9 © 2019 Elsevier Inc. All rights reserved.

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Baek et al., 2015; Mou et al., 2015). In particular, metal nanoparticulate composites as anticancer agents have been attracting attention due to their tunable size, readiness of synthesis, and unique anticancer efficacy (Alexiou et al., 2000; Jain and Stylianopoulos, 2010; Sun et al., 2014; Lee et al., 2015; Sao et al., 2015; Carregal-Romero et al., 2015; Baek et al., 2015; Mou et al., 2015). However, determining how to get these advances to the clinic remains a significant challenge (Mitragotri et al., 2015; Venditto and Szoka, 2013; Park, 2013). As another class of hybrid metal composites, transition metal complex compounds have been extensively considered as an anticancer drug since they are suitably to be designed for extraordinary biological activity and selectivity by adjusting the substituents of their ligands (Romero-Canelo´n and Sadler, 2013; Jungwirth et al., 2011; Wani et al., 2016; Renfrew, 2014). Numerous iron complexes with different ligand systems have demonstrated exciting therapeutic benefits in comparison to the market-selling anticancer metallodrugs such as cisplatin, oxaliplatin, and photofrin. However, many concerns are yet to be resolved. Several examples of iron complexes [e.g., Fe(Salen)] highlighted in this chapter have overcome cancer cell resistance, display high cytotoxicities with selectivity, fewer toxic effects, and act via different mechanisms of action [i.e., reactive oxygen species (ROS) generation]. Very few studies have demonstrated the potential of iron complexes in in vivo tumor models, however, there are extremely limited studies available on the in vivo explorations of iron complexes as anticancer agents. Thus, exploring the promising potential of the potent iron complexes highlighted above in in vivo antitumor models is highly encouraged. In this chapter we consider how organometallic materials can help to address the aforementioned problems. The role of anticancer metal complexes will be explored as a means to target cancer drug therapy, with the aim of employing the results in the clinical setting, especially the designing of a local drug-delivery system (DDS) via magnetic metal complexbased composites. Magnetically guided DDSs based on magnetic metal composites could save lives and enrich the quality of cancer targeting by making it possible to tailor therapy to the individual patient and minimize time and costs, as well as raising considerations for future applications. With these aims in mind, we will look in more detail at a magnetic metal complex drug system as a versatile cancer targeting agent through a wide range from diagnosis (imaging) to therapy (chemotherapy, drug delivery, and hyperthermal treatment), as illustrated in Fig. 13.1. We begin by briefly reviewing the challenges in conventional anticancer treatments, following by advantages of intrinsic magnetic drug design, and its application to advanced cancer chemotherapy. In the final section, the novel methods for synthesizing magnetic- and pHresponsive (smart) DDSs are discussed, as a leading-edge synthetic paradigm of targeted DDS. Finally, this chapter is an exquisite reference source for both materials scientists and biomedical engineers who desire to learn more about how metal complex-based nanocomposites are engineered and used in the design of advanced magnetic drug-delivery nanosystems.

13.2 Conventional Chemotherapy

FIGURE 13.1 Schematic illustration of the use of magnetic metal composite nanomaterials for simultaneous quadruple modality in chemotherapy/drug delivery/imaging/hyperthermia.

13.2 CONVENTIONAL CHEMOTHERAPY Alkyl antineoplastic (ANP) drugs is a generic term for antimetabolites or antitumor agents containing an alkyl group (
CH2
CH2
) (Scott, 1970). The structure is similar to those of nucleic acids or metabolites in a protein synthesis process, which is impaired by cells. For example, ANP drugs are effective for alkylating DNA, which leads to inhibition of DNA replication, resulting in cell death. These effects are regardless of cell cycles, are attributed to the Go period that is responsible for an active cell growth phase, and often damage many organs, for example, bone marrow, alimentary canal mucosa, germ cells, or hair roots. Moreover, antitumor antibiotics are chemical substances produced by microorganisms, and associated with DNA synthesis inhibition, DNA strand breaking, and potential antitumor activity (Galm et al., 2005). Also, microtubule inhibitors have antitumor effects by directly acting on microtubules that serve important roles in maintaining normal functions of cells (Perez, 2009), for example, by forming spindles during cell division, locating cell organelles, and transporting substances. The microtubule inhibitors act on cells, which divide actively, and nerve cells. Furthermore, parathormone antineoplastic drugs are effective against hormonedependent tumors (Herington et al., 2010). Female hormones or antiandrogen

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drugs are administered to androgen-dependent prostatic cancers. Topoisomerase (TOPOS) inhibitors are nuclear enzymes that temporarily affect breaks in DNA and change the number of tangles in DNA strands. TOPOS inhibitor I is an enzyme that causes breaks in one strand of a circular DNA, allowing the other strand to pass, and then closes the breaks; a TOPOS inhibitor II temporarily breaks both strands of circular DNA, allowing the other two DNA strands to pass between the former two strands, and reconnecting the broken strands. Recently, these agents have been considered as potential commercial anticancer agents (Khadka and Cho, 2013). Furthermore, nonspecific immune potentiators inhibit growth of cancer cells by activating the immune system (Mokyr and Dray, 1987). Cisplatin was discovered in 1845 and licensed for clinical use in 1978 as a first-generation anticancer compound (Janos and Robin, 2006), a most successful commercially available metal complex-based anticancer agent. A cis geometric isomer of the metal complex is associated with anticancer properties by inhibiting DNA synthesis by forming DNA strands, interchain bonds, or DNA
protein bonds. However, it causes severe nephropathia and requires a large amount of fluid replacement, which has been limited by many clinical applications. Topical anesthetics also have the same kind of problem side effects. Topical anesthetics are used to treat topical itches and pains from, for example, mucosa or skin caused by hemorrhoidal disease, stomatitis, gum disease, cavities, tooth extraction, or typical operations (Sobanko et al., 2012). Lidocaine/tetracaine cream (Pliaglis, Galderma Laboratories, Texas, USA) is known as a representative topical anesthetic, showing prompt pain alleviation activity (Sobanko et al., 2012). However, this agent is attributed to an antiarrhythmic effect (“Lidocaine Hydrochloride Antiarrhythmic,” 2015). Furthermore, if lidocaine is injected into the spinal fluid when giving spinal anesthesia, lidocaine often spreads through the spinal fluid; in a worst-case scenario, there is a fear that lidocaine might reach a cervical part of the spinal cord and thereby cause the respiratory function to stop and bring about critical adverse effects (Adelaide, 2006). First-generation nanotherapeutics [e.g., Cremophor EL (CrEL)] have been commonly used as a formulation vehicle for encapsulating various poorly watersoluble drugs, including the anticancer agent paclitaxel (Taxol), which arose from an urgent need to address the limitations of conventional DDSs (Gelderblom et al., 2001). However, in contrast to earlier reports, CrEL is not an inert vehicle, but employs a range of cytotoxic effects, some of which have significant clinical implications that resultein fast and unselective tissue distribution as well as vehicle-associated toxicities. Thus, they have yet to translate to substantially improved patient outcomes (Gelderblom et al., 2001). Consequently, it is expected that cancer treatment can be provided effectively while inhibiting the adverse reactions by using the drug delivery to guide the antitumor agent to cancer cells and have the pharmacological effects exhibited and focused on the cancer cells. It is also expected that drug delivery can prevent the diffusion of topical anesthetics, maintain the pharmacological effects, and reduce adverse reactions.

13.4 Metal
Ligand Complexes as a Composite Anticancer Drug

13.3 STRIVING TOWARD TARGETED CHEMOTHERAPY Conventional therapeutic drug-delivery platforms involved pharmacokinetic limitations associated with conventional drug administration/formulations, for example, low efficacy, intracellular instability, poor solubility, low biodistribution, and rapid clearance by an immune system, for example, reticuloendothelial systematic (RES) uptake (Wang et al., 2016; Ranade and Cannon, 2011; Tiwari et al., 2012). It is of particular significant how to efficiently guide the drug to the desired region. After a drug dosage is administered to a living organism, it reaches the affected site and employs its pharmacological effects on-site, thus exerting its therapeutic effects. Instead, if the drug reaches tissue other than the affected site (e.g., healthy tissue), it may not be safe and even worsen rather than better the clinical situation. A technique to guide a drug to the affected site is called a “targeted” drug-delivery system (TDDS), which has been extensively focused on in the past decade as an up-to-date medication technology (Wang et al., 2016). Generally speaking, the TDDS holds at least two key advantages. One is that a sufficiently high drug dose can be concentrated at the targeted site tissue: pharmacological effects will not be achieved unless the drug concentration at the affected site is appropriate or constant. A sufficient therapeutic effect cannot be relied upon if the drug level is low. The other merit is that the drug molecules can be directed to only the affected site tissue, allowing adverse reactions to normal tissue to be reduced, which is ideal for cancer treatment by antitumor chemotherapeutic drugs. Most antitumor agents inhibit cell growth at the tumor site, where cells divide aggressively, while the agents also prevent the growth of even healthy tissue in which cells divide actively, such as bone marrow, hair roots, or alimentary canal mucosa. Eventually, cancer patients to whom the antitumor agents are administered may suffer from unfavorable side effects such as anemia, hair loss, vomiting, etc. Since such adverse reactions impose a heavy burden on patients, the fact is that the dosage needs to be limited and the pharmacological effects of antitumor agents may be insufficient.

13.4 METAL
LIGAND COMPLEXES AS A COMPOSITE ANTICANCER DRUG In coordination chemistry, a ligand is a functional molecular group that links to a metal ion core to form a coordination complex. The coordination interaction between a metal atom and a ligand molecule can lead to creating a precise molecular architecture with distinct properties, rather than random particulate composites. Since a platinum-aminoligand-based complex, cisplatin, has been developed (Sobanko et al., 2012), many comprehensive studies using other metal complexes have been performed to reveal the diverse medicinal features attributed to the organometallic structures and functions (Romero-Canelo´n and Sadler, 2013;

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Jungwirth et al., 2011; Wani et al., 2016; Renfrew, 2014). Currently, metal complexes of dissimilar transition metals are favored candidates for dealing with a wide variety of cancers due to their major role in tunable biological activities, simply by switching ligand-based building block components (Romero-Canelo´n and Sadler, 2013; Jungwirth et al., 2011; Wani et al., 2016; Renfrew, 2014). Such a “molecular compositing” technique as a drug design at the molecular level can orchestrate the predetermined combination of coordination chemistry and medicinal inorganic chemistry, and thus, employ diverse strategies in the development of exceptional properties of metal ions for the design of new anticancer drugs.

13.4.1 IRON COMPLEXES Until now, investigations into the anticancer properties of iron complexes with various ligand frameworks have revealed that several classes of iron complexes are promising for anticancer treatments against different human cancer cell lines (Romero-Canelo´n and Sadler, 2013; Jungwirth et al., 2011; Wani et al., 2016). In particular, it has been reported that Schiff base ligand systems play a key role in forming metal complexes with iron that present exciting anticancer properties. Some of these complexes have displayed anticancer activities even higher than cisplatin and thus hold potential for further investigation (Wani et al., 2016). While exhibiting better activities than cisplatin, several studies have also confirmed better selectivity and the potential to overcome drug resistance in human cancer cell lines (Gust and Posselt, 2008; Mandal et al., 2009; Hille et al., 2011; Lange et al., 2009; Wu¨rtenberger et al., 2015). An iron complex based on orthovanillin and 1,2-phenylenediamine Schiff base has overcome the resistance of specific human cancer cell lines (Lee et al., 2011; Kim et al., 2011b; Azani et al., 2010; Ansari et al., 2011a,b; Vanco et al., 2015; Shaaban et al., 2012). A series of methoxy-substituted iron(III)
salophene complexes have shown a very desirable anticancer activity against MCF-7, MDA-MB-231, and HT-29 cancer cell lines (Hille and Gust, 2010). The anticancer activity of the complexes was shown to be subjected on the location of the methoxy substituents in the salicylidene moieties, according to a time-dependent chemosensitivity assay (Wani et al., 2016).

13.4.2 QUANTITATIVE STRUCTURE
FUNCTION RELATIONSHIP OF IRON-SALEN COMPLEXES The biological activities of iron
salen complexes have been explored since 1931, when it was shown that salen complexes of Fe(III) possess anticancer features against MCF7 cells (Herchel et al., 2009). Iron
salen derivatives generate hydroxyl radicals in the presence of a reducing agent, for example, dithiothreitol (DTT), leading to damage to DNA in vitro (Ansari et al., 2009, 2011a,b). The probability of providing a defined compound as a medication is very rare.

13.4 Metal
Ligand Complexes as a Composite Anticancer Drug

Now, chemical computing models are employed in designing new medications, which has resulted in saving cost and time and designing more potential medications. Among the numerous computational methods, QSFR has a noteworthy role in designing anticancer medications. Moreover, the underlying basis of the structure
function relationship focuses on the interpretation of the molecular structure and cytotoxic effects, while QSAR endeavors to identity a quantitative relationship between them (Selassie, 2003; Mohajeri and Dinpajooh, 2008; Nikolic, 2007; Gao and Cao, 2006). Recently, Ghanbari et al. reported that the fuzzy logic and artificial neural network model with high statistical significance has outstanding capability to address the biological nature of the substituent of the salen ligand on the anticancer activity based on its geometrical parameters and position (Ghanbari et al., 2014). For instance, the nature of the substituent has a major effect on biological activity. The influence of Cl ligand replacing the heterocyclic N donor ligands exhibits that 1H-tetrazol-5-amin(Hatz) increases in activity. Results confirm that modification of Cl ligand on the heterocyclic N-donor ligands has a minor effect in contrast to replacement of the aromatic ring group on anticancer activity, as presented in Fig. 13.2. Nonetheless, to date, no established common rules in designing new effective metal complex-based anticancer drugs, in particular: (1) the selection of desirable ligand complex with safety and efficacy still remains enigmatic; (2) ideal therapeutic strategies are still awaited. Consequently, it is very critical to expedite the development of new therapeutic agents against cancer.

13.4.3 MAGNETIC NANOPARTICLES (MNPs) AS AN ESSENTIAL CARRIER FOR MAGNETIC DDS MNPs are a class of engineered particulate materials of less than 100 nm that can be directed under the guidance of an external magnetic field. MNPs are usually composed of magnetic elements, such as iron, nickel, cobalt, and their oxides such as magnetite (Fe3O4), maghemite (Fe2O3), cobalt ferrite (Fe2CoO4), and chromium dioxide (CrO2) (Wu et al., 2015). For years, MNPs have triggered enormous research activities in both the academic and industrial communities in the physical, chemical, environmental, and medical areas due to boundless scope of their unique magnetic, electronic, and biomedical properties (Wu et al., 2015; Mohammed et al., 2016). MNPs were first used in wastewater treatment and pollutant removal in early 1940s, but the concept of MNPs as magnetic drug carrier particles has existed for over 30 years (Mohammed et al., 2016). Specifically, in order to realize direct therapies and diagnostics to targets in the body in a minimally or noninvasive fashion, MNPs were used as a fundamental carrier to be loaded with therapeutic agents, such as drugs or genes; instead of surgery or chemotherapy, the use of MNP carriers could possibly permit clinicians to use external magnets to focus treatment to the

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FIGURE 13.2 Structure
activity of Fe
salen derivatives. Adapted from Ghanbari, Z., Housaindokht, M.R., Izadyar, M., Bozorgmehr, M.R., Eshtiagh-Hosseini, H., Bahrami, A.R., et al., 2014. Structure-activity relationship for Fe (III)-Salen-like complexes as potent anticancer agents. Sci. World J. 745649, 10. Copyright 2014 by the Hindawi Publishing Corporation.

13.4 Metal
Ligand Complexes as a Composite Anticancer Drug

precise positions of a disease within a patient. Recent progress with MNPs has implemented the creation of magnetic composite nanoarchitectures for local DDS, which stand at the cutting-edge of nanomedicine, and afford enhanced diagnostic and therapeutic efficacy, while diminishing side effects (Lee et al., 2015; Carregal-Romero et al., 2015; Baek et al., 2015; Mou et al., 2015). MNPs have been used as the “one and only” of their type, an essential component for magnetic DDS: no magnetic DDSs other than metal oxide-based ones have been explored. MNPs often comprise iron oxide NPs (IONPs) that can be drawn and guided by a magnet or external magnetic field, which is harmless for brief exposures, hence allowing one to locally aim them in living organisms in a noninvasive manner. Due to their magnetically guidable role, IONPs have not only been used as magnetic separation tools, but have also emerged as potential systems for local organ diagnosis and treatment, such as in imaging contrast agent, drug-delivery platforms, and hyperthermia (Benelmekki, 2015; Benelmekki et al., 2014, 2015; Vernieres et al., 2014; Kim and Benelmekki, 2016). The preparation methods of IONPs consist primarily of the chemical process by thermal decomposition or coprecipitation of inorganic salts, hydrothermal and solvothermal syntheses, sol
gel synthesis, microemulsion, ultrasound irradiation, and biological synthesis (Wu et al., 2015). As the most conventional method, the thermal reduction process can be used to acquire IONPs from a mixture of iron precursors like FeCl2 and FeCl3 in very basic solutions at room temperature or at an elevated temperature. However, as a main drawback of the solution-based method, the use of toxic solvents and reagents is necessary in all synthetic routes of NPs, which is challenging, not only provoking potential environmental hazards, but also it is subject to have considerable limitations in physical and chemical properties (e.g., uniform size/shape, boiling point, solubility). Other remarkable preparation methods are physical methods that comprise mainly the production of gas-phase nanoclusters by sputtering of the bulk inorganic material, where a high-purity metal target is bombarded with Ar ions, followed by the consequent deposition of the sputtered metal atoms on the surface (substrate) support to yield a uniform dispersion of MNPs (Wu et al., 2015). Basically, the nanoclusters can be formed continuously in size from a molecule up to 105 atoms per cluster. Compared with the chemical methods, this technique can generate MNPs in a higher rate of monodispersity, while the production yield is relatively very low. Various MNPs with different morphologies have been manufactured, including NPs, nanowires, and nanorods (Lee et al., 2015; Wu et al., 2015). Very recently, unlike singular NP fabrication, multiplexed and heterogonous hybrid MNPs were designed using a gas-phase synthetic method (Benelmekki, 2015; Benelmekki et al., 2014, 2015; Vernieres et al., 2014; Kim and Benelmekki, 2016; Kim et al., 2014; Kim and Lu, 2016). For example, a wide variety of hybrid nanocomposites, ranging from coreshells to nanoscrolls, can be synthesized by sputtering multitargets including diverse material precursors, such as Fe, Ag, Au, Al, Si, Mg, Pd, C, etc. Despite the attractive versatility and preclinical potential of MNPs, very few NPs that only address one or a few biological barriers advance to the clinical

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stage. Commercial superparamagnetic IONPs such as Feridex, Resovist, and Endoderm are well-known magnetic resonance (MR) imaging NP agents that have been approved by FDA, and studied in a wide variety of precise determination/tracking of tissue locations (Li et al., 2015; Wang, 2011), tumor sites (Hachani et al., 2013), and stem cells (Luo et al., 2014). The MNP-enabled translatable insight has led many experts to approach the field of NP-based drug delivery in the hope of transitioning the discipline from platforms with broad potential to those capable of bringing positive clinical products. Marrying macromlecular-based formulation technology (Hadjichristidis et al., 2011; Gheybi and Adeli, 2015; Kataoka et al., 2001) with MNPs has led to the innovation of various theranostic magnetic TDDS-vehicles (Lee et al., 2015; Carregal-Romero et al., 2015; Baek et al., 2015; Mou et al., 2015), such as polymeric nanocarriers, into which prodrugs are often coloaded with magnetic nanoparticles (MNPs) into the nanocarrier, and which help to shield the drug and to empower its magnetically guided imaging, following by local release in response to an alternating magnetic field (AMF) (Fig. 13.1A). Drug loading using MNPs can be attained using different techniques. These include loading onto the polymeric carrier matrix adjoining the MNPs, encapsulation into core
shell stimuli-responsive polymers, covalently bonding to activated surface MNPs, and trapping within magneto-liposomes. The primary goal of drug release control is to preserve the drug concentration in the blood and/or the target site at an effective level. Drug release kinetics aspires to maintaining a balance between the minimum effective concentration and the minimum toxic concentration. Administration of a single large dose can elevate the drug level above the minimum toxic concentration, which results in initial toxic adverse effects and minimal effective time. Multiple dosing in a timely manner can decrease these variations, but it could result in some concerns for the patient (Lee and Yeo, 2015). Thus, it is important to achieve sustained release profiles and low dosing frequency. Mathematical modeling of drug delivery is important for understanding the physical mechanisms that govern drug release and to predict its temporal release. Different models have been developed to describe drug release from polymer-coated MNPs. In recent year, controlled architecturing technology of inorganic
organic NPs has been enabled by “hybridizing” at the nanoscale (Lee et al., 2015; CarregalRomero et al., 2015; Baek et al., 2015; Mou et al., 2015; Kim and Benelmekki, 2016; Kim et al., 2014; Kim and Lu, 2016). The integrated nanohybrid composite systems may have surfaces and interfaces with dissimilar chemical nature, enabling the creation of new properties, and allow us to explore a wide diversity of composite materials with foci on anisotropic properties. Among the many drug-delivery approaches, magnetically guidable DDS platforms have demonstrated the advantage that the drug delivery can be locally directed with a magnet, even after drug administration, simply by applying a magnetic field. For example, as an anisotropic composite, an MNP-based DDS formulation, such as micelles, liposomes, and macromolecular assemblies, has confirmed benefit in encapsulating/solubilizing

13.4 Metal
Ligand Complexes as a Composite Anticancer Drug

therapeutic cargos, extensively prolonging the circulation lifetimes of drugs (Lee et al., 2015; Carregal-Romero et al., 2015; Baek et al., 2015; Mou et al., 2015). Their efficacy has been proposed for a broad range of biological applications, including cancer chemotherapy, tissue repair, hyperthermal therapy, or MR imaging contrast enhancement (Lee et al., 2015; Carregal-Romero et al., 2015; Baek et al., 2015; Mou et al., 2015). However, such systems have rarely been applied clinically because of their inherent limitations, as follow. (1) The synthesis of MNPs with desirable magnetic property for a TDDS platform demands copious steps to manipulate the NP structure (core and diameter), anisotropy, chemical composition, and surface coating (Lee et al., 2015; Carregal-Romero et al., 2015; Baek et al., 2015; Mou et al., 2015). (2) Substantial attention is required to avoid adverse outcomes in vivo, that is, an excessive dose of MNPs can trigger oxidative stress-induced cell damage (Naqvi et al., 2010; Soenen et al., 2012), and gradual degradation can result in accumulation in the liver and other organs (Mou et al., 2015; Wang et al., 2010) potentially leading to fatal effects such as cardiovascular injury, blood clots, and hypersensitivity. (3) Instability of micelle formulation: ionic/nonionic conjugation with tethering between drug molecules and nanocarrier materials may be readily dissociated, and micelles can be heat-denatured or lost during systemic circulation in vivo (Cong et al., 2016). (4) A major geometrical obstacle in DDS construction is that MNPs themselves are typically bulky, so that coloading of drugs is spatially constrained, and encapsulation efficiency can be low. Also, the nanocarrier itself displays limited morphological tunability and poor drug loading (, 20%) due to the high proportion of carrier materials, for example, polymers, required for efficient drug loading (Mou et al., 2015; Luo et al., 2014). To address these problems, we require a single low-molecular-weight drug component with multifunctional capabilities. On the other hand, if a drug compound is inherently magnetic, most of these problems may be solved because no inorganic metal-based magnetic carriers, including MNPs, would be required, as illustrated in Fig. 13.3B. It can reduce the

FIGURE 13.3 Comparative illustration of magnetic drug-delivery systems. (A) Conventional method of coloading magnetic particles and drugs with a carrier; (B) intrinsic magnetic drug-based (magnetic particle-free) method.

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size of the loading portion that magnetic components occupy, and, eventually, increase drug loading capacity. For instance, relatively less bulky and lighter systems than NPs, such as metal complexes, can be promising candidates for this purpose, as they themselves are not only small, but can be therapeutically active also. However, to the best of our knowledge, there are no anticancer magnetic metal complex systems, and some systems with optical properties were studied previously (Renfrew, 2014; Cong et al., 2016).

13.4.4 MOLECULAR MAGNETIC IRON COMPLEX FOR MAGNETO-DDS 13.4.4.1 Synthesis of iron salen In the last few decades, refined design of molecular catalysts for medical and industrial applications has been a long-standing goal in the field of catalytic chemistry. For instance, in 1969, N,N0 -bis(salicylidene)ethylenediamine, or salen, was employed as a ligand for iron complexation, and mainly studied as an intermediate catalyst for asymmetric chemical synthetic procedures (Venkataramanan et al., 2005; Cozzi, 2004; Ford and Wecksler, 2005; Murray, 1974). As exemplified by chloro(N,N0 -ethylenebis(salicylideneaminato))iron ([Fe(Salen)]Cl), this metal salen derivative is now considered as a potential anticancer compound (Wani et al., 2016). The synthetic technique of iron salen has some advantages over other preparation methods using toxic heavy metals (e.g., Co, Ni, Cr, etc.), due to the reduced environmental contamination during synthetic procedures. Also, the process is economically cost-effective since the iron excess can be recoverable from the chamber, without producing liquid wastes. The magnetic property of such metal salen derivatives, possessing a Schiffbase complex, has been intensively investigated due to its unique molecular magnetic ligand complex structure (Herchel et al., 2009; Jancso´ et al., 2005). However, it is impossible for them to be practically applicable due to the very low critical temperature, at which they exhibit positive values of magnetization in response to a room temperature magnetic field (Gatteschi et al., 2006, 2012; Hao et al., 2015). A fundamental magnetic salen complex system can be produced by emerging a transition metal (M) atom (i.e., M 5 Mn, Cr, Fe) with a salen ligand (1:1 ratio), as presented in Fig. 13.3. While the metal complexes are a single inorganic
organic unit system as a primary composite, an oxidized form via M-O-M configuration (μ-oxo) can lead to the creation of a dimeric system, μ-oxo N,N0 - bis (salicylidene)ethylenediamine iron [referred to as diFe(Salen)] (Fig. 13.3C), in which their magnetic property can be manipulated by altering the geometric parameters (e.g., angle, length, direction, etc.). The magnetic features of the magnetic ion
ligand
magnetic ion angle interactions are usually explicated by a superexchange in terms of the so-called “Goodenough
Kanamori
Anderson” rules (Ferlay et al., 1995; Anderson, 1963; Goodenough, 1963). Based on these rules, a 180 degrees superexchange (the angle of M-O-M is 180 degrees) of two magnetic

13.4 Metal
Ligand Complexes as a Composite Anticancer Drug

FIGURE 13.4 Synthetic scheme of (A
C) metal
ligand complexes (primary composites) and (D) a simple self-assembly (secondary composites) by Fe(Salen) building blocks.

ions with partially filled d shells is strongly antiferromagnetic, while a 90 degrees superexchange interaction is ferromagnetic. The nature of metal complex molecules is usually hydrophobic and majorly dissolved in nonpolar organic solvents (e.g., dichloromethane, chloroform, methanol, etc.); in an aqueous solution, they can form more extended (larger) structures as secondary composites by taking a self-assembly or aggregation process (Fig. 13.4D).

13.4.4.2 Design of magnetic iron salen Recently, a medical-industrial interdisciplinary research team at Yokohama City University (YCU) School of Medicine and IHI Inc. in Japan, tackled a question and challenge as follows: “Can we design metal complex as a single drug agent that inherently possesses multiple functions such as chemotherapeutic activity and magnetic property?” They have claimed that various metal complexes can be designed to become anticancer-active and can be engineered as a magnetic metal complex (MMC) (Ishikawa and Eguchi, 2012). The conceptual material design of MMC addresses the abovementioned issues, with the object of realizing a magneto-DDS which is capable of solving conventional technical problems and is easy to put into practical clinical applications. The MMC structure comprises an organic and an inorganic compound: the inorganic component can be a divalent metallic element composed of Fe, Cr, Mn, Co, Ni, Mo, Ru, Rh, Pd, W, Re, Os, Ir, Pt, Nd, Sm, Eu, or Gd, while the organic entity includes a metal chelating ligand

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such as salen (an anticancer agent) or forskolin (a composition effective in the treatment of male erectile dysfunction) (Sugawara et al., 2011). The MMC can be intrinsically magnetic by engineering molecular coordination of a metal-ligand structure (Ishikawa and Eguchi, 2012). A mathematical molecular simulation having modified side chains and/or crosslinked side chains can be used to determine the magnetic properties (e.g., ferromagnetic or antiferromagnetic) from a spin-charge density distribution with magnetic spin states (S) obtained by a numerical calculation for the molecular model (Ishikawa and Eguchi, 2012). In principle, since drugs themselves behave ferromagnetically, it is possible to guide the drugs to a predetermined target site in the body using an external magnetic field without using additional supports made from magnetic bodies as in conventional cases. As a result, conventional problems, such as difficulties in oral administration, the large size of carrier molecules in general, or technical problems in bond strength and affinity with the drug molecules can be resolved. Furthermore, it is possible to realize a magneto-DDS, which is easy to address the limits of existing MNP-based DDS described above. N.M. Gallagher et al. reviewed organic molecules with high-spin ground states (S $ 1), which creates room temperature magnetism from polymeric materials with more parallel spins than conventional metal magnetic substances (Gallagher et al., 2015). Ideally, molecules with strong paramagnetic properties should not only increase the sensitivity of organic paramagnetic relaxation reagents, such as magnetic resonance imaging (MRI) contrast agents (Davis et al., 2011; Rajca et al., 2012), but also are critical fundamental building blocks for the future development of spintronics (Sanvito, 2011; Sugawara et al., 2011). However, some challenging problems in real-world high-spin research are how to synthesize stable organic magnets at room temperature, as well as how to validate theoretical models for ferromagnetic coupling strength in very large (Sc1) systems. Eguchi et al. studied the theoretical relationship between the magnetic property of dimeric μ-oxo Fe (Salen) and the chemical structure in silico by first principles calculations (Ishikawa and Eguchi, 2014). They have also demonstrated that the diFe(Salen) shows both cytotoxicity and magnetization, which was useful in diverse animal cancer models for magnet-guided drug delivery and visualization of the accumulated drug by MR imaging (Eguchi et al., 2015). These aspects are discussed in detail in the following sections.

13.4.4.3 Theoretical investigation of anticancer iron salen by first principles calculations The first principles calculations (Segall et al., 2002) have been widely used as a potent simulation tool to define metal nanostructures in a wide range of fields from condensed matter physics to aerospace industry (Drebov et al., 2013). For example, this approach is to identify a metal structure among various candidate materials, following by final selection of a material with specific physical function to assign a spacecraft assembly (Lan et al., 2014). This process is similar to a common drug screening procedure. It tracks for a drug compound(s) in a large

13.4 Metal
Ligand Complexes as a Composite Anticancer Drug

compound library through virtual-docking computational simulation, and thus, the selected compound may have the desired pharmacological function for an aimed at medical therapy. In this regard, a biomedical engineer can apply it to drug screening, leading to identify a therapeutic compound. Therefore, such a crossindustrial use of a screening approach can be beneficial to design a drug with a novel property in translatable pharmacological research, for example, an anticancer agent with magnetism. To verify the molecular magnetism of diFe(Salen), Eguchi et al. used computational techniques such as generalized gradient approximation (GGA) 1 U (Chu et al., 2012) and the CASTEP program package (Segall et al., 2002) by the experimental data of Fe complex crystallography (Eguchi et al., 2015). The energy gap between the ferromagnetic ordering and two different antiferromagnetic orderings of the Fe spins in the four Fe atom unit cell was identified. The calculated energy values indicated that ferromagnetism was favored in the complex structure (Eguchi et al., 2015). Based on Perdew
Burke
Ernzerhof (PBE)-GGA calculations that were conducted using a linearized augmented planewave method (Ishikawa and Eguchi, 2012), an Fe atom is present as a high-spin Fe21, though with substantial covalency involving the ligands, indicating a hypoligated complex (Eguchi et al., 2015). This finding is correlated with the cyclic voltammogram analyses (Eguchi et al., 2015), demonstrating that the oxidation potential of diFe(Salen) was greater than that of the ferrocene (a standard) redox potential (Eguchi et al., 2015), confirming that native Fe(Salen) per se was in the divalent state, at least in its electrochemical behavior. Interestingly, the geometrical distance between Fe-O-Fe was relatively short and implied a trivalent interaction. The evident difference in valence state is almost certainly due to the nature of the hypoligated complex, which possesses partly covalent bonding and also unpaired electrons in its structure, as has been described in various complexes (Eguchi et al., 2015). We discovered that the structure of diFe(Salen) has a large covalent element based on the first-principles studies and this fact was also reinforced by X-ray crystallographic analysis. The calculated spin moment of the compound was 4 μB per Fe atom, which was the expected value for fully polarized high-spin Fe21. This high spin moment, together with the covalent bonding, results in an extremely strong spin-dependent hybridization of the Fe 3d orbitals with the adjacent ligand (O and N) p orbitals. Hybridization with the ligands is strong in the majority spin channel, leading to a substantial magnetic polarization on the N and O atoms that are bonded to Fe, which is parallel to the Fe moments. Substantially induced polarizations at other atoms of the salen moieties were also parallel to the Fe moments. Moreover, the polarization of N and O that are bound to Fe atom is followed by polarization of C in the salen units. This occurs in a pattern, where a substantial polarization on every second C but not on the intervening atom lies, and hence it allows all of these magnetic polarizations to be parallel to the Fe moments. This atomic alternation with and without moments is a common feature of structures built from sp2-hybridized C and is also represented in long-range ferromagnetic interactions in graphene (Pisani et al., 2008). These

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mathematical analyses reveal that the magnetic moments are distributed over the salen units by the Fe-induced spin-dependent sequential hybridization of N, O, and C, and the magnetic moment is not simply localized at the Fe ions. The details of the intermolecular exchange interactions would depend on the completely defined structure and configuration of the hypoligated diFe(Salen), including the H positions, which must be confirmed in the future.

13.4.4.4 Crystallographic analysis It is very unusual but exciting that an organometallic complex is magnetic above room temperature. To better understand the structural foundation of this finding, Eguchi et al. performed single crystal X-ray diffraction analysis of diFe (Salen) (Table 13.1) (Eguchi et al., 2015) followed by a synchrotron accelerator-based X-ray structural analysis. The result revealed that the diFe (Salen) compounds have a crystalline structure, showing that two Fe centers of iron-salen units are bonded by a single O atom (Fig. 13.4A and C)—dimeric Fe (Salen) or [Fe(salen)]2O as a strict designation. It is presumed that monomeric Fe(Salen) was oxidized in air to give dimers as reported in the literature (Murray, 1974). Remarkably, the angle of Fe-O-Fe was determined as 146.359 degrees, which is theoretically consistent with a ferromagnetic interaction, which also agrees with the classic Goodenough
Kanamori
Anderson rule (Anderson, 1963; Goodenough, 1963). This is also associated with common magnetic interactions of a transition metal ion, such as Fe or Ti, through O, which implies the Fe-O-Fe angle structure plays an important role in producing magnetism in the diFe(Salen) molecule. The diFe(Salen) powder is poorly dissolved in water and is often shown as sharp and irregular particulates with size of a few hundred nanometers (mesoscopic) to a few micrometers, as shown in TEM imaging (Fig. 13.5E), whereas sonicated particles displayed more smoothened and thinner morphology (Fig. 13.5F) (Eguchi et al., 2015). As the particle size is reduced, the edges of the particles were smoothened compared with those of the unsonicated particles.

13.4.4.5 Purity analysis Based on the elemental analysis of diFe(Salen) powder, the experimental values were close to the expected ones: C (58.21 vs 57.73), H (4.27 vs 4.42), Fe (16.92 vs 17.2), and N (8.49 vs 8.49). As shown in Table 13.2, there is no substantial impurity in the sample compound, showing high purity ( . 95%) (Eguchi et al., Table 13.1 Crystallographic Parameters of Dimeric μ-oxo Fe(Salen) Compound Compound

Crystal System

Fe2C32H28O5N4

Triclinic

2 ( )

nm A 1.748

b 1.76

c 1.3768

α 66.49

β 81.10

γ 73.12

13.4 Metal
Ligand Complexes as a Composite Anticancer Drug

Table 13.2 Impurity Analysis of diFe(Salen) DiFe(II)Salen Li Be B Na Mg Al K Ca Sc Ti V Cr Mn Co Ni Cu Zn Ga Ge As Rb Sr Y Zr Nb Mo Ru Rh Pd Ag Cd In

,0.0001 ,0.0001 a

0.004 0.0004 0.002 ,0.0001 0.0002 ,0.0001 ,0.0001 ,0.0002 ,0.0001 a

0.001 0.002 0.0001 0.0002 ,0.0001 ,0.0005 a

,0.0001 ,0.0001 ,0.0001 0.0001 ,0.0001 ,0.0001 ,0.0001 ,0.0001 ,0.0001 ,0.0001 ,0.0001 ,0.0001

DiFe(II)Salen Sn Sb Te Cs Ba La Ce Pr Nd Sm Eu Gd Tb Dy Ho Er Tm Yb Lu Hf Ta W Re Ir Pt Au Tl Pb Bi Th U

,0.0001 ,0.0001 ,0.0001 ,0.0001 ,0.0001 ,0.0001 ,0.0001 ,0.0001 ,0.0001 ,0.0001 ,0.0001 ,0.0001 ,0.0001 ,0.0001 ,0.0001 ,0.0001 ,0.0001 ,0.0001 ,0.0001 ,0.0001 ,0.0001 ,0.0001 ,0.0001 ,0.0001 ,0.0001 ,0.0001 ,0.0001 ,0.0001 ,0.0001 ,0.0001 ,0.0001

The contents of F, Cl, S, and Br were below the limit of detection. a Indicates values impossible to measure: B and As evaporated during measurement, and Mn was not measured due to the interference of other coexisting elements.

2015). On FT-IR spectral analysis, diFe(Salen) particles showed the IR bands (neat) as follow: 1619, 1384, 1336, and 1302 cm21. Inductively coupled plasma (ICP) mass spectroscopy and X-ray fluorescence analysis did not detect the presence of any metals, other than iron, which could account for the exclusive magnetism in the diFe(Salen) samples.

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13.4.4.6 Anticancer properties The development of therapeutic iron complexes is currently an immature arena of investigation and more efforts are required to have suitable insights into a realistic basis for the effects of the molecular structure of iron complexes on their biological targets and the resulting biological properties, for example, anticancer activity. Thus, it is urgent to uncover for their molecular behavior to optimize their cytotoxic effects. Eguchi et al. confirmed that some metal salen derivatives, such as N,N0 -bis (salicylidene) ethylenediamine chromium (Cr-salen) exhibited potent anticancer properties similarly to cisplatin, while N,N0 -bis(salicylidene) ethylenediaminemanganese (Mn-salen) did not show any cytotoxic activity (Eguchi et al., 2015). In turn, the diFe(Salen) NPs exhibited induced substantial cellular apoptosis in MAT-Lu prostate cancer cells, similarly cytotoxic to cisplatin (Eguchi et al., 2015). However, its apoptosis-inducing efficacy was varied among other cell types, such as melanoma (Clone M3), squamous cell carcinoma (VX2), and osteosarcoma cells (POS-1). It also exhibited an antiproliferative effect in a notably reduced degree over healthy cells, such as fibroblasts, and rat and mouse smooth muscle cell (Eguchi et al., 2015). In particular, the sonicated compounds showed more reactive oxygen species (ROS) generation in MAT-Lu prostate cancer cells. The ROS generation effect of sonicated Fe(salen) was greater on cancer cells than normal cells (rat aorta smooth muscle cells) (Eguchi et al., 2015). Consequently, ROS may play a significant role in inducing cytotoxicity of diFe(Salen). Due to the poor water-solubility of the iron-salen NP samples, the NPs were suspended in saline after sonication. Transmission electron microscopy (TEM) showed that sonication for 6 hours allowed particle size to be reduced, with diminished density, and the edges of the NPs were smoothened, compared with those of the unsonicated ones (Fig. 13.5E and F) (Eguchi et al., 2015). Based on the size distribution of the colloidal suspension of diFe(Salen) NPs using dynamic light-scattering measurements, the unsonicated and sonicated NPs presented size distributions from 1.2
3 μm and 60
800 nm, respectively, in good agreement with the TEM results (Eguchi et al., 2015). Zeta potential measurements of the diFe(Salen) NPs revealed a zeta potential value of
24.1 mV, indicating a stable colloidal dispersion (Eguchi et al., 2015). To validate the rational mechanism of cytotoxicity of diFe(Salen) NPs, firstly, the cell uptake event of diFe(Salen) NPs was examined by TEM, as well as energy-dispersive X ray spectroscopy (EDS) (Eguchi et al., 2015). TEM confirmed that a substantial amount of diFe(Salen) NPs were located within VX2 cells, followed by the elemental analysis via EDS which confirmed that such NPs were attributed from diFe(Salen) (Eguchi et al., 2015). It is commonly believed that Fe ion, either divalent or trivalent, can react with H2O2 to produce ROS via the Fenton or Fenton-like reaction, respectively (Inoue and Kawanishi, 1987; De Laat and Le, 2006). Because the diFe(Salen) NPs contain iron, this is a key factor that can lead to ROS production and potentially cytotoxicity. Taking into consider

13.4 Metal
Ligand Complexes as a Composite Anticancer Drug

FIGURE 13.5 Structural characterizations of dimeric μ-oxo Fe(Salen) composite. (A) Chemical molecular structure, (B) projections of the spin-polarized density of states onto Fe d orbitals within the LAPW sphere radius of 1.6 bohr. The results of PBE GGA calculations with relaxed atomic positions on a per-atom basis are shown. The majority spin is shown above the axis, and the minority is shown below. Note the broader majority spin features reflecting strong spin-dependent hybridization. (C) ORTEP-crystal structure of diFe(Salen). Atomic anisotropic B-factors for nonhydrogen atoms are shown as ellipsoids. Blue, red, purple, and orange atoms represent carbon, oxygen, nitrogen, and iron, respectively. Hydrogen atoms were omitted for clarity. (D) Three-dimensional simulation model. Size-controlled diFe(Salen) nanocrystallines by TEM analyses of (E) unsonicated and (F) sonicated diFe (Salen) samples, a photo of associated sample solution and the estimated structural model. Adapted from Eguchi, H., Umemura, M., Kurotani, R., Fukumura, H., Sato, I., Kim, J.-H., et al., 2015. A magnetic anti-cancer compound for magnet-guided delivery and magnetic resonance imaging. Sci. Rep. 9194. Copyright 2015 by the Nature Publishing Group.

the fact that sonicated fine diFe(Salen) NPs displayed greater cytotoxicity than unsonicated NPs, it is presumed that the production of ROS was increased as the NP size became smaller by sonication. This seems reasonable because the fine diFe(Salen) NPs would have a larger surface area and be more diffusive when the large particles are broken into smaller ones after sonication treatment. Moreover, the ROS activity of diFe(Salen) NPs was greater on cancer cells than normal cells, in good agreement with the previous investigations that tumor cells usually produce larger amounts of hydrogen peroxide than normal cells (Manda et al., 2009) in cellular mitochondria and peroxisomes (Bai et al., 1999). The mechanism of diFe(Salen) NP-induced ROS production was investigated by cyclic voltammetry (CV) (Eguchi et al., 2015) (Fig. 13.6). The oxidization

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FIGURE 13.6 Cyclic voltammogram (CV) of diFe21(Salen). Redox ROS validation by CV (A). CV of (i) 0.1 M TBAP in ACN (black line), (ii) 1 mM H2O2 in 0.1 M TBAP in ACN (red line), (iii) 1 mM diFe21(Salen)0.5H2O in 1 mM H2O2 and 0.1 M TBAP in ACN (blue line) (scan rate 5 50 mV/s). (B) CV of 1.4 mM diFe21(Salen) in 0.1 mM TBAP ACN solution (scan rate 5 50 mV/s), showing a nonsymmetrical redox peak as a standard Fe(Salen) peak. Adapted from Eguchi, H., Umemura, M., Kurotani, R., Fukumura, H., Sato, I., Kim, J.-H., et al., 2015. A magnetic anti-cancer compound for magnet-guided delivery and magnetic resonance imaging. Sci. Rep. 9194. Copyright 2015 by the Nature Publishing Group.

current of B140 μA at 1.2 V versus Ag/Ag1 was detected (red line) when H2O2 was added into acetonitrile (CAN) medium with 0.1 M TBAP as an electrolyte, while the oxidization current was immediately decreased when Fe21(Salen) was added (blue line). It is of note that the reduced current is identical with monomeric Fe21(Salen) alone, instead of dimeric Fe(Salen) as an oxidixed form in a powder state. This decrease was attributed to the depletion of H2O2 via Fe21, for example, Fenton reaction, as shown in reaction (13.1). In the same way, the reduction current peak of H2O2 at a potential below
0.6 V versus Ag/Ag1 was observed (B on red line), which the current (B) also decreased (blue line). Fe21 1 H2 O2 -Fe31 1 OH2 1  OH

(13.1)

CV of the control buffer solution (black line) showed mostly zero current. When H2O2 was added, H2O2 was readily decomposed at the electrode, as shown in the oxidation process (reaction (13.2)), yielding a large oxidization current (A on red line). H2 O2 -O2 1 2H1 1 2e2

(13.2)

The large oxidization current was also decreased (A on blue line) when diFe21(Salen) was added, reflecting that the H2O2 was mostly consumed by the Fenton reaction, and the resultant Fe31(Salen) was produced as the current decreased. This current also mimics that with Fe21(Salen) alone, with the same peak current value (approximately 20 μA).

13.4 Metal
Ligand Complexes as a Composite Anticancer Drug

The consumption of H2O2 was also reflected by a decrease in the reduction current (B on blue line versus red line). H2O2 was decomposed at the electrode, producing the reduction current (reaction (13.3)), indicating that this reduction current was also decreased as the amount of H2O2 decreased. H2 O2 1 e- -1=2H2 1 HO2 2

(13.3)

As shown in Fig. 13.6B, a nonsymmetrical peak current was observed, which indicates a native monomeric Fe(Salen). Accordingly, Fe(Salen) was oxidized at the upward curve at 10.5 V versus Ag/Ag1. Because this oxidation potential of Fe(Salen) (0.5 V) was greater than a ferrocene (standard) redox potential, it was found that Fe(Salen) is in the divalent state at an initial potential of 0 V. Note that the oxidation peak, that is, the upward peak of Fe(Salen), was much broader than that of ferrocene, indicating that the Fe(Salen) was oxidized in a dissimilar fashion from ferrocene. Fe(Salen) was most likely decomposed after the oxidization process because the reduction peak (downward peak) disappeared in the Fe (Salen) sample.

13.4.4.7 Magnetic property One of the most beneficial assets of a magnetic anticancer agent is its inherent magnetism, allowing us to magnetically guide/deliver drugs to a desirable disease location. Not only are they capable of targeting specifically, but also they would inhibit possible resistance development due to long-term exposure of anticancer agents (because the drugs on treated site and can be magnetically moved or removed). The first principles calculation-based in silico analysis was found to be implicated in the presence of magnetisms in diFe(Salen) (Eguchi et al., 2015): the diFe(Salen) particles were able to be rapidly attracted by a rare earth magnet in air. The movement of crystalline particles of the diFe(Salen) particles can also be observed under a microscope (Eguchi et al., 2015): similar-sized particles with differently shaped grains were completely drawn to the magnet with a similar rate under microscopic monitoring. Consequently, the magnetic property does not depend upon the shape of particles. Based on an observation of the attachment on a magnet in a simulated blood (water) flow, the particles were accumulated at the edges of a permanent magnet, where the calculated magnetic field was maximized. However, when the grain size of metal-salen complex NPs is too small, that is, dissolved in an organic solvent, the required magnetism to draw the NPs cannot be employed. On the other hand, when the grain size of the metal-salen complex is large, that is, dissolved in water, the metal-salen complex cannot readily pass through blood capillaries. Thus, it is important that a process control (e.g., ultrasonication) to achieve desirable grain size, and to be applicable for a living body system without clogging the capillaries, as well as guidable ability to a target region with a magnetic field. To further characterize the magnetic properties, magnetization versus magnetic field curves using a superconducting quantum interference device (SQUID) system, that is, a standard technique to identify magnetism, was performed

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(Eguchi et al., 2015). Magnetization versus magnetic field plots from 5 K to 310 K revealed that the compound presented as magnetically active (ferromagnetic) with a hysteresis loop (Fig. 13.7A). The magnetization of this diFe(Salen) NP sample was found to be stable in air, as well as in solution, and remained unaffected for at least 3 years. Combined with the chemical purity analyses, these results illustrated that the iron-salen nanoparticle sample is inherently magnetic! Therefore, visualization of drug accumulation was able to be conducted using magnetic resonance (MR) imaging. Due to its ferromagnetism, diFe(Salen) NPs can be visualized by MR imaging. An in vitro sample of the diFe(Salen) NPs exhibited a concentration-dependent (0
1.94 mM) negative signal alteration on a T2-weighted image (Fig. 13.7B), indicating that this anticancer compound has the capability for clinical MRI visualization. It also demonstrated that slight longitudinal relaxation rate increment and moderate transverse relaxation rate were enhancement determined quantitatively, showing a linearity at 0.12
0.97 mM (Fig. 13.7C). To examine MR imaging of the diFe(Salen) NPs after magnetguided drug delivery in mouse, the diFe(Salen) NPs were intravenously administered to a melanoma-grafted model on the tails and a stationary magnet was put on the side of half of the tumor (Fig. 13.7D). The stationary magnet induced signal reduction at the side of the tumor on T2 -weighted MRI implying local accumulation of Fe(Salen) NPs (Fig. 13.7E). The diFe(Salen) dispersion was injected into the proximal tail vein of the mice, and a magnet was applied to the distal tail site where the melanoma was grafted; this was to avoid the immediate trapping of diFe(Salen) NPs by the magnet after injection. The magnet was applied to the edge of the expected tumor growth area on the tail, and then swept multiple times, thus the magnetic guidance was able to spread over an extended tail region. Efficient accumulation of diFe(Salen) NPs was confirmed by chemical staining (green color) of Fe(Salen) NPs in tail tissues (Fig. 13.7F). The melanoma in mice was dramatically reduced (9.1% 6 3.4%) in the presence of diFe(Salen) NPs, in response to the AMF irradiation (n 5 6
10 per group), confirming its anticancer effect in vivo, whereas the melanoma extension was worst in the control group (100% 6 17.2%) (Fig. 13.7G).

13.4.4.8 Cancer hyperthermia Hyperthermia is a promising cancer therapy technique, in which cancer tissues can be targeted when exposed to a slightly raised temperature (42 C
45 C), thus increasing their susceptibility to radio- or chemotherapy (Radiofrequency, 1984; Suit and Shwayder, 1974). For instance, capacitive heating with a radiofrequency electric field is the most commonly used in clinical practice (Abe et al., 1986; Oura et al., 2007). However, a main practical drawback of this method is difficulty in heating the target tumor site to the desired temperature without damaging the surrounding healthy tissues: an electromagnetic field wave from an external source must penetrate through normal tissues, leading to cell ablation via an inevitable temperature gradient. In spite of the advance of hyperthermia modalities, including radiofrequency ablation and ultrasound hyperthermia

13.4 Metal
Ligand Complexes as a Composite Anticancer Drug

FIGURE 13.7 Magnetic characterization and medical performances of di(FeSalen) nanoparticles. Magnetic susceptibility analysis using SQUID (A). MR imaging of in vitro (B, C) and in vivo settings. (D
G), T1- and T2-weighted images (B). Quantitative proton longitudinal (R1) and transverse relaxation rate (R2) (C), MR imaging in vivo study: a scheme of magneto application to half of the melanoma tumor is displayed (D). MR imaging was performed placing a permanent magnet (630 mT, surface magnetic flux density) in contact with half of the tumor for 3 h, Sagittal slices of T2 -weighted MR images at the tail site were achieved (T2 ) (E). On the diFe(Salen) NP administration with magnet application, signal reduction at half of the tumor on T2 -weighted MRI was detected. The scheme illustrates the sliding method with a stationary magnet along the mouse tails (F): by locally placing the magnet, the AMF was applied to the edge of the expected melanoma growth area for 10 min. The magnet was slid 5 mm along the tail (arrows) and the AMF equally applied for 10 min. The magnet sliding was repeated five times over the length of 30 mm for 3 h after each injection. Note that the magnet was moved by 5 mm at each sliding because the AMF generated by this magnet was strongest within 5 mm of its edge. Corresponding photos of mouse tails with melanoma pigmentation (G). Adapted from Eguchi, H., Umemura, M., Kurotani, R., Fukumura, H., Sato, I., Kim, J.-H., et al., 2015. A magnetic anti-cancer compound for magnet-guided delivery and magnetic resonance imaging. Sci. Rep. 9194. Copyright 2015 by the Nature Publishing Group.

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(Oura et al., 2007; Mardynskiı˘ et al., 2007), the current efficacy is limited to the tumor size and depth, resulting in a failure to precisely target a specific tumor site by focusing heat exposure. Nowadays, 48% of tumors are located in the oral cavity, and 90% of these are oral squamous cell carcinoma. Oral malignancy, including tongue cancer, is associated with severe sickness that has a long-term survival rate of less than 50%, in spite of advances in the treatment of oral cancer by surgery, chemotherapy, and radiation. The survival ratio of patients remains very low, mainly due to lymph node metastasis. Surgical elimination of the cancer tissues is the gold standard, but involves numerous complications, such as dysphagia or dysarthria. Therefore, a more effective treatment for oral cancer, with fewer complications, needs to be established. MNPs produce heat when exposed to an alternating magnetic field (AMF) as a result of hysteresis and relaxation losses, leading to heat production (Chatterjee et al., 2011; Jordan et al., 1993; Thiesen and Jordan, 2008; Qun Zhao et al., 2012). By guidance with a magnet, MNPs can be directed at the tumor site, and subjected to AMF exposure to generate heat within the tumor, enabling local thermal ablation in the target site. MNPs and anticancer drugs can be accompanied by a nanocarrier formulation such as micelles, and the drugs may be rendered therapeutically ineffective by heat-induced degradation. As discussed in the previous sections, diFe(Salen) NPs showed both stable magnetic and anticancer properties when exposed to an AMF. The magnetic measurement by SQUID suggested that diFe(Salen) NPs could produce thermal energy due to hysteresis losses when exposed to an AMF. Recently, Sato et al. demonstrated that the threefold approach of anticancer chemotherapy with diFe(Salen) NPs, magnetically guided delivery of the nanoparticles to the tumor, and AMF-induced heating of the nanoparticles to induce local hyperthermia exhibited a potent anticancer effect in a rabbit tongue cancer model in vivo (Sato et al., 2016). Because tongue cancer cells are squamous in nature, various squamous cancer cell lines were cultured, including VX2 (rabbit), HSC-3 (human), and OSC-19 (human), followed by investigating the mechanism of cell death and the cellular uptake of diFe(Salen) particles. The diFe(Salen) NPs exhibited potent, dose-dependent anticancer effects on these cell types. The IC50 values were similar among these cell types (approximately 7 μM). Remarkably, the diFe(Salen) NP sample allowed substantial heat generation by an AMF with a vertical coil, driven by a transistor inverter. In the dry powder state, the temperature of an AMF-exposed diFe(Salen) NP sample rose to . 80 C within a few minutes (Fig. 13.8A and B), in contrast to the cisplatin sample that tiggered no heat generation (Fig. 13.8B). In the wet dispersion state, diFe(Salen) NP suspension in culture medium illustrated that the temperature increment was less than the dry mode, because of heat loss through the liquid medium, and the temperature increase was both electric current (EC)-dependent and concentration-dependent (Fig. 13.8C). Local injection of diFe(Salen) NPs significantly reduced the tumor volume (223% 6 80.6%) on a mouse leg in a timely manner, whereas AMF

13.4 Metal
Ligand Complexes as a Composite Anticancer Drug

FIGURE 13.8 diFe(Salen) NP-mediated heat generation under AMF irradiation. Thermography of diFe (Salen) nanoparticle dry powder in a tube before (Pre) and 5 min after AMF exposure (5 min) (A). Heat production by cisplatin or diFe(Salen) upon AMF exposure (B). The AMF was applied at a frequency of 308 kHz and EC 250 A. The effect of diFe(Salen) concentration (15 or 30 μM) and electrical current (200, 250, or 300 A) on the temperature in the culture medium (C). Effect of diFe(Salen) nanoparticle injection and AMF exposure on tumor size (D, E). The photo and graph show the tumor volume changes over time. diFe(Salen) inhibited tumor growth, while AMF exposure of diFe (Salen)-injected mice further inhibited the tumor growth. In vivo fluorescence imagingbased observation of effect of Fe(Salen) nanoparticle injection and AMP exposure over tumor size, determined by IVIS (F). Photos of an oral cancer model (rabbit tongue tumors) in each group before (upper) and after (lower) AMF treatment in the presence/absence of diFe(Salen) (G). The mean tumor volume (mm3) of each group is also presented. Control (cont), intravenous injection of diFe(Salen) nanoparticles (i.v.), diFe(Salen) nanoparticle injection and electromagnet application (i.v. 1 DDS), and diFe(Salen) nanoparticle injection, electromagnet application, and AMF exposure (i.v. 1 DDS 1 AMF). Adapted from Sato, I., Umemura, M., Mitsudo, K., Fukumura, H., Kim, J.-H., Hoshino, Y., et al., 2016. Simultaneous hyperthermia-chemotherapy with controlled drug delivery using single-drug nanoparticles. Sci. Rep. 6:24629. Copyright 2015 by the Nature Publishing Group.

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exposure in the absence of the particles had no effect on the tumor volume (Fig. 13.8D
F). It is notable that a high drug dose for in vivo treatments was not required for successful results in either application of the electromagnet or AMF exposure. Moreover, the tongue is readily accessible within the oral cavity, and thus it is easy to apply a magnet and an AMF. In this study, an electromagnet was utilized instead of a permanent magnet, to enhance the hyperthermal induction in an oral cancer model, because a permanent magnet was difficult to apply onto the tongue surface for a long time. Intravenous injection (i.v.) of diFe(Salen) NPs (5 mg/L) every day for 7 days significantly reduced the tumor size (144.3% 6 51.3%: i.v. group). By applying an AMF through the electromagnet, the tongue tumor had almost vanished after 1 week (25.7% 6 8.4%: i.v. 1 EM 1 AMF group) (Fig. 13.8G). At this dose level, there was no increase in kidney or liver enzymes in any of the groups. As a comparative metal salen species, Cr-salen and Mn-salen also exhibited positive magnetization, however, they did not show hysteresis, suggesting that there was no possibility of magnetic heat generation in these materials (Eguchi et al., 2015). Additionally, the hyperthermal performance has also been demonstrated in a brain cancer model, for example, glioblastoma (Ohtake et al., 2017).

13.5 HYBRID METAL SALEN
POLYMER NANOCOMPOSITES AS NANO-DDS Although diFe(Salen) possesses exceptional functions in magnetic and anticancer properties, there are some drawbacks, such as its poor water solubility, limited processibility, and nonspecific binding/clogging with surface issues, due to its hydrophobic interface, which can further inhibit clinical advances. Very recently, to address these issues, Kim et al. (2016, 2017) developed a straightforward, onestep method to formulate magneto-drug vehicles by encapsulating diFe(Salen) using biodegradable smart block-copolymers [e.g., polycaprolactone (PCL)-polyethylene glycol (PEG) or PCL-b-polypyrrole (PPy)] (Fig. 13.9) (Kim et al., 2016, 2017). In principle, block copolymers can be designed as heterofunctional or bifunctional, in which they can convey more than one function, enabling highly functional tasks in response to external stimuli, such as heat, magnetic field, light, pH, molecules, and so on (Kim et al., 2016; Singh and Amiji, 2018). For example, in PCL-b-PPy, PCL polymers are heat-sensitive, while the PPy segment associates with electroconductivity or pH changes. Notably, these nanocomposite agents did not contain additional magnetite particles, as required in traditional methods, but utilized the enhanced magnetism of diFe(Salen), which also holds inherent antitumor activity. This novel synthetic method is a “green” process that does not require toxic solvents and reagents, and provided unique core
shell nanostructures with a high loading capacity (B90%). Moreover, by using natural biocoating agents such as bovine serum albumin or gum arabic, it enables core-size

13.5 Hybrid Metal Salen
Polymer Nanocomposites as Nano-DDS

FIGURE 13.9 Synthesis of “inorganic metal salt-free” magneto-nano-DDS by self-assembling molecular magnets [diFe(Salen)] and smart copolymers, which is responsive to multiple stimuli (magnetic field, heat, and pH), toward multifunctional theranostics, including magnetic smart drug delivery, MRI, and magneto-hyperthermic therapy. Adapted from Kim, J.-H., Eguchi, H., Umemura, M., Sato, I., Yamada, S., Hoshino, Y., et al., 2017. Magnetic metal-complex-conducting copolymer nanoassemblies as versatile single-drug anticancer nanoplatform. NPG Asia Mater. 9:e367. Copyright 2017 by the Nature Publishing Group.

tuning from multicores to single cores. The obtained hybrid composite NPs can simultaneously perform multiple tasks in theranostics—magnetically guided DDS, MRI contrast, magneto-hyperthermal effect, and triggered release of antitumoractive diFe(Salen) NPs, in response to multiple stimuli, for example, temperature, magnetic field, and pH. Distinguishing features of this interdisciplinary work included the simplicity and green method, fine control of the core-size transition, and biocompatibility, which will be appropriated for a wide-range of “minimally invasive” theranostic clinics, including targeted DDS, as noted above. Hence, it is proposed that the novel method for synthesizing smart magneto-DDS will open a new synthetic paradigm of DDS and be of interest to scientists working on applications of magnetic particles and nanocarriers in various biomedical fields. Ideally, unlimited opportunities are available for the composite approach of integrating the organic materials, for example, smart polymers, in the form of bio-active nanomaterials, whether synthetic or natural. Strategies to offer smart capabilities to the composite nanomaterials primarily pursue attaining matrices that are inductive to biological entities, for example, cells, or that stimulate/trigger target cell responses that are crucial in the tissue regeneration processes. Responsiveness to internal or external stimuli, including pH, temperature, ionic

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strength, and magnetism, is a promising means to improve the multifunctionality in smart scaffolds with on-demand delivery potential. These approaches will deliver the next-generation platforms for designing three-dimensional matrices and delivery systems for tissue-regenerative applications.

13.6 CONCLUSION It is obvious that the anticancer arena is shifting toward more rational approaches that pay attention to complications such as biological barriers that may constrain clinical translation. The discussion in this chapter has addressed the current barriers to conventional chemotherapy platforms and common magnetite-based local delivery methods. Although the distinct hurdles that hamper adequate delivery of therapeutics to tumors are indeed complex, there have been limitless and significant efforts to realize local (targeted) nanocarrier systems as a suitable anticancer strategy, and which potentially offer a solution to the severe side effects associated with direct drug delivery to tumor cells. In particular, a magnetic particlebased drug-delivery method could transform the way deep-tissue tumors and other diseases are targeted. As highlighted here, innovative strategy implementations, such as the design of nontraditional magnetic drug materials, including iron complexes containing salen ligand derivatives, have shown distinct advantages over preexisting conventional MNP platforms. Rather than the direct use of conventional drugs accompanyied by magnetites, novel diFe(Salen) NPs are bestowed with both potential anticancer properties and appropriate magnetism at room temperature, which do not need to include additional magnetic supports and common drugs. Therefore, diFe(Salen) NPs allow us to enable magnet-guided delivery, MR imaging, and hyperthermia, and thus improve anticancer efficacy, whereas they allow the required dose for chemotherapy to be decreased. Such anticancer therapeutic and imaging (theranostic) strategies could alter our concept of future pharmacotherapy and anticancer drug development. However, diFe(Salen) NPs also have some drawbacks, such as poor water-solubility, loss of magnetic property in solvents, and potential cytotoxicity when accumulated in organs. Therefore, to develop water-soluble and safe iron complex-based drugs for the treatment of all cancer types, a single-step fabrication method for a magnetic metal complex-based local DDS platform in response to multiple (magnetic, thermo, pH) stimuli, was demonstrated. This technology could enable a new therapeutic multimodality that combines spatial precision and local selectivity to target specific tumors, which could shift the synthetic paradigm to realize advanced clinical translations. In addition, due to their molecular ligand-controlled assembly in a programmable fashion, the design, synthesis, and structural characterization of metallonanocomposites would be extensively applicable in other fields, such as biosensing, single-molecule optical detection, bioinformatics, and heterogeneous

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14

Nanocoatings and thin films

Valentina Grumezescu1 and Irina Negut1,2 1

National Institute for Lasers, Plasma, and Radiation Physics, Magurele, Romania 2Faculty of Physics, University of Bucharest, Magurele, Romania

14.1 INTRODUCTION The development of deposition processes and materials has witnessed a surge in interest among researchers to apply coatings on different substrates and for many applications, as most of the interactions (mechanical, thermal, chemical, and electrochemical) of a material with/in an environment commence from its surface (De Hosson and Cavaleiro, 2006). For example, in the biomedical domain, the upper layer of an implant is responsible for the interaction with surrounding tissues (Chan et al., 2017). Consequently, the surface of a material is the most important part to be engineered. Although standards are constantly developing for production and manufacturing processes, surface adjustment technologies in the manufacturing process are unavoidable. Nowadays, the assembling of nanoparticles (NPs) onto surfaces is intensively studied with the purpose of obtaining functional nanocoatings (De Hosson and Cavaleiro, 2006). However, it is still a challenge to construct uniform nanocoatings on surfaces by means of micro- and/or nanostructures. For planar surfaces, many approaches and techniques have been advanced to fabricate nanocoatings with well-controlled thickness, such as dip-coating (Hurd and Brinker, 2011) and laser deposition (Limban et al., 2014). The use of any method that can guarantee the reproduction of features such as size, shape, as well as the chemical structure of initial nano- and microstructures onto solid substrates would be valuable for all application requests. Moreover, supplementary requests for nanostructured/thin films are thickness control, uniformity, adherence to the substrate surface, limitation in the use of dangerous chemicals, and high treating temperatures. Consequently, during recent years there has been growing interest in the advance of deposition techniques, optimized for the immobilization of nanomaterials onto diverse surfaces in the form of both nanostructures and thin films. The knowledge of the relationship between deposition/processing parameters and conditions, as well as material structural and chemical properties, is challenging for novel applications.

Materials for Biomedical Engineering: Inorganic Micro- and Nanostructures. DOI: https://doi.org/10.1016/B978-0-08-102814-8.00016-0 © 2019 Elsevier Inc. All rights reserved.

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This chapter discusses the basic outlines of some well-known coating processes, namely dip-coating, and laser deposition, with a special emphasis on materials and state-of-the-art applications. As the choice of deposition technique depends on the type of coating materials, desired surface morphology, and available budget, in the first section we refer to dip-coating as a low-cost process for obtaining different types of materials with different compositions. The section dedicated to laser deposition introduces examples of laser nanostructuring/processing of hybrid NPs, pointing out the importance of obtained thin films in biomedical domains, mostly for the control and prevention of microbial colonization.

14.2 NANOCOATING FABRICATION METHODS 14.2.1 DIP-COATING METHOD Among the available deposition techniques, dip-coating is the most widely applied for industrial and, in particular, laboratory applications which are essentially unfounded on the simple processing, the low cost, and the high coating quality. Nevertheless, other techniques like spin coating, spraying, or meniscus coating are practical for some applications (Feng et al., 2018; Balzarotti et al., 2017). The industrial origins of dip-coating trace back to the seminal works at Schott in the 1940s (Dislich and Hinz, 1982) and ever since the late 1950s it has been used in the production of automotive rear mirrors. The technological relevance of the dip-coating technique is indicated by several elaborations that have been presented in the meantime (Brinker et al., 1991). In recent years optical area coatings like antireflective (Guldin et al., 2013; Willey, 2016) and solar control glasses (Nielsen et al., 2014) have also been coated using this technique. Dip-coating represents a promising technique applied for obtaining nanocoatings and consists of three stages: dipping, withdrawing, and drying. This technique offers numerous advantages such as inexpensive setup, process simplicity, uniformity of deposition, low processing temperature, and the ability to coat complex shapes and patterns (Aksakal and Hanyaloglu, 2008; Grosso, 2011). In addition, wet chemical sol
gel processing, due to its adaptability and ease of liquid film deposition, becomes a beneficial technique for coating with a variety of inorganic and hybrid materials. In general, liquid film deposition (Dislich and Hinz, 1982; Yusoff et al., 2014), comprehends the use of a liquid precursor film on a substrate which then is transformed/adapted to the wanted coating material in a succeeding post-treatment step. For all thin-film processes, cleanliness and control of atmospheric parameters are essential to obtain high-quality films. Taking into account the above-mentioned, the dip-coating technique is demonstrated to be a reliable and powerful resource for the deposition of optical coatings.

14.2 Nanocoating Fabrication Methods

Additionally, the coating volume and thickness can be controlled by altering the concentration of suspension, the number of dips, and varying the withdrawal speed. Heat treatment of coated substrate is often required to densify the coating layer, to increase coating
implant bonding, and eliminate porosity (Xiao and Liu, 2006). Furthermore, thermal stresses originating from the difference in thermal coefficient between the ceramic coating and metallic implant result in the formation of microcracks and delamination of the coating from the substrate (Sridhar et al., 2003). The main advantage of the dip-coating technique in combination with sol
gel processing is that it allows the application of a variety of oxide and inorganic
organic hybrid materials on both large areas and complex-shaped substrates. With the sol
gel dip-coating technique method, a variety of materials can be deposited as thin films but metal oxides are the most widely employed. Such inorganic coatings can be deposited from monomeric or low-molecular-weight polymeric precursors, but NPs are also increasingly involved. In addition to these inorganic coatings, inorganic
organic hybrid materials can be deposited (Yusoff et al., 2014). The solvent should have a moderate volatility, a boiling point at 50 C
120 C, to give the liquid film enough time to level out, but keeping the short drying times. To obtain a thin film, it is essential to have low surface tension to allow complete wetting of the substrate and a homogeneous liquid flow. For these reasons, mostly short-chained aliphatic alcohols (ethanol, propanol, isopropanol, nbutanol) are preferred, but other solvents like esters or glycole ethers can also be used. Moreover, mixtures of different solvents can often help to tailor the deposition performance but a phase separation by selective evaporation has to be avoided as this normally leads to flow instabilities and wetting problems. To obtain the final coating material, normally a further curing or sintering step (post-treatment) is then necessary. The consolidation step represents the actual sol
gel transition with concomitant processes of draining, evaporation, and hydrolysis. In experimental cases this becomes apparent in a receding drying line that is moving downwards with colorful parallel interference lines, leaving behind the consolidated gel film. In contrast to the bulk sol
gel process, the complete transition passes in only a few seconds or less if volatile solvents are used. In this stage of the deposition, any turbulence or variation in the atmospheric conditions will inevitably lead to inhomogeneities in the film properties. Atmospheric parameters, like humidity and temperature, have to be set carefully, as atmospheric water is an essential reactant in the formation of the sol
gel film and the temperature decisively influences the drying rate. But the air flow conditions can also affect the film formation by governing the solvent evaporation and ensuring continuous replacement of the atmosphere. As a matter of fact, the best results are obtained with a laminar flow around the substrate, whereas any turbulence or drought could cause homogeneities. Basically, the coating thickness can be controlled by the withdrawal speed and the concentration and viscosity of the coating liquid. For a given coating system,

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however, there is normally an operational range that allows the preparation of smooth and homogeneous films. Typical withdrawal speeds range between 1 and 15 mm/s but these are still dependent on the employed solvent and precursor system. In contrast, lower withdrawal speeds are difficult to control and wet films are more sensitive to variations due to the radical association and resulting coatings are usually inhomogeneous; this is the case of a sol
gel film.

14.2.1.1 Nanocoatings prepared by dip-coating An interesting application of the dip-coating technique is to make a longer life for implants in the human body; by using this technique one can reduce the porosity and improve the biocompatibility of implants with the immiscible medium (Aksakal and Hanyaloglu, 2008). As the heat treatment of coated substrates is often required to densify the coating layer, to increase coating
implant bonding, and eliminate porosity (Zhitomirsky, 2002), a high sintering temperature can lead to degeneration of the metallic substrate and also phase transformation of HA into a noncrystalline phase which increases the dissolution rate in body fluid. The coating of bioactive ceramics on metal implants is an effective approach to solve corrosion-related problems and improve the biocompatibility of metallic implants. It is known that hydroxyapatite is a synthetic material, which promotes osseointegration and accelerates tissue fixation at the implant surface during the early stages of implantation (Li et al., 1996). The dip-coating technique can be successfully used to obtain homogeneous crack-free composite coatings on titanium implants (Sollazzo et al., 2008; Jokinen et al., 1998). In another work (Catauro et al., 2017), three hybrids from organic
inorganic nanocomposites, containing polyethylene glycol (PEG) embedded in matrices of SiO2, ZrO2, and TiO2, were produced by the sol
gel method. Materials found in the sol phase were applied for dip-coating of titanium grade 4 (the material of interest for orthopedic and dental implants) in order to transfer their biological activity to the inert material. The hybrids were achieved by the addition of different weight percentages of PEG to each glass matrix. After the hybrids (sols) were prepared by the sol
gel method they were used for the dip-coating procedure on Ti substrates 24 hours after synthesis and with withdrawal speeds of 25 mm/min for silica and titania and 35 mm/min for zirconia. After coating, Ti substrates were heat-treated at 45 C for 24 hours in order to promote film densification without polymer degradation. Tests confirmed that an increase in the PEG content results in crack-free coatings. After soaking the coated Ti into simulated body fluid, biological properties of coatings were assessed in vitro by growing NIH 3T3 murine fibroblasts on coated and uncoated surfaces. The bioactivity tests suggested that the presence of the polymer makes the films more biocompatible and this type of coating can be used for improving the osseointegration ability of Ti implants. Titanium and its alloys are widely used for dental and orthopedic prostheses owing to their favorable mechanical properties, low cytotoxicity, good corrosion

14.2 Nanocoating Fabrication Methods

resistance, and biocompatibility (Mohseni et al., 2014; Lopez-Esteban et al., 2003). One of the main requirements for implant longevity is high corrosion resistance. The formation of an oxide layer on the surface of Ti occurs rapidly upon contact with the air. This oxide layer is considered instrumental in decreasing the dissolution rate of metal implant in the biological environment (Marino et al., 2001). Moreover, the presence of an oxide layer also enhances the bioactivity of Ti in body fluids as it provides a site for the deposition of calcium and phosphate compounds via an ionic exchange process with the apatite from the bone tissue (Li and de Groot, 1993). Several studies have reported excessive levels of Ti ions in the vicinity of the implanted Ti-based implants. Moreover, the released Ti ions can combine with biomolecules from the host environment and result in adverse biological reactions (Bessho et al., 1995). Another popular metallic material that is used for temporary implants and prostheses is the stainless steel 316L. This material has the ability to withhold significant loads and undergoes plastic distortion prior to failure. However, the main disadvantage of using 316L in the human body is the fact that it is susceptible to localized corrosion when in contact with the aggressiveness of the human physiological environment (Cie´slik et al., 2009). This results in the release of toxic elements such as iron, chromium, nickel, and molybdenum into the human organism. Therefore, it is important to develop methods to increase the performance and the service life of stainless steel. In this respect, PLA/nanosized hydroxyapatite composite (PLA/nHA) coatings made by the sol
gel method have been deposited onto 316L material (Mohammadzadeh Asl et al., 2017
2018). To prepare the suspension, PLA was dissolved in extra pure chloroform, heated at 60 C and then subjected to stirring, until a jelly-like material was formed. Then, 5 wt.% of nanosized hydroxyapatite was gradually added to this solution under continued heating and stirring. Steel sheets were dip-coated in a suspension for different time intervals. After performing tests, it was found that the coatings were homogeneous and with a thickness of about 5 μm. The toxicity performed on fibroblast cells demonstrated that coated sheets had acceptable cell viability, in addition, the corrosion rate of samples decreased after the coating process. Process parameters and polymer concentration can be controlled to achieve the desired coating thickness to prevent corrosion when compared with uncoated implants, thus making a potential candidate for biomedical application (Catauro et al., 2015; Hume et al., 2011; Baker et al., 2009). In another study, a nanocomplex composed of metallocene polyethylene (mPE) amalgamated with nanohydroxyapaptite nanorods (mPE-nHA) was produced and dip-coated with Aloe vera extract after subjecting it to a microwave treatment (Wang et al., 2017). The coating of A. vera onto mPE-nHA samples was made by the dip-coating procedure. At first, mPE-nHA samples were dipped in the A. vera extract for a period of 12 and 24 hours, respectively. After the coating procedure, the samples were dried at room temperature for 24 hours in order to eliminate the moisture from the surface. The results of A. vera
coated mPEnHA samples were compared with the pristine mPE-nHA, which was the control.

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By means of the 3D-Hirox digital microscope model (KH-8700, Hirox Technologies, Hackensack, NJ, United States), the authors studied, at different magnifications, the topography and the A. vera coating on samples. The thickness of the A. vera coating was found to be 94.8 μm for the 12-hour dipped samples and the thickness increased for the 24-hour samples. Moreover, after testing the antithrombogenic nature of the mPE-nHA A. vera
coated samples, the authors concluded that this original nanocomposite could be used with success in bone tissue engineering applications. Dip-coating was also used as an efficient technique to increase the biocompatibility of fibrous materials. HA/chitosan nanohybrid coatings (HA/CS/CFs) on porous carbon fiber felts (PCFFs) have been achieved by depositing chitosan/calcium phosphate precursors on PCFFs by the dip-coating method (Liu et al., 2014). After the chitosan powder was dissolved in acetic acid solution (2 vol.%) to obtain a homogeneous solution, Ca(NO3)24H2O and NaH2PO42H2O were added. PCFFs samples were immersed into the above mixed solution for 5 minutes. HA/CS/CFs exhibit exceptional biocompatibility, which can increase the adhesion, distribution, and proliferation of human bone marrow stromal cells. Therefore, HA/CS/CFs have great prospects for bone scaffold materials. Poly(methyl methacrylate)/carbonated hydroxyapatite (PMMA/CHA) (Morales-Nieto et al., 2013) composite was prepared in the form of a solution to be applied on ultrahigh-molecular-weight polyethylene (UHMWPE) substrate. The coatings were prepared by dipping UHMWPE substrates in the PMMA/CHA solution through a mechanical device that kept an immersion speed of 70 mm/ min for 5 minutes. After the immersion, samples were left in air for 5 minutes, and then at 120 C for 3 hours. The measurements by image analysis presented average thickness of this rough coating of 29 μm. By testing the wear and friction behavior of the PMMA/CHA coating, one may conclude that the coating could be appropriate for medical domains where the tribological behavior and high mechanical properties are needed.

14.2.2 MATRIX-ASSISTED PULSED LASER EVAPORATION METHOD Laser techniques used for the deposition of materials are central areas of experimental studies with encouraging prospects toward the domains of nanofabrication and/or nanostructuring. Laser techniques are reputable alternatives to conventional procedures since they can guarantee complete control of the thickness of the deposited material and good adherence to the substrate surface. Moreover, materials can be deposited by laser methods on any type of substrate (Chrisey and Hubler, 1994). A modern technique, entitled matrix-assisted pulsed laser evaporation (MAPLE) (Pique´, 2011), was developed with the aim of constructing thin films from organic and/or bio-organic materials. The distinctive phase of MAPLE is that the material of concern is diluted or suspended in a volatile solvent (matrix); the matrix is expected to shield the material to be deposited from the direct and

14.2 Nanocoating Fabrication Methods

damaging action of the incident laser radiation (Pique´, 2011). The most used lasers in MAPLE are excimer (Oprea et al., 2016) or Nd:YAG (Yang and Zhang, 2019), for the reason that the ultraviolet radiation connects to almost any target material. Also, infrared laser wavelengths can be used (e.g., Er:YAG lasers) (Ge et al., 2015). Hence, matrices must be picked to have high absorption at the laser radiation wavelength, in order to prevent photo-decomposition of the material of interest (Pique´, 2011). The matrix and the material to be deposited are then cooled until they become solid (named “target”) and kept frozen at liquid nitrogen temperature (by means of a liquid nitrogen reservoir coupled to the target holder) during the laser deposition procedure. Usually, the concentration of the solution in the target is in the range of 0.5 2 10 wt.%. During laser deposition the target is rotated in order to allow an even erosion of the frozen solution.

14.2.2.1 Nanocoatings prepared by MAPLE Materials can be identified as nanoscaled when their sizes (at least one of their external sizes) are in the region of 1
100 nm (ISO/TS 80004-4:2011(E)). At the nanometer scale, besides the chemical composition, the functional properties of materials are dependent on the aspect ratio, specific surface-to-volume-area, size, and shape. Accordingly, NPs are demarcated in the literature as particles having dimensions within the nanometer domain (typically ,100 nm) and are known to hold properties (optical, electronic, magnetic, and thermal) that are not experienced by their bulk equivalents (Naito et al., 2018). A special type of NP, magnetic NPs, not only have the all-purpose features of NPs but also magnetic properties. Magnetic NPs have unique magnetic sensitivity when a magnetic field is applied onto them; this function is especially valuable for MRI (Kim, 2018). Correspondingly, the magnetocaloric outcome of these NPs can be used for tumor treatment (Xue et al., 2017). Magnetic NPs consist of: metal (Au, Ag, Fe, Co, and Ni), metal oxide (iron oxides such as γ-Fe2O3 and Fe3O4), ferrites (such as CoFe2O4), and metal alloy NPs (e.g., FeCo, FePt) (Naito et al., 2018). From the above-mentioned metal oxide NPs, Fe2O3 and Fe3O4, are the most used as they can be prepared with ease and are size- and shapecontrollable (Akbarzadeh et al., 2012). Nevertheless, in many domains (e.g., medical), properties of adjusted magnetic NPs in their powder-like formulation cannot be completely used. These requirements had been of support to scientists for designing hybrid NPs, which can be composed of two or more nanomaterials (including metals or polymers); one of them could act as a core and the other as a shell (Lo´pez-Lorente et al., 2011; Zaleska-Medynska et al., 2016). As a result of the ability to manipulate magnetic NPs’ structure, their properties can be adjusted to be adapted for particular applications such as electronics (Liu et al., 2018), optoelectronics (Sanfelice et al., 2017), energy production and storage (Ong et al., 2018), sensing (Qin et al., 2018), and medical and pharmaceutical activities (Samiei et al., 2016).

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After being surface-modified, mostly with polymeric materials, magnetic NPs can possess excellent biocompatibility and can be used as routes for the transport of drugs, essential oils, or genes directly to the diseased area of the human body (Salatin et al., 2015), and, under the action of a magnetic field, to realize targeted therapy (Kang et al., 2017). However, due to their small size, NPs interact with biological structures resulting in health-associated risks (Salatin et al., 2015), therefore an important issue in constructing products comprising NPs is to degrade or recuperate them from the environment. Therefore, the immobilization of NPs onto surfaces is beneficial due to the fact that they can be effortlessly removed from the substrates and from the environment. Current studies prove that the MAPLE method could represent an unconventional way to immobilize NPs on different substrates by selecting a suitable laser regimen, such as the laser wavelength and pulse intensity, fluence, reactive atmosphere, etc. Herein we present some recent studies on thin nanostructured films based on magnetite NPs, deposited by the MAPLE technique and with applications in medicine. Moreover, a special emphasis is put on the bioevaluation of these films and their application in the medical field. In particular, in Holban et al. (2015), the authors fabricated with success a nanostructured surface having B10 nm average diameter magnetite and carvone (Fe3O4@CAR) NPs and evaluated the biological activity of this material deposited as a thin film by MAPLE. For this purpose, Fe3O4@CAR NPs were dispersed in dimethyl sulfoxide (DMSO) as a 1.2% (w/v) solution and immersed in liquid nitrogen for 30 minutes. MAPLE deposition was performed using a KrF (λ 5 248 nm and τ FWHM 5 25 ns) excimer laser source with a repetition rate of 15 Hz and a laser spot area of 36 mm2. The laser fluence was within the range of 300
500 mJ/cm2, but only the fluence of 500 mJ/cm2 did not break the functional groups of carvone. The in vitro results demonstrated that surfaces containing the fabricated bioactive nanostructured film have pronounced antimicrobial activity, impeding the colonization and biofilm formation of both Gram-positive and Gram-negative tested species. Furthermore, it was found that the obtained thin film is not cytotoxic, permitting the normal development of human endothelial cells. Interesting results were accomplished using a thin layer of kanamycin functionalized 5-nm magnetite (Fe3O4@KAN) NPs deposited by MAPLE onto silicone and glass substrates (Grumezescu et al., 2015). A laser fluence study was performed in order to establish the optimum deposition settings with respect to the number of applied pulses (30,000
60,000) and the preservation of the chemical structure. The tested fluences were of 300, 400, and 500 mJ/cm2, respectively. The Fourier-transform infrared spectroscopy spectra showed that the fluence of 400 mJ/cm2 was the best compromise between laser pulses and the stoichiometric transfer of kanamycin. From scanning electron microscopy (SEM) images (Fig. 14.1, left), it can be observed that the surface was constituted from aggregates that formed a porous coating; this topology can be considered advantageous

14.2 Nanocoating Fabrication Methods

FIGURE 14.1 SEM micrograph of Fe3O4@KAN thin-film MAPLE-deposited onto silicone substrate. Reprinted from Grumezescu, V., Andronescu, E., Holban, A.M., Mogoanta, L., Mogo¸sanu, G.D., Grumezescu, A.M., et al., 2015. MAPLE fabrication of thin films based on kanamycin functionalized magnetite nanoparticles with anti-pathogenic properties. Appl. Surf. Sci. 336, 188
195, with permission from Elsevier.

when used as a coating for biomimetic implants, due to the larger interface of the active surface with the adjacent cells. Moreover, in Fig. 14.1 (right) it can be seen that the surface presented a nanometric morphology thanks to Fe3O4@KAN NPs coalescence. Additionally, in vivo and in vitro tests proved that such nanostructured morphology was favorable in the control of antibiotic release in nontoxic doses. These results uncovered the potential of a Fe3O4@KAN-functionalized nanostructured surface to be used as an implant coatings. In another study, the authors obtained nanostructured thin films with good biocompatibility and resistance to microbial colonization and biofilm formation (Grumezescu et al., 2018). They prepared magnetite NPs functionalized with gentamicin (Fe3O4@G) and embedded them into poly(lactic-co-glycolic acid) (PLGA) spheres by an oil-in-water emulsion recipe (Grumezescu et al., 2014). The PLGA-Fe3O4@G spheres were deposited by MAPLE on glass and silicone substrates. The target made from a solution of 1% (w/v) PLGA-Fe3O4@G spheres in n-hexane matrix was frozen at liquid nitrogen temperature. A KrFT (λ 5 248 nm, τ FWHM 5 25 ns) excimer laser beam impacted the target at fluences of 200, 300, and 400 mJ/cm2, a repetition rate of 20 Hz, and for 20,000
40,000 subsequent pulses. During the deposition, the cryogenic target was rotated with 0.4 Hz and maintained at a temperature of B173K by constant cooling with liquid nitrogen. After conducting a fluence study by means of infrared mapping, second derivative infrared micrographs (Fig. 14.2) showed that the nanocoating synthesized at a laser fluence of 400 mJ/cm2 presented the best uniformity.

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FIGURE 14.2 Second derivate IR mappings of drop-cast (A) and the thin film (B
D), F 5 200 mJ/cm2 (B), F 5 300 mJ/cm2 (C), and F 5 400 mJ/cm2 (D). Reprinted from Grumezescu, V., Negut, I., Grumezescu, A.M., Ficai, A., Dorcioman, G., Socol, G., et al., 2018. MAPLE fabricated coatings based on magnetite nanoparticles embedded into biopolymeric spheres resistant to microbial colonization. Appl. Surf. Sci. 448, 230
236, with permission from Elsevier.

The shapes, sizes, and integrity of PLGA/Fe3O4@G thin films after the laser transferral were examined by SEM. SEM micrographs at different magnifications given in Fig. 14.3(A and B) show that the morphological features (spherical shape) of initial PLGA/Fe3O4@G nanostructures were preserved after the laser transfer. The size distribution of deposited PLGA-Fe3O4@G varies from submicron up to a few microns, with an average of B3 μm. The antimicrobial and antibiofilm competence of coatings was tested against Gram-positive (Staphylococcus aureus) and Gram-negative (Pseudomonas aeruginosa) clinical microorganisms by viable cell counts assay, performed at different time intervals. The obtained results proved that coatings of PLGA-Fe3O4@G displayed proficient antimicrobial activity against both adherent and sessile bacterial cells. In addition to their antiadherence and antibiofilm effect, MAPLE thin films were highly biocompatible, agreeing with the regular development and growth of cultured human amniotic fluid stem cells. This approach could be successfully applied for the optimization of medical surfaces in order to control and prevent microbial colonization and further development of biofilm-associated infections.

References

FIGURE 14.3 SEM micrographs of PLGA-Fe3O4@G thin film, at different magnifications (A-10.000X; B-200.000X), deposited by MAPLE onto silicone substrate. Reproduced and adapted from Grumezescu, V., Negut, I., Grumezescu, A.M., Ficai, A., Dorcioman, G., Socol, G., et al., 2018. MAPLE fabricated coatings based on magnetite nanoparticles embedded into biopolymeric spheres resistant to microbial colonization. Appl. Surf. Sci. 448, 230
236 with permission, Copyright 2018, Elsevier.

14.3 CONCLUSION This chapter presents two different and conventional processes for the deposition of thin films as well as nanoparticle deposition in the form of nanocoatings and their current progress. The surface modification by implementing synthesis of coatings by laser processing and dip-coating methods represents an appropriate approach for enhancing the performances of medical devices.

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317.

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Index Note: Page numbers followed by “f” and “t” refer to figures and tables, respectively.

A Acalypha indica, 8
9 Accelerometers, 368 Acetylcholinestrase (AChE), 362 Acetylsalicylic acid, 299 AC magnetic susceptibility, 246 Actinomyces spp., 156
157 Active pharmaceutical ingredients (API), 198 Alkyl antineoplastic (ANP) drugs, 429
430 Alternating magnetic field (AMF), 450 Aluminosilicate-based drug carriers, 288
303 cationic surfactants, 288
294 pharmaceutical application of, 291
294 physicochemical properties of, 290
291 surfactant-modified aluminosilicates functionality of, 298
303 preparation and characterization of, 294
298 Aluminosilicate/chitosan composites for drug delivery, 307 Aluminosilicates, 285, 316 as drug carriers, 286
287 3-Aminopropyl triethoxysilane (APTS), 215 4-Aminothiolphenol (4-ATP), 215 Amperometric biosensors, 377
378, 383 Anesthetics, topical, 430 Antibacterial test using silver nanoparticles, 222
223 Antibiotic therapy, 154
155 Anticancer chemotherapeutic agents, 427
428 Anticancer effects, 148 Anticancer iron salen, theoretical investigation of by first principles calculations, 440
442 Antimicrobial agent, 21
22, 119
120, 153
156, 413 Antimicrobial resistance, 168 Antimicrobial testing, metal nanoparticles for, 413
416 Arsenic sulfide, 135
137 Artabotrys odoratissimus, 13
14 Atomic emission spectroscopy with inductively coupled plasma (ICP-AES), 297

B Bacillus licheniformis, 8
9 Bacteria, 153
154 Bacterial contamination, 168 Bacterial infectious diseases, 21
22

Basal cell carcinoma (BCC), 215 fingerprinted detection, 219
222 Batch adsorption method, 296 Bentonite, 286 Benzalkonium chloride (BC), 296
297 Bimetallic nanoparticles, 189
190 Bioactive ceramics, 50, 63, 331, 466 Bioactive glass nanofibers, 329
330, 351 conventional methods to produce, 332
334 methods to produce, 335
338 bottom-up methods, 335
336 top-down methods, 336
338 production, by laser spinning technique, 342
351 in tissue engineering, 330
332 for tissue engineering and composites, 338
342 Biofilm, 156
158 Biofilm treatment and prevention, 154
155 Bioglass 45S5 fibers, 329
330, 338
340, 346, 346f Biological sensors, 379
382 DNA, 379 protein, 379
382 Biomaterial-based treatments in orthopedics, 63 Biomimetic in vitro models, 259
260 versus classical cell cultures, 263
269 perspectives of, 267
269 representing the bridge between in vitro and in vivo research, 262
263 Bionanocomposites, 403 Biosensing applications, of metal nanoparticles, 412
413 Biosensors, 357, 368, 387f mixed metal oxides-based nanocomposites for. See Mixed metal oxides-based nanocomposites for biosensors Bisphenolglycidyl dimethacrylate (Bis-GMA), 167 Block copolymer micelles as nanoreactors, 177 bimetallic nanoparticles, 189
190 biomedical applications, 195
198 comparison of poloxamers and poloxamines, 190
195 future perspectives, 202
203 micelle architecture and mixed micelles, 181
183 poloxamers and poloxamines, 178
180 study results, 199
202

479

480

Index

Block copolymer micelles as nanoreactors (Continued) synthesis of various morphologies of gold nanoparticles, 183
189 icosahedral gold nanoparticles, 184
186 nanoplates, 187
189 Bone cements, 161 Breast cancer cell detection, gold nanoparticles application for, 218

C C14E8, 195 Cacumen platycladi, 9 Calcium phosphate, 24, 63, 349 Cancer hyperthermia, 448
452 Cancer immunotherapy/drug delivery metal nanoparticles’ applications in, 407
408 Capsicum annuum L. extract, 8
9 Captopril, 376 Carbon-fiber-reinforced polyetheretherketone (CFR-PEEK), 70 Carboxydextran-coated iron oxide MNPs, 255
256 Cationic acrylates and cationic silanes, 156
161 Cationic poly(ethylene imines) (PEI), 162 Cationic surfactants, 288
294 pharmaceutical application of, 291
294 physicochemical properties of, 290
291 Caveolae, 252
253 CD4 count, 226 Cell labeling, 249 Cellular labeling using NPs, 408
409 Ceramic matrix, composites based on, 58
59 Cetylpyridinium (CP), 298 Cetylpyridinium chloride (CPC), 300 Cetyltrimethylammonium bromide, 212 Chemical (bio)sensors, 374
376 Chemical vapor deposition, 336 Chemotherapy conventional, 429
430 targeted, 431 Chinese hamster ovarian (CHO) cells, 86 Chitooligosaccharides, 304 Chitosan, 303
307 physical and chemical properties of, 304
306 safety and regulatory status of, 306
307 Chitosan
aluminosilicate composites for drug-delivery and wound-healing applications, 311t functionality of, 310
316 Chitosan-modified aluminosilicates preparation and characterization of, 307
310 Chlorhexidine digluconate, 161

Chloride-containing quaternized amine polyurethanes, 165 Cholesterol detection, 383
384 Cisplatin, 430 Clinical biosensors, 382
386 cholesterol, 383
384 glucose, 382
383 immunology, 385
386 urea, 385 CO2 laser, 343
344 Composites based on ceramic matrix, 58
59 based on polymer matrix, 57
58 classification of, 48f properties of, 59
61 Compression molding, 58 Conducting polymers (CPs), 364
365, 366t Conductor, 359f Contact-active materials, 155 Continuous-fiber composites, 58 Conventional chemotherapy, 429
430 Copper, 362 Copper nanoparticles, 11
17 Coprecipitation, 212
213 Core
shell iron
gold nanoparticles, 19 Core
shell structured nanoparticles, 216 Corona, 245 Cremophor EL (CrEL), 430 CTAB, 184 CuNPs, 413
414 Cytocompatibility test, 111
112 Cytotoxicity, 119
120, 167

D Dental caries, 156
157 Devitrification, 332 Diabetes, 22 Diclofenac diethylamine (DDEA), 299 Diclofenac sodium (DS), 299 Diltiazem, 376 Dimethylaminododecyl methacrylate (DMADDM), 157, 167 Dip-coating method, 464
468 nanocoatings prepared by, 466
468 Dithiothreitol (DTT), 432
433 DNA biosensors, 379 DNA sequence determination, 411
412 Drug-delivery system (DDS), 230
231, 427
428 Drug-incorporated micellar systems, 201 Dynamic light scattering (DLS), 244
245, 244f

Index

E Electroactive polymers, 365 Electrochemical immunosensors, 385 Electrospinning, 336
338 Endocytic pathways, 252
253 Endocytosis, 251 of magnetic nanoparticles, 257
258 Energy-dispersive X ray spectroscopy (EDS), 444
445 Enhanced permeability and retention (EPR) effect, 249
250 Enterococcus faecalis, 14 Environment biosensors, 376
378 heavy metals, 376
377 pesticide and dust, 377
378 EpiDerm, 263
264 Epstein
Barr virus (EBV), 228 Ethanol, 373 Ethylcarbodiimide (EDC), 218 Euphorbia hirta (L.), 8
9 Exocytosis, 257 External cation exchange capacity (ECEC), 295 Extracellular polymeric substance (EPS), 154 Extrinsic semiconductor, 359f

F F-68 NPs, 201 F-127, 186f, 187
189, 201 Fabrication, 58
59, 387
388 Fiber-forming temperature, 332 Fibers, 54
56 Fibroblast-like synoviocyte (FLS) cells, 362 Filament winding, 58 Fluorescent biological labeling, 410
411 Fluorescent metal NPs, 410
411 5-Fluorouracil, 10
11 Fourier-transform infrared (FTIR) spectra, 121, 297 Fragile melts, 333 Free-volume elements (FVEs), 120 Fusarium oxysporum, 8
9, 14

G Garcinia mangostana, 4
5, 9 Gas (bio)sensors, 368
374 ethanol, 373 NOx, 369
373 oxygen, 373
374 water (humidity), 374 Gastrointestinal tract models, 265
266 Gel electrophoresis, 246
247 Genapol, 178

Ginko biloba L., 13
14 Glass transformation range, 333 Glass transition temperature, 333 Glucose biosensors, 382
383 Gold nanoparticles (AuNPs), 2
5, 177
178, 404
406 biomedical applications of, 3f for breast cancer cell detection, 218 as carrier for immune therapies, 407 for detection of proteins, 412 fluorescence imaging, 409
411 synthesis of various morphologies of, 183
189 “Goodenough
Kanamori
Anderson” rules, 438
439 Gram-negative bacteria, 166 Gram-positive bacteria, 16, 164 Graphene/zinc oxide nanocomposites, 362, 363f “Green” synthesis metal NPs, 402 Green synthesis of gold nanoparticles, 4
5

H Halloysite, 286 Halloysite nanotubes (HNT), 307, 314
315 Heavy metal biosensors, 376
377 Hepatitis virus type B (HBV), 228 HER2, 218 Herpes simplex virus, 224
225 Hexadecyltrimethylammonium bromide (HB), 296
297, 299
300 High-resolution TEM (HRTEM) characterization, 121
122 Humidity sensors, 374 Hybrid metal complex nanocomposites for targeted cancer diagnosis and therapeutics, 427 conventional chemotherapy, 429
430 hybrid metal salen
polymer nanocomposites as nano-DDS, 452
454 metal
ligand complexes as a composite anticancer drug, 431
452 iron complexes, 432 iron
salen complexes, 432
433 magnetic nanoparticles (MNPs), 433
438 molecular magnetic iron complex for magneto-DDS, 438
452 targeted chemotherapy, 431 Hybrid metal salen
polymer nanocomposites as nano-DDS, 452
454 Hydrophilic
lipophilic balance (HLB), 198 Hydrothermal method, 213
214 Hydrolyzable groups, 158
159 Hydroxyapatite (HAp), 23
29, 81, 329
330, 466 biological studies, 86
87

481

482

Index

Hydroxyapatite (HAp) (Continued) cell culture, 86 MTT assay, 86
87 carbonation in biological apatites, 83 confirmation of carbonation in HAP by FTIR analysis, 90
92 cytocompatibility test, 111
112 HRTEM analysis, 100
103 importance of Zn, Mn, and Mg as trace elements present in bone, 83
84 mechanical alloying (MA), 84 sample preparation by, 84
85 method of analysis, 87
90 microstructural analysis, 87
89 physical and mechanical property measurement, 89
90 modification in HAP structure due to Mn/Mg/ Zn substitution, 94
100 phase confirmation of unsintered HAP samples from XRD patterns, 90 quantitative phase estimation of unsintered samples using Rietveld’s method, 92
93 sample characterization, 86 sintered HAP samples, mechanical properties of, 107
111 spark plasma sintered samples, microstructure characterizations of, 103
107 spark plasma sintering, 85
86

I Ibuprofen (IBU), 299 ICIE16 glass nanofibers, 349 Icosahedral gold nanoparticles, 184
186 Image sensors, 368 Immunosensors, 385
386 Immunotherapy carriers, metal nanoparticles as, 407 Impedimetric immunosensor, 385
386 Inorganic composites in biomedical engineering, 47 anomalies, 62
65 fracture and fatigue failure, 62
65 applications in biomedical engineering, 66
71 dentistry, 66
68 orthopedic, 70
71 prosthetics and orthotics, 68
69 tissue engineering, 69
70 background, 47
52 biological response, 65
66 categorization, 52
53 ceramic matrix, composites based on, 58
59 components, 53
57 fibers, 54
56

interface, 57 matrices, 53
54 particles, 56 polymer matrix, composites based on, 57
58 properties of composites, 59
61 Inorganic nanoparticles, biomedical, 1, 2f copper nanoparticles, 11
17 gold nanoparticles, 2
5 hydroxyapatite nanoparticles, 24
29 iron nanoparticles, 17
19 selenium nanoparticles, 10
11 silver nanoparticles, 5
10 zinc oxide nanoparticles, 19
24 In situ quaternization of tertiary amines, 162
164 Insulators, 359f Interface, 57 International Organization for Standardization (ISO), 211
212 Intracellular trafficking, of internalized nanoparticles, 255
257 Intrinsic semiconductor, 359f Inverse microemulsion, 213 Inviscid melts, 333
334, 337
338 Iodide quaternary ammonium methacryloxy silicate (IQAMS), 158 Ionic/molecular mimicry, 413
414 Iron complexes, 432 Iron nanoparticles, 17
19 Iron oxide MNPs, 256
257 Iron oxide NPs (IONPs), 435 Iron salen, synthesis of, 438
439 Iron
salen complexes, 432
433

K Klebsiella pneumonia, 8
9

L Langmuir waves, 398 Laser deposition, 463 Laser spinning technique, glass nanofibers production by, 342
351 Lauroyl sarcosinate (SR), 298 Leaching materials, 155 Ligand and metal charge transfer (LMCT) complex, 181, 194
195 Liquidus temperature, 332
333 Localized surface plasmon resonance (LSPR), 6 Lung models, 264
265

M Macromlecular-based formulation technology, 436 Macropinocytosis, 252
253

Index

MagForce, 251 Magnesium aluminum silicate, 286 Magnetic contrast agents, 249 Magnetic fluid hyperthermia, 251 Magnetic iron salen, design of, 439
440 Magnetic metal complex (MMC), 439
440 Magnetic nanoparticles (MNPs), 223
230, 241
242 advantages, perspectives, and limitations of biomimetic in vitro models versus classical cell cultures, 263
269 gastrointestinal tract models, 265
266 lung models, 264
265 perspectives of biomimetic in vitro models, 267
269 placenta models, 266 skin models, 263
264 urothelium/urinary bladder models, 266
267 arsenic removal from water, 224 biological treatment targeting Mycobacterium tuberculosis, 229
230 CD41 cell separation, 226 characterization of mobility in 3D gels and in the artificial extracellular matrix, 246
247 current biomedical applications of, 247
251 cell labeling, 249 magnetic fluid hyperthermia, 251 magnetic nanoparticles as contrast agents for magnetic resonance, 249
250 magnetofection, 250 molecular isolation and magnetic separation, 248 perspectives, 251 as delivery vectors, 248 detection of pathogenic viruses, 226
228 endocytic pathways, 252
253 endocytosis of magnetic nanoparticles, 257
258 as an essential carrier for magnetic DDS, 433
438 herpes DNA separation, 224
225 in vivo and in vitro models, 258
263 biomimetic in vitro models representing the bridge between in vitro and in vivo research, 262
263 comparison of, 258
260 routes and model organisms of magnetic nanoparticle administration, 260
262 intracellular trafficking and fate of internalized nanoparticles, 255
257 properties of, 242
244 changed in physiological fluids, 244
245 physicochemical properties, 245
246 silica coating of, 215

specific and rapid tuberculosis detection, 228
229 uptake pathway depending on the properties of nanoparticles and the cell type, 253
255 Magnetic resonance (MR) imaging, 249, 435
436, 447
448 Magnetic targeting, 247 Magnetofection, 250 Magnolia kobus, 13
14 Matrices, 53
54 Matrix-assisted pulsed laser evaporation method, 468
472 nanocoatings prepared by, 469
472 Mechanical alloying (MA), 84 sample preparation by, 84
85 Menschutkin reaction, 156 Mesenchymal stem cells (MSCs), 64, 329
330 Metal
ligand complexes as a composite anticancer drug, 431
452 iron complexes, 432 iron
salen complexes, 432
433 magnetic nanoparticles (MNPs), 433
438 molecular magnetic iron complex for magnetoDDS, 438
452 anticancer iron salen by first principles calculations, 440
442 anticancer properties, 444
447 cancer hyperthermia, 448
452 crystallographic analysis, 442 design of magnetic iron salen, 439
440 magnetic property, 447
448 purity analysis, 442
443 synthesis of iron salen, 438
439 Metallocene polyethylene (mPE), 467
468 Metal nanoparticles, 177, 397
399 applications in biology, 410
416 antimicrobial testing, 413
416 biodetection of proteins, 411
412 biosensing applications, 412
413 fluorescent biological labeling, 410
411 functionalization of, 403
406 medical applications, 407
409 cancer immunotherapy/drug delivery, 407
408 imaging of tissues and cells/nanoparticles in diagnostics, 408
409 properties of, 399
401 synthesis approaches to, 402
403 Metal NPs (Cu and Au)
polyaniline (PANI) nanocomposites, 402
403 Metal oxides nanostructures (MONs), 360 Methacryloxyethyl cetyl dimethyl ammonium chloride (DMAE-CB), 157 Methacryloyloxydecyl phosphate (MDP), 167

483

484

Index

Methacryloyloxydodecyl pyridinium bromide (MDPB), 157 Meticillin-resistant Staphylococcus aureus, 154
155 1-Methylimidazole (MIA), 224
225 Micelle architecture and mixed micelles, 181
183 Microemulsion, 12
13, 213 Microemulsion/inverse microemulsion, 212
213 Micrometric glass fibers, production of, 333
334, 334f Microwave-assisted chemical reduction, 13 Minimum inhibitory concentration (MIC) test, 162 Mixed metal oxides-based nanocomposites for biosensors application of sensors, 368
386 biological sensors, 379
382 chemical (bio)sensors, 374
376 clinical biosensors, 382
386 environment biosensors, 376
378 gas (bio)sensors, 368
374 fabrication, 387
388 nanocomposites/particles, 365
367 polymers, 365 recommendations for future work, 390 selectivity, 389 semiconducting (nano)materials, 358
365 sensitivity, 389 sensors and biosensors, 367
368 sensing measurement, 367
368 time factors, 389 Mohs micrographic surgery (MMS), 219
220 Molecularly imprinted polymers (MIPs), 365 Montmorillonite (MMT), 286, 315 Montmorillonite/chitosan nanocomposite, 307
309, 315
316 Morganella morgani RP42, 14 Morganella psychrotolerans, 14 Motion detectors, 368 Multidrug-resistant (MDR) cancer cells, 197 Multifunctional nanoparticles, 215
218 applications of, 230
232 Multinanoparticles, 215
216 Mycobacterium tuberculosis (MTB), 228
230

N N,N’-bis(salicylidene)ethylenediamine, 438 Nanocoating fabrication methods, 464
472 dip-coating method, 464
468 nanocoatings prepared by dip-coating, 466
468 matrix-assisted pulsed laser evaporation method, 468
472 nanocoatings prepared by MAPLE, 469
472

Nanocoatings prepared by dip-coating, 466
468 Nanocomposites/particles, 365
367 Nanocrystallization, 198 Nanofiber, definition of, 329
330 Nanoparticle (NP)-guided functionality in AS4S4/ ZnS NCs, 127
146 atomic-deficient structure of AS4S4/ZnS NCs, 130
140 compositional evolution of FVEs in AS4S4/ ZnS NCs, 132
140 expected channels of mixed positron-Ps trapping in NP-based composites, 130
132 biological activity of AS4S4/ZnS NPs, 142
146 dissolution of As from mixed AS4S4/ZnS NPs, 142
143 in vitro anticancer functionality of AS4S4/ ZnS-PX407 NSs, 144
146 characterization of AS4S4/ZnS NCs prepared in a dry-milling mode, 127
130 characterization of AS4S4/ZnS-PX407 NSs prepared in a wet-milling mode, 140
141 Nanoparticles, 177, 211 applications of, 218
232 antibacterial test using silver nanoparticles, 222
223 basal cell carcinoma fingerprinted detection, 219
222 for breast cancer cell detection, 218 magnetic nanoparticles, 223
230 multifunctional nanoparticles, 230
232 in diagnostics, 408
409 functionalization of, 214
215 magnetic nanoparticles, silica coating of, 215 multifunctional, 215
218 perspectives, 232
233 synthesis of, 212
214 chemical reduction, 212 coprecipitation, 212
213 hydrothermal method, 213
214 microemulsion and inverse microemulsion, 213 seeding, 213 sonoelectrodeposition, 214 Nanoplates, 187
189 Nanostructured hydroxyapatite (nano-HA), 23 Nanostructurization, 124
125, 137 Natural factories, 8
9 Natural polymers, 363
364, 403 Nonsteroidal antiinflammatory drugs (NSAIDs), 299 NOx gases detection, 369
373

O Ocimum sanctum, 9 Oligochitosans, 304
305

Index

Organic nanoparticles, 216
217 Orthopedics, biomaterial-based treatments in, 63 Osteoblasts, 64 Ostwald ripening, 184 Oxygen sensors, 373
374

P p-aminothiolphenol (PATP), 215 Parathormone antineoplastic drugs, 429
430 Particles, 56 Pathogenic Streptococcus mutans, 156
157 PEG-functionalized metal NPs, 406 PEG molecules, 407 Pesticide and dust, detection of, 377
378 pH effect on synthesis of AuNPs, 190
192 Phagocytosis, 252
253 Phosphate glass fibers, 341 Photon correlation spectroscopy, 245 Photothermal therapy (PTT), metal nanoparticles for, 407
408 Pinus eldarica, 9 Placental BeWo b30 Transwell model, 262
263 Placenta models, 266 Planetary ball mill, 84
85 Plasma oscillations, 398 Plasmonic NPs, 409 Plasmonics, 398 Pluronics, 178
179, 184, 186f, 187
189, 200 Pluronics L61, 201 Poloxamer-407, 198 Poloxamers, 178
180, 197 comparison of poloxamines and, 190
195 Poloxamines, 178
180, 197, 199 comparison of poloxamers and, 190
195 Poly(2-hydroxyethyl methacrylate) (pHEMA), 69 Poly(α, γ, L-glutamic acid), 9
10 Poly(DL-lactide) (PDLLA) composites, 338
339 Poly(glycerol sebacate) (PGS), 338
339 Poly(p-acryloylaminobenzylphosphonic acid), 8 Polyethylene glycol (PEG), 466 Polyethylene oxide (PEO), 177
180, 190 Polyglycolic acid, 338
339 Polylactic acid (PLA), 21
22, 338
339 Poly lactic-co-glycolic acid (PLGA), 21
22 Polymerase chain reaction (PCR), 226
227 Polymer-based nanocomposites, applications of, 406
416 Polymeric micelles, 202 Polymeric surfactants, 288 Polymer matrix composite (PMC), 57
58 Polymers, 365 Polymethylmethacrylate (PMMA), 66
68, 71, 338
339

Polyol, 212
213 Polypropylene oxide (PPO), 177
180, 190 Polysaccharides, 404 Polyurethane, 50
51 Polyvinylpyrrolidone (PVP), 216
217 Porous carbon fiber felts (PCFFs), 468 Porphyra vietnamensis, 8
9 Positron annihilation lifetime (PAL) spectroscopy, 120 Positron e1 trapping formalism, 123
124 Precipitation, 212
213 Prevascularization, 64 Primary human peripheral blood mononuclear cells (PBMNCs), 65 Protein biosensors, 379
382 Proteins, biodetection of, 411
412 Proton-sponge effect, 256
257 Pseudomonas stutzeri, 14 Ps trapping formalism, 124 Pultrusion, 58

Q Quaternary ammonium compound derivatives, 291
292, 294 antimicrobial resistance, 168 background, 153
154 biofilm treatment and prevention, 154
155 cationic acrylates and cationic silanes, 156
161 cytotoxicity, 167 disinfectants and preservatives, 161
162 in situ quaternization of tertiary amines, 162
164 variables influencing the antimicrobial properties of, 164
167 Quaternary ammonium methacrylate-modified nanosilica (QMSNs), 164 Quaternary ammonium silanes (QASs), 158
160 QAS-functionalized methacrylate (QAMS) groups, 160 Quercetin (QN), 199

R Raman spectroscopy, 6, 220 Reactive oxygen species (ROS), 256, 259 generation, 444 Resinous composites for dental applications, 156 Reuss model, 60 Rietveld analysis, 100 Rietveld’s method quantitative phase estimation of unsintered samples using, 92
93 Rotary jet spinning, 336, 337f

485

486

Index

S Salbutamol, 376 Salicylic acid (SA), 376 Scaffolds and composites, 330
332 Scanning electron microscope (SEM), 344 Seed growth method, 177
178, 184 Seeding method, 213 Selected area electron diffraction (SAED) pattern, 100
103 Selectivity, 389 Selenium nanoparticles, 10
11 Semiconducting (nano)materials, 358
365 Semiconducting polymers (SCPs), 365 Semiconductors, 358
360, 359f Sensitivity, 389 Sensors and biosensors, 367
368 sensing measurement, 367
368 Shigella flexneri, 21
22 Silane-based QAC, 160
161 Silica coating of magnetic nanoparticles, 215 Silver nanoparticles (AgNPs), 5
10 antibacterial test using, 222
223 Skin biomimetic in vitro models, 263
264 Skin cancer, detection of, 219
220 Smart and intelligent polymers, 183 Sol
gel process, 158
160, 159f Sonochemical method, 13 Sonoelectrodeposition, 214 Spark plasma sintered samples, microstructure characterizations of, 103
107 Staphylococcus aureus, 154
155, 349 Static models, 265
266 Stimuli-responsive polymers, 197 Streptococcus gordonii, 156
157 Streptococcus mitis, 156
157 Streptococcus mutans, 156
157 Streptococcus oralis, 156
157 Streptococcus sanguinis, 156
157 Structured reducing agents, 180 Superconducting quantum interference device (SQUID) system, 447
448, 450
452 Superparamagnetic iron oxide nanoparticles (SPIONs), 242 Surface-enhanced Raman spectroscopy (SERS), 220
222, 398, 409 Surface plasmon resonance (SPR), 397
398, 400
401, 400f Surfactant-modified aluminosilicates functionality of, 298
303 preparation and characterization of, 294
298 Surfactant-modified zeolites (SMZs), 296, 298, 302 Surfactants, 288, 289f

Synperonics, 178
179 Synthetic polymers, 363
364, 403 Syzygium aromaticum, 13
14

T T7 bacteriophages, 412
413 Tao
Eldrup equation, 124 Targeted chemotherapy, 431 “Targeted” drug-delivery system (TDDs), 431 Tertiary amines, in situ quaternization of, 162
164 Tetra-arsenic tetrasulfide As4S4 polymorphs, 119 AS4S4/ZnS NC characterization methodology, 121
126 atomic-deficient structure, 123
126 atomic-relevant structure, 121
123 biological activity, 126 mechanochemical synthesis of As4S4/ZnS NCs in a dry-milling mode, 121 of As4S4/ZnS-PX407 NSS in a wet-milling mode, 121 nanoparticle (NP)-guided functionality in AS4S4/ZnS NCs, 127
146 atomic-deficient structure of AS4S4/ZnS NCs, 130
140 biological activity of AS4S4/ZnS NPs, 142
146 characterization of AS4S4/ZnS NCs prepared in a dry-milling mode, 127
130 characterization of AS4S4/ZnS-PX407 NSs prepared in a wet-milling mode, 140
141 Tetronics, 190, 192 T304, 199 T904, 194
195, 200 T1107, 201
202 Theranostics, 452
454 Therapeutic proteins, 197
198 Thin films, nanocoatings and, 463 nanocoating fabrication methods, 464
472 dip-coating method, 464
468 matrix-assisted pulsed laser evaporation method, 468
472 Thiolate-protected gold nanoparticles, 4 3D extracellular matrix (ECM), 246
247 Time factors, 389 Tissues and cells, imaging of metal nanoparticles in, 408
409 Titanium, 466
467 Titanium implants, 466 Toll-like receptor (TLR9), 408 Topical anesthetics, 430 Topoisomerase (TOPOS) inhibitors, 429
430

Index

Transmission electron microscope (TEM), 344, 444 Triblock polymer (TBP), 180
181, 183 Trichoderma koningiopsis, 14 Triethylene glycol dimethacrylate (TEGDMA), 167 3-(Trimethoxysilyl)-propyldimethyloctadecyl ammonium chloride (SiQAC), 158
159 Triple junctions (TJs), 130 Tuberculosis (TB), 228
229 Tulsi (Ocimum sanctum), 9

U Urea biosensor, 385 Urothelium/urinary bladder models, 266
267

V Vapor
liquid
solid method (VLS), 335 Voight model, 60

W Water vapor concentration, monitoring of, 374

X Xanthine molecules, 361
362 X-ray diffraction (XRD) analysis, 297, 310

Z Zeolite, 287, 294
295 Zeta potential (ZP), 122, 444 Zinc (Zn), 362 Zinc oxide nanoparticles, 19
24 Zinc sulfide (ZnS), 137 Zingiber officinale, 13
14 Zirconium (Zr), 362

487