Recent Developments in Bio-Nanocomposites for Biomedical Applications 9781617615139

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BIOTECHNOLOGY IN AGRICULTURE, INDUSTRY AND MEDICINE

RECENT DEVELOPMENTS IN BIO-NANOCOMPOSITES FOR BIOMEDICAL APPLICATIONS

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BIOTECHNOLOGY IN AGRICULTURE, INDUSTRY AND MEDICINE

RECENT DEVELOPMENTS IN BIO-NANOCOMPOSITES FOR BIOMEDICAL APPLICATIONS

ASHUTOSH TIWARI EDITOR

Nova Science Publishers, Inc. New York

Copyright © 2011 by Nova Science Publishers, Inc. All rights reserved. No part of this book may be reproduced, stored in a retrieval system or transmitted in any form or by any means: electronic, electrostatic, magnetic, tape, mechanical photocopying, recording or otherwise without the written permission of the Publisher. For permission to use material from this book please contact us: Telephone 631-231-7269; Fax 631-231-8175 Web Site: http://www.novapublishers.com NOTICE TO THE READER The Publisher has taken reasonable care in the preparation of this book, but makes no expressed or implied warranty of any kind and assumes no responsibility for any errors or omissions. No liability is assumed for incidental or consequential damages in connection with or arising out of information contained in this book. The Publisher shall not be liable for any special, consequential, or exemplary damages resulting, in whole or in part, from the readers’ use of, or reliance upon, this material. Any parts of this book based on government reports are so indicated and copyright is claimed for those parts to the extent applicable to compilations of such works. Independent verification should be sought for any data, advice or recommendations contained in this book. In addition, no responsibility is assumed by the publisher for any injury and/or damage to persons or property arising from any methods, products, instructions, ideas or otherwise contained in this publication. This publication is designed to provide accurate and authoritative information with regard to the subject matter covered herein. It is sold with the clear understanding that the Publisher is not engaged in rendering legal or any other professional services. If legal or any other expert assistance is required, the services of a competent person should be sought. FROM A DECLARATION OF PARTICIPANTS JOINTLY ADOPTED BY A COMMITTEE OF THE AMERICAN BAR ASSOCIATION AND A COMMITTEE OF PUBLISHERS. Additional color graphics may be available in the e-book version of this book. LIBRARY OF CONGRESS CATALOGING-IN-PUBLICATION DATA Recent developments in bio-nanocomposites for biomedical applications / editor, Ashutosh Tiwari p. ; cm. Includes bibliographical references and index. ISBN 978-1-61761-513-9 (eBook) 1. Nanomedicine. I. Tiwari, Ashutosh, 1945[DNLM: 1. Nanocomposites. 2. Nanomedicine. QT 36.5] R857.N34R43 2010 610.28--dc22 2010028626

Published by Nova Science Publishers, Inc. † New York

CON TEN TS Preface Chapter 1

Chapter 2

Chapter 3

Chapter 4

ix Polysaccharides Based Amphiphilic Nanocarriers for Potential Drug Delivery Applications Ashutosh Tiwari, Ajay K. Mishra, Shivani B. Mishra, Rajeev Mishra, S.K. Shukla, and Anjali M. Rahatgaonkar

1

Bionanocomposites – Current Status and Prospects in Drug Delivery Fields M. Prabaharan, R. Jayakumar and Ashutosh Tiwari

17

Design of a Biocompatible Nanocomposite Particulate and Drug Delivery System Roli Mishra and Satyendra Mishra

41

Recent Progress in Ceramic Nanomaterials for Biomedical Application Shivani B. Mishra, Ajay K. Mishra, Ashutosh Tiwari, Radhe Shyam Rai and Anjali M. Rahatgaonkar

Chapter 5

Biomedical Applications of ZnO Nanostructures Ahsanulhaq Qurashi

Chapter 6

Sol-Gel Derived Sio2-Chitosan/Carbon Nanotubes- Promising Matrices for Bio-Recognition Events Ashutosh Tiwari, Ajay K. Mishra, Radhe Shyam Rai, Shivani B. Mishra, Shunsheng Cao, Rajeev Mishra, S.K. Shukla, Smarti Bhadoria, Premlata Kumari and Mani Prabaharan

Chapter 7

Iron Nano-Structure: A New Dimension in Biomedical Science Mainak Ganguly, Anjali Pal and Tarasankar Pal

Chapter 8

Polysaccharide-Silica Nanocomposites: Synthesis, Characterization and Potential Applications Vandana Singh and Pramendra Kumar

57

73

91

107

133

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Contents

Chapter 9

A Perspective on Toxicology of Nanoparticles Smrati Bhadauria, Rajeev Mishra, Ashutosh Tiwari

Chapter 10

Fabrication of Bionanocomposites from Natural Biopolymer Matrices and Inorganic Nanofillers Ajay K. Mishra, Shivani B. Mishra, Ashutosh Tiwari, and Radhe Shyam Rai

Chapter 11

Nanomaterials for Cancer Diagnostics and Therapeutics Rajeev Mishra, Smrati Bhadauria, Jyoti Mishra and Ashutosh Tiwari

Chapter 12

Synthesis of Novel Stimuli Responsive Nanocomposites for Biomedical Applications Emilio Bucio, H. Iván Meléndez-Ortiz, Takashi Isoshima and Javier Macossay

153

173

191

207

Chapter 13

Surface Plasmons in Biomedicine Miguel Angel Garcia

233

Chapter 14

Biocompatible Nanocomposites for Bone Tissue Engineering Chunyan Wang, Minghui Yang, Zhiyong Qian and He Li

261

Chapter 15

Composites of Chitosan for Biomedical Applications Nazma Inamdar and V.K.Mourya

277

Chapter 16

Nanomaterials in the Advancement of Electrochemical DNA Biosensors Anees A. Ansari

Chapter 17

Development in Diagnosis and Treatment with Nanotechnology Rajiv Lochan Gaur, Rajeev Mishra, Richa Srivastava, Smriti Bhadauria and Ashutosh Tiwari

Chapter 18

Bio-Nanocomposites Based on Naturally Occurring Common Polysaccharides Chitosan, Cellulose and Starch with their Biomedical Applications Hassan Namazi and Mohsen Mosadegh

Chapter 19

Development of Bio-Friendly Energy Harvesting Materials Radhe Shyam Rai, Ashutosh Tiwari, Ajay K. Mishra and Shivani B. Mishra

Chapter 20

Recent Advances in Biomedical Applications of Multifunctional Nanocomposites Avinash C. Pandey, Prashant K. Sharma and Ranu K. Dutta

Chapter 21

Bio-Nanomaterial Quantum Dots: A Boon for Medical Sciences Anjali M. Rahatgaonkar, Ashutosh Tiwari, Mukund S. Chorghade, Shivani B. Mishra and Ajay K. Mishra

345 369

379 399

409 433

Contents

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Chapeter 22

Hydrogel Nanocomposites in Biology: Design and Applications Premlata Kumari, Ashutosh Tiwari, Ajay K. Mishra, Radhe Shyam Rai and Shivani B. Mishra

459

Chapter 23

Cellulose Based Bio- Nanocomposites: Tailoring and Applications S.K. Shukla, N.G. Giri, V.K. Singh and Ashutosh Tiwari

467

Index

498

P REFACE Bio-nanocomposites form a unique class of a research area that integrates biology, chemistry, materials science, engineering and nanotechnology to present an interdisciplinary approach for solving of problems. In today’s world, bio-nanocomposites are becoming increasingly prevalent due to the extraordinary properties that they possess. Scientists learn to select suitable matrices (e.g. aliphatic polyesters, polypeptides and proteins, polysaccharides, and polynucleic acids) and fillers (e.g. nanotubes, nanofibers, clay nanoparticles, hydroxyapetite and metal nanoparticles) and alter their chemistry and structure to suit the target field. This book provides the most recent research made in the field of bionanocomposites as applied to biomedical fields including drug-delivery, biosensors, cancer diagnosis, and tissue engineering. Chapter 1 - The amphiphilic systems based on polysaccharides and their derivatives as drug delivery nanocarriers have recently gained much interest due to their biocompatibility, biodegradability, ability to form hydrophobic clusters, drug loading and controlled release properties. They are mostly used as nano-sized micelles which are supramolecular core-shell structures formed by self-aggregation of individual amphiphilic macromolecules comprised of hydrophobic and hydrophilic domains. This book chapter discuss about the polysaccharides and their important amphiphilic derivatives that have been studied and exploited as carriers for drug delivery applications. Specific attention has been drowning on the preparation amphiphilic nanocarriers with some important polysaccharides viz. alginate, dextran, carrageenan, hyaluronic acid, guar gum and xanthan and their derivatives Chapter 2 - Bionanocomposites (BNCs) have established themselves as a promising class of hybrid materials derived from natural and synthetic biodegradable polymers and organic/inorganic fillers. A critical factor underlying biomedical nanocomposite properties is interaction between the chosen matrix and the filler. This chapter discusses current efforts and key research challenges in the development of these composite materials for use in potential drug delivery applications. BNCs discussed here include layered BNCs, layered double hydroxides (LDHs), inorganic BNCs, polymeric BNCs, chitosan-based hydrogels and magnetic BNCs. From this review, it is concluded that BNCs would be promising materials for drug delivery applications. Chapter 3 - Nanocomposite biocompatible hydrogels (NCHG) are an important class of biomaterials that can be utilized in applications such as drug delivery, tissue engineering, and hyperthermia treatment. The composite consists of the following components:nanoparticles (NPs), matrix gel, and chlorhexidine (CHX) as antibacterial drug. The NPs were obtained by

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free radical initiated copolymerization of the monomers, 2- hydroxyethyl methacrylate (HEMA) and polyethyleneglycol dimethacrylate (PEGDMA), in aqueous solution. The same monomers were used to prepare crosslinked matrices by photopolymerization. NCHGs were obtained by mixing NPs, monomers, and drug in an aqueous solution then crosslinked by photopolymerization. The incorporation of nanoparticles into a hydrogel matrix can provide unique properties including remote actuation and can also improve properties such as mechanical strength. Since hydrogel nanocomposites have been proposed as implantable biomaterials, it is important to analyze and understand the response of the body to these novel materials. This chapter covers the background, definitions, and potential applications of hydrogels and hydrogel nanocomposites. It also covers the various types of hydrogel nanocomposites as defined by the nanoparticulates embedded in the systems which include clay, metallic, magnetic, and semiconducting nanoparticles. The specific concerns of the biocompatibility analysis of hydrogel nanocomposites are discussed along with the specific biocompatibility results of the nanoparticulates incorporated into the hydrogel matrices as well as the biocompatibility of the hydrogels themselves. The limited data available on the biocompatibility of hydrogel nanocomposites is also presented. Overall, currently investigated hydrogel systems with known biocompatibility may have the potential to provide a “shielding” effect for the nanoparticulates in the hydrogel nanocomposites allowing them to be safer materials than the nanoparticulates alone. Chapter 4 - Fine tuning of the material at atomic, molecular and macromolecular level touching the nanoscale, significantly improves the properties of the material in nanotechnology. This is a multidisciplinary field that encompasses various disciplines of fundamental and applied sciences viz. physics, material science, colloid and interface science, chemical engineering, mechanical engineering, electrical engineering. Some other fields that are benefitting from the nanotechnology are supra-molecular chemistry, robotics, photonics, optoelectronics, and biotechnology. An improvement in safety, quality of life and health, potential possible solution to industrial challenges, environment protection and pollution control are some of the great expectations of nanotechnology. Chapter 5 - Nanostructures have attracted tremendous attention from researchers in various disciplines because their high surface-to-volume ratio and high crystal quality are highly desirable for many technological applications including biosensors, tissue engineering and drug delivery system. Several synthetic methods have been used to fabricate various nanostructures. These synthetic approaches are mainly categorized into two main classes according to how the nanostructure is formed: dry and wet chemical synthesis. Both the methods have their pros and cons respectively. Various methods were developed for functionalization of nanostructures to modify their surfaces. Different techniques for the effective biofunctionalization of one-dimensional (1D) ZnO nanostructures were illustrated in this chapter. Various biomolecules like human serum albumin, bovine serum albumin, angiotensin II and DNA molecules were effectively immobilized by modifying the surface of 1D ZnO nanostructures. Molecular functionalization of ZnO nanobelts demonstrated which improved the optoelectronic and electrical properties. ZnO nanostructures were also studied for advanced biological applications like fluorescence detection, cellular biocompatibility, biosafety, biosensor, and mammalian cell adhesion. Chapter 6 - The performance of enzyme based biosensors usually depends on the physicochemical properties of the electrode materials as well as process of the enzyme immobilization and also enzyme concentration on the electrode surface. Although, various

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matrices are reported in the literature for the immobilization of enzymes to use in biosensors, the method of immobilization and electrode matrices, both are considered promising factor during the determination of the operational and storage stability of the biosensors. The intrinsic stability of enzymes has encouraged for applying biomaterials engineering to improving stability. In this sequence, the use of nanomaterials such as carbon nanotubes (CNTs) to fabricate matrices for biosensors is one of the most exciting approaches because nanomaterials have a unique structure and high surface-to-volume ratio. The present chapter deals the preparation of SiO2-chitosan/CNTs (SiO2-CHIT/CNTs) bio- nanocomposite, enzyme immobilization, characterization, electrochemical behavior, bio- recognition, interference, stability and response study of bio- analytes for the potential biosensor applications. Chapter 7 - The iron oxide nanostructures have been extensively used in biomedical science. After certain modifications, they can play very promising roles in Magnetic resonance imaging (MRI), bioseparations, drug delivery, hyperthermia, biosensors etc. As T2 contrast agents, they are very well accepted. Super paramagnetic behavior added a new dimension in medical science. How various coating and doping help the iron nano particles in lot of biomedical applications have been discussed with examples. Detection of targeted cells by different methods is also stated. The recent development of iron nanobio composite is certainly surprising. But, they have some roles also regarding environmental toxicity which may affect the health of humans and animals. Chapter 8 - Polysaccharides are abundant and renewable biological materials having interesting physical properties e.g. film-forming, gelling and thickening. They can be easily processed in different forms such as beads, films, capsules and fibers. Their incorporation in silica gels is of great interest in the context of biotechnological applications towards the development of device and in the preparation of high performance and multifunctional materials. A large number of polysaccharides/modified polysaccharides silica nanocomposites have been designed using sol-gel technology where interaction between inorganic and organic components range from strong covalent chemical bonds to weak hydrogen/Vander Waals bonds, though electrostatic interactions have been also witnessed in some cases. Such polysaccharides-silica nanocomposites are being successfully used as matrices for immobilization of living prokaryotic cells in the biodegradation processes of toxic environmental pollutants, as effective adsorbents in the removal of metal ions from solutions and for sensor applications. Present chapter elaborates the sol-gel synthesis, characterization and application of a number of polysaccharide-silica nanocomposites. Different schematic presentation herein will give an insight that how the polysaccharide and silica precursor type effect the composition and the final properties of the composites. Chapter 9 - Nanotechnology involves the manipulation of matter on a near atomic scale to produce new structures, devices, and materials. Consequently nanoparticles are particles that have at least one dimension in the range of 1–100 nm. Nanomaterials can be broadly categorized in two classes (a) carbon-based materials such as fullerenes (b) carbon nanotubes and inorganic nanoparticles including those based on metal oxides (iron oxide, cerium oxide, titanium dioxide, silicon dioxide, etc), metals (gold and silver), and semiconductor nanoparticles or so-called quantum dots (typically cadmium sulfide and cadmium selenide). Some experts consider the advent of nanotechnology to be a revolution that will pave way for many exciting developments and applications Nanomaterials are expected to have significant applications in diverse areas such as consumer and industrial products, energy, medicine etc.

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Nanomaterials are being explored to be used for drug delivery and for medical imaging purposes [1-3]. Such a diverse application potential emerges from the fact that nanomaterials have unique physical properties and quantum mechanics that vary significantly with changes in the size to the range of nanoscale (1-100 nm). Interestingly at the nanoscale, fundamental mechanical, electrical, optical, and other properties can significantly differ from their bulk material counterparts. These novel properties of nanomaterials offer great promise to provide new technological breakthroughs. However such varied applications also demand extensive knowledge of potential adverse effects and associated health hazards. It is also an important consideration is to determine whether nanoparticles cause new types of effects not previously seen with larger particles or bulk chemicals? Furthermore, if this is indeed the case, how can one measure and predict such nano-specific effects? Various studies have been conducted to assess toxicity potential of nanoparticles and results have raised concerns regarding the health hazards of certain nanoparticles, such as carbon nanotubes [4-6] and quantum dots [7].The health risk posed by a nanoparticle is an outcome of its hazard potential to human health and its exposure potential. The hazard or toxicity of a nanoparticle is the ability for the substance to affect adversely or cause severe morbidity and/or mortality. The exposure potential of a nanoparticle is determined by its bioavailability to humans through different routes such as inhalation, ingestion, and dermal pathways. Exposure potential also includes its ability to accumulate, persist, and translocate within the environment and the human body. Both these parameters are a function of the physicochemical properties of any toxicant including nanoparticles. In order to be able to asses these effects accurately, one must have foolproof assay systems and methodology with full knowledge about the existing limitations of the system. Keeping this in view the current chapter will briefly describe the physicochemical characteristics of nanomaterials that may impact toxicological potential various target organ systems, cellular targets of toxicity, methods of evaluating toxicity and the limitations of these methods. Nanoparticles may pose toxicological hazards due to their enhanced reactivity (for example, chemical, electrical, and magnetic) and potential for systemic availability. The difference in physicochemical properties of nanomaterials than that of bulk material can significantly modify cellular uptake, protein binding, translocation from sitel-of-entry to the target site, and the potential for causing tissue injury [8;9]. The theoretical ability for nanomaterials to cause adverse effects on biological systems is a major point of concern for toxicologists, and regulatory agencies. Several studies addressing health and safety issues associated with the development and use of nanomaterials have been published in the scientific literature [8;10-14]. Apart from vulnerabilility of the biological system to exogenous agents, the nanoparticles themselves may have certain inherent attributes that may render them toxic. The following section describes various attributes of nanoparticles that can contribute to its toxic potential Chapter 10 - Fabrication of biodegradable type of composites have been made and utilized for various purposes when even the term science and technology did not even come into existence. It is surprising although that how our forefathers with very few resources were able to develop the materials that are existing till date. Some of the examples of these antique composites made from biopolymer in famous Great Wall of China whose construction started initially in 121 B.C. as earth works were connected and made strong by clay bricks-made of local materials initially using red willow reeds and twigs with gravel during the Han dynasty (209 B.C.), and later with clay, stone, willow branches, reeds and sand during the Qin dynasty (221–206 B.C.) [1-3]. Other examples include, bows made with adhesively bonded laminates

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of animal horns and tendons, wood or silk used by the Mongolians in 1200 A.D. Composite materials have gone through significant developments in terms of use of different raw materials, processes and even applications. Similarly, use of natural polymers is not new, since paper, silk, etc., have been used from historical times. The use of natural polymers was superseded in the 20th century as a wide-range of synthetic polymers was developed based on raw materials from low cost petroleum. Chapter 11 - Nanotechnology is a multidisciplinary field, which covers a vast and diverse array of devices derived from engineering, biology, physics and chemistry with broad application for molecular imaging, diagnosis, and targeted therapy. Recently it has emerged as one of the most propitious field in cancer treatment. Nanotechnology is definitely a medical boon for diagnosis, treatment and prevention of cancer disease. It supports and expands the scientific advances in genomic and proteomics and builds on our understanding of the molecular underpinnings of cancer and its treatment. The promise of nanotechnology lies in the ability to engineer customizable nanoscale constructs that can be loaded with one or more payloads such as chemotherapeutics, targeting units, imaging and diagnostic agents. This chapter addresses some of the major milestones in the integration of nanotechnology and cancer biology, and the future of nanoscale approaches for cancer management. Chapter 12 - Recent advances in the fields of polymers and nanocomposites have been attracting both academic and industrial attention because the dramatic improvement exhibited in the properties of these composites even at low filler contents. Some examples of the technologies developed over the last few years utilizing the scientific advances in nanocomposites and nano-biotechnology can be found in bio-mimicry, diagnostics, and therapeutics. This chapter reviews the general structure, preparation and properties of polymers and nanocomposites used in biomedical applications. Furthermore, the chapter discusses state of the art smart polymers obtained by gamma rays, using direct method and pre-irradiation method, and their nanocomposites with potential biomedical applications. Chapter 13 - Surface plasmons (SPs) consist on collective oscillations of conduction electrons in metallic nanoparticles (NPs). The capability of SPs to concentrate the light and enhance locally the associated electric field, provides optical properties not achievable with any other optical process. Those effects generate a large number of applications in biomedicine as direct nanoparticles-based detection and imaging techniques, local heating and amplification of other processes. This chapter presents an overview of SPs in NPs for non specialist in the field. It describes the fundamentals of the SP and the relationship between the morphological features of the NPs (size, shape, surrounding media, nanoparticles ensembles) and the SP. Finally, it also surveys the main applications of the SP in biomedicine which has been recently developed. Chapter 14 - With the increasingly aging population, paramount people need the repair of bone fractures or defects. To achieve the restoration of native tissue architecture, biocompatible nanocomposite scaffold with biological elements to stimulate cell proliferation and differentiation and eventually osteogenesis is prominence for the bone repair. Natural bone is a composite which comprising inorganic and organic materials. The biomimetic nanocomposites prepared by polymers including natural and synthetic polymers and different inorganic bioactive materials have been extensively studied for bone tissue engineering. The 3D nanocomposite scaffolds can be organic/inorganic composites in the form of sponge-like, nanofibrous and sintered microspheres or inorganic/inorganic composites.

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Chapter 15 - The nanocomposites are multiphase materials or hybrids in which at least one of the components has dimension in the nanometer scale. These materials exhibit extraordinary mechanical and physical properties than the cocstituent phases. The composites can be tailored to meet the requirements of a particular application with manipulations of these properties. For biomedical applications the polymer composites are of value. Chitoasn is a much appreciated polymer in this respect because of its biocompatbility, biodegrability, nontoxicity and cationic nature. The composites of chitosan can be elaborated with inorganic materials as well as with polyionic materials with polyelectrolyte complex formation. The comopites with calcium phosphate based materials, clays are evaluated for tissue engeneering applications along with their drug eluting properties. The composites of chitosan with metal oxide nanoparticles, metal nanoparticles, carbon nanotubes are maneuvered as biosensors. Similar operations can be developed with chitosan/quantum dots composites in addition to the bioimaging functions. The plethora of biomedical applications is put forword by chitosan/polyion complexes. The future potential of chitosan composites in biomedical applications is very promising for therapeutics and diagnosis. The awareness and the emergence of the analytical protocols for quality assessment of chitosan will see to these applications without delay. Chapter 16 - Nanomaterials are increasingly used for the construction of electrochemical DNA biosensors. Nano-scale materials offer excellent prospects for interfacing biological recognition events with electronic signal transduction for designing a new generation of bioelectronic devices exhibiting novel functions. Particularly, nanomaterials such as noble metal nanoparticles (Au, Ag, Pt, Pd), carbon nanotubes (CNTs), magnetic nanoparticles, quantum dots and metal oxide nanoparticles have been actively investigated for their applications in electrochemical DNA biosensors, which have become a new interdisciplinary frontier between biological sciences and material science. In this article, discussed some main advances and explore the application prospects and discuss the issues, approaches, and challenges, with the aim of stimulating a broader interest in the development of nanomaterialbased electrochemical DNA biosensors. Chapter 17 - Our survival on the planet depends on statistics about smart and efficient dealing with diseases. We are always exposed and encountering to old and new emerging disease. The law of survival (survival of fittest) implies on every living creature including parasites and they have way better strategy to endure the adverse because of simple structure and easy gene modification. Besides these they compensate by other mutations to the modification accrued in process to protect themselves from human strategies (like pressure of eliminate as drug). Considering all these together, we are dealing with disease causing organisms which are changing rapidly. On the other hand we have environmental stress which changes our own milieu rapidly. The changes may end up with disease like cancer. We are exposed to face these unwanted changes and are bound to develop the disease. With all the disease and there consequences, we need earliest diagnosis and best treatment to combat it. Till last few decades we have had traditional diagnosis and treatment. As we moved forward to present scenario we can rely on technological advancement to deal with disease. Finer detail and mechanisms can help us to design new and best effective strategies against these lethal threats to humankind. The technological revolution has significantly improved our approach towards diagnosing and treatment of diseases. With recent advancement, now we have better device to observe phenomenon behind the disease at molecular level. This will be the base of our future diagnosis and treatment. With this strategy we can go to the level of

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atom and reach to better understanding. The finer details enable us to look at nanostructures and find out the markers in different body fluids (like blood plasma or tissue fluid). We have equipped ourselves with all these advance tools and technologies but these are not used as efficiently as they should. There are several problems to be considered like distribution of technology, efficiency of use and above all the cost. Considering all these consequences, we are dealing with two aspects of nanotechnology in this book chapter; diagnosis and treatment. Chapter 18 - This review paper reports recent advances in the field of naturally occurring polysaccharide-based nanocomposites and their biomedical applications. These types of materials have attracted a lot of attentions in both academic and industrial sector. While organic biomedical agents show good inhibition efficiency and a broad spectrum of activity however, their relative low stability (e.g., low decomposition temperature and short life expectancy) cannot be ignored. As a result, there is urgent need to develop biopolymer based nanocomposited materials provided with dual bioactive advantages of organic biomedical agents and inorganic biomedical agents as they will become more important in the future. To achieve this goal polysaccharide-based (such as chitosan, cellulose and starch) nanocomposites have been developed and clinically tested. Since, they combine the structure, physical and chemical properties of both inorganic and organic materials. Most work with polysaccharide nanocomposites have concentrated on biomedical applications due to their non toxic, drug delivery, biodegradable, biocompatible, wound dressing, and etc. properties. In addition to these properties the chitosan has excellent antibacterial activity. Therefore, polysaccharide nanocomposites as bio-nanocomposites are widely used for biomedical applications such as tissue engineering scaffolds, drug delivery, wound dressing and antibacterial film. For instance, both, poly(ethylene glycol) (PEG) and chitosan (CS) played vital roles in the reduction of metal ions into nanoparticles (NPs) as well as provided good stability to the formed bio-nanoparticles. These biopolymers not only help in reducing the metal ions into nanoparticles but also provide distinguished stability for a sustained release of nanoparticles for antibacterial applications. The developed porous nanocomposite film has exhibited superior antibacterial properties and good mechanical properties than the chitosan and chitosan–silver nanocomposites, Chapter 19 - Jacques and Pierre Currie discovered the phenomenon of piezoelectricity in 1880, category of smart materials exhibiting unique and interrelated properties. Application of stress to a piezoelectric crystal generates a corresponding electric charge. Conversely the application of an external voltage will induce a shape change. Many materials display piezoelectric properties, some of which are naturally occurring e.g. Quartz, whilst others are engineered to display the properties, e.g. lead zirconate titanate (PZT), polyvinylidene fluoride (PVDF), Sodium Potassium Niobate and etc. The polymer materials are soft and flexible; however have lower dielectric and piezoelectric properties than ceramics. Monolithic piezoelectric ceramics are rigid, heavy and produced in block form; therefore add additional mass and stiffness to the host structure, especially when bonding to flexible/lightweight materials. Chapter 20 - Nanocomposites are a subset of composites that take advantage of unique materials properties on the small scale. The definition of nano-composite material has broadened significantly to encompass a large variety of systems such as one-dimensional, two-dimensional, three-dimensional and amorphous materials, made of distinctly dissimilar components and mixed at the nanometer scale. A nanocomposite is similar to a multiphase solid material where one of the phases has one, two or three dimensions of less than 100

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nanometers (nm), or structures having nano-scale repeat distances between the different phases that make up the material [1]. In the broadest sense this definition can include porous media, colloids, gels and copolymers, but is more usually taken to mean the solid combination of a bulk matrix and nano-dimensional phase(s) differing in properties due to dissimilarities in structure and chemistry. The mechanical, electrical, thermal, optical, electrochemical, catalytic properties of the nanocomposite will differ markedly from that of the component materials. Chapter 21 - Bionanomaterials constitute a fascinating class of hybrid materials derived from natural and synthetic biodegradable polymers and organic/inorganic fillers. Bionanocomposites are part of an emerging and promising interdisciplinary scientific arena that bridges biology, chemistry, materials science, and nanotechnology. Recent applications of nanomaterials impact numerous diverse areas, particularly, electronics, catalysis and biomedical research. This review summarizes key concepts of current nanotechnology that can be utilized for biomedical research or can be elaborated for use in biological systems. Chapter 22 - Hydrogels are three dimensional polymeric networks with the ability to swell several times their dry weight by absorbing water and other biological fluids. Hydrogel nanocomposites are new class of biomaterial that has recently attracted a lot of attention for applications in medical and pharmaceutical areas. The nanopaticles such as clay, ceramic, metallic or metal oxides dispersed in hydrogel matrix to form hydrogel nanocomposites. In this chapter, rapidly emerging field of hydrogel nanocomposites are introduced and hydrogels nanocomposites containing different types of nanoparticles like hydroxyapatite and tricalcium phosphate are highlighted. In particular, tissue engineering applications of few hydrogels have been highlighted. Chapter 23 - The biomedical and environmental problem poses a considerable interest in the development of renewable resource based nanocomposites materials. They are formed by the combination of natural polymers and inorganic solids, with at least one dimension on the nanometer scale. These biohybrid materials exhibit improved structural and functional properties of great interest for different applications. The properties inherent to the biopolymers, that is, biocompatibility and biodegradability, open new prospects for these hybrid materials with special incidence in regenerative medicine and in environmentally friendly materials (green nanocomposites). The cellulose based nanocomposites (CBNC) are widely used in automotive industry, building materials, packaging materials and in biomedicines. CBNC have established themselves as a promising class of hybrid materials derived from natural and synthetic biodegradable polymers and organic/inorganic fillers. Different chemistries and compositions can lead to applications in tissue engineering, loadbearing composites for bone reconstruction. A critical factor underlying biomedical nanocomposite properties is interaction between the chosen matrix and the filler. This article discusses current efforts and key research challenges in the development of these materials for use in potential biomedical applications.

In: Recent Developments in Bio-Nanocomposites… Editor: Ashutosh Tiwari

ISBN 978-1-61761-008-0 © 2011 Nova Science Publishers, Inc.

Chapter 1

POLYSACCHARIDES BASED AMPHIPHILIC NANOCARRIERS FOR POTENTIAL DRUG DELIVERY APPLICATIONS Ashutosh Tiwari1,2, Ajay K. Mishra3, Shivani B. Mishra3, Rajeev Mishra4, S.K. Shukla5, and Anjali M. Rahatgaonkar5 1

School of Materials Science and Engineering, Jiangsu University, Zhenjiang 212 013, China 2 National Institute for Materials Science, Tsukuba, Ibaraki 305 0047, Japan 3 Department of Chemical Technology, University of Johannesburg, Doornfontein, Johannesburg 17011, South Africa 4 Department of Cancer Genetics, School of Medicine, Nihon University, Tokyo 173 8610, Japan 5 Department of Polymer Science, Bhaskaracharya College of Applied Science, University of Delhi, New Delhi 110 075, India 6 Department of Chemistry, Institute of Science, Civil lines, R.T. road, Nagpur, MS 440001, India

ABSTRACT The amphiphilic systems based on polysaccharides and their derivatives as drug delivery nanocarriers have recently gained much interest due to their biocompatibility, biodegradability, ability to form hydrophobic clusters, drug loading and controlled release properties. They are mostly used as nano-sized micelles which are supramolecular core-shell structures formed by self-aggregation of individual amphiphilic macromolecules comprised of hydrophobic and hydrophilic domains. This book chapter discuss about the polysaccharides and their important amphiphilic derivatives that have been studied and exploited as carriers for drug delivery applications. Specific attention has been drowning on the preparation amphiphilic nanocarriers with some important 1

Corresponding author: E-mail: [email protected]; Tel: (+86) 511-8879-0191; Fax: (+86) 511-8879-0769.

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Ashutosh Tiwari, Ajay K. Mishra, Shivani B. Mishra et al. polysaccharides viz. alginate, dextran, carrageenan, hyaluronic acid, guar gum and xanthan and their derivatives

1. INTRODUCTION Micelles formed from amphiphilic block copolymers have been paid wide attention in the field of controlled drug delivery because they can solubilize various hydrophobic drugs, increase bioavailability, and stay unrecognized during blood circulation [1]. Polymeric micelles used for drug delivery in intravenous administration must be non-toxic to human body. In this reverence, polysaccharides and their derivatives with amphiphilic characters have recently gained much interest due to their biocompatibility, ability to form hydrophobic clusters, drug loading and controlled release ability [1,2]. The presence of hydrophobic groups bound to the polysaccharide chain can lead to the formation of large aggregates [3], which can change solution properties such as viscosity [1], surface tension [4] and solubility [5]. Charged associative polysaccharides permit to conjugate high amount of grafted hydrophobic moieties together with good water solubility. Their solution behavior is governed, especially in the dilute regime, by the occurrence of both long range electrostatic repulsions and hydrophobic attractions. In recent years, a number of studies have reported on polysaccharide based nanocarriers for drug delivery. In addition, more recent progresses have been made on nanocarriers based on polysaccharides as matrices for controlled release of drugs. Concerning the topic, physically cross-linked amphiphiles are of great interest, particularly because the micelles formation in the nano size ranges can be often carried out under mild conditions and in the absence of organic solvents. The number of polysaccharides that have been investigated for the preparation of micelles as delivery systems is extremely large; therefore including all the polysaccharides in one chapter would be difficult task. Thus, in this chapter, attention has been focused only on the recent developments of nanocarriers based on polysaccharides such as alginate, dextran, carrageenan, hyaluronic acid, guar gum, xanthan and their derivatives for drug delivery and related applications.

2. ALGINATE Alginate is generally extracted from marine brown algae or it can also be produced by bacteria. It consists of a backbone of (1→4) linked β-D-mannuronic acid (M) and α-Lguluronic acid (G) residues of widely varying composition and sequence (Figure 1). The physico-chemical properties of alginate have been found to be highly affected by the M/G ratio as well as by the structure of the alternating zones. An enzymatic pathway was recently used to obtain sequences with a known M/G ratio and this procedure is potentially very helpful in rationalizing the conformational, and therefore the micellar properties, of the alginate samples [6]. For example, in the pharmaceutical field, alginate has been used as excipient in tablets, with modulated drug delivery dosage forms being developed from its micellar forms [7, 8]. Due to the intrinsic properties of alginate micelles such as biocompatibility, mucoadhesion, biodegradability and ease of manipulation much attention

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has recently been focused on the delivery of proteins, drug encapsulation, and tissue regeneration.

Figure 1. Chemical structure of Alginate.

In terms of drug/protein delivery, numerous applications of alginate micelles or microand/or nano-spheres have been proposed and here we report the most representative studies and their results. In order to optimize the modulation of drug release from such systems, most of these studies have centered on improving the stability and the resistance to erosion in different organic fluids. With this aim, alginate was modified by means of macromolecules, which are able to establish ionic interactions with the alginate carboxylate ions, thus forming a shell around the alginate micellar systems that, in turn, become more resistant and suitable for numerous applications. In this respect, chitosan, chitosan derivatives, and poly-L-lysine were used as typical surface modifying agents [9-12]. Another approach for the surface modification of alginate micelles and spheres has been the chemical modified around the alginate core. For instance, micelles with a multilayer shell of chitosan linked with tripolyphosphate were able to modulate the release of ampicillin in in vitro experiments, showing an improved resistance of the micelles [13]. Genipine, a natural cross-linker for chitosan, was used in the preparation of alginate-chitosan beads resulting in a chemical micelles formation occurring in the core of the bead or onto the outer shell, according to the specific procedure followed for the preparation [14]. The resulting systems, loaded with indomethacine or bovine serum albumin (BSA), as a model protein drug, showed a modulated release that was dependent on the specific applied procedure and on the pH of the release medium. Specifically, when the outer shell was made up of a multilayer of poly(allylamine hydrochloride)/poly(styrene sulfonate) such micelles showed the peculiar behavior of “attracting” positively charged macromolecules inside their structure. This technique is potentially very promising for the macromolecular drug delivery in biomedical applications [15]. Likewise, alginate derivatives have been prepared in order to obtain chemical or physical micelles with properties different from those observed when the natural polymer was used [16]. Alginate was chemically modified into low molecular weight oligomers that were then cross-linked with a biodegradable spacer, such as adipic dihydrazide, to form biodegradable micelles [17]. These systems, loaded with antineoplastic agents (methotrexate, doxorubicin, and mitoxantrone), showed three different delivery mechanisms occurring simultaneously, and according to the authors and improvement of drug targeting was obtained together with the reduction of side effects. Chemically introduced hydrophobic modifications (e.g., alkyl chains by esterification of carboxylate groups) can vary dramatically the alginate behavior,

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leading to a new type of physical micelles essentially based on the hydrophobic interactions among alkyl chains, being reinforced by calcium ions [18]. Release rates of model drugs from such micelles matrices show a remarkable sensitivity to the presence of surfactants and enzymes capable of cleaving the ester linkages. In situ formed micelles loaded with cisplatin and rhenium-188 were tested for regional radiochemotherapy of mammary tumors [19]. Alginate cross-linked with ethylene glycol diglycydyl ether and calcium chloride was also loaded with mitomycin-C, a pharmacotherapeutic agent used in the chemotherapy after a transurethral resection; in vivo studies demonstrated the feasibility of such micelles system [20]. In the resent, several micelles systems exist in which proteins are successfully incorporated into micelles and then released. One of the most extensively studied proteins is the Vascular Endothelial Growth Factor (VEGF), which has been incorporated into ionically cross-linked alginate micelles [21] and delivered both by diffusion and by stimulation. It has been pointed out that the bioactivity of VEGF delivered from alginate micelles was greater than that obtained when VEGF was administered without the micelles, the effect being due to the stabilization of the growth factor via an alginate interaction. The efficacy of this system in driving the angiogenesis around the implant site has been demonstrated both in vitro and in vivo [22]. The release of another angiogenesis promoting protein, basic Fibroblast Growth Factor (bFGF), was also studied from heparin-alginate micelles and also in this case an enhanced angiogenesis was observed [23].

3. DEXTRAN Dextrans can be defined as glucose homopolysaccharides that feature a substantial number of consecutive α-(1→6) linkages in their major chains, usually more than 50% of the total linkages. These α-D-glucans possess also side chains stemming from α-(1→2), α-(1→3), or α-(1→4) branch linkages (Figure 2). Dextran and its derivatives are among the main promising candidates for the preparation of micelles capable of giving a sustained release of bioactive materials such as proteins and peptides.

Figure 2. Structure of dextran polysaccharide.

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In fact, in the last few years, a great variety of bioactive proteins and peptides have been produced and since they present low bioavailability after oral administration, they are generally delivered parenterally. Thus, to achieve a therapeutic effect, repeated administrations are necessary due to the rapid elimination from the circulation. For these reasons, new systems for protein delivery are being widely investigated. One of the first approaches was to introduce reactive double bonds by functionalizing the polymer with glycidyl acrylate [24]. With a subsequent exposure to -irradiation the dextran formed crosslinked micelles, which could be degraded by dextranase. The network was examined as a potential delivery system for invertase that was chosen as a model protein and was incorporated into the micelles before exposure to the irradiation. Systems obtained with different doses of irradiation showed slower release rates as the cross-linked units increased. Furthermore, the experimental delivery profiles indicated that, in the presence of dextranase, the release was mainly controlled by degradation rather than by diffusion. The release was not monotonic but followed a pulsatile trend; such a peculiar profile can be explained by the dextranase-induced surface degradation on the micelles. Indeed, invertase is a large molecule whose diffusion is very limited inside the micellar system but, as the chains are cleaved by dextranase, the pore size gradually increases and, at a certain point, a large amount of invertase was released. This quite unusual pulsatile release may be useful in the delivery of drugs which do not have to be released at a constant rate. Dextran chemical micelles, containing methacrylate moieties in the side chain and obtained by means of irradiation of dextran derivatives, have been widely investigated because the photocross-linking reactions allows to avoid the usual disadvantages of the chemically cross-linked matrices [25]. The micelles were able to swell in aqueous medium and to dissolve in the presence of dextranase. Furthermore, the irradiation of the dextran derivative in the presence of a polyaspartamide derivatized with glycidyl methacrylate yielded innovative micelles that were only partially hydrolyzed by dextranase. It is worth pointing out the versatility of these matrices: by introducing a polyaspartamide moiety and varying the drug loading procedure that changed the network chemical structure, it was actually possible to modulate the rate of drug release in the gastrointestinal tract. Another derivative of dextran, the hydroxyethyl methacrylated dextran, was prepared as both cylindrical micelles and microspheres that were loaded with immunoglobulin G (IgG) [26]. The micelles were degradable under physiological conditions due to the presence of carbonate esters in the cross-links of the micellar system. The micelles showed a biphasic release as a result of the progressive swelling followed by the dissolution of the chains: the delivery was always faster during the second phase. The release of a protein from methacrylated dextran and lactate-hydroxyethyl methacrylated dextran with different crosslinks density has been studied [27]. The first derivatives were stable under physiological conditions whereas the second ones degraded because of the ester groups present in the micellar system. The release profiles of the guest molecule showed that the release decreased by increasing the cross-link density of the micelles. With the dextran methacrylate with high initial water content the release followed a Fickian diffusion indicating that the pore sizes of the matrix were larger than the hydrodynamic radius of the protein. In the case of lower water content the guest molecule resulted fully entrapped within the micellesal meshes. On the other hand, the degradable micelles (lactate-hydroxyethyl methacrylate dextran) with similar network characteristics produced an almost zero order release of the loaded molecule. Thus, the delivery of varying the cross-link density and/or the degradation is a characteristic of the

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micelles. These results appear to be of great interest as interleukin-2 is an important mediator for the immune responses and, because of its biological function; it has been already tested in clinical trials for the treatment of tumors. The dextran derivatives-based micelles can therefore be actually considered promising protein delivery systems for tumor immunotherapy. Sulfopropylated dextran microspheres, loaded with a water-soluble drug, were recently included in cellulose acetate butyrate microcapsules [28]. Another interesting system is the self forming micelles obtained by mixing oppositely charged cross-linked dextran [29]. Rheological studies showed that micelles occurred when equal volumes of oppositely charged microspheres were mixed. Furthermore, the micelles had a reversible yield point: above a certain applied stress the system started to flow whereas the micelles formation occurred as the stress was removed. The advantages of physical micelles are various when compared to the chemical ones. Chemical crosslinking can often damage the entrapped substance leading to a loss of activity and the cross-linking agents in most cases are toxic, necessitating removal before in vivo applications. On the other hand physical micelles, where micelles formation is not instantaneous, can be administered by injection as liquid formulations that can micellesify in situ. The dextran-derived micelles as carriers for BMP have also been tested for the periodontal regeneration of the tissue lost through chronic infective disease caused by bacteria present in dental plaque [30]. The matrix was prepared by polymerizing a dextran derivative in an aqueous system, thus avoiding the use of organic solvents and preserving the biological activity of the loaded BMP. The release from the micelles of the BMP performed in vitro in phosphate buffer solution indicated two-phase release kinetics. The growth factor present inside the microspheres showed an effect similar to that of concentrated solutions but retaining its activity for a prolonged period of time. The in vivo biocompatibility is an important parameter that has been investigated in the case of micelles obtained by mixing aqueous solutions of dextran grafted with L-lactic acid oligomers and dextran grafted with Dlactic acid oligomers [31]. The physical micelles were due to stereo complex formation of the oligomers of opposite chirality. The micelles, implanted subcutaneously in rats, did not significantly activate the immune system thus showing a reasonable compatibility. Since no adverse cellular response was observed, this kind of stereo complexed dextran-oligolactic micelles seems to be suitable for controlled release of pharmaceutically active proteins. Furthermore, the micellar masses degraded completely after several days leaving a fibrotic area, indicating that the micelles were replaced by a connective tissue. Another very promising field is that of the so-called bioresponsive, “intelligent” or “smart” micelles that can actually regulate drug release responding to environmental stimuli by swelling and deswelling. For this purpose, pH-sensitive micelles for drug delivery applications have been developed by cross-linking the carboxymethyl dextran with a derivative of carbodiimide and with hydroxysuccinimide leading to the formation of ester bonds [32]. The micelles, bearing a certain number of -COOH groups, were stimuli-responsive; the porosity of the matrix increased in response to changes in pH and ionic strength. micellar system was pHdependent, resulting in an increase in the diffusion of a tested protein as pH and ionic strength increased. In particular, the micelles exhibited a reversible response to pH change under conditions of constant ionic strength. The system showed several advantages: variation of carboxylic group density and crosslinking reagent concentration allowed the monitoring of

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the charge density and the degree of cross-linking so that the balance between degradation rates and properties could be easily controlled. A novel type of dextran-based micelles with thermoresponsive and biodegradable properties has been developed for the encapsulation and controlled release of hydrophilic drugs in response to temperature changes [33]. The micelles showed a Lower Critical Solution Temperature (LCST) at 32 °C: as the temperature reached the LCST value, a significant amount of water was dispelled from the micelless and a phase separation was observed while, below the LCST, the micelles started to disintegrate and later dissolved completely. In contrast, above the LCST, the degradation was much slower and there was no detectable disintegration during the first days. Two hydrophilic model drugs of different size loaded into the micelles showed release rate profiles with an opposite temperature-dependent trend: the smaller molecule was delivered according to a simple diffusion process, while the delivery of the bigger molecule was controlled by the degradation of the micelles. The preparation of an IPN with dextran and gelatin using lipid microspheres as a drug microreservoir has been exploited in the field of biodegradable systems for drug delivery [34]. The morphology of the system was heavily dependent on the preparation temperature, i.e., above or below the sol-gel transition of the gelatin component. The release of micelles was studied in the presence of two enzymes. The micelles prepared below the transition temperature were not degraded if only one enzyme was present while it was completely degraded in the presence of both enzymes. On the other hand, the network prepared at a temperature above the critical value, showed a release from the response to dual enzymes was achieved. Dextran polymers have been also evaluated for colon drug delivery [35]. In particular, some authors have investigated the stability of dextran micelles in vitro models, simulating human small intestinal and colonic environments to evaluate the suitability of dextran matrices as carriers for colonic specific drug delivery [36]. The degradation of the micelles was estimated by the evaluation of the rate and extent of polysaccharide breakdown in comparison with several chemically different polysaccharides. Indeed, colon specific drug delivery is of great interest for the local treatment of colonic diseases (e.g. Crohn's disease, ulcerative colitis and cancer) as well as for the oral delivery of peptides and vaccines, owing to their instability in the upper gastrointestinal tract. The majority of the anaerobic bacteria in the colon are saccharolytic, deriving their energy from the fermentation of carbohydrates, which results in the production of short chain fatty acids and gasses. The micelles, prepared by cross-linking reaction between the dextran samples with different amounts of hexamethylenedisocyanate, were studied in simulated small intestinal juices by incubation with the enzymes present in the human small intestine, amyloglucosidase, invertase and pancreatin. The micelles were stable while they were completely degraded in a human colonic fermentation model after a fairly long period that could be shortened by changing the structure or the thickness of the micelles, e.g. by preparing a thin layer as a coating of a tablet. After a single day, the systems showed no weight loss while an appreciable reduction in the mass appeared later, related to the degree of cross-linking. To higher cross-linking values, a corresponding lower mass loss was estimated. Dextrans have also been evaluated in the form of azodextrans for colon drug delivery [37]. In this case the matrix was degraded both by the reduction of the azogroups in the crosslinks as well as by enzymatic breakdown of the polysaccharide backbone. The in vitro degradation study showed that no release of sugars and weight loss occurred without dextranase. Apart from micellar approach, prodrug systems have also been found suitable for colon targeting:

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r intactt and unabsorrbed in the thhe drug moleccule linked too the polar deextran chain remains sttomach and in the small inteestine but wheen the prodrugg enters into thhe colonic micrroflora it is accted upon by dextranase which w cleavess the dextran chain random mly and at thhe terminal linnkages releasiing the molecuules into the colon [38].

4. CARRAGEENA A AN Carrageenaan is the genneric name foor a family of o micellar forming f polyssaccharides obbtained by exttraction of cerrtain species of o red seaweedds (Rhodophyyceae), in partiicular from C Chondrus crisppus, Euchema,, Gigartina sttellata and Iriidaea. The maajor constituennts of such allgae are the soo-called carraggeenans, co-poolysaccharidess with a linearr backbone built up by D D-galactose and d 3,6-anhydroo-α-D-galactosse partially sullphated (Figurre 3).

Fiigure 3. Structu ure of Carrageennan.

Three com mmercially impportant carrageenans namelly -(mono-sullfate), -(di-suulfate), and --carrageenan (three-sulfate)) are identifieed based on thheir sulfate coontent. The firrst two are m micellar formin ng systems, whereas w -carrrageenan is a thickener aggent. -Carraggeenan has beeen used to prrepare micelles with two naatural polymers, agar and geelatine, with thhe resulting diisc-shaped maatrices being tested for the release r of theoophylline. In these t mixed syystems, the foormation of phhysical cross-llinks betweenn the chains off the different molecules sloowed down thhe diffusion raate of the guest molecule through the matrix. m Drug release mechhanism was reelated to the network structuure that actuallly depended on o the startingg polymer [39]]. Different reelease rates of o the loadedd molecules have been obtained o in reelation to thee different hyydrophobicity y that allowed a selective innteraction withh -carrageenaan. The more the drug is hyydrophobic, the t stronger the adsorptioon is in the chains, leadding to lowerr diffusion cooefficient. Thee decrease in delivery rate is even more evident whenn two hydrophhobic drugs (cchlorpromazinne and amitripptyline) are loaaded together into the matrix, giving the possibility off designing du ual drug systems based onn hydrophobicc cooperative effects for suustained or coontrolled releaase of some grroups of drugss [40]. It is reportted in the liteerature that regularity of thhe carrageenann spherical matrices and thheir drug deliv very rate couldd be influenceed by the polyymers concentration, the drrug loading annd the crosslin nking density.. Compare to mono and divvalent counterrions, trivalentt ions were leess effective in n the preparattion of the sppheres which presented p big cracks and channels on thheir surface, probably due too the lack of a good cohesioon. This aspecct clearly show ws how the knnowledge of the t stereo-chemical and eneergetic aspectss of the interaactions betweeen ions and

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macromoleculees could leadd to a bettter understanding of the macroscopicc systems. m U Unfortunately t this aspect is not often invvestigated withhin the field of o drug deliveery, mainly beecause multi-component syystems show a degree of complexity thhat is rather difficult to raationalize and d whose hierrarchy is selddom explainaable in termss of single and a simple coomponents.

5. HYAL LURONIC ACID Hyaluronicc acid micelles have gaiined recent interest i as new n biocomppatible and biiodegradable materials m withh applications in drug deliveery. Hyaluronnic acid is an unbranched u unnsulfated glyccosaminoglycaane composed of repeating disaccharide d u units of D-glucuron nic acid and N-acetylgluco N osamine linkedd α-(1→4) annd -(1→3) reespectively (F Figure 4) [41]. While hyaluuronic acid is produced p from m streptococcii for industriaal purposes, it is also preseent in human connective tissues, t wheree it plays an important rolle in many biiological mech hanisms [42].

Fiigure 4. Structu ure of Hyaluroniic acid.

Hyaluronicc acid is usedd as a viscoelaastic material in ophthalmoologic surgeryy and as an innjectable solu ution for the treatment off joint diseases in orthopaaedics. The remarkable r viiscoelastic pro operties of hyaaluronic acid and a its compleete lack of imm munogenicity make it an atttractive biom material. Hyaluuronic acid alsso possesses several s pharm macological prooperties, as it inhibits plateelet adhesion and a aggregatioon, and stimuulates angiogennesis, makingg it suitable d t through the foor vascular appplications [43]. Its efficacy as drug carrieers has been demonstrated veestibular deliv very of gentam micin, ocular delivery of pilocarpine, p inntranasal insullin release, annd vaginal dellivery of calcittonin [44]. As hyaluro onic acid alonne is unable too give micellees, chemical reeactions mustt be carried ouut in order to obtain chemically cross-liinked networkks. Different approaches a caan be used, suuch as via divvinylsulfone [445], via glyciddyl ether [46], and via gluthharaldehyde [47]. Stable hyyaluronic acidd micelles haave also been prepared usinng a water-sooluble carbodiiimide that foorms ester bonnds between thhe hydroxyl annd carboxyl grroups of hyaluuronic acid. Fuurthermore, am mide bonds can c be introduuced by crosss-linking the polymer in thhe presence of o L-lysine m methyl ester [448] to obtain micelles m with lower water content c and ann increased reesistance to deegradation. Siimilarly, crosss-linking was carried c out wiith 1,3 diamm minopropane, leeading to a hyydrogel that was w used for loocal administrration in the osteoarthritic o k knee of ibuproofen-lysine

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[49]. The same anti-inflammatory drug was tested using a cross-linked sulphated derivative of hyaluronic acid in order to increase the hemocompatibility of the material. Furthermore, the release rate from this micelle could be modulated by treatment with CO2, leading to microstuctured micelles. While in most cases drugs have been covalently attached to soluble hyaluronic acid, bioactive molecules have also been chemically bound to preformed hyaluronic acid micelles, which can be obtained by different cross-linking methods [50]. Other hyaluronic acid micelles were formed by crosslinking thiol-modified hyaluronic acid chains with poly(ethylene glycol) diacrylate and mitomycin C [51]. Though films prepared with this type of micelles were cytotoxic in in vitro experiments, no severe peritoneal fluid leukocyte response was elicited when implanted in vivo into a rat peritoneal cavity. The slow release of mitomycin C by hydrolysis has led to the system being proposed as an alternative route for cancer bladder treatment, when crosslinking was carried out in situ onto the bladder wall. Controlled delivery of DNA complexes from biomaterials can enhance gene transfer by maintaining an elevated concentration of DNA within the cellular microenvironment via sustained release or substrate immobilization. This goal was recently obtained [52] by developing a procedure based on the immobilization of plasmid DNA complexed with poly(ethylene imine) and immobilized to a hyaluronic acid-collagen micelles by means of non covalent interactions. An interesting topic, due to its wide variety of applications, is related to the hyaluronic acid thermo-responsive micelless that can be obtained using Pluronic® and its derivatives. Hyaluronic acid modified with a vinyl group has been chemically crosslinked with di-acrylated Pluronic F127 by a photo-polymerization [53]. Further, micelles formation ability has been studied as a function of temperature and tested for the release of human growth hormone or plasmid DNA at different temperatures and cross-linking conditions [54]. Poloxamers have been grafted onto hyaluronic acid chains [55] by coupling mono amine-terminated poloxamers using 1-ethyl-3-(3dimethylaminopropyl)-carbodiimide and N-hydroxylsuccinimide. The lower critical solution temperature of the resulting polymer was affected both by the grafted hyaluronic acid concentration and the drug content. The release of ciprofloxacin was tested in vitro, leading to the polymeric micellar system being proposed as an in situ drug carrier for a prolonged ocular delivery. Another thermo-sensitive injectable micelles was recently prepared by grafting semitelechelic poly(N-isopropylacrylamide) with amino groups at the end of each main chain, onto the carboxyl groups of hyaluronan chains using carbodiimide chemistry [56]. The drug release properties of this new micellar system were tested using riboflavin and BSA as model molecules for in vivo and in vitro experiments respectively. Recently, micelles based on adipic dihydrazide derivative of hyaluronic acid, poly(ethylene glycol)-propiondialdehyde and the growth factors were prepared. The capability of such systems to elicit angiogenesis was tested on mice. Obtained results showed that the loaded hyaluronic acid micelles were stable and biocompatible [57].

6. GUAR GUM Guar gum belongs to the large group of seed gums, widely diffused in nature. Being principally made up of mannose and galactose sugar units, seed gums are also known as galactomannans. They differ from each other in their mannose/galactose ratio and distribution

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paattern of the galactose g residdues along thhe mannan chaain. Guar gum m is a hydrophylic, nonioonic polysacccharide extraacted from thhe endosperm mic seed off the plant Cyamopsis teetragonalobus. Guar gum beelongs to the large l family of galactomannnans and consists of a Dgaalacto-D-mann nan [58]. The formula of the guar gum is shown in Figuure 5.

Fiigure 5. Chemiccal structure of guar gum.

Guar gum hydrates in cold water to form f a highlyy viscous soluution in whichh the single poolysaccharide chains interaact with eachh other in a complex c way.. Guar gum has h proven paarticularly useeful for colon delivery as it can be degradded by the speecific enzymess present in thhe tract of inteestine [59]. Likke other polym mers used for colon targetinng, guar gum can protect thhe drug in thee stomach andd small intesttine environm ment, while deelivering the drug d to the coolon where itt undergoes assimilation a b specific microorganismss or degradation by the by ennzymes, leading to the finaal delivery of the drug. Hennce, it is usedd to form proddrugs, as a cooating materiaal or as micellees entrapping drugs inside its i network. The T utility of guar g gum in thhe preparation n of chemicall micelles to be degraded in the large intestine has also been innvestigated. A problem arisses in its exceellent swellingg properties annd hydrophiliccity, which reequires its protection in the upper part off the GI tract. Cross-linking with glutaralddehyde has beeen proven to o decrease its swelling propperties [60]. Guar G gum as micelles m was found f to be noot suitable forr delivery of highly h water-ssoluble drugs due to its fasst delivery. Onn the other haand, it is poteentially useful with poorly water-soluble drugs, to act as a specific carrier for coolon delivery, with or without specificc enzymes. By B introducinng modificatioons on the poolymeric chaiin of guar guum the dosagge forms werre prepared, which w were capable c for caarrying the looaded hydrophhobic drug thrrough the prooximal portionns of the GI tract, t while m maintaining thheir susceptibiility to degraadation by thhe colonic ennzymes. Anotther useful appplication of guar g gum hass been evidencced in the treeatment of opeen-angle glaucoma [61], w where guar gum m was blendedd with carboppol 940 and soodium alginatee. The system was a free flowing liquid with low visscosity beforee use, turningg into a hydrrogel upon coontact with v of thhe blend, a arrtificial tears fluid. Here, thhe guar gum specifically inncreased the viscosity crrucial parametter in obtaininng the controlleed drug deliveery.

7. XANTHAN Xanthan, a microbial exxopolysaccharride consisting of a celluloosic backbonee with two m mannose and one o glucuronicc acid side chaains on every second glucoose residue is considered ann anionic poly yelectrolyte (F Figure 6). Thhe molecular weight w of xannthan can reach up to 6

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million Daltonss, which makkes it possible to create exttremely viscouus solutions at m a very low cooncentrations In addition to t its enzymatic resistance,, xanthan gum m is stable ovver a wide raange of tempperatures and pH, which finds f many applications a inn food, pharm maceutical, coosmetic, and oil-drilling o inddustries [62].

Fiigure 6. Structu ure of xanthan building b blocks.

Though xanthan has beeen widely studdied and used as a tablet excippient to increaase the drug raate of delivery y, not much work w has beenn reported conncerning the use u of this pollymer for a suustained drug release. In thiis sense, an innteresting studdy has been caarried out on thhe micellar coomplexes baseed on xanthann and chitosann for the delivvery of bioacttive moleculess [63]. The obbtained micellles showed a homogeneouus porous nettwork with fibrillar structuures. These fibrillar micellees formed channels that alllowed the paassage of polyymeric substrrates to the i e enzymes weree lodged. The polyionic p miccelles have thee advantage reegions where immobilized off creating an ionic i microsystem which faavors the stabilization of prroteins. Furtheermore, the poorous structurre, facilitatingg the diffusioon of both the t substrate and the prodduct of an ennzymatic reacction, appears to be suitablee for enzyme immobilizatioon, and also for f loading biioactive substaances for pharrmaceutical foormulations.

CON NCLUSIONS S d out throughhout this chaapter, polysacccharides and their derivattives show As pointed vaariability and versatility duue to their com mplex nanostrructures whichh are not founnd in other cllasses of polyymers. Due to their micellaar properties, these t polymerrs have a widde range of appplications inn the biomediccal fields parrticularly in controlled c druug delivery. As A a result, reecently an inccreased numbber of publicaations and pattents concerniing micelles preparation p frrom the nativee and derivatiized polysacchharides have been b reportedd. However, itt should be nooted that a litttle work has been b focused on the physico-chemical annd rheologicall properties off these polym mers in order to t understand how these poolymers behavve at a nanosccopic level thhat finally deteermine the druug delivery meechanism. In fact, f parameters such as origgin, culture

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conditions, and percentages of minor groups on the macromolecules can dramatically influence several properties of polysaccharides; these factors are very seldom taken into consideration in publications related to drug delivery. This overview on polysaccharide micelles for drug delivery and closely related applications could help researchers to develop novel types of micelles based on polysaccharides and their derivatives with peculiar properties that make them suitable for a wide variety of applications in the field of pharmaceutics.

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In: Recent Developments in Bio-Nanocomposites… ISBN 978-1-61761-008-0 Editor: Ashutosh Tiwari © 2011 Nova Science Publishers, Inc.

Chapter 2

BIONANOCOMPOSITES – CURRENT STATUS AND PROSPECTS IN DRUG DELIVERY FIELDS M. Prabaharan1,1, R. Jayakumar2 and Ashutosh Tiwari3,4 1

Department of Chemistry, Faculty of Engineering and Technology, SRM University, Kattankulathur 603 203, India 2 Center for Nanosciences, Amrita Institute of Medical Sciences and Research Centre, Amrita Vishwa Vidyapeetham, Kochi 682 026, India 3 School of Materials Science and Engineering, Jiangsu University, Zhenjiang 212 013, China 4 National Institute for Materials Science, Tsukuba, Ibaraki 305 0047, Japan

ABSTRACT Bionanocomposites (BNCs) have established themselves as a promising class of hybrid materials derived from natural and synthetic biodegradable polymers and organic/inorganic fillers. A critical factor underlying biomedical nanocomposite properties is interaction between the chosen matrix and the filler. This chapter discusses current efforts and key research challenges in the development of these composite materials for use in potential drug delivery applications. BNCs discussed here include layered BNCs, layered double hydroxides (LDHs), inorganic BNCs, polymeric BNCs, chitosan-based hydrogels and magnetic BNCs. From this review, it is concluded that BNCs would be promising materials for drug delivery applications.

1. INTRODUCTION Bionanocomposites (BNCs) form a fascinating interdisciplinary area that brings together biology, materials science, and nanotechnology. New BNCs are impacting diverse areas, in particular, biomedical science. Generally, polymer nanocomposites are the result of the 1 Corresponding author. E-mail: [email protected].

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combination of polymers and inorganic/organic fillers at the nanometer scale. The extraordinary Versatility of these new materials springs from the large selection of biopolymers and fillers available to researchers. Existing biopolymers include, but are not limited to, polysaccharides, aliphatic polyesters, polypeptides and proteins, and polynucleic acids, whereas fillers include clays, hydroxyapatite, and metal nanoparticles [1]. The interaction between filler components of nanocomposites at the nanometer scale enables them to act as molecular bridges in the polymer matrix. This is the basis for enhanced mechanical properties of the nanocomposite as compared to conventional microcomposites [2]. BNCs add a new dimension to these enhanced properties in that they are biocompatible and/or biodegradable materials. For the sake of this review, biodegradable materials can be described as materials degraded and gradually absorbed and/or eliminated by the body, whether degradation is caused mainly by hydrolysis or mediated by metabolic processes [3]. Therefore, these nanocomposites are of immense interest to biomedical technologies such as tissue engineering, bone replacement/repair, dental applications, and controlled drug delivery. Current opportunities for bio-nanocomposites in the biomedical arena arise from the multitude of applications and the vastly different functional requirements for each of these applications. For example, the screws and rods that are used for internal bone fixation bring the bone surfaces in close proximity to promote healing. This stabilization must persist for weeks to months without loosening or breaking. The modulus of the implant must be close to that of the bone for efficient load transfer [4,5]. The screws and rods must be noncorrosive, nontoxic, and easy to remove if necessary [6]. Thus, a bio-nanocomposite implant must meet certain design and functional criteria, including biocompatibility, biodegradability, mechanical properties, and, in some cases, aesthetic demands. The underlying solution to the use of bio-nanocomposites in vastly differing applications is the correct choice of matrix polymer chemistry, filler type, and matrix–filler interaction. This article discusses current efforts and focuses on key research challenges in the emerging usage of BNCs for potential drug delivery applications. In drug therapy, it is important to provide therapeutic levels of pharmaceutically active agents to the site of action and maintain them during the treatment. Furthermore, it is desired to minimize temporal variations in drug concentration that can lead to periods of overdosing. Modified release technologies are employed to deliver active ingredients in a controlled manner, providing some actual therapeutic temporal and/or spatial controls of drug release. In this respect, BNCs could be potential carriers for controlled drug delivery due to their enhanced functionality and multifunctional properties in contrast with their more-limited single-component counterparts.

2. DRUG DELIVERY 2.1. Layered BNCs For the past four decades polymer layered silicate nanocomposites have attracted intense research interest with the promise of applications across many industries. However, the proposed widespread commercial use of this technology has not been fully realized, with a few notable exceptions in automotive, barrier (packaging), fire retardant and structural

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applications. This slow progress is in part due to the difficulties in melt blending nanoclays and polymers at elevated temperatures to achieve a homogeneous dispersion of nanoclay platelets in the polymer matrix without degradation of both polymer and nanoclay. This constraint is in part eliminated for low melting point biodegradable polymer/biopolymer layered silicate nanocomposites, such as those with poly(caprolactone) [7,8], poly(ethylene glycol) [9], poly(ethylene-oxide)PEO [10], polylactides [11,12] and poly(vinylpyrrolidone) [13], where improved mechanical and functional stability of biopolymers can be achieved. Concomitant with the development of polymer nanocomposites, there has been renewed interest in the area of hot-melt extrusion as a technique for the preparation of polymer/biomolecule composite materials for drug delivery [14]. The main advantage of using extrusion technology is the ability to move from batch processing normally used for polymer/drug manufacture to a continuous process. This enables consistent product flow at relatively high throughput rates, such that a drug loaded polymer can be extruded, for example, into a sheet or thin film for patches or into a tube for catheters or other medical tubing. Furthermore, the bioactive molecule loaded extrudate can be pelletized followed by secondary melt processing, such as injection moulded into heart valves. A number of researchers have examined the use of hot-melt extrusion to prepare biomedical polymer–drug mixtures, for example, hydroxypropylcellulose [15], PEO [16] and a commercially available melt-extruded formulation, Kaletra [17]. Other groups have used solution cast methods to produce drug loaded polymer nanocomposites in the form of hydrogels [18], nanoparticles [19] and solution cast nanocomposites [20] although the latter has limitations with regard scale-up of the process and is not environmentally friendly. Drug release from polymeric matrices has been modeled by different researchers [21], and the mechanism of drug release from solid dispersions in water-soluble polymers has been reviewed by Craig [22]. Molecular interactions between the drug molecule and polymer chains also affect the mechanism of drug release. Layered silicates alone have also been used in pharmaceutical applications as adsorbents, thickeners and excipients [23] and recent research has outlined the use of layered silicates [24], mesoporous silicates [25] and double layered hydroxides as drug and gene delivery vehicles [26]. Recently, composites of paracetamol loaded poly(ethylene glycol) (PEG) with a naturally derived and partially synthetic layered silicate (nanoclay) were prepared using hot-melt extrusion [27]. The extent of dispersion and distribution of the paracetamol and nanoclay in the PEG matrix was examined using a combination of field emission scanning electron microscopy (Figure 1), high resolution transmission electron microscopy (HR-TEM) and wide-angle X-ray diffraction (WAXD). The paracetamol polymorph was shown to be well dispersed in the PEG matrix and the nanocomposite to have a predominately intercalated and partially exfoliated morphology. The form 1 monoclinic polymorph of the paracetamol was unaltered after the melt mixing process. The crystalline behavior of the PEG on addition of both paracetamol and nanoclay was investigated using differential scanning calorimetry (DSC) and polarised hot-stage optical microscopy. The crystalline content of PEG decreased by up to 20% when both drug and nanoclay were melt blended with PEG, but the average PEG spherulite size increased by a factor of 4. The time taken for 100% release of paracetamol from the PEG matrix and corresponding diffusion coefficients were significantly retarded on addition of low loadings of both naturally occurring and partially synthetic nanoclays. The dispersed layered silicate platelets encase the paracetamol molecules,

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retarding diffusion and altering the dissolution behavior of the drug molecule in the PEG matrix.

Figure 1. FE-SEM images of fractured surfaces of (a) PEG, (b) PEGP5, (c) PEGP5M1 and (d) PEGP5S5.

Kevadia et al. reported the intercalation of procainamide hydrochloride (PA), an antiarrythmia drug in montmorillonite (MMT), as a new drug delivery device [28]. Optimum intercalation of PA molecules within the interlayer space of MMT was achieved by means of different reaction conditions. Intercalation of PA in the MMT galleries was confirmed by XRD, FT-IR, and thermal analysis (DSC). In order to retard the quantity of drug release in the gastric environment, the prepared PA–MMT composite was compounded with alginate (AL), and further coated with chitosan (CS). The surface morphology of the PA–MMT–AL and PA–MMT–AL–CS nanocomposites beads was analyzed by SEM. The in vitro release experiments revealed that AL and CS were able to retard the drug release in gastric environments, and release the drug in the intestinal environments with a controlled manner (Figure 2). The release profiles of PA from composites were best fitted in Higuchi kinetic model, and Korsmeyer–Peppas model suggested diffusion controlled release mechanism. Thin films and coatings that sustain the release of DNA from surfaces could play an important role in the development of localized approaches to gene therapy [29]. For example, polymer-coated intravascular stents have been used to localize the delivery of DNA to the vascular wall and could lead to innovative gene-based treatments for vascular diseases or related conditions [30].

PA  release %

Bionanocomposites – Current Status and Prospects in Drug Delivery Fields

PA‐MMT PA‐MMT‐AL PA‐MMT‐AL‐CS

21

A

Time, h

PA  release %

B

PA‐MMT PA‐MMT‐AL PA‐MMT‐AL‐CS Time, h Figure 2. Release profiles of PA in (A) gastric (pH 2.2) and (B) intestinal fluid (pH 7.4) at 37±0.5 ◦C.

Likewise, plasmid-eluting polymer matrices have been applied to the localized delivery of DNA to cells in the context of tissue engineering [31]. Although degradable polymer matrices can be used to sustain the release of encapsulated DNA, general methods for the localized, efficient, and sequential delivery of DNA from thin films and surfaces do not yet exist. Recently, Multilayered polyelectrolyte assemblies 100 nm thick fabricated from alternating layers of a degradable cationic polymer and plasmid DNA was used to localize the delivery of DNA to cells growing in contact with or in the vicinity of macroscopic film coated objects (Figure 3) [32]. In addition to the localization of DNA at the surface of film-coated substrates, it was found that these films present DNA in a condensed form that may influence and enhance the internalization and processing of DNA by cells. The layer-by-layer assembly approach used in this study provides control over the location and distribution of plasmid DNA in thin, nanostructured films that can be fabricated onto a variety of complex macroscopic substrates. As such, the materials and approaches reported here could represent an attractive framework for the local or non-invasive delivery of DNA from the surfaces of implantable materials or biomedical devices. The design principles outlined above introduce new opportunities to design more advanced layered materials that enhance or influence further the mechanisms through which DNA and other biological materials are internalized.

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Figure 3. (A) Idealized scheme showing layer-by-layer fabrication of a multilayered film fabricated from alternating layers of degradable polymer 1 (gray) and a plasmid DNA encoding a fluorescent protein (green). Incubation of this material under physiological conditions results in the gradual release of DNA. (B) General scheme illustrating the direct and localized transfection of cells using a quartz slide coated with a polymer 1/DNA film. Coated quartz slides are placed manually on top of cells growing on the surface of a tissue culture dish.

2.2. Layered Double Hydroxides (LDHs) LDHs have been known for many decades as catalyst and ceramic precursors, traps for anionic pollutants, catalysts, and additives for polymers, but they recently attracted attention as potential nano-sized carriers for therapeutic/bio-active molecules and genes. Among the many different nanoparticles that have been shown to facilitate gene and/or drug delivery, LDH nanoparticles are particularly well suited for this purpose due to their many desirable properties. Recently, Mg2Al(OH)6NO3 LDH nanoparticles of varying lateral sizes were synthesized by altering the synthesis conditions [33]. The synthesis conditions particularly influencing the particle size distribution of the LDH suspensions are (a) the temperature during the co-precipitation step and (b) the duration and the temperature of the hydrothermal treatment. The association of these nanoparticles with plasmid DNA was studied and it was established that–in contrast to previously published reports–for the plasmid sizes used no significant intercalation occurs. The plasmids wrap around individual particles instead and aggregation of particles is observed. However, due to the observed strong interaction between LDH nanoparticles and DNA, the particles were nonetheless evaluated as transfection agents for mammalian cells. Considerable transfection efficiencies when transfecting adherent cell lines (i.e., HEK293T, NIH 3T3, COS-7, and CHO-K1) were observed, while the transfection of suspension CHO-S cells remained unsuccessful. This is attributed to the formation of aggregates upon DNA–LDH complex formation which settle on top of adherent cells but due to the constant agitation of suspension cultures not on the surface of e.g., CHO-S cells. LDHs consist of cationic brucite-like layers and exchangeable interlayer anions. Because of their biocompatibility, some LDHs, such as Mg/Al, Zn/Al, Fe/Al and Li/Al-LDH, can be used as host materials for drug-LDH host–guest supramolecular structures [34]. The antiinflammatory drug fenbufen has been intercalated into layered double hydroxides for the first time by co-precipitation under a nitrogen atmosphere. The product has been characterized by

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powder XRD, FT-IR spectroscopy, elemental analysis and thermogravimetry (TG) and shows an expanded LDH structure, indicating that the drug has been successfully intercalated into LDH. In addition, the dependence of the nature of the fenbufen intercalation process on conditions such as pH value and chemical composition of the host has been systematically investigated. The interlayer distance in the intercalated materials increases with increasing pH value, resulting from a change in the arrangement of interlayer anions from monolayer to interdigitated bilayer. Drug release characteristics of the pillared LDH materials were investigated by a dissolution test in a simulated intestinal fluid (buffer at pH 7.8). The results show that the drug release of supramolecular LDH materials was a slow process, especially in the case of Mg/Al intercalated materials, suggesting that these drug-inorganic hybrid materials can be used as an effective drug delivery system. Recently, PPT–LDH nanohybrid composites were prepared and compared their tumor inhibition effects with that of free PPT [35]. Anticancer drug podophyllotoxin (PPT) was encapsulated in the galleries of Mg–Al LDHs by a two-step approach. Tyrosine (Tyr) was first incorporated into the interlayer space by co-precipitation with LDH, prop opening the layers of Mg–Al/LDH and creating an interlayer environment inviting drug molecules. PPT was subsequently intercalated into the resulting material lamella by an ion exchange process. The intermediate and final products, which can be termed drug-inorganic nanocomposites, have been characterized by powder XRD, UV-VIS spectrophotometer, TEM and in cell culture. The results demonstrate that the interlayer spacing distance of the PPT–LDH nanohybrids (34% w/w of drug/material) is 18.2 A°. LDHs do not harm normal cells (293T) based on toxicity tests. Ex-vivo anticancer experiments reveal that the PPT–LDH nanohybrids have higher tumor suppression effects than intercalated PPT. The authors conclude that the higher tumor inhibition effects of PPT–LDH hybrids result from the inorganic drug delivery vehicle, LDHs. Naproxen (NP) and flurpibrofen (FB) are well-known examples of non-steroidal antiinflammatory drugs (NSAIDs) of 2-arylpropionic acid derivatives, and they are widely used for pathophysiological diseases such as headache and fever [36] based on their analgesic and antipyretic properties [37]. One of serious problems in the application of NP and FB is their poor water-solubility and readily absorbing characteristics. It is known that the more rapidly absorbed drug the faster onset of activity and the rate at which a drug dissolves in the gastrointestinal tract controls the rate of the drug appearance in the blood [38], so for NP, to improve its dissolution behavior, several approaches have been reported; solid dispersions with polyethylene glycol [39] and with polyvinylpyrrolidone [40], complexation with cyclodextrins [41], solid binary systems with chitosan [42], and compaction process using hydroxypropyl methylcellulose [43]. To overcome the problem of drug solubility, Berber et al. have chosen LDH [44]. These materials provide many interesting applications; anion exchange properties due to the weak interactions between the interlayer anions and the positively charged layers [45], as additives in polymers [46], bio-nanocomposites based on the fact that most of biopolymers interact strongly with calcium ions and host materials to create drug—LDH supramolecular structures as drug delivery system based on their biocompatibility. Figure 4 shows the schematic diagram of FB-LDH intercalation process. From drug loading, thermal analysis and X-ray measurements it is found that coprecipitaion technique is better than reconstruction technique to obtain intercalated monophase nanocomposites. In acidic medium LDH dissolved and the intercalated drug starts to release in a molecular form which is suitable for adsorption. The

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drug solubility has been investigated before and after intercalation. It has been found that LDH improves the drug solubility and its dissolution rate.

Figure 4. Schematic diagram of FB-LDH intercalation process.

Enalaprilate (Enal), an active pharmaceutical component, was intercalated into a LDH (Mg/Al-LDH) by an ion exchange reaction [47]. The use of a LDH to release active drugs is limited by the low pH of the stomach (pH ~1.2), in whose condition it is readily dissolved. To overcome this limitation, xyloglucan (XG) extracted from Hymenaea courbaril (jatoba) seeds, Brazilian species, was used to protect the LDH and allow the drug to pass through the gastrointestinal tract. All the materials were characterized by XRD, Fourier transform infrared spectroscopy, elemental analyses, transmission electronic microscopy, thermal analyses, and a kinetic study of the in vitro release was monitored by ultraviolet spectroscopy. The resulting hybrid system containing LDH–Enal–XG slowly released the Enal. In an 8 h of test, the system protected 40% (w/v) of the drug. The kinetic profile showed that the drug release was a co-effect behavior, involving dissolution of inorganic material and ion exchange between the intercalated anions in the lamella and those of phosphate in the buffer solution. The nanocomposite coated protection with XG was therefore efficient in obtaining a slow release of Enal.

2.3. Inorganic BNCs The lanthanide ions (Ln3+, Ln = Er, Tm, and Yb) doped up conversion nanocrystals have attracted significant interest due to their many potential biological applications [48]. Compared with conventional down conversion fluorescent labels [49] which require an ultraviolet or blue excitation wavelength, up conversion nanocrystals have many conceivable advantages as fluorescence labeling materials including an improved signal-to-noise ratio due to the absence of auto fluorescence and reduction of light scattering [50], carrying out easily in vivo imaging upon the NIR (near infrared) irradiation with noninvasive and deep penetration to the tissue or cell [51], low photobleaching [52] and feasibility of multiple labeling with different emissions under the same excitation. A compact, power-rich, and

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inexpensive 980 nm near infrared laser may be used as the excitation source. Up to now, among the various host materials for up conversion, hexagonal NaYF4 ( -NaYF4) has been reported as the most efficient host matrix for green and blue up conversion when activated by Yb3+ and Er3+/ Tm3+ ions due to the very low phonon energy of its lattice [53]. Many methods have been used to prepare hexagonal NaYF4 [54], but the products prepared by those methods were usually big or hydrophobic, having adverse effect on biologic applications. Cubic NaYF4 can turn into hexagonal NaYF4 through annealing [55], but in general, annealing makes nanoparticles aggregate and grows up. Novel up conversion nanocomposites, silica coated cubic NaYF4:Yb3+, Tm3+ nanoparticles, with nonporous structure were presented recently [56]. After annealing, monodisperse cubic/hexagonal mixed phases NaYF4:Yb3+, Tm3+@SiO2 nanoparticles were obtained, and the NaYF4:Yb3+, Tm3+ cores became nanoporous (Figure 5). They demonstrate increased up conversion emission compared with un-annealed dense NaYF4:Yb3+, Tm3+ nanoparticles due to the appearance of the hexagonal NaYF4:Yb3+, Tm3+. The silica shell not only makes the nanocomposites possess bio-affinity but also protects the NaYF4:Yb3+, Tm3+ cores from aggregating and growing up. Thus the up conversion, nanoporous and bio-affinity properties were combined into one single nanoparticle. These multifunctional nanocomposites are expected to find applications in biological fields, such as biolabels, drug storage and delivery.

Figure 5. Synthesis of porous NaYF4:Yb3+, Tm3+@SiO2 nanoparticles.

Hydrothermal synthesized Fe3O4 microspheres have been encapsulated with nonporous silica and a further layer of ordered mesoporous silica through a simple sol–gel process [57]. The surface of the outer silica shell was further functionalized by the deposition of YVO4:Eu3+ phosphors, realizing a sandwich structured material with mesoporous, magnetic and luminescent properties. Figure 6 shows the formation process of the multifunctional Fe3O4@nSiO2@mSiO2@YVO4:Eu3+ composite microspheres. The multifunctional system was used as drug carrier to investigate the storage and release properties using ibuprofen (IBU) as model drug by the surface modification. The results reveal that the material shows typical ordered mesoporous characteristics, and have monodisperse spherical morphology with smooth surface and narrow size distribution. Additionally, the multifunctional system shows the characteristic emission of Eu3+ (5D0–7F1–4) even after the loading of drug molecules. Magnetism measurement reveals the super paramagnetic feature of the samples. Drug release test indicates that the multifunctional system shows drug sustained properties. Moreover, the emission intensities of Eu3+ in the drug carrier system increase with the released amount of drug, thus making the drug release be easily tracked and monitored by the change of the luminescence intensity.

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Figure 6. The formation process of the multifunctional Fe3O4@nSiO2@mSiO2@YVO4:Eu3+ composite microspheres.

In recent years, hydroxyapatite (HA) a biocompatible and bioactive material derived from the chemical precipitation methods, has been incorporated into calcium sulfate to form calcium sulfate/HA composite [58] and overcome disadvantages of pure calcium sulfate bone substitutes, i.e. too fast resorption rate, resulting in an intense granulomatous response in the surrounding tissues [59] and transient cytotoxic effect leading to inflammatory reactions [60]. Drug-release properties of calcium sulfate/HA composites have been successfully investigated using various antibiotics such as gentamicin, cephalexin and vancomicin [61]. Recently, Nanocomposite of 50 wt% calcium sulfate and 50 wt% nanocrystalline apatite was produced and its biocompatibility, physical and structural properties were compared with pure calcium sulfate cement [62]. Indomethacin (IM), a non-steroidal anti-inflammatory drug, was also loaded on both calcium sulfate and nanocomposite cements and its in vitro release was evaluated over a period of time. The effect of the loaded IM on basic properties of the cements was also investigated. Biocompatibility tests showed a partial cytotoxicity in calcium sulfate cement due to the reduced number of viable mouse fibroblast L929 cells in contact with the samples as well as spherical morphologies of the cells. However, no cytotoxic effect was observed for nanocomposite cement and no significant difference was found between the number of the cells seeded in contact with this specimens and culture plate as control. Other results showed that the setting time and injectability of the nanocomposite cement was much higher than those of calcium sulfate cement, whereas reverse result obtained for compressive strength. In addition, incorporation of IM into compositions slightly increased the initial setting time and injectability of the cements and did not change their compressive strength. While a fast IM release was observed from calcium sulfate cement in which about 97% of the loaded drug was released during 48 h, nanocomposite cement showed a sustained release behavior in which 80% of the loaded IM was liberated after 144 h. Thus, the nanocomposite can be a more appropriate carrier than calcium sulfate for controlled release of IM in bone defect treatments.

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Encapsulation of imaging agents and drugs in calcium phosphate nanoparticles (CPNPs) has potential as a nontoxic, bioresorbable vehicle for drug delivery to cells and tumors. Recently, 20-30 nm diameter organically doped CPNPs were prepared using a variety of fluorescent dyes [63]. Specifically, Cascade Blue, SAB, fluorescein sodium salt, rhodamine WT, and Cy3 amidite were successfully encapsulated displaying the ability to encapsulate several varieties of small organic molecules. Comparisons of the diameter by FCS and TEM confirm solution-phase colloidal stability of the particles, as well as their spherical morphology. Additionally, the fluorescence quantum efficiency exhibited nearly a 4-fold increase from 0.045 to 0.202 for the free and encapsulated dye, respectively. Dissolution of the particles at low pH was proven by a shift in the diffusion coefficient to larger values, indicating a release of encapsulated contents in environments similar to that of endolysosomes. As further proof of this property, bovine aortic cells were effectively stained with the particles, and dissolution was inhibited through the use of cytochalasin-D. Preliminary drug delivery results show that a hydrophobic chemotherapeutic, such as ceramide, can be delivered in Vitro to human vascular cells. Both carboxy- and aminofunctional nanoparticles were prepared, and the carboxy-terminal particles were successfully used as a platform for PEG functionalization. Paradigm-shifting modalities to more efficiently deliver drugs to cancerous lesions require the following attributes: nanoscale-size, targetability, and stability under physiological conditions. Often, these nanoscale drug delivery vehicles are limited due to agglomeration, poor solubility, or cytotoxicity. Thus, Kester et al. have designed a methodology to encapsulate hydrophobic antineoplastic chemotherapeutics within a 20-30 nm diameter, pHresponsive, nonagglomerating, nontoxic calcium phosphate nanoparticle matrix. In this study, they report on CPNPs that encapsulate both fluorophores and chemotherapeutics, are colloidally stable in physiological solution for an extended time at 37 °C and can efficaciously deliver hydrophobic antineoplastic agents, such as ceramide, in several cell model systems [64]. This report demonstrates that CPNPs are colloidally stable for extended times in physiological conditions and can be used to deliver fluorophores and lipophilic drugs for a variety of cell types. The metabolic pathways for Ca2+ uptake and membrane transport are not perturbed and do not elicit acute toxicity in neural cells. Thus, CPNPs have broad potential applicability as ex vivo and in vivo imaging agents as well as for drug delivery platforms. Validation of CPNPs as highly efficacious, nontoxic, broadly based drug delivery platforms has been detailed in this study. The pH changes occurring during endocytosis leads to dissolution of CPNP and subsequent cytosolic or perinuclear release of encapsulated agents. Thus, drugs with little or no solubility in physiological liquids can be delivered using the CPNP approach. Cancer therapeutics provides an excellent demonstration of the efficacy of cellular delivery of a hydrophobic bioactive agent via the CPNPs.

2.4. Polymeric Bncs Natural biodegradable polymers such as dextran and chitosan have been considered for targeted drug delivery [65] and quantum dots are being explored for imaging the distribution of drug in vivo [66]. The unique integration of drug targeting and visualization has high potential to address the current challenges in cancer therapy. Thus, it is attractive to consider the possibility of investigating a system that combines the biodegradable material, chitosan,

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and the semiconductor quantum dots (QDs). Monodispersed ZnO QDs of size 2-4 nm were successfully synthesized by a chemical hydrolysis method that exhibited a strong blue emission at ~ 440 nm as a drug delivery carrier [67]. The experimental methodology scheme depicted in Figure 7 enabled fabrication of water dispersed ZnO-QD-chitosan-folate carrier loaded with anticancer drug, doxorubicin. The experimentally observed drug loading efficiency was ~ 75%. Chitosan enhances the stability of the QDs because of the hyrophilicity and cationic charge characteristics of chitosan. The drug release response of DOX-loaded ZnO-QD-chitosan-folate carrier was characterized by an initial rapid drug release followed by a controlled release. The study point toward the application of water-dispersed ZnO with long term fluorescence stability for design of new drug release carrier. OH O

OH N CH2-NH-

N

O

O

NH-CH-COOH

+ CH2-CH2-COOH NH2

O

H2N

N

N

Folic acid

Chitosan EDC, DMSO pH, 4.7

OH O OH

O

O O

N CH2-NH-

N

NH-CH-COOH CH2-CH2-CO NH

H2N

N

N

Figure 7. Encapsulation of quantum dots with folate conjugated chitosan.

The structural arrangement of molecules, ions, and other molecular species in the confined spaces of nanoscale pores and mineral interlayer are a key factor in understanding transport and reactivity in many technological and biological systems. In this respect, considerable research efforts have been focused on the design of nanoscale oral sustainedand controlled-release drug delivery systems. Biodegradable polymers have long been of interest in controlled release technology because of the ability of these polymers to be reabsorbed by the body. There are not very many polymers which intercalate Cloisite, forming a good nanocomposite. Singhal et al. carried out some preliminary investigations on the synthesis of Cloisite and biodegradeable poly hydyroxyethyl methacrylate (PHEMA) nanocomposites for drug delivery application [68]. The nanocomposite were prepared by in situ intercalative polymerization of PHEMA within the Cloisite galleries The nanocomposites were found to exhibit intercalation and hydrogen bonding between Si - O of cloisite and C+O of HEMA. In the last decades, hybrid systems composed of clay particles dispersed in a polymer matrix have been designed to obtain polymer–clay nanocomposites (PCNC) with new and

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interesting properties. In these systems, the dispersed particles have at least one dimension in the nanometre range and consequently there are strong interfacial interactions between the polymer matrix and clay particles as opposed to conventional composites. PCNC have attracted great interest due to the wide range of advantageous properties compared to the free polymers, such as increased mechanical strength, thixotropy, reduced gaseous permeability and higher heat resistance, even though the quantity of clay may be 5% or less. As a result of these advantageous properties, PCNC have been proposed for several applications, in biochemical and pharmaceutical fields [69]. Both biopolymers and clay minerals have been proposed as adequate supports to obtain new drug delivery systems. The use of PCNC for drug delivery purposes appears as an interesting strategy to improve the features of both clays and polymers alone [70]. The properties of the drug delivery systems based on PCNC may be modulated by suitable choice of nanocomposite component materials (i.e. the polymers and clays used) and/or manufacturing conditions. Several methods have been described to load the drug into PCNC. For example, the drug can be adsorbed on the composite surface by suspending the nanocomposites in an active solution and allowing the drug molecules to interact with the PCNC [71]. Another possible loading process may be provided by preparing the polymer– clay nanocomposite in the presence of the active ingredient, both by mixing the polymer matrix, clay particles, and the active ingredient [72]. Coating methods are an interesting possibility to obtain the modified drug delivery PCNC systems. As for example, drug granules of pellets may be partially or completely coated with a solution of the PCNC by using a piece of fluid bed equipment [73]. The release pattern of the encapsulated drug particles may be controlled by the thickness of the PCNC coating and the size of the active ingredient particles. Otherwise, nanoparticles may be obtained by coating of inert particles with a first drug layer (reservoir) and then with a PCNC layer (diffusion layer). Regarding the mechanisms of drug release from the PCNC, diffusion of the drug molecules through the PCNC matrices, swelling or erosion of the PCNC, and ionic interaction between the drug molecules and the polymer and/or clay have been proposed. Frequently, drug release results have been explained on the basis of different mechanisms. For example, the complexation of MAS with alginate beads improved the entrapment efficiency of diclofenac.84 The release rate of the beads followed different kinetics depending on the release medium, being controlled by interaction between the clay and the polymer, which led to an increase in the tortuosity of the swollen beads. Additionally, this interaction caused the stronger gel matrix and the slower disintegration of the beads, compared to non-clay reinforced beads, which led to the slower release rate of diclofenac. In a recent work, Lu and Mai examined most of the existing models for interpreting the permeability of PCNC and proposed a tortuosity based model, taking into account the possible PCNC morphologies, i.e. intercalating, flocculating, and exfoliating and modifying the previous models to consider the random motion of gas/liquid molecules in the constrained environment of the PCNC [74]. In recent years, there has been intense interest on carbon nanotubes (CNTs) because of their unusual physical properties and large application potential, covering a broad range in nanotechnology [75]. With its remarkable tensile strength, high resilience, flexibility, and other superlative electrical, and physico-chemical properties, CNTs are of paramount importance to researchers in the recent years [76]. The large surface area together with the above said properties has also made CNTs and their derivatives very attractive and potential candidates for nanoelectronics, nanolithography, composite materials, sensors, optical

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actuators, biomolecular recognition, and biomedical applications including DNAmodification, drug delivery, and gene delivery [77]. In general, it is widely acknowledged that the chemical modification of CNT surfaces with functional monomers and polymers or physical wrapping of the polymers over the surface of the nanotubes are the methods for making CNT-polymer hybrid materials with tailor made properties and functionalities [78]. Kumar et al. reported a simple method for the functionalization of multi-walled carbon nanotubes (MWNTs) with a biomedically important polymer, poly(2-hydroxyethyl methacrylate) (poly(HEMA)), by chemical grafting of HEMA monomer followed by free radical polymerization (Figure 8). The nanotubes were first oxidized with a mixture of conc. nitric acid and sulfuric acid (1:3), in order to obtain carboxylic acid functionalized MWNTs. Then the grafting of HEMA on to the surface of MWNTs was carried by chemical functionalization of HEMA with acid chloride-bound nanotubes by esterification reaction [79]. FT-IR was used to identify functionalization of –COOH and HEMA groups attached to the surface of the nanotubes. The presence of poly(HEMA) on the nanotubes was confirmed by FESEM, TEM, and TGA analyses. Additionally, the dispersibility of the polymer functionalized nanotubes in methanol was also demonstrated. Considering the biomedical importance of poly(HEMA) and the recent successful in vivo studies on CNTs, in future, these materials are expected to be useful in the pharmaceutical industry as novel biomaterials composites with potential applications in drug delivery. CH

3 CH3

H 2C O C OH

SOCl2 reflux

O

O

C

C Cl

C

O

O O

HEMA Pyridine, THF

AIBN C H

CH 3 3 CH2

C

n

O C

O

O

O

Figure 8. Reaction scheme for the synthesis and preparation of polyHEMA-f-MWNTs.

A specially designed CNT is developed for use in the early detection and treatment of cancer. The key functionalities for biomedical diagnosis and drug delivery are incorporated into the CNTs. Guo et al. assembled the nanotubes with different properties and functionalities based on a unique nanoscale design [80]. An idealized representation of the

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nanostructure design for in vivo imaging and drug storage is schematically illustrated in Figure 9. The hollow core and polymer-coated surfaces of the nanotube can be used to store antitumor agents such as paclitaxel as a consequence of non-covalent adsorption. For deep tissue imaging, the outer surface of the nanotube is conjugated with luminescent materials such as QD. In this study, in vivo imaging of live mice is achieved by intravenously injecting QD-conjugated CNT. With near infrared emission around 752 nm, the CNT with surfaceconjugated QD (CNT-QD) exhibit a strong luminescence for non-invasive optical in vivo imaging. CNT surface modification is achieved by a plasma polymerization approach that deposited ultra-thin acrylic acid or poly(lacticco-glycolic acid) (PLGA) films (~3 nm) onto the nanotubes. The anticancer agent paclitaxel is loaded at 112.5±5.8mg mg-1 to PLGAcoated CNT. Cytotoxicity of this novel drug delivery system is evaluated in vitro using PC3MM2 human prostate carcinoma cells and quantified by the 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT) assay. The in vivo distribution determined by inductively coupled plasma mass spectrometry (ICP-MS) indicates CNT-QD uptake in various organs of live animals.

Carbon nanotube Plasma polymer coating Quantum dot Anti‐cancer drug loaded Figure 9. Schematic diagram illustrating the concept of a CNT functionalized with plasma polymer coating, luminescent QD, and loaded with anticancer drugs. The functionalized CNT can be used as biomarkers and drug carriers.

For most biomedical applications, it is necessary to use biomacromolecules to impart biological functionality and biocompatibility [81]. However, it remains a challenge to incorporate biomacromolecules, such as proteins, into core/shell nanoassemblies due to their fragility. On the other hand, bionanoparticles (BNPs), such as viruses and viruslike biogenic assemblies, are promising building blocks for materials development since they are

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monodisperse in size and shape, and can be functionalized in a robust, well-defined manner [82]. Various functional structures can be obtained through hierarchical self-assembly of BNPs [83]. Recently, a versatile strategy based on noncovalent interactions of bionanoparticles and polymers has been developed to obtain raspberry-like core/shell biocomposites [84]. To enhance the polymer/oligomer–virus interactions, functional groups promoting hydrogen bonding and electrostatic interactions are necessary. Therefore, poly(4vinylpyrindine) (P4VP) was employed in this study because it is well known that P4VP and block copolymers comprising P4VP can assemble with other polymers or nanoparticles to form various morphologies. As shown in Figure 10, the CPMV-co-P4VP nanocomposite was obtained by mixing aqueous solutions of CPMV and P4VP to prepare biocomposite spheres. The structures have very good coverage of the NPs on the surface of polymeric spheres, as characterized by TEM and FESEM analyses. Since bionanoparticles, such as viruses and viruslike particles, ferritins, heat shock protein cages, and enzyme complexes, are highly organized scaffolds with robust chemical and physical properties and fascinating structural symmetries, myriad bionanoparticles have drawn great attention in the past decade in functional materials development [85]. This method allows the synthesis of hierarchically assembled composite colloids using BNPs as building blocks, which will lead to broad potential applications including drug delivery and tissue engineering. Moreover, this approach should be applicable to other types of polymers and biomacromolecules.

Figure 10. Formation of CPMV-co-P4VP raspberry-like nanocomposites through noncovalent interactions.

Micro- and nanoparticles of bio-compatible and bio-degradable polymers such as polylactic-co-glycolic acid (PLGA) are widely used as delivery devices for the administration of sensitive biopharmaceuticals such as proteins, peptides and genes. PLGA composite particles combine at least two different materials, i.e. the polymeric excipient PLGA and one or more active pharmaceutical ingredients. This leads therefore to a number of quality criteria, e.g. drug loading (i.e. the drug fraction in the drug–polymer co-formulation), encapsulation efficiency (i.e. the fraction of the drug used in the process that is encapsulated in PLGA), or homogeneity and stability of the produced co-formulations. Finally, there are further aspects of product quality related to pharmaceutical activity that may be assessed in specific testings either in vitro or in vivo. Kluge et al. prepared PLGA micro/nanocomposite using supercritical fluid extraction of emulsion process [86]. By variation of PLGA concentration and stirring rate during emulsion preparation, particles of pure PLGA with average sizes ranging between 100 nm and a few µm with very narrow size distributions have been produced in controlled and reproducible manner (Figure 11). Moreover, lysozyme has been

Bionanocomposites – Current Status and Prospects in Drug Delivery Fields

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used for the formation of composite particles with PLGA. Three different encapsulation methods have been investigated and evaluated by determining the corresponding encapsulation efficiencies. With the method of in situ suspension emulsions, an encapsulation efficiency of up to 48.5% has been achieved. The current study highlights the potential of supercritical fluid extraction of emulsion as an attractive and scalable process for the manufacturing of drug–PLGA composite particles for pharmaceutical applications.

1. Raw materials

and

2. Drug incorporation

(soluble)

or

3. Emulsion formation

O/W emulsion

Insoluble drug

or

or In‐situ precipitation Immiscible solvents

S/O/W suspension emulsion

and

Alternative processing: ‐Spray drying ‐ co‐acervation W/O/W double emulsion Figure 11. Strategies for drug encapsulation into PLGA particles.

2.5. Chitosan-Based Hydrogels Chitosan, a polysaccharide derived from naturally abundant chitin, is currently receiving a great deal of interest for biomedical application because of good biocompatibility, biodegradability, and bioactivities [87]. The chitosan-based thermosensitive hydrogel systems

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have been extensively studied for biomedical applications, e.g. drug delivery [88] and tissue engineering [89]. All of them are injectable liquid at low temperature and transform to semisolid hydrogels at body temperature. It is mainly because that the temperature responsive hydrogels do not require organic solvents, copolymerization agents, or externally applied trigger for gelation suitable for biomaterial applications [90]. Chenite et al. [91] first developed a novel approach to produce thermosensitive neutral hydrogel based on chitosan/polyol salt combinations that could undergo sol–gel transition at a temperature close to 37 ºC. Other researchers also evaluated the hydrogel for use in pharmaceutical applications [92] and cartilage repair [93]. Many modified chitosan polymers also have thermosensitive characteristics, such as the PEG-grafted chitosan [94], hydroxybutyl chitosan [95], N-isopropylacrylamide-grafted chitosan [96] and quaternized chitosan [97]. All of them are injectable liquid at low temperature and transform to semisolid hydrogels at body temperature. Therefore, they have a broad range of medical applications, particularly for sustained in vivo drug release and tissue engineering. However, the burst delivery of drug-loaded gels is obvious. This disadvantage can restrict their applications as biomaterials. Nanoparticles have been proposed as drug delivery systems with potential applications such as prolonging the residence time of drugs in the blood circulation [98] or improving transmucosal transport of macromolecular bioactive compounds [99]. In recent years, nanoparticles based on polyelectrolyte complexes from oppositely charged macromolecules as controlled drug release formulations, especially for peptide and protein drug delivery, have attracted considerable attention [100]. Polyelectrolytes are macromolecules carrying a relatively large number of functional groups that either are charged, or under suitable conditions, can become charged [101]. The macromolecules may constitute either polycations or polyanions, depending on their functional group type. Polyelectrolyte complexes are therefore formed by the reaction of one polyelectrolyte with another oppositely charged polyelectrolyte in an aqueous solution. The process is simple, feasible, and usually performed under mild conditions. Another advantage of this system is that since preparation of the complex is through physical crosslinking by electrostatic interactions instead of chemical crosslinking; the possibility of toxicity associated with crosslinking reagents involved in chemical crosslinking processes can be eliminated. Among these polymers, polysaccharides have been frequently studied for drug delivery and medical applications. A few papers [102, 103] also reported the quaternized chitosan nanoparticles were biocompatible and non-toxic according to cytotoxicity assay and used as antibacterial agent, drug, and gene delivery system. Through the electrostatic effect of –N+(CH3)3 and –COO-, the nanoparticles of N-(2-hydroxyl) propyl-3-trimethyl ammonium chitosan chloride –carboxymethyl chitosan were prepared. The nanoparticles with different charges were obtained by the different ratio of –N+(CH3)3 and –COO-, which were suitable for drug delivery with opposite charges, such as propranolol and diclofenac sodium. Recently, the synthesis and characterization of a thermosensitive chitosan/poly(vinyl alcohol) (PVA) composite hydrogel containing nanoparticles with different charges for drug delivery were reported [104]. The release of the positive drug was the slowest with the hydrogels containing negative nanoparticles. Similarly, the release of the negative drug was the slowest with the hydrogels containing positive nanoparticles. However, the releases of the two drugs were both the fastest with the pure hydrogels. It indicated the addition of nanoparticles was helpful to slow the suitable drug release. Though the nanoparticles did not reinforce the gel strength, the

Bionanocomposites – Current Status and Prospects in Drug Delivery Fields

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electrostatic effect between nanoparticles and drugs reduced the burst release. Therefore, the composite gels are attractive for applications as carriers for drug delivery.

2.6. Magnetic BNCs With the growing interest in nanocomposites and their applications in biology and medicine, studies examining the biocompatibility of those materials are critical. Magnetic hydrogel nanocomposites based on poly (N-isopropylacrylamide) and iron oxide nanoparticles were fabricated via UV-polymerization with tetra(ethylene glycol) dimethacrylate acting as the crosslinking agent [105]. In vitro biocompatibility analysis via NIH 3T3 murine fibroblast cytotoxicity was investigated. The fibroblasts in both direct and indirect contact with the hydrogels exhibited favorable cell viability indicating minimal cytotoxicity of the systems. In addition, swelling studies indicated that hydrogels with lower crosslink densities yield higher swelling ratios and that the presence of magnetic nanoparticle did not affect the swelling response of the hydrogel systems. Upon exposure to an alternating magnetic field, the hydrogel nanocomposites with iron oxide nanoparticles showed the capability for remote heating (Figure 12). This evaluation shows that these hydrogels have the potential to be used in biomedical applications such as drug delivery and hyperthermia for cancer treatment.

Figure 12. Schematic illustrating the effect of an alternating magnetic field on a NIPAAm/iron oxidebased hydrogel nanocomposite. The collapsing response of the hydrogel system upon the increase in temperature from the heating of the magnetic particles in the AC magnetic field can be seen at the nano-, micro-, and macroscopic levels.

Surface control of magnetic nanoparticles is gaining in importance since surface modification has been proven useful in a wide range of technological applications including electronics and photonics, heterogeneous catalysis, chemical sensing, water remediation,

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information storage and medical diagnosis [106]. Compared to polymer coatings, silicacoated nanoparticles represent one alternative to increase relaxivity and expand modularity through different chemistries [107]. Recently, monodisperse iron oxide/microporous silica core/shell composite nanoparticles, core( -Fe2O3)/shell(SiO2), with a diameter of approximately 100nm and a high magnetization were synthesized by combining sol–gel chemistry and supercritical fluid technology [108]. The chemical process to fabricate the material is shown in Figure 13. This one-step processing method, which is easily scalable, allows quick fabrication of materials with controlled properties and in high yield. The particles have a specific magnetic moment (per kg of iron) comparable to that of the bulk maghemite and show superparamagnetic behavior at room temperature. The nanocomposites are proven to be useful as T2 MRI imaging agent. They also have potential to be used in NMR proximity sensing, theranostic drug delivery, and bioseparation.

Figure 13. Processing pathway for obtaining the nanocomposite material. 1) Colloidal dispersion of Fe2O3 nanoparticles in hexane. 2) Initial sol with silicon precursor, water, solvents and iron oxide NPs at ambient conditions. 3) Expanded sol under supercritical conditions with gel composite particles. 4) Dry composite particles.

CONCLUSIONS In this chapter, we have discussed an emerging group of BNCs based on various natural as well as synthetic polymers and nanofillers that are either used extensively or show promise in the area of biomedical fields. These novel materials vary from inorganic/ceramicreinforced nanocomposites for magnetic and mechanical enhancement to peptide-based nanomaterials in which peptides are both the filler and the matrix, with the chemistry designed to render the entire material biocompatible. Interest in these BNCs varies from application-oriented design to understanding a multitude of structure–property relations. Requisite functional criteria include mechanical strength, biocompatibility, biodegradability,

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morphology, and a host of other parameters, depending on end use. However, at the basis of the performance of these BNCs are interactions between the biopolymer or synthetic polymer and the filler, which can be tuned and perfected to suit specific needs. We hope that further research into these interactions will prove valuable in contemplating the design of novel BNCs for biomedical applications including drug delivery and tissue engineering.

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[91] Chenite, A., Chaput, C., Wang, D., Combes, C., Buschmann, M. D., Hoemann, C. D., Leroux, J. C., Atkinson, B. L., Binette, F., Selmani, A., 2000, Biomaterials 21, 2155. [92] Gariepy, E. R., Leclair, G., Hildgen, P., Gupta, A., Leroux, J. C., 2002, J. Controlled Release 82, 373. [93] Hoemann, C. D., Sun, J., Legare, A., McKee, M. D., Ranger, P., Buschmann, M. D., 2001, Trans. Orthop. Res. Soc. 26, 626. [94] Bhattarai, N., Ramay, H. R., Gunn, J., Matsen, F. A., Zhang, M. Q., 2005, J. Controlled Release 103, 609. [95] Dang, J. M., Sun, D. N., Ya, Y. S., Sieber, A. N., Kostuik, J. P., Leong, K. W., 2006, Biomaterials 27, 406. [96] Chung, H. J., Bae, J. W., Park, H. D., Lee, J. W., Park, K. D., 2005, Macromol. Symp. 224, 275. [97] Wu, J., Su, Z. G., Ma, G. H., 2006, Int. J. Pharm. 315, 1. [98] Gref, R., Minamitake, Y., Peracchia, M. T., Trubetskoy, V., 1994, Science 263, 1600. [99] Mathiowitz, E., Jacob, J. S., Jong, Y. S., Carino, G. P., Chickering, D. E., Chaturvedi, P., Santos, C. A., Vijayaraghavan, K., Montgomery, S., Bassett, M., Morrel, C., 1997, Nature 386, 410. [100] Xu, Y. M., Du, Y. M., Huang, R. H., Gao, L. P., 2003, Biomaterials 24, 5015. [101] Spalla, O., 2002, Curr. Opin. Colloid Int. Sci. 7, 179. [102] Shi, Z. L., Neoha, K. G., Kanga, E. T., Wang, W., 2006, Biomaterials 27, 2440. [103] Wang, X. Y., Du, Y. M., Luo, J. W., 2008, Nanotechnology 19(6), 65707. [104] Tang, Y., Zhao, Y., Li, Y., Du, Y., 2010, Polym. Bull. Doi: 10.1007/s00289-009-02140. [105] Meenach, S. A., Anderson, A. A., Suthar, M., Anderson, K. W., Hilt, J. Z., 2009, J. Biomed. Mater. Res. 91A, 903. [106] Jeong, U., Teng, X. W., Wang, Y., Yang, H., Xia, Y. N., 2007, Adv. Mater. 19, 33. [107] Barbe, C., Bartlett, J., Kong, L., Finnie, K., Lin, H. Q., Larkin, M., Calleja, S., Bush, A., Calleja, G., 2004, Adv. Mater. 16, 1959. [108] Taboada, E., Solanas, R., Rodriguez, E., Weissleder, R., Roig, A., 2009, Adv. Funct. Mater. 19, 2319.

In: Recent Developments in Bio-Nanocomposites… ISBN 978-1-61761-008-0 Editor: Ashutosh Tiwari © 2011 Nova Science Publishers, Inc.

Chapter 3

DESIGN OF A BIOCOMPATIBLE NANOCOMPOSITE PARTICULATE AND DRUG DELIVERY SYSTEM Roli Mishra and Satyendra Mishra Department of Medicinal Chemistry, University of Minnesota Minneapolis, MN 55455 Minnesota, USA

ABSTRACT Nanocomposite biocompatible hydrogels (NCHG) are an important class of biomaterials that can be utilized in applications such as drug delivery, tissue engineering, and hyperthermia treatment. The composite consists of the following components: nanoparticles (NPs), matrix gel, and chlorhexidine (CHX) as antibacterial drug. The NPs were obtained by free radical initiated copolymerization of the monomers, 2hydroxyethyl methacrylate (HEMA) and polyethyleneglycol dimethacrylate (PEGDMA), in aqueous solution. The same monomers were used to prepare crosslinked matrices by photopolymerization. NCHGs were obtained by mixing NPs, monomers, and drug in an aqueous solution then crosslinked by photopolymerization. The incorporation of nanoparticles into a hydrogel matrix can provide unique properties including remote actuation and can also improve properties such as mechanical strength. Since hydrogel nanocomposites have been proposed as implantable biomaterials, it is important to analyze and understand the response of the body to these novel materials. This chapter covers the background, definitions, and potential applications of hydrogels and hydrogel nanocomposites. It also covers the various types of hydrogel nanocomposites as defined by the nanoparticulates embedded in the systems which include clay, metallic, magnetic, and semiconducting nanoparticles. The specific concerns of the biocompatibility analysis of hydrogel nanocomposites are discussed along with the specific biocompatibility results of the nanoparticulates incorporated into the hydrogel matrices as well as the biocompatibility of the hydrogels themselves. The limited data available on the biocompatibility of hydrogel nanocomposites is also presented. Overall, currently investigated hydrogel systems with known biocompatibility may have the potential to provide a “shielding” effect for the nanoparticulates in the hydrogel nanocomposites allowing them to be safer materials than the nanoparticulates alone.

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INTRODUCTION Hydrogels are an important class of polymeric materials that have been utilized in a wide variety of biomedical and pharmaceutical applications. Hydrogels are threedimensional, hydrophilic, polymeric networks that can absorb up to thousands of times their dry weight in water or biological fluids [1,2]. They consist of polymer chains with either physical or chemical crosslinks that prevent the dissolution of the hydrogel structure and instead result in swelling of the material upon interaction with aqueous solutions. Hydrogels are advantageous for many biomedical applications due to their resemblance of natural living tissue and inherent biocompatibility which can be partially attributed to their soft, flexible nature and high water content [1]. Hydrogel systems such as poly(hydroxyethyl methacrylate) (PHEMA), poly(N-isopropylacrylamide) (PNIPAAm), poly(vinyl alcohol) (PVA), and poly(ethylene glycol) (PEG) have been widely investigated for a wide variety of biomedical and pharmaceutical applications.

Hydrogel Nanocomposites Despite the many advantages of using conventional crosslinked hydrogels, their applications are often limited due to their poor mechanical and limited response properties [3]. The random nature of the crosslinking reactionsinvolved in hydrogel fabrication and the resulting morphological inhomogeneity can induce these limitations [4]. Recently, work has been done to improve hydrogel properties (e.g.,mechanical strength) and to add unique properties (e.g., response to novel stimuli) through the fabrication of hydrogel nanocomposites [5]. Hydrogel nanocomposites involve the incorporation of various nanoparticulate materials within a versatile hydrogel matrix which can provide easy, straightforward methods for enhancing the properties (e.g., improving the mechanical properties) of hydrogels. Although a number of fabrication techniques have been used to create such systems, in situ polymerization of particles into a monomer solution is a common way to create a hydrogel nanocomposite. Nanocomposite hydrogels have been shown to modify and improve a variety of material properties, including magnetic and optical properties. For example, it is possible to tune a temperature-responsive system with electrochemical responses of a conducting polymer through the addition of electroactive, conducting particles in a hydrogel matrix [6]. Thus far, a number of nanoparticulates have been utilized in nanocomposite hydrogel systems including metallic nanoparticles, carbon nanotubes (CNTs), clay, ceramics, magnetic nanoparticles, hydroxyapatite (HA), and semiconducting nanoparticles. A hydrogel-drug system that relies on the consecutive action of two trigger mechanisms to release a drug has the potential to target specific sites within the body, according to the scientists in the Netherlands who developed the system. The triggers are a stimulus that converts the gel into a solution and an enzyme that cleaves the hydrogelator-drug link. The system has a number of advantages over other two-stage drug delivery systems such as polymeric gelator systems, according to the team. First, the gels have rapid response times—on the order of a few seconds—that are not attainable by polymeric systems. In addition, the enzymatic cleavage of the gelator-drug linker is specific for the linker bonds.

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Figure 1. HYDROGEL DRUG: System comprises a gelator scaffold (pink), linker (green), and model drug (blue).

Figure2. CLEAVAGE Release of drug from gel fiber requires two stimuli: first heat or a pH decrease, then an enzyme.

“Since certain enzymes are present only in specific sites within the human body—for example, at tumor sites—it is possible to use enzymes to selectively cleave bonds and thus release drugs exclusively in target areas or tissues,” van Bommel says.

Clay-Based Nanocomposite One of the most widely studied classes of hydrogel nanocomposites involves the addition of nanoparticulate clay to the hydrogel system. Thermoresponsive PNIPAAm systems have been the most commonly used, however, systems involving poly(acrylic acid) (PAA), poly(methyl methacrylate) (PMMA), and poly(N, N-dimethylacrylamide) (PDMAA) have also been studied. One of the main advantages of the addition of clay to hydrogels is that the clay has been shown to act as a crosslinking agent, increasing the mechanical properties of the composites [7,8]. The improvement in mechanical strength is primarily driven by good dispersion and/or the ability of the clay to exfoliate in the polymer [7]. The types of clay nanoparticulates incorporated into hydrogel nanocomposites include montmorillonite (MMT), bentonite, and other silicate clays. These systems have been widely characterized regarding changes in mechanical strength, swelling properties, drug release rates, and thermal transitions. The subject of hybrids based on layered inorganic compounds such as clays has been studied for a considerable time, but the area is enjoying a resurgence of interest and activity

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as a result of the exceptional properties which can be realised from such nanocomposites. Materials variables which can be controlled and which can have a profound influence on the nature and properties of the final nanocomposite include the type of clay, the choice of clay pre-treatment, the selection of polymer component and the way in which the polymer is incorporated into the nanocomposite. The last of these may be dictated by the available processing methods and whether the prospective user is an integrated polymer manufacturer or a specialist processor.

Clays and Clays Modification Common clays are naturally occurring minerals and are thus subject to natural variability in their constitution. The purity of the clay can affect final nanocomposite properties. Many clays are aluminosilicates, which have a sheet-like (layered) structure, and consist of silica SiO4 tetrahedra bonded to alumina AlO6 octahedra in a variety of ways. A 2:1 ratio of the tetrahedra to the octahedra results in smectite clays, the most common of which is montmorillonite. Other metals such as magnesium may replace the aluminium in the crystal structure. Depending on the precise chemical composition of the clay, the sheets bear a charge on the surface and edges, this charge being balanced by counter-ions, which reside in part in the inter-layer spacing of the clay. The thickness of the layers (platelets) is of the order of 1 nm and aspect ratios are high, typically 100-1500. The clay platelets are truly nanoparticulate. In the context of nanocomposites, it is important to note that the molecular weight of the platelets (ca. 1.3 x 108) is considerably greater than that of typical commercial polymers, a feature which is often misrepresented in schematic diagrams of clay-based nanocomposites. The clays often have very high surface areas, up to hundreds of m2 per gram. The clays are also characterised by their ion (e.g. cation) exchange capacities, which can vary widely. One important consequence of the charged nature of the clays is that they are generally highly hydrophilic species and therefore naturally incompatible with a wide range of polymer types. A necessary prerequisite for successful formation of polymer-clay nanocomposites is therefore alteration of the clay polarity to make the clay ‘organophilic’. An organophilic clay can be produced from a normally hydrophilic clay by ion exchange with an organic cation such as an alkylammonium ion. For example, in montmorillonite, the sodium ions in the clay can be exchanged for an amino acid such as 12-aminododecanoic acid (ADA): Na+-CLAY + HO2C-R-NH3+Cl- Æ.HO2C-R-NH3+-CLAY + NaCl The way in which this is done has a major effect on the formation of particular nanocomposite product forms and this is discussed further below. Although the organic pretreatment adds to the cost of the clay, the clays are nonetheless relatively cheap feedstocks with minimal limitation on supply. Montmorillonite is the most common type of clay used for nanocomposite formation; however, other types of clay can also be used depending on the precise properties required from the product. These clays include hectorites (magnesiosilicates), which contain very small platelets, and synthetic clays (e.g. hydrotalcite), which can be produced in a very pure form and can carry a positive charge on the platelets, in contrast to the negative charge found in montmorillonites.

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Synthetic Processing of Clays-Based Nanocomposites The synthetic route of choice for making a nanocomposite depends on whether the final material is required in the form of an intercalated or exfoliated hybrid (Figure 3). In the case of an intercalate, the organic component is inserted between the layers of the clay such that the inter-layer spacing is expanded, but the layers still bear a well-defined spatial relationship to each other. In an exfoliated structure, the layers of the clay have been completely separated and the individual layers are distributed throughout the organic matrix. A third alternative is dispersion of complete clay particles (tactoids) within the polymer matrix, but this simply represents use of the clay as a conventional filler.

Figure 3. Illustration of the supercritical carbon dioxide process. Polymer and clay are mixed together followed by a soaking period in scCO2. The system is depressurized, and the expanding CO2 delaminates platelets

Factors Affecting the Tpye of Organo-Clay Hybrid Formed In recent years, there has been extensive study of the factors which control whether a particular organo-clay hybrid can be synthesised as an intercalated or exfoliated structure. Since clay nanocomposites can produce dramatic improvements in a variety of properties, it is important to understand the factors which affect delamination of the clay. These factors include the exchange capacity of the clay, the polarity of the reaction medium and the chemical nature of the interlayer cations (e.g. onium ions). By modifying the surface polarity of the clay, onium ions allow thermodynamically favourable penetration of polymer precursors into the interlayer region. The ability of the onium ion to assist in delamination of the clay depends on its chemical nature such as its polarity. For positively charged clays such as hydrotalcite, the onium salt modification is replaced by use of a cheaper anionic surfactant. Other types of clay modification can be used depending on the choice of polymer, including ion-dipole interactions, use of silane coupling agents and use of block copolymers. An example of ion-dipole interactions is the intercalation of a small molecule such as dodecylpyrrolidone into the clay. This is similar in concept to compatibilisation of polymer blends. A typical block copolymer would consist of a clay-compatible hydrophilic block and a polymer-compatible hydrophobic block (figure 4).

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Figure 4. Structure of a typical polymer-compatible hydrophobic block.

The block length must be controlled and must not be too long. High degrees of exfoliation are claimed using this approach.

Polymer Incorporation The correct selection of modified clay is essential to ensure effective penetration of the polymer or its precursor into the interlayer spacing of the clay and result in the desired exfoliated or intercalated product. Indeed, further development of compatibiliser chemistry is undoubtedly the key to expansion of this nanocomposite technology beyond the systems where success has been achieved to date. Polymer can be incorporated either as the polymeric species itself or via the monomer, which is polymerised in situ to give the corresponding polymer-clay nanocomposite. The second of these is the most successful approach to date, although it probably limits the ultimate applicability of these systems. Polymers can be introduced either by melt blending, for example extrusion, or by solution blending. Melt blending (compounding) depends on shear to help delaminate the clay and can be less effective than in situ polymerisation in producing an exfoliated nanocomposite.

Clays-Nylon Nanocomposite The earliest example of the in situ polymerisation method was work by Toyota on synthesis of clay-nylon nanocomposites and this remains probably the most studied system, including work by Bayer and Ube. In a typical synthesis ADA-modified clay is dispersed in the monomer caprolactam, which is polymerised to form the nylon-6-clay hybrid as an exfoliated composite. Complete exfoliation may be preceded by intercalation of the monomer in the clay. Generally, low concentration of clay (a few %) are incorporated in these nanocomposites, partly because this is often sufficient to modify the desired properties significantly, but also because higher levels of clay can adversely increase the system viscosity leading to poor processability, although the viscosity increase is shear rate dependent. Other nylons and copolyamides (e.g. nylon-6/6,6) have also been incorporated in clay nanocomposites. Functionality such as hydroxyl groups can be introduced into the onium salt modifiers to improve compatibility with the nylon via hydrogen bonding – this can lead to an enhancement of desirable nanocomposite properties.

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Similar modification of the chemistry may be required for successful exfoliation with other types of polymer system. In the case of ethylene-vinyl alcohol (EVOH) copolymers, use of hydroxylated quaternary ammonium ions improves compatibility between the clay and the EVOH by introducing favourable hydroxyl group interactions. In polypropylene (PP) nanocomposites, maleic anhydride grafted PP is used as a compatibiliser. Polymerisation initiators can be anchored to the clay platelet surface and this approach has been extended to living free radical polymerisation of styrene, where an initiating species bonded to the TEMPO free radical is attached to the surface of the clay platelets via ion exchange.

High Temperature Thermoplastics in Clay-Based Nanocomposites For preparation of nanocomposites from high temperature engineering thermoplastics, a major limitation in the use of conventional onium ion modified clays is the thermal instability of the alkylammonium species during processing. The block copolymer route developed by TNO (vide supra) offers one potential solution to this problem. Imidazolium salts such as that depicted in Figure 3 are also more thermally stable than the ammonium salts.

Figure 5. Structure of an imidazolium salts.

A further alternative is the use of phosphonium salts in place of ammonium salts – this can lead to an increase in the degradation temperature of the organo-clay from 200-300°C to >300°C. Wholly synthetic organo-clays clays, on the other hand, can exhibit thermal stability to beyond 400°C. By using these last two approaches, Triton Systems has succeeded in producing nanocomposites from high temperature resins such as polyetherimide (PEI). Forming the nanocomposite provides a potential route to producing ‘speciality’ engineering resin performance from cheaper ‘standard’ engineering resins.

Controlled Release Drug Delivery from Hydrogels Controlled release drug delivery is a new way to treat illnesses. The term controlled release refers to the ability of a drug delivery system to release a drug over an extended period of time at a controlled rate. Over the last 20 years, it has become more popular as a way to treat diseases such as cancer and diabetes. It generally involves implanting an engineered polymer directly into the organ or system that is affected by a disease. Since the polymer is implanted directly into the tissues affected by disease, the side effects are often small compared to systemic drug delivery (i.e. taking a pill or getting a shot). Brain diseases

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are particularly good candidates for controlled release techniques because of a physiological feature known as the blood-brain barrier. The blood- brain barrier refers to a tight sheath of cells that surround the blood vessels in your brain. These cells make sure that only specific types of molecules get into the brain. More specifically, only small (molecu- lar weight less than 1000 Da), water insoluble molecules can get into the brain. Consequently, the types of drugs that are developed for brain disease must fit this criterion, which is unfortunate because many promising drugs are water soluble or large. The use of controlled re- lease techniques has led to tremendous breakthroughs in treating people brain diseases.

An Example of a Novel Drug Delivery System In 1997, chemical engineers at Purdue University in West Lafayette, IN, under the direction of Nicholas A. Peppas, reported the synthesis of a glucose-sensitive hydrogel that could be used to deliver insulin to diabetic patients using an internal pH trigger. This system features an insulin-containing “reservoir” formed by a poly[methacrylic acid-g-poly(ethylene glycol)] hydrogel membrane into which glucose oxidase has been immobilized. The membrane itself is housed between nonswelling, porous “molecular fences”. Unlike hydrogel systems that release their entrapped drug entities upon swelling, this system works oppositely by shrinking the membrane “gates”. The exact trigger for this mechanism involves creating an acidic environment around the gel. This is accomplished when a body produces high sugar levels; glucose interacts with the immobilized glucose oxidase in the gates, yielding gluconic acid, which in turn lowers the body’s pH and triggers the gate opening. An individual’s own glucose levels thus determine and direct the insulin delivery. At present, researchers are studying ways to precisely control the drug delivery rate by considering the effects of varying the size of the gates, the concentration of the entrapped insulin, and the rate at which the gates can open and close[9].

Drug-Carryingmagnetic Nanocomposite Particles for Potential Drug Delivery Systems Recently, researchers have been trying to develop targeted therapeutic systems by using external forces, including magnetic fields, ultrasound, electric fields, temperature, light, and mechanical forces to concentrate drugs within tumors [10-13]. In these systems, the drug is localized at a specific targeted area by externally generated forces, and then activated them [14]. Of the type considered, magnetic particles carrying drug molecules are targeted to specific sides of the body by external magnetic fields. Shortly after concentration on targeted region, drug molecules are gradually released, thus improving the therapeutic efficiency of the drugs by lowering the collateral toxic site effects on the healthy cells or tissues [15-18]. Magnetic targeted system with fields generated between 100 and 2500 Oe seems more promising as the basis of a drug localization system due to their effectiveness, lower risk, cost, and practical use, as compared to other systems [19-21]. The three main mechanisms for releasing drug molecules from the polymeric magnetic spheres into a blood vessel or tissue are diffusion, degradation, and swelling followed by diffusion [22,23]. Diffusion occurs when drug molecules dissolve in bodily fluids around or

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within the particles and migrate away from the particles. Degradation takes place when the polymer chains hydrolyze into lower molecular weight species, effectively releasing drug molecules that were trapped by the polymer chains. Swelling-controlled release systems are initially dry. When they are placed in the body, they swell to increase inside pressure and porosity, enabling the drug molecules to diffuse from the swollen network. The release of active drug molecules can also be varied over a certain period based on external and internal parameters [20-24]. Figure 6 shows the diffusion, degradation, and swelling release mechanisms of drugcarrying magnetic spheres. Poly (D,L-lactide-co-glycolide) (PLGA), approved by the Food and Drug Administration (FDA) for drug delivery purposes [24-27], is used as a host material because of its biodegradability.

Figure 6: Schematic illustrations of diffusion, degradation, and swelling release mechanisms of drugcarrying magnetic PLGA spheres

Poly(Amidoamine)( PAMAM) /CMS Dendritic Nanocomposite for Controlled Drug Delivery The invention of the dendrimer is attributed to Donald Tomalia, who first published his report of poly(amidoamine) dendrimer synthesis in 1979 from his laboratory at the Michigan Molecular Institute in Midland, Michigan. His first poly(amidoamine) dendrimer was the result of reacting three methylacrylate molecules to an ammonia core, followed by the addition of three ethylenediamine molecules to form the G0 amidoamine. By continuing this two-step process of methylacrylate/ethylenediamine addition, successive amidoamine generations are produced, doubling the number of terminal amine groups each time [28] . Concurrently, Fritz Vögtle of the University of Bonn published his own dendrimer synthesis consisting of ammonia derivatives with acrylonitrile. Later, George Newkome would publish an alternative synthesis of similar molecules that he called “arborols,” after the tree-like symmetry, but his 1985 discovery would be largely overshadowed four years later when a collaboration between Cornell University and ATandT Laboratories would revolutionize dendrimer synthesis, setting off an explosion of dendrimer research around the globe [29] . In 1989, Jean Fréchet of Cornell University and Timothy Miller of ATandT Laboratories jointly developed a convergent synthesis for producing dendrimers. Rather than иeginning with a core molecule and building each generation onto the core outwardly, Fréchet and

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Miller were able to begin with the dendrimer periphery and inserted the molecular core as the last step [30]. In this manner, high-purity dendrons of the desired generation could be synthesized, and then by reacting these dendrons with the core molecule, dendrimers could be produced with the same high purity [31]. Prior to the development of convergent dendrimer synthesis, only a handful of scientific papers had been published on dendrimer research; in the five years that followed, dendrimer research literally exploded within the scientific community[32]. Progress in nanoscience and nanotechnology has led to an explosive growth in the young and immature, inter-disciplinary field of nanomedicine. Syntheses of precise, biocompatible macromolecules with nanometer dimensions have been reported. Perhaps, one of the most monodispersed polymers that is commercially available is the poly(amidoamine) (PAMAM) dendrimer . This molecule has a core from which branches emanate in a regular, well-defined fashion. The resulting water-soluble macromolecule has a high density of reactive groups on its surface with cavities in its interior. The unique architecture of a dendrimer allows attachment of various molecules on its surface or inclusion within its cavities. Attachment of molecules such as fluorescent probes, drugs, imaging agents, oligonucleotides, cell-specific targeting agents, or antibodies to a dendrimer creates nanodevices. These nanoparticles can be controlled and manipulated for numerous potential applications in medicine. The availability of a wide variety of biocompatible linkers for construction of nanodevices is critical for their successful application in nanoscience and nanomedicine. A number of commercially available heterobifunctional bioconjugation linkers have been used in preparation of various nanodevices. Because of their early discovery and thus the amount of research that has been conducted with them, PAMAM dendrimers are among the very few commercially available dendrimers, available in generations 0 to 10 from Aldrich, Inc. The PAMAM dendrimers available from Aldrich, Inc., are prepared by a divergent synthesis [33]. Dendrimers are one of the emerging delivery systems with the capability to present such hydrophobic agents in a formulation with better prospective .These dendritic macromolecules with a large number of suface terminal groups and interior cavities offer a better opportunity for delivery by becoming charged and acting as static covalent micelles.These are biocompatible, nonimmunogetic , and water- soluble and possess terminal functional groups for binding various targeted or guest molecules [34]. The host – guest properties of dendrimers based on hydrophobic and ionic interactions apart from physical entrapment have been thoroughly studied [35, 39]. Polyamidoamine (PAMAM) dendrimers are highly hyperbranched synthetic polymers with well-defined spherical structure and nanometer scale size [40]. Their diameters increase with each generation while molecular weight and number of functional surface groups double [41]. The functional surface groups make them very hydrophilic and highly water soluble [42]. These surface functional groups are often used to covalently attach drugs, targeting ligands or imaging agents for targeted delivery, controlled release, or imaging applications [43]. Small drug–dendrimer conjugates aim to carry therapeutic agents to specific tissues in order to reduce systemic effects and increase efficacy at the targeted site. For such a strategy to be effective the conjugates should be stable until they reach the site of interest and then release the drugs within the target tissue before they are eventually cleared from the body [44]. Premature release or high stability of the conjugate will render the conjugate delivery

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system ineffective since most small drugs will not be active in conjugated form. Therefore one key challenge in the preparation of dendrimer–drug conjugates is to design systems that can release their payloads specifically at the desired tissue in a predetermined fashion. In order to predict the efficacy of dendrimer conjugates in vivo, determination of drug release profiles in conditions that conjugates would go through is necessary. Better understanding of the release mechanisms and profiles of PAMAM dendrimer–drug conjugates will help design effective delivery systems. Amide and ester bonds are most commonly used for the conjugation of small drugs to polymers whereas other linkages such as disulfide bonds have also been demonstrated [4549]. Preparation and in vitro efficacy of PAMAM dendrimer conjugates of ibuprofen, methylprednisolone, and N-acetyl cysteine were recently reported [50-51].

 

Figure 7. Synthesis of G4-GFLG–ibuprofen conjugate.

Drug release from polymer–drug conjugates plays a crucial role on the efficacy. This is especially true for dendrimers where there is a steric crowding at the surface. The drug release characteristics of G4- polyamidoamine (PAMAM) dendrimer–ibuprofen conjugates with ester, amide, and peptide linkers were investigated, in addition to a linear PEG–ibuprofen conjugate to understand the effect of architecture and linker on drug release. Ibuprofen was directly conjugated toNH2-terminated dendrimer by an amide bond and OH-terminated dendrimer by an ester bond. Understanding these structural and steric effects on their drug release characteristics is crucial for the design of dendrimer conjugates with high efficacy. Drug release profiles of ester- and amide-bonded conjugates of 4th generation PAMAM dendrimers in various media are reported. The hydrolysis of ester conjugates showed clear pH-dependent rates, whereas the amide conjugates were very stable at all pH buffers. PAMAM dendrimer–ibuprofen conjugates without linkers were found to be stable against enzymatic hydrolysis due to steric effects. The steric blockage of esterase activity in plasma by the dendrimer structure makes ester linkage useful for dendrimer-based sustained release systems. Such ester-linked PAMAM dendrimer conjugates are promising drug carriers that

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could provide controlled release for various drugs, in addition to their inherent targeting capabilities. Alternatively, amide bonds in peptidyl linkerscan be employed for conjugation if an enzymatic cleavage scheme can be identified. Steric hindrance plays a key role in enzymatic cleavage of drugs from dendrimer conjugates, since esterase and protease enzymes are macromolecules of comparable size. Better understanding of such steric effects and appropriate choice of linkage leads to effective drug delivery formulations and have important implications on designingPAMAMdendrimer–drug conjugates.

 

Figure 8: Synthesis of PAMAM–S–S–NAC (1).

A PAMAM–(COOH)46–(NAC)18 conjugate has been prepared using a disulfide linker, that enables relatively rapid intracellular release of the drug. The FITC-labeled anionic dendrimer is rapidly taken up by microglial cells, despite the anionic surface charge. PAMAM–(COOH)46–(NAC)18 conjugate is non-toxic even at the higher concentrations tested in vitro. PAMAM–(COOH)46–(NAC)18 conjugate is a more effective anti-oxidant and anti-inflammatory agent when compared to free NAC in vitro. The conjugate showed significant efficacy even at the lowest dose (0.5mM NAC), where the activity was comparable or better than that of Eunice KennedyShriver free drug at 8mM (16× higher dosage). This suggests that dendrimer–NAC conjugates could be effective nanodevices in decreasing inflammation and injury, induced by activated microglial cells in disorders such as cerebral palsy. Dendrimers are emerging as potential intracellular drug delivery vehicles. Understanding and improving the cellular efficacy of dendrimer–drug conjugates, can lead to significant in vivo benefits. This study explores efficacy of anionic polyamidoamine (PAMAM–COOH) dendrimer–N-acetyl cysteine (NAC) conjugates for applications in neuroinflammation. The anti-oxidative and anti-inflammatory effects of PAMAM–(COOH)46–(NAC)18 conjugate is evaluated on microglial cells in vitro. Anionic PAMAM dendrimer–NAC conjugatewas synthesized with a glutathione sensitive linker for intracellular release. The non-toxic conjugate is a more effective anti-oxidant and anti-inflammatory agent when compared to free NAC in vitro.

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Figure 9. NAC release mechanism of PAMAM–S–S–NAC in the presence of excess GSH.

CONCLUSION Hydrogel nanocomposites are novel biomaterials that can be used in a wide variety of applications including tissue engineering, drug delivery, and hyperthermia treatment.They are often vantageous to conventional hydrogels in that they provide improved properties such as increased mechanical strength and unique properties such as remote control actuation. These improvements can allow for hydrogel systems to be used in areas (e.g. bone tissue engineering) where they would not have been used before. The safety of hydrogel nano composites can be evaluated through their biocompatibility responses. Although it would be ideal to assume that since each of the constituents of a hydrogelnanocomposite is biocompatible that the entire system would also be, however, this is not necessarily a wise assumption. It is necessary to evaluate each composite based on the specific application, it will be used in whether that is tissue engineering, biosensors, or drug delivery. Only at this point can a hydrogel nanocomposite be deemed safe for biomedical use. Dendrimers are emerging as potential intracellular drug delivery vehicles. Understanding and improving the cellular efficacy of dendrimer–drug conjugates, can lead to significant in vivo benefits. Drug release from polymer–drug conjugates plays a crucial role on the efficacy. This is especially true for dendrimers where there is a steric crowding at the surface. The drug release characteristics of G4- polyamidoamine (PAMAM) dendrimer–ibuprofen conjugates with ester, amide, and peptide linkers were investigated, in addition to a linear PEG– ibuprofen conjugate to understand the effect of architecture and linker on drug release. Ibuprofen was directly conjugated toNH2-terminated dendrimer by an amide bond and OHterminated dendrimer by an ester bond. A tetra-peptide-linked dendrimer conjugate and a linear mPEG–ibuprofen conjugate were also studied for comparison to direct linked dendrimer conjugates.The study explores efficacy of anionic polyamidoamine (PAMAM– COOH) dendrimer–N-acetyl cysteine (NAC) conjugates for applications in neuroinflammation. Understanding these structural and steric effects on their drug release characteristics is crucial for the design of dendrimer conjugates with high efficacy.

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[19] S.A. G´omez-Lopera, R. C. Plaza, and A. V. Delgado, “Synthesis and characterization of spherical magnetite/biodegradable polymer composite particles,” Journal of Colloid and Interface Science, vol. 240, no. 1, pp. 40–47, 2001. [20] R. Asmatulu, M. A. Zalich, R. O. Claus, and J. S. Riffle, “Synthesis, characterization and targeting of biodegradable magnetic nanocomposite particles by external magnetic fields,” Journal of Magnetism and Magnetic Materials, vol. 292, pp. 108–119, 2005. [21] R. Asmatulu, Biomaterials—Class Notes,Wichita State University, Wichita, Kan, USA, 2008.http://www.devicelink.com/mpb/archive/97/11/003.html, October 2008. [22] P.A.R. Glynn, B.M.E. vander Hoff, and P.M. Reilly “A general model for prediction of molecular weight distributions of degraded polymers, development and comparison with ultrasonic degradation experiments,” Journal of Macromolecular Science, Part A, vol. 6, pp. 1653–1664, 1976. [23] C. N. O’Brien and A. J. Guidry, “Formulation of poly(D,Llactide- co-glycolide) microspheres and their ingestion by bovine leukocytes,” Journal of Dairy Science, vol. 79, no. 11, pp. 1954–1959, 1996. [24] Lima K. M. and J. M. Rodrigues, “Poly(D,L-lactide-coglycolide) microspheres as a controlled release antigen delivery system,” Brazilian Journal of Medical and Biological Research, vol. 32, pp. 171–180, 1999. [25] S.-J. Lee, J.-R. Jeong, S.-C. Shin, et al., “Nanoparticles of magnetic ferric oxides encapsulated with poly(D,L lactideco- glycolide) and their applications to magnetic resonance imaging contrast agent,” Journal of Magnetism and Magnetic Materials, vol. 272–276, part 3, pp. 2432–2433, 2004. [26] J. Emami, H. Hamishehkar, A. R. Najafabadi, et al., “Particle size design of PLGA microspheres for potential pulmonary drug delivery using response surface methodology,” Journal of Microencapsulation, vol. 26, no. 1, pp. 1–8, 2009. [27] Saboktakin,M.R.,A., Maharramov, M.,A.,Ramazanov, Nature and Science,2007,5(3), 30-36. [28] Saboktakin,M.R.,A., Maharramov, M.,A.,Ramazanov, Poly(amidoamine)( PAMAM) /CMS Dendritic nanocomposite for controlled drug delivery Journal of American Science,2007, 3(4),40-45. [29] Armstrong ,J.,A.,N., Bloembergen, et al., Interactions between Light Waves in a Nonlinear Dielectric, Physical Review,127(6),1918-1939. [30] Betley,T.,A.,M.,M., Holl, et al. , [31] Langmur,2001,17(9),2768-2773. [32] Bloembergen, N.,and Pershan, P.,S., Light waves at the boundary of nonlinear mediaPhysical review,1967,128(2),606-622. [33] Bloembergen, N., Surface nonlinear optics: a historical overview Applied physics,B, Lasers and optics , 1999,68(3),289-293. [34] Chen,H.,G., and P. Knochel, A new mild oxidation of amines to aldehydes and ketonesTetrahedron Letters,1988,29(51),6701-6702. [35] Chen,W.,D.,A.,Tomalia, et. Al. , [36] Macromolecules,2000,33(25),9169-9172. [37] Dvornic, P.,R.,A. M. ,de Leuze-Jallouli,et.al., Macromolecules, 2000,33(15), 5368-378. [38] Esfand , R., D.,A.,Tomalia , Poly(amidoamine) (PAMAM) dendrimers: from biomimicry to drug delivery and biomedical applications [39] Drug Delivery Today,2001,6(8),427-436.

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[40] Ghosh,S. and A.K. Banthia , Synthesis of photoresponsive polyamidoamine (PAMAM) dendritic architecture Tetrahedron Letters , 2001,42(3),501-503. [41] Kolhe, P., E.,Misra,R.,M., Kannan,Int. J. Pharm.,2003,259,143-160. [42] Esfand, R., Tomalia, D.A., 2001. Poly(amidoamine) (PAMAM) dendrimers: from biomimicry to drug delivery and biomedical applications. Drug Discov. Today 6, 427– 436. [43] Svenson, S., Tomalia, D.A., 2005. Dendrimers in biomedical applications reflections on the field. Adv. Drug Deliv. Rev. 57, 2106–2129. [44] Gillies, E.R., Frechet, J.M.J., 2005. Dendrimers and dendritic polymers in drug delivery.Drug Discov. Today 10, 35–43. [45] Lee, C.C., MacKay, J.A., Frechet, J.M.J., Szoka, F.C., 2005. Designing dendrimers for biological applications. Nat. Biotechnol. 23, 1517–1526. [46] Vicent, M.J., Duncan, R., 2006. Polymer conjugates: nanosized medicines for treating cancer. Trends Biotechnol. 24, 39–47. [47] Najlah, M., Freeman, S., Attwood, D., D’Emanuele, A., 2006. ynthesis, characterization and stability of dendrimer prodrugs. Int. J. Pharm. 308, 175–182. [48] Gurdag, S., Khandare, J., Stapels, S., Matherly, L.H., Kannan, R.M., 2006. Activity of dendrimer–methotrexate conjugates on methotrexate-sensitive and resistantcell lines. Bioconjugate Chem. 17, 275–283. [49] Navath, R.S., Kurtoglu, Y.E., Wang, B., Kannan, S., Romero, R., Kannan, R.M.,Dendrimers–drug conjugates for tailored intracellular drug release based on glutathione levels. Bioconjugate Chem. 2008, 19, 2446–2455. [50] Khandare, J., Kolhe, P., Pillai, O., Kannan, S., Lieh-Lai, M., Kannan, R.M.,2005. [51] Synthesis, cellular transport, and activity of polyamidoamine dendrimer– methylprednisolone conjugates. Bioconjugate Chem. 16, 330– 337.

In: Recent Developments in Bio-Nanocomposites… ISBN 978-1-61761-008-0 Editor: Ashutosh Tiwari © 2011 Nova Science Publishers, Inc.

Chapter 4

RECENT PROGRESS IN CERAMIC NANOMATERIALS FOR BIOMEDICAL APPLICATION Shivani B. Mishra11, Ajay K. Mishra1, Ashutosh Tiwari2,3, Radhe Shyam Rai4 and Anjali M. Rahatgaonkar5 1

Department of Chemical Technology, University of Johannesburg, Doornfontein, Johannesburg 17011, South Africa 2 School of Materials Science and Engineering, Jiangsu University, Zhenjiang 212 013, China 3 National Institute for Materials Science, Tsukuba, Ibaraki 305 0047, Japan 4 Departmento de Engenharia Cerâmica e do Vidro and CICECO, Universidade de Aveiro, 3810-193 Aveiro, Portugal 5 Department of Chemistry, Institute of Science, Civil lines, R.T. Road, Nagpur, MS 440001, India

1. INTRODUCTION Fine tuning of the material at atomic, molecular and macromolecular level touching the nanoscale, significantly improves the properties of the material in nanotechnology. This is a multidisciplinary field that encompasses various disciplines of fundamental and applied sciences viz. physics, material science, colloid and interface science, chemical engineering, mechanical engineering, electrical engineering. Some other fields that are benefitting from the nanotechnology are supra-molecular chemistry, robotics, photonics, optoelectronics, and biotechnology. An improvement in safety, quality of life and health, potential possible solution to industrial challenges, environment protection and pollution control are some of the great expectations of nanotechnology. Research in the field of nanoscience and nanotechnology based thin films coating, is widely explored for developing nanocoating systems for food, health and biomedical industry. Nanocomposites and nanostructure thin films are the emerging domains of 1

Corresponding author: E-mail: [email protected]; Tel.: (+27) 11-5596163, Fax: (+27) 11-5596425.

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naanotechnologyy that are siggnificantly inffluencing the food packagiing, bio-implaants, novel poolymeric conttainers for foood, medical suurface instrum ments and coateed nanoparticlles for bionaanotechnologyy. Nanotechnnology offers a platform for high outtput for somee common teechnological problem p like bacteria sellectivity, longg-term instabbility of micrroorganism reesponse and long time too reach the steady s state response r in the t field of biosensors. b Innnovations lik ke “laboratoryy-on-a-chip” iss no more a dream d for varrious scientist across the w world for sampple preparations together with w biochemiccal reactions and detectionn steps in a siimple and autoomated manneer.

2. NAN NOTECHNO OLOGY AND D ITS BIOM MEDICAL APPLICATIO ONS b easily integgrated and inncorporated innto medical Nano-strucctured materiaals can thus be deevices as the biological b sysstems like viruus and proteinns exhibit natuural nanostructtures [1-2]. M Medicine and biomedical b enngineering are one of the moost promising fields of nanoostructured m materials [3-4]. The most recent r advances includes novel n tissue engineered scaaffolds and deevices, site sppecific drug deelivery system ms, non viral gene g carriers, biosensors b andd screening syystems and cliinical and bio--analytical diaagnostic and thherapeutic [5-66]. Various ou utcome of nannotechnology such s as nanoccomposites havve been used to stabilize annd regeneratee bone matrrices [7–10]; nanotubes and nanowirres have demonstrated unnprecedented sensitivity for biomoleculee detection [11–13]. Besidees, nanoscale assemblies annd particles with w high bio-aaffinity to speccific host sitess for precise drug d administrration were ussed to deliverr high concenttrations of theerapeutic druggs and/or biom molecules [144–16]. Still, thhere is need too modify the existing nanoostructured maaterials for bioomedical appllications to fuurther improv ve and optimize the properrties biomediccal devices, to t meet the demands d of biiomedical app plications onn materials, and/or a to enllist and adappt more nanoostructured m materials/devic ces into the biomedical b syystems [17–199]. In generaal, there are three t basic doomains wheree nanotechnollogy plays a huge role in the field of biomedical b appplications. These are diagn nostic techniqque, drugs, proostheses and implants. i Effoorts are made by several coompanies usin ng nanotechnoology to devellop anticancerr drugs, implaanted insulin pumps p and geene therapy.

G Graphical presenntation of nanoteechnology and its biomedical applications a

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3. CERAMIC NANOMATERIALS Ceramics or creamers are chemical compounds from the group of oxides, nitrides, borides and silcides. The research and development in the field of ceramics together with nanotechnology is forming a bridge between the material science and life science especially in the medical field. A few of these are, hard and super hard ceramics form abrasive and cutting devices, ceramic membranes for liquid or gas hyper filtrations at molecular level, ion conducting ceramics, semi conductors and super conductors for batteries and sensor. Some other ceramics which are worth to mention are ferroelectric ceramics for capacitors, sensors, piezoelectric transducers, electro-optic devices and thermistors, ferromagnetic ceramics for telecommunication and television [20]. In this section we will discuss about ceramics in nano-forms that are investigated across the world for various biomedical applications.

3.1. Ceramic Nanocomposites Ceramic nanocomposites can be better understood as the composite material where ceramic component can behave either as matrix forming ceramic matrix nanocomposite or it can also act as a filler/reinforcement in a polymer, thus giving rise to polymer ceramic nanocomposite. In both the forms, ceramic part is mostly present in nano-scale range. For an easy understanding, graphical presentation is given below. Thus, this section can further be categorized into two parts i.e. ceramic matrix nanocomposites and polymer-ceramic nanocomposites.

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Ceramics are usually brittle and easily fractured as a consequence of crack propagation. There have been attempts to make ceramics suitable for engineering applications through the incorporation of a ductile metal phase or another ceramic into the matrix. This leads to improved mechanical properties such as hardness and fracture toughness, which occur as a result of the relationship between the different phases, matrix and reinforcements, at the phase boundaries throughout the material.

3.1.1. Ceramic Matrix Nanocomposites In a typical ceramic matrix nanocomposite (CMCs), ceramic matrices are reinforced with ceramic or metal fillers which provide toughness to otherwise brittle ceramic, electrical and thermal conductivity, thermal expansion and hardness. The desirable properties however are high temperature stability, high thermal shock resistance, high hardness, high corrosion resistance, light weight, nonconductive and non magnetic behaviour and versatility in finding the unique engineering solutions. Some of the potential applications are given in table 1. Ceramic matrices are of two types i.e. oxide matrices and non-oxide matrices and might contain residual metal after processing. Oxide matrices are considered as more mature and environmental friendly. A few common oxide matrices are alumina, silica, mullite, barium aluminosilicate, lithium aluminosilicate and calcium aluminosilicate. Out of these, alumina and mullite have been the most widely used because of their in-service thermal and chemical stability and their compatibility with common reinforcements. On the other hand, nonoxide ceramics with superior structural properties, hardness, and, in some environments, corrosion resistance are rapidly entering the marketplace. Some of the more common nonoxide ceramics include silicon carbide (SiC), silicon nitride (Si3N4), boron carbide, and aluminium nitride (AlN). Of these, SiC has been the most widely used, with AlN of increasing interest where high thermal conductivity is required and Si3N4, where high strength is desired [21]. Table 1. Various ceramic matrix nanocomposites and their application CMCs SiO2 / Fe ZnO / Co

Potential Applications High performance catalyst and data storage Field effect transistor for optical femto-second study of inter particle interaction Metal oxide/metal

BaTiO3/SiC, ZTA/Ag SiO2/ Co SiO2/Ni Al2O3 / SiC Si3N4 / SiC Al2O3 / NdAlO3 and

Catalysts, sensors and optoelectronic devices Electronic industries / ferroelectric devices Optical fibers Chemical sensors Structural materials Solid state laser media, phosphors Al2O3 /LnAlO3 and electrical media

TiO2 / Fe2O3 Al2O3/Ni

High density magnetic recording media Engineering parts

PbTiO3 / PbZrO3

Microelectronics and micro-electromechanical systems

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Various scientists have explored CMCs for biomedical applications. Here, we would focus on some of the recent work carried out. Nano-crystals of synthetic hydroxyapatite (HAP) have much more bioactive surface and reasonable resorption rate; but after implantation they are easily carried away with blood, unsuitable for handling and have zero mechanical strength [22]. Duvok et al. proposed a new bioactive ceramics for full bone tissue restoration-inorganic composites consisting of modified phosphate ceramics, bioactive glasses and bioactive glass-ceramic-so-called SYNTHETBONE materials. The SYNTHETBONE composite was made up of well known components like HAP, trichlorophosphate (TCP), biphase HAP–TCP ceramics, bioactive glasses and bioactive glassceramics. The chemical composition of bioactive glass-ceramics used in the SYNTHETBONE composite was based on silicate compositions with maximal bioactivity but contained also the crystallizing nucleus and other additions.

Figure 1. Filling bone defect after tumor surgery with SYNTHETBONE porous granules.

Figure 1 show the transmission electron micrograph of filling bone defects after tumour surgery with SYNTHETBONE. The SYNTHETBONE composite was produced by mixing, milling, moulding and sintering. This composite had excellent properties in terms of strength, shrinklessness and high efficiency. Jurczyk and Niespodziana developed new generation titanium-ceramic bionanocomposites [24]. Titanium-HA and titanium-silica nanocomposites were synthesized by ball milling and then compacted at 830 MPa. These compacts were then put to heat treatment at around 1200 °C in inert atmosphere. Cytotoxic tests on these composites show superior cytocompatibilty when compared to conventional titanium. Zirconia toughened alumina (ZTA) were prepared and tested for ceramic joint prostheses [25]. ZTA was prepared by colloidal route synthesis whereby doping a stable suspension of high-purity alumina with zirconium alkoxide was done. The microstructure showed (Figure2) the zirconia nanoparticles uniformly distributed in alumina matrix. The ZTA composite was

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tested on human osteoblasts isolated from patients undergoing orthopaedic surgery and produced no significant differences were observed for wear behaviour after 7 million cycles. This result offered an option for long term reliability of ceramic joint prostheses.

Figure 2. Microstructure of NZTA ceramic materials obtained from nonaqueous colloidal slurries.

Song et al. [26] produced nanofibrous web constituted of collagen and HA from electrospinning method. The electro-spun web showed a well-developed nanofibrous structure with HA contents of up to 20 wt%. The internal structure of the collagen–20 wt% HA nanofibers showed highly elongated apatite nano-crystalline precipitated within the collagen matrix. The nanocomposite provided matrix conditions for cells to adhere and populate, in a comparable level to the pure collagen nanofibers, and to direct cells to elicit osteoblastic marker, alkaline phosphatase.

3.1.2. Polymer-Ceramic Nanocomposites In composites where the matrices are primarily the polymers with ceramic fillers or reinforcement are better known as polymer ceramic nanocomposites. Polymer-ceramic nanocomposites can further be classified into layered and non-layered types. The reinforcing materials employed in the production of polymer nanocomposites can are categorized according to their dimensions [27]. For example, when the three dimensions are in the nanometer scale, they are called iso-dimensional nanoparticles such as spherical silica, metal particles and semiconductor nanoclusters [28]. The second type of reinforcement is formed by nanotubes or whiskers, which contain two dimensions in the nanometer scale and one larger, forming an elongated structure. Carbon nanotubes and SiC whiskers, extensively studied as reinforcing nanofillers, is be included in this second category. Finally, a third type of reinforcement is characterized by only one dimension in the nanometer range [29-31]. In this group, the filler contains sheets one to a few nanometers thick and hundreds to thousands nanometers long. Figure 3 shows the transmission electron micrographs of one, two and three dimensional nanomaterials. This family is called polymer-layered nanocomposites. These materials are obtained by intercalation of the polymer (or a monomer subsequently polymerized) inside the galleries of

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the layered host. Many synthetic and natural crystalline hosts that are able, under specific conditions, to intercalate a polymer have been described. However, the biodegradability of polymer nanocomposites primarily depends upon nature of polymer matrices. Composite fabrication research has focused on developing polymer/ceramic blends, precipitating ceramic onto polymer templates [32] and coating polymers onto ceramics [33].

Figure 3. Transmission electron micrographs of one, two and three dimensional nano-materials

Bakumov et al. prepared functional ceramic composites consisting of a dispersion of silver nanoparticles in a silicon (carbon) nitride matrix (nc-Ag/Si(C)N) via the polymer– ceramic route [34]. The obtained materials consisted of silver nanoparticles distributed in a silicon (carbide) nitride matrix. These materials were found to have a very high bactericide activity against Gramnegative (E. coli) and Gram-positive (S. aureus) bacteria. Figure 4 shows the silver ceramic nanocomposites and its bactericidal activity. In another study, to produce and analyse properties of a polymer nanocomposite materials in which silica (SiO2) nanoparticles were applied as a modifier. A matrix of the composites consisted of the resorbable P(L/DL)LA polymer. Nano-metric dispersion of the nano-filer particles was confirmed by a thermal analysis and mechanical tests. The presence of the nanoparticles influenced hydrophility and surface topography of the nanocomposites. Bioactivity test was performed by incubation of all materials in simulated body fluid (SBF), which is an artificial body fluid with pH and ion concentration similar to the natural one, but free from cells and proteins

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Silver ceraamic nanocom mposite Bacteriicide activity for f Control Fiigure 4. Silver ceramic c nanocoomposites and itts bactericide acctivity

In vitro testing of αω-hydroxyl (or triethooxysilane) pooly-caprolactoone (PCL) naanocomposites were investtigated. The biodegradable b nanocomposiite was preparred by solgeel technique by adding αω-hydroxyl α (or triethoxyssilane) encappped PCL [355]. Atomic fluorescence micrograph m of the nanocom mposites is shoown below inn Figure 5a-b. “In vitro” biiodegradation was carried out o at 37 °C by b immersing the ceramer (50% PCL coontent) in a 0..1M sodium phosphate bufffer solution (pH H = 8) added with porcine esterase e (23 unnits/mL).

(a)

(b)

Fiigure 5a. (a) 800 × 800 nm2 taapping mode AF FM picture of ceeramer containiing 50 wt % PC CL. (b) 200 × 200 nm2 zoom m area of Biodeggradabilty of PC CL.

(a)

(b)

Fiigure 5b. 800-800 nm2 tappingg mode AFM im mage of ceramer containing 500 wt% PCL: (a) after 8 days off enzymatic deggradation; (b) affter 10 days of enzymatic e degradation.

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Blackson and Harris worked on developing conformal alumina films on the external and internal particle surface of highly porous poly (styrene-divinylbenzene) using atomic layer deposition technique [36]. The ultrathin alumina films obtained were expected to increase the biocompatibility and bioactivity of the polymer to promote cell adhesion and proliferation for tissue engineering. Recently, a novel bioanalogue hydroxyapatite (HAP)/chitosan phosphate (CSP) nanocomposite was fabricated by a solution-based chemical methodology with varying HAP contents with an appreciable improved mechanical properties and minimal surface defects. The use of CSP acted as a coupling/anchoring agent and provided a significant platform for better dispersion of nanoparticles in the polymer matrix through its pendant phosphate groups. Cytotoxicity test confirmed that the developed composite is cytocompatible. Primary murine osteoblast cell culture study showed that the HAP/CSP nanocomposite was osteocompatible and highly osteogenic in vitro. Therefore, the developed HAP/CSP nanocomposite may be potentially applied in bone tissue engineering applications [37].

3.2. Ceramic Nanostructures Rapid advancements of ceramic based nanostructured materials have been explored for wide variety of biomedical application which focuses on novel tissue engineered scaffolds and devices, sites specific drug delivery systems, non-viral gene carriers, biosensor and screening systems, and clinical bio-analytical diagnostics and therapeutics [38-39]. Among these nanostructures, nanoscale assemblies particles have been identified which are applied to deliver high concentrations of therapeutic drugs or biomolecules. These nano-assemblies had high bio-affinity for the specific host sites for precise drug administration [40-42]. Architectural micro- and nano-shell sensors based on smart location design of fluorescent indicators (usually in the core) and enzymatic detection layers (usually in the outer shell) have allowed production of simple bio-colloids, capable of semi-quantitative detection of glucose, lactose, urea, and other materials which are specific substances for the corresponding biocatalytical reactions. It is building upon the possibilities afforded by coating micro/nanoscale templates. With functional materials such as enzymes and dyes, sensors for chemicals and biochemicals are being developed, as tools for biological research, medical diagnostics and monitoring, and bio-defence applications [43]. Using nanoparticles coated with fluorescent materials that respond selectively to specific species by binding or other interactions, ratiometric nanoscale probes have been developed for intracellular and extracellular measurements of ions and oxygen. These nano-devices have advantages over standard liquid-phase small molecule indicators in that they provide a protective package for the chemistry, separating the dyes from the biological environment, and in doing so, reducing nonspecific responses, dyeprotein binding, and toxic effects. Goodrich and Winter, successfully prepared α-Chitin nano-crystals from shrimp shells. They isolated α-Chitin from shrimp shells which was then subjected to acid hydrolysis and mechanical disruption to yield the nano-crystals. The TEM image of these nano-crystals is shown in Figure 6.

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Figure 6. TEM micrograph of chitin nano-crystals from shrimp shells formed after hydrolysis and mechanical dispersion.

A dye adsorption method was used to assess the specific surface areas of chitin and cellulose nano-crystals. The morphological similarity to the collagen fibres in the extracellular matrix of natural tissue, nanofibers scaffolds are also accepted to prove to be a biologically preferred scaffold/substrate for proliferation and phenotype maintenance of chondrocytes and chondrogenic differentiation of mesenchymal stem cells. Electro-spun nanofibers scaffolds were shown to support chondrocytic phenotype of fetal bovine chondrocytes and chondrogenic induction and maintenance of TGF-b1 treated MSCs [45-47]. Nanofibers scaffolds have also been implemented in ligament and tendon reconstruction research.

3.3. Ceramic Nanotubes / Nanofibers Although, much work had not been reported in the application of bio-ceramic nanotubes and nanofibers, still it is one of targeted domain of ceramic nanomaterials for various biomedical applications. Very recently, Patel et al. [48] reported report silica formation onto nanofibers of polyethylene imine (PEI) blended with poly (vinyl-pyrrolidone) (PVP) obtained via electrospinning of their 50:50 (w/w) blend. The active component, PEI, catalyzes rapid silica formation, within minutes, upon immersion of the PEI/PVP nanofibers in silica precursor tetramethylorthosilicate (TMOS). Scanning electron micrographs of these nano fibres is shown in Figure 7. This finding was in agreement with the proposed mechanisms of the silica formation by polyamines where the formed silica closely interacts with the polyamines and results in 2:1 silica/polyamine by weight. Calcinations of the silicified fibre mats led to porous ceramic nanofibers consisting of porous nano-structured silica fibrils. This bio-mimetic route to rapid synthesis of hybrid composite nanofibers could have widespread use, including diverse applications as catalysts, engineered tissues, and structural materials.

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Figure 7. SEM images of the fibers of the 50:50 blends of linear PEI and PVP in Ethanol electro-spun.

Using simple techniques like electrospinning allow workers to generate nanofibers from natural biopolymer like chitin and thus can be applied to tissue engineering, wound dressing, drug delivery systems and many other medical applications [49].

4. METHODS OF SYNTHESIS OF CERAMIC NANOMATERIALS 4.1. Ball Milling Ball milling, which is also better described as mechanical crushing, has been a traditional method of fabricating fine powders. This method was first used to produce nanomaterials, although today is very confined since new more effective methods were developed. Ball milling breaks down the material into nano-crystallites and can be used to synthesise a variety of new types of materials. In this process, small balls are allowed to rotate around the inside of a drum and drop under gravitational forces onto a solid, enclosed in the drum. Its significant advantage is that it can readily be implemented commercially. Notwithstanding, ball milling can hardly reduce the filler particle size below 100 nm. To circumvent this roadblock, chemical processes are used instead to produce building blocks on a molecular scale [50].

4.2. Plasma Arching Plasma is an ionised gas and is achieved by making gas conduct electricity, providing a potential difference across two electrodes, so that the gas yields up its electrons and thereby ionises. Plasma arching is basically used to synthesise deposits on surfaces rather than new solid structures. As a surface deposit, the nano-material can be as little as few atoms layers and is not characterised as a nano-material, unless at least one dimension of the bulk particle is of the nanometre scale. Otherwise, it is characterised as a thin film and not a nano-material.

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A variation on plasma arching is flame ionisation; if a material is sprayed into a flame, ions are produced which can also be collected and deposited in nano-crystallite form [50-51]. Plasma spray coatings on maxillofacial implants exhibit several limitations e.g., unpredictable chemistry, porosity, inherent fractures. The coatings are subjected to fracture or fragmentation during insertion and service, and have unpredictable rates of dissolution. One of the primary reasons for the lack of control with the use of this process is that extremely high temperature must be employed in melting the initial powder. That renders it difficult to obtain the proper chemistry and structure of the resulting coating.

4.3. Chemical Vapour Deposition Chemical Vapour Deposition (CVD) involves depositing nano-particulate material from its gas phase. The material is heated to form a gas that is afterwards allowed to deposit as a solid coating on a surface, usually under vacuum. There may be direct deposition or deposition by chemical reaction to form a new product, which differs from the initial volatilised material. CVD can also be used to grow surfaces; the object to be coated is allowed to stand in the presence of the chemical vapour. The first layer of atoms or molecules deposited may react with the surface. Nevertheless, these first formed depositional species can act as a template on which material can grow. The structures of these materials are often aligned, because the way in which atoms and molecules are deposited, is influenced by their neighbours. This works best if the host surface is extremely flat. During deposition, a site for crystallisation may form in the depositional axis, so that aligned structures grow vertically; therefore, this is an example of self-assembly, which gives the surface unique characteristics. Additionally, CVD can be utilised to form partial surface coatings [50].

4.4. Pulsed Laser Deposition In the Pulsed Laser Deposition (PLD) technique, there is a vacuum chamber containing the coating material in a sintered/ pressed form which is bombarded with laser beam. In PLD there can also be a blender containing solution where a solid disk rotates. The solid disk is subjected to laser beam pulses creating hot spots on its surface. The size of the nanoparticles can be controlled by the energy of the laser and the rotation speed of the disk [7]. A similar to the PLD technique is also the ion beam sputtering process, where argon beam is usually used for the bombardment [52].

4.5. Electrodeposition Electrodeposition has been used for a long time to synthesise electroplated materials. In nanotechnology, the aim is to place only a single molecular layer of coverage on a surface in a highly controlled way. Electrodeposition can be addressed to fill holes to synthesise dispersed nanomaterials. Nano-holes have strategically been placed in membranes. Filling nano-sized holes in polymer membranes with various combinations of metals produces nanocomposites, which have different uses. For instance, if some holes are filled with a

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conducting metal like gold they can be charged and this can influence the nature of ions that will go through the unfilled holes. If there is a device at the other end that responds to charge, the device becomes a specific ion detector. Other nano-composites, if compound specific, they can be used as the active sensing units in so-called intelligent biomaterials. The most important development is the manufacture of multipurpose chips, which will be able to sense a host of substances at once and so provide very specific and effective diagnoses [50].

4.6. Sol–Gel Sol–gel is a useful process of self-assembly for the synthesis of nanoparticles. Colloids are suspensions with molecules of 20–100 lm in diameter in a solvent. The colloid that is suspended in a liquid is the ‘sol’, and the suspension that keeps its shape is the ‘gel’. Thus, ‘sol–gels’ are suspensions of colloids in liquids that keep their shape. The sol–gel process involves the evolution of networks through the formation of a colloidal suspension and gelation of the sol to form a network in continuous liquid phase. The precursors for synthesising these colloids normally consist of ions of a metal, but also sometimes of other elements surrounded by various reactive species, i.e., the ‘ligands’. The sol–gel formation occurs in four stages: (a) hydrolysis,(b) condensation and polymerisation of monomers to form particles, (c) growth of the particles, (d) agglomeration of the particles followed by the formation of networks that extend throughout the liquid medium resulting in thickening, which forms a gel. Upon drying, trapped volatiles are driven off and the network shrinks as further condensation may occur. These processes are basically affected by the initial reaction conditions. By controlling these factors, it is possible to vary the structure and the properties of the sol-gel derived inorganic network. For instance, with hydrolysis under controlled conditions, dispersed spherical nanoparticles can be synthesised [50].

4.7. Precipitation Precipitation of a solid from a solution is a common method for the fabrication of nanoparticles. In the precipitation process, the salts of various elements are taken in the required proportion and are dissolved in water or together with suitable solvents to acquire complete mixing on an atomic scale. A precipitating reagent is added, which results in the precipitation of the components at the required ratio. The precipitate is dried and manipulated in the same way as powders, except that normally there is no further need for finer grinding. Particles size and morphology can be controlled by changing different reaction parameters. For obtaining the precipitate of well-defined stoichiometry, the factors that have to be taken into consideration are: (a) the chemical conditions, i.e., pH and anion concentration-, (b) the hydrodynamic conditions, -i.e., vigorous mixing- and (c) the counter ions. Precipitation technique can provide uniform nucleation, growth and aging of the nanoparticles throughout the solution [53].

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5. POTENTIAL RISKS OF NANOTECHNOLOGY FOR BIOMEDICAL APPLICATIONS Nanotechnology applications have not been marketed long enough for claims to be corroborated about risks to human health or the environment. Still, small nanoparticles can enter the human body through pores and may accumulate in cells. Being biocompatible and biodegradable, the ceramic nanomaterials are least expected to have health hazards. However, historical experience with unintended consequences of technologies, such as drug resistance to antibiotics or the persistence of chemicals such as DDT in the environment, teaches us to take precautions.

CONCLUSIONS AND FUTURE PROSPECTS Medical nanotechnologies are entering industrial production, mainly for diagnostics, drugs, and therapies. In the longer term, nanotechnology is able to improve implants and even let blind people see. Tailor-made structure, topographies and bio-mimetic behaviour of ceramic nanomaterials are achievable that are able to simulate the natural structures and mechanisms. These are promising alternatives to conventional materials, because they can potentially be designed to match the requirement of a medical applications viz. tissue engineering, sensor, implants, drug delivery etc. With no health hazard expected, bio nanoceramics is a blessing to medical field.

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[14] S.M. Moghimi, J. Szebeni, Progress in Lipid Research 42 (2003) 463. [15] H. Takeuchi, H. Yamamoto, Y. Kawashima, Advanced Drug Delivery Reviews 47 (2001) 39. [16] R.H. Muller, C. Jacobs, O. Kayser, Advanced Drug Delivery Reviews 47 (2001) 3. [17] Q.A. Pankhurst, J. Connolly, S.K. Jones, J. Dobson, Journal of Physics, Applied Physics 36 (2003) 176. [18] M.O. Oyewumi, R.J. Mumper, International Journal of Pharmaceutics 251 (2003) 85. [19] S.M. Moghimi, A.C. Hunter, J.C. Murray, Pharmacological Reviews 53 (2001) 283. [20] R. Pampuch, Constitution and Properties of Ceramic Materials, Elsevier, Amsterdam (1991). [21] K.J. Bowman, S.K. El-Rahaiby, and J.B. Wachtman, Jr., Handbook on Discontinuous Reinforced Ceramic Matrix Composites, AmericanCeramic Society. Eds. 1995. [22] J. Planell, S. Best, D. Lacroix, Bone Repair Biomaterials, Woodhead Publishing, Abington, 2009. [23] V. Duboka,, A. Shynkaruk, O. Atamanenko, V. Protcenko, V. Kindrat, E. Shynkaruk, V. Kischuk, E. Buzaneva, Material science and engineering B, [In Press]. [24] M. Jurczyk, K. Niespodziana, Obróbka Plastyczna Metali t. XIX (2008) nr 4. [25] Affatato, R. Torrecillas, P. Taddei, M. Rocchi, C. Fagnano, G. Ciapetti, A. Toni, Journal of Biomedical Materials Research Part B: Applied Biomaterials (2005) 76. [26] Ju-Ha Song, Hyoun-Ee Kim, Hae-Won Kim, J Mater Sci: Mater Med 19 (2008) 2925. [27] M. Alexandre, P. Dubois, Materials Science and Engineering. 28 (2000) 1. [28] N. Herron, D.L. Thorn, Advanced Materials 10 (1998)1173. [29] V. Favier, G.R. Canova, S.C Shrivastava, J.V Cavaille, Polymer Engineering Science 37 (1997)1732. [30] Chazeau, J.Y. Cavaille, G.Canova, R. Dendievel, B. Boutherin, Journal of Applied Polymer Science. 1999; 71(11):1797-1808. [31] M. Ogawa, K. Kuroda, Bulletinn of the Chemical Society of Japan. 70 (1997) 2593. [32] A.Bigi, E. Boanini, S. Panzavolta, N. Roveri, K. Rubini, J Biomedical Material Research 59 (2002) 709. [33] A.F Tencer, P.L. Woodard, J. Swenson, K.L.Brown, J Orthopeadic Research 5 (1987) 275) 32. [34] V. Bakumov, K. Gueinzius, C. Hermann, M. Schwarz, E. Kroke, Journal of the European Ceramic Society 27 (2007) 87. [35] D. Tian, P. Dubois, C. Grandfils, R.obert Je´roˆme, Chemistry Mateials 9 (1997) 870. [36] Xinhua Liang, Steven M. George, and Alan W. Weimer Chemistry. Materials 19(2007) 5388. [37] N. Pramanik, D. Mishra, I. Banerjee, T. K. Maiti, P. Bhargava, P. Pramanik, International Journal of Biomaterials [In Press]. [38] L.A. Bauer, N.S. Birenbaum, G.J. Meyer, Journal of Materials Chemistry 14 (2004) 517. [39] L. Mazzola, Nature Biotechnology 21 (2003) 1137. [40] S.M. Moghimi, J. Szebeni, Progress in Lipid Research 42 (2003) 463. [41] H. Takeuchi, H. Yamamoto, Y. Kawashima, Advanced Drug Delivery Reviews 47 (2001) 39. [42] R.H. Muller, C. Jacobs, O. Kayser, Advanced Drug Delivery Reviews 47 (2001) 3.

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[43] M. McShane, J. Brown, K. Guice, Y. Lvov, J. Nanoscience and Nanotechnology, 2(2002) 41. [44] J. D. Goodrich, W. T. Winter, Biomacromolecules 8 (2007) 252. [45] L. Song, D Baksh, R.S. Tuan, Cytotherapy 6 (2004) 596. [46] R. Tuli, W.J. Li, R.S. Tuan, Arthritis Res Ther., 5 (2003) 235. [47] S. Sahoo, H. Ouyang , J. Goh , Tissue Engineering 12 (2006) 91. [48] P.A. Patel, J. Eckart, M.C. Advincula , A. J. Goldberg, P. T. Mather, Polymer 50 (2009) 1214. [49] C. K. S. Pillai, C. P. Sharma Trends in Biomaterial Artificial Organs 22 (2009)179. [50] M. Wilson, K. Kannangara, G. Smith, M. Simmons, B. Raguse, in Nanotechnology: Basic Science and Emerging Technologies (Australia: Chapman and Hall/CRC (2002), p. 56. [51] C. P. Poole Jr, F. J. Owens, in Introduction to Nanotechnology (USA: WileyInterscience Publication, 2003), p. 72. [52] S. A. Catledge, M. D. Fries, Y. K. Vohra, W. R. Lacefield, J. E. Lemons, S. Woodard, R. Venugopalan, J. Nanosci. Nanotechnol. 2(2002) 293. [53] A.G. Walton, in The Formation and Properties of Precipitates (New York/USA: Interscience Publishers, 1967).

In: Recent Developments in Bio-Nanocomposites… ISBN 978-1-61761-008-0 Editor: Ashutosh Tiwari © 2011 Nova Science Publishers, Inc.

Chapter 5

BIOMEDICAL APPLICATIONS OF ZnO NANOSTRUCTURES Ahsanulhaq Qurashi 1 21 1

School of Engineering, Toyama University, 3190 Gofuku, Toyama 930-8555, Japan Chemistry Department and Center of Research Excellence in Nanotechnology, King Fahd University of Petroleum and Minerals, Dhahran, Saudi Arabia

2

ABSTRACT Nanostructures have attracted tremendous attention from researchers in various disciplines because their high surface-to-volume ratio and high crystal quality are highly desirable for many technological applications including biosensors, tissue engineering and drug delivery system. Several synthetic methods have been used to fabricate various nanostructures. These synthetic approaches are mainly categorized into two main classes according to how the nanostructure is formed: dry and wet chemical synthesis. Both the methods have their pros and cons respectively. Various methods were developed for functionalization of nanostructures to modify their surfaces. Different techniques for the effective biofunctionalization of one-dimensional (1D) ZnO nanostructures were illustrated in this chapter. Various biomolecules like human serum albumin, bovine serum albumin, angiotensin II and DNA molecules were effectively immobilized by modifying the surface of 1D ZnO nanostructures. Molecular functionalization of ZnO nanobelts demonstrated which improved the optoelectronic and electrical properties. ZnO nanostructures were also studied for advanced biological applications like fluorescence detection, cellular biocompatibility, biosafety, biosensor, and mammalian cell adhesion.

1. INTRODUCTION Nanotechnology represents a new and enabling platform that promises to provide a range of innovative technologies for biological applications. The anticipation is that organic/inorganic hybrids will be able to perform some specific functions better than either 1

Email: [email protected].

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purely organic or purely inorganic systems. The fascinating example is novel biosensors based on protein-functionalized semiconductor devices. In such devices, the proteins provide the desired biological sensitivity, specificity, and access to the large field of genetic engineering, whereas the semiconductor technology takes care of the fast and reliable data acquisition and provides the interface to state-of-the-art data processing and transmission technologies. Nanotechnology also allows the manipulation of materials at the nanometer scale, which facilitates precision engineering to study physiochemical properties as well as their typical interactions with biological systems [1-12]. An appropriate modification in morphological, structural, electrical, magnetic, chemical, and physical properties of nanomaterials, makes them predominantly attractive and pioneering choice for technological and biological applications [13-16]. ZnO nanostructures have received substantial interest in recent years due to their potential use in electronic and optoelectronic and sensor device applications originating from their wide band gap (3.36 eV) semiconductor properties [16-30]. From the biomedical perception, ZnO nanostructures have been used in the cosmetic and sunscreen industry because of their transparency and capability to reflect, scatter, and absorb UV radiation and as food additives [31, 32]. ZnO nanostructures are considered to be an excellent choice for next generation biological applications including antimicrobial agents, drug delivery, bioimaging probes, and cancer treatment [33-36]. In this chapter we will demonstrate the comprehensive biomedical applications of ZnO nanostructures.

2. 1D ZnO NANOSTRUCTURES One-dimensional (1D) nanostructures embody a group of nanomaterials with exceedingly anisotropic morphologies, the minimum dimension falling in the range of 1–100 nm [37-40]. Emblematic examples of 1D nanostructures include nanowires, nanotubes, nanobelts and nanorods [41-47]. Metal-oxide semiconductor nanowires are illustrated by the proficient transport of electrons and excitons, and have been regarded as the most promising building block for nanoscale electronic and optoelectronic devices. Nanosystems can be assembled from these elements using such nanowires as interconnects. Nanoscale 1D semiconducting metal-oxides have attracted much attention due to their importance in understanding the fundamental properties at low dimensionality as well as for their applications in optoelectronic nanodevices and biological applications. Among 1D metal-oxides nanostructures, ZnO were broadly investigated for biomedical applications [4860]. Here we present one such example of patterned 1D ZnO nanostructures [20]. Figure 1 shows low (left) and high (right) magnification FESEM images of the various patterns of dimension-controlled ZnO nanorod arrays (NRAs), grown selectively on the desired areas of substrates with the help of electron beam lithography. Different patterns of nanorod arrays like circular (a), square (b), conical bundle (c), and single or a couple of nanorods (d) were created successfully to grow 1D nanostructures. The distance between arrays was periodically spaced and the lengths and diameters of as-grown nanorods were 1.0–1.5 m and ~50 nm, respectively. These patterned NRAs displayed an outstanding homogeny in terms of density, diameter and length.

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Figure 1. Low (left) and high (right) magnification FESEM images of ZnO nanorod arrays grown selectively on pre-patterned Si substrates with diameters of 2 m ((a), (b)), 500 nm (c), and 50–100 nm (d), respectively Reprinted from Ref. [20], copyright permission from IOP 2010.

These results demonstrate that tailoring the pattern size of the substrate through the electron beam process readily controls the dimensions and density of NRAs. It is interesting to note that the flourishing synthesis of periodic single or a couple of ZnO nanorods was accomplished by decreasing the pattern size of the substrate to sub-100 nm (d). The intermittently missing spots in the 100 nm patterns were most likely caused by either poor electron beam patterning.

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2.1. Biofunctionalization of ZnO Nanostructures In order to engineer the biosensors based on ZnO nanostructures, it is imperative to realize the essential mechanism involved in the adsorption of proteins or biomolecules on surfaces of ZnO nanostructures. The functionalization step is critically important for the realization of ZnO-based sensor technology with high sensitivity and selectivity. Various different approaches were utilized to activate the surface of ZnO nanostrutcures through multistep organic reactions. Here in this chapter we have illustrated few significant efforts made by different researchers for effective biofunctionalization of ZnO nanostructures for possible and efficient biosensor applications. To demonstrate the biosensing capability of the ZnO NRAs, a biosensing protocol has been developed [61]. Strip patterned ZnO NRAs shown in Figure 2 were used to develop biofunctionalization strategy. Figure 3 demonstrates the stepwise biofunctioanlization of ZnO NRAs. Briefly, the surface of the ZnO NRAs was functionalized with ATES (step I). Then, the amino groups on the 3-aminopropyltriethoxysilane (ATES)-silanized ZnO NRAs are reacted with the aldehyde group of glutaraldehyde (GA), yielding an ATES/GA surface (step II), subsequently followed by the immobilization of angiotensin II and bovine serum albumin (BSA) on the surface (step III) [61]. Each step of surface biofunctionalization was characteriized by X-ray photoelectron spectroscopy (XPES) and MiniSims to know the surface reaction. The immobilizing densities of angiotensin II and BSA proteins were determined by Coomassie assay. Liu et al, reported the biofunctionalization of ZnO NRAs on flexible thermoplastic polyurethane (TPU) substrate, using dimercaptosuccinic acid (DMSA), and 1-ethyl-3-(3dimethylaminopropyl) carbodiimide), (EDC) [62]. Finally human serum alibumin ‘(HAS) and bovine serum albumin were immobilized on the DMSA and EDC modifed ZnO NRAs. The schematic chemical reaction is shown in Figure 4. Each reaction step was analyzed by XPS. Also the photoluminescence (PL), and Coomassie assay techniques were employed to detect the surface density of immobilized protein molecules. One can expect the conjugation of specific biomolecules on the functionalized ZnO NRAs for the detection of complementary biomolecules by advanced techniques like PL. Recently Taratula et al, developed an advanced surface functionalization protocol to study the ZnO nanotips biosensors integrated with microelectronics [63]. They used two types of carboxylic acid linkers for the functionlization of 500 nm long ZnO nanotips grown by MOCVD with single stranded DNA (ss DNA), followed by hybridization with complementary ssDNA tagged with flourecein illustrated in Figure 5. This ZnO biofunctionalization protocol was devised for the fabrication of ZnO-biomolecules with bulk acoustic wave (BAW) biosensors. This typical scheme was accomplished in three major steps. First, or N-(15-carboxypentadecanoyloxy) succinimide, or 16-(2-pyridyldithiol) hexadecanoic acid both bifunctional C16 carboxylic acids, were attached to ZnO nanotips via COOH group, leaving at the converse end of the alkyl chain a thiol group protected as a 2pyridyl disulfide, or a carboxylic group protected as a N-succinimide, respectively. In the second step, ssDNA was covalently linked to each type of ZnO-linker film: the 2-pyridyl disulfide end group was substituted with 16 bases 5 -thiol-modified DNA (SH-ssDNA), and the N-succinimide ester end group was substituted with 16 bases 5 -amino-modified DNA (NH2-ssDNA). At last in the third step, the DNA-functionalized ZnO nanotips were hybridized with paired 5 -fluorescein ssDNA.

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Fiigure 2. ZnO naanorods arrays: optical microsccope (a) and low w magnificationn FESEM imagee (b); high m magnification FE ESEM images (c, d);

Fiigure 3. Schemaatic illustration of immobilizattion process witth angiotensin II on ZnO NRA As.

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Reprinted from Ref. [62], with permission from copyright 2010 American Chemical Society. Figure 4. Chemical scheme of DMSA-self-assembled and protein-conjugated ZnO nanorods, and the sensitive optical quality observed by PL detecting while proteins bound.

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Figure 5: ZnO Nanotips Surface Modification via Route A (Thiol-Disulfide Exchange Reaction) and Route B (NHS-Ester Hydrolysis), Reprinted from Ref. [63], with permission from copyright 2010 American Chemical Society.

In order to assemble the effective biosensor which can replace the existing biosensors, still we need to develop straight forward and most convenient route for the biofunctionalization of metal-oxide nanostructures.

2.2. Electrical Properties of Surface Molecular Functionalization of ZnO Nanobelts Molecular functionalization of 1D nanostructures provides new ways for the fabrication of high performance chemical and biosensor devices. In the recent past a single nanowire, nanobelt or nanorod devices have attracted significant interest in advanced applications. Various research groups have investigated the performance of individual nanowire device for diverse applications. However there are very rare reports of molecular functionalization of such type of nanodevices. Lao et al, functionalized the surfaces of ZnO nanobelts with a thin self-assembled molecular layer and studied their electrical and optoelectronic properties [64]. It was found that the electrical and optical properties of single nanobelt device significantly enhanced by molecular functionalization. Figure 6 shows I-V characteristics of ZnO nanobelts functionalized with different end group molecules. They observed negative differential resistance (NDR) as indicated in Figure 6. It was found that about 80% molecule coated nanobelts displayed NDR behavior regardless of type of end group. These results revealed wide variety of applications of functionalized nanostructures.

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Reprinted from Ref. [64], with permission from copyright 2010 American Chemical Society. Figure 6:Typical I-V characteristics of the molecular-functionalized ZnO NBs with different end-group molecules, which shows a typical negative differential resistance effect at room temperature. The current for the NB with molecule functionalization layer, HOOC(CH2)10COOH (blue line), was demagnified by 5 times for comparison purpose.

This work also showed possibility of various kinds of molecular functionalization and variety of enhanced biosensors can be made by choosing related molecular functionalization. The peak to valley ratios in the positive bias I-V curves at room temperature ranged from 1.56 (dicarboxylic acids, HOOC-(CH2)10COOH, green line) to 1.08 (lysine, H2N(CH2)4CH(NH2)COOH, red line). The coating of molecular layer has efficiently decreased the contact resistance between a nanobelt and the Au electrode, resulting in a change of a Schottky contact into an Ohmic contact. These functionalized molecules greatly reduce the dissolution rate of ZnO nanobelts in biofluid; and it can interestingly enhance the life time of device for biomedical applications.

2.3. Protective Antigen and Fluorescence Detection Using Stacked ZnO Hexagonal Nanoplatlets and ZnO Nanoplatforms: Park et al demonstrated a novel strategy for ultrasensitive detection of a protective antigen (PA83) of anthrax via ZnO horizontally stacked hexagonal platelets (VRHHPs) in combination with a FITC-labeled PA affinity peptide [65]. The nanorods were made of horizontally stacked hexagonal platelets which are consistently spaced and grown unidirectionally upon a glass substrate via an innovative and straightforward procedure. The microgrpahs taken under UV emission revealed fluorescence sensitivity to PA as a function of antigen concentration, and a negative control using BSA could not produce any fluorescence signal. Figure 7 (panels a-f) represents the biomolecular fluorescence images in the presence of ZnO VRHHPs at PA concentrations ranging from 1.5 nM to 15 aM. Their results showed that the detection sensitivity of the ZnO VRHHPs-peptide system approaches attomolar concentrations.

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Figure 7. Optical ultraviolet emission images of ZnO VRHHPs-protein-peptide system for the detection limit of PA concentration: (a) 1.5 nMPA, (b) 15 pM PA, (c) 0.15 pM PA, (d) 1.5 fM PA, (e) 0.15 fM PA, (f) 15 aM PA, (g) BSA. (h) The intensities measured at various PA concentrations were normalized with respect to the intensity measured when using 1.5 nM PA. The normalized intensity is plotted against various PA concentrations. Fluorescence intensity at 15 aM PA concentration was significantly lower than intensities at PA concentrations g 1.5 fM by P < 0.001. Fluorescence intensity at 0.15 fM PA concentration was significantly lower than intensities at PA concentrations g 1.5 pM by P < 0.001, intensity at 1.5 fM PA by P < 0.01, and intensity at 15 fM PA by P < 0.05. The values are the average of five independent experiments, Reprinted from Ref. [65], with permission from copyright 2010 American Chemical Society.

The fast diagnosis of anthrax is crucial and preliminary step for its proficient treatment and cure. In this experiment they used an intentionally imperative binding capability of Protective antigen and fluorescence-enhancing properties of ZnO VRHHPs to assist the biomolecular detection, as indicated by the attomolar detection sensitivity of PA. ZnO VRHHPs could be prospective biosensors with the capability to detect enormously low-level concentrations, which can be employed in research, disease diagnosis, and other nanobiological systems. Florescence detection is one of the most interesting research field of biological systems including biotechnology, biological research, celluar imaging, drug delivery, disease

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diagnosis etc. Recently Dorfman et al, studied that by using model proteins and nucleic acid systems, ZnO nanoplatform can effectively enhance the detection ability of biomolecular florescence [66]. It is found that without using any physiochemical amplification process ZnO nanostructures facilitated the enhanced florescence detection of biomolecules and ZnO substrate contrasted with other traditionally used substrates such as quart, glass, polymer and silicon. The ZnO NRAs as fluorescence substrate in this biomolecular detection allowed subpicomolar and attomolar sensitivity detection of proteins and DNA through traditional flourescence microscope. Figure 8 (a-e) depicts enhanced fluorescence detection facilitated by the use of ZnO nanoplatforms. The ZnO nanosplatforms will be highly useful in accomplishing highly sensitive and exact detection of biological samples like nucleic acids, DNA, RNA, proteins, cells, by using negligible sample volumes of ultratrace-level concentrations.

2.4. Cellular Biocompability and Biosafety Of ZnO Nanostructures In past few years various researchers made tremendous efforts to study the interaction of ZnO nanostructures with the biological systems. Recently Li et al, for the first time studied the biocompatibility of two different cell lines from different origins tissues using ZnO nanostructures [67]. In their investigation they found that Hela cells showed a complete biocompatible to ZnO nanostructures from low to high nanowire concentrations beyond a couple of production periods. The L929, demonstrated good reproduction behavior at lower nanowire concentrations, however, when the concentration was close to 100 μg/ml, the viability reduced to ~50 %. SEM images of the samples were demonstrated in Figure 9 (a, b). Figure 9 (a) shows the growth of two Hela cells began on the surface of ZnO nanowires. Hela cells phagocytosed few broken ZnO nanowires into the cell membrane (Figure 9 b). They found that no external force was required for the phagocytosis, due to the self-activity of the cells. This study revealed the biocompatibility and biosafety of ZnO nanowires when they are applied to biological system at normal concentration range.

2.5. Detection of Phenol by Tyrosinase Immobilization on ZnO Nanorods ZnO nanostructures have unique advantages including the high specific surface area, nontoxicity, chemical stability, electrochemical activity, and high electron communication features. Hence, they are promising tool for biosensor applications. Gu et al, demonstrated one excellent example of biosensor based on ZnO nanorods [68]. They immobilized tyrosinase on gold electrode to check the electrochemical response of ZnO nanorods to detect the phenol through electrobiochemical reaction. They treated the gold electrode by a typical procedure to form a rough sphere head covered with Au-Zn alloy layer which made the ZnO growth easy and convenient. This was a very rare experiment where alloy was formed on the electrode to make the growth of ZnO nanorods and their convenient geometry. Zinc powder was used as source material to carry out the hydrothermal reaction on the surface of the electrode. The grown nanorods were with smooth surfaces and reliable size.

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Figure 8. Enhanced fluorescence detection facilitated by the use of ZnO nanoplatforms: model protein system. (a) When confocal microscopy was employed to measure fluorescence emission, no distinctive contrast was observed in the confocal images taken from nonspecifically adsorbed FITC-antiIgG molecules on Si wafers, SiNRs, and patterned PMMA substrates. (b) Markedly strong fluorescence emission was detected from FITC-anitIgG molecules that were adsorbed on the surfaces of single ZnO nanostructures. Owing to the strong fluorescence signal, topological profiles of each ZnO nanomaterial are clearly visible in the confocal image. Confocal images of (a) and (b) are taken from a 25 x 25 μm2 area. (c) The same fluorescence enhancing effect was observed from FITC-antiIgG that was deposited on regularly patterned ZnO nanoplatforms on the micrometer scale. The repeat spacing of the underlying ZnO nanoplatforms is 20 μm and the concentration of FITC-antiIgG used in the confocal measurement was 200 μg/mL. (d) Relative fluorescence intensity from nonspecifically adsorbed FITCantiIgG molecules prepared on various control substrates such as PMMA film, quartz, glass, Si, ZnO thin film, and SiNRs. The plot displays fluorescence intensity measured from 200 μg/mL FITC-antiIgG on these control substrates which was normalized with respect to that on ZnO nanoplatforms. (e) The plot of fluorescence intensity versus protein concentration displays the detection sensitivity limit of protein molecules on ZnO nanoplatforms using a conventional confocal microscope. Fluorescence data from protein molecules of the same composition and concentration on patterned PMMA platforms are plotted for comparison. ZnO data points at low concentration are rescaled and displayed in the inset for clarity. The lowest concentration of FITC-antiIgG on ZnO nanomaterials which were readily detectable using a 40 mW Ar laser was 2 ng/mL. Reprinted from Ref. [66], with permission from copyright 2010 American Chemical Society.

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Figure 9. SEM images of Hela cells on ZnO NWs arrays. (a) Two Hela cells are growing on the surface of ZnO NWs arrays. (b) Cells are upheld by the NWs. Some ZnO NWs are phagocytosed into the Hela cell (pointed out by the red arrow). The diameter and the length of the nanowires are ~100 nm and ~1.5 m, respectively, Reprinted from Ref. [67], with permission from copyright 2010 American Chemical Society.

The successfully assembled direct bridge for electron transfer during the tyrosinase catalyzed the detection of phenol. Electrochemical characterization revealed that the tyrosinase with low ionization electron potential (IEP) was firmly absorbed on the surface of ZnO nanorods with high IEP, and that the absorbed enzyme retain its bioactivity to a great extent. The biosensor exhibited the tremendous performance. Figure 10 (a) demonstrates an emblematic current-time plot for the sensor on successive step addition of phenol. The calibrated association between the reduction current and the concentration of the phenol was plotted in Figure 10 (b), which can be separated into two linear regions corresponding to lower and higher concentration, as shown in Figure 10 (c) and (d), respectively. The response time was less than 5 s upon adding phenol to the buffer solution. The sensitivity and detection limit were improved to 103.08 A/mM and 0.623 M.

2.6. ZnO Nanorods for Reduced Adhesion of Mammalian Cells Cellular interaction with nanostructures became interesting field of study recently due to their prominent role in tissue engineering and biomedical implants. Lee et al, demonstrated the silicon oxide (SiO2) coated ZnO nanorods virtually eliminate cell adhesions in fibroblasts and endothelial cells [69]. They found that the lack of adhesion was not due to a decrease in matrix protein adsorption on the nanostructures, but rather an incapability of cells to accumulate crucial adhesions. The diameter of each circle was 50 mm and spacing between the circle 40-60 mm. The ZnO nanorods have diameter 50 nm and their length 500 nm. Figure 11 (a) and (b) shows fibroblasts preferably adhered to the flat surface rather than to the nanorods after culture 48 h culture. Similar patterning was also seen with ZnO nanorods without SiO2 coating. Therefore, these results suggest that densely packed nanorods have excellent anti-fouling potential by virtue of their topology.

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3. SUMMARY Hetero-interfaces between organic and inorganic matter have been of enormous significance in basic and applied sciences because of probable applications for optoelectronics and biosensors.

Figure 10: The current-time response (a) and the calibrated current-concentration relationship (b) for successively increasing phenol concentration in 2 M steps at -200 mV; Panel b was separated into two linear regions corresponding to Cphenol < 20 M (c) and Cphenol > 20 M (d), respectively. Reprinted from Ref. [68], with permission from copyright 2010 American Chemical Society.

The anticipation is that organic-inorganic hybrids will be able to present some unambiguous functions better than either purely organic or purely inorganic systems. An excellent example is novel biosensors based on protein-functionalized semiconductor devices. Extensive advancement has been made regarding the functionalization of carbon nanotubes with proteins through bioconjugation. Studies to understand nanomaterials with bioconjugated biomolecules for biological applications have been reported in the literature. In this chapter efforts have been made to study various biological applications of 1D ZnO nanostructures. Four unique and successful methods for biofunctionalization of 1D ZnO nanostructures have been illustrated in details. The improved electrical and optical properties of single ZnO nanobelt devices by molecular functionalization is also demonstrated. A novel approach for ultrasensitive detection of a protective antigen (PA83) of anthrax via ZnO VRHHPs in permutation with a FITC-labeled PA affinity peptide is stated.

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Figure 11. NIH 3T3 fibroblasts on patterned SiO2 coated nanorods. Fluorescent microscopic images showing that NIH 3T3 fibroblasts preferably attached on glass. Cells are stained for actin (red), vinculin (green) and nucleus (blue). Cells were confined on the flat circular regions. Dashed lines indicate the edge of patterns. Reprinted from Ref. [69], with permission from copyright 2010 elsevier.

The nanorods are made of horizontally stacked hexagonal platelets which are consistently spaced and grown unidirectionally upon a glass substrate via an pioneering and straightforward method. The ZnO NRAs as a fluorescence substrate is included where the biomolecular detection allowed sub-picomolar and attomolar sensitivity detection of proteins and DNA though traditional flourescence microscope. The biocompatibility and biosafety of ZnO nanowires were demonstrated and it was observed that by using this strategy one can study different type of metal oxide nanostructures for future biological applications. An electrochemical biosensor device based on ZnO nanorods were illustrated to detect phenol by using tyrosinase enzyme. Finally a reduced cell adhesion of mammalian cells were studied by comparing pristine and and SiO2 coated ZnO nanorods.

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In: Recent Developments in Bio-Nanocomposites… ISBN 978-1-61761-008-0 Editor: Ashutosh Tiwari © 2011 Nova Science Publishers, Inc.

Chapter 6

SOL-GEL DERIVED SIO2-CHITOSAN/CARBON NANOTUBES- PROMISING MATRICES FOR BIO- RECOGNITION EVENTS Ashutosh Tiwari1,2, Ajay K. Mishra3, Radhe Shyam Rai4, Shivani B. Mishra5, Shunsheng Cao1, Rajeev Mishra5, S.K. Shukla6, Smarti Bhadoria7, Premlata Kumari8, and Mani Prabaharan9 1

School of Materials Science and Engineering, Jiangsu University, Zhenjiang 212 013, China 2 National Institute for Materials Science, Tsukuba, Ibaraki 305 0047, Japan 3Department of Chemical Technology, University of Johannesburg, Doornfontein, Johannesburg 17011, South Africa 4 Departmento de Engenharia Cerâmica e do Vidro and CICECO, Universidade de Aveiro, 3810-193 Aveiro, Portugal 5 Department of Cancer Genetics, School of Medicine, Nihon University, Tokyo 173 8610, Japan 6 Department of Polymer Science, Bhaskaracharya College of Applied Science, University of Delhi, New Delhi 110 075, India 7 Division of Toxicology, Central Drug Research Institute, Lucknow 262 001, India 8 Applied Chemistry Department, S. V. National Institute of Technology, Surat, Gujarat, India. 9 Department of Chemistry, Faculty of Engineering and Technology, SRM University, Kattankulathur 603 203, India

ABSTRACT The performance of enzyme based biosensors usually depends on the physicochemical properties of the electrode materials as well as process of the enzyme

1 Corresponding author: E-mail: [email protected]; Tel: (+86) 511-8879-0191; Fax: (+86) 511-8879-0769.

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Ashutosh Tiwari, Ajay K. Mishra, Radhe Shyam Rai et al. immobilization and also enzyme concentration on the electrode surface. Although, various matrices are reported in the literature for the immobilization of enzymes to use in biosensors, the method of immobilization and electrode matrices, both are considered promising factor during the determination of the operational and storage stability of the biosensors. The intrinsic stability of enzymes has encouraged for applying biomaterials engineering to improving stability. In this sequence, the use of nanomaterials such as carbon nanotubes (CNTs) to fabricate matrices for biosensors is one of the most exciting approaches because nanomaterials have a unique structure and high surface-to-volume ratio. The present chapter deals the preparation of SiO2-chitosan/CNTs (SiO2CHIT/CNTs) bio- nanocomposite, enzyme immobilization, characterization, electrochemical behavior, bio- recognition, interference, stability and response study of bio- analytes for the potential biosensor applications.

1. INTRODUCTION Organic-inorganic nanocomposites based on polymer and silica are an important class of advanced materials. The combined organic-inorganic characteristics of the composites represent an improvement in their physical properties. The silicate-filled polymer composites often exhibit remarkable improvement in mechanical, thermal, and physicochemical properties when compared with their pure one. Sol-gel synthesis of composite materials allows the production of materials by which it is possible to control particle size, shape and sometimes even final packing of the colloidal particles [1-4]. Sol-gel derived nanobiomaterials have recently been used for the fabrication of biosensors [5, 6]. In general, biosensors provide easy operation, accuracy, sensitivity and selectivity for bioanalytes. Several nanomaterials including sol-gel films [7-10] and selfassembled monolayers [11] have been used for biosensors fabrication using layer-by-layer technique [12], physical adsorption [13], hydrogel or sol-gel entrapment [14], cross-linking and covalent techniques [15]. It is known that sol-gel derived silica (SiO2)-based nanomaterials possess high mechanical strength, tunable porosity, chemical inertness, thermal stability and negligible swelling in aqueous as well as non-aqueous medium [16, 17]. The sol-gel derived silica prepared using tetraethyl orthosilicate (TEOS) precursor has been found to be more reactive towards the condensation reaction and has high affinity towards enzymes as compared to other precursors such as tetramethyl orthosilicate (TMOS), etc. [18]. Silica and biopolymer composites are attractive for the preparation of high performance and multifunctional materials using sol-gel method [19]. TEOS has been extensively used as silica precursor for the preparation of various biopolymer-silica hybrid composites [20-21]. The biopolymer silica composites prepared by the sol-gel processes in aqueous solutions, owing to their promising properties, are of special interest [22-26]. It was observed that polysaccharides can serve as a template for silica generated in situ by the sol-gel processes, thus manipulating its synthesis as well as properties and structure. The polysaccharides promoted silica polymerization through acceleration and catalysis of processes [27]. Their effect is explicable by the formation of hydrogen bonds between hydroxyl groups of macromolecules and silanols generated by the hydrolysis of precursor. In this context, chitosan (CHIT) is a cationic polysaccharide which is attracted much interest owing to its interesting properties such as biocompatibility, non-toxicity, low-cost,

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good film forming ability, high mechanical strength and high hydrophilicity [28]. Moreover, presence of amino and hydroxyl groups in CHIT can facilitate covalent enzymes immobilization for biosensor application [29, 30]. However, sol-gel-derived silica-CHIT (SiO2-CHIT) biocomposite exhibits interesting biosensor characteristics, yet very small response current is observed. The incorporation of carbon nanotubes (CNTs) into SiO2-CHIT nanocomposite film is likely to result in improved sensing characteristics. CNTs enhance electro-catalytic activity due to presence of edge-plane-like sites located at both ends and in the defect region [31]. Besides this, these provide high surface-to-volume ratio and faster electron-transfer kinetics due to curvature of CNTs that produce changes in energy bands close to Fermi level [32]. Keeping this in view, CNTs have recently been utilized for fabrication of electrochemical biosensor [33-36]. The aim of this chapter is to focus on preparation of sol-gel derived SiO2-CHIT/carbon nanotubes (SiO2-CHIT/CNTs) bionanocomposite, characterization, enzyme immobilization techniques and biosensor applications. In this review, strategies involved in the preparation of cholesterol, urea and creatinine biosensors based on SiO2-CHIT/CNTs bio- nanocomposite and their properties such as electrochemical, morphology and biosensing behavior are discussed in details.

2. PREPARATION OF SIO2-CHIT/CNTS BIO- NANOCOMPOSITE The use of nanomaterials such as CNTs to fabricate matrices for biosensors is one of the most exciting approaches because nanomaterials have a unique structure and high surface-tovolume ratio [37]. The surfaces of nanomaterials can also be tailored in the molecular scale in order to achieve various desirable properties [38]. The diverse properties of nanocomposite materials such as unique structure and good chemical stability enable them to provide a wide range of applications in sensor technology [39]. Further, nanocomposites do not suffer from the drawback of sensing complications, synthesis complexities; have a long shelf life, and efficiency. In addition, the fundamental electronic characteristics of CNTs could also be used to facilitate the uniform current within the nanocomposite biosensing electrodes. There are many reports on integration of CNTs with sol-gel derived SiO2-CHIT to fabricate biosensors to gain synergistic action using organic-inorganic bio- nanocomposites. The sol-gel SiO2CHIT is prepared by mixing of alcoholic silica precursor such as TEOS and CHIT solution under magnetic stirring at room temperature. To this mixture, homogeneously dispersed CNTs in ethanol are added. The mixture initially comprising of two phases is made uniform by stirring vigorously until -SiO2 is distributed evenly in the aqueous solution while the hydrolysis reaction occurs. After certain time period, the opaque and black sol is formed (Figure 1). In a control process, tetraethoxysilane undergone hydrolysis and formed tetrahydroxy silane (silanol) at acidic pH [40]. The resulted silanol then reacted with CHIT via a condensation reaction between the -OH groups and led to the formation of a CHIT-SiO2 composite network, in which CNTs are homogeneously dispersed. Both CNTs and SiO2 improve the mechanical properties of the CHIT-SiO2-CNTs bio- nanocomposite, primarily CNTs enhance the electrical conductivity of the bio- composite.

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Si

OH

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OC2H5

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OH

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n O

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Condensation

Si O

OH O

O

Tetrahydroxy silane O

n

Si

O OH NH2

NH

n O

CHIT

CH3

CHIT-SiO2 -CNT composite

Figure 1. Reaction scheme for the preparation of SiO2-CHIT/CNTs bio- nanocomposite.

3. FABRICATION OF ENZYME-SIO2-CHIT/CNTS BIOELECTRODES The SiO2-CHIT/CNTs sol thin film is fabricated by spread it uniformly onto a substrate such as ITO glass plate using spin coating technique and subsequently dried at room temperature. SiO2-CHIT/CNTs/substrate electrode is washed with deionized water followed by phosphate buffer saline of pH 7.0 in order to maintain pH over the electrode surface. SiO2CHIT/CNTs electrode is treated with aqueous glutaraldehyde as a cross-linker. The freshly prepared enzyme solution is uniformly spread onto glutaraldehyde treated SiO2-CHIT/CNTs electrode and is kept in a humid chamber for 12 h at 4 oC, Figure 2.

Figure 2. Schematic diagram of the fabrication of CHIT-SiO2-CNTs/ITO and ChOx/CHIT-SiO2MWNT/ITO electrodes.

The enzyme-SiO2-CHIT/CNTs bioelectrode is immersed in phosphate buffer solution of pH 7.0 in order to wash out unbound enzyme from the electrode surface. A stable enzyme-

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substrate coupling is achieved with gluteraldehyde as a cross-linking agent [41]. At one end gluteraldehyde is attached to the –NH2 side of the CHIT-SiO2-CNTs/ITO electrode through a reaction between the -CHO end group of glutaraldehyde, while other end of the gluteraldehyde is attached to enzyme such as creatine amidinohydrolase (CAH) through a reaction between the -CHO group of gluteraldehyde and the terminal -NH2 group of enzyme, which resulted in a enzyme/CHIT-SiO2-CNTs/ITO bioelectrode as shown in the Figure 3. O

O H

O H

N

N

H

H H NH2

H

NH2

N

N

O

Glutaraldehyde CHIT-SiO 2-CNT matrix having free NH 2 groups

One end of Glutaraldehyde coupled with CHIT-SiO 2-CNT

N

H

N

H

NH2

CAH CAH covalent bonding of free -CHO end of coupled glutaraldehyde-CHIT-SiO2-CNT

Figure 3. Covalent immobilization of the creatine amidinohydrolase (CAH) enzyme on SiO2CHIT/CNTs bio- nanocomposite matrices using gluteraldehyde as a linker.

4. CHARACTERIZATIONS The bare and enzyme immobilized SiO2-CHIT/CNTs bio- nanocomposite can be characterized with Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM) and cyclic voltammetry (CV). In Figure 4, the infrared peaks of CHIT (curve A) in SiO2-CHIT (curve B) become wider and sharper due to overlap of functional groups of CHIT and SiO2, i.e., stretching vibration bands of Si-O-Si, Si-O-C and C-O bond. In addition, two new peaks appear at 1300 and 785 cm-1 pertaining to stretching vibration of C-Si and bending vibration of C-H corresponding to CH3-Si group [42]. On incorporation of CNTs in CH-SiO2 hybrid, the infrared band corresponding to SiO2 becomes broader and a new infrared band appears at 890 cm-1 revealing presence of CNTs that affects vibration mode of CHIT and SiO2 resulting in the formation of SiO2-CHIT/CNTs bio- nanocomposite. After immobilization of enzyme, FTIR bands corresponding to -NH/-OH group in nanobiocomposite become broader suggesting interaction between amino and hydroxyl group of CHIT with. However, presence of 1672 and 1442 cm-1 peaks (corresponding to amide bands) indicates immobilization of enzymes [43]. The surface morphology, Figure 5 of SiO2-CHIT/CNTs (image A) reveals the monodispersed rope like structure of CNTs surrounded with globular appearance of SiO2 particles into CHIT matrix indicating that CNTs and SiO2 are uniformly dispersed into the backbone of CHIT. We speculate that CNTs are rapped with CHIT and SiO2 via electrostatic interactions. The surface morphology of SiO2-CHIT/CNTs nanobiocomposite further changes after the immobilization of enzyme revealing attachment of enzymes over the electrode surface (image B-C).

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% Transmittance

(B)

(A)

0

5 500

10000

1500

2000

2500

30000

3500

Waven number (cm m -1 ) Fiigure 4. FTIR sp pectra of the (A A) CHIT-SiO2/C CNTs nanocom mposite and (B) CAH/CHIT-SiO O2/CNTs.

Fiigure 5. SEM im mages of the (A A) CHIT-SiO2/C CNTs and (B-C) Enzyme/CHIT T-SiO2/CNTs.

It may be noted that apppearance of CNTs C is less predominant p d to immobilization of due ennzymes onto nanobiocom mposite via electrostatic interactions and covalennt binding. M Multifunctional l matrix pressumably provvides mesopoorous surface resulting inn enhanced ennzyme loadin ng at the enzyyme loaded SiO S 2-CHIT/CN NTs bioelectrrode. Figure 6 shows a prrobable mechaanism of enzyyme immobilizzation onto naanobiocompossite using gluttaraldehyde ass linker. It apppears that available -NH2 grooups of CHIT get covalentlyy attached witth aldehyde grroup of glutarraldehyde at one o end and another a aldehyyde group getss linked with available N 2 groups off enzymes via covalent boonding and ellectrostatic innteractions bettween bioNH naanocomposite and enzymes.

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Fiigure 6. Proposeed mechanism for f preparation of ChEt-ChOx//CNTs/SiO2-CH HIT/ITO bioeleectrode.

5.. ELECTRO OCHEMICAL STUDY Electrochem mical study measures m the movement m andd separation of o charge in matter, m i.e., thhe study of thee transfer of electrons. e Mosst of the chem mical reactions involve chargge transfer, annd matter may y hold chargee, either positiive or negativve. The chargees could be discrete d and m measureable or partial and diffuse; chaarged matter can maintainn separations leading to innteresting effeects. Figure 7 shows the CV Vs of the elecctrochemical cells c using eitther CHITSiiO2-CNTs/ITO O or CAH/CH HIT-SiO2-CN NTs/ITO electrrode at a consstant 20 mVs-1 scan rate inn 50 mM phossphate buffer solution (pH 7.0, 0.9% NaaCl) containing 5 mM Fe(C CN)63−/4−. The current of the electrocheemical cell usiing the electroode CHIT-SiO O2-MWCNTs//ITO (2.5 x 100-3 A) was ab bout five timess of that usingg the CAH/CH HIT-SiO2-CNT Ts/ITO bioeleectrode (0.5 x 10-3 A). Thuus, immobilizing CAH ontoo the bare elecctrode reducedd the current. A decrease inn current afterr the immobillization of CA AH may be attributed a to a slower redox behavior w when compareed with the bare CHIT-SiO O2-CNTs/ITO O electrode. The T covalent binding of C CAH on the CHIT-SiO2-C CNTs/ITO electrode contrrols the mom ment of the supporting ellectrolytes [44]. Also, thee non-conduccting nature of o the CAH molecules might m have coontributed to the t decrease inn current wheen using the CAH/CHIT-SiO O2-CNTs/ITO O electrode. The study indiccates a high affinity a of CA AH to the CHIIT-SiO2-CNT Ts nanocompoosite matrix b attributed to: t (1) the addvantageous nanoporous n ovver the electrrode surface, which may be suurface of the CHIT-SiO2--CNTs matrixx for the enzzyme immobiilization that can favor

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conformational changes of the enzyme, and (2) the high surface-to-volume ratio, which can help to effectively immobilize CAH onto the CHIT-SiO2-CNTs nanocomposite [45]. 10 10

0.003 (A)

Current (A)

0.002 (B)

0.001 0.000 -0.001 -0.002

-0.003 -0.3 -0.2 -0.1 0.0 0.1 0.2 0.3 0.4 0.5

0

0.6

Potential (V) Figure 7. Cyclic voltammograms of the (A) CHIT-SiO2-CNTs/ITO and (B) CAH/CHIT-SiO2CNTs/ITO electrodes in PBS (50 mM, pH 7.0, 0.9% NaCl, 5 mM Fe(CN)63-/4-) at a 20 mVs-1 scan rate.

The CV studies of CHIT/ITO, SiO2-CHIT/ITO, CHIT-CNTs/ITO, SiO2CHIT/CNTs/ITO, ChEt-ChOx (cholesterol esterase-cholesterol oxidase)/SiO2CHIT/CNTs/ITO electrodes have been conducted to understand the synergy between the various components in phosphate buffer saline containing [Fe(CN)6]3−/4− at scan rate of 50 mV/s, Figure 8A (a-c). The CV of pure CHIT (curve a) shows well defined reversible redox behavior attributed to highly positive charged species on matrix indicating that electrons originate from the negatively charged medium. After addition of -SiO2- in CHIT, magnitude of current decreases (curve b) due to formation of complex between SiO2 and -OH/-NH2 group of CHIT. In the case of CHIT and CNTs (curve c), magnitude of current decreases and the potential is shifted towards the lower side at 0.213 V as compared to that of CHIT (0.301 V) (curve a). It appears that CHIT-CNTs bio- composite has increased number of electrons compared to that of CHIT since ΔEp is inversely proportional to the number of transferred electrons (ΔEp α 1/n) [46], while curve (d) exhibits highest voltammetric response on SiO2CHIT/CNTs/ITO electrode because SiO2/CNTs provides an electroactive surface area that enhances electron conduction pathway and promotes electron transfer between the enzymes and electrode. The redox potential of ChEt-ChOx/SiO2-CHIT/CNTs/ITO bioelectrode (curve e) is much less as compared to the other electrode due to slow redox process during the biochemical reaction. The inset in Figure 8A shows oxidation (Ipa) and reduction (Ipc) peak current against ʋ1/2 (ʋ is the scan rate) for SiO2-CHIT/CNTs/ITO and ChEt-ChOx/SiO2CHIT/CNTs/ITO bioelectrodes. The diffusion control process yields linear response obtained by plotting peak current versus square root of the scan rate and it depends on value of the diffusion coefficient. The values of slope of the peak oxidation current obtained using

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d(I)/d(ʋ1/2) (square root of the scan rate is proportional to D1/2 for electrode and nanobiocomposite) have been found to be as 94.6 and 99.3 A (mV/s)1/2, respectively. The high value of slope of the electrode compared to other bioelectrode is probably due to the binding of ChEt and ChOx onto electrode and possibly controls transport of the ions. Figure 8B exhibits CV of SiO2-CHIT/CNTs/ITO bioelectrode as a function of scan rate varying from 10 to 100 mV/s. The magnitudes of cathodic peak and anodic peak currents increase with increasing scan rate. The increased peak-to-peak separation reveals electron transfer between ChEt-ChOx/SiO2-CHIT/CNTs/ITO bioelectrode and the medium. The variation of potential difference (ΔEp) between cathodic (Epc) and anodic (Epa) peaks for SiO2-CHIT/CNTs/ITO nanobiocomposite matrix and ChEt-ChOx/SiO2-CHIT/CNTs/ITO bioelectrode is shown in Figure 8C. The decrease in ΔEp value for the bioelectrode indicates faster kinetics of electron transfer on the surface [47]. The surface concentrations of CHIT/ITO, SiO2-CHIT/CNTs/ITO; ChEt-ChOx/SiO2-CHIT/CNTs/ITO bioelectrode have been estimated from the plot of current verses potential (CV) using equation: Ip = n2F2I*AV/4RT (Brown-Anson model) where n is the number of electrons transferred which is 1 in the case of CHIT/CNTs/ITO electrode, F is the Faraday constant (96,584 C/mol), I* is the surface concentration (mol/cm2) obtained for the SiO2-CHIT/CNTs/ITO nanobiocomposite matrix, A is the surface area of the electrode (0.25 cm2), V is the scan rate (50 mV/s), R is the gas constant (8.314 J/mol K), and T is the absolute temperature (298 K). The values of surface concentrations for CHIT/ITO electrode and SiO2-CHIT/CNTs/ITO nanobiocomposite matrix have been found as 11.8 × 10-8 and 13.0 × 10-8 mol/cm2, respectively [48]. These results indicate that SiO2-CHIT/CNT1s/ITO matrix provides increased electroactive surface area for loading of enzymes (ChEt and ChOx). Figure 8D demonstrates the Faradaic impedance spectra, presented as Nyquist plots obtained from real (Z ) and imaginary (-Z ) in the frequency range 0.01-105 Hz for CHIT/ITO electrode, SiO2-CHIT/CNTs/ITO nanobiocomposite and ChEt-ChOx/ SiO2-CHIT/CNTs/ITO in phosphate buffer saline (pH 7.0, 0.9% NaCl) containing 5mM [Fe(CN)6]3−/4− that yields information about electrical properties at desired interfaces. The values of electron-transfer resistance (RCT) derived from the diameter of semicircle of impedance spectra are obtained as 4.59 × 103 Ω for CHIT/ITO electrode (curve a), 3.35 × 103 Ω (curve b) for SiO2CHIT/CNTs/ITO electrode and 7.15 × 103 Ω for ChEt-ChOx/SiO2-CHIT/CNTs bioelectrode, respectively. The semicircle of CHIT/ITO (curve a) exhibits charge transfer phenomena between electrode and medium. Compared to CHIT/ITO electrode, charge-transfer resistance (RCT) value obtained for SiO2-CHIT/CNTs/ITO electrode (curve b) decreases resulting in enhanced electron transfer or conductive pathway towards electrode. This suggests that presence of both SiO2 and CNTs enhance ionic transport in CHIT results in the formation of complex and improved charge transfer. It can be seen that RCT increases after immobilization of ChEt and ChOx onto SiO2-CHIT/CNTs/ITO nanobiocomposite (curve c) due to the hindrance provided by macromolecular configuration of ChEt and ChOx to electron transport between electrode and redox mediator indicating immobilization of ChEt and ChOx onto SiO2-CHIT/CNTs/ITO surface. The value of RCT is dependent on electrochemical reaction time constant, τ( τ = ½π fmax = Rp·Cdl, where fmax is the frequency at which maximum Z obtained, Rp is the polarization resistance and Cdl is the double layer capacitance). DPV studies have been carried out on SiO2-CHIT/CNTs/ITO electrode (Figure 8E).

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Fiigure 8. (A) Cyclic voltammoggram of (a) CHIIT/ITO electrodde; (b) SiO2-CH HIT/ITO electroode; (c) CHIT/CNTs/ITO O electrode; (d) SiO2-CHIT/CN NTs/ITO electroode; (e) ChEt-C ChOx/SiO2CHIT/CNTs/ITO O bioelectrode at a 50 mV/s in PB BS containing 5 mM Fe[(CN)66]3−/4−. The innset shows oxxidation and red duction peak cuurrent with squaare root of scan rate for SiO2-C CHIT/CNTs/ IT TO ellectrode; (e) Ch hEt-ChOx/SiO2--CHIT/CNTs/IT TO bioelectrodee. (B) CVs of ChEt-ChOx/SiO C O2CHIT/CNTs/ITO O bioelectrode at a as a function of scan rate (100-100 mV/s). (C C) Square root of o scan rate e (b) ChhEt-ChOx/SiO22-CHIT/CNTs/IITO bioelectrodde and foor (a) SiO2-CHIIT/CNTs/ITO electrode; diifference between the cathodic and anodic peaak shifts. (D) Nyquist N plot of (aa) CHIT/ITO ellectrode, (b) SiiO2-CHIT/CNT Ts/ITO electrodde, and (c) ChEtt-ChOx/SiO2-C CHIT/CNTs/ITO O bioelectrode. (E) DPV of (aa) CHIT/ITO eleectrode, (b) SiO O2-CHIT/CNTss/ITO electrode, and (c) ChEt-C ChOx/SiO2CHIT/CNTs/ITO O bioelectrode inn PBS containinng 5 mM Fe[(C CN)6]3−/4− at potential p height as 0.4995 V potential periood 0.07 ms and interval periodd as 0.14 ms. V,

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DPV experiments have been conducted in phosphate buffer saline (50 mM, pH 7.0) containing 5mM [Fe(CN)6]3−/4− in the range -0.1 to 0.6 V. The DPV measurements have been carried out at 0.49 V, potential period as 0.07 ms and interval period as 0.14 ms. The value of maximum response current obtained as 7.58 × 10-5 A for CHIT (curve a) increases to 8.19 × 10-5 A on incorporation of SiO2 particles and CNTs into CHIT. This suggests that conducting nature of CNTs results in increased ionic transport in CHIT enhancing electron transfer towards the electrode. The magnitude of current decreases to 5.30 × 10-5 A for ChEtChOx/SiO2-CHIT/CNTs/ITO bioelectrode indicating slow redox process at bionanocomposite due to insulating characteristics of ChEt and ChOx revealing immobilization of ChEt-ChOx/SiO2-CHIT/CNTs/ITO bio- nanocomposite. The electro active molecule, H2O2 is resulted from the enzymatic reaction between the cholesterol oxidase and the cholesterol (Figure 9).  

H3C CH3 H2O2

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Figure 9. Libration of electrically active H2O2 as a sensing element resulting from the reaction of cholesterol and ChOx.

In the biochemical reaction, the positive charge on the SiO2-CHIT/CNTs/ITO nanobiocomposite accepts electrons generated during re-oxidation of ChEt and ChOx prior to the evolution of oxygen resulting in enhanced current response of the SiO2-CHIT/CNTs/ITO nanobiocomposite matrix. The enhancement in peak current suggests that this nanobiocomposite provides favorable microenvironment to enzyme wherein CNTs provide enhanced electron transfer to the electrode. Direct acceptance of electrons by the matrix is attributed to enhanced charge transport in the SiO2-CHIT/CNTs/ITO film due to electrons hopping via conductive CNTs that mediate electron transfer via bio- nanocomposite in presence of [Fe(CN)6]3−/4− as mediator. The value of the enzyme-substrate kinetics parameter (Michaelis-Menten constant, Km) estimated using the Lineweaver-Burke plot reveals affinity of enzyme for desired analyte. It is noted that Km is dependent both on matrix and the method of immobilization of enzymes that often results in their conformational changes resulting in different values of Km. Besides this; the value of Km for the bound enzyme can be lower or higher than that of purified enzyme. In

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this study the value of Km is obtained for the ChEt-ChOx/SiO2-CHIT/CNTs/ITO bioelectrode as 3.4 mg/dL (0.052 mM) that is smaller than the reported value [49]. The lower Km value indicates high affinity for cholesterol oleate attributed to the immobilization of ChOx and ChEt onto SiO2-CHIT/CNTs/ITO bio- nanocomposite for faster biochemical reaction. The value of sensitivity of ChEt-ChOx/SiO2-CHIT/CNTs/ITO bioelectrode estimated from the slope of curve has been found to be 3.8 A/mM. The values of standard deviation and correlation coefficient obtained from the linear regression analysis for the bioelectrode have been found to be 1.23 A and 0.994, respectively. Table 1 compares the characteristics of CNTs and sol-gel silica-based biosensors as reported in the literature. Hence, the CHIT-SiO2CNTs bio-nanocomposite matrices provided a longer shelf life, higher selectivity, and shorter response time to immobilized enzyme. Table 1. Comparison of the characteristics of sol-gel CHIT-SiO2-CNTs bio- nanocomposites based biosensors CNTs Bionanocomposite electrode ChOx/CHITSiO2-CNTs/ITO

ChOx/CHITSiO2-CNTs/ITO ChOx/CHITSiO2-CNTs/ITO ChOx/CHITSiO2-CNTs/ITO Prussian blueglass carbon/chitosan/ silica/MWCNTs

Sensing element

Immobilization technique

Linearity with [enzyme]

Response time

Shelf-life

Sensitivity

Reference

Cholesterol esterase and Cholesterol oxidase Urease

Covalent linkage

10-500 mg/dL

10s

2 months

3.8 μA/mM

[41]

Creatine amidinohy drolase Cholesterol oxidase Cholesterol oxidase

Covalent linkage Covalent linkage Covalent linkage Covalent linkage

8 months

[45]

-

-

8 months

-

[50]

50- 650 mg/dL 4 x 10-3 0.7 mM

5s

6 months

[51]

13 s

450 °C), the materials are purely inorganic in nature. Biopolymers/silica composites represent a new category of environmentally safe materials for applications to explore [8]. The use of synthetic polymers [9-10] is related to environmental problems and materials produced using synthetic polymers are not biodegradable. Production costs for such materials are high as the polymer synthesis itself requires expensive organic solvents and petrochemicals. There has been recent interest in using polysaccharides/modified polysaccharides as templates in the nanocomposite synthesis [8,11] because they are economical, abundantly available and biodegradable materials [12] with different structures and characteristics. To make an optimal choice, the composites differing in polysaccharide composition are desired to be compared. Advancements in the field of material research, specifically porous materials [13] have facilitated their use in variety of applications such as catalysts [14-16], adsorbents [17], sensors[18], optical devices[19-20], drug carriers[21-22] and biomedical materials [23]. Variety of silica precursors have been used [8] for the composite synthesis such as tetraethylorthosilicate (TEOS), tetramethylorthosilicate (TMOS), tetrakis(2 hydroxyethyl)orthosilicate (THEOS), ethoxtrimethylsilane (ETMS), vinyltriethoxysilane (VTES), methyltrimethoxysilane (MTOS) and -glycidoxypropyltrimethoxysiloxane (GPTMS) and depending upon the precursor/template type and conditions used, materials of different characteristics and performances have been synthesized. The following sections describe and discuss different polysaccharide silica nanocomposites that have been synthesized and exploited for variety of applications.The discussion has been categorized polysaccharide wise for an easy understanding of the subject.

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(I) Acacia Gum Acacia gum is a complex arabinogalactan polysaccharide [24-25], an exudate from acacia trees which are widely available in tropical and sub-desert regions of India. It is light weight, biodegradable, biocompatible material with good flexibility, excellent ionic mobility and electrical conductivity [26,27] but poor mechanical and thermal properties. Acacia gum–silica nanocomposites of have been synthesized [28] by pouring silica sol produced by tetraethylorthosilicate (TEOS) in acacia gum dispersions. Combination of biological, chemical and physical properties of acacia gum and mechanical and thermal properties of silica resulted biomaterials suitable for technological applications [29-31]. The composition of silica to biopolymer was tailored in order to get high electrical conductivity [32]. The nanocomposite gels were dried at different temperatures to form aerogels. With the increase in the silica content, the electrical conductivity of the hybrid increased because of the silica crystallization in the biopolymer medium and also due to the anionic nature of native material [27]. The composite synthesized using 1:2.5 gum to silica ratio had maximum electrical conductivity and was comparable to synthetic conducting polymers. The electrical conductivity of the hybrids decreased on heat treatment [33] due to the creation of the voids at layer surface and pore enlargement in the bulk that creates large band gaps restricting the mobility of electrons and ions in continuous material (Figure 1) AFM topographic analysis (Figure 2) of Acacia gum-silica nanocomposite showed the development of mesopores (43-61 nm) in the continuous phase of the composite on heat treatment and thus the material is useful for biosensor applications [30]. The encapsulation of silica network in acacia gum resulted inter-hydrogen bonding in the nanocomposite that increased the polarity.

Figure 1. Effect of thermal curing on electrical conductivity of the nanocomposite material [1:2.5; Acacia gum–silica ratio] (Ref. Tiwari et al Mater. Lett. 2007, 61, 4587–459).

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Figure 2. AFM picture of Acacia gum-silica nanocomposite treated at 120 °C ((Ref. Tiwari et al Mater. Lett. 2007, 61, 4587–459).

(II) Chitosan and Chitin Among the polysaccharides, chitosan is a copolymer of linked β(1→4), 2-amino-2deoxy-D-glucan and 2-acetamidodeoxy-D-glucan. It is mainly used in medicine and food industries [34]. The presence of hydroxyl and amino groups, which are excellent functional groups for the anchoring of a large variety of organometallic complexes, makes chitosan a good candidate as a precursor for heterogeneous molecular catalysts [35-36], while Chitin (poly-N-acetyl glucosamine) have unique multifunctional engineering mechanical properties and biocompatibility [37-40]. Chitosan/silica hybrids have been synthesized and shaped as microspheres [41], which depending upon the experimental conditions developed a core/shell structure having homogeneous hybrid with shell of pure silica (Figure 3). The amino groups of the biopolymer still remain accessible as active sites for heterogeneous catalysis. The materials have been prepared by mixing chitosan in acetic acid with TEOS [41] and the resulting gel was dried under supercritical conditions followed by calcination (Figure 4). Blank corresponding to the pure silica and a hybrid with sodium dodecyl sulfate were also prepared under similar conditions. Nanoparticles were obtained in the case of silica alone while hybrid showed long fibers. It was observed that chitosan organized the silica and that the sol-gel transcription was efficient. The nitrogen adsorption/desorption isotherms of the samples indicated that the materials were mesoporous. Although surface areas were very high, two phases were observed, one was constituted of pure silica, and the other was a hybrid containing 30 wt % chitosan. Unfortunately, the results suffered from a lack of reproductively in the composition of the hybrid which could not be improved by modifying experimental parameters.

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Figure 3. SEM picture of hybrid bead synthesized using four grams of chitosan microspheres (in ethanolic media) in an excess of a mixture of 75% of tetraethoxysilane (TEOS) (6 cm3) and 25% of water (2 cm3) (Ref. Karine et al, Chem. Mater. 2004, 16, 3367-337).

Figure 4. SEM images of chitosan/silica hybrid (a) before calcination (b) after calcinations (Ref. Karine et al, Chem. Mater. 2004, 16, 3367-3372).

Organosilica-chitosan crosslinked nanospheres [42] have been also developed through self-assembly of amphiphilic copolymers synthesized by concurrent grafting polymerization and sol–gel reaction. The core ‘organo-silica’ was formed by hydrolysis and condensation of an alkyloxosilane-3-(trimethoxysilyl) propyl methacrylate (TMSPM) that was simultaneously polymerized using chitosan/tert-butyl hydroperoxide as redox-pair initiator. The well-defined nanospheres had adjustable sizes below 100 nm, and had not harmful residues impeding medical applications. This synthesis method simplified the preparation of silica-polymer spheres by eliminating the previous core-forming step.

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Hybrid nanocompositional chitosan/silica sorbent [43] has been prepared by hydrolytic polycondensation of tetraethoxysilane (TEOS) using chitosan as template. The resulting hybrid chitosan/silica sorbent was tested for high-performance liquid chromatography. Suitable for tissue engineering 3D hybrid scaffolds based on silica and chitosan in various compositions are reported [44]. Various unique shapes of silica/chitosan hybrid particles [45] were obtained by biomimetic synthesis of silica with chitosan where the chitosan solution was first incubated for different periods of time, leading to different aggregation states and then some amount of prehydrolyzed TEOS was added and reacted. A novel sol-gel nanocomposite material [46] has been produced by silica sol-gel process in micro emulsion organogels prepared by a percolating droplet network containing chitosan. The structural features of the obtained chitosan-silica composites have been described and compared with silica-gelatin nanocomposites. With sucrose and polyethylene glycol 4000 (PEG 4000) being synergic imprinting molecules, covalent surface coating on silica gel was achieved by using polysaccharide incorporated sol–gel process starting from chitosan and an inorganic epoxy-precursor eg. glycidoxypropyltrimethoxysiloxane at room temperature [47]. The dosage of epoxy-siloxane agent was a key parameter to control both the availability and the accessibility of adsorption sites. The sample having 1:2 epoxy group to NH2 (siloxane/glucosamine) ration was evaluated to be most efficient sorbent in wastewater treatment. The dynamic adsorption in column underwent a good elimination of Cu2+ in treating electric plating wastewater (Figure 5.). The prepared composite sorbent exhibited high surface area, reusability, easy preparation in absence of organic solvents, cost-effectiveness and high stability. The dynamic adsorption behavior of the prepared sorbent in treating electric plating wastewater was studied in column where the concentration of Cu2+ was reduced to less than 1mg L−1.

Figure 5. In-column dynamic adsorption of Cu2+ using prepared porous sorbent for treating electric plating wastewater.(Ref. Li et al, F. Analytica Chimica Acta 2007, 585, 211–218)

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Sol–gel/organic hybrid composite material [48] film was fabricated using TMOS (as silica precursor) and chitosan to immobilize horseradish peroxidase on a carbon paste electrode for the fabrication of an amperometric H2O2 biosensor. The fabrication procedure was systematically optimized to improve the biosensor performance. The biosensor had a fast response of less than 10 s. The biosensor retained ca. 85% of its original activity after 30 days of storage in a phosphate buffer at 4 ºC. Chitosan/silica hybrid composite film from chitosan and MTOS on the surface of Prussian blue modified glass carbon electrode [49] gave an improved amperometric glucose biosensor based on glucose oxidaze for determining the glucose concentration in real human blood samples. Sol–gel/organic hybrid composite material of chitosan and tetramethoxysilane has also been developed for the bovine serum albumin (BSA)-encapsulated monolithic column for capillary electrochromatography [50]. The composite monolith was used to immobilize BSA in a fused-silica capillary. The addition of gelatin and chitosan to the alkoxysilane enabled the enantioseparation of Tryptophan (Trp). The monolithic column prepared from chitosan with tetramethoxysilane showed a high enantioselectivity for Trp enantiomers. Furthermore, the composite materials exhibited a higher stability compared to the silica sol–gel column. A series of chitosan-silica hybrids have been synthesized using different weight ration of TEOS/VTES and chitosan through a sol–gel process and the relevant physical and chemical properties have been tested. Thermal studies indicated the thermostability of the hybrids increase with increase in the amount of VTES and TEOS as reticular inorganic SiO2 is formed. The hybrid material that was made of TEOS was higher in thermostability than that of VTES because for SiO2 to take shape is hard in the poorly soluble VTES. The tensile strength and elongation of the hybrid materials were studied which were found superior than those of pure chitosan. Table 1 Thermal and mechanical properties of chitosan and hybrid materials (Ref. Kato, et al, Journal of Chromatography A, 2004, 1044, 267–270) Weight of Thermal properties Mechanical properties VTES/TEOS (g)

(Td)a (ºC)

(Tm)b (ºC)

Char yield (%)

Tensile strength

Elon-

gation (MPa)

245 303 34.1 32.03 0/0c 0.0.8 249 306 37.2 33.19 0.8/0.8 253 308 40.2 36.65 0.8/1.6 257 310 43.8 38.41 0.8/2.4 260 313 45.6 31.16 263 315 47.1 30.25 0.8/3.2 247 304 36.3 33.23 1.2/0 a b Temperature at 10% weight loss, Temperature at maximum weight loss, cChitosan

(%)

19.3 20.1 21.7 22.7 18.5 17.7 19.8

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Silica formation due to the sol–gel processing using THEOS [52] has been preferred over TMOS and TEOS, respectively due to its complete compatibility with biopolymers [53-55] and it can be used at conditions at which others do not work. THEOS is useful where the phase state of a system is strongly dependent on the pH and temperature. Use of THEOS enables the gelling of chitosan at a concentration beginning from a few wt % though otherwise chitosan is nongelable. The hybrid gelled at cirumneutral pH and the polysaccharide was shown to catalyze the process. Phase separation or precipitation was not observed after their mixing and the gelled solutions were homogeneous. The hydrogels remained stable and no phase separation or change in the optical properties of the hydrogels was observed with time. Chitosan-silica hybrids [18] have also been synthesized for the fabrication of an amperometric hydrogen peroxide biosensor. The synthesis was done in absence of any catalyst or organic solvents taking THEOS as silica precursor. The gelation time for the solgel transition and dynamic rheological properties of the resultant gel matrix was modulated by the amount of added THEOS. The structure of the hybrid gel was made up of a network and spherical particles, as confirmed by SEM observation. By electrochemical experiments, it was found that such a hybrid gel matrix could retain the native biocatalytic activity of the entrapped horseradish peroxidase and provide a fast amperometric response to hydrogen peroxide. Under the optimized experimental conditions, the fabricated biosensor was found to have good analytical performance, reproducibility, and storage stability. Again the catalytic effect of chitosan on the sol-gel process of THEOS was established. A sensitivity-enhanced glucose biosensor [56] was fabricated using multi-walled carbon nanotubes (CNT), Pt nanoparticles and sol-gel derived chitosan (CS)/silica hybrid. New organic-inorganic hybrids SiGCX (X = 1 to 3) were prepared from the biopolymer chitosan with a degree of the deacetylation of 86% and three distinct silylating agents of the type (CH3O)3Si-R-NH2 [R = -(CH2)3-, -(CH2)3NH(CH2)2- and -(CH2)3NH(CH2)2NH(CH2)2-] [57]. Both chitosan and silylating agents had the amine groups crosslinking through linear glutaraldehyde units. Carbamated chitosan [58] and butyrylchitosan [59] have also been used for preparing silica hybrid films for immobilizing horseradish peroxidase on a carbon paste electrode. Cuttlebone β-chitin [60] acts as a highly organized organic template with macroscopic porosity for the preparation of silica-polysaccharide composites having 3-D interconnected box structures.

(III) Carageenan Hybrid nanocompsites containing TEOS/ ETMS and k-carrageenan were synthesized via sol–gel method at room temperature which were useful as biocatalyst in enzyme immobilization [61]. FTIR studies revealed that in the synthesized hybrid nanocomposites, strong chemical covalent bonds, as well as weak forces—Hydrogen bonding, Vander Waals or electrostatic interactions existed. Nanostructure with well-defined nanounits and their aggregates was evidenced by AFM studies. The size of nanoparticles varied from 6 to 12 nm and the dimensions of their aggregates were about 25–86 nm. The application of the synthesized silica hybrids as carriers for obtaining active biocatalysts have been studied and

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their enzyme activity towards different aliphatic and carboxylic nitriles was established (Figure 6).

Figure 6. Nitrilase activity as a function of k-carrageenan concentration (Ref Samuneva et al, J. Sol-Gel Sci. Technol. 2008, 48, 73-79)

As in the case of chitosan, THEOS has been used to synthesize monolithic hybrid biomaterials with three main types of carrageenans, -, -, and - carrageenans. It was found that they promoted the mineralization by acting as a template for the inorganic component. The material properties were regulated by both the precursor and carrageenans. The increase in silicate concentration led to a rise in the stiffness and brittleness of the material, whereas the polysaccharide addition made it softer and more elastic. It was shown that the formation and properties of mixed gels were determined by the nature of carrageenan. -Carrageenans brought about shrinkage of hybrid materials that led to water separation, while - and carrageenans did not induce the syneresis. Furthermore, - and -carrageenans experienced a thermoreversible phase transition in the hybrid materials owing to the helix–coil transition. This resulted in a step like change in the mechanical properties of mixed systems in the corresponding temperature range while -Carrageenan hybrid gel remained unchanged with the temperature. It was found that the polysaccharides modified the structure of silica-based materials. CO2 supercritical drying allowed to prepare mesoporous carrageenan aerogels and represents a significant improvement upon classical drying methods. However, a significant shrinking takes place during the drying process and the macroporosity of the gel is lost. Silica-pillaring provides an effective method to preserve the texture of a carrageenan hydrogel upon supercritical drying. Particles of amorphous silica, a reasonably inert phase, are retained

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in the net of carrageenan fibrils and prevent the collapse of the gel when the solvent is removed [62]. Composite aerogels in which the carrageenan network is stabilized by silica nanoparticles present several potential advantages for drug-delivery systems e.g control of the bead size through the synthesis process, ability of carrageenan to complex cationic drugs and improved diffusion properties due to the stabilization of the gel volume. Biosilica-coated carrageenan microspheres for yeast alcohol dehydrogenase encapsulation are also reported [63].

(v) Alginate Silica/alginate biocomposites [64] haves been synthesized by gelation of the trapped alginate on addition of a divalent metal cation using TMOS as silica precursor. The formation of silica-based biocomposites relied on the interaction between the mineral and the organic components. At pH 7, alginic acid (pKa≈5) exhibits negatively charged carboxylate and neutral hydroxyl groups whereas silica precursors are neutral or negatively charged. Therefore, only weak hydrogen bonds may arise between the two components of the materials. This is illustrated by the fact that alginate gels can be almost completely removed from the composite. Thermogravimetric analysis, SEM and nitrogen adsorption-desorption experiments indicated that the polymer is located in the macroporosity of the biocomposites so that it can be partially removed by citrate treatment. Even though only weak hydrogen bonds are expected, the morphological features of the composites revealed to depend on the biopolymer content of the initial solution. Three dehydrogenases: formate dehydrogenase, formaldehyde dehydrogenase, and alcohol dehydrogenase were encapsulated in an alginate-silica (ALG-SiO2) hybrid gel [65] for converting carbon dioxide to methanol. The hybrid was prepared through in situ growth of the silica precursor within an alginate solution, which was followed by Ca2+ cross-linking. The significantly improved catalytic properties of the dehydrogenases in the ALG-SiO2 composite were attributed to the creation of the appropriate immobilizing microenvironment: high hydrophilicity, moderate rigidity and flexibility, ideal diffusion characteristics, and optimized cage confinement effect.

(IV) Cellulose Cellulose is the most naturally abundant renewable polymer. It possesses several unique properties required in different technical areas and biomedicine [67]. Generally, cellulose does not easily form bio-composite materials, as it is almost insoluble in most of the solvents and even also does not melt at elevated temperatures. Silica derivatives of cellulose are of special interest since these substantially improve the thermal stability of the parent polymer matrix and improve its lipophilic behaviour and the affinity towards specific substrates [66,67]. Mimicking bio-mineralization, silica hybrids of cellulose/modified cellulose have been synthesized via sol-gel route by taking different silica precursors eg TEOS, TMOS, THEOS, silicic acid, etc [68-70].

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For the hybrid synthesis, cellulose requires preactivation for which air-dried pulp of cellulose is disintegrated in water and then washed with ethanol to remove the excess of water. To this preactivated pulp, a mixture with predetermined proportion of TEOS, distilled water, ethanol and catalyst are added and constant suspension stirring result the hybrid in 24 h at room temperature (~20°C). Different mineral acids and several heteropoly acids (HPAs) have been used as catalyst [71] in the preparation of the hybrids. HPAs showed a higher catalytic efficiency than that of conventional mineral acids (HCl, HNO3, H3PO4, H2SO4). Tungstophosphoric acid (H3PW12O40) was the best catalyst among HPAs used. The effect of sol–gel synthesis conditions on the yield of hybrids and the structure of the silica counterpart was established. Hydrophobic character of cellulose enhanced on hybrid formation. FTIR spectra was (Figure 7A) used as a evidence for silica incorporation but the band corresponding to the Si– O cellulose vibration confirming the covalent bonding between silica and cellulose (1000– 1150 cm-1) has not been clearly detected.

Figure 7. (A) FTIR spectra of bleached pulp and hybrid material series (BP/silica ); (B) 29Si MAS NMR spectra of hybrid series (a) and (b) obtained with 3 x 10-4 mol/l concentration of PW12 and hybrid series (c) obtained with 6 x 10-4 mol/l concentration of PW12. (Ref. Sequeira, et al, Mater. Sci. Eng. C, 2007, 27, 172-179).

The efforts in the detection of cellulose linkage with siloxy moiety using differential 13C CP-MAS NMR spectra of initial pulp and hybrids have also failed. These facts, however, do not signify that these linkages do completely not exist. Probably, the abundance of cellulose-

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silica covalent bonds is very low (80% highly porous. This biocomposite scaffold exhibits directing the growth of human fetal osteoblasts. The unique nanoscale biocomposite system with inherent surface functionalization for human fetal osteoblasts adhesion (hFOB), migration, proliferation and exhibited higher proliferation compared to PCL and PCL/HA , while the mineralization even higher than collagen [57]. The PLLA/ collagen/HA shows fiber diameter 310 ±125 nm which is much smaller than pure PLLA 860 ±110 nm and PLLA/HA 845 ±140 nm as shown in the Figure 4. In addition, the tensile strength of PLLA/ Collagen/HA fibrous scaffold reduced to 2.05 ±0.10 MPa compared to PLLA scaffold 4.69 ±0.19 MPa and PLLA/HA scaffold 3.10±0.15 MPa. However, the synergetic effect of the presence of collagen and HA in PLLA/ collagen/HA/provided cell recognition sites together with apatite for cell proliferation and osteoconduction necessary for mineralization and bone formation [58].

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Figure4. SEM images of electrospun (a) PLLA, (b) PLLA/HA, and (c) PLLA/collagen/ HA nanofibers [58].

The growth factor such as the bone morphogenetic protein (BMP) can also be incorporated into the composite to help the bone formation. The bone morphogenetic protein 2(BMP-2) silk fibroin/HA fiber scaffolds were prepared via electrospining the aqueous based suspension for in vitro bone formation. This scaffold with BMP-2 and nano-HA resulted in the highest calcium deposition and enhanced transcript levels of bone-specific markers [59].

Sintered Microspheres/Nanoparticles Nanocomposites Recently selective laser sintering (SLS) technique has been taken to prepare porous tissue engineering scaffolds based on the synthetic polymer/HA nanocomposite microspheres [6063]. This kind of scaffold mimics the composition, hierarchy, and interconnected porosity of bone and the poor crystalline of calcium phosphate in native bone. SLS for tissue engineering scaffold fabrication is advantageous because it can process a wide range of biocompatible and biodegradable materials. SLS builds up 3D objects layer-by-layer of thin 2D layers (~0.1 mm thick) to the required size, shape and internal structure by laser-induced fusion of small particles [62]. For example, PLLA/carbonated HA nanocomposite microspheres with a size

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range of 5-30 um were prepared by emulsion techniques. The optimized porous scaffolds were fabricated from PLLA/HA nanocomposite microspheres by SLS via adjusting the laser power, scan spacing and part bed temperature as shown in the Figure 5.

Figure 5. An isometric view of a PLLA/HA nanocomposite scaffold [62].

INORGANIC/INORGANIC NANOCOMPOSITES High porosity, interconnected pore structure generally favors tissue regeneration. However, because of their natural brittleness, ceramics such as HA and TCP, in a porous form, have very low strength and toughness. Some studies have shown that biphasic calcium phosphate scaffolds can lead to fast bone formation for bone reconstruction. The biphasic calcium phosphate plants have been shown to be surrounded by new bone tissue within a few weeks after implantation at bony sites [7, 64]. Recently a novel nanocomposite using nanofiber HA as a filler phase in -TCP porous scaffolds were fabricated by Ramay and Zhang and proved that second phase particulates in porous ceramic scaffolds could similarly enhance mechanical properties [65]. HA nanofibers were prepared with a biomimetic precipitation method. The composite scaffolds were fabricated by a method combining the gel casting and polymer sponge techniques. The mechanical property of the scaffold was significantly enhanced by the inclusion of HA nanofibers. The porous composite scaffold attained a compressive strength of 9.8±0.3 MPa, comparable to the high-end value (2–10

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MPa) of cancellous bone. The toughness of the scaffold increased from 1.00±0.04 to 1.72±0.02 kN/m, as the concentration of HA nanofibers increased from 0 to 5 wt %. Adiposederived stromal cells (ASCs) harvested from Sprague-Dawley (SD) rats were induced to osteogenesis before seeded into porous HA/TCP scaffold. These constructs were implanted in nude mice subcutaneously exhibited obvious ectopic bone formation. HA/TCP scaffold containing osteogenic ASCs filled in rat critical-size cranial defects showed complete repair. These results imply that combining osteogenic ASCs with HA/TCP stimulate bone regeneration and repair for the large size bone defects [66].

CONCLUSION The biocompatible nanocomposites consisting of a natural or synthetic polymer reinforced with an inorganic phase are increasingly preferred for bone tissue engineering because they more closely mimic the natural composite structure of bone. The nanocomposites scaffold can be made of different forms including sponge-like, fibrous, sintered microspheres depending on the manufacturing methods. In the future, more complicated nanocomposite scaffold with biological elements to stimulate cell proliferation and differentiation and eventually osteogenesis will be conducted for the bone repair in order to achieve the restoration of native tissue architecture.

ACKNOWLEDGMENT This work is financially supported by National High-Tech Project of China (863-Project, 2007AA021902) and the Science and Technology Research Project of Shandong Provincial Education Department (Grant No. J08LC54).

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[38] Katti KS, Katti DR, Dash R. Synthesis and characterization of a novel chitosan/montmorillonite/hydroxyapatite nanocomposite for bone tissue engineering. Biomedical Materials. 2008;3. [39] Kim HW, Kim HE, Salih V. Stimulation of osteoblast responses to biomimetic nanocomposites of gelatin-hydroxyapatite for tissue engineering scaffolds. Biomaterials. 2005;26:5221-30. [40] Kim HW, Song JH, Kim HE. Nanoriber generation of gelatin-hydroxyapatite biomimetics for guided tissue regeneration. Advanced Functional Materials. 2005;15:1988-94. [41] Wang L, Nemoto R, Senna M. Effects of alkali pretreatment of silk fibroin on microstructure and properties of hydroxyapatite-silk fibroin nanocomposite. Journal of Materials Science-Materials in Medicine. 2004;15:261-5. [42] Wang L, Nemoto R, Senna M. Microstructure and chemical states of hydroxyapatite/silk fibroin nanocomposites synthesized via a wet-mechanochemical route. Journal of Nanoparticle Research. 2002;4:535-40. [43] Costa HS, Stancioli EFB, Pereira MM, Orefice RL, Mansur HS. Synthesis, neutralization and blocking procedures of organic/inorganic hybrid scaffolds for bone tissue engineering applications. Journal of Materials Science-Materials in Medicine. 2009;20:529-35. [44] Wang SF, Kempen DHR, Yaszemski MJ, Lu LC. The roles of matrix polymer crystallinity and hydroxyapatite nanoparticles in modulating material properties of photo-crosslinked composites and bone marrow stromal cell responses. Biomaterials. 2009;30:3359-70. [45] Nejati E, Firouzdor V, Eslaminejad MB, Bagheri F. Needle-like nano hydroxyapatite/poly(L-lactide acid) composite scaffold for bone tissue engineering application. Materials Science and Engineering C-Biomimetic and Supramolecular Systems. 2009;29:942-9. [46] Douglas T, Pamula E, Hauk D, Wiltfang J, Sivananthan S, Sherry E, et al. Porous polymer/hydroxyapatite scaffolds: characterization and biocompatibility investigations. Journal of Materials Science-Materials in Medicine. 2009;20:1909-15. [47] Fu SZ, Guo G, Wang XL, Zhou LX, Liu TT, Dong PW, et al. Preparation and Characterization of n-Hydroxyapatite/PCL-Pluronic-PCL Nanocomposites for Tissue Engineering. Journal of Nanoscience and Nanotechnology. 2010;10:711-8. [48] Fu SZ, Gun G, Gong CY, Zeng S, Liang H, Luo F, et al. Injectable Biodegradable Thermosensitive Hydrogel Composite for Orthopedic Tissue Engineering. 1. Preparation and Characterization of Nanohydroxyapatite/Poly(ethylene glycol)Poly(epsilon-caprolactone)-Poly(ethylene glycol) Hydrogel Nanocomposites. Journal of Physical Chemistry B. 2009;113:16518-25. [49] Jose MV, Thomas V, Johnson KT, Dean DR, Nyalro E. Aligned PLGA/HA nanofibrous nanocomposite scaffolds for bone tissue engineering. Acta Biomaterialia. 2009;5:305-15. [50] Thomas V, Dean DR, Jose MV, Mathew B, Chowdhury S, Vohra YK. Nanostructured biocomposite scaffolds based on collagen coelectrospun with nanohydroxyapatite. Biomacromolecules. 2007;8:631-7.

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[51] Thomas V, Jagani S, Johnson K, Jose MV, Dean DR, Vohra YK, et al. Electrospun bioactive nanocomposite scaffolds of polycaprolactone and nanohydroxyapatite for bone tissue engineering. Journal of Nanoscience and Nanotechnology. 2006;6:487-93. [52] McCullen SD, Zhu Y, Bernacki SH, Narayan RJ, Pourdeyhimi B, Gorga RE, et al. Electrospun composite poly(L-lactic acid)/tricalcium phosphate scaffolds induce proliferation and osteogenic differentiation of human adipose-derived stem cells. Biomedical Materials. 2009;4. [53] Zhang Y, Venugopal JR, El-Turki A, Ramakrishna S, Su B, Lim CT. Electrospun biomimetic nanocomposite nanofibers of hydroxyapatite/chitosan for bone tissue engineering. Biomaterials. 2008;29:4314-22. [54] Gupta D, Venugopal J, Mitra S, Dev VRG, Ramakrishna S. Nanostructured biocomposite substrates by electrospinning and electrospraying for the mineralization of osteoblasts. Biomaterials. 2009;30:2085-94. [55] Ngiam M, Liao SS, Patil AJ, Cheng ZY, Chan CK, Ramakrishna S. The fabrication of nano-hydroxyapatite on PLGA and PLGA/collagen nanofibrous composite scaffolds and their effects in osteoblastic behavior for bone tissue engineering. Bone. 2009;45:416. [56] Venugopal J, Vadgama P, Kumar T, Ramakrishna S. Biocomposite nanofibres and osteoblasts for bone tissue engineering. NANOTECHNOLOGY. 2007;18:-. [57] Prabhakaran MP, Venugopal J, Ramakrishna S. Electrospun nanostructured scaffolds for bone tissue engineering. Acta Biomaterialia. 2009;5:2884-93. [58] Li CM, Vepari C, Jin HJ, Kim HJ, Kaplan DL. Electrospun silk-BMP-2 scaffolds for bone tissue engineering. Biomaterials. 2006;27:3115-24. [59] Khan YM, Katti DS, Laurencin CT. Novel polymer-synthesized ceramic compositebased system for bone repair: An in vitro evaluation. Journal of Biomedical Materials Research Part A. 2004;69A:728-37. [60] Duan B, Wang M, Zhou WY, Cheung WL. Synthesis of Ca-P nanoparticles and fabrication of Ca-P/PHBV nanocomposite microspheres for bone tissue engineering applications. Applied Surface Science. 2008;255:529-33. [61] Zhou WY, Lee SH, Wang M, Cheung WL, Ip WY. Selective laser sintering of porous tissue engineering scaffolds from poly(L)/carbonated hydroxyapatite nanocomposite microspheres. Journal of Materials Science-Materials in Medicine. 2008;19:2535-40. [62] Chua CK, Leong KF, Tan KH, Wiria FE, Cheah CM. Development of tissue scaffolds using selective laser sintering of polyvinyl alcohol/hydroxyapatite biocomposite for craniofacial and joint defects. Journal of Materials Science-Materials in Medicine. 2004;15:1113-21. [63] Nery EB, Legeros RZ, Lynch KL, Lee K. TISSUE-RESPONSE TO BIPHASIC CALCIUM-PHOSPHATE CERAMIC WITH DIFFERENT RATIOS OF HA/BETATCP IN PERIODONTAL OSSEOUS DEFECTS. Journal of Periodontology. 1992;63:729-35. [64] Ramay HRR, Zhang M. Biphasic calcium phosphate nanocomposite porous scaffolds for load-bearing bone tissue engineering. Biomaterials. 2004;25:5171-80. [65] Lin YF, Wang T, Wu L, Jing W, Chen XZ, Li ZY, et al. Ectopic and in situ bone formation of adipose tissue-derived stromal cells in biphasic calcium phosphate nanocomposite. Journal of Biomedical Materials Research Part A. 2007;81A:900-10.

In: Recent Developments in Bio-Nanocomposites… ISBN 978-1-61761-008-0 Editor: Ashutosh Tiwari © 2011 Nova Science Publishers, Inc.

Chapter 15

COMPOSITES OF CHITOSAN FOR BIOMEDICAL APPLICATIONS Nazma Inamdar and V.K.Mourya1 Department of Pharmaceutics, Government College of Pharmacy, Vedant Road, Aurangabad (M.S.) 431005, India

ABSTRACT The nanocomposites are multiphase materials or hybrids in which at least one of the components has dimension in the nanometer scale. These materials exhibit extraordinary mechanical and physical properties than the cocstituent phases. The composites can be tailored to meet the requirements of a particular application with manipulations of these properties. For biomedical applications the polymer composites are of value. Chitoasn is a much appreciated polymer in this respect because of its biocompatbility, biodegrability, nontoxicity and cationic nature. The composites of chitosan can be elaborated with inorganic materials as well as with polyionic materials with polyelectrolyte complex formation. The comopites with calcium phosphate based materials, clays are evaluated for tissue engeneering applications along with their drug eluting properties. The composites of chitosan with metal oxide nanoparticles, metal nanoparticles, carbon nanotubes are maneuvered as biosensors. Similar operations can be developed with chitosan/quantum dots composites in addition to the bioimaging functions. The plethora of biomedical applications is put forword by chitosan/polyion complexes. The future potential of chitosan composites in biomedical applications is very promising for therapeutics and diagnosis. The awareness and the emergence of the analytical protocols for quality assessment of chitosan will see to these applications without delay.

INTRODUCTION The composites are multifunctional materials having unprecedented mechanical and physical properties that can be tailored to meet the requirements of a particular application. 1

Email: [email protected].

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However there is no universally accepted definition of a composite material to encompass the versatility of its meaning. We can consider a composite to be a material consisting of two or more distinct constituent phases bonded together. The constituent phases are considered as matrix and fillers. Based on matrix, composites are classified into four primary categories as polymer matrix composites, metal matrix composites, ceramic matrix composites, and carbon/carbon composites. Currently, polymer matrix composites are the most widely used class. The reinforcement of composites occurs due to presence of fillers which might be in various geometries as aligned continuous fibers, discontinuous fibers, whiskers (elongated single crystals or nanoaparticles also termed as nanocrystals, monocrystals, microfibrils, microcrystals, or microcrystallites, despite their nanoscale dimensions), particles (flakes, sphere, others) and numerous forms of fibrous architectures produced by textile technology, such as fabrics and braids. Conceptually the nanocomposites are multiphase materials or hybrids in which at least one of the components has at least one dimension in the nanometer scale (below 100 nm). Depending on how many dimensions are in the nanometric range, distinction of phase/composite can be made as: (i) Isodimensional nanoparticles which have three nanometric dimensions. (ii) Nanotubes or whiskers which are elongated structures with two dimensions in the nanometer scale and third dimension in larger scale. (iii) Polymer-layered crystal nanocomposites when only one dimension is in the nanometer range and almost exclusively obtained by the intercalation of the polymer (or a monomer subsequently polymerized) inside the galleries of layered host crystals [1]. The presence of nanoscale components imparts nanocomposites intrinsically new properties and characteristics that are not present in conventional composites or the pure components. The structure-property relationship of nanocomposites is very much influenced by these geometries and dimensions. It has been known for a long time that various biological composites exist in nature. These are made from organic matrix and inorganic fraction and fulfill the required structural and functional properties in their role as the skeleton, teeth or shells of organisms [2]. These are hierarchically structured composites in which soft organic materials are organized on length scales of 1–100 μm used as frameworks for specifically oriented and shaped hydroxyapatite, CaCO3, SiO2, and Fe3O4 crystals [3]. With the clue from nature the scientific fraternity has always been in search of the composites to be exploited in biomedical field. If the composites are biocompatible and biodegradable their utility increases many fold. The biopolymers chitosan and its precursor chitin may provide an answer to this search. Although chitosan composites are relatively new and have not been commercialized yet, a number of scholarly research publications have intensively discussed them from the point of view of fundamental property determination and the development of new materials to meet a variety of applications. The several aspects of biomedical applications of chitosan composites include tissue engineering, drug delivery, gene delivery, medical imaging and biosensors for clinical and environmental diagnostics.

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CHITOSAN AS A POLYMER Chitin exists as a natural composite with proteins and minerals in invertebrates preserving the structural integrity as in the shells of arthropods [4, 5]. Contrary to chitin, chitosan is not widespread in the nature. It is industrially obtained by partial deacetylation of chitin. Its chemical structure, represented in Figure 1, is a random linear chaining of N-acetyl-Dglucosamine units (acetylated unit) and D-glucosamine (deacetylated unit) linked by β-(1-4)) linkages. The unique chemical feature of chitosan is its primary amine in the glucosamine residues. It imparts interesting properties to molecule as Lewis base activity (nucleophilicity due to amine), polyelectrolyte properties (due to protonated amines), and complexation ability. OH *

NH2 O

HO

O

O

O

HO

O NH

H3C O

m

OH

n

Figure 1. Repeat residues for chitin and chitosan. Chitin is composed predominantly of (m) units and chitosan is composed predominantly of (n) units distributed in random fashion.

The presence of groups as amine, acetamide, and hydroxyl functions in the molecules leads to hydrogen bonds electrostatic interactions controlling the stiffness and aggregation of chitosan molecule [6-8]. Chitosan is a large molecule with a typical molecular weight of 105– 106, a radius of gyration of 10–100 nm,[7,9-11] and a contour length approaching microns [12, 13]. In solution, chitosan behaves as a stiff worm-like chain with persistence lengths reported to be 5–15 nm [9-11, 14]. The aqueous solubility of chitosan at low pH (below 6) is due to protonation of primary amines conferring a positive charge and cationic polyelectrolyte properties to it [15]. Besides the properties of the solvent and the pH of a given medium, there are intrinsic factors such as the molecular weight, the degree of deacetylation, and the distribution of acetyl groups which strongly affect the conformation of the chitosan molecule [16, 17]. As pH increases, the amines are deprotonated and chitosan undergoes a transition from a soluble cationic polyelectrolyte to an insoluble polymer. At high pH, chitosan’s electrostatic repulsions are reduced allowing the formation of inter-polymer associations (e.g., liquid crystalline domains or network junctions) that can yield fibers, films, or hydrogels, depending on the conditions used to initiate the soluble-insoluble transition [18]. The pHresponsive switch lies near neutrality (chitosan’s apparent pKa has been reported to range between 6 and 7, particularly convenient range for biological applications). Boucard and coworkers proposed three conditions necessary for chitosan to undergo a sol–gel transition to form a homogeneous gel [19]: (i) the chitosan concentration must exceed C*, the critical concentration for chain overlap (C* ~ 0.1% [7, 20] (ii) there must be a critical balance between attractive and repulsive interactions; and (iii) the conditions that initiate gel formation must be uniformly percolating. If these conditions are not satisfied, then chitosan

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chains that undergo the soluble-to insoluble transition can precipitate as small aggregates without forming a 3-D elastic network.

Figure 2. Schematic illustration of chitosa’s versatility for composite elaboration. At low pH (less than about 6), chitosan’s amines are protonated conferring polycationic behavior to chitosan. At higher pH (above about 6.5), chitosan’s amines are deprontonated and reactive. Also at higher pH, chitosan can undergo interpolymer associations that can lead to fiber and network (i.e., film and gel) formation.

With these insights and framework it is possible to employ chitosan into self-organized form either alone or along with other components by various processes as complexation with polyanions, layer by layer assembly, casting, spinning, and electrodeposition.(Figure2) In addition microcontact printing methods have been used to stamp materials onto chitosan films or to stamp chitosan onto activated glass surfaces [21]. In the presence of other components chitosan serves as a length-scale interconnect for the hierarchical assembly of nano-scale components into macro-scale systems [22]. (Figure3)The primary amines (atomic length scale) of the glucosamine repeating units (molecular length scale) provide sites to self assemble or connect pre-formed nano-scale components to the polysaccharide backbone (macromolecular length scale). Connections to the backbone can be formed by exploiting the electrostatic, nucleophilic, or metal binding capabilities of the glucosamine residues. The applicability may be manipulated since the reactive groups as amines and hydroxyls provide means of covalent linkages and so to functionalize, graft, and crosslink the polymer.

Figure 3. Chitosan as a length-scale interconnect for hierarchical assembly. The figure shows that nanoscale assemblies of small molecules (e.g., a vesicle) or other nano-components (e.g., a protein or nanoparticle) can be connected to sites along a chitosan chain. These structures, in turn, can be connected into larger supramolecular assemblies and deposited at specific device addresses or formed as free standing films.

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The incorporation/association of other components into assembly of chitosan or of chitosan into other assemblies generates composites of potential applications. The accompanying constituents of these composites can be inorganic or organic. The inorganic constituents include as ceramics, calcium orthophosphate based cements, clays, metal oxides, metals nanoparticles, CaCO3, carbon nanotubes where as organic constituents include natural and synthetic materials as genetic materials, proteins (collagen, gelatin, silk fibroin, keratin etc), glycosaminoglycan (heparin, chondroitin sulphate, hyaluronan, dermatan sulphate etc) natural anionic polymers (alginate,pectin, carrageenan, chemically modified chitosan, glucomannan, xanthan etc) synthetic anionic polymers (poly(acrylic acid) (PAA) poly(methacrylic acid) (PMAA), polyethyleneoxide (PEO) etc.

CHITOSAN–INORGANIC MATERIAL COMPOSITES CALCIUM ORTHOPHOSPHATE BASED MATERIAL Tissue engineering applies methods from materials engineering and life sciences to create artificial constructs that restore, maintain, or improve tissue function [23]. In tissue engineering research, the challenging factors are the seeding cells, growth factors, cytokines, tissue construction and scaffold materials [24]. The scaffold materials have an essential function concerning cell infiltration, guided migration, anchorage, recruitment, proliferation, differentiation and tissue formation in three dimensions [25]. These functional aspects demand the materials which are biomimetic with respect to compatibility, degradability, mechanical strength and porosity. The scaffold features of macroporosity as larger pore size, greater interpore connectivity etc. are necessary for accelerated tissue regeneration and are application specific.

Bone Tissue Engineering The ceramics as based on calcium orthophosphate compounds are regarded as highpotential scaffolds due to their osteoconductive properties and are used for orthopedic and dental restorative applications [26]. To apply in this way, self-setting calcium orthophosphate cement uses calcium orthophosphate compounds such as monocalcium phosphate monohydrate (MCPM Ca(H2PO4)2·H2O), dicalcium phosphate dihydrate (DCPD, CaHPO4·2H2O, brushite), dicalcium phosphate anhydrous (DCPA, CaHPO4, monetite), betatricalcium phosphate (Β-TCP, Ca3(PO4)2, whitlockite), tetracalcium phosphate monoxide (TTCP, Ca4(PO4)2O, hilgenstockite), octacalcium phosphate (OCP, Ca8(HPO4)2(PO4)4·5H2O), pentacalcium hydroxylapatite (HA, Ca10(PO4)6(OH)2, hydroxyapatite), calcium-deficient hydroxyapatite (CDHA, Ca10-x(HPO4)x(PO4)6-x(OH)2-x (0< x < 1), biphasic calcium phosphate, (HA and β-TCP), carbonateapatite (Ca5(PO4,CO3)3X) etc. The cement might be supplemented with calcium carbonate (to improve cement mechanical properties and compliance), calcium oxide (as an accelerator) or zinc oxide (as coagulant) etc [27]. During setting of calcium phosphate cement (CPC), the formation of HA occurs in vivo at body temperature and pH without immune response. However they are washed-out due to their long setting time and to the penetration of body fluids before setting completely. Moreover

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these materials do not appear to be very attractive because of poor processability into highly porous structures, brittleness, slow degradability, low cell seeding ability due to limited supply of nutrition at the inside of the implant or less than optimal cell–cell interactions, and strong binding to growth factors. To circumvent these problems, useful alternatives are fast setting CPC (which sets much faster than conventional CPC materials) and the preparation of composite cements by the addition of polymeric viscous gels (which reduce the liquid penetration to the cement paste) into the liquid component of the cements [28, 29]. The combination of both approaches is being studied increasingly. Chitin and chitosan are interesting polymers in this aspect. The generated chitosan composites can be analyzed for the physical and chemical by various techniques as porosity measurement, mechanical strength, scanning electron microscopy, TEM, thermogravimetric analysis, X-ray diffraction, X-ray photoelectron spectroscopy, and Fourier transformed infrared spectroscopy etc. The biological activities studied included their in vitro and in vivo ability to sustain and facilitate cell growth, functioning as expression of alkaline phosphatase, biocompatability etc.

Process of Apatite Deposition The polymer matrix of chitosan can be deposited with HA-based calcium phosphate compound in situ or ex situ. In commonly used in situ method, HA is precipitated in matrix polymer in solution state followed by ageing and solvent removal (lyophilization etc). For this, at suitable pH, aqueous acidic solution of chitosan is reacted with solution containing calcium and phosphate ions precipitating HA and polymer simultaneously. The components used can be H3PO4, (NH4)2HPO4, KH2PO4, NaH2PO4, Na2HPO4, Na3PO4, Ca(OH)2, CaCl2, CaCO3, Ca(NO3)2·4H2O, CaSO4·0.5H2O) Yamaguchi et al. used such co-precipitation method to prepare the composites by using chitosan dissolved in organic acids as acetic, lactic, citric and malic acid [30]. Transmission electron microscopy images showed that HA forms elliptic aggregations. The growth of HA crystals was inhibited by organic acids with more than two carboxyl groups, which strongly bind to HA surfaces via a COO⎯Ca+2 bond. In case of pure HA, the crystallites did not form aggregations. The in situ formation of HA maintains the high porosity, uniform dispersion, avoids reaggregation of particles [31]. In ex situ process the prepared HA-based calcium phosphate material is added to polymer matrix of either chitosan alone or chitosan/composite candidate combination. In such composites the HA nanocrystallites were well intact with the chitosan macromolecules. FTIR results indicated the existence of hydroxyl and amide groups in addition to the characteristic peaks of HA in the composite [32]. Wang et al. handled the mixing of HA nanoparticles and chitosan by ball milling and with pore forming agent and dichlorobenzene in 1:1:1 ratio and pressure molded the composite in required shape by dispersing in citric acid solution [33]. The scaffolds can be fabricated with layer by layer technique by dispensing composite material into precipitating solvent as sodium hydroxide–ethanol [34]. Another procedure used to prepare composites was developed by Akashi and co-workers by alternately soaking chitosan/poly(vinyl alcohol) hydrogels in calcium and phosphate solutions sequentially for about 2hrs each, and repeating this cycle usually 5 times, in order to produce HA on/in the hydrogels [35]. The procedure could be applied to chitosan; the 3-D

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scaffolds or membranes [36, 37]. The 3-D shape of the resulting composite was controlled by the shape of the starting chitosan hydrogel and the swelling was reduced with increase of the amount of incorporated HA (up to 70% by weight of ceramic could be added to the composites). The deposition of apatite can be made by supersaturated solution of calcium phosphate. The porous chitin scaffolds obtained by freeze drying were developed into composite by in situ precipitation of calcium phosphate from supersaturated solution since the polar surface of the scaffold favored apatite nucleation and growth with continuous carpet nature accommodating up to 55% by mass mineral [38]. Mineralisation of the chitosan sponges or scaffolds can be carried out using a double diffusion technique mimicking the biological system. For this Manjubala et al. used a chamber which contained two parts separated by a circular hole at the centre, at which the polymer scaffold could be fixed so that the different solutions (containing ions as calcium in one and phosphate in other) placed in the two parts could diffuse through the polymer [39]. The XRD and FTIR analyses of composite prepared so confirmed the phase purity of the apatite and showed formation of no other calcium phosphate phases. The aggregation of HA in presence of chitosan occurs due to presence of amino groups in chitosan molecule working as the nucleation sites [30]. The chemical interactions between calcium ions on polymer surface and amino groups of molecule, probably via coordination bond, nucleate HA nanocrystals and aligned them along the molecule. The hydration studies of calcium phosphates using X-ray diffraction, infrared spectroscopy, SEM and XPS analysis showed that the organic compounds with the charged functional groups (–OH, –NH2, –COO⎯) had a great effect on the hydration reaction of calcium phosphates. The bonding energies of primary element (Ca, P) in hydration product were changed affecting hydration morphologies, self-setting properties, rheological properties and mechanical strength. In the composites of β-chitin from pen of squid (Loligo sp) with octacalcium phosphate or hydroxylapatite prepared by precipitation of the mineral into scaffold via a double diffusion system exhibited only oriented precipitation of octacalcium phosphate [40]. The crystals with the usual form of (001) blades grow inside chitin layers preferentially oriented with the {100} faces parallel to the surface of the squid pen and were more stable to the hydrolysis than that precipitated in solution. It was thought that the mechanical factors were the predominant cause for the orientation of the octacalcium phosphate crystals with the a-axis almost normal to the chitin fibers and the compartmentalized space in the chitin governed the orientation of the crystals, even if epitaxial factors might have played a role in the nucleation processes. When the role of small amounts of chitosan (ppm) in the precipitation of calcium phosphate was studied, the results indicated that polymer may have interactions, but just enough to conduct ions to molecular recognition and correct epitaxy to orient crystals. Strong interactions would lead to the binding of species and make them unavailable for further oriented growth [41]. Modified chitosans such as with phosphate or aspartic acid function promote growth of HA with assistance of chelating or anchoring sites provided by these pendants [42, 43]. It is logical to get the observation that chitosan composites with calcium orthophosphate compound could support the apatite formation more readily during the biomimetic process [44]. The encouragement of growth of calcium deficient apatite was reported with Ca(OH)2 treated chitosan phosphate [45]. Phosphorylated and carboxymethylated chitin on the other hand exerted a potent inhibitory effect on the seeded growth of HA from a supersaturated solution, reducing the initial rate of crystallization by more than 90% at a solution concentration of 10–4 M. Both

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chitin derivatives also retarded the rate of spontaneous calcium phosphate precipitation [46]. The type of calcium phosphate precipitated was poorly crystallized, calcium-deficient apatite. The chitin derivatives were found to be incorporated into the precipitate and influenced both the phase and morphology of calcium phosphate formed.

Physical Behavior The nanocomposites of high- and medium-molecular-weight (MW) chitosan scaffolds with 0.5, 1 and 2 wt % fraction of HA fabricated by freezing and lyophilization had a highly porous structure and the pore size (~50 to 120 μm) was in a similar range for the scaffolds with different content of HA. A combination of XRD, FTIR and electron microscopy indicated that HA particles were uniformly dispersed in chitosan matrix and there was a chemical interaction between chitosan and HA. The compression modulus of hydrated chitosan scaffolds was increased on the addition of 1 wt % HA from 6.0 to 9.2 kPa in highMW scaffold. The water uptake ability of composites decreased with an increase in the amount of HA, while the water retention ability was similar to pure chitosan scaffold. After 28 days in physiological condition, nanocomposites indicated about 10% lower degree of degradation in comparison to chitosan scaffold. The biological response of pre-osteoblasts (MC 3T3-E1) on nanocomposite scaffolds was superior in terms of improved cell attachment, higher proliferation, and well-spread morphology in relation to chitosan scaffold [47]. The transparent and slight yellow rods of chitosan/HA nanocomposite with well dispersed HA nanoparticles was prepared by in situ hybridization. The initial mechanical properties of bending strength and bending modulus of composite were 86MPa and 3.4GPa, respectively, which was 2–3 times stronger than that of polymethymethacrylate and bone ceramics. The high mechanical properties obtained were due to the layered structure confirmed by the SEM photographs of composite [48]. Xu et al. observed a reinforcement of TTCP cement by the addition of chitosan lactate [49]. An increase in flexural strength, work of fracture and strain-at-peak load was observed, which was explained by the difference in microstructure of the chitosan composite. Also macroporosity was added to the composite by addition of mannitol crystals [50]. This led to a dramatic decrease of the elastic modulus and flexural strength, though macroporosities up to 65% were obtained and cells show infiltration into the scaffolds. Addition of chitosan malate to CPC (TTCP with dicalcium phosphate anhydrous) reduced setting time from 87 min for the control without chitosan to 13 min at 20% chitosan and increased the flexural strength from 4 to 14 MPa without compromising the resorbability of the cement. The dissolution rates (fraction of mass loss per day, %/day) were 1.05 for CPC control and 1.08 for CPC/chitosan. Dissolution studies indicated the CPC with 20% chitosan were dissolved by nearly 30% for 28 d at a pH of 4.5 with the formation of a microporous surface, compared to the formation of large cracks and exfoliations for the CPC control [51]. Chitosan was also used as the matrix for the incorporation of -tricalcium posphate and calcium phosphate invert glass by a solid–liquid phase separation of the polymer solution and subsequent sublimation of the solvent [52]. The composites had improved compressive modulus and strength. These composites were suggested as suitable materials for tissue engineering with macroporous structures and properties being adjusted by β-TCP/glass ratio and ceramic/polymer ratio. The

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in situ hardening of chitosan/HA composite could be synergized by use of absorbable fibers as silk or polygalactin [53]. Fiber reinforcement by polygalactin substantially increased the resistance of CPC to cyclic fatigue and fracture; and supported human umbilical cord mesenchymal stem cells (hUCMSC) attachment, proliferation, viability excellently [54]. (Table 1) Chitosan added to a CPC makes the cement more injectable as was observed by Leroux et al. without substantially modifying the setting reaction [55]. Similar results were observed by Takagi et al. who concluded that CPC-chitosan composites exhibited a better cohesion in water than regular TTCP cement [56]. Calcium sulfate hemihydrate enhanced the injectability, crystallinity, compressive strength of chitosan citrate/TTCP cement [57]. Chitosan-bonded self-hardening bone-filling composite paste containing HA granules had compressive strength comparable to cancellous bone. The composite powder produced very little heat during kneading, and had a pH value similar to that of human plasma [58]. Such composite could be manually inserted or injected under pressure into a bone defect where it set in situ. The presence of HA does not limit the moldability of the composite. A high flexible chitosan-HA composite film was prepared using homogeneously dispersed solutions of microscale HA powder and chitosan at various concentrations using a molding method. The films could hold up to 70 % HA without microcracks, phase separation and brittleness [59]. The membranes of chitosan/HA composite developed via dynamic filtration had uniformly embedded needle-like HA nanocrystals with low crystallinity. As the HA content was increased, the tensile strength of the membranes exhibited a steady decrease, while the elastic modulus increased by a factor of 2 when 20% HA was added [60]. Such membrane on application over the defected area can act as barrier to prevent the in-growth of cells from some undesirable tissues at the same time allowing the migration of cells with osteogenic potential into the secluded space so that fast osteogenesis can proceed unimpeded within this space. Takechi et al. studied the soft tissue response to anti washout fast setting CPC/chitosan composite and tried to understand the origins of the good response to CPC[29, 61]. In spite of the fast transformation to apatite being claimed as the origin of the good tissue response to these cements, they showed, by comparison with composite cements with citric and acrylic acids, that the dominant role was played by the fast setting (that is, high initial mechanical strength). However, this is a necessary, but not sufficient, condition for this behavior. When implanted in tibia, the composite was surrounded by thin fibrous tissue, while particles of the conventional CPC were scattered and surrounded by foreign body reaction giant cells [28]. However it did not promote bone formation when compared to conventional CPC. One possible reason could be the very low amounts of chitosan used (approximately 0.14%), what could not have sufficient pharmacological effect to regenerate bone. Water-soluble phosphorylated chitin and phosphorylated chitosan when added to CPC as monocalcium phosphate monohydrate/calcium oxide cement and dicalcium phosphate dehydrate/calcium hydroxide cement enhanced the mechanical strength considerably, while only prolonging the setting time slightly [43, 62]. The composite of phophorylated chitosan with of 10-60 wt % HA nanoparticles imparted appreciable improvement in mechanical properties with minimal surface defects and were cytocompatable (murine L929 fibroblast), osteocompatible (Primary murine osteoblast cell culture) and highly osteogenic in vitro [63]. Polyanionic derivative of chitosan obtained by C-6 carboxylation and 2-sulfonation enhanced

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the mechanical strength of the dicalcium phosphate dihydrate and calcium hydroxide cements by increasing the dissolubility of the cement start materials and binding the calcium ions strongly afterwards [64]. In vivo evaluation of implants of these cements into preformed radial defects in rabbits showed formation of a thin layer of fibers between the newly formed bone and the implant with higher 6-carboxy,2-sulfonated chitosan samples after four weeks. The degradation rates of the reinforced cement were significantly lower than the original CPC alone [65].

Cellular Response, Bioresorption, Biodegradation Chitosan is found to potentiate the differentiation of osteoprogenitor cells and support the expression of extracellular matrix proteins by human osteoblasts and chondrocytes [60, 6668]. The osteoclsast (bone resorbing cells) which work together in a tightly regulated cycle along with osteoblsts can also be cultured on chitosan [69, 70]. Osteogenesis is augumented by chitosan derivatives too [71-73]. The studies of implants of HA/chitin matrix loaded with mesenchymal stem cells induced to osteoblasts into bone defects of the rabbit femur displayed proliferation and recruitement of surrounding tissue to grow in [74]. N,Ndicarboxymethyl chitosan with its improved chelation ability yielded the chelates of calcium acetate and with disodium hydrogen phosphate in appropriate ratios (molar ratio Ca/DCMC close to 2.4) and favoured osteogenesis while promoting bone tissue regeneration in sheep, leading to complete healing of otherwise non-healing surgical defects. The bone regeneration was observed in human patients undergoing apicectomies and avulsions. The safety of such derivatives was proven with composite implanted in rat femur [75]. The alleviation of demerits of hydroxyl carbonateapatite like particulate migration into surrounding tissues and low bioresorption had been reported with chitosan composite [76]. Preliminary histological examination showed no inflammation and appearance of new bone around the composite [77]. At the molecular and cellular levels the biocompatibility of CPC/Chitosan has been studied mostly on cells of mesenchymal origin such as bone marrow stromal cells [78, 79], chondrocytes [80], primary osteoblasts [81], or osteoblastic cell lines [27, 82-85], and also muscle derived stem cells [86].

Nerve tissue engineering The chitosan tubes derived from crab tendons form a hollow tube structure, which is useful for nerve regeneration. The mechanical strength of the tubes is quite high along the longitudinal axis, but is somewhat low for a pressure from side and tube walls swell to reduce the inner space of the tubes in vivo. The properties limit the clinical use of the chitosan tubes. These problems can be eluded with use chitosan/apatite composite [87]. The chitosan tubes on repeated reaction with CaCl2 and Na2HPO4 had apatite crystals in the walls of the chitosan tubes. The c-axis of the crystals aligned well in parallel with chitosan molecules. The thermal annealing of the chitosan/apatite composite tubes at 120°C to prevented it from swelling, and simultaneously formed into a triangular shape to enhance the stabilization of the tube structure and retain their shape even in vivo after implantation. Animal tests using SD rats

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further showed that the chitosan tubes effectively induced the regeneration of nerve tissue, and were gradually degraded and absorbed in vivo.

Engineering of other Tissues The potential of HA/chitosan scaffold as a good substrate candidate in periodontal tissue engineering has been demonstrated with scaffolds containing 1% HA [88]. The results indicated that the porosity and pore diameter of the HA/chitosan scaffolds were lower than those of pure chitosan scaffold. However when human periodontal ligament cells were seeded onto the scaffold, and implanted subcutaneously into athymic mice it helped not only to proliferate these cells but also to recruit surrounding tissue to grow in the scaffold. The degradation of the scaffold significantly decreased in the presence of HA. Assessment of physical properties of chitosan/HA indicated that a ratio of HA to chitosan sol of 4/11 by weight is optimal in the preparation of the composite membrane that can be of value in guided tissue regeneration and in localizing HA at implantation sites in periodontal bony defects or over extensively resorbed edentulous alveolar ridges. Subperiosteal implantation of the membranes over rat calvaria revealed that the membranes were well tolerated, with fibrous encapsulation and occasional areas of osteogenesis extending into the connective tissue that surrounded the implant. Increasing the hydroxyapatite content above ~35 wt % seems to enhance membrane degradation [89].

Drug Delivery The chitosan-composites may function as an effective delivery vehicle for osteoinductive growth factors, antibiotics and other molecules necessary to promote bone regeneration. The composites were loaded with growth factors, cytokines for promoting the tissue generation as bone morphogenic protein, platelet-derived growth factor, basic fibroblast growth factor, Nell-1 [Nel-like molecule-1; Nel (a protein highly expressed in neural tissue encoding epidermal growth factor like domain)] etc [33, 90, 91]. Chitosan/HA composite with released the loaded basic fibroblast growth factor (bFGF) in sustained pattern for 7 days after initial burst in contrast to complete release in 10-20 hrs by plain chitosan scaffold [92]. The protein release can be controlled further via tailoring the CPC microstructure with composite preparation method, scaffold generation method, constituent profile. For e.g. the lyophilized scaffolds posses more porosity [93]. In the CPC-chitosan/polygalactin composite, porosity was controlled by the ratio of CPC–mannitol powder:chitosan lactate solution [94, 95]. The combination of fast- and slow dissolution porogens/fibers in scaffolds modulated macropore formation rates to match bone healing rates. Such scaffold of CPC with 10% chitosan at a powder:liquid ratio of 3:1 loaded with 100 ng/mL of protein-A released about 0.4 (or 40%) protein mass fraction at the end of 1200h of immersion. The release of retained protein is expected to occur as the hydroxyapatite matrix is gradually resorbed. The localized and beneficial release profile were achieved in a brushite–chitosan system loaded with Vascular endothelial growth factor and Platelet derived growth factorDGF to enhance bone healing after implanting in rabbit femur [96].

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The composites can be good carrier for small drug molecules. Takechi et al. added flomoxef sodium to the liquid phase of the TTCP cement/chitosan composite and measured release from preset disks for 3 days [97]. Results showed a release pattern that was characterized by an initial burst, followed by a more sustained release. The total % of drug released in 24 h was 24–35%, where the addition of chitosan in different amounts did not influence total release after 72 h. Even more, the release from these chitosan enriched cements did not differ significantly from the normal TTCP cement, though the maximum amount of chitosan used was 1.0%. Lee et al. added TCP particles to chitosan sponges by freeze-drying and crosslinking a mixture of a chitosan solution and TCP [98]. Zhang et al. added HA and calcium phosphate invert glass to a chitosan sponge by crosslinking [99]. These scaffolds were also loaded with gentamicin by immersing them in gentamicincontaining phosphate buffer solutions [100]. Release patterns showed a high burst release that was diminished by addition of the ceramic particles and a total release of 90% after 3 weeks in release medium. A more sustained release from the particle-containing composite observed, which was suggested to occur due to a higher extend of chitosan crosslinking. With higher chitosan content in nano HA/chitosan (70:30 wt % HA:chitosan) sustained drug delivery was achieved only with 1.0 wt % berberine. The release profiles followed the Higuchi equation at the infusion stage. The drug loaded pellets can inhibit bacterial growth (Staphylococcus aureus) at the standardized berberine minimum inhibitory concentration of 0.02 mg/mL during berberine release from 1 to 28 days [101]. A controlled release drug delivery system was developed with chitosan/HA scaffold prepared by mixing their coprecipitates with the drug (tetracycline) and freeze-drying. To get a controlled release, a porous core of chitosan/HA composite was loaded with drug and coated with a porous drug-free layer (~1 mm) of composite so that a concentration gradient of drug could be created between the core and shell of the scaffold. Further gradation was achieved by deposition of a dense layer via the molding and air-drying processes, thereby generating a structure gradient between the inner and outer layers around the core. The in vitro drug-release test demonstrated that the porous layer without drug on the outer surface of the scaffold significantly reduced the initial burst of drug release and extended the release period. Finally, a successive and dense chitosan/HA composite layer endowed the scaffold with a sustained, drug release pattern without any initial drug burst [102]. The deposition of HA particles in the polymer matrix helps to control strength and release of loaded material. The matrix of chitosan-polyvinyl alcohol provides a thermosenstitive gel structure suitable for protein delivery but the gel strength is weak and burst release is observed [103]. The gel can be stabilized by deposition of HA into matrix by in situ or ex situ process especially by the former [104]. The composites were liquid solutions at low temperature (about 48°C), but gels under physiological conditions. The gels containing 0.1 mM hydroxyapatite synthesized through in situ process, the swelling degree was the lowest, and the speed of bovine serum albumin protein release was the slowest. (Figure4)

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a

289

b

C

Figure 4. Proposed gel formation structures (A) pure CS/PVA gel; (B) HA/CS/PVA composite gels ex situ; (C) HA/CS/PVA composite gels in situ.

Table 1. Composites of chitosan/apatite and other constituents Composite of Chitosan/poly(vinyl alcohol)/hydoxyapatite

Chitosan/collagen/HA Chitosan/silk fibroin/HA

Chitosan/polyacrylic acid/HA

Chitosan/phosphorylated chitosan/HA

Comment Chitosan/poly(vinyl alcohol) blend containing hydoxyapatite nanoaprticles electrospuned into nanofibrous mats had HA nanoparticles filled in the nanofibers or dispersed on the surface of fibers In situ HA synthesis in the Collagen/Chitosan system provided a feasible route for bone grafting nanocomposites The composite obtained via a coprecipitation method at room temperature had carbonate-substituted HA with low crystallinity (needle-like 20–50 nm in length and around 10 nm in width) deposited in the matrix, the composite had a higher compressive strength Polyelectrolyte complexation by solutions of HA containing chitosan and PAA gave uniformly distributed HA particles in PEC. Chitosan/ polyacrylic acid /HA compositions of 50/50/70 showed optimum compressive strength, of 50/50/80 showed minimum swelling, of 40/60/80 showed better Human osteosarcoma cell adhesion and viability Polyelectrolyte complexation by solutions of Calcium/phospahte containing chitosan and phosphorylated chitosan gave uniformly distributed carbonate-containing, lowcrystallinity HA particles in PEC and promoted osteoblast functioning

Ref [105]

[106] [107]

[108-110]

[111-112]

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Nazma Inamdar and V.K.Mourya Table 1. (Continued)

Composite of Poly 2hydroxyethylmethacryl ate grafted chitosan/gelatin/HA or HA/titania Chitosan/konjac glucomannan/HA

Chitosan/carboxymethy l cellulose/Nano-HA

Carboxymethyl citin/apatite

Carboxymethyl chitin/HA

Carboxymethyl chitin/tricalcium phosphate

Polyacrylamidoglycolic acid grafted chitosan/HA Chitosan/polymethylme thcrylate/HA Chitosan/alginate/HA

Chitosan/HA

Comment HA filler containing 10% titania had enhanced affinity to copolymer and compressive properties close to cancellous bone, formed apatite

Ref [113]

Composite was preapard by coprecipitation of solution of konjac glucomannan chitosan solution in H3PO4 with Ca(OH)2 solution. The composite revealed a high degradation in SBF and the rate and route could be different from chitosan, konjac glucomannan Improved the compressive strength compared than nano-HA/chitosan composite and had controllable degradation rate via adjusting the chitosan/carboxymethyl cellulose weight ratio A non-woven fabric of chitin short fibres ~8 mm in diameter treated with saturated Ca(OH)2 aqueous solution had a bonelike apatite layer formed on the surface within 3 days on soaking in a simulated body fluid The composite (HA=57.0 wt %)when injected into the calvarial bone of rats had biocompatibility, osteoconductivity. May be especially useful in facial bone augmentation because it can be injected with only a small skin stab. Rabbit tibia defects (4 mm in diameter) were repaired after 4 weeks more effectively by the composite and composites were cytocompatable in in vitro genotoxicity and carcinogenicity Complexes of HA were generated by addition of HA to the polymerization medium

[114, 115]

Chitosan/polyacrylic acid /HA compositions of 10/40/50 was more compatible for cell attachment, proliferation, osteoconductible than polymethylmethcrylate The composite prepared by co-precipitation method had the decrease of pore diameter with increase of HA content up to 30 wt.%, compressive strength increased with increase of HA content. Amount and size of HA particles affected the mechanical strength, with the highest strength obtained when 10 wt% nano-sized HA Porous chitosan-hxdroxyapatite hybrids developed by partial enzymatic degradation of the chitin/chitosan

[116, 117]

[118]

[119]

[120]

[121]

[122]

[123, 124]

[125, 126]

Composites of Chitosan for Biomedical Applications Composite of

Chitosan/apatite/Silicon apatite Chitosan/calcium silicate Silanol modified chitosan/calcium nitrate tetrahydrate Chitosan/gelatin/HA

Chitosan/gelatin/tricalci um phosphate

HEMA grafted Chitosan/gelatin/calciu m pyrophospahte Chitosan/HA/Polyethyl eneoxide

Chitosan/polylactic acid/HA

Chitosan/HA/Bi4Ti3O12 Genipin crosslinked chitin chitosan/HA

291

Comment surface using lysozyme. The L929 mouse fibroblastic lung cell adhesion and proliferation on composite depends on substrate roughness, porosity and stability The molecular weight of chitosan and silicate doping affected the nucleation and growth of apatite nanocrystallites Growth of a bonelike apatite on chitosan microparticles after a calcium silicate treatment Silanol groups and calcium ion acted in a synergy to form apatite crystals

Ref

Enhanced protein and calcium ion adsorption properties of composite improved cell adhesion, progenicity, multi-lineage differentiation potentials with enhanced osteogenic differentiation of human mesenchymal stem cells. The size of deposited HA depended on gel, ion concentration and temperature Hybrid polymer network developed via co-crosslinking with glutaraldehyde hadimproved compressive properties. A mild inflammatory response was observed over 12 weeks in subcutaneous biocampatibilty studies in rabbits Composites with good bioactivity and suitable mechanical properties

[130132]

In situ co-precipitation synthesis approach with an electrospinning process gave homogenous dispersion of the spindle-shaped HA nanoparticles (ca. 100 x30 nm) within the chitosan matrix. The composite cold be drawn into fibers which supported human fetal osteoblast functioning Homogeneous nanocomposites synthesized by in situ precipitation method with improved mechanical strength had HA nanoparticles in a rod-like shape (diameter ~ 50 nm, length ~300 nm) composed of randomly orientated subparticles of ~10 nm in diameter. The formation of nanoparticles could be controlled by a multiple-order template effect Chitosan/50% BIT/ 50% HA had dielectric constant K ~3360 at 100 Hz Scaffold sustained the nonuniform pore size of the scaffolds. Improved mechanical strengths, attachment of bovine knee chondrocytes, chondrocyte grew with fewer fibrosis in the junction between the newly formed tissues and the scaffold matrices

[127]

[128] [129]

[133]

[134]

[135]

[136]

[137] [138]

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Nazma Inamdar and V.K.Mourya Table 1. (Continued)

Composite of Glutaraldehyde crosslinked chitosan/HA Chitosan/HA/Ti6Al4V

HA/collagen/PLLA/chi tin Chitosan/Hydroxyapatit e

Comment The proliferation and morphology of MC 3T3-E1 cells seeded on the composite scaffolds showed better biocompatibility than pure chitosan scaffolds. The composite prepared by copreicipitation method allowed coating of Ti6Al4V with film thicknesses of ~ 1 μm with an adhesion strength superior to 15MPa. Supported growth of bone marrow stromal cells Chitin reinforced composite had better mechanical properties and decrease degradation rate in vitro. Injectable, porous microspheres were prepared with tripolyphosphate as coagulating agent. Microspheres can be developed into scaffold swith large pore size

Ref [139]

[140, 141]

[142] [143, 144]

CLAYS The phyllosilicate crystal structure is based on the pyrophyllite structure Si4Al2O10(OH)2 and can be described as a crystalline 2:1 layered clay mineral with a central alumina octahedral sheet sandwiched between two silica tetrahedral sheets corresponding to seven atomic layers superposed [145]. (Figure5, Table 2) This structure becomes (Si8)(Al4+ + yMgy)O20(OH)4,Mx for the montmorillonite (MMT) or (Si8)(Al6-yLiy)O20(OH)4,My for the 3+ + hectorite or (Si8-xAlx)(Mg6-y My )O20(OH)4,Kx+y for vermiculite. These differentiations are mainly due to the isomorphic substitutions that take place inside the aluminum oxide layer. These substitutions induce a negative charge inside the clay platelet, which is naturally counter balanced by inorganic cations (Li+, Na+, Ca2+, K+, Mg2+, etc.) located into the interlayer spacing or gallery. The global charge varies depending on the phyllosilicates. For the smectite and the mica families, this charge varies from 0.4 to 1.2 and from 2 to 4 per unit cell, respectively. Because of the weak interactions between the stacking layers, the cations inside the gallery can be easily exchanged by other cations. The maximum capacity for the cation exchange is known as the cation exchange capacity (CEC) and is expressed as mequiv/100 g. For instance, the MMT CEC varies from 70 to 120 meq/100 g depending on their extraction site [146]. The distance observed between two platelets of the primary particle, named interlayer spacing or d-spacing (d001), depends on the silicate type. This value does not entirely depend on the layer crystal structure, but also on the type of the counter-cation and on the hydration state of the silicate. For instance, d001 = 0.96 nm for anhydrous MMT with sodium as counter ion, but d001 = 1.2 nm in usual conditions. This increase is linked to the adsorption of one layer of water molecules between the clay platelets [1].

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Figure 5: Structure of 2:1 phyllosilicates.

Table 2. Classification of 2:1 phyllosilicates Charge per unit cell Smectites 0.4–1.2

1.2–1.8 Micas 2 4

Di-octahedral phyllosilicate

Tri-octahedral phyllosilicate

Montmorillonite (Si8)(Al4yMgy)O20(OH)4,Mx+ Beidellite (Si8-y Alx)Al4O20(OH)4,Mx+ Illites (Si8-x Alx)(Al4-y My2+O20(OH)4,Kx+y+

Hectorite (Si8)(Al6yLiy)O20(OH)4,My+ Saponite (Si8-x Alx)(Mg6)O20(OH)4,Mx+ Vermiculite (Si8-xAlx)(Mg6-y My3+)O20(OH)4,Kx+y+

Muscovite (Si6 Al2)(Al4)O20(OH)2,K2+ Margarite (Si4 Al4)(Al4)O20(OH)2,Ca2+

Phlogopite (Si6 Al2)(Mg6)O20(OH)2,K2+ Clintonite (Si4 Al4)(Mg6)O20(OH)2,Ca2+

Chitosan is a cationic polyelectrolyte and therefore it can penetrate into the interlayer region and exchanges those interlayer cations to compensate the silicate charge.(Figure6) Thus, the intercalation of chitosan into MMT and hectorite layered silicates can be carried out mainly through ion exchange processes [147], i.e. following the usual mechanism governing the intercalation of cationic polymers into 2:1 charged phyllosilicates, with additional contributions of hydrogen bonding or entropic phenomena [148-152]. The assembly between polymers and clays could be achieved by various processes: (i) intercalation, i.e. by the insertion of the polymer in solution or in liquid phase into the interlayer region of the inorganic host solid (ii) coprecipitation, i.e. by the synthesis of the 2D inorganic solid in the presence of the polymer in solution (iii) delamination (exfoliation) and restacking, i.e. by the random separation by several nanometers between consecutive layers of the inorganic substrate followed by the aggregation of lamellae entrapping polymeric species.

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(Figure 7) Nanocomposites elaboration protocol for chitosan mainly includes solvent intercalation process. This elaboration process is based on a solvent system in which the polymer is soluble (aqueous acid) and the silicate layers are swellable (water for MMT). The polymer solution and clay dispersion are mixed together leading to a polymer chains intercalation. Then, the solvent is evaporated to obtain nanocomposite materials.

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Figure 6. Schematic representation of intercalation of chitosan into Na-montmorillonite.

Depending on the process conditions and on the polymer-clay affinity, different morphologies can be obtained. These morphologies are (i) microcomposites, (ii) intercalated nanocomposites or (iii) exfoliated nanocomposites. At low MMT content, several authors have shown the formation of an exfoliated nanostructure [153-155]. At higher MMT content, namely more than 5 wt%, the formation of intercalated/flocculated structure was observed [156]. Different clays employed as composite phase with chitosan are montmorillonite, vermiculite, rectorite, sepiolite, attapulgite, etc. The nanofillers/chitosan interactions modify the thermal, mechanical, swelling– deswelling behavior, porosity, permeability, absorption capacity, and fatigue properties in response to external stimulus etc. The increase in glass transition temperature by DSC was linked to the ionic interactions established between the chitosan and the nanofiller, which reduced the chains mobility. The increases in the tensile strength correlated to a small decrease in the strain at break were observed in the different chitosan nano-biocomposite. These raises were induced by the nanofillers/chitosan interactions, which enhance the stress transfer at the interface. The strain at break decrease was related to the morphology of the chitosan-MMT hybrid materials which displayed, in the best case, an intercalated/exfoliated structure. Such an increase in stiffness is already well reported into the literature and is correlated to the clay rigidity and dispersion state [157]. Other layered ceramics as zirconium phosphonate containing sulfophenyl groups in the interlayer region Zr(O3P–C6H4SO3H)2, can be tried as nanofiller in chitosan matraix . Such nanocomposite with 0.6 wt % zirconium

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sulfophenyl phosphonate exhibited improvement in tensile strength, elongation at break and water resistance by 60.0%, 69.7%, and 41.8%, respectively against parent chitosan [158].

Figure 7. Types of composite derived from interaction between clays and polymers: (a) phase-separated microcomposite the polymer chains have not penetrated into the inter-layer spacing and the clay particles are aggregated; (b) intercalated nanocomposite polymer chains have diffused between the platelets and (c) exfoliated nanocomposite the clay layers are individually delaminated and homogeneously dispersed into the polymer matrix.

The composite prepared through the reactions of chitosan in acidic solution with potassium persulfate incorporated along with MMT gave exfoliated nanocomposite where exfoliation as well as chitosan degradation was triggered by potassium persulfate. The film casted with the composite solution had increased tensile strength and impeded degradation in vitro[159]. The in situ grafting of lactic acid as polylactic acid on chitosan while compositing with MMT imparted the hydrophilicity, swelling behavior and plasticity to casted film [160]. Water absorption by composites increased with degree of grafting of lactic acid and decreased with increasing content of clay. The introduction of clays as MMT/attapulgite/sepiolite/kaolin to chitosan-g-polyacrylic acid prepared by in situ intercalative polymerization among chitosan, acrylic acid, and clay in aqueous solution generated superabsorbent nanocomposite [161-164]. The nanocomposites had higher swelling ability and pH-responsivity as compared to those prepared by grafting of acrylic acid on chitosan/MMT composites. The incorporation of unexpanded vermiculite to chitosan-g-polyacrylic acid also enhanced the swelling rate and the water absorbency with formation of more porous and loose composite [165]. Chitosan-gpolyacrylic acid with acid-activated, ion-exchanged, vermiculite modified with hexadecyl trimethyl ammonium bromide had not only enhanced water absorbency but also improved the swelling rate, water-holding capacity, and salt-resistant ability [166]. Vermiculite modified with a bit shorter organic compound cetyl trimethyl ammonium bromide improved thermal performance of chitosan polymer [167] The composite of poly(butyl acrylate) grafted chitosan with tricetadecylmethyl ammonium bromide displayed resistance to water absorption with substantial improvements in mechanical and thermal properties so as their potential as packaging films and seed coating can be foreseen [168].

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Chitosan-clay nanocomposites appear to be a way to improve applicability needing robust and stable form of chitosan. The weak-base anion exchange ability of pure chitosan has been applied in the development of surface-modified sensors for anion detection, based on the casting of chitosan films onto the surface of glassy carbon electrodes [169, 170]. Nevertheless these devices lack long-term stability probably because of alteration of the characteristics of chitosan films. Chitosan in Na+- MMT or hectorite provides compact and robust three-dimensional nanocomposites with anionic exchange sites due to the special arrangement of the biopolymer as a bilayer when the amount of intercalated chitosan is higher than the cationic exchange capacity (CEC) of the clay. During clay-chitosan interaction, the first chitosan layer is adsorbed through a cationic exchange procedure, while the second layer is adsorbed in the acid salt form. Since the de-intercalation of the biopolymer is very difficult, the -NH3 +X- species belonging to the chitosan second layer act as anionic exchange sites (the exchange capacity 57.1 mequiv/ 100 g for MMT) [150]. These materials have been successfully used in the development of bulk modified electrodes just by the addition of graphite powder necessary to confer the required electronic conductivity to the system. These electrodes exhibit numerous advantages as easy surface renewal, ruggedness, and long-time stability. When applied in the potentiometric determination of several anions these sensors showed a higher selectivity toward monovalent anions. The best potentiometric response towards nitrate ions was observed [158]. This selectivity behavior could be explained by the special conformation of the polymer as a nanostructured bidimensional system in the clay interlayer space. The chitosan-sepiolite naocomposite modified with graphite powder was assessed as active phase of potentiometric sensors in a way similar to that reported for chitosan/MMT materials [152]. However, in this case the chitosan-sepiolite based carbon paste electrode showed a low stability in performance, and therefore, this electrode design could not be employed for such applications. It could be appointed that alternative electrode designs based in epoxy or PVC matrixes, which give more robust devices, can be a potential solution for the proposed electroanalytical applications. For sensor development one can make use of improved porosity as done with CaCO3-chitosan nanocomposite film fabricated by one-step co-electrodeposition method immobilized with acetylcholinesterase for detection of methyl parathion [171]. The assembly of chitosan/clay hybrid can be exploited as a drug carrier since the introduced clay can improve provide a slower and more continuous release of the drug in comparison with pure polymers in addition to improving the physicochemical properties of the polymer. During the interaction between clay and polymer, the drug molecules can accommodate themselves as guest in the interlayer region of the lamellar host and get released by diffusion and/or de-intercalation process. Such behavior was studied with chitosan-g-polylactic acid/MMT nanocomposite for carriage of ibuprofen sodium [167, 172] The composites were prepared by drying vacuum drying and lyophilization of the mix of chitosan solution in lactic acid and aqueous MMT solution exhibited different morphologies such as smooth films and porous scaffolds, respectively and were found to be stable regardless of pH of the medium. The MMT reinforcement controlled the ibuprofen release rate in phosphate buffer saline solution (pH7.4). The porous scaffolds had higher and faster drug release than films. Incorporattion of other polymers to basic composite can help to attain the desired characters in the drug delivery system. The hydrogel beads for controlled release of diclofenac were developed using composite of chitosan-g-polyacrylic acid-attapulgite supplemented with calcium crosslinked sodium alginate [173]. Attapulgite helped in reducing

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swelling ratio and burst effect of hydrogel where as alginate provided pH sensitivity. The drug release was minimum (~4%) and maximum (100%) at pH 2.1 and 6.8 respectively within 24 hrs. The drug release was swelling-controlled at pH 6.8. The deterioration of the responsiveness and reversibility of chitosan upon repeated on–off electrostimulation switching operations are major limitations for its use in electrically controlled drug delivery as it suffers from too much structural instability for the precise control of the release of drug [174]. The hurdle has been shown to be overcome by chitosan/MMT nanocomposite. With lower MMT content, the crystallinity of the chitosan was slightly reduced, resulting in a decrease in the mechanical properties and an increase in the swelling ratio of the hybrid. [175]. With higher clay concentration, the adverse effect caused by the small addition of clay were overcome and there was increased cross-linked bonding mechanically reinforcing the hybrid to show improved tensile strength and a decrease in the swelling ratio. After repeatedly switching the electric field on and off, the higher clay concentration (> 0.5 wt.%) of the hybrid composites maintained the same capability of deswelling and swelling after more than 10 cycles, compared with both the pure chitosan film and the hybrid composites with lower clay content (e.g. 0.5 wt %). The betterment of fatigue properties was utilized to achieve a stable pulsatile drug release by electrostimulation [176]. The chitosan 2 wt % MMT nanohydrogels demonstrated mechanically reliable, practically desirable pulsatile release profile of Vit B12 and excellent anti-fatigue behavior compared with pure chitosan. With a lower MMT concentration (1 wt %), the release kinetics of vitamin B12 from the nanohydrogel showed a pseudo-zero-order release, and the release mechanism was changed from a diffusion-controlled mode to a swelling-controlled mode under electrostimulation. Further increase in the MMT content reduced both the diffusion exponent n and the responsiveness of the nanohydrogel to electrostimulation but its anti-fatigue behavior was considerably improved. Wang et al. prepared such composites of chitosan/OREC (organically modified rectorite) for variety of studies. Rectorite Na0.6Ca0.3K0.1Al6Si6O20(OH)4.2(H2O) is a kind of layered silicate with the structure and characteristic much like those of MMT but with poor affinity between rectorite and polymer. So rectorite modified by organic reagent as cetyltrimethyl ammonium bromide is employed in chitosan nanocomposite formation. The characteristics of films of chitosan/OREC nanocomopsite were related to the amount and the interlayer distance of OREC in them. Loading of model drug bovine serum albumin and its in vitro release studies showed a slower and more continuous release for the nanocomposite films in comparison with pure chitosan film, and cumulative release was proportional to the amount and the interlayer distance of OREC [177]. The enhancement in antimicrobial activity is exhibited by chitosan-OREC and N-(2-hydroxyl) propyl-3-trimethyl ammonium chitosan chloride/OREC [156, 178] on the same line as N-(2-hydroxyl) propyl-3-trimethyl ammonium chitosan chloride and cetyltrimethyl ammonium bromide modified MMT composite [179]. It was attributed to inherent antibacterial properties of chitosan, hydrophobicity and cationic charge of the introduced quaternary ammonium group, adsorption of negatively charged bacteria and of toxins on clay. The nontoxic nature of the composite to cells and the utility of quaternized chitosan/rectorite composite as a non-viral gene delivery system was affirmed [180, 181]. Effect of chitosan structure and modification upon formation of chitosan/OREC composite employed for DNA delivery was studied with low- and high molecular weight fully deacetylated chitosans and their glycosylated derivatives substituted with the trimer GlcNAc–GlcNAc–Mannose [182]. The interlayer distance of rectorite depended on the

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amount of chitosan and the structure of intercalated chitosan. The largest interlayer distance of 3.35 nm was obtained when the mass ratio of low molecular weight non-substituted chitosan to rectorite was 2:1. The colloidal stability, DNA retention ability of the chitosan/DNA/rectorite nanocomposites increased with the increasing molecular weight of the intercalated chitosan and was improved by glycosylation. The chitosan/rectorite composites deliver DNA to human cells in vitro albeit with reduced efficacy compared to chitosan/DNA nanoparticles. The composites find plenty of applications in separation sciences basedon their sorption properties. (Table 3) Such sorption properties can be explored in biomedical field. In the study done by Pathk and Bajpai, the chitosan/magnetite nanoparticles were mixed into blood stream to carry out sorptive removal of urea during the dialysis process which could finally be removed by moderate field generator [183]. The FTIR spectral studies confirmed the binding of Fe3+/2+ with oxygen of urea owing to 6 co-ordination number of Fe3+/2+. Table 3. Uses of chitosan/ceramic composite in separation sciences Composite Chitosan/montmorillonite Chitosan/montmorillonite N,O-carboxymethylchitosan/montmorillonite Chitosan-g-poly (acrylic acid)/montmorillonite Chitosan-g-poly (acrylic acid)/rectorite Chitosan-g-poly(acrylic acid)/attapulgite Chitosan/TiO2 Chitosan/Al2O3

Comment /Used for Coagulation/flocculation of metals Cu2+ Co2+, Ni2+ Adsorption of congo red, tannic acid, cationic dyes, tungsten Removal of Congo red

Ref [184] [185-188]

Removal of mehtylene blue

[190]

Removal of ammonium ion

[191]

Removal of ammonium ion, copper

[192, 193] [194, 195] [196]

Remotion (adsorption plus degradation) of methylene blue, benzopurpurin, Pb (II) from aqueous solution Removal of Copper(II)

[189]

CALCIUM CARBONATE The understanding of the biomineralization can be a great source of biological inspired synthetic strategies for the design of new mineral/organic hybrids. Calcium carbonate plays important functions in biominerals and biomineralization with ordered crystal nucleation, phase and growth dynamics. It exists in various polymorphic forms as aragonite, calcite, vaterite and amorphous calcium carbonate (ACC) etc. Recent research show the crystallization of aragonite in the nacre occurs in a gelling environment of silk-like proteins into a preformed scaffold built of β-chitin [197]. With this lead, composite material made of chitin and calcium carbonate were obtained by precipitation of the mineral into a chitin scaffold by means of a double diffusion system. The supersaturation inside the compartmentalized space in the chitin governed the location and polymorphism of the crystals [198]. The acetyl group of polymer induced the formation of organized calcium carbonate

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better than phosphate [199]. The strong influence of chitosan on the stabilization of a determined polymorph and also on the morphology of the calcium carbonate crystals and their stabilization was confirmed [200]. Carboxymethyl chitosn was shown to affect the crystal morphology of calcium carbonate precipitated by reaction of CaCl2 and Na2CO3. Spherical particles in calcite morph were deposited at 10,000 ppm concentration of carboxymethyl chitosan [201]. However recent studies on effect of carboxymethyl chitosan aqueous solution on polymorph selection and nanocrystallite rearrangement of calcium carbonate at different temperatures (25–95°C) declared formation of vaterite with traces of calcitite and with trace of calcite aragonite formation [202]. Vaterite was predominant polymorph with trace of calcite at 25 °C, whereas pure aragonite was obtained at 95°C. As a heterogeneous nucleator and stabilizing agent, probably carboxymethyl chitosan changed the nucleation and growth of calcium carbonate from thermodynamic into kinetic control. Under kinetic limitation, the reaction rate of aragonite increased along with the elevating of temperature and surpassed the rate of vaterite above 327 °K. The polymorphs argonite and vaterite were deposited in the thin-film matrix of chitosan when it was immersed into the supersaturated aqueous solution of CaCO3 containing poly(aspartate), 4.4 x10-4 to 1.0 x10-2 wt % [203] Vaterite formation became dominant when the concentration of poly(aspartate) was increased. The films of amorphous calcium carbonate were formed by use of a maleic chitosan through a particle buildup process, with aggregation and coalescence occurring simultaneously [204].

METAL OXIDE NANOPARTICLES The combination of nanomaterials of metal oxides or metals with polymeric architecture seems to have potential for the design of novel materials and their innovative applications. An exciting opportunity for such nanocomposites is waiting in the area of biosensors as a means of modification of electrodes. The combined and synergic properties arising from polymer chitosan and metal or metal oxide nanoparticles or II–VI colloidal semiconductor nanoparticles (also called quantum dots or QDs) facilitate the working of modified electrode. Electrochemical biosensors for clinical diagnosis and environmental analysis are usually fabricated by immobilizing reactive/responsive biomolecules on the surface of electrodes. The biocompatibility, film-forming and adhering abilities of chitosan provides avenue for immobilization of biomolecules like antibodies, enzymes, toxins, DNA, cells etc. Chitosan was initially used to immobilize biomolecules for development of electrochemical biosensors to detect the presence of hydrogen peroxide, glucose and urea [205-207]. The porous morphology of chitosan retains activity of immobilized molecules. It however has nonconducting nature, limited porosity/surface area and may not offer uniform distribution of immobilized material needed for sensory purpose. This is not surprising, as the polymer backbone of chitosan is highly polar and capable of forming hydrogen bonds with adjacent chains producing rigid, low density, nonporous structure. These limitations can be overcome by modifying chitosan with fillers as clay, metal oxide or metal nanoparticles etc. Amperometric biosensor for monitoring phenol (or glucose) based on a polyphenol oxidase (or glucose oxidase) entrapped in clay/chitosan film spread on glassy carbon electrode was developed where clay was laponite (Mg5.5Li0.5)Si4O10(OH)2(Na+ 0.73·nH2O) [208, 209]. A

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highly stable biological film was formed on the functional glassy carbon electrode via stepby-step self-assembly of chitosan, laponite, hemoglobin for catalysis of hydrogen peroxide reduction [210]. Several types of metal oxide and metal nanoparticles Fe3O4, TiO2, ZnO, ZrO2, Al2O3, CeO2, SiO2, gold, platinum can be employed similarly. The metal or metal oxide nanoparticles reveal good dispersing properties, large surface area, high protein loading capacity and in addition provide fast electron transfer kinetics. The nanoparticles deposited on electrode surface assists the functioning (direct electrochemistry) of proteins immobilized on electrode with chitosan. The establishment of satisfactory electrical communication between the active site of the most oxidoreductase enzymes and the electrode surface is difficult due to insulation of the redox center of enzyme by a protein shell. Because of this shell, the protein cannot be oxidized or reduced at an electrode at any potential. In order to achieve this task, mediator (discrete, electroactive intermediaries between electrodes and solution couples) have been utilized. However direct electron transfer of some redox-proteins can also take place with the help of nanoparticles without need of additional mediators. Chitosan helps the nanoparticles to entrap, distribute and assemble into higher-order structures, a requirement that is necessary to exploit many of the unique properties and potential applications of nanoparticles such as in direct electron transfer [211]. The help extended by chitosan is due to its pH responsive self-assembling properties. Chitosan’s pH-responsive properties allow itself to be directed to assemble (i.e., to electrodeposit) in response to locally applied electrical stimuli [21]. When the applied voltage is sufficient for protons present in solution to be reduced at the cathode surface then a localized pH gradient is generated. If this localized gradient is created with immersion of cathode in the aqueous acidic solution of chitosan, then chitosan chains that experience the high localized pH at the cathode surface get deposited there. (Figure8) Depending on the stimulus and generated localized pH gradient it is possible to deposit either thin film or thicker hydrogel on the cathode surface. The directed assembly of chitosan can be controlled spatially and temporally. The deposition of chitosan is not confined to two-dimensional surfaces, but can be extended to the base and sidewalls of microfluidic channels. The chitosan can be conjugated with proteins which can be bioactive or chemoactive as chemiluminescent. The chitosan’s potential for the sequential, signal-guided assembly of proteins from solution to individual addresses has also been demonstarated. (Figure 9). The sensitivity and selectivity of the biosensor vary depending on the choice of immobilized component, nanomaterial and chitosan type. However, the selectivity of these chitosan/inorganic nanocomposite biosensors can be improved by introducing a third component, as nanoparticles of alloy or room temperature ionic liquid or carbon nanotubes or quantum dots. (Table 4).

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Figure 8. (a) Directed assembly of chitosan in response to locally applied electrical stimuli. Mechanism for chitosan’s directed assembly (i.e.electrodeposition). Chitosan can be conjugated with proteins. (b) Chitosan’s electrodeposition can mediate nanoparticle assembly. Mechanism of chitosan-mediated nanoparticle deposition at the cathode.

Figure 9. Hybrid materials can be directed to assemble if one component (e.g., the nano-component) can respond to an externalstimulus. Microfabrication allows devices to be constructed that can apply a variety of external stimuli with spatial and temporal control.

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The sol–gel chemistry based on the hydrolysis and polycondensation of metal alkoxides M(OR)x, where M= Al, Ce, Mo, Si, Sn, Ti, V, W, etc present convenient method to afford the hybrid materials with or without covalent bonds between the polymer and inorganic components. In this respect Tao et al. had prepared stable chitosan/TiO2 hybrid films by sol– gel method tetrabutyl titanate as precursor and acetylacetone as chelating agent by in situ sol– gel process, where Ti–O–Ti inorganic network was bonded with chitosan macromolecules by hydrogen bonding as well as covalent bonding [212]. (Fig 10)

Figure 10. Hypothetic scheme of interaction between tetrabutyl titanate and chitosan

The film had good tensile strength, thermal stability, and its potential use as a bioactive electrode has been discussed by Khan and Dhayal [213]. The complexation capability of chitosan with transition metal oxides allows the growth of layer by-layer films as chitsan/WO3/chitosan/TiO2 [214] or of microspheres as Fe3O4 nanoparticles incorporated into silica spheres coated with chitosan [215]. Adoption of the sol-gel technique to elaborate silica/chitosan composite involves solubilization of tetetraalkylsiloxane as methyl or ethyl in acidic solution of chitosan, subsequent gelation and removal of solvent (solvent displacement, conventional drying, supercritical drying etc) [216]. The composites show properties of aerogel or xerogel which are dependent on chitosan to silica ratio. These materials observed to be biocompatible [217], may find use in diverse applications such as bone tissue engineering [218,219], bioactive coating materials on titanium-based medical implants [220], carbon silica composites via pyrolysis [217], catalyst [221], etc. The enhancement of surface area may prove advantageous at variety of applications as use of chitosan in wound healing. A highly porous silica and chitosan-based hemostatic dressing (TraumaStat, OreMedix) was superior in controlling hemorrhage in a severe groin injury model in swine than Chitoflex (Hem-Con), or standard gauze [222]. The combination of chitosan and MRI negative contrast agent (Fe3O4 nanoparticles) give a new biocomposite for medical imaging [223, 224]. The combination of chitosan with stable fluorescent semiconductor nanocrystal (quantum dots) enables the composite nanoparticles to

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become a targeting fluorescent vesicle for drug and gene delivery. The preparation of three component composite (chitosan/Fe3O4/quantum dots) has also been investigated for creating multifunctional delivery and diagnostic systems [225].

METAL NANOPARTICLES Gold nanoparticles (GNPs) are of particular interest because of their unique optical and electronic characteristics as well as their excellent biocompatibility. GNPs with different shapes and sizes have been widely used in fundamental research, catalysis, biosensing, and very recently, the diagnosis and treatment of cancer. The versatility of GNPs can be supported by its elaboration as composite with biocompatible polymer as chitosan. So far, there are mainly two kinds of approaches for this. The first approach comprises direct chitosan coating on preformed GNPs or adsorption of GNPs on chitosan coated surface [226] by taking advantage of opposite electrostatic charge [227]. Chitosan has been used as a stabilizing or protecting agent by Esumi in preparation of gold nanoparticles [228]. This property can be optimized with fuctionalization of chitosan as with thiol group or oleoyl group as reported by Nandanan et al. [229]. Huang and Yang found that chitosan was more than a protecting agent and that gold salt can be reduced to zero valent gold nanoparticles by chitosan itself without any additional reducing agent [230]. Hence the second approach of GNPs synthesis includes reduction of HAuCl4 auric acid with chitosan solution alone [231, 231], or with an additional mild reducing agent as sodium borohydride [228, 233], citrate [234, 235], EDTA [236]. The method can be adopted to synthesize GNPs on solid surface or thin films [237]. Modification of electrode surfaces with the GNPs will provide a microenvironment similar to that of the redox-proteins in native systems and gives the protein molecules more freedom in orientation, thereby reducing the insulating effect of the protein shell for the direct electron transfer through the conducting tunnels of GNPs. In 1996, Brown and co-workers have reported a reversible electrochemistry of horse heart cytochrome c at SnO2 electrodes modified with 12 nm-diameter GNPs. Since then, a great deal of literatures have been reported to complete the direct electron transfer of redox-proteins using GNPs as promoter [238]. Electrodes modified with chitosan/gold nanocomposites have been investigated for a wide range of biomedical applications including glucose sensing [239] and electrochemical coding of single nucleotide polymorphisms [240]. With well defined size of small metal nanoparticles, the polydispersity of the nanocomposites is low (i.e. high homogeneity). Hence with the proper optimization, stable aqueous dispersion of the composite nanoparticles can be obtained with high surface positive charge (>30 mV) that have been investigated for drug delivery and gene delivery [233, 241]. Oh et al made use of ionic interaction between the anionic gold nanoparticles and cationic chitosan to induce the loading of paclitaxel in composite nanospheres through the mixing drug suspension containing GNPs and chitosan solution [242]. Loading of peptide drug as insuln for nasal delivery was also tried considering the absorption enhancement properties of chitosan by transient opening of the tight epithelial junctions due to its positive charge [243]. The cationic nature of chitosan helps in visualisation of anionic sites on several microorganisms by electron microscopy when chitosan/gold nanocomposites. Such reports

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are among the earliest ones of using chitosan/inorganic composites in biomedical imaging [244, 245]. Later, the strategy has been extended to imaging of mammalian cells [227]. Modification of the chitosan matrix by gold nanoparticles provides the mechanical strength to composite so that conduits can be drawn. These conduits can be used for bridging the two nerve stumps with directing axonal growth properly. The preliminary outcome obtained after 6 weeks of conduits implanted to bridge the rat sciatic nerve across a 10-mm– long defect demonstrated that the biodegradable micropatterned conduits preseeded with NSC provided a combination of physical and biological guidance cues for regenerating axons at the cellular level and offered a better alternative for repairing sciatic nerve transactions [246]. The advantage can be taken of specific property of nanoparticles as done with antibacterial activity of the silver nanoparticles. Silver exerts its biocidal effects by interfering with the respiratory chain at the cytochromes or with components of the microbial electron transport system, and by binding DNA and inhibiting DNA replication. Silver also appears to have anti-inflammatory properties and can increase reepithelialisation rates by over 40%. These properties of silver can be accrued with inherent antimicrobial properties of chitosan for benefit. Antimicrobial results showed that the silver nanoparticle/chitosan nanocomposite systems display a very effective bactericidal activity toward both Gram positive and Gram negative bacteria. However, the composite hydrogel did not show any cytotoxic effect toward the eukaryotic cell lines as mouse fibroblast (NIH-3T3), human hepatocarcinoma (HepG2), and human osteosarcoma (MG63) cells [247]. This is due to the fact that the nanoparticles, immobilized in the gel matrix, can exert their antimicrobial activity by simple contact with the bacterial membrane, while they can not be uptaken and internalized by eukaryotic cells. Potential of such composites in wound dressing has been readily suggested [248, 249]. The structural reinforcement of the composite can be done with crosslinking or complexing of the composite with genipin [250]. The nanocomposite polydimethylsiloxane/clay/chitosan/silver showed excellent antibacterial properties and can be utilized to improve the antibacterial function of biomedical catheter materials [251]. MnO2 nanoparticles was introduced to chitosan film containing amperometric glucose biosensors based on the direct oxidation of hydrogen peroxide where MnO2 effectively oxidize ascorbic acid to electrochemically inactive product and its interference on the sensors [252].

CARBON NANOTUBES Carbon nanotubes (CNTs) are a manmade form of carbon that did not exist until the 1990s. They have one-dimensional nanoscale structure, very high aspect ratio (i.e., length to diameter ratio), and high surface-to-volume ratio excellent flexibility, electrical conductivity, thermal conductivity, strength, and stiffness [253]. The enhanced electro-catalytic activity is due to presence of edge-plane-like sites located at both ends and in the defect region and faster electron-transfer kinetics due to curvature that produce changes in energy bands close to Fermi level [254, 255]. This offers their utility in electrochemical sensing applications. However manipulation and applications of CNTs is difficult because of their poor solubility in usual solvents, difficulty in dispersion in any solvents due to nanotube–nanotube or van der Waals interactions and tendency to aggregate [256]. To solve the problem polymers or surfactants be used to noncovalently encase/wrap nanotubes that ease the surface tension

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between the hydrophilic aqueous phase and the hydrophobic surface of the CNTs or to covalently attach onto the nanotube surface[257-259]. Chitosan appears to be advantageous over other polymers as peptides, amylase, polyacrylate, polyacrylonitirile, poly(ethyleneimine), polyvinylalcohol etc. due to endowment of biocompatibility by chitosan and mild conditions required for handling [260]. The dispersion of CNTs can be attained with chitosan of optimum degree of deacetylation [261]. Wang and coworkers prepared the CNTs/chitosan nanocomposites by mixing an acetic acid-added aqueous solution of chitosan with a CNTs-suspended water solution, followed by homogenizing and casting to obtain dry uniform films [258]. Liu and coworkers used a pH dependent controlled surface-deposition and glutaraldehyde crosslinking process to prepare anion-destroyable chitosan-decorated CNTs [257]. With use of chitosan derivatives as O-carboxymethylchitosan, and O-carboxymethylchitosan modified by poly(ethyleneglycol) at the -COOH position Yan et al. were able to produce highly effective debundling and dispersion of single walled CNTs in neutral pH aqueous solution. They found that trimethyl ammonium chitosan was unable to disperse single walled CNTs. They concluded the possibility that the –NH2 contributes to adsorption of the polymer on the nanotubes and the free electron pair in the amine groups of chitosan and derivatives plays a role in finely dispersing the nanotubes. With quaternization of –NH2 group possibly cation-π interaction with nanotubes is diminished due to steric hindrance and so is the ability to disperse nanotubes [262]. Chitosan disperses CNTs over the surface as of electrodes maintaining the homogeneous layer of CNTs and does so without compromising structure-properties of the pristine CNTs as either mediator based or direct electron transfer system based electron transfer ability [257, 263, 264]. Gorski and coworkers developed an electrochemical sensing platform based on the integration of redox mediators and CNTs in chitosan matrix [265, 266]. By incorporating dehydrogenase, microperoxidase-II or glucose oxidase in the multilayer film, it was demonstrated to promote protein electron transfer with the electrode indicating that CNTs could wire the protein to the electrode [267, 268]. The promotion of direct electron transfer by CNTs between redox enzymes and electrode surfaces enables reagentless detection. Moreover CNTs containing electrode limits the interference of other electroactive species from biological samples (e.g. ascorbic acid, uric acid etc.) since the electrodes can be operated at a low applied potential. High mechanical strength and high water stability of the CNT-interspersed chitosan are other attractive features. In practice, performance of biosensors especially that of enzyme based usually depends on the physicochemical properties of the electrode materials as well as process of the enzyme immobilization, enzyme concentration, protection of the immobilized enzyme, conservation of biosensing ability etc on the electrode surface. The use of matrix made up of chitosan/SiO2/MWCNT proved to be efficient with these respect for covalent immobilization of creatinine amidinohydrolase [269]. Owing to these striking properties, many CNT/chitosan nanocomposite based electrochemical bionsensors have been investigated in the detection of soluble redox-active biomolecules or to facilitate enzyme-catalysed redox reactions. (Table 4) With use of functionalized CNTs, the chemical linkages between chitosan and CNTs can be employed to warrant the homogenous dispersion of CNTs. For example –SO3H groups of poly(styrene sulfonic acid)-functionalized CNTs [270, 271] or –COOH group of carboxylated CNTs [172], –COCl group of acyl-chlorinated CNTs [273, 274] react with amino groups of chitosan. Modified chitosan can be employed to handle the chitosan at different pH

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environment as carboxymethyl chitosan, (with –CH2COOH group), N-succinyl chitosan (with –CH2CH2COOH group), 2-hydroxypropyltrimethylammonium chloride chitosan (with – CH2CHOHCH2CN+(CH3)3Cl group) [275, 276]. The nnaocomposites elaborated by covalent decoration of fuctionalized CNT with chitosan or noncovalent stacking of modified chitosan on CNTs too retain the electroconductive properties [276, 277]. The composite of chitosan/CNTs display improved mechanical properties in various physical forms as films enabling them to withstand rigorous conditions faced during utilization as sensors. When compared with neat chitosan, the mechanical properties, including the tensile modulus and strength, of the nanocomposites are greatly improved by about 93% and 99%, respectively, with incorporation of only 0.8 wt % of MWNTs into the chitosan matrix [258]. The dispersion of 2 wt% of CNTs in chitosan gave an increased tensile modulus (or strength) from 1.08 GPa (or 37.7 MPa for strength) for neat chitosan to 2.15 GPa (or 74.3 MPa for strength) for the 2 wt% CNTs containing chitosan. With incorporation of clay 3 wt % and CNTs 0.4 wt %, the tensile strength and Young’s modulus of the nanocomposites are significantly improved by about 171 and 124%, respectively, compared with neat chitosan [278] or by about 50% with 1.0 wt% MMT and 1.0 wt% MWCNTs [279]. For chitosan/CNT hydrogel beads manufactured by dispersing CNTs0.01 wt.% with cetyltrimethylammonium bromide 0.05 wt.% into chitosan solution 1 wt%, maximum endurable force at complete breakdown of beads increased from 1.87 to 7.62 N with incorporation of CNTs and its adsorption capacity increased from 178.32 to 423.34 mg g_1 for adsorption of congo red as compared to strength of beads by a conventional reinforcing method of cross-linking with epichlorohydrin [280]. Such mechanical improvement was seen in fibers scaffold drawn of the chitosan/CNT composite [281]. Increase in strength may envisage the use of chitosan/CNT in tissue engineering. However, some controversy exists in regards to the biocompatible character of CNT, with some in vitro studies reporting that CNT are cytotoxic while other ones showing CNT to be good substrates for cellular growth [282, 283]. Meanwhile different studies are performed for their potential for biomedical utility. For e.g. the membrane of chitosan/MWCNT with CNT 0–4 wt % were treated to deposit nanoscopic apatite. With low MWCNT concentrations apatite was formed on the composites, the smallest crystallite size being 9 nm at 1 wt% MWCNT. The dispersion of the MWCNT affects the crystallite size and the Ca/P molar ratio of the composite [284]. Abarrateg et al. have studied the use of MWCNT/chitosan scaffolds composed of a major fraction of MWCNT (up to 89 wt %) and a minor one of chitosan with a well-defined microchannel porous structure (extended to the whole monolithic dimensions) as biocompatible and biodegradable supports for culture growth [283]. For this purpose, they performed in vitro assays of cell adhesion, viability and proliferation onto the external surface of MWCNT/chitosan scaffolds with C2C12 cell line (myoblastic mouse cell), which is a multipotent cell line able to differentiate towards different phenotypes under the action of some chemical or biological factors. They observed the behavior of the evolution of the C2C12 cell line towards an osteoblastic lineage in presence of the recombinant human bone morphogenetic protein-2 (rhBMP-2) in vitro (e.g., following the appearance of alkaline phosphatase activity) and in vivo, by implantation of MWCNT/chitosan scaffolds adsorbed with rhBMP-2 in muscle tissue and evaluation of the ectopic formation of bone tissue after cofactor release. Under these circumstances, scaffolds could direct cellular behavior and

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function, repopulating and resynthesizing new natural matrixes and hence, opening promising perspectives for 3D construction of different tissues. One of the final applications of these materials includes their use as neural recording/stimulating electrodes. Along with this function they may act as carrier of beneficial molecules. Hence neurotrophoin 3 was incorporated into CNT/chitosan biogels as a possible means of the controlled release of the NT-3 via electrical stimulation [285]. The hydrogels of nanocomposite prepared as nano hydroxyapatite coated CNTs and chitosanglycerophosphate-citrate were thermosensitive. These gels were cytocompatible with L L929 cell-culture [286]. Modulated release of dexamethasone by electrical stimulation was investigated using chitosan and SWCNT host carrier films [287]. Drug-loaded SWCNT/chitosan composite films were solution-cast on carbon paper substrates. Unstimulated and stimulated release of dexamethasone in phosphate buffer saline solution at pH 7.4 was measured. Accelerated and complete dexamethasone release was observed upon potentiostatic application of a negative potential (−0.8V vs. Ag/AgCl) to the composite film, which was attributed to the electrostatic repulsions of SWCNTs and dexamethasone during charging. It was also found that the dexamethasone release can be slowed down during passive release (unstimulated) through the addition of SWCNTs. Further control can be achieved upon application of a positive potential (+0.15V vs. Ag/AgCl) in which the release rate was slower than in the passive case. The supramolecular architectures of β-Cyclodextrinmodified chitosan and adamantane–modified pyrene mediated by carbon nanotubes improved the DNA condensing efficiencies of chitosan [288]. The cooperation between cationic and aromatic groups as well as the dispersion of chitosan agglomerates by MWCNTs was the key factors to enhance DNA condensation of cationic polymers. Table 4. Biosensors developed with composites of chitosan Activity Glucose

Carried out with GOX/Fe3O4 NPs/chitosan on ITO GOX/Fe3O4 NPs/chitosan/nafion on platinum electrode GOX/ZrO2 NPs/chitosan on platinum electrode GOX/ferrocene-doped silica NPs /chitosan on GCE GOX/silica sol–gel/chitosan on prussian blue modified GCE GOX/silver NPs/chitosan on platinum electrode GOX/gold NPs/chitosan on gold electrode GOX-HRP/silver NPs/CNT/o-phenylenediamine/chitosan on ITO GOX/gold NPs/CNT/chitosan on GCE GOX/platinum nanowires/CNT/chitosan on GCE GOX/gold-platinum NPs/MWCNT/chitosan on GCE GOX/graphene/chitosan on GCE GOX/chitosan/ionic liquid (1-Butyl-3-methyl-imidazolium tetrafluoroborate) on nano-gold electrodes GOX/MWCNT/chitosan on Prussian blue modified ITO GOX/ferrocene modified MWCNT/chitosan on GCE GOX/MWCNT/chitosan on poly (allylamine) and polyvinylsulfuric

Ref [289] [290] [291] [292] [293] [294] [295, 296] [297] [298300] [301] [302] [303] [304] [305] [306] [307]

Composites of Chitosan for Biomedical Applications Activity

H2O2

Galactose Lactate

Cholesterol

Esterified cholesterol DNA hybridization

Carried out with acid potassium salt deposited platinum electrode GOX/MWCNT/chitosan-EDC–NHS on graphite-epoxy resin composite electrode GOX/CdS/Chitosan/aligned CNT/Platinum nanoelectrode on GCE GOX /Prussian blue NPs/MWCNT/chitosan GOX /gold NPs/thiol functionalized MWCNT/Ionic liquid /chitosan Glucose dehydrogenase/MWCNT/chitosan/glutaraldehyde on GCE HRP/ZnO NPs/chitosan on GCE HRP/gold NPs/chitosan on gold electrode HRP/clay/gold NPs/chitosan on GCE HRP/gold NPs/sol–gel carboxymethyl chitosan HRP/silica sol–gel/chitosan on carbon paste electrode HRP/organosilica@chitosan/MWCNT on GCE HRP/gold nanoelectrode/MWCNT/chitosan on GCE Hemoglobin/SiO2 NPs/chitosan on GCE HRP/ionic liquid of 1-butyl-3-methyl-imidazolium tetrafluoroborate/chitosan on GCE Hemoglobin/gold NPs/L-cysteine/gold NPs/Pt NPs/chitosan on platinum disk electrode Gold-platinum NPs/ionic liquid (trihexyltetradecylphosphonium bis(trifluoromethylsulfonyl)imide/chitosan on GCE and ITO Platinum nanowires/CNT/chitosan on GCE HRP/gold NPs/chitosan on screen printed carbon electrode HRP/MWCNT/chitosan/glutaraldehyde on GCE Prussian blue modified-SWCNT/chitosan on GCE MWCNT/chitosan/glyoxal or glutaraldehyde or epichlorohydrin and EDC–NHS on graphite-epoxy resin composite electrode Galactose oxidase/SWCNT/chitosan/ glutaraldehyde on GCE Lactate dehydrogenase/MWCNT/ chitosan on GCE Lactate dehydrogenase/MWCNT/ polyvinylimidazole-Os/chitosan on GCE ChOX/ZnO NPs/chitosan on indium-tin-oxide glass plate ChOX/gold NPs/MWCNT/chitosan/room-temperature ionic liquid (1butyl-3-methylimidazolium tetrafluoroborate) on ITO ChOX/Platinum NPs/MWCNT/chitosan on GCE Platinum NPs/CNT/poly(sodium-p-styrenesulfonate) salt/ chitosan on gold electrode ChOX/MWCNT/SiO2/chitosan on Prussian blue modified GCE ChOX and cholesterol esterase/silica NPs/multi-walled CNT/chitosan on ITO ssDNA/ZnO NPs/chitosan on GCE ssDNA/ZnO NPs/MWCNT/chitosan on GCE

309 Ref [308] [309] [310] [311] [267] [312, 313] [314] [315] [316] [317] [318] [319] [320] [321] [322] [323] [324] [325] [326] [327] [328] [329] [330] [331] [332] [333] [334] [335] [336] [337] [338] [339, 340]

310

Nazma Inamdar and V.K.Mourya Table 4. (Continued)

Activity

DNA methylene blue interaction Reduction of H2O2/nitrite/tr i-chloroacetic acid

Choline chloride Acetyl thiocholine Phenolic compounds Ethanol Organophosp hate

Paroxon Nitric oxide, O2, H2O2 Ferritin α-fetoprotein Human IgG Ochratoxin-A Chemilumine scence sensor for itopride, triprolylamin

Carried out with ssDNA/ ZrO2 NPs/CNT/chitosan on GCE ssDNA/CeO2 NPs/chitosan on GCE dsDNA/MWCNT/chitosan on graphite electrode dsDNA/MWCNT/chitosan on a screen printed carbon electrode ssDNA/V2O5 nanobelt/MWCNT/chitosan on N-hexylpyridinium hexafluorophosphate-graphite electrode dsDNA/CNT/chitosan on GCE

Ref [341] [342] [343] [344] [345]

Hemoglobin/silica NPs/chitosan on GCE

[347]

Myoglobin/gold NPs/clay/chitosan on GCE Hemoglobin/TiO2 NPs/room temperature ionic liquid of 1-butyl-3methylimidazolium hexafluorophosphate/chitosan on carbon paste electrode Myoglobin/ poly(dimethyldiallylammonium chloride)/MWCNT/ chitosan on gold electrode Choline oxidase/MnO2 NPs/chitosan on GCE

[348] [349]

[351]

Acetylcholinesterase/MWCNT/chitosan/glutaraldehyde on GCE

[352]

Tyrosinase/Fe3O4 NPs/chitosan on GCE

[353]

Alcohol dehydrogenase/MWCNT/chitosan on GCE AChE/gold NPs/chitosan on gold electrode

[354] [355]

AChE/gold NPs/CdTe QDs/chitosan on GCE AChE/MWCNT/chitosan/glutaraldehyde on GCE Chitosan/gold-TiO2 NPs on GCE Organophosphorus hydrolase /thioglycolica cid capped CdSe QDs/chitosan Fe2O3 nanotubes/chitosan on gold electrode

[356] [357] [358] [359, 360] [361, 362] [363] [364] [365] [366] [367, 368]

Ferritin antibody/Fe3O4 NPs/chitosan on GCE α-fetoprotein antigen/gold NPs/CNT/chitosan on GCE Goat-anti-human IgG antibody/ZnO NPs/chitosan on GCE Rabbit IgG/TiO2 NPs/chitosan on ITO Ru(bpy)32+-doped silica NPs/chitosan on GCE

[346]

[350]

Composites of Chitosan for Biomedical Applications Activity e Chemilumine scence sensor for pyrogallol Chemilumine scence sensor for H2O2 Electrochemil uminescence sensor for IgG Direct electron transfer reaction with cytochrome c Electrochemi cal study of K562 Leukemia cells Electroreducti on of pbenzoquinone or H2O2 NADH

Nitrite, bromide Nitrite 2,2’-azinobis-(3ethylbenzthia zoline-6sulfonic acid) diammonium salt, catechol, and O2 Reduction of cimetidine Niclosamide

311

Carried out with

Ref

Luminol-doped silica NPs/chitosan on graphite electrode

[369]

Luminol modified gold nanostructures/chitosan

[370]

CdS QDs-CNT combined with gold NPs/Chitosan

[371]

Cytochrome c/gold NPs/CNT/ chitosan on GCE

[372]

K562 leukemia cells/gold NPs/chitosan

[373]

GOX/MWCNT/chitosan on gold electrode

[374]

MWCNT/chitosan on GCE MWCNT/Azure dye/chitosan on GCE MWCNT/dopamine/chitosan on gold electrode Ionic liquid of 1-butyl-3-methylimidazolium tetrafluoroborate / MWCNTs/chitosan on GCE Chitosan/carboxylated MWCNT on GCE

[375] [266] [376] [377]

Cytochrome c/ multi-walled CNT/poly(amidoamine)/chitosan on GCE Laccase/CNT/chitosan on GCE

[378, 379] [380] [381]

Myoglobin/ SWCNT/chitosan on gold electrode

[382]

Carbon nanoparticle/chitosan on GCE

[383]

312

Nazma Inamdar and V.K.Mourya Table 4. (Continued)

Activity Sulfite Copper Glutamate

Carried out with MWCNTs/ferrocene-branched chitosan on GCE Functionalized CNT paste electrode modified with crosslinked chitosan Glutamate dehydrogenase/ MWCNT/chitosan/meldola’s blue on GCE

Ref [384] [385] [386]

Activity= Detection or determination of, if not mentioned NPs= Nanoparticles QDs= Quantum dots CNT= carbon nanotubes, MWCNT= Multi walled carbon nanotubes, SWCNT= single walled carbon nanotubes GOX= Glucose oxidase, HRP= Horseradish peroxidase, ChOX= Cholesterol oxidase, AChE = Acetylcholinesterase GCE= glassy carbon electrode, ITO= indium-tin oxide electrode EDC= 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, NHS= N-hydroxysuccinimide

QUANTUM DOTS II–VI colloidal semiconductor nanoparticles in the range of 2-6 nm also called quantum dots (QDs) unique because of theirs inimitable excellent optical properties, such as wide absorption and narrow more symmetrical emission spectra, large extinct coefficients, resistance to photobleaching, long fluorescence lifetime, and size-tunable emission [387]. In particular, QDs have offered great potential applications in molecular fluorescence probes, cancer detection and biological imaging and host of other applications as gene expression studies, high-throughput screening, and medical diagnostics based on optical coding technology. As most QDs are prepared in an organic solvent and have a layer of hydrophobic organic ligands on their surface they are not dispersible in water and thus not suited for any bioapplications. For biological application, the QDs must be coated with a biocompatible polymer to improve solubility, photostable properties as well as to minimize the toxic side effects. As a polymer chitosan does envelop the QDs. The wrapping can progressed in situ while the QDs are generated or on preformed QDs. (Figure 11)Synthesis of QDs in situ with chitosan in aqueous system through a -radiation route at room temperature under ambient pressure, chitosan assists the synthesis as well as coating of QDs [388]. In synthesis of chitosan-coated CdSe QDsand CdS QDs the mechanism discussed by the authors Kang et al. says: the Cd2+ ions in solution are chelated by the amino and hydroxyl groups on the chitosan chains (a) Then owing to the Coulomb force, the Se2– ions move towards Cd2+ ions, resulting in the formation of CdSe core on chitosan chains (b). With the particle growth, the CdSe nanocrystals are formed and coated by chitosan (c). The diameter of the resulting QDs prepared in this way were about 4 nm with narrow size distribution with absorption peak at 460nm and an emission peak at 535 nm. Without any physical assistance ZnS QDs with average size of 3.4nm were prepared in situ in chitosan film [389].

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Figure 11. Development of chitosan coated CdSe QDs (a) Chelation of Cd2+ (b) Movement of Se2- by Coulomb forces (c) Particle growth.

The positively charged chitosan could bind with CdSe QDs to form a network structure aggregates via electrostatic attraction and hydrophobic forces. The interaction lead to the remarkable enhancement of Resonance Rayleigh scattering (RRS) and the enhancements were in proportional to the concentration of chitosan in a certain range. The reason for the enhancement of RRS intensity were assumed to be: resonance enhanced that this interaction can be developed as a method for the determination of chitosan using thioglycolic acid and Lcysteine capped CdSe QDs as probes [390]. The association between chitosan and QDs can be employed in tandem for gene/therapeutic molecule delivery and tracking. The chitosan based nonviral delivery system for siRNA was developed where chitosan was doped with QDs. Chitosan NPs doped with fluorescent QDs as a selftracking and nonviral vehicle to deliver human epidermal growth factor receptor 2 (HER2) siRNA was developed. By labeling of chitosan/QD nanoparticle surface with HER2 antibody, targeted delivery of HER2 siRNA to HER2-overexpressing SKBR3 breast cancer cells was shown to achieve through tracking of the delivery construct [391]. The CdSe-ZnS core-shell QDs encapsulated with chitosan produced nanoparticles in the range of 60 nm [392]. These nanoparticles were small enough to be internalized into the myoblast cells. In vitro 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide cytotoxicity tests on primary myoblast cells suggested that the cytotoxicity of these QDs was greatly reduced after chitosan encapsulation. The film of CdSe-ZnS core-shell QDs with chitosan was developed using layer-by-layer self-assembly technique with good control over the composition, architecture of the film. This leads to large third-order optical nonlinearity suitable for further development of highly efficient third-order nonlinear devices that can be used in optical communication and optical informationprocessing for, e.g., all-optical switching and optical data storage. The employment of QDs in composite may help in the electron transfer abilities of the composite.

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The biosensor has been developed with immobilization of acetylcholinesterase in composite of CdTe QD/GNP/chitosan [393]. There are a few other reports of biosensing applications of QD/chitosan composites. (Table 4)

CHITOSAN–POLYIONIC MATERIAL COMPOSITES Chitosan with reactive –NH2 functions gets protonated in weakly acidic aqueous solutions. It hence is a weak base with an intrinsic pKa near 6.5 and a low charge density, with a maximum of only one charge per residue i.e. every ~5.15A°. At pH 4.0, the protonation of –NH2 to –NH3+ is complete. Hence the molecule of chitosan present in appropriate solutions is a cationic polyelectrolyte, which opens the possibility for interactions with negatively charged molecules (anions and polyanions). The strong electrostatic interactions between charged microdomains of at least two oppositely charged polyelectrolytes results in formation of polyelectrolyte complex (PEC). The most predominant molecular forces for PEC assembly are strong electrostatic interactions between opposite charges of macromolecules. Other forces as hydrogen bonding, hydrophobic interactions and van der Waals forces complement PEC formation. As a unique natural cationic polymer, chitosan has been studied as polyelectrolyte partner of several PECs with natural or synthetic anionic molecules. The composites of chitosan with other polyelectrolytes provide a number of application based opportunities depending on the nature of constituent in the composite, and preparation parameters as method of preparation, concentrations, mixing ratios, pH, nature of ions, ionic strength, temperature, feeding order, duration of the interaction, and mixing intensity [394]. In accordance to their final uses, PECs can be prepared as powders, membranes or films, sponges, fibers, gels, spheres or in solutions. Host of naturally present polyionic polymers as nucleic acids, proteins and glycosaminoglycans as well as synthetic polymers offers a wide choice suitable for applications. The possibilities of the final uses of these PEC composites are so immense that their detail discourse pleads a dedicated chapter. To get a glimpse, an example of chitosan/protein PEC is discussed briefly here. Due to its biocompatibility and biodegradability, chitosan have a wide range of applications as or in artificial skin, artificial blood vessels, cell implantation, nerve regeneration, superficial wound-healing, and bone healing treatment etc [395, 396]. However, a few of the important limitations of chitosan as a material is its relatively poor mechanical properties, and required rate of degradation. Hence, the addition of other polymers is necessary to overcome this problem. Composites of chitosan made with other polymers, such as starch [397], silk fibroin [398], polylactide [399], and cellulose [400] have been developed and studied for various applications in the past several years. When chitosan is mixed with proteins (gelatin, collagen, silk fibroin, keratin, laminin, peptides) as in the medium of pH above the isoelectric point of protein where it gets net negative charge, there occur electrostatic interactions between the protonated ammonium ions of chitosan and carboxylate groups of protein forming PECs [401]. The proteins abundant in cystein residues as keratin support the complex characteristics by additional intra and inter disulphide linkage. The crosslinking of chitosan and protein with agents for e.g. as genipin, proanthocyanidin helps to modulate the properties of complexes [402, 403]. The basic pH

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dependent complexation of chitosan/proteins imply that stabilization, decomplexation will also be pH dependent and will imparting pH responsive properties to the complex [404]. Such behavior can be harnessed to develop pH dependent drug delivery system. Mao and colleagues had developed chitosan–gelatin hybrid polymer network as a pH sensitive matrix for drug delivery [405, 406] and later on explored their use in tissue engineering [407-409]. The complexes of chitosan-gelatin are not only restricted to delivery of drugs any more [410-412] but extended to the DNA delivery [413] and macromolecules as recombinant human bone morphogenetic protein-2 [414]. The chitosan/protein complexes can intensify cytocompatibility via shielding the positively charged chitosan to a suitable charge density, enhance cell proliferation and decline cell apoptosis [415]. The role of chitosan/protein PECs equivalent to extracellular matrix for cell survival has been exhibited for many types of cells (kerationocyte [416-418], Cos-7 fibroblast [419], L929 fibroblast [420], C3A cells [421], rat hepatocytes [422], human umbilical vein endothelial cell [423], human MG-63 osteoblast-like cells [424], buffalo embryonic stem [425], adipose tissue derived stem cells [426] rat mesenchymal stem cells [427]) endorsing their use as scaffold for tissue engineering. The microcarriers made of chitosan and gelatin have been fabricated and found to be promising for hepatocyte culture [428]. Encapsulation of cells in such compatible composites may extend their functional longevity provide immunoisolation beneficial during xenotransplantation such as of pancreatic cells [429, 430]. In the film form the composite of chitosan/gelatin was evaluated as artificial skin since the composite has better water uptake properties and flexibility in addition to cell compatibility. The mechanical and biological properties of the chitosan/gelatin needed for such purpose can be improved by elaborating ternary composites of chitosan/gelatin with PEG [431], hyaluronic acid [432], fibrin [433], keratin[434] or glass ceramic [435], βtricalcium phosphate [436]. Rationally the PEC of chitosan/gelatin had been evaluated for wound healing [437, 438], for cartilage tissue engineering [439, 440]. Zhang et al. found that the soft and flexible film from chitosan and gelatin composites had better nerve cell affinity compared to chitosan [441]. The use of membrane protein as laminin with chitosan as conduit provides guided nerve tissue generation ability. This property can be augmented with incorporation of nerve growth factors [442]. The oxygen permeability, optical transmittance, water absorptivity and mechanical properties of chitosan/gelatin complex are such that it can be used as contact lens as well as an agent for ophthalmic drug delivery agent [443, 444]. Collagen, a parent protein of gelatin, has been assessed for many of the applications analogous to gelatin. The PECs of chitosan with glycosaminoglycan (heparin, chondroitin sulphate, hyaluronan, dermatan sulphate etc) are evaluated mainly for tissue engineering and drug delivery purposes where as the PECs with natural anionic polymers (alginate, pectin, carrageenan, chemically modified chitosan, glucomannan, xanthan etc) are evaluated for drug delivery, and biotechnological applications. The complexes of chitosan with lipids provides vesicle forming ability where as the PECs with synthetic polymers bestow the properties to the complex such that if loaded with drug its release can be controlled by pH or temperature dependent nature, swelling properties, erosion ability of the polymer. The PEC of chitosan and chitosan derivatives with nucleic acids is an interesting area of nonviral delivery of genetic material. Development, properties and applications of such complexes are already well documented [394].

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CHALLENGES AND PERSPECTIVES The main biomedical applications of chitosan composites can be catergorized into four classes as tissue engineering, drug and gene delivery, bisensors and bioimaging. Chitosaninorganic composites and chitosan polyion composites are extensively researched for realization of these applications in conventional manner (termed as first generation composites). The integration of nanotechnology had renewed the interst in these complexes especially for biosensor and bioimagig functions. Two or more areas can be integrated into multifunctional tools for the improved applications such as drug eluting scaffold, targeting of combined imaging and drug delivery vesicle, live cell censors, cell arrays and lab on chip microfluidic devices (the putative second and third generation composites). The data related to their development, properties and applications is growing and one can expect exciting discoveries in these areas. The major drawback or challenge is the lack of general consensus in analysing the physicochemical properties of chitin and chitosan and their quality criteria. A significant contribution towards this goal is still awaited. The science fraternity many a times rely on the information provided in the product specifications by the manufacturers. Even the two most fundamental characteristics of chitin and chitosan, the deacetylation level and molecular weight, are still determined by several analytical methods and the correlations among these methods are poorly understood. The formal attempt to standardize the analytical protocols for the quality assessment of chitomaterials is needed. This will give an impetus to chtiomaterial applicability and accepatability. With respect to the further development in biomedical applications of chitosan based composites, a good potential is anticipated in the field of chitosan–inorganic nanocomposite in the second and third generation applications.

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[369] Zhang LL, Zhang XW. Novel electrogenerated chemiluminescence sensor for pyrogallol with core-shell luminol-doped silica nanoparticles modified electrode by the self-assembled technique. Anal Chim Acta 2006; 570: 207-213. [370] Wang W, Cui H. Chitosan-luminol reduced gold nanoflowers: from one-pot synthesis to morphology-dependent SPR and chemiluminescence sensing. J Phys Chem C 2008; 112: 10759-10766. [371] Jie G, Liu P, Wang L, Zhang S. Electrochemiluminescence immunosensor based on nanocomposite film of CdS quantum dots-carbon nanotubes combined with gold nanoparticles-chitosan. Electrochem Commun 2010; 12: 22-26. [372] Xiang CL, Zou YJ, Sun LX, Xu F, Direct electrochemistry and electrocatalysis of cytochrome c immobilized on gold nanoparticles chitosan carbon nanotubes modified electrode. Talanta 2007; 74: 206-211. [373] Ding L, Hao C, Xue Y, Ju H. A bio-inspired support of gold nanoparticles-chitosan nanocomposites gel for immobilization and electrochemical study of k562 leukemia cells. Biomacromolecules 2007; 8: 1341-1346. [374] Zhou Q, Xie Q, Fu Y, Su Z, Jia X, Yao S. Electrodeposition of carbon nanotubeschitosan-glucose oxidase biosensing composite films triggered by reduction of pbenzoquinone or H2O2. J Phys Chem B 2007; 111: 11276-11284. [375] Zhai X, Wei W, Zeng J, Gong S, Yin J. Layer-by-Layer assembled film based on chitosan/carbon nanotubes, and its application to electrocatalytic oxidation of NADH. Microchim Acta 2006; 154: 315-320. [376] Ge B, Tan Y, Xie Q, Ma M, Yao S. Preparation of chitosan-dopamine-multiwalled carbon nanotubes nanocomposite for electrocatalytic oxidation and sensitive electroanalysis of NADH. Sens Actuators B 2009; 137: 547-554. [377] Wang Q, Tang H, Xie Q, Tan L, Zhang Y, Li B, Yao S. Room-temperature ionic liquids/multi-walled carbon nanotubes/chitosan composite electrode for electrochemical analysis of NADH. Electrochim Acta 2007; 52: 6630-6637. [378] Jiang L, Wang R, Li X, Jiang L, Lu G. Electrochemical oxidation behavior of nitrite on a chitosan-carboxylated multiwall carbon nanotube modified electrode. Electrochem Commun 2005; 7: 597-601. [379] Zeng Y, Zhu Z-H, Wang R-X, Lu G-H. Electrochemical determination of bromide at a multiwall carbon nanotubes-chitosan modified electrode. Electrochim Acta 2005; 51: 649-654. [380] Chen Q, Ai S, Zhu X, Yin H, Ma Q, Qiu Y. A nitrite biosensor based on the immobilization of Cytochrome c on multi-walled carbon nanotubes-PAMAM-chitosan nanocomposite modified glass carbon electrode. Biosens Bioelectron 2009; 24: 29912996. [381] Liu Y, Qu X, Guo H, Chen H, Liu B, Dong S. Facile preparation of amperometric laccase biosensor with multifunction based on the matrix of carbon nanotubes-chitosan composite. Biosens Bioelectron 2006; 21: 2195-2201. [382] Su S, Zhang L, Pan Y, Cai Y, Zhang Y. Direct electron transfer of Mb on chitosan/single-wall carbon nanotubes film modified Au electrode and its interaction with cimetidine. Russ J Electrochem 2008; 44: 218-225. [383] Ghalkhani M, Shahrokhian S. Application of carbon anoparticle/chitosan modified electrode for the square-wave adsorptive anodic striping voltammetric determination of Niclosamide. Electrochem Commun 2010; 12: 66-69.

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[384] Zhou H, Yang W, Sun C. Amperometric sulfite sensor based on multiwalled carbon nanotubes/ferrocene-branched chitosan composites. Talanta 2008; 77: 366-371. [385] Janegitz BC, Marcolino-Junior LH, Campana-Filho SP, Faria RC, Fatibello-Filho O. Anodic stripping voltammetric determination of copper(II) using a functionalized carbon nanotubes paste electrode modified with crosslinked chitosan. Sens Actuators B 2009; 142: 260-266. [386] Chakraborty S, Retna Raj C. Amperometric biosensing of glutamate using carbon nanotube based electrode. Electrochem Commun 2007; 9: 1323-1330. [387] Xu CJ, Xing BG, Rao JH. A self-assembled quantum dot probe for detecting betalactamase activity. Biochem Biophys Res Commun 2006; 344: 931-935. [388] Kang B, Chang S-Q, Dai Y-D, Chen D. Synthesis of green CdSe/chitosan quantum dots using a polymer-assisted g-radiation route. Radiat Phys Chem 2008; 77: 859-863. [389] Wang X, Du Y, Ding S, Fan L, Shi X, Wang Q, Xiong G. Large two-photon absorbance of chitosan-ZnS quantum dots nanocomposite film. Physica E 2005; 30: 96-100. [390] Peng J, Liu S, Wang L, Liu Z, He Y. Study on the interaction between CdSe quantum dots and chitosan by scattering spectra. J Colloid Interface Sci 2009; 338: 578-583. [391] Tan WB, Jianga S, Zhang Y. Quantum-dot based nanoparticles for targeted silencing of HER2/neu gene via RNA interference Biomaterials 2007; 28: 1565-1571 [392] Tan WB, Huang N, Zhang Y. Ultrafine biocompatible chitosan nanoparticles encapsulating multi-coloured quantum dots for bioapplications. J Colloid Interface Sci 2007; 310: 464-470. [393] Dua D, Chen S, Song D, Li H, Chen X. Development of acetylcholinesterase biosensor based on CdTe quantum dots/gold nanoparticles modified chitosan microspheres interface. Biosens Bioelectron 2008; 24: 475-479. [394] Inamdar NN, Mourya VK. Chitosan and anionic polymers - Complex formation and applications. In: Polysaccharides: Development, Properties and Applications. (Tiwari A. Ed.) 2010, Nova Science Publisher, NewYork , pp. (In Press) [395] Muzzarelli RAA. Chitins and chitosans for the repair of wounded skin, nerve, cartilage and bone. Carbohydr Polym 2009; 76: 167-182. [396] Senel S, McClure SJ. Potential applications of chitosan in veterinary medicine. Adv Drug Deliv Rev 2004; 56: 1467-1480. [397] Bourtoom T, Chinnan MS. Preparation and properties of rice starch-chitosan blend biodegradable film. LWT – Food Sci Technol 2008; 41: 1633-1641. [398] Gobin AS, Froude VE, Mathur AB. Structural and mechanical characteristics of silk fibroin and chitosan blend scaffolds for tissue regeneration. J Biomedl Mater Res Part A 2005; 74A: 465-473. [399] Peesan M, Supaphol P, Rujiravanit R. Preparation and characterization of hexanoyl chitosan/polylactide blend films. Carbohydr Polym 2005; 60: 343-350. [400] Phisalaphong M, Jatupaiboon N. Biosynthesis and characterization of bacteria cellulose-chitosan film. Carbohydr Polym 2008; 74: 482-488. [401] Taravel MN, Domard A. Collagen and its interaction with chitosan: II. Influence of the physicochemical characteristics of collagen. Biomaterials 1995; 16: 665-671. [402] Chiono V, Pulieri E, Vozzi G, Ciardelli G, Ahluwalia A, Giusti P. Genipin-crosslinked chitosan/gelatin blends for biomedical applications. J Mater Sci: Mater 2008; 19: 889898.

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[403] Kim S, Nimni ME, Yang Z, Han B. Chitosan/gelatin–based films crosslinked by proanthocyanidin. J Biomed Mater Res Part B: Appl Biomater 2005; 75B: 442-450. [404] Chen X, Li W, Zhong W, Lu Y, Yu T pH sensitivity and ion sensitivity of hydrogels based on complex-forming chitosan/silk fibroin interpenetrating polymer network. J Appl Polym Sci 1997; 65: 2257-2262. [405] Yao KD, Yin YJ, Xu MX, Nong YF. Investigation of pH sensitive drug delivery system of chitosan–gelatin hybrid polymer network. Polym Int 1995; 38: 77-82. [406] Yao KD, Xu MX, Yin YJ, Zhao JY, Chen XL. pH sensitive chitosan–gelatin hybrid polymer network microspheres for delivery of cimetidine. Polym Int 1996; 39: 333-337. [407] Yin YJ, Yao KD, Cheng GX, Ma JB. Properties of polyelectrolyte complex films of chitosan and gelatin. Polym Int 1999; 48: 420-432. [408] Shen F, Cui YL, Yang LF, Yao KD, Dong XH, Jin WY, Shi HD. A study on the fabrication of porous chitosan–gelatin network scaffold for tissue engineering. Polym Int 2000; 49: 1596-1599. [409] Mao JS, Zhao LG, Yin YJ, Yao KD. Structure and properties of bilayer chitosan– gelatin scaffolds. Biomaterials 2003; 24: 1067-1074. [410] Xiaoyan A, Jun Y, W Min, Haiyue Z, Li C, Kangde Y, Fanglian Y. Preparation of chitosan–gelatin scaffold containing tetrandrine-loaded nano-aggregates and its controlled release behavior. Int J Pharm 2008; 350: 257-264. [411] Foda NH, El-laithy HM, Tadros MI. Implantable biodegradable sponges: Effect of interpolymer complex formation of chitosan with gelatin on the release behavior of tramadol hydrochloride. Drug Dev Ind Pharm 2007; 33: 7-17. [412] Lei M, Liu S, Liu Y. Resveratrol protects bone marrow mesenchymal stem cell derived chondrocytes cultured on chitosan–gelatin scaffolds from the inhibitory effect of interleukin-1 . Acta Pharmacol Sin 2008: 29: 1350-1356. [413] Guo T, Zhao J, Chang J, Ding Z, Hong H, Chen J, Zhang J. Porous chitosan–gelatin scaffold containing plasmid DNA encoding transforming growth factor- 1 for chondrocytes proliferation. Biomaterials 2006; 27: 1095-1103. [414] Shi S, Cheng X, wang J, Zhang W, Peng L, Zhang Y RhBMP-2 microspheres-loaded chitosan/collagen scaffold enhanced osseointegration: An experiment in dog. J Biomater Appl 2009; 23: 331-346. [415] Mao JS, Cui YL, Wang XH, Sun Y, Yin YJ, Zhao HM, Yao KD. A preliminary study on chitosan and gelatin polyelectrolyte complex cytocompatibility by cell cycle and apoptosis analysis. Biomaterials 2004; 25: 3973-3981. [416] Mao JS, Zhao LG, Yao KD, Shang QX, Yang GH, Cao YL. Study of novel chitosan– gelatin artificial skin in vitro. J Biomed Mater Res 2003; 64A: 301-308. [417] Park KE, Jung SY, Lee SJ, Min B-M, Park WH. Biomimetic nanofibrous scaffolds: Preparation and characterization of chitin/silk fibroin blend nanofibers. Int J Biol Macromol 2006; 38: 165-173. [418] Yoo CR, Yeo I-S, Park KE , Park JH, Lee SJ, Park WH, Min B-M. Effect of chitin/silk fibroin nanofibrous bicomponent structures on interaction with human epidermal keratinocytes. Int J Biol Macromol 2008; 42: 324–334. [419] Kathuria N, Tripathi A, Kar KK, Kumar A. Synthesis and characterization of elastic and macroporous chitosan–gelatin cryogels for tissue engineering. Acta Biomater 2009; 5: 406-418.

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[420] Tanabe T, Okitsu N, Tachibana A, Yamauchi K. Preparation and characterization of keratin–chitosan composite film. Biomaterials 2002; 23: 817-825. [421] Yu B-Y, Chou P-H, Chen C-A, Sun Y-M. Kung S-S. C3A Cell behaviors on micropatterned chitosan–collagen–gelatin membranes. J Biomater Appl 2007; 22; 255274. [422] Jiankang H, Dichen L, Yaxiong L, Bo Y, Hanxiang Z, Qin L, Bingheng L, Yi L. Preparation of chitosan–gelatin hybrid scaffolds with well-organized microstructures for hepatic tissue engineering. Acta Biomater 2009; 5: 453-461. [423] Zhu A, Zhao F, Ma T. Photo-initiated grafting of gelatin/N-maleic acyl-chitosan to enhance endothelial cell adhesion, proliferation and function on PLA surface. Acta Biomater 2009; 5: 2033-2044. [424] Nagahama H, Divya Rani VV, Shalumon KT, Jayakumar R, Nair SV, Koiwa S, Furuike T, Tamura H. Preparation, characterization, bioactive and cell attachment studies of achitin/gelatin composite membranes. Int J Biol Macromol 2009; 44: 333-337. [425] Thein-Han WW, Saikhun J, Pholpramoo C, Misra RDK, Kitiyanant Y. Chitosan– gelatin scaffolds for tissue engineering: Physico-chemical properties and biological response of buffalo embryonic stem cells and transfectant of GFP–buffalo embryonic stem cells. Acta Biomater 2009; 5: 3453-3466. [426] Altman AM, Gupta V, Ríos CN, Alt EU, Mathur AB. Adhesion, migration and mechanics of human adipose-tissue-derived stem cells on silk fibroin–chitosan matrix. Acta Biomater 2009; doi:10.1016/j.actbio.2009.10.034. [427] Yang M-C, Wang S-S, Chou N-K, Chi N-H, Huang Y-Y, Chang Y-L, Shieh M-J, Chung T-W The cardiomyogenic differentiation of rat mesenchymal stem cells on silk fibroin–polysaccharide cardiac patches in vitro. Biomaterials 2009; 30: 3757-3765. [428] Li K, Wang Y, Miao Z, Xu D, Tang Y, Feng M. Chitosan/gelatin composite microcarrier for hepatocyte culture Biotechnol Lett 2004; 26: 879-883. [429] Yang K-C, Wu C-C, Lin F-H, Qi Z, Kuo T-F, Cheng Y-H, Chen M-P, Sumi S. Chitosan/gelatin hydrogel as immunoisolative matrix for injectable bioartificial pancreas. Xenotransplantation 2008; 15: 407-416. [430] Yang K-C, Wu C-C, Cheng Y-H, T-F Kuo, Lin F-H. Chitosan/gelatin hydrogel prolonged the function of insulinoma/agarose microspheres in vivo during xenogenic transplantation. Transplantation Proceedings 2008; 40: 3623-3626. [431] Hong H, Liu C, Wu W. Preparation and characterization of chitosan/PEG/gelatin composites for tissue engineering. J Appl Polym Sci 2009; 114: 1220-1225. [432] Liu H, Yin Y. Yao K. Construction of chitosan–gelatin–hyaluronic acid artificial skin in vitro. J Biomater Appl 2007; 21: 413-430. [433] Natarajan N, Shashirekha V, Noorjahan SE, Rameshkumar M, Rose C, Sastry TP. Fibrin-Chitosan–gelatin Composite Film: Preparation and Characterization. J Macromol Sci Part A: Pure Appl Chem 2005; 42: 945-953. [434] Bazargan-Lari R, Bahrololoom ME, Nemati A. Preparatoion asnd mechanical evaluations of a novel keratain-chitosan–gelatin film. World Appl Sci J 2009; 7: 763768. [435] Peter M, Binulal NS, Nair SV, Selvamurugan N, Tamura H, Jayakumar R. Novel biodegradable chitosan-gelatin/nano bioactive glass ceramic composite scaffolds for alveolar bone tissue engineering. Chem Eng J 2008: doi:10.1016/j.cej.2010.02.003

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In: Recent Developments in Bio-Nanocomposites… ISBN 978-1-61761-008-0 Editor: Ashutosh Tiwari © 2011 Nova Science Publishers, Inc.

Chapter 16

NANOMATERIALS IN THE ADVANCEMENT OF ELECTROCHEMICAL DNA BIOSENSORS Anees A. Ansari1 King Abdullah Institute for Nanotechnology, King Saud University, Riyadh-11451 P.O Box-2455, Saudi Arabia

ABSTRACT Nanomaterials are increasingly used for the construction of electrochemical DNA biosensors. Nano-scale materials offer excellent prospects for interfacing biological recognition events with electronic signal transduction for designing a new generation of bioelectronic devices exhibiting novel functions. Particularly, nanomaterials such as noble metal nanoparticles (Au, Ag, Pt, Pd), carbon nanotubes (CNTs), magnetic nanoparticles, quantum dots and metal oxide nanoparticles have been actively investigated for their applications in electrochemical DNA biosensors, which have become a new interdisciplinary frontier between biological sciences and material science. In this article, discussed some main advances and explore the application prospects and discuss the issues, approaches, and challenges, with the aim of stimulating a broader interest in the development of nanomaterial-based electrochemical DNA biosensors.

1. INTRODUCTION Since last decade, nanomaterials have emerged as a new frontiers of multidisciplinary science (with roots in biology, chemistry, and engineering), and is presenting an array of both opportunities and challenges across all areas of biomedical sciences. In particular, rapid progress with the use of engineered nanomaterials with diameters from 10 to 100 nanometers has led to significant new advances in detection of biological molecules, organism and in bioprocess [1-3]. Because of the nature of their targets, biosensors need to be faster, smaller, more sensitive, and more specific than nearly all of their physicochemical counterparts or the 1 E-mail: [email protected].

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traditional methods that they are designed to replace [4]. Rapid and accurate detection of biomolecules are essential parameters in medical diagnosis as it permits for fast treatment and does not allow patients to be lost to follow-up. Small size and greater sensitivity mean lessinvasive sampling and detection of molecules such as neurotransmitters or hormones at biologically-relevant levels [2,4]. Greater specificity allows assays to be performed in complex fluids such as blood or urine without false negative or false positive results. Nanotechnology promises to improve biosensing on all of these fronts [1]. Nanofabricated materials can bind directly to biomolecules and/or act as transducers to extremely small and sensitive detectors. The unique electrical, chemical, thermal and catalytic properties of namomaterials offer excellent prospects in the development of electrochemical DNA biosensors [1-4]. These electrochemical biosensing devices have received considerable recent attention in connection to the detection of DNA hybridization [3]. The high sensitivity of such devices, coupled to their compatibility with modern microfabrication technologies, portability, low cost (disposability), minimal power requirements, and independence of sample turbidity or optical pathway, make them excellent candidates for DNA diagnostics. Nanomaterials are opening new horizons for the application of electrochemical DNA biosensors. The applications of nanomaterials in electrochemical biosensors have been reviewed recently [1-4]. Wang et al., suggested that nanomaterials are extremely useful in the fabrication of electrochemical DNA biosensing devices [3]. There are many reports are available in literature on direct electrochemistry of redox active probe single strand DNA (ssDNA) immobilized onto nanoparticle-modified electrodes [2, 4-6]. These nanostructured modified electrodes not only improve the catalytic activity of the transducer but also promote the enzymatic reaction on the electrode surface. The enhanced electrochemistry is due to the ability of the small nanoparticles to reduce the distance between the redox site of a protein and the electrode, since the rate of electron transfer is inversely dependent on the exponential distance between them. A range of nanostructured including nanotubes, nanobelt, nanofibers, nanorods, nanocomb and nanowires, prepared from metals, semiconductor, carbon or polymeric species, have been widely investigated for their ability to enhance the response of biosensors [2,4]. Nanoparticles can be used in a variety of ways, such as modification of electrode surfaces, or to modify biological receptor molecules such as enzymes, antibodies or ODNs. Some successes of nanostructured materials have been ascribed due to their ability to improve the features of bioassays, allowing miniaturization and speed, reducing reagent and sample consumption, and facilitating the performance of heterogeneous formats [2]. This article highlighted the recent developments of nanomaterials based electrochemical DNA biosensensing devices for detection and quantification of biomacromolecules or disease cause pathogens and discusses future considerations and opportunities for advancing the use of electrochemical sensors for clinical diagnostics.

2. ELECTROCATALYTIC PROPERTIES OF NANOMATERIALS Electrocatalytic properties of novel nanomaterials display a fundamental role in the organization of electrochemical biosensing devices. A variety of nanostructures have been investigated to determine their properties and proposed their possible applications in the

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development of electrochemical biosensing devices [1-5]. These nanomaterials include metal nanoparticles, oxide nanoparticles, semiconductor nanoparticles, polymeric nanomaterials, carbon nanotubes and even nanocomposite materials have been widely used in the construction of electrochemical biosensors because their unique electrocatalytic properties. Such as metal-nanoparticles (e.g.,platinum) catalyze the redox process of some biomolecules with analytical interest, which can be monitorized using electroanalytical techniques. Nanomaterials have high surface-to-volume ratio, electro-catalytic activity as well as good biocompatibility and novel electron transport properties make them highly attractive materials for ultra-sensitive detection of biological macromolecules via bio-electronic devices [4]. Some nanoscale materials exhibited remarkable electron transport properties, which are strongly depend on their nanocrystalline structure. Morphological based nanomaterials show new capabilities that are generated by combination of novel nanobuilding units and strategies for assembling them. These extraordinary electrocatalytic characteristics of the nanomaterials are exploiting in the fabrication of an efficient electrochemical bio-recognition devices. The surface features of nanoscale materials such as shape, size, diameter, surface condition, crystal structure and its quality, chemical composition, crystallographic orientation along the axis etc are very important parameters, all of which influence the electron transport mechanism of the nanomaterials [3,4]. Particular morphology of nanomaterials include nanotubes, nanofibers, nanorods, nanowires, nanocombs, nanodisc, nanorings nanoparticles have different electrical conductance and tune the surface behavior of the deposited thin film on the substrate [2-4]. Moreover, the electron transport properties of the nanomaterials can also be altered by introduced some doping materials into the matrix, which enhance the surface properties and electrical conductance of the nanomaterials. The small change in the surface properties of the nanomaterials can cause remarkable change in the transport behavior. It has been reported that the change in electrical conductivity of the bio-electrode is influenced by minor surface perturbations such as binding of bio-macromolecular species on a long conduction channel. 1D semiconductor electronic nanomaterials, in particular, have active surfaces that can easily be modified for immobilization of numerous biomolecules [4]. Additionally, the sizes of biological macromolecules, such as proteins and nucleic acids are comparable to nanoscale building blocks. Therefore, any interaction between such molecules should induce significant changes in the electrical properties of 1-D nanostructures. Due to the extreme smallness of these nanomaterials, it is possible to pack a large number of biomacromolecule-functionalized nanomaterials onto a remarkably small footprint of an array device. These nanometer-scale electronic transducers reduce the pathway for direct electron communication between redox biomolecule to the electrode for sensitive and speedily detection of analyte without any hindrance [2,3]. In this article illustrate the usefulness of nanoscale materials for the designing of efficient electrochemical DNA sensing device and also highlight the potential analytical applications in terms of nanostructured based electrochemical biosensors and bioreactors. All these properties of the nano-scale materials strongly depend on the synthesis procedures used to grow them. Therefore, extensive efforts have been made to synthesized novel morphological based nano-size materials such as nanowires, nanorods, nanotubes, nanofibers, nanobelts and nanorings, etc., because these morphological nano-size materials based electrochemical biosensing devices show higher performance (sensitivity, selectivity, and real time detection limit) compared to those fabricated from other forms of the nanomaterials [4]. These novel

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nanomaterials with control size, shape and structure can be tuned by altering the physical, chemical and biological routes.

3. PREPARATION METHODS OF NANOMATERIALS Various preparation techniques have been proposed for the deposition of thin film on the conductive electrode surface for the fabrication of electrochemical bio-transducers. Extensive and successful efforts have been made for deposition of thin film using by numerous techniques including electrochemical, electrophoretic, pulse laser deposition, Rf-magnetron sputtering, reactive evaporation, spray pyrolysis, solution growth, molecular beam epitaxy, metal organic chemical vapor deposition (MOCVD) and plasma-enhanced chemical vapor deposition (PECVD) have been employed for the growth of nanostructured thin films [7]. Among numerous deposition techniques of thin film on the electrode surface, electrochemical deposition is an attractive choice for the preparation of stoichiometric and high quality nanomaterials film for biosensing applications. Because electrochemical deposition can control of the surface properties of the film such as morphology, size, thickness, length, diameter, orientation, and alignment on electrode surfaces (especially this last property fundamental to control the final analytic response). This is possible because all the electroanalytic parameters-such as the potential value, the current density, the deposition time, the electrical charge required for the growth, the supporting electrolyte and its ionic strength, the properties of the doping agents (due to the presence of specific functional groups, acting as stabilizing agent toward the polymeric films), and the pore membrane dimensions-can be controlled. These parameters played a crucial role in the designing of sensitive electrochemical biosensing devices.

4. METHODS OF DNA IMMOBILIZATIONS To prepare electrochemical DNA biosensor, the immobilization of DNA probe in a predictable manner on the substrate play a crucial role in a successful development of biosensors. The basic requirement for construction of a successful electrochemical biosensing transducer is that the biological material should bring the physico-chemical changes in close proximity of a transducer [8]. In this direction, immobilization strategy of biomacromolecules on the nanomaterials substrate has played a major role. Immobilization not only helps in forming the required close proximity between the biomaterial and the transducer, but also helps in stabilizing it for reuse. Biological material has been used for direct immobilization on the surface of fabricated transducer or in most cases a membranes, which can subsequently be mounted on the transducer [9]. Biomacromolecules can be immobilized either through adsorption, entrapment, covalent binding, cross-linking or a combination of all these techniques. A large number of techniques have been proposed in research literature for the immobilization of viable and non-viable cells as well as cell–enzyme conjugates. Selection of a technique and/or support would depend on the nature of the biomaterial and the substrate and configuration of the transducer used. The choice of support and technique for the preparation of membranes has often been dictated by the low diffusion resistance of the

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membrane. Moreover, the choice of immobilization technique mainly depend on the probe DNA to be immobilized, the nature of the solid surface and the transducing mechanism. Therefore, a successful transducing surface required some important parameters including selection of electrode materials, biocompatibility, nontoxicity, absence of diffusion barriers, stability with changes in temperature, pH, ionic strength or macro-environment, sufficient sensitivity and selectivity for the analyte of interest as well as low cost and ease of mass production [8,9]. The immobilization of the sensing bioelement (probe), which specifically recognizes the analyte (target), onto a transducing surface, is the key-step in the construction of biosensing devices. There are many methods to immobilize the bio-macromolecules such as adsorption, physical entrapment in gels or membranes, cross-linking, covalent binding, entrapment, encapsulation and others as use of solid binding matrices. The immobilization matrix may function purely as a support or may also be concerned with mediation of the signal transduction mechanism. The purpose of any immobilization method is to retain maximum activity of the biological component on the surface of the transducer. The selection of an appropriate immobilization method depend on the nature of the biological element, type of the transducer used, physic-chemical properties of the analyte and operating conditions for the biosensor [10]. Physical adsorption of the bio-component based on vander-Waals attractive forces is the oldest and simplest immobilization method. Generally, the adsorption of bio-macromolecules directly onto naked surfaces of bulk materials may frequently result in their denaturation and loss of bioactivity. However, the adsorption of such biomacromolecules onto the surfaces of nanosized materials can retain their bioactivity because of the biocompatibility of nanoparticles. Since most of the nanosized materials carry charges, they can electrostatically adsorb biomolecules with different charges [11]. Besides the common electrostatic interaction, some nanosized materials can also immobilize biomolecules by other interactions. For example, it is reported that gold nanoparticles can immobilize ssDNA through the covalent bonds formed between the gold atoms and the amine groups and cysteine residues of proteins [12].

5. APPLICATIONS OF NANOMATERIAL FOR ELECTROCHEMICAL DNA BIOSENSORS Recent advances in the development of such electrochemical biosensing devices open new opportunities for DNA diagnostics. DNA biosensors, based on nucleic acid recognition processes are rapidly being developed towards the assay of rapid, simple and economical testing of genetic and infectious diseases. Electrochemical detection of DNA hybridization usually involves monitoring of a current response, resulting from the Watson–Crick base-pair recognition event into a readable analytical signal, under controlled potential conditions. A basic DNA biosensor is designed by the immobilization of a single stranded (ss) ODN (probe) on a transducer surface to recognize its complementary (target) DNA sequence via hybridization [2,3]. The probe-coated electrode is commonly immersed into a solution of a target DNA whose nucleotide sequence is to be tested. When the target DNA contains a sequence which matches that of the immobilized ODN probe DNA, a hybrid duplex DNA is formed at the electrode surface is known as a hybrid. Such hybridization event is commonly

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detected via the increased current signal of an electro-active indicator (that preferentially binds to the DNA duplex), in connection to the use of enzyme labels or redox labels, or from other hybridization-induced changes in electrochemical parameters (e.g. capacitance or conductivity). In the following sections, highlight the nanomaterials application involved in construction of portable electrochemical DNA biosensing devices. As illustrated the success of such devices requires a proper combination of nanomaterials surface chemistries, DNArecognition and electrical detection protocols.

5.1. Polymeric Nanomaterials for Electrochemical DNA Biosensors Mostly polymeric nanomaterials used for the bio-macromolecule immobilization are conducting polymers including polyaniline, poly(phenylenevinylene), polypyrrole, polythiophene polyacetylene and polyindole [10]. The unique electronic structure of polymeric nanomaterials which is responsible for their remarkable high electrical conductivity, ease of processibility, low ionization potentials, good environmental stability and high electron affinity [10,13]. Conductivity exhibits a strong dependence on solution pH and oxidation state. Conducting polymeric materials retain the exclusive properties of nanomaterials like as large surface area, size, and quantum effect, which further increase the merit of conducting polymers in designing and making novel biosensors [13,14]. In terms of biological applications, the thickness and shape of the polymeric film which is most important factor to control the electrochemical characteristics of the transducers can be easily controlled in the nanometer to micrometer range by the modification in deposition method. These excellent properties of the polymeric nanomaterials provide better signal transduction, enhanced sensitivity, selectivity, durability, biocompatibility, direct electrochemical synthesis and flexibility for the immobilization of biomolecules, including DNA [10]. Versatility of these polymers are determined by the following: its biocompatibility; capability to transduce energy arising from interaction of analyte and analyte-recognizing-site into electrical signals that are easily monitored; capability to protect electrodes from interfering materials; easy ways for electrochemical deposition on the surface of any type of electrodes. Nowadays polymeric nanomaterials are becomes major tools for nanobiotechnological applications. Thin films of polymeric materials having both high conductivity and fine structure in nanoscale are a suitable substrate for immobilization of single strand-ODNs to electrochemical DNA hybridization detection. Nie et al., [15] presented simple and label-free electrochemical sensor for detection of DNA hybridization based on nanostructured conducting polymer, poly(indole-6-carboxylic acid). Covalently grafted 18-mer aminosubstituted ODN probe onto the polymer surface displayed dynamic determination range for complementary target ODN from 3.5×10−10 mol L−1 to 2.0×10−8 mol L−1 and the corresponding detection limit was 5.79 pmol L−1[15]. Sexually transmitted disease (Neisseria gonorrhoeae) biosensor was developed on electrochemically polymerized nanostructured polyaniline(nsPANI) film deposited onto ITO(ITO) electrode. The probe ssDNA was covalent attached to the functionalized nanostructured polyaniline surface through crosslinking agent avidin–biotin coupling agent. The nsPANI amplify DNA recognition and transduction events, which is applied for ultrasensitive electrochemical detection of target DNA. Author observed improved detection limit of complementary target ODN upto

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0.5×10−15M within 60 s of hybridization time at 25oC.The proposed approach is highly sensitive and selective for detection of specific nucleic acid and can be distinguish presence of N. gonorrhoeae from Neisseria meningitidis and Escherichia coli culture and spiked samples from the urethral swabs of the patients. This biosensor was used for clinical samples [16]. Ghanbaria et al., [17] have applied electrochemically deposited nano-structured polypyrrole film onto Pt electrode for DNA sensing. Scanning electron microscopy (SEM), cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) were used to analyze the surface morphology and analytical characteristics of the electro polymerized polypyrrol film deposited on the Pt electrode. The proposed biosensor has good dynamic range, correlation coefficient (0.05–1.0 μM and 0.9983, respectively) and low detection limit (0.02μM)[17]. In addition to nanowires, nanofibers, nanotubes and nanorods of polymeric materials has growing interest in the design and development of electrochemical transducers. The ease of fabrication and ability to manipulate their electrical, magnetic, and optical properties make them attractive for the construction of DNA biosensing devices. Nanotubes of conducting polymers make a channel for transferring the electron from the redox active site of ssDNA molecule to electrode surface. Chang et al., [18] have been grow highly organized conducting polyaniline nanotube under a well-controlled nanoscale dimension on the graphite electrode using a magnetron sputtering method, followed by two-step anodization in oxalic acid at 40 V and 4 °C to create an alumina template of nanopore array. This process allows orientation and location control of the nanotubes, which are applied to immobilized 21-mer oligonucleotides (ODN) probes for the fabrication of electrochemical DNA biosensor. The analytical characteristics of the resulting biosensors were optimized using by differential pulse voltammetry (DPV). Conducting PANI nanotube arrays have signal enhancement capability, allowing the DNA biosensor to readily detect the target ODN at a concentration as low as 1.0 fM (~300 zmol of target molecules). The fabricated biosensor display good capability of differentiating the perfect matched target ODN from one-nucleotide mismatched ODNs even at a concentration of 37.59 fM [18]. Due to high mechanical and chemical stability and good electrical conductivity polymeric materials amplify the electrochemical signal for sensitive detection of analyte. In this context a number of reports have been published in the literature [19-25]. The majority of approaches detecting hybridization events involve the covalent attachment of appropriate ODNs at conductive electrode substrates including inherently conducting polymers. For example, it has been shown that ODNs can be covalently attached to polyaniline monomers forming electrochemically conductive electroactive copolymers. Alternatively, ODNs directly covalently attached to polymeric surface after polymer synthesis. According to them, the conductance of the polymeric materials was changed upon interaction with the complementary DNA, enabling the sensing of the ODN. The specific hybridization of grafted ODNs with the complementary nucleotide target induces a modification of the electrochemical behavior of the conductive polymer backbone. When non-complementary DNA (one base mismatch) was introduced in the sample, no response was observed, verifying the specificity of the sensor [25]. Additionally, in comparison to metal nanoparticles, conducting polymeric nanomaterials have some advantages including low-temperature synthesis, tunable conductivity, and no need for purification, endopening, or catalytic deposition processing. Unfortunately, polymeric nanomaterials are usually less favorable as the element in biosensor construction

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because of their relative low conductivity than the carbon nanotube as well as their nonoriented nanofiber morphology, leading to low detection sensitivity.

5.2. Metal Nanoparticles for Electrochemical DNA Biosensors Noble metal nanoparticles are opening new horizons in the application of analytical chemistry. Due to their special sizes, noble metal nanomaterials display novel physical and chemical properties, such as the nanoscale surface effect etc. Catalysis effect is another outstanding characteristic of the transition metal nanomaterials, especially the noble metals, which extensively applied for many chemical synthesis reactions [2-4,12]. Similarly, metal nanoparticles are also both heterogeneous and homogeneous catalysts. The catalysis takes place on the active sites of the surface of metal nuclei (i.e. the mechanism is similar to conventional heterogeneous catalysis). Owing to superior stability and complete recovery in biochemical redox processes, noble metal nanoparticles have been applied as catalysts in numerous biomedical applications [12]. These noble metal nanoparticles are widely recognized as an ideal support in fabrication of electrochemical biosensors. Metallic nanoparticles not only improve the sensing property of the biomolecules but also enhanced the electron communication rate in between redox active ssDNA species and electrode surface. Many researchers have explored the properties of Au, Ag, Pt and Pd nanoparticles for the designing of amperomatric bioelectronic device [26-29]. Mirkin reported on gold nanoparticle-based electrochemical DNA chips [64]. Wang and his coworkers [26] were developed powerful stripping voltammetry electroanalytical technique based on metal nanoparticles for determination of trace amount of target DNA hybridization. This technique is highly sensitive and offers remarkably low detection limits (picomolar). Recent activity has led to highly sensitive nanoparticle-based stripping electrical bioassays applied for electrochemical DNA sensors with sensitivities in the pico- and femtomolar range [27, 28]. Similar group in another approach have applied nanoparticlebased protocol for detecting DNA hybridization based on a magnetically induced solid-state electrochemical stripping detection of metal tags [29]. Zhu et al.,[30] employed multi-walled carbon nanotubes(MWCNTs) and Pt nanoparticles dispersed in Nafion modified onto GCE for construction of sensitivity-enhanced electrochemical DNA biosensor. Nafion is a biopolymer has excellent film forming ability enhanced the higher loading of the ODN on the bioelectrode for lower range determination of target DNA. The performance of the biosensor showed sensitive determination of DNA hybridization with a linear concentration from 2.25×10−7 to 2.25×10−11 mol l−1with detection limit 1.0×10−11 mol l−1. Qing et al.,[31] electrodeposited Pt nanoparticles on GCE surface for electrochemical hybridization determination of specific deoxyribonucleic acid sequence in genetically modified soybean. A linear calibration graph was observed for the complementary DNA over a concentration range of 2.14 × 10–9–2.14 × 10– 7 M and detection limit 1.0 × 10–9 M. Chang et al.,[32] applied palladium(Pd) nanoparticles combined with MWCNTs dispersed in Nafion modified on GCE showed much enhanced signal for the sensitive determination of target DNA hybridization. The DPV of the electrode before and after hybridization was determined in the presence of a MB as an indicator at -0.32 V. Due to large surface area of Pd nanoparticles and MWCNTs accelerate the electron transfer rate of redox MB for selective and sensitive determination of target DNA hybridization signal. The resulting electrochemical biosensor showed linearity for

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target DNA from 7.5x10-13 to 2.3x10-9 M and detection limit 1.2-10-13 M. Gold nanoparticles are another important nanomaterial using for DNA hybridization detection. In their approach, thiol molecules are used to stabilize gold nanoparticles by covalent Au–S bonds. In addition, strong covalent bond between gold nanoparticles and –SH groups could offer an opportunity to construct multilayer films using cross-linkers with these functional groups. The chemical bonds formed between the Au nanoparticles and the enzymes facilitate the redox process and enhance the performance of the biosensor. At solid electrode surfaces, the electrochemical oxidation of DNA is associated with the irreversible oxidation of guanine and adenine residues, with a great enhancement of the analytical signal. A novel method for selective and sensitive recognition of complementary DNA by chemically grafting probe ssDNA onto functionalized gold nanoparticles were presented by Glynou et al.[33]. Gold nanoparticles amplify DNA recognition and transduction events, which may be used as an ultrasensitive method for electrical biosensing of DNA or proteins. Multilayered uniform self-assembled structure have formed for co-adsorption of probe ssDNAfunctionalized gold nanoparticles as scaffolds for the construction of a via the hybridization of complementary target DNA. Self assembled monolayer required very small amount of ODN for covalent attachment to the surface functional groups, resulting in the binding of desired molecule in the near vicinity of the electrode surface and act as a molecular wire between biomolecule and the electrode surface. The resulting biosensor showed an enhanced peak current due to the multilayer gold nanoparticles not only provide a biocompatible microenvironment for the protein to undergo direct electron transfer reactions but also amplify the electrochemical signal by increasing the binding sites for the protein immobilization. The proposed biosensor was linear in the concentration range from 2 × 10−9 to 1 × 10−7 M with a detection limit of 6.7 × 10−10 M [34]. Hu et al.,[35] developed nanoporous gold electrode and multifunctional encoded Au nanoparticle for designing of sensitive electrochemical DNA sensor. The multifunctional encoded Au nanoparticles amplify the detection signal efficiently and could detect the DNA target quantitatively, in the range of 8.0 × 10-17-1.6 × 10-12 M and low detection limit upto 28 aM. Yang et al.,[36] described electrochemical impedance measurements for detection of sequence-specific DNA, related to phosphinothricin acetyltransferase (PAT) trans gene in the transgenic plants, based on electro-polymerized poly-2,6-pyridinedicarboxylic acid film on GCE. A layer of Au nanoparticles were assembled on the fabricated electrode for covalent adsorption of probe ssDNA on the electrode surface. The hybridization events were monitored by CV and DPV measurements of the immobilized probe ssDNA using by MB as indicator and EIS. The hybridization event led to a decrease of impedance values (Ret) reflecting the reduction of the electrode resistance. The difference of Ret value between the ssDNA/NG/PDC/GCE and hybridization DNA-modified electrode (dsDNA/NG/PDC/GCE) was used as the signal for detecting the PAT gene fragment with the dynamic range from 1.0x10-10 to 1.0x10-5mol/L with detection limit of 2.4x10–11 mol/L [36]. The high efficiency of the biosensor arises from the combination of the electrocatalytic properties of Au nanoparticles with the biocompatibility and flexibility of the polymeric materials. The high sensitivity, selectivity and long lifetime of DNA sensors depends on the immobilization procedure of DNA probes onto electrode surfaces. Zhang et al.,[37] described electrochemical entrapped ssDNA molecules into polymeric film subsequently gold nanoparticles introduced using for DNA sensor. The polymeric materials enhanced the enzyme loading and stability of the bioelectrode and amplify the DNA hybridization signal efficiently. Whereas, gold

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nanoparticles promote the electron transfer reaction on the electrode surface for fast response time of the analyte detection. A glassy electrode modified gold nanoparticles/cysteamine /poly glutamic acid applied for immobilization of probe ssDNA linked covalently to the gold nanoparticles through 5 thiol-linker. DPV technique was used for monitoring the DNA hybridization events. The measured results indicated that the reduction peak current was linearly increased with increasing the concentration of complementary target DNA from 9.0×10-11 to 4.8×10−9 M with a detection limit of 4.2×10-11 M [37]. In another approach carboxylic group functionalized MWCNTs were assembled on to electropolymerized aminobenzoic acid film on the surface of the GCE for the detection of target DNA [38]. The biosensor showed linear response within the concentration range of complementary ODN from 1.0×10−12 to 5.0×10−9 M with a detection limit of 3.5×10−13 M [38]. Similar group developed the DNA biosensor based on by layer-by-layer covalent attachment of gold nanoparticles (GNPs) and thiol group functionalized MWCNTs on an Au electrode [39]. The electrostatic layer-by-layer selfassembly onto CNTs carriers maximizes the ratio of DNA tags per binding event to offer the greatest amplification factor reported to date (showing that the probe DNA activity increases with the number of DNA layers). SEM, FTIR and CV were used for confirmation of the alteration in surface morphology after immobilization of probe ssDNA to the carboxylic group and formation of product in the appropriate form. Due to the ability of CNTs to promote electron-transfer reactions, the high catalytic activity of gold nanoparticles and the sensitivity of presented electrochemical DNA biosensors are remarkably improved. The proposed DNA biosensor has excellent selectivity, reproducibility and stability in DNA hybridization assay. The detection limit of the method for target DNA was 6.2 pM and response current showed linearity in a wide concentration range of target DNA from 5.0×10−10 to 1.0×10−11M [39]. In another approach of this group presented amperomatric DNA biosensor based on silver nanoparticles/poly(trans-3-(3-pyridyl) acrylic acid) (PPAA)/ with (MWCNTs–COOH)modified GCE [40]. The electropolymerized film onto carboxyl groups functionalized MWCNTs electrode and electrodeposited silver nanoparticles on the composite film modified onto GCE. The hybridization events were monitored by DPV measurements of the intercalated adriamycin. This biosensor showed excellent electrochemical performance during DNA hybridization assay such as high sensitivity, reproducibility, stability and long linear concentration range from 9.0 x 10-12 to 9.0 x 10-9 M with a detection limit of 3.2 x 10-12 M [40]. Polyamidoamine and 3-mercaptopropionic acid modified Au electrode was used for immobilization of DNA on gold nanoparticles to obtain a stable recognition layer through biotin–avidin combination to detect complementary target, using signal amplification with Au nanoparticles and [Ru(NH3)6]3+ as redox electro-active indicators [41]. The resulting biosensor showed a dynamic detection range of the sequence-specific DNA from 1.4×10−11 to 2.7×10−14 mol L−1 and the detection limit 1.4×10−14 mol L−1. This DNA biosensor revealed low detection limit and excellent selectivity against two-base mismatched DNA [41]. Electrochemically deposited gold nanoparticles and then zirconia (ZrO2) film modified onto GCE was used for electrochemically detection of DNA hybridization. MB used as redox intercalator for identification of DNA hybridization. The sequence-specific detection of DNA hybridization of PAT gene in the transgenic plants was detected with a detection range from 1.0 x 10-10 to 1.0 x 10-6 mol/L, and detection limit of 3.1 x 10-11 mol/L [42]. Electrochemical performance of probe DNA assembled onto colloidal gold nanoparticles and carboxyl group-

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functionalized CdS nanoparticles modified Au electrode was well preserved [43]. Due to the high surface energy of Au nanoparticles increase the electrode surface area for more binding amount of CdS and finally enhance the electrochemical responses. CdS nanoparticles were used for simple covalent linking method of carboxyl acid group functionalized CdS with amino group of cysteine. The DNA immobilization and hybridization on the exterior CdS nanoparticles was characterized with the use of Co(phen)2 2+ as an electrochemical indicator. The biosensor quantified at a linear range from 2.0×10−10 to 1.0 ×10−8 M, with a detection limit of 2.0×10−11 M [43]. Ding et al.,[44] utilized highly sensitive bioelectronic protocols for sequence specific detection of target DNA based on thiol-functionalized capture DNA immobilized on the modified GCE-electrode. Modified gold nanoparticles with CdS nanoparticles were applied to amplify the detection signal by an amidization reaction between bio-bar code binding DNA on the surface of Au NPs and mercapto acetic acid on the surface of CdS NPs. Electrochemical performance of the resulting biosensor was optimized within the concentration range of target DNA from 1.0×10−14 to 1.0×10−13 M. A detection limit of4.2×10−15 M of target DNA was achieved [44]. Du et al. [45] reported a novel and sensitive sandwich electrochemical DNA biosensor based on the amplification of magnetic microbeads and Au nanoparticles modified with bio bar code and PbS nanoparticles. This involves a sandwich bioassay based on magnetic microspheres were coated with 4 layers polyelectrolytes in order to increase carboxyl groups on the surface of the magnetic microbeads, which enhanced the amount of the capture DNA. The modified magnetic microbeads shows improved sensing performance of the bioelectrode and amplify the electrochemical signal also, such as amount of DNA loading, sensitivity, selectivity and low detection limit. The present DNA biosensor showed linear relationship of the target DNA within the concentration range from 2.0×10−14 M to 1.0×10−12M and detection limit upto 5.0×10−15M [45]. A novel strategy was proposed by Hu et al., [46] based on electrochemical stripping assay for ultrasensitive detection of target DNA hybridization. Semiconductor PbS nanoparticles used as a tag for DNA hybridization detection and electrochemical stripping measurement of the lead ions. The fabricated nanoporous gold electrode modified with single-stranded DNA (ssDNA) and Au nanoparticles co-loaded with two kinds of ssDNA could detect target DNA upto femtomolar and exhibited excellent selectivity against one-base mismatched DNA and non-complementary DNA. The resulting DNA biosensor demonstrated a good linear relationship with the target DNA concentration in the range of 9.0×10−16 to 7.0×10−14 M with detection limit 2.6×10−16 M [46]. Electrochemical detection of short DNA ODN of the avian flu virus H5N1 with sequence 5’-CCA AGC AAC AGA CTC AAA-3’ on gold electrode surface was performed by Ting et al., [47] in connection with silver nanoparticles as a label conjugated with a well-known DNA intercalator, doxorubicin. The observed Ag/AgCl redox process signal of the silver nanoparticle labels was subsequently used to quantify the amount of DNA. The proposed DNA biosensor achieved the detection limit upto1 pM [47]. Kong et al.,[48] described ultrasensitive electrical detection method of nucleic acids based on interdigited microelectrodes. The observed results suggested that the attached hematin molecules with hybridized DNA act as a catalyst to accelerate reducing ammoniacal silver ion to form silver nanoparticles. Alteration in conductance of the silver nanoparticles directly correlated with the number of the hybridized DNA molecules. Under optimized conditions the biosensor was

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sensitive upto1 fM. The proposed biosensor was also applicable to the detection of RNA. Zhang et al.,[49] have reported an attractive ultrasensitive electrochemical DNA biosensor based on highly characteristic solid state process. Functionalized silver nanoparticles with typical size 3-5 nm were used as electroactive label on the surface of gold electrode modified with thiolated natural probe PNA and 6-mercapto-1-hexanol as linker for detection of ODN from the H5N1 bird flu virus. The proposed biosensor has good response to DNA over a wide concentration range from 10 fM to 10 nM with detection limit upto 10 fM. Silver nanoclustermodified gold electrode has been constructed and used for the detection of DNA hybridization. The resulting biosensor showed highly linear calibration plot over the entire DNA concentration range from 500–2500 ng/ml [50].

5.3. Metal Oxide Nanoparticles for Electrochemical DNA Biosensors One-dimensional semiconductor metal oxide nanoparticles are new class advanced materials using in the design and fabrication of electrochemical biosensors [3,4,7,8]. These materials improve the analytical capacities of sensor devices which are highly desired. The inorganic ceramics exhibit relatively high mechanical strength, enhanced thermal stability and negligible swelling in both aqueous and organic solutions compared to most conventional materials. Nanometer-scale metal oxides based electronic biosensors offer high sensitivity and real-time detection. For example, due to the high surface-to-volume ratio of the metal oxide nanoparticles, the detection sensitivity of the constructed transducers may be increased to a single-molecular detection level by monitoring the very small change in conductance caused by binding of biomolecular species on a long conduction channel [7]. Nanostructured metal oxide electronic biosensors, in particular, have active surfaces that can easily be modified for immobilization of numerous biomolecules [8]. However, this advantage may not apply to many non-oxide semiconductor nanomaterials because their surfaces are not stable in an air environment, which leads to formation of an insulating native oxide layer and may degrade device reliability and sensitivity. Metal oxide nanoparticles based electrode solved this problem [7]. Although, many metal oxide based electrodes have been fabricated for detection of DNA hybridization. Feng et al., utilized CeO2/Chitosan composite matrix to immobilization of probe single-stranded DNA (ssDNA) for construction of DNA biosensor related to the colorectal cancer gene [51]. Chitosan introduced CeO2 nanocomposite matrix represented good biocompatibility, nontoxicity and excellent electronic conductivity, showing the enhanced loading of ssDNA probe on the surface of electrode. DPV was used to analyze the signal response of internal hybridization indicator MB and amount of colorectal cancer target DNA sequence. The proposed biosensor shows satisfactory reproducibility, selectivity and linearity in a wide concentration range from 1.59×10−111.16×10−7 mol L−1 with high detection sensitivity and observed highest hybridization efficiency at 45 oC [51]. The DNA hybridization sensitivity was dramatically enhanced by doping the SWCNTs and 1-butyl-3-methylimidazolium hexafluorophosphate hydrophobic room temperature ionic liquid into the nano-CeO2 nanocomposite modified onto GCE due to the synergistic effect. The SEM micrographs were used for recognition of the nanosized shuttle-shaped cerium oxide and immobilization of DNA on the electrode surface. DPV and CV were employed to

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examine the surface properties and electrochemical characteristics of the constructed transducers. In the range of 1.0×10−12 mol/L to 1.0×10−7 mol/L, and detection limit 2.3×10−13 mol/L was checked for detection of sequence specific DNA of phosphoenol pyruvate carboxylase gene [52]. Zhu et al.,[53] detected DNA hybridization onto zirconia (ZrO2) thin films modified gold electrode as a sensing platform using by DPV technique. ODN probes were covalently attached via phosphate group at 5’end to the electrodynamically deposited zirconia thin films onto the bare gold electrode. Methylene blue was utilized as a electroactive labeling indicator to investigate the electrochemical DNA hybridization assay. The linearity of the biosensor was estimated under the target DNA concentration ranging from 2.25×10−10 to 2.25×10−8 mol l−1 with detection limit of 1.0 × 10−10 mol l−1[53]. MWCNTs, ZrO2 nanoparticles doped chitosan-modified onto GCE was employed to immobilization of ODNs for sensitive detection of DNA hybridization using by electroactive daunomycin as an indicator [54]. Chitosan was chosen as the material to form the membrane due to its excellent film-forming and adhesion abilities, together with its nontoxicity and biocompatibility. Moreover, chitosan contains amino groups, thus providing a hydrophilic environment, which is compatible with the biomolecules. SEM analysis confirmed the presence of MWCNTs and ZrO2. Coupling of MWCNTs with chitosan and ZrO2 nanoparticles provides enhanced electroactive surface area for higher amount loading of probe DNA and excellent electron transfer ability between the ODNs and the electrode surface. The response of the fabricated biosensor was linear under the logarithm target DNA concentration range from 1.49×10−10 to 9.32×10−8 mol L−1 with detection limit 7.5×10−11 mol L−1[54]. Another strategy was proposed for the construction of DNA biosensor based on chitosan doped ZnO nanoparticles for voltammetric detection of DNA hybridization. The immobilization of the probe ssDNA is based on the absorption of the nanostructured ZnO [55]. Nanostructured ZnO greatly enhances the active surface available for ssDNA binding over the geometrical area. The resulting nanobiocomposite provides a shelter for the ODNs to retain its bioactivity under considerably extreme conditions and the ZnO nanoparticles in the biocomposite offer excellent affinity to probe DNA. The established biosensor was effective to discriminate the complementary target sequence and two-base-mismatched sequence, with a detection limit of 1.09 x 10-11 mol L-1 of complementary target [55]. Unfortunately, owing to some drawbacks of doped nanomaterials films for construction of bioelectronic transducers, especially their thickness and brittleness, the practical applications of ceramic materials may be improved by the alteration in the fabrication methods. Efforts have been made to seek for new process which could overcome their advantage for biomolecule immobilization in biosensor construction. In recent years, some investigators have developed sol-gel derived matrices for immobilization of desired biomolecules to construct the electrochemical biosensors. Sol–gel materices can be prepared under ambient conditions and exhibit tunable porosity, high surface area, biocompatibility, optical transparency, excellent thermal stability, chemical inertness and negligible swelling in aqueous and non-aqueous solutions. Besides this, a sol– gel derived nano-porous film can retain its bioactivity in a given micro-environment and can be used for direct electron transfer between DNA active sites and the electrode. The high biomolecule loading per unit area and the optical transparency of the glass makes this approach particularly suitable for electrical signal transduction methodologies. Ansari et al.,[56] exploited sol-gel derived nanostructured zinc oxide (ZnO) film deposited onto ITO glass substrate to immobilization of 20-mer thiolated ODN probe (th-ssDNA) for detection of

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target DNA(sexually transmitted disease (Neisseria gonorrhoeae)) using a hybridization technique. X-ray diffraction, UV-Visible and SEM were applied to confirm the crystalline nature and morphology of the nanostructured ZnO film before and after probe ssDNA immobilization. The response of the proposed biosensor was linear in the concentration range of target DNA from 0.000524 fmol–0.524 nmol, with a detection limit of 0.000704 fmol and hybridization time of 60 s[56]. A new approach have been developed by Zhu et al., based on Cu2O hollow microspheres consisted of Cu2O nanoparticles for fabrication of electrochemical DNA biosensor of hepatitis B virus [57]. Electrochemical performance of the biosensor showed sensitive determination of complementary target DNA sequences concentration ranging from 1× 10-10 to 1 × 10-6 mol.L-1, with a detection limit of 1.0 ×10-10mol.L-1. Hollow Cu2O microspheres were greatly enhance the immobilization of the DNA probe on the electrode surface and improve the sensitivity of DNA biosensors [57]. A biosensor was fabricated by drop coating technique of carboxylic group functionalized magnetic nanobeads mixture onto the surface of GCE followed by the deposition of MWCNTs and 5’-NH2 capped probe sequence ODN by EDC solution based chemistry. High electron communication ability of the MWCNTs and magnetic enrichment improves the detection sensitivity of the proposed biosensor. In the range of 1.0 × 10–13–1.0 × 10–6 M, the concentration of the complementary sequence was linear with the response of the electrochemical signal of MB and the detective limit of target ODN was 43 fM [58]. Some organic-inorganic hybrids nanocomposite materials also attracted substantial attention to many researchers in the development of electrochemical DNA biosensors. Owing to combining potential distinct properties of organic and inorganic components within a single molecular composite [4,7]. Organic materials offer structural flexibility, convenient processing, tunable electronic properties, photoconductivity, efficient luminescence and the potential for semiconducting and even metallic behavior. Inorganic compounds provide the potential for high electron carrier mobilities, band gap tunability, a range of magnetic and dielectric properties, and thermal and mechanical stability [59,60]. In addition to combining distinct characteristics, new or enhanced phenomena can also arise as a result of the interface between the organic and inorganic components. These hybrid nanocomposites materials provide enhanced dual characteristics which are efficiently retain the bioactivity of immobilized probe ssDNA for construction of biorecognition transducers. Fe2O3 microspheres and self-doped PANI nanofibers (copolymer of aniline and maminobenzenesulfonic acid) modified carbon ionic liquid electrode used to immobilization of probe ssDNA for sensitive impedomatrically detection of sequence-specific DNA of phosphoenol-pyruvate carboxylase (PEPCase) gene [61]. The [Fe(CN)6]3−/4− was employed as a internal indicator. Strong adsorption ability of Fe2O3 microspheres and excellent conductivity of self-doped PANI nanofibers (copolymer of aniline and maminobenzenesulfonic acid) enhanced the sensitivity of DNA hybridization recognition. DNA hybridization events was monitored with a label-free EIS strategy. The response of the optimized biosensor was measured under the wide concentration range from 1.0×10−13 to 1.0×10−7 mol/L, with detection limit 2.1×10−14 mol/L [61]. Shrestha et al.,[62] have applied a new biosensing strategy based on modified rare earth semiconductor oxide followed by surface-immobilized single-stranded ODN for label free rapid detection of DNA hybridization by impedomatricaly, change in electrical impedance curve used as a detection signal. A significant shift in impedance curve was measured because of change in the interfacial electrical properties of the adsorbed single stranded

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nucleic acid and its complementary partner upon hybridization with the complementary oligonucelotide strand [62]. In a similar report thiol-modified ODN was immobilized on the surface of praseodymium oxide for impedomatric detection of unlabeled DNA hybridization [63]. Atomic force microscopy image were used to investigate the surface topographical features of the deposited film before and after immobilization of probe DNA. The proposed electrochemical AC impedomatric biosensor showed ultrasensitivity for the detection of complementary ODNs in solution without the use of label reagent.

5. 5. Quantum Dots for Electrochemical DNA Biosensors Semiconductor nanomaterials (CdS, ZnS, PbS, GaN) were used for the designing of an amperometric DNA biosensor. Owing to their unique (size-tunable fluorescent) properties, the intrinsic redox properties and the sensitive electrochemical stripping analysis of the metal components of semiconductor nanoparticles cause the labels in the electrochemical biosensor to be very sensitive. The concept was first demonstrated by Wang’s group using semiconductor nanoparticle labels for the electrochemical DNA hybridization assay. Semiconductor nanoparticles maintained the bioactivity and the structure of probe ssDNA molecules and also electrocatalyzed the reduction of dissolved oxygen, resulting in a significant increase of the reduction peak current. Recent years, several inventive designs for electrochemical DNA biosensors based on semiconductor quantum dots have appeared. Wang et al., [64, 65] reported on electrochemical assays based on quantum dot nanocrystals as tracers. These quantum dots exhibit sharp and well resolved stripping voltammetry signals proportional to the concentration of corresponding DNA targets due to the well defined oxidation potentials of the metal components. The calibration plots were linear of the resulting biosensor with the lowering detection limit 2.7 pM, correlation coefficients, 0.979(T1) and 0.975(T2)) [65]. In a similar strategy Hansen et al., [66] utilized CdS nanoparticles for label free electrochemical sensing of the target DNA. The proposed stripping voltammetry method offer excellent sensitivity up to 0.1 fmol of target DNA. Ding et al., [67] reported a sandwich electrochemical immunoassay protocol for quantitative detection of target DNA or other proteins based on the use of different semiconductor nanoparticle tracers (CdS, ZnS, and PbS). The fabricated sandwich electrochemical biosensor offer reliable low detection limit of 9.6 pg/mL [67]. Chen et al., [68] have developed GaN nanowires for label free electrochemical detection of target DNA (anthrax lethal factor sequence) using dual routes EIS and photoluminescence (PL) measurements. The resulting GaN nanowires biotransducer showed enhanced sensitivity to surface-immobilized DNA molecules due to nanowires provides high surface binding energies for more binding sites to probe DNA and surface-enhanced charge transfer capability to the analyte. This novel biosensor revealed excellent selectivity and specificity, down to picomolar concentration, high response sensitivity and a low detection limit useful for potential applications [68].

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5.6. Carbon Nanotubes for Electrochemical DNA Biosensors Carbon nanotubes (CNTs) are the promising carbonaceous materials which received considerable attention many researchers because of their unique structure-dependent electrical, chemical and mechanical properties. The unusual properties of the CNTs owing to the covalent sp2 bonds and tubular structure with large length/diameter ratios renders them excellent candidates for biosensor or bioreactor applications. CNT can be divided into singlewall carbon-nanotubes (SWCNT) and multi-wall carbon-nanotubes (MWCNT). SWCNT possess a cylindrical nanostructure (with a high aspect ratio), formed by rolling up a single graphite sheet into a tube [69-74]. SWCNT can thus be viewed as molecular wires with every atom on the surface. CNTs are used in composite materials in electronic devices, as sensors, actuators, field emitters, energy storage media and biomaterials. The high stability of the CNTs in an oxidative environment renders them not only excellent catalyst support materials but also high-performance catalysts for hydrocarbon oxidation. In addition, processing in oxidative environment is one of the most widely used methods for purificating or reshaping the original structure of carbon-based materials or for tailoring their physical, chemical, and electronic properties by introducing oxygenated groups in the C cage. The oxygen functional groups can convert the metallic CNTs into semiconducting, improve the adhesive properties, or selectively functionalize the surface to meet the application demands. Furthermore, the use of CNTs as analytical tools, and the construction of nanodevices and nanosensors based on CNTs are other exciting areas of development for modern analytical science. The general role of CNTs in analytical chemistry was recently reviewed [69-76]. Considering particular role of CNTs in electroanalytical chemistry, properties such as good biocompatibility, huge high surface area, wide electrical windows, flexible surface chemistry, ease to functionalization for biomolecule co-adsorption, enhanced electronic conductivity and a high mechanical resistance have driven an impressive research effort in electroanalytical applications. Recently, the important feature and possible potential applications of CNTs were extensively reviewed [69-83]. The electrode fabrication techniques using by CNTs and the hybridization indication techniques are both play important roles in developing ultrasensitive, selective and miniaturized electrochemical DNA biosensor for quick and reliable DNA sequence analysis in practical application, such as early cancer detection and point-to-care use. Due to their huge surface energy and flexible surface chemistry to functionalize the biomolecules on the CNTs surface accelerate electron-transfer rate between the redox active ssDNA molecule and electrode surface. CNTs increase the attached DNA amount on the CNTs-based substrate surface, it also can concentrate a great number of enzyme or electroactive nanoparticles to indicate DNA hybridization, and the good electro-transfer property of CNTs can amplify the hybridization signal when detecting the electrochemical signal of purine or hybridization indicators at CNTs-based transducer as hybridization signal. All of these amplification factors have offered promising prospects for fabricating highly sensitive electrochemically DNA biosensing protocols. A biosensor based on chitosan doped with CNTs, successfully used to detect salmon sperm DNA [69-80]. Chitosan doped CNTs matrix deposited onto graphite electrode coimmobilized fish sperm DNA for detection of salmon sperm DNA. Chitosan is a biopolymer mechanically and chemically stable and having strong adhesive nature to the substrate. Chitosan was widely used as an effective dispersant of CNTs, provides large surface area for the covalent immobilization of ODNs, that is enhanced the higher DNA loading and longer

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detection range of the analyte. MB was employed as a redox active indicator for electrochemically quantitative detection of DNA hybridization signal. It was found that CNTs can enhance the electroactive surface area threefold (0.28+0.03 and 0.093+0.06 cm2 for chitosan–CNT- and chitosan-modified electrodes, respectively) and can accelerate the rate of electron transfer between the redox-active MB and the electrode. A low detection limit of 0.252 nM fish sperm DNA was achieved, and no interference was found in the presence of human serum albumin. The DPV signal of MB was linear over the fish sperm DNA concentration range of 0.5–20 nM [84]. Another interesting approach consists in the assembling of a DNA electrochemical biosensor based on chitosan doped MWCNTss deposited onto SPCE [85]. Analytical performance of the bionanocomposite transducer was investigated using by DPV technique with the DNA redox marker [Co(phen)3]3+, CV and EIS with [Fe(CN)6]3– as a redox probe in a phosphate buffer solution (PBS),respectively. A comparative study between DNA/MWNT-CHIT/SPCE and DNA/CHIT/SPCE matrices have been proposed to confirm the deep DNA damage using by CV and electrochemical impedance spectroscopy techniques [85]. The remarkable electrical properties of CNT suggest the possibility of developing superior electrochemical sensing devices, ranging from amperometric enzyme electrodes to label-free DNA hybridization biosensor. The tailored electronic conductivity of CNTs, coupled with their ease of processing/modification and rich chemistry, make them extremely attractive as 1-D sensing materials. Hembram et al.,[86] studies the electrical and optical properties of MWCNTs/DNA nanocomposite. CNTs were covalently bonded to DNA at the ends of defects sites and the wrapping of DNA on the CNTs is due to van der Waals force. An enhanced conductivity of the CNTs nanocomposite was examined when increase DNA concentration [86]. CNTs facilitate the electrochemical oxidation of DNA guanine nucleotide, which allows direct detection of DNA on the modified electrodes. The chemical structure and molecular weight of the DNA altered the electrochemical properties of the nucleic acids. Furthermore, the denaturation of native DNA improves the adsorption of biopolymer on CNTs and results in an increase in DNA oxidation current on the modified electrode. The resulting CNT-modified bio-electrodes demonstrate the feasibility of direct detection and characterization of DNA and DNA damaging factors [87]. Self-assembled MWNTs layer was developed onto gold substrate to covalent immobilization of probe ssDNA. DPV technique was applied to examine the alteration of hybridization between the probe and target DNA with the help of MB as an internal indicator. Biosensing results were compare in between self-assembled MWNTs and random MWNTs, found that self-assembled MWNTs based biosensor had higher hybridization efficiency like as high selectivity and long range hybridization detection limit [88]. In a similar report, carboxylic SWCNTs were self-assembled on an amine-modified platinum electrode surface and followed by the assembly of NH2-DNA with the carboxylamine coupling for co-adsorption of DNA oligoneucleotides [89]. Field Emission Electron Microscopy (FEG-SEM) images demonstrated the covalent immobilization of the probe DNA on the fabricated electrode surface. Cyclic voltammetry and UV–Vis spectroscopy were employed to investigate the mechanism in between probe DNA and riboflavin (VB2). The resulting biosensor exhibited high sensitivity and low detection limit for the tested riboflavin [89]. CNTs in order to obtain a fine dispersion in the selected solutions or matrices. Several methods have been developed, including covalent or non-covalent modifications. Depending

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on the methods used, functional groups can be introduced onto the surface of nanotubes. Meanwhile, it would endow CNTs with multifunctional applications by integrating other functional groups or materials onto their surface. A functionalized nanotube might have mechanical, optical or electrical properties that are different from those of the original nanotube. Therefore, it is an interesting area to functionalize CNTs for all kinds of applications. Gong et al., [90] prepared DNA–thionine–carbon nanotube (DNA–Th–CNT) nanocomposites to immobilization of DNA on the surface of CNTs via thionine (Th). The fabrication process of nanocomposite was characterized using by Raman spectroscopy, UV– vis spectroscopy, AFM, and SEM. Thionine has excellent electron facilitating properties and efficiently accelerate the electron communicate rate between the redox active species and electrode surface. In addition of Thionine can retain the native secondary conformational structure of DNA molecules after it was immobilized on the bioelectrode. Electrochemical characteristics of the functionalized CNTs have good quality electrochemical response with a long-term stability for the potential use in the fields of DNA biosensors [90]. Tam et al.,[91] have studied the covalently immobilized probe DNA onto MWCNTs for direct and label-free detection of influenza virus (type A). FTIR and Raman spectra was used for confirmation of covalent bonding in between amine and phosphate groups of the DNA sequence. The fabricated DNA biosensor can detect target DNA up to 0.5 nM and response time of DNA sensor is approximately 4 min. The change in electrical conductivity of the modified bioelectrode was used as a response signal of the biosensor, which was altered by hybridization of the DNA [91]. Thus, a novel strategy of altering the electronic properties of nanotubes are done either by chemically functionalizing them with a moiety or altering the structure whose intrinsic properties are electrically configurable. Wahab et al., have used functionalized CNTs interacted with nano-size hydroxyapatite to examined the interaction of bio-molecules (deoxyribonucleic acid (DNA) and nano-hydroxyapatite (HA)with the nanocompote for sensitive development of electrochemical transducer. NMR and FTIR technique were applied for the confirmation of interaction and functionalization of CNTs with DNA molecules [92]. Zhu and co-workers [93] presented a very attractive work about non-covalent functionalization of MWCNTs sidewalls to immobilization of poly(amidoamine) dendrimer for the fabrication of efficient electronic transducers to form the DNA biosensors. They found that G2-PAMAM dendrimer attached with MWNTs electronic transducer having a large number of amino groups on the surface increase the covalent bonding of probe DNA, resulting an increase the sensitivity of the impedomatric biosensor for sensitive and selective detection of target DNA with a low detection limit down to 0.1 pM. The [Fe(CN)6]3- plays as an indicator, and the function based on electrostatic repulsion of [Fe(CN)6]3- anion with negatively charged DNA phosphate backbone. The interfacial charge-transfer resistance of the bioelectrode was altered as the concentration of the target DNA was change, indicated the response signal of the transducer. The constructed biosensor exhibited linearity of the target DNA within a concentration range from0.5 to 500 pM with a detection limit of 0.1 pM (S/N = 3). The proposed method is simple, sensitive and reliable and could be reasonably useful for practical applications [93]. In other application, MWCNTs dispersed in dimethylformamide or aqueous sodium dodecyl sulfate solution mixed into colloidal gold nanoparticles in PBS were deposited on SPCE used as the signal transducer of a dsDNA-based biosensor. MWNTs in SDS solution based transducer revealed substantial enhancement in the electrochemical response. The fabricated biosensor was tested on berberine and isoquinoline plant alkaloid.

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Author evaluated anticancer agent berberine effect on target DNA which depends on berberine concentration in the range 75 and 50 g mL−1, using by voltammetric technique [94]. Carboxyl groups functionalized MWCNTs modified onto electropolymerized aminobenzoic acid on the surface of the GCE was applied for fabrication of sensitive electrochemical DNA biosensor for the detection of target DNA hybridization [38]. SEM, CV and EIS were used to investigate the electrode surface texture and electrochemical characteristics before and after enzyme immobilization. For covalent immobilization of DNA molecules Au nanoparticles layer was introduced on the nanocomposite electrode surface. Gold nanoparticles promote the electron transfer rate between the redox active DNA species and the electrode surface. Under optimized conditions, DNA hybridization current was monitored by DPV technique. The biosensor had linearity in a wide concentration range of the complementary ODNs from 1.0×10−12 to 5.0×10−9M with a detection limit of 3.5×10−13 M [38]. Ye and Ju [95] reported the use of screen printed carbon electrode modified with MWCNTs for the fast and sensitive detection of DNA and RNA from the electrooxidation of guanine and adenine residues catalyzed by MWCNTs. The proposed transducer could be detect calf thymus ssDNA concentration ranging from 17.0 to 345μgml−1 with a detection limit of 2.0μgml−1 at 3 and yeast tRNA ranging from 8.2μgml−1 to 4.1 mg ml−1[96]. Wang et al.,[96] employed CNTs modified GCE electrochemical transducers. Examined the attractive performance of the enzyme based electrochemical biassaays of DNA hybridization. CNT based electrochemical transducers was used for ultrasensitive electrical bioassays of proteins and DNA. The unique electronic, chemical, and mechanical properties of CNTs make them extremely attractive for electrochemical sensors. Most CNT-sensing work has focused on the ability of surface-confined CNTs to promote electron-transfer reactions involved in biocatalytic devices [97,98]. In another approach, CNT amplification platform combined with CdS particles have been reported by Wang et al.,[99]. CNTs utilized as supporting materials to concentrate nanoparticles or enzyme molecules on it as a new and more powerful DNA hybridization indicator than using a single nanoparticle or enzyme molecule indicating DNA hybridization. Due to large surface area of the CNTs, a larger number of octadecanethiolcapped CdS nanoparticles can be attached onto acetone-activated CNTs under hydrophobic force, the whole complex is then used as a hybridization indicator to be labeled at probe 2 DNA. After hybridization in a sandwich manner (probe 1-target-probe 2), these CdS nanoparticles are dissolved into Cd2+ for stripping voltammetry detection. Because 500 CdS particles can load on an individual CNT, the detection limit is consequently improved to 500fold as compared with single CdS nanoparticle labeling technique.

CONCLUSIONS Nanomaterials are opening a new horizon in the development of electrochemical DNA biosensing devices. Such DNA biosensing devices could be useful for diagnosing and monitoring infectious disease, monitoring the pharmokinetics of drugs, detecting cancer, and disease biomarkers, analyzing breath, urine and blood for drugs of abuse, detecting biological and chemical warfare agents, and monitoring pathogens in food. The unique and attractive properties of nanostructured materials present new opportunities for the designing of highly

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sophisticated electroanalytical DNA biosensing devices. Due to the high surface area, nontoxicity, biocompatibility and charge-sensitive conductance of nanomareials act as effective transducers in nanoscale biosensing and bioelectronic devices. These nanostructured materials based electrochemical DNA devices have a number of key features, including high sensitivity, exquisite selectivity, fast response time and rapid recovery (reversibility), and potential for integration of addressable arrays on a massive scale, which sets them apart from other sensors technologies available today. The sensitivity of the sensor depends on the dimensions and morphological shape of the nanomaterials involved. Therefore, some morphological (nanotube, nanowires, nanofibers, nanorods) based biosensing transducers could function as effective mediators and facilitate the electron transfer between the active site of probe DNA and surface of the electrodes. The resulting nanostructures could be substantially stronger and lighter than conventional nanomaterials which are currently used in the construction of biosensing devices. It is an urgent need to develop an efficient and reversible effective electrochemical DNA biosensing device, which are capable in detecting of analytes (target DNA) up to small concentrations. In the near future, we argue that these advances could and should be developed at molecule level detection in simple nanosensor devices. To fully realize the potential applicability of nanostructures in electrochemical sensors, several issues related to their fabrication methods need to be addressed.

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In: Recent Developments in Bio-Nanocomposites… ISBN 978-1-61761-008-0 Editor: Ashutosh Tiwari © 2011 Nova Science Publishers, Inc.

Chapter 17

DEVELOPMENT IN DIAGNOSIS AND TREATMENT WITH NANOTECHNOLOGY Rajiv Lochan Gaur1, Rajeev Mishra2, Richa Srivastava3, Smriti Bhadauria4, and Ashutosh Tiwari5,6 1

Pathology Department, Stanford School of Medicine, Stanford University, Palo Alto, California 94304, USA 2 Department of Cancer Genetics, School of Medicine, Nihon University, Tokyo 173 8610, Japan 3 GRB Division, Central Institute of Medicinal and Aromatic Plants, Lucknow 226 015, India 4 Division of Toxicology, Central Drug Research Institute, Lucknow 262 001, India 5 School of Materials Science and Engineering, Jiangsu University, Zhenjiang 212 013, China 6 National Institute for Materials Science, Tsukuba, Ibaraki 305 0047, Japan

1. INTRODUCTION Our survival on the planet depends on statistics about smart and efficient dealing with diseases. We are always exposed and encountering to old and new emerging disease. The law of survival (survival of fittest) implies on every living creature including parasites and they have way better strategy to endure the adverse because of simple structure and easy gene modification. Besides these they compensate by other mutations to the modification accrued in process to protect themselves from human strategies (like pressure of eliminate as drug). Considering all these together, we are dealing with disease causing organisms which are changing rapidly. On the other hand we have environmental stress which changes our own milieu rapidly. The changes may end up with disease like cancer. We are exposed to face these unwanted changes and are bound to develop the disease. With all the disease and there consequences, we need earliest diagnosis and best treatment to combat it. Till last few decades we have had traditional diagnosis and treatment. As we moved forward to present scenario we can rely on technological advancement to deal with disease. Finer detail and

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mechanisms can help us to design new and best effective strategies against these lethal threats to humankind. The technological revolution has significantly improved our approach towards diagnosing and treatment of diseases. With recent advancement, now we have better device to observe phenomenon behind the disease at molecular level. This will be the base of our future diagnosis and treatment. With this strategy we can go to the level of atom and reach to better understanding. The finer details enable us to look at nanostructures and find out the markers in different body fluids (like blood plasma or tissue fluid). We have equipped ourselves with all these advance tools and technologies but these are not used as efficiently as they should. There are several problems to be considered like distribution of technology, efficiency of use and above all the cost. Considering all these consequences, we are dealing with two aspects of nanotechnology in this book chapter; diagnosis and treatment.

2. DIAGNOSIS Human and disease remain in near vicinity since the life originated. [1]. Our knowledge to treat the disease depends on its diagnosis. Technically, diagnosis means identification of disease by its signs and/or symptoms; and clinically this is advance stage of already established disease. At this particular stage it might be lethal for the subject. In most of the cases at the level of diagnosis (high load of active pathogens or dysfunction of organ), the disease is curable and till now this is basis of identification of disease worldwide. There are two basic problems related to our present diagnostic approach 1) in most of physiological disorder at the level of conventional diagnosis the condition is irreversible and the organs are not repairable (the only way is to maintain the function of organ with continuous consumption of available drugs) and 2) in case of pathogenic microorganisms at the level of conventional diagnosis the patient may be treated till elimination of organisms but by that time, threat of spread of organism to vicinity is very high and in some cases the organism remain in patients body in latent stage for years (can develop as active disease in near future). In present scenario, the most desirable and appropriate to deal with these complications are the early diagnosis, which has never been an easygoing task. Technical advancement with all the tools available is not even sufficient to diagnose the oldest disease known to mankind like TB (70% confidence level with advance test). At present we have biochemical, microbiological, immunological, chemical and physical devices to diagnose the disease. All together are performing well at certain level with some reasonable flaws. The major drawbacks with these methods are that they are responsive after development of disease at certain level and that may be too late to cure. The other downside is, how to diagnose latent or non-responsive patient who may serve as reservoir to spread the disease. Sometimes relapse of latency make it worst and that might be too late for treatment. In light of the entire enigma; early and accurate diagnosis is most desirable task in battle against disease. To achieve this we have fusion of advance technical and biological phenomenon. We are discussing some of these advance diagnostic tools. Although, most of them are in process of refinement in terms of technology, cost effectiveness, easy to use and reach to the least approachable population who are badly hit. It is not possible and also beyond the scope of this chapter to cover finer details of every aspect. We are discussing some examples and trying to cover major aspects and strategy of early diagnosis.

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Diagnosis is the technique based on detection of bio-molecules present at certain concentration. Assays used should sensitive enough to detect this least desired concentration. The common most targets are bio-molecules like proteins and genes. The conventional approach to attain high sensitivity is target-based amplification and signal-based amplification for example polymerase chain reaction (PCR)[2] and enzyme-linked immunosorbent assay (ELISA) [3] respectively. These techniques are defined in detail by numerous authors in terms of their reliability, sensitivity and robustness for diagnosis. PCR is capable in detection by few copy numbers and ELISA is sensitive enough to detect the concentration till picogram level [3]. Our main resource for disease detection has been based on signal based techniques in last few decades. Scientific innovation leads us to added specific detection level i.e; detection at nanoscale. In few cases we can move to further drop the detection limit (i.e. high sensitivity). High sensitivity enhances the accuracy and detection at least concentration. Newer technique like ‘scanometric’ permits the sensitivity to attomolar (10–18) concentrations of proteins and nucleic acids in busy biological samples (plasma sample) [4]. DNA-functionalized gold nanoparticles (in terms of their binding, catalytic and optical properties) are being used in a variety of detection approaches as fluorescent [5], colorimetric, chemiluminisence, surfaceplasmonresonance, scanometric, and Raman-spectroscopy based detection methods. In terms of protein detection assay if we compare ELISA (most efficient till few years back) and assays using protein-modified gold nanoparticles [6], we can end up with lower level of detection by several orders of magnitude. There are numerous newer discoveries related with diagnosis using nanomaterial are available and are in process of scaling up. One such example is carbon-nanotubes [7] (modifies biomolecule) and nanowires made of silicon [8]. These arrangements work on different in electrical conductance on target binding and thus translated into electrical or spectroscopic signal. These tactics mixed with nanoparticle can culminate to better sensitive way of detection of disease. Cancer is one of the most studied and worrisome disease considered worldwide. The problems associated with this disease are the lack of early diagnosis [9] and precise localization of metastatic cells10. Several approaches have been used to deal with cancer (Figure1).

Figure 1. Nano-technological approach towards cancer.

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Early diagnosis is in demand since several years and in light of present knowledge will certainly help in control of cancer, prevention of metastasis and halt on adverse effect of anticancer chemotherapy (can prevent toxicity based problem). [11] With our best tools and techniques we can only diagnose cancer at advance stages and even after surgical removal [12] (most appropriate treatment of the solid cancers) there is no track of metastasis [13] (even micro-metastasis is still debatable for the researchers). In view of all possible methods available, the best approach seems to be the identification of specific biomarkers at cells surface in least concentration (Figure 2). One such approach is to use whole cell as target by technique named ‘Systematic Evolution of Ligands by Exponential enrichment’ (SELEX) [14]. In this process aptamer-conjugated nanoparticle enrichment can be detected and on cancerous cell bound through specific marker. Nanoparticles are having versatile application in biomedical imaging. Various inimitable characteristics like unique magnetic property, assorted surface chemistry, manageable absorption and emission, flexibility to engineer are some unique accountability. Efforts are continued to use these properties together and develop probe to detect cancer and other disease. If we look at other approach like advance live imaging system, cancer can be visualized by Fluorescence [15] and UV-absorption [16] methods, frequently called as optical detection technique. This system is based on technique to probe cancer cells with designed nanoparticles, after being activated these particles have capability to change their optical property and detected and used as cancer cell marker. “Quantum Dots” (nano-sized crystals of CdSe/ZnS) [17] are used to detect the cancer cell or tissue based on optical detection technique. Quantum dots are found suitable to detect migratory and invasive cancer cells. The same principle was used with some modification for detection of individual DNA molecules (multicolor quantum dots) [18] and made possible to detect oncogenes. These newer techniques have been modified further by incorporation of quantum dots into cationic liposome enable to be imaged efficiently with an intracellular label. Such amalgams of nanoparticles are in growing phase and can be used to study cancer cells at intracellular level which may be useful in rapid screening of various chemotherapeutic candidates. The further expansion of this technique is to improve the properties by providing near infra-red florescence and can be used for lymph node mapping [19], essentially important for different surgery including breast surgery. There is always a possibility to detect bacteria by the product they release; nanoaptasensor [20] is a method for detecting of these molecules at low level, like anthrax toxin through the detection of its polypeptide entity protective antigen (PA toxin) using a PA toxin ssDNA aptamer functionalized single-walled carbon nanotubes (SWNTs) [21] device. Aptasensor has a detection limit as low as 1 nano-mole. These sensors are reusable and are a promising tool for rapid and accurate diagnosis of bacterial product. Till date, we are aiming to improve the assay and to detect the bio-molecules at least available concentration. There are always possibilities of some pitfalls like over sensitivity and falls positive cases. The process of refinement needs more samples and resources to make optimum cut-off point, improve assay quality, more efficient detection method and conclusive result. These assays can’t come to practice without support of health worker, health agencies, hospitals and common man as it needs a lot of trials to reach from bench to clinic.

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TREATMENT All the advances we attempt to make in diagnosis of disease will lead us to early and accurate treatment. It is certain that we have a lot of gains to trounce health problems. At this stage we can’t use our regular methodologies of treatment particularly the medicines delivery system has to be improved. We have to consider some of common lead from early diagnosis like requirement of least quantity of drug at lower concentration; such minimum amount is not easy to reach protected to site of action. The drug or compound at this low concentration may be equivalent to physiological or bio-available level. A whole bunch of literature is already available on different delivery system. All have their shortcomings and advantages and most of them are under trail. Here we are discussing few of them to cover general criteria and demand of future treatment approach. The physically examinable structure to be analyzed at nano-scale was possible after introduction of Atomic Force Microscope (AFM) [22] by Binnig, Quate, and Gerber (1986). Very soon AFM expended its popularity because of its ability to capture topographical images of object surface kept in air of liquid at nanometer resolution. AFM has capacity to measure elasticity of samples like living cells, which are too fragile otherwise. AFM has a major role in tissue engineering. The surface structure of cell and its interface are important details needed for different investigations. The knowledge of topological 3D structure and compositions of cell was always in demand. With the help of AFM, combined with confocal microscopy it is now possible to check the arrangement and structure of cells or organisms with added details. In further advancement of AFM, we now have next generation Scanning Thermal Microscopy (SThM) [23]. This system enables us to have image maps by change of thermal conductivity across the surface area of sample. The technology is still at developing stage and in near future we may hope for a better and more accurate image system. The recent advance therapeutic approach is combination of gene vaccine and nanotechnology. Most of the emphasis has been focused on cancer and some infectious disease where gene vaccine has some possibility. The approach can be useful with other diseases once we have some countable leading edge. The delivery of gold nanoparticle bind with DNA via protamine can deliver nucleic acid to desired destination, [24] and even RNA transportation is possible with same mechanism. The advantage with RNA is to provide more accurate therapeutic consequences. These techniques can successfully introduce nucleic acids to destination for intern utilization by the cells. The emerging biomimetic techniques can be applied in tissue engineering, bio-recognition, marked therapeutics and drug/gene delivery. There are plenty of methods describing bio-recognition using particle at nano-scale. DNAfunctionalized nano-particle [25] is one of the recent nano-molecule that can be assembled in controlled way in predesigned structures. It has been assumed that this nano-particle is useful in molecular sensing due to its new adjustable pattern and controlled optical and electrical and magnetic response, chemical heterogeneity and high biomolecular concentration. These precise bio-recognition DNA and its physical/chemical characteristics permits for an exploitation of DNA-functionalized nanomaterials for detection of nucleic acids, while a broad tunability of DNA interactions warrants extending their use for detection of proteins, small molecules and ions. Cancer diagnosis ends up with chemotherapy in more than fifty percent cases. As a consequence, chemotherapeutic drugs are exposed to all cells of body (both cancerous and

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non-cancerous); in turn tumor cells develop drug resistance and rest of cells end up with adverse effect of chemotherapy. Nono-diagnosis will make the case worse without coupled with better drug delivery system, because in that case cancerous cells would little in number and more and more healthy cells will be exposed to anti-cancer drugs. Other drawbacks with routine treatment are poor stability of drugs with which craft treatment to inadequate end. The only appropriate way to overcome these problems seems to find a better delivery system. Nano-particles emerge as promising delivery system without any adverse degradation or chemical exposure to other cells. Solid Lipid Nanoparticles (SLN)[26] for drug delivery is further evolved and promising system emerges in last few years. These particles have advantage over other nanoparticle system and are capable of encapsulating both hydrophobic and hydrophilic drugs. SNL are smaller than micron in magnitude and composed of lipid matrix. These particles are specially designed to remain solid at room and physiological temperature. These SNLs are physiologically compatible and biodegradable. Above all they provide protection to the drug from oxidative or photochemical degradation and prevent them from other chemicals and physiological events. These properties make SNLs, a choice of delivery system in cancer treatment but a long journey still remain to make this system affordable at lower cost. The other budding drug delivery system for cancer treatment is liposome based nanoparticles. The system not only provides a controlled and sustained drug release but also accesses the improved pharmacokinetics and reduced systemic toxicity. Few drugs are already available in market (albumin-nanoparticle-based Abraxane) [27]. Newer improvement in this technology can help in multi-drug resistance tumor. Research showed that this system offers enhanced precision in targeting prostate cancer and open new avenues for breast cancer treatment. Cryosurgery (minimally invasive freezing therapy) is newly evolved and an accurate way of impairment cancerous cells. There are several hindrance remain to overcome for successful and regular use of cryosurgery. The most crucial flaw is incomplete freezing; that ends with inadequate destruction of cancer tissue. The small number of surviving cancerous cells can multiply and give rise to a new tumor structure. With the fear of regeneration of tumor the other apparent problem is damage of healthy tissue near the vicinity of cancer by release of frost from probe. To overcome these problems, cryo-nano-surgery [28] has been proposed. The principle involves is the delivery of nanoparticles as functional suspension that would have promising physical and chemical properties into the target tissues. This will serve in numerous ways; like adjuvant or drug carrier and can maximize the freezing heat transfer to the target tissue. Eventually it will regulate freezing scale and have control on ice-ball formation. The process will end up by holding avoidable heat leak and prevent the surrounding tissue from freezing. This strategy is helpful for cancer as well as normal tissue due to physical border created by nanoparticles. Research is still going on for expansion of this technology for other surgical processes than cancer. The complete understanding of heterogeneous cancer like breast tumor will help in improvement of treatment and further understanding about their behavior. Quantum dot (QD)-based immuno-fluorescent (QD-IHC) [29] nanotechnology has potential advantages to explain this special physiology. Chosen cancer markers can be displayed more efficiently by this technique and the information can be quantified and use to define and understand the heterogeneous nature of tumor. More markers can be taken together to understand the process of formation of assorted cancer.

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Insulin is the only remedy to fight against diabetes. Researchers have been investigating the possibility of its oral intake and it seems to be an elusive aim. Some countable advantages are discovery of biocompatible and biodegradable polymers combine with synthesis of nanopeptide delivery systems. Even till date the system is not fully evolved for patient use but initial results are encouraging in terms of half-life of particle and insulin is found to be delivered in systemic circulation. This may be expected in few years for the patient use. In some complications like neurodegenerative disorders (NDs), it is almost impossible to deliver the drug across the blood brain barrier. Any drug delivery system do not offer ample cyto-architecture and network pattern to do the job successfully. There is a little hope with nano-technological (engineered) material as they can interact with biological systems at a molecular level and enhance the effect several fold [30]. This possibility will open the horizon for drug to interact with target site and make them more responsive physiologically. The approach has its importance in treatment of Alzheimer’s and Parkinson’s diseases. Pathogenic microorganisms have a special way to survive by releasing free radicals to damage the immune cells (eg. Pseudomonas aeruginosa releases phycocynin which give rise the reactive oxygen species (ROS) and provoke the immune cell damage. Antioxidant nanoparticles are devised to offset the effect of ROS and biofilm formation [31]. These nanoparticles are also useful in dealing with condition like ventilation associated pneumonia, a rare condition of compromised respiratory tract immunity by oxidative stress caused due to high levels of oxygen. We are at a platform to develop the new strategies for treatment of diseases with appropriate drug and delivery system. The real challenges are still face to transfer these finding from bench to clinics and implementation of these strategies to regular use.

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[29] Chen C, Peng J, Xia H, Wu Q, Zeng L, Xu H, Tang H, Zhang Z, Zhu X, Pang D, Li Y. Quantum-dot-based immunofluorescent imaging of HER2 and ER provides new insights into breast cancer heterogeneity. Nanotechnology. 21(9):95101 (2010). [30] Modi G, Pillay V, Choonara YE, Ndesendo VM, du Toit LC, Naidoo D. Nanotechnological applications for the treatment of neurodegenerative disorders. Prog Neurobiol. 88(4), 272-85, (2009). [31] Elswaifi SF, Palmieri JR, Hockey KS, Rzigalinski BA. Antioxidant nanoparticles for control of infectious disease. Infect Disord Drug Targets. 9(4), 445-52 (2009).

In: Recent Developments in Bio-Nanocomposites… ISBN 978-1-61761-008-0 Editor: Ashutosh Tiwari © 2011 Nova Science Publishers, Inc.

Chapter 18

BIO-NANOCOMPOSITES BASED ON NATURALLY OCCURRING COMMON POLYSACCHARIDES CHITOSAN, CELLULOSE AND STARCH WITH THEIR BIOMEDICAL APPLICATIONS Hassan Namazia,b1 and Mohsen Mosadegha *a

Research Laboratory of Dendrimers and Nanopolymers, Faculty of Chemistry, University of Tabriz, Tabriz EA, Iran b Research Center for Pharmaceutical Nanonotechnology, Tabriz University of Medical Science, Tabriz, Iran

ABSTRACT This review paper reports recent advances in the field of naturally occurring polysaccharide-based nanocomposites and their biomedical applications. These types of materials have attracted a lot of attentions in both academic and industrial sector. While organic biomedical agents show good inhibition efficiency and a broad spectrum of activity however, their relative low stability (e.g., low decomposition temperature and short life expectancy) cannot be ignored. As a result, there is urgent need to develop biopolymer based nanocomposited materials provided with dual bioactive advantages of organic biomedical agents and inorganic biomedical agents as they will become more important in the future. To achieve this goal polysaccharide-based (such as chitosan, cellulose and starch) nanocomposites have been developed and clinically tested. Since, they combine the structure, physical and chemical properties of both inorganic and organic materials. Most work with polysaccharide nanocomposites have concentrated on biomedical applications due to their non toxic, drug delivery, biodegradable, biocompatible, wound dressing, and etc. properties. In addition to these properties the chitosan has excellent antibacterial activity. Therefore, polysaccharide nanocomposites as bio-nanocomposites are widely used for biomedical applications such as tissue engineering scaffolds, drug delivery, wound dressing and antibacterial film. For instance,

1 P.O. Box 5166616471, Tabriz EA, Iran.Tel.: +98 411 3393121; Fax: +98 411 3340191. E-mail: [email protected].

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Hassan Namazi and Mohsen Mosadegh both, poly(ethylene glycol) (PEG) and chitosan (CS) played vital roles in the reduction of metal ions into nanoparticles (NPs) as well as provided good stability to the formed bionanoparticles. These biopolymers not only help in reducing the metal ions into nanoparticles but also provide distinguished stability for a sustained release of nanoparticles for antibacterial applications. The developed porous nanocomposite film has exhibited superior antibacterial properties and good mechanical properties than the chitosan and chitosan–silver nanocomposites,

1. CHITOSAN 1.1. Chitosan Structure Chitin is the second most abundant agro-polymer produced in the nature after cellulose. It appears in nature as ordered crystalline micro fibrils forming structural components in the exoskeleton of arthropods or in the cell walls of fungi and yeast [1, 2]. It is an acetylated polysaccharid possesed from N-acetyl-D-glucosamine units linked through (1

4)linkages.

Chitosan (CS) is obtained with the deacetylation reaction of chitin. In contrary to chitin, chitosan is not widespread in the nature. It is found in some mushrooms (zygote fungi) and into the termite queen’s abdominal wall. It is industrially obtained by partial chitin deacetylation [3]. As shown in Figure 1 its chemical structure is a random linear chaining of N-acetyl-D-glucosamine units (acetylated glucopyranose unit) and D-glucopyranose amine (deacetylated unit) linked by (1

4)linkages. In acid conditions, when the amino groups

are protonated, it becomes a water soluble polycationic compound. The chitosan is characterized by its acetylation degree and by its molecular weight. These last parameters influence its viscosity and solubility. According to the bioresource, industrial chitosan shows acetylation degrees from 2 to 60%. In solid state, chitosan is a semi-crystalline polymer morphology has been investigated and many allomorphs have been described. [4, 5]. This natural polysaccharide possesses useful characteristics such as non toxicity, high biocompatibility and non-antigenicity that offer the possibility of clinical use and many biomedical applications [6-10]. In fact, the combination of good biocompatibility, intrinsic antibacterial activity, ability to bind to growth factors and to be processed in a variety of different shapes makes CS a promising candidate scaffold material for cartilage, intervertebral disc and bone tissue engineering in clinical practice [11-13]. Due to its unique chemical structure a lot of investigations have been carried out as drug delivery systems with chitosan hydrogels [14-18].

1.2. Chitosan Nanocomposites and Their Biomedical Application 1.2.1. Antibacterial Behavior Antimicrobial agents can be divided into inorganic and organic ones according to their chemical composition. Inorganic antimicrobial agents show long life expectancy and high heat resistance. However, they exhibit weak mould proof activity and large dosage when they are needed to be used [19, 20].

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Figure 1. Chemical structure of chitosan.

While organic antibacterial agents show good inhibition efficiency and a broad spectrum of activity, more importantly they display blending compatibility with organic matrixes such as textile, paints, polymer, etc., however, their relative low stability e.g., low decomposition temperature and short life expectancy cannot be ignored [21]. As a result, there is an urgent need to develop organic–inorganic hybrid compounds provided with dual antibacterial advantages of organic antimicrobial agents and inorganic antimicrobial agents as they potentially will become more important in the antimicrobial applications. Wang et al. [22] have studied on preparation chitosan/rectorite nanocomposite and their antimicrobial activity. In order to get better solubility of chitosan in different pH conditions it has modified into N-(2-hydroxyl) propyl-3-trimethyl ammonium chitosan chloride (HTCC). The biopolymer HTCC entered into the interlayer of rectorite (REC) and nicely distributed in the HTCC matrix despite the high content of REC (25–50 wt%). It was the high affinity and the strong interaction between HTCC and REC that resulted in the excellent antimicrobial activity of the obtained bio-nanocomposites due to the adsorption and immobilization capacity of modified REC and the antimicrobial activity of HTCC. In these kinds of dual actions, the nanocomposites could permeate into the cell membrane, damage the cell wall, disturb the natural processes of the cell and finally result in the fast death of microorganisms. The lowest minimum inhibition concentration values of the nanocomposites against Staphylococcus aureus and Bacillus subtilis were less than 0.00313% (w/v) in all media tested, and the killing rate on S. aureus reached more than 90% in 30 min. In other work Wang et al. [23] showed the antibacterial property of quaternized chitosan/organic montmorillonite nanocomposite. Montmorillonite (MMT) modified with cetyltrimethyl ammonium bromide (CTBA) as an organically modified layered silicate and modified chitosan as HTCC were used. Antimicrobial studies pointed out that the bio-nanocomposites could strongly inhibit the growth of a wide variety of microorganisms, including Grampositive bacteria, Gram-negative bacteria, and fungi; more importantly, they demonstrated good antimicrobial capacity in whichever medium, in weak acid, water, or weak base [23]. As the amount of MMT is increased, the bio-nanocomposites showed better inhibitory effect on microorganisms, especially Gram-positive bacteria. This difference of antimicrobial effect on Gram-positive bacteria and Gram-negative bacteria is due to the cell wall of each bateria. The Gram-positive bacteria consist of a thick peptidoglycan layer and the cytoplasmic membrane. Its peptidoglycan layer is extensively crosslinked in three dimensions forming a solid mesh. Despite its thickness, the peptidoglycan layer of Grampositive bacteria is not a barrier to the

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diffusion of foreign molecules. But Gram-negative bacteria have a small layer of peptidoglycan and an outer membrane. The outer membrane is made of a liposaccharides layer that is very toxic. Because of its structure, Gram-negative bacteria are unusually permeable to foreign molecules [23]. Therefore, Gram-negative bacteria are generally less susceptible to antibiotics and antibacterial agents than Gram-positive bacteria. The lowest minimum inhibition concentration (MIC) value of the nanocomposites against Staphylococcus aureus and Bacillus subtilis were less than 0.00313% (w/v) under all the conditions. According to result of this research that the long chain alkyl with hydrophobicity in both HTCC and CTAB are easy to permeate into the cell membrane. In this way, the strong effects on microorganisms arise [23]. Sanpui et al. have reported antibacterial properties of a chitosan–Ag-nanoparticle composite [24]. This composite was synthesized by adding prepared AgNO3 solution and then NaOH solution to chitosan solution, with constant stirring at 95 °C. The appearance of a yellow color about 1 min after addition of the NaOH solution, indicated formation of Ag nanoparticles (AgNPs). Various amounts of this composite were used to treat bacteria, keeping the ratio of the Ag to chitosan concentrations constant at 1:46. Chitosan has strong affinity towards metal ions because of the presence of numerous amine and hydroxyl groups [25]. Under alkaline condition chitosan can reduce Ag+ ions to AgNPs [26]. And the nanoparticles product is attached to the polymer, thus providing a single-step synthesis and stabilization of AgNPs. The separated composite was found to have significantly higher antimicrobial activity than its components at their respective concentrations. Variations in the bactericidal efficiency of chitosan can be attributed to the fact that antimicrobial activity of chitosan depends on a number of factors such as the molecular weight and degree of deacetylation of a chitosan preparation process, the viscosity, ionic strength, pH and presence of metallic ions in the medium, and the temperature. The concentration of chitosan in composite preparations that completely inhibited the growth of E. coli was 0.012% [26]. As a result, this investigation indicates that the composite was more efficient than either AgNPs or chitosan alone for inactivating bacteria, probablly due to synergistic effect of both the AgNPs and chitosan in the composite. The outer membrane (OM) of Gram negative bacteria such as E. coli consists of lipopolysaccharides (LPS) having phosphate and pyrophosphate groups which render the cell surface negatively charged. As chitosan is a cationic polymer, it could be attached to the E. coli cell wall by electrostatic interaction [26]. Vimala et al. [27] have introduced a method to synthesis of porous chitosan–silver composite as bio-nanocomposite films in view of their increasing areas of application in wound dressing, antibacterial application and water purification. The entire process consists of three-steps including silver ion-poly(ethylene glycol)(PEG) matrix preparation, addition of chitosan matrix, and removal of poly(ethylene glycol) from the film matrix. Films were immersed in hot distilled water (80 ◦ C) over a period of 1 hour. During this process, most of the PEGs were extracted into distilled water since PEG is highly soluble in water at this temperature and the resulted films were left with highly porous structures. Uniform porous and brown color chitosan films impregnated with silver nanoparticles (AgNPs) were successfully fabricated by this facile approach. Both, poly(ethylene glycol) (PEG) and chitosan (CS) played vital roles in the reduction of metal ions into nanoparticles (NPs) as well as provided good stability to the formed nanoparticles [27]. These polymers not only help in reducing the metal ions into nanoparticles but also provide excellent stability for a sustained release of nanoparticles for antibacterial applications. The developed porous nanocomposite film has exhibited superior

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antibacterial properties and good mechanical properties than the chitosan and chitosan–silver nanocomposites, suggesting that it can be applied for wound dressings and water purification purpose. The antibacterial activity of developed chitosan/silver and PEG/chitosan/silver nanocomposites was determined by disc diffusion method for E. coli, Bacillus, and K. pneumoniae. It was found that the foresaid samples exhibited an inhibition zone while the chitosan film does not involve in the inhibition zone process [27]. Rhim et al. [28] have reported the preparation of four different types of chitosan-based nanocomposite films which were prepared using a solvent-casting method by incorporation with four types of nanoparticles, that is, an unmodified montmorillonite (Na-MMT), an organically modified montmorillonite (Cloisite 30B), a Nano-silver, and a Ag-zeolite (AgIon). The antimicrobial activity of chitosan and chitosan-based bio-nanocomposite films was tested. In this four different food pathogenic bacteria including Staphylococcus aureus, Leuconostoc monocytogenes, Salmonella typhimurium, and Escherichia coli were utilized for testing the antimicrobial activity of the films. Chitosan and chitosan/Na-MMT nanocomposite films did not show clear microbial inhibition zones, whereas nano-silver- and Ag-Ion incorporated nanocomposite films exhibited distinctive microbial inhibition zones against all four test microorganisms in the disk method. Iinterestingly, Cloisite 30B-incorporated nanocomposite film exhibited antimicrobial activity against the two Gram-positive bacteria studied, S. aureus and L. monocytogenes, but did not show any antimicrobial activity against the two Gram-negative bacteria, S. typhimurium and E. coli. The increased antimicrobial activity of Ag-incorporated films may be increased through high infiltration of the Ag component with high bactericidal effect. Ag ions reportedly submit to the negatively charged bacteria cell wall, changing the cell wall permeability. This action coupled with protein denaturation induces cell lysis and death [29]. By compositing with nanoparticles mechanical and water vapor barrier properties of chitosan-based bio-nanocomposite films were significantly increased (P < 0.05) in comparison to those of controled chitosan films [28]. Mechanical and barrier properties of chitosan films were affected through intercalation of nanoparticles, that is, tensile strength increased by 7-16%, whereas water vapor permeability decreased by 25-30% depending on the nanoparticle material tested. Chen et al. [30] also reported the preparation of thiourea chitosan using reaction of chitosan with ammonium thiocyanate in ethanol, then thiourea chitosan–Ag+ complex was prepared. XPS confirmed that in thiourea chitosan–Ag+ complex, S atoms coordinated to silver ions and were the major electron donors; O atoms were another electron donors next to S atoms. Thiourea chitosan– Ag+ complex improved the instability of Ag+. In vitro antimicrobial activities of the complex were evaluated against six species of bacteria and molds. The complex showed a wide spectrum of effective antimicrobial activities. Its MIC values were much lower than those of chitosan, sodium diacetate and sodium benzoate and their complex have better antibacterial activity than antifungal activity.

1.2.2. Drug Release Behavior Wang et al. have reported preparation of modified chitosan/montmorillonite nanocomposites [31]. The hot intercalation method was applied to prepare these HTCC/MMT nanocomposites. The nanocomposites with initial HTCC/MMT weight ratios of: 1:2, 1:1, 2:1 and 4:1 were obtained and then HTCC and different HTCC/MMT nanocomposite solutions (1.0 mgml-1) were slowly added to the above calcium alginate pre-gel under stirring, and then HTCC and HTCC/MMT nanoparticles were obtained and investigation showed that their size

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are around 200 nm then drug delivery behavior of nanoparticles were introduced. Bovine serum albumin (BSA), as a model protein drug, was incorporated into the nanoparticles, and their drug-controlled release properties were tested. Results show that the amount incorporated MMT in nanoparticles has significant effect on release behavior. Montmorillonite with having a large specific surface area, exhibits good adsorbability, cation exchange capacity, standout adhesive ability and drug-carrying capability [32]. However, when the MMT content is too large, the collapse of the structure may be induced so that the nanoparticles cannot load enough BSA. Therefore, nanoparticles (HTCC/MMT mass ratio of 1:1) with the largest interlayer distance show the highest drug loading capacity and slowest drug release results. Wang et al. hve studied on drug delivery behavior of chitosan/organic rectorite nanocomposite films [33]. Drug-controlled release in vitro studies showed a slower and more continuous release for the nanocomposite films in comparison with pure chitosan film, and the drug-delivery cumulative release was proportional to the amount and the interlayer distance of organic rectorite OREC. Pure rectorite is hydrophobic and the affinity between rectorite and polymer is not enough, so rectorite must be modified in order to increase the affinity between rectorite and polymer. Organic rectorite (OREC) is calcium rectorite modified by cetyltrimethyl ammonium bromide. Chitosan and chitosan/OREC nanocomposite films and corresponding drug-loaded films were prepared by a casting/solvent evaporation method in which chitosan and chitosan/OREC nanocomposite powder with different chitosanOREC mass ratios (2:1, 6:1, 12:1, 20:1, 50:1). These bio-nanocomposite films were also successfully loaded with bovine serum albumin (BSA), as a kind of model drug and the drugcontrolled delivery behavior of the films was studied. It is worthwhile to note that the bionanocomposite films, especially bio-nanocomposite sample with the largest interlayer distance gallery of OREC clay exhibit the subsequent slowest sustained release, and with the increase of the amount of OREC, the releases are slower [33]. Liu et al. have studied on the role of polymer–filler interaction in the drug release behaviors of CDHA (Ca-deficient hydroxyapatite)/chitosan nanocomposite membranes, in situ incorporation of CDHA nanoparticles, i.e. CDHA synthesized in the presence of chitosan, was employed [34]. For comparison, ex situ synthetic process was also used. In other words, CDHA nanofiller was synthesized first and then added into the chitosan solution. Membrane permeability and diffusion exponent of CDHA/CS nanocomposites with various CDHA contents at 95/5, 90/10, 70/30 and 50/50 for both in situ and ex situ processes were systematically investigated to explore the influence of CDHA nanofillers on the drug release behavior and the corresponding release mechanism. For ex situ processes, CDHA nanoparticles were first prepared by mixing Ca(CH3COO)2 aqueous solution with H3PO4 aqueous solution. Vitamin B12 is a water-soluble agent with low molecular weight (1355Da), small molecular size and negligible interaction with CDHA, selected for drug release tests. The results indicated that the drug diffusion mechanism is altered by the CDHA–chitosan interaction which is strongly influenced by both the synthesis process and the concentration of the CDHA nanofiller in the membrane. On the other hand, a lower permeability (P) value of the membranes was observed for those prepared via the in situ process. The membrane permeability was decreased with the incorporation of CDHA from 0% to 10%. However, further increase in the CDHA from 10% to 30% caused an increase in the permeability, which became more pronounced when the CDHA content was higher than 30%. Zhang et al. [35] reported the fabrication of macroporous chitosan scaffolds reinforced by Beta tricalcium phosphate ( -TCP) and calcium

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phosphate invert glasses using a thermally induced phase separation technique. Results indicated that incorporating -TCP and glass into the chitosan matrix effectively reduced the initial burst release of gentamicin- sulphate (GS) from the composite chitosan scaffolds. In comparison with the GS loaded pure chitosan scaffolds, the initial burst release of GS was decreased through incorporating calcium phosphate crystals and glasses into the scaffolds, and the sustained release for more than 3 weeks was achieved. Zhou et al. have studied preparation of silver–chitosan, silver–chitosan/clay, and polydimethyloxane(PDMS)/silver– chitosan/clay. The resulted nanocomposites applied in indwelling biomedical catheter materials are synthesized and their bacteriostasis to Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and Candida albicans [36]. Inhibition Ring Test showed that PDMS/silver–chitosan/clay nanocomposites kill the mass of predominant urinary bacteria compared with chitosan and AgNO3, the rate of killing bacteria is greatly improved. Also Ag (I) releasing from samples was studied. The silver ion is a biocompatible metal and is widely used as an antibacterial agent [37]. On comparing the outcome of the drug controlled releasing system test on PDMS/silver–Chitosan with PDMS/Clay–Chitosan–Ag nanocomposites, the results show that the rate of Ag (I) release in PDMS/Clay–Chitosan–Ag nanocomposites is apparently slower.

1.2.3. Wound–Dressing It is well known that wound-dressing materials should be durable, stress resistant, flexible, pliable, and elastic with reasonable tensile properties, which could bear the stresses exerted by different parts of the body having varying contours. It was reported that the increase in flexibility could improve the contact between the film and the tissue, hence promoting penetration of the polymeric chains into the tissue to form a strong bonding, leading to an increase in the adhesion strength, Depan et al. [38] have prepared organic– inorganic hybrids of layered silicates and chitosan-g-lactic acid (LA) by grafting of lactic acid on chitosan in the presence of layered silicates without using any catalyst using lactic acid itself as solvent. Polylactic acid is a natural intermediate in carbohydrate metabolism. Owing to their biodegradability, homopolymers and copolymers of lactic acid have been widely used in biomedical applications. Depan et al. have showed that grafted poly(lactic acid) chains may act as internal plasticizers to reduce the brittleness of chitosan films/to obtain more soft and elastic film. Watthanaphanit et al. have showed that enhanced antibacterial behavior and mechanical property of sodium alginate nanocomposite fibers with chitosan nanowhisker as nanofiler [39]. This nanocomposite was fabricated with wet-spun method and the weight ratios of chitosan whiskers to alginate ranging from 0.2% to 1.0% w/w. Alginate is a biopolymer derived from cell walls of certain brown algae [40] It is a linear block copolymer of 1

4-linked -D-mannuronic and α-L-guluronic acid residues. Available in various

fibrous and hydrogel products, alginate-based materials are extensively used in wound-care applications, because they offer many advantages, e.g., biocompatibility, haemostatic capability and gel-formability upon subjected to an aqueous environment [41]. Antibacterial property of the neat and the alginate/chitosan whiskers nanocomposite yarns was evaluated based on the colony count method. The test was conducted against Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli. Evidently, the bacterial reduction rate (BRR) values of the nanocomposite yarns containing 0.6% and 1.0%

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w/w of chitosan whiskers against S. aureus were about 40% and 43%, respectively, while those against E. coli were greater at about 66% and 84%, respectively [39].

1.2.4. Tissue Engineering Chitosan (CS) can be utilized in combination with other bioactive inorganic ceramics, especially hydroxyapatite (HA) to further enhance tissue regenerative efficacy and osteoconductivity. In this composite, chitosan just plays in a role of adhesive to dissolve the problem of difficulty of HA specific shape and migration of HA powder when implanted. Hu et al. [42] have studied on preparation homogenous, transparent and high-strength CS/HA nanocomposite, in which the matrix CS is precipitated and the filler of HA synthesized simultaneously by in situ hybridization. A transparent and slight yellow CS/HA nanocomposite with high strength was prepared by in situ hybridization. The high mechanical properties are obtained due to the layered structure confirmed by the SEM photographs of composite. The initial mechanical properties of bending strength and bending modulus of composite are 86MPa and 3.4GPa, respectively, which is 2–3 times stronger than that of PMMA and bone ceramics. Nano-size HA particles identified by TEM and XRD was dispersed well in CS/HA composites, which can also be proved by the transparent apparent of composite rod. The addition of HA can reduce the water absorption, which postponed the retention of mechanical properties of CS/HA composite under moisture condition [42]. Preparation methods of HA/chitosan composites have been reported, such as mixing of a HAp powder in a chitosan solution [43] and coating of HA particles onto a chitosan sheet [44]. However, the composites obtained by these means were microscopically inhomogeneous and often caused inflammation when implanted. Yamaguchi et al. have reported of preparation homogeneous HA/chitosan composites using a coprecipitation method. In the composites obtained, calcium phosphate formed crystalline HA when acetic acid and lactic acid were used in the preparation solvent of chitosan while it was found to be amorphous when organic acids having more than two carboxyl groups were used. The mechanical strength could be enhanced by heat treatment in a saturated steam and was ascribed to the formation of hydrogen bonds among chitosan molecules [45]. Chen et al. [46] have reported the preparation of HA nano-particle and HA/chitosan nanocomposite. Nano-sized hydroxyapatite powder was synthesized successfully in aqueous solutions. The particle size is about 20–30 nm in width and 50–60 nm in length. Using this nano-powder, hydroxyapatite/chitosan nano-composites are also prepared by the precipitation. The aqueous solution of NH4H2PO4 was added to the mixture of the aqueous solution of Ca (NO3)2 and chitosan whose degree of deacetylation is 84%. Nano hydroxyapatite (HA) has wide range of medical applications, particles mobilization and slow resorbable nature limits its use in certain applications particularly, periodontal and alveolar ridge augmentation. To enhance its usage, Murugan et al. [47] have prepared composite bone paste using chitosan and nano HA through wet chemical method to facilitate particulate immobilization and to boost the bioresorbability of nano HA. The physical nature of paste form implies that it would be highly beneficial for the particle immobilization upon implantation. In vitro physiological stability and solubility of the composite was performed in phosphate buffered saline under physiological condition and found that the rate of resorbability of composite was quite higher than nano HA. In the n-HA/Chitosan scaffold prepared by freeze-drying has a poor compressive strength although it has better cell biocompatibility than the CS scaffold in vitro. To solve these

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problems, many researchers have added another organic polymer as a reinforcing phase. Carboxymethyl cellulose (CMC) obtained from natural cellulose by chemical modification is a water soluble cellulose ether derivate [48] and is very similar to CS in structure. Jiang et al. have shown describe the preparation of three n-HA/CS/ CMC composite scaffolds are prepared with weight percentage of CMC of 40, 30 and 15 wt% [49, 50]. In this studies, nHA/CS/CMC composite scaffolds by a simple and effective freeze-drying method without introducing any other poisonous cross-linking agents. The composite scaffold was formed by the main ion cross-linking interaction resulting from the [NH3+ ] of CS and [COO-] of CMC. In addition, the SBF soaking experiment showed the scaffold of 30 wt% CMC had an acceptable degradation rate and good bioactivity in vitro. Yin et al. [51] have reported that Chitosan–Gelatin/ -TCP composite scaffolds have fabricated through a freeze-drying technique. With -tricalcium phosphate, the compressive modulus and yield strength of the scaffolds were greatly improved. A mild inflammatory response was observed over 12 weeks. The biocompatibility evaluations revealed that the composite scaffolds have good biocompatibility. The preliminary biocompatibility was evaluated subcutaneously on rabbits. Its intensity declined along with the scaffolds gradually being degraded. The results suggested that the scaffolds can be utilized in nonloading bone regeneration. Zhang et al. have reported the similar work [52]. Incorporation of hydroxyapatite (HA) into the matrix of collagen (Col) and chitosan (Cs) by in situ synthesis was introduced to prepare nanocomposites [53]. Preparation of Col–Cs–HA nanocomposites, calcium and phosphate-containing solutions were added dropwise into the neutral blend for in situ HA synthesis in Col–Cs system (Col:Cs = 5:4). The Ca/P ratio was set to 1.67. Structural investigations of the pure Col–Cs mixture validated the influence of Cs on Col assembly, but the molecular interactions between Col and Cs was partially depressed during the intervention of in situ HA synthesis, as revealed by FTIR and DSC analyses. A series of Col–Cs–HA (CCHA) nanocomposites with varying HA content were thereby prepared by a sequential method, involving in situ synthesis in the Col–Cs system, then gelling at 25 °C and subsequently washing the resultant elastic gel followed by dehydration consolidation. Formation of a well integrated microstructure of organic fibers (ca. 90 nm in size) and dense matrix including inorganic aggregates (less than 30 nm in size) was found in these nanocomposites. These results indicated that in situ HA synthesis in the Col–Cs system provided a feasible route for bone grafting nanocomposites. Kim et al. [54] have prepared a new bioactive bone cement (BBC), composed of natural bone powder (hydroxyapatite; HA), chitosan powder, and the currently available polymethylmethacrylate (PMMA) bone cement, for utilization in orthopedic surgeries such as vertebroplasty or as bone filler. Three types of BBCs (BBC I (50:40:10), BBC II (40:50:10), and BBC III (30:60:10)) were prepared with different composition ratios of PMMA:HA:Chitosan . In vitro tests and animal studies were performed with the new BBCs, and with currently available commercial PMMA bone cement. The results showed that the water absorbency, weight loss, and porosity of the BBCs were higher than those of pure PMMA, but the compressive Young’s modulus and the ultimate compressive strength (UCS) of the BBCs were lower than those of pure PMMA. The exothermic temperatures of the BBCs were considerably lower than that of pure PMMA. BBC II and III required longer times to solidify than did pure PMMA. Intrusion tests showed that the BBCs were more interfering than was pure PMMA. Cell proliferation tests demonstrated that BBC II was preferable to pure PMMA for cell attachment and proliferation. No cytotoxic characteristics were found associated with any of the BBCs. In animal tests,

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BBC II was moore biocompatible and osteoconductible than was puree PMMA. Thee results of B inn vitro and animal a studiees indicated that the propposed BBCs have potentiial clinical appplication as replacements r f the pure PM for MMA bone ceements currenttly in use [54]].

2. CELLULOSE E 2. 1. Cellulosse Structuree w lignins Cellulose is the most abuundant biopolyymer in the biiosphere. Often associated with (ligno-cellulosee products), thhis carbohydraate polymer is the main coonstituent of wood, w flax, i a linear maacromolecule constituted c off D-glucose raamie, hemp orr cotton. This biopolymer is unnits linked by y 1

4linkaages and show ws a semi-cryystalline structture (see Figuure 2). The

gllucose monom mers units in cellulose form m both intra- and inter-moolecular hydroogen bonds geenerating celllulose microfiibrils. These hydrogen h bonnds lead to thhe formation of a linear crrystalline structure with a high h theoretical tensile strength [55]. Deepending on thheir origin, ceellulose microofibrils have diameters in the range 2––20 nm whilee their length can attain seeveral tens off microns [56]]. The microfi fibrils cellulosse chains are aligned a in parrallel in an allmost perfect crystalline array. a Some defects d arise from f dislocations at the innterface of m microcrystallin e domains aloong the microffibril length [557]. These defects are advaantageously ussed to produce, by acid treeatment, rod-liike mono-crysstals called whiskers w havingg the same diiameter as th he starting microfibrils m buut shorter lenngth. Thanks to these chaaracteristics (m microscopic diimensions, forrm and excepttional mechannical propertiess), these whiskkers can be inncorporated ass a reinforcinng component into polymerr matrices to produce nanoocomposite m materials with enhanced e propperties for a wide w range of potential p applications [58].

2..1.1. Bacteriaal Cellulose Bacterial cellulose c is onne of type celllulose that haas bacterial orrigin. BC is secreted s by G Gluconacetobac cter xylinus (= =Acetobacter xylinum) andd has unique properties p inclluding high w water holding capacity, c highh crystallinity, a fine fiber network, n and high h tensile strength [59, 600]. BC has reccently been stuudied for use as blood vessels [61]. Beinng similar to human h skin, baacterial cellulo ose can be appplied as skin substitute in treating extennsive burns [62]. BC has pootential to be used u as a subsstrate for tissuue engineering of cartilage due d to its high strength in thhe wet state as a well as its moldability inn situ, biocom mpatibility andd relatively siimple, cost effficient producction [63-66].

Fiigure 2. Chemiccal structure of cellulose.

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2. 2. Cellulose Nanocomposites and their Biomedical Application 2.2.1. Antibacterial Behavior Bacterial cellulose is an interesting material for using as a wound dressing since it can control wound exudates and can provide moist environment to a wound resulting in better wound healing. Maneerung et al. have reported preparation of BC/silver nanocomposite for this purpose [67]. However, bacterial cellulose itself has no antimicrobial activity to prevent wound infection. To achieve an antimicrobial activity, silver nanoparticles were impregnated into bacterial cellulose through the chemical reduction by immersing bacterial cellulose in the silver nitrate solution. Sodium borohydride was then used to reduce the absorbed silver ion (Ag+) inside of bacterial cellulose to metallic silver nanoparticles (Ag°). The freeze-dried silver nanoparticle-impregnated bacterial cellulose exhibited strong the antimicrobial activity against Escherichia coli (Gram-negative) and Staphylococcus aureus (Gram-positive). The antibacterial activity of samples was measured by the disc diffusion method and the colony forming count method. A recent study showed that impregnation, instead of coating the wound dressing with silver nanoparticle or nanocrystal improved the antimicrobial activity of the wound dressing and lowered possibility of the normal human tissue damage. In other work Son et al. have reported preparation of cellulose acetate (CA) nanofibers electrospun from CA solutions with 0.5 wt% of AgNO3 were irradiated with UV light at 245 nm [68]. The Ag nanoparticles were generated only on the surface of the CA nanofibers and their average size was 21 nm after UV irradiation for 240 min. The CA nanofibers incorporating Ag nanoparticles with an average size of 21 nm exhibited strong antimicrobial activity. 2.2.2. Tissue Engineering Hydroxyapatite (HAp) and bacterial cellulose (BC) are both excellent materials for use in biomaterial areas. Preparation of HA/BC composites has been proposed in an attempt to make a new class of biomaterials with high mechanical performance and good osteoconductivity and biodegradation for tissue engineering and orthopaedic surgery. Wan et al. [69, 70] have reported the preparation of HA/BC nanocomposites with a 3-dimensional (3-D) network were synthesized via a biological route by soaking both phosphorylated and unphosphorylated BCs in 1.5 simulated body fluid (SBF). Results indicated that the hydroxyl groups of bacterial cellulose are not enough reactive to trigger HAp growth. Phosphorylation treatment of BC greatly enhanced its capability of inducing HA formation. FTIR results showed that the obtained HA crystals were partially carbonate substituted. Therefore, the prepared HA by the current biomimetic process was similar to natural bone in structure, crystallinity and crystal size. In other work Millon et al. have used bacterial cellulose nanofibers as nanofiller thrugh PVA polymer for biomedical application [71]. Polyvinyl alcohol (PVA) is a biocompatible hydrogel with characteristics desired for biomedical applications. It can be crosslinked by a low temperature thermal cycling process. By using a novel thermal processing method under an applied strain and with the addition of a small amount of bacterial cellulose (BC) nanofibers, an anisotropic PVA-BC nanocomposite was created. The stress–strain tensile properties of porcine aorta were closely matched in both the circumferential and the axial directions by one type of anisotropic PVA-BC nanocomposite (10% PVA with 0.3% BC at 75% initial strain and cycle 2) within

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physiological range, with improved resistance to further stretch beyond physiological strains. The PVA-BC nanocomposite gives a broad range of mechanical properties, including anisotropy, by controlling material and processing parameters. PVA-BC nanocomposites with controlled degree of anisotropy that closely match the mechanical properties of the soft tissue it might replace, ranging from cardiovascular to other connective tissues, can be created.

3. STARCH 3.1. Starch Structure Starch is mainly extracted from cereals (wheat, corn, rice, etc.) and from tubers (potatoes, manioc, etc.). It is stocked into seeds or roots and represents the main plant energy reserve. Depending on the botanical origin of the plant, starch granules can have very different shapes (sphere, platelet, polygon, etc.) and size (from 0.5 to 175 mm). These granules are composed of two α- D-glucopyranose homopolymers, the amylose and the amylopectin. Their proportions into the granules depend directly on the botanical source [72]. The amylose is mainly a linear polysaccharide composed of D-glucose units linked by α(1

4) linkages .

These chains are partially ramified with some α (1՜6) linkages (see Figure 3). Depending on the botanical source and the extraction process, the amylase molecular weight varies from 105 to 106 g mol-1 with a polydispersity ranging from 1.3 to 2.1 [73-75]. The amylase chains

show a single or double helix conformation with a rotation on the α (1՜4) linkage [76]. The amylopectin is the main starch component and has the same monomeric unit as amylose. It shows 95% of α (1՜4) and 5% of α (1՜6) linkages.

Figure 3. Chemical structure of starch (amylopectin)

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These latter are found every 24–79 glucose units [77] and bring to the amylopectin a highly branched structure. Consequently, the amylopectin structure and organization can be seen as a grape with pending chains [78]. The starch granule organization consists in an alternation of crystalline and amorphous areas leading to a concentric structure [79]. The amorphous areas are constituted of the amylose chains and the amylopectin branching points. The semi-crystalline areas are mainly composed of the amylopectin side chains. Some cocrystalline structures with the amylose chains have been also identified [80, 81]. Depending on the botanical origin, starch granules present a crystallinity varying from 20 to 45%.

3. 2. Starch Nanocomposites and Their Biomedical Application 3.2.1. Tissue Engineering Silva et al. have studied the synthesis and bioactivity of polymer soluble potato starch and composite (with Bioactive Glass 45S5) micron-size particles [82]. A result is that although with different properties, both polymer and composite particles were able to form a calcium phosphate layer at their surface, which is a clear indication of their bioactivity. The cytotoxicity and the ability to support cell attachment and growth of the developed materials were also studied, and both polymer and composite materials were shown to be noncytotoxic. The MTT test is a biochemical test widely used to assess cytotoxicity by measuring cell viability and proliferation in a qualitative way. This biochemical test is based in the reduction of (3-(4, 5-dimethylthiazol-2-yl)-2,4-diphenyltetrazolium bromide) (which is watersoluble and has a yellow tonality) by the cell mitochondrial enzyme succinate dehydrogenase, yielding a purple color salt insoluble in water. The salt absorbs at a wavelength of 570 nm and since only living cells have the capability of metabolizing the MTT, it gives a measurement of the viable cells Preliminary. The results show that both types of materials were found to allow rat bone marrow cells to attach and to proliferate on their surface and to express osteogenic markers, such as alkaline phosphatase and osteopontin. The obtained results indicate that the developed carriers might be used as substrates for cell culture in vitro, in order to form constructs that might be used as a part of a tissue engineering strategy. Biomaterials used as a bone substitute should be a temporary material serving as a scaffold for bone remodelling [83, 84]. This type of material must degrade in a controlled fashion into nontoxic products that the body can metabolise or excrete via normal physiological mechanisms [85, 86]. Short-term cytotoxicity testing shows that cells cultured with both polymer and composite leachable remain clearly viable. Preliminary studies on the ability of these particles to support attachment and growth of undifferentiated rat bone marrow cells have shown that cells do adhere to the materials, and that they express osteopontin both in the presence or absence of dexamethasone in the culture medium. Marques et al. [87, 88] have studied to determine which, from a range of the starch-based biomaterials, would be more suitable to be used in orthopaedic applications. This included blends of corn starch and ethylene vinyl alcohol (SEVA-C), corn starch and cellulose acetate (SCA), corn starch and polycaprolactone (SPCL) and its composites with increasing percentages of hydroxyapatite (HA). Osteoblast-like cells (SaOs-2) were cultured in direct contact with the polymers and composites and the effect of the incorporation and of increasing percentages of the ceramic in osteoblast adhesion/proliferation was assessed in the evaluation of cell adhesion and proliferation rate. The proliferation rate was found to differ

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from blend to blend as well as with the time of culture and with the presence of HA depending on the material. SEVA-C and respective composites systematically presented the higher number of cells comparatively to the other two blends. SPCL composites were found to be less suitable for cell proliferation. The amount of cells quantified after 7 days of culture, both on the surface and on the wells showed a delay in the proliferation of the cells cultured with SPCL composites comparatively to other materials and to TCPS. SCA composites, however, did support cell adhesion but also induce a slight level of toxicity, which results in delayed proliferation on the cells adhered to the wells. In fact, cells were well adhered and spread on the majority of the surfaces. Thus, starch-based biomaterials can be seen as good substrates for osteoblast-adhesion and proliferation that demonstrates their potential to be used in orthopaedic applications and as bone tissue engineering scaffolds. Sousa et al. [89] have studied on processing and properties of bone-analogue composites aimed to be used in temporary or permanent orthopaedic applications. The studied matrices were biodegradable starch based blends (with ethylene-vinyl alcohol copolymer and with cellulose acetate). Composites of these materials with hydroxyapatite (HA-the main inorganic constituent of the human bone) were produced by extrusion compounding and subsequently injection moulded. A non-conventional injection moulding technique known as shear controlled orientation in injection moulding (SCORIM) was used deliberately to induce a strong anisotropic character to the processed composites. It was possible to produce, both biodegradable and bioinert matrix composites, with properties that might allow for their application in the orthopaedic field.

3.2.2. Diagnostic Applications Magnetic nanoparticles have been proposed for use as biomedical purposes to a large extent for several years. The development of techniques that could selectively deliver drug molecules to the diseased site, without a concurrent increase in its level in healthy tissues, is currently one of the most active areas of cancer research. Saboktakin et al. [90] have reported the using of carboxymethyl starch(CMS) as the coating material for SPIONs (superparamagnetic iron oxides nanoparticles) to achieve the stabilization and drug delivery of ferrofluid. The CMS-coated SPIONs of about 10 nm diameter having a core–shell structure with magnetic core and polymer shell have been successfully prepared. The FT-IR experimental results proved that the CMS is adsorbed onto the surface of SPIONs through the hydrogen bonding between polar functional alcohol groups of CMS and hydroxylated and protonated surface sides of the oxide. Hence the resultant nanoparticles possess an excellent solubility and stability in ferrofluid. Therefore, CMS as a coating material not only prevented the aggregation between SPIONs in physiological medium but also provided a capacity to be delivered in cancer tissue specifically, which suggests the potential utility of CMS-coated SPIONs as a contrast agent for cancer diagnosis [90].

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In: Recent Developments in Bio-Nanocomposites… ISBN 978-1-61761-008-0 Editor: Ashutosh Tiwari © 2011 Nova Science Publishers, Inc.

Chapter 19

DEVELOPMENT OF BIO-FRIENDLY ENERGY HARVESTING MATERIALS Radhe Shyam Rai11, Ashutosh Tiwari2,3, Ajay K. Mishra4, and Shivani B. Mishra4 1

Departmento de Engenharia Cerâmica e do Vidro and CICECO, Universidade de Aveiro, 3810-193 Aveiro, Portugal 2 School of Materials Science and Engineering, Jiangsu University, Zhenjiang 212 013, China 3 National Institute for Materials Science, Tsukuba, Ibaraki 305 0047, Japan 4 Department of Chemical Technology, University of Johannesburg, Doornfontein, Johannesburg 17011, South Africa

ABSTRACT Jacques and Pierre Currie discovered the phenomenon of piezoelectricity in 1880, category of smart materials exhibiting unique and interrelated properties. Application of stress to a piezoelectric crystal generates a corresponding electric charge. Conversely the application of an external voltage will induce a shape change. Many materials display piezoelectric properties, some of which are naturally occurring e.g. Quartz, whilst others are engineered to display the properties, e.g. lead zirconate titanate (PZT), polyvinylidene fluoride (PVDF), Sodium Potassium Niobate and etc. The polymer materials are soft and flexible; however have lower dielectric and piezoelectric properties than ceramics. Monolithic piezoelectric ceramics are rigid, heavy and produced in block form; therefore add additional mass and stiffness to the host structure, especially when bonding to flexible/lightweight materials.

1 Corresponding Author: E-mail: [email protected].

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1. INTRODUCTION Now in these days the trends in technology allow the decrease size and power consumption of complex digital systems. This decrease in size and power gives rise to new concepts of computing and use of electronics, with many small devices working collaboratively or at least with strong communication capabilities and medical application. One ambient vibration energy source is human movement, with energy rife in breathing, blood pressure and walking. Approximately 60–70Wof power is consumed during walking and a piezoelectric material in a shoe with a conversion efficiency of 12.5% could produce 8.4 W of power. Intelligent clothing with flexible piezoelectric materials integrated into fabrics such as gloves, may be capable of collecting a portion of the mechanical energy associated with daily activities. Wearable devices will undoubtedly multiply in years to come due to a constant decrease in size and power requirements of electronic systems. Piezoelectric materials respond to almost any type and magnitude of physical stimulus, including but not limited to pressure, tensile force, and torsion. Secondly solar energy is a very attractive source for powering the sensor array and the solar technology has matured over the years. But the light intensity can drop the efficiency of this system. One of the major challenges in the implementation of solar technology on the “energy on demand” platform has been the requirement of bulky electronics. The variation in the light intensity (cloudy vs. sunny day) can drop the efficiency. Air flow and mechanical vibration are the other attractive alternatives in locations where these are available. Mechanical energy can be converted into electrical energy using piezoelectric, electromagnetic or electrostatic mechanism. Piezoelectric transducers are more suitable mechanical to electrical energy converters. The piezoelectric materials are the first prime factor affecting the performance of generator. Commonly used piezoelectric materials are based on lead zirconate tanate (PZT) ceramics, relaxor ferroelectric, PVDF. The primary factor for the selection of piezoelectric materials for energy harvesting devices or sensor application is the transduction rate. Electricity is increasingly consumed by electronic products and appliances. The development of an energy-efficient, stable, cheap and convenient power source has been the focus of research. Meanwhile, the energy issue has always been a potential threat to modern society. The biggest problem is the environmental and health pressure caused by generating electricity using atomic or coal or other sources. Emissions of primary small particles (less than 2.5 m), secondary small particles (less than 10 m), sulphur dioxide and nitrogen oxides directly cause pneumoconiosis, progressive massive fibrosis, emphysema, chronic bronchitis, and accelerated loss of lung function1. Greenhouse gases are also contributing to the warmer climate and increasing number of floods, tornadoes and other forms of disastrous weather. According to a WHO study [1], 1500000 people were killed by greenhouse gasrelated diseases from 1900 to 2000. Although energy efficiency is improving and recoverable reserves are increasingly being adopted, exhaustion of traditional energy is just around the corner. It is now recognized that renewable and clean energy sources are among the best solutions on a large scale. The market of piezoceramic components is dominated by Lead Based PZT containing more than 60wt% lead. Lead is a heavy metal, and its toxicity is well known. Some of the symptoms of Lead poisoning are headaches, constipation, nausea, anemia and reduced

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fertility. Continuous uncontrolled exposure could cause more serious symptoms such as nerve, brain or kidney damage. Some other piezoceramic compounds are based on bismuth and /or barium, which are also heavy metals with expected problems of toxicity. But due to its high-performance piezoelectric and ferroelectrics properties, this material has been extensively used in sensors, energy harvesting, actuators, memory display and other electronic devices [2-4]. In view of the environmental point, the accumulation of lead compounds has been developed into a social problem in the worlds; e.g. the volatization of lead during firing process and the final disposal of electronic parts including lead compounds. Thus Lead has recently been expelled from many commercial applications and materials owing to concerns regarding its toxicity. The exclusion of electronic parts including lead compounds has been restricted gradually and will be prohibited by WEEE (Waste Electrical Electronic Equipment) in the EU and world in near future. But it is in practice, considered to be impossible to collect all lead compounds from disposed electronic devices with the present technology. In electronic parts, it is just in piezoelectric materials that concentration of lead is the highest, and majority of those are PZT systems. This is because PZT includes lead as its main components at more than 50%. There is, therefore, a strong demand to develop a substitution of PZT system materials. Several kinds of non-lead based materials have been reported; BaTiO3 (BT) system [5], (Na,K)NbO3 (NKN) system [6-7], (BiNa)TiO3 system [8-9], Bismuth layered structural ferroelectrics [10-11], langasite system [12], BiMeO3 (Me = Sc, Fe, In etc.) [13-14]. However there are no suitable materials that could perfectly substitute PZT and this has been the stimulant for growing research on this subject. Among the oxide perovskite-structured ferroelectric materials, sodium potassium niobate or Na0.5K0.5NbO3 (NKN) has recently emerged as one of the most promising materials in radio frequency (rf) and microwave applications due to high dielectric tunability and low dielectric loss. The NKN is a promising candidate material for FRAM device, because it has high remnant polarization, low processing temperature and fatigue-free characteristics. A large number of researcher are working on to replace PZT (Complete / partially) by some other ferroelectric and piezoelectric systems. Similar to PZT, Sodium Potassium Niobate (Na1-xKx)NbO3, is a combination of ferroelectric (KNbO3) and antiferroelectric (NaNbO3) [15], which makes it promising base materials for lead free piezoelectrics. Among all the non Lead ferroelectrics materials (Na,K)NbO3 (NKN) systems has a similar curie temperature and high electromechanical coupling constant compared with PZT. Even though Lead based materials have better properties than NKN based materials, considering the fact of lead being non environment friendly, researchers in NKN based materials have gained a new importance. Moreover a lot of desirable changes in the property of ceramics are expected when prepared from nano sized powders. This gave us an insight to investigate NKN and doped NKN system for their use in electronic industries. Here we explore the development of a novel system which will be suitable for micro and macro electronic industry. Generally these novel systems act as passive elements that simply conduct electricity between power sources (e.g., batteries) and various devices such as high-brightness light-emitting diodes (HBLEDs) in an integrated and flexible substrate. Even when the phrase "energy-harvesting" has been used, the system is a simply a passive component that conducts electricity generated by solar cells or by thermoelectric generator chips (i.e., convert body heat to electricity). Jacques and Pierre Currie discovered the phenomenon of piezoelectricity in 1880, a category of smart materials exhibiting unique and interrelated properties. Application of stress

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to a piezoelectric crystal generates a corresponding electric charge. Conversely the application of an external voltage will induce a shape change. Many materials display piezoelectric properties, some of which are naturally occurring e.g. Quartz, whilst others are engineered to display the properties, e.g. lead zirconate titanate (PZT), polyvinylidene fluoride (PVDF), and cellular or porous polymer electrets. Probably the most common piezoelectric materials are polymers (PVDF) and ceramics (PZT). The polymer materials are soft and flexible; however have lower dielectric and piezoelectric properties than ceramics. Monolithic piezoelectric ceramics are rigid, heavy and produced in block form; therefore add additional mass and stiffness to the host structure, especially when bonding to flexible/lightweight materials. This and their fragile nature limit possibilities for wearable devices. The field of piezoelectric polymers have long been dominated by ferroelectric polymers of the PVDF family discovered in 1969 [16-17]. Piezoelectric ceramic materials are generally physically strong, chemically inert, and relatively inexpensive to manufacture. Ceramics manufactured from formulations of lead zirconate/lead titanate exhibit greater sensitivity and higher operating temperatures. A traditional piezoelectric ceramic is a mass of perovskite crystals. Each crystal consists of a small tetravalent metal ion in a lattice of larger, divalent metal ions giving each crystal a dipole moment. Above the Curie temperature, each pervoskite crystal in a fired ceramic exhibits a simple cubic symmetry with no dipole moment. At temperatures below the Curie temperature each crystal has a tetragonal or rhombohedral symmetry and a dipole moment. Adjoining dipoles form regions of local alignments called domains. The alignment gives a net dipole moment to the domain and thus a net polarization. The direction of polarization among the neighboring domains in a piezoelectric ceramic is random so the ceramic has no overall polarization. Poling aligns the domains in the ceramic giving a net polarization. Recently, lead-free ferroelectric K1-xNaxNbO3 (KNN) has attracted interest as an electrically tunable material for piezoelectric, optoelectronic etc. Lead-free KNN-based ceramics will be prepared onto various substrates by Pulsed Laser Deposition method, using pure and doped targets and thermal treatments of crystallization under high-intensity excimer laser irradiation. The appropriate processing conditions will be determined with the aim of obtaining ceramics, with ferroelectric responses comparable to those of corresponding ceramics. A complete characterization of the materials will include the test of capacitors, piezoelectric and ferroelectric, trying to relate these properties with the processing conditions and to establish their use in real devices. The objective of this chapter is concerned with the preparation and determination of piezoelectric and ferroelectric properties in a pure and rare-earth doped lead-free NKN ferroelectric material. We focus on NKN, which is a solid solution of the anti-ferroelectric NaNbO3 and the ferroelectric KNbO3. The study on piezoelectric harvesting began in 1984, when polyvinylidene fluoride patch was used to get energy during inspiration of a human being [18]. However, the power generated was insufficient to power the desired electronics. This study was performed in the early stages of low-power electronics and properties of a piezo-material were not optimized. The energy taken from walking of human being can reach 67 W and thus used for harvesting [19]. The first use of piezoelectrics for harvesting energy from a human body was described in several papers [20-21] and they also reported that it can be improved by piezoceramic material itself and circuitry. Some design study was conducted by Ramsey and Clark [22], who investigated the feasibility of using a piezoelectric transducer for in vivo MEMS

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applications. Their research confirmed a possibility of bio-MEMS powering from vibration energy existing in a human body during operation. Some authors [23] studied the efficiency of commercial multilayer piezoelectric actuators for energy harvesting and showed that the frequency of vibrations is a critical factor for efficiency. The design of the multilayer actuator has to be adjusted to a specific frequency range. Some papers [24-27] are reported to different types of piezoelectric materials for energy harvesting. The power densities for various environmental sources are available in literature [28-33]. Solar energy has the capability of providing higher power of magnitudes compare to other sources. The solid solution of KNbO3 and NaNbO3 displays a good piezoelectric response, particularly for the Morphotropic phase boundary composition Na0.5K0.5NbO3 (NKN), with piezoelectric properties of d33 = 80 pC/N, kp = 36–40%, Qm = 130, and εr = 290, when it was prepared by ordinary sintering. We optimized NKN ceramics to be a promising candidate for piezoelectric transducer, tunable microwave components. Now in these days the world is moving towards a technological way of living. More and more people are carrying portable electronic devices or more and more sensors are attached to remote locations. But, as the technology for portables has grown tremendously, battery and energy storage technology has not kept up. Piezoelectric materials are capable of converting mechanical excitations into electrical outputs and vice-versa. Studies have shown that, in the case of ceramic/ polymer composites, when the volume fraction exceeds a critical value, the effective piezoelectric coefficients of the composite become comparable to, or even larger than the bulk ceramic material. Piezoelectric materials have been used extensively for energy harvesting, relying essentially on mechanical vibrations of structures to which they are attached. The magnitude of the transduction is governed by the effective piezoelectric stress constant d, and the effective piezoelectric voltage constant g. The primary factor for the selection of a piezoelectric material for energy harvesting device or sensor application is the transduction rate and figure of merit and also our research also explored the relationship between the mechanical quality factor, the coupling coefficient and the transformation efficiency of mechanical to electrical energy in the lead free piezoelectric disc. We noted that high efficiency for piezoelectric conversion devices requires large quality and electromechanical coupling factors, so we provide the discussion of the impact of Q and k2 on efficiency or guidelines for device development. A critical component of any energy harvesting system is the electrical circuit that connects the piezoelectric element to the device to be powered. Optimized circuits for these specific types of application have been constructed and successfully tested. In energy harvesting circuits mainly two functions (i) to effectively transfer the electrical charge from piezoelectric transducer into an effective storage mechanism such as super-capacitor, and (ii) to match the impedance of the generator to the external load) were considered. The commonly used converter circuit is called a buck converter and it operates in the discontinuous current mode (DCM). Several other techniques are used in conjunction with the circuit for reducing the matching impedance such as multilayer the piezoelectric transducer structure and increasing the area aspect ratio (area/thickness). The literature review show that all previous efforts were mainly concentrated on lead based devices and the lead based efficient piezoelectric materials. So it’s needed for further progress in lead free devices and lead free piezoelectric materials. NKN is an attractive candidate because of its large piezoelectric coefficients and high rare-earth solid solubility. It

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can be doped with rare earth ions like neodymium, erbium, ytterbium, thulium and praseodymium to incorporate laser active properties. Of these ions the erbium has gained most interest. Finally, the scientific activity in energy harvesting is motivated strongly by possible applications in power section and energy conservation.

2. METHOD The work will be divided into two major tasks devoted to different stages/priorities of the work. 1) Optimization of materials for devices application and all the characterization for samples with different techniques. 2) Design and build prototype systems for energy harvesting. Depending on the progress in first tasks and results on feasibility (or proof-of-concept) study of the materials developed, a more detailed investigation will be undertaken into each particular piezoelectric material and devices on their base.

2.1. Materials Synthesis and Characterization _ _ _ _ _ _

Synthesis of NKN based compounds using physical and chemical methods. Doping of Alkali metals and with rare-earth ions like at A or B site in NKN. Optimize the Morphotropic phase boundary. High-quality dense samples for achieving single-phase compound of correct structure. Samples with correct stoichiometry and density as close as possible to the theoretical density for a single crystal of the material. -Optimization of materials properties for devices application and all the characterization for samples with different techniques.

2.2. Doping with Different Alkali and Rare Earth Materials _ _ _

- Optimization of different properties with doping materials. - Study doping ratio with different properties. - Study of sintering temperature and time.

Bulk ceramics in the amorphous and crystalline form will be used for optimizing mole% of the doping, piezoelectric, ferroelectric properties. We can also study the sintering temperature and time with different properties. Sintering studies will allows us to study the effects of oxygen stoichiometry, the change in density, and possibly oxygen adsorption at grain boundaries in on the material properties.

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In the view of the above, it is highly relevant to try and find alternatives and/ or replacement for the conventional lead based materials and therefore several lead free and low lead systems have been suggested. NKN is one of the most important classes of non lead ferroelectric compounds which have received significant attention by researcher in the last decade. The present proposal will begin with the synthesis of NKN compound by solid state reaction method followed by detail structural and electrical characterizations. As the miniaturization of electronic devices continues, desired characteristics and processing of the starting powders become a critical issue. For improved properties it is desired to have smaller (nanosized) particles (i.e. bigger surface area) of the starting NKN powder with low loss and conductivity with controlled particle morphology and dispersibility. The sol-gel emulsion process has been successfully used for synthesis of single to multi component ceramic powders with controlled particle size. The proposed sol-gel emulsion process will be used to prepare NKN and different metal ion incorporated NKN powders in nano size, having tailored size and shapes. The various process parameters like surfactant and support solvent ratio and the nature of the surfactant are known to affect the powder size and morphology. This process has the potential to synthesis nano structured powders with controlled chemical composition at low calcination temperatures. For development of energy harvesting materials we carry out a detailed investigation of the above mentioned physical/chemical routes for synthesis of ultra fine NKN based powders. The effect of the particle size, presence of different dopants in the NKN structure has to be critically investigated to achieve the desired electrical and electronics properties of the final product that can be competitive alternative to PZT for application. 1) To synthesis NKN and/or metal doped NKN compound by physical route. 2) To synthesis nano sized NKN using chemical method. 3) Structural characterization of the NKN and metal doped NKN compounds (synthesis by method 1 and 2) 4) Electrical, piezoelectric and ferroelectric characterization of the NKN and metal doped NKN. 5) Study of the effect of processing parameters on the size and shape of NKN particles. 6) Study the effect of particle size of the starting powders on the properties of the dense ceramics developed. 7) Comparison of the properties of materials developed with existing materials as available from literature.

3. SUMMARY This main objective will be achieved by the choice of effective piezoelectric materials, development of the devices on their base and testing the devices together with the available electronics in real vibration conditions. Strain in piezoelectric material causes a charge separation. So Stress/strain on piezoelectric material can develop electric charge. This transformation from mechanical to electrical energy is obtained through the direct piezoelectric effect, and using a storage circuit the generated electrical energy is stored. There are three primary steps in power generation as outlined in this schematic: trapping the

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mechanical AC stress from an available source, converting the mechanical energy into electrical energy with a piezoelectric transducer, and processing and storing the generated electrical energy. The mechanical output can be in the form of a burst or continuous signal, depending on the cyclic mechanical amplifier assembly. In this transformation, there is mechanical loss because of mismatch in mechanical impedance represented by damping factor and reflection ratio, and there is electromechanical loss depending upon the magnitude of coupling factor. Depending on the frequency and amplitude of the mechanical stress, one can design the required transducer, its dimensions, vibration mode, and desired piezoelectric material. In a piezoelectric generator, the charge generation is directly related to the extent to which the ceramic element is deformed. So we will focus on these problems during the materials synthesis, and development of energy harvesting devices. Materials prepared from Non lead based materials (NKN, NKN-BT), commercial powder with high piezoelectric coefficients and minimum porosity. Following subtasks will be focused in research. (i) Identification of suitable powder, binder, and material of internal electrodes (Ag, Ag/Pd). (ii) Fabrication of piezoelectric multilayers by screen printing of metal electrodes and laminating the composite structure. (iii) Choosing proper annealing strategies to obtain dense sample without second phases. The work will be concentrated on polishing, electroding, bonding of single crystals and investigation of their piezoelectric performance. Both the geometrical dimensions and poling strategies will be varied to obtain the high piezoelectric response and reliability.

REFERENCES [1] [2] [3]

J. Dewei, L. Jing Front. Energy Power Eng. China 2009, 3(1): 27–46 K. Uchino Ferroelectric Devices, Marcel Decker, New York 2000. K. Yamashita , H. Katata, M. Okyama., G. Kato, S. Aayagi and Y. Suzuki, Sensors and Actuators A, 97, 30, 2002. [4] S. Priya, R. Taneja, R. Mayers, and R. Islam Piezoelectric and Acoustic Materails for Transducer Applications, Chapter 18, 373, 2008. [5] T.Sugawara, m. Shimizu, T. Kimura, K. Kaktori and T. Tani, Ceramic Tranc, 136, 389, 2003. [6] Y. Saito. H. Takao, T. Tani, T. Kimura, K. Takatori, and T. Tani. Ceramic Tranc, 136, 389, 2003 [7] M. Ichiki, L. Zhang, m. Tanaka and R. Maeda, J. Europen Ceramic soc. 24, 1693, 2004. [8] G. A. Smolenski, V. A. Isupov, A. I. Agranovskaya, and N.N. Kainik, Sov. Phys. Solid State 2, 2651, 1961. [9] T. Takenaka, K. maruyama and K. Sakata, Jpn. J. appl. Physc. 30 2236, 1991. [10] T. Takenaka, T. Gotoh., S. Mutoh. And T. Sasaki, Jpn. J. Appl. Phys. 34, 5384, 1995.

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[11] 11.M. Hirose, T. Suzuki, H. Oka, k. Itakura, Y. Miyaauchi and T. Tsukada, Jp. J. Appl. Physics 38, 5561, 1999. [12] K. Shimamura, h. Takeda, T. Kohno. And t. Fukuda, J. of Crystal growth. 163, 288, 1996. [13] R. E. Eitel, C. A. Randall, T. R. Shrout. And S. E. Park. Jpn. J. Appl. Physics. 41, 2099, 2002. [14] S. Zhang, C. A. Randall and T. R. Shrout. Appl. Phys. Lett. 83. 3150, 2003. [15] R. Wang, . R.Xie, T. Sekiya and Y. Shimojo, Materials Research Bullettin, 39, 1709, 2004. [16] H. Kawai, Japan. J. Appl. Phys. 8, 975, 1969, [17] L. M. Swallow, J. K. Luo, E. Siores, I. Patel and D Dodds, Smart Mater. Struct. 17, 025017 , 2008. [18] E. Hausler, E. Stein, Ferroelectrics 60, 277, 1984. [19] T. Starner, IBM Systems J. 35, 618, 1996. [20] T. Kymissis, C. Kendall, J. Paradiso, N. Gershenfeld, Proc. IEEE Symp. on Wearable Computers Oct. 1998, Pittsburg, pp. 132-139. [21] N. S. Shlenk, McS Thesis and references therein, MIT, Massachusets, 1998. [22] M. J. Ramsey, W. W. Clark, Proc. of the SPIE 8th Conf. on Smart Materials and Structures, Newport, CA, 4332, 429, [23] M. Goldfarb, L. D. Jones, ASME J. of Dynamics Systems and Control, 121, 566, 1999. [24] A. H. Sodano, E. A. Magliula, G. Park, D. J. Inman, Proc. 13th Intl. Conf on Adaptive Structures and Technologies, Oct. 2002, Potsdam, pp. 132-139. [25] H. A. Sodano, G. Park, D. J. Leo, D. J. Inman, Proc. of the SPIE 10th Conf. on Smart Materials and Structures, San Diego, CA, Vol. 5050, p. 101-108. [26] H. A. Sodano, G. Park, D. J. Inman, Mech. Syst. and Sign. Proc. 18, 683, 2004. [27] A. H. Sodano, G. Park, , D. J. Leo, D. J. Inman, Proc. of ASME Intl. Mech. Engineering Congress and Expo, Nov. 2004, V. 40, 2. [28] S. Roundy, P.K.Wright, J.M. Rabaey Energy Scavenging for Wireless SensorNetworks. Kluwer Academic Pub., Boston. (2004). [29] Calaway Jr EH (2004) Wireless Sensor Networks. Auerbach Pub., NY. [30] N. Shenck, J.A. Paradiso, IEEE Micro. 21, 30, 2001. [31] S. Meninger, J.Q. Mur-Miranda, R. Amirtharajah, A.P. Chandrakasan, and J.H. Lang JH. IEEE Trans. VLSI Syst. 9, 64, (2001). [32] S. Roundy, P.K. Wright and J. Rabaey Computer Comm. 26, 1131 (2003). [33] J. Paradiso and T. Starner, Energy Scavenging for Mobile and Wireless Electronics.Pervasive Computing, Jan – March, 18 (2005).

In: Recent Developments in Bio-Nanocomposites… ISBN 978-1-61761-008-0 Editor: Ashutosh Tiwari © 2011 Nova Science Publishers, Inc.

Chapter 20

RECENT ADVANCES IN BIOMEDICAL APPLICATIONS OF MULTIFUNCTIONAL NANOCOMPOSITES Avinash C. Pandey1, Prashant K. Sharma and Ranu K. Dutta Nanotechnology Application Centre, University of Allahabad, Allahabad 211 002, India

1. INTRODUCTION Nanocomposites are a subset of composites that take advantage of unique materials properties on the small scale. The definition of nano-composite material has broadened significantly to encompass a large variety of systems such as one-dimensional, twodimensional, three-dimensional and amorphous materials, made of distinctly dissimilar components and mixed at the nanometer scale. A nanocomposite is similar to a multiphase solid material where one of the phases has one, two or three dimensions of less than 100 nanometers (nm), or structures having nano-scale repeat distances between the different phases that make up the material [1]. In the broadest sense this definition can include porous media, colloids, gels and copolymers, but is more usually taken to mean the solid combination of a bulk matrix and nano-dimensional phase(s) differing in properties due to dissimilarities in structure and chemistry. The mechanical, electrical, thermal, optical, electrochemical, catalytic properties of the nanocomposite will differ markedly from that of the component materials. In mechanical terms, nanocomposites differ from conventional composite materials due to the exceptionally high surface to volume ratio of the reinforcing phase and/or its exceptionally high aspect ratio. The interface area between the matrix and reinforcement phase(s) is typically an order of magnitude greater than for conventional composite materials. The matrix material properties are significantly affected in the vicinity of the reinforcement. Ajayan et al. [1] showed that with polymer nanocomposites, properties related to local chemistry, degree of thermoset cure, polymer chain mobility, polymer chain conformation, degree of polymer chain ordering or crystallinity can all vary significantly and continuously 1 Corresponding Author: E-mail: [email protected], [email protected].

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from the interface with the reinforcement into the bulk of the matrix. This large amount of reinforcement surface area means that a relatively small amount of nanoscale reinforcement can have an observable effect on the macroscale properties of the composite. For example, adding carbon nanotubes improves the electrical and thermal conductivity. Other kinds of nanoparticulates may result in enhanced optical properties, dielectric properties, heat resistance or mechanical properties such as stiffness, strength and resistance to wear and damage. In general, the nano reinforcement is dispersed into the matrix during processing. The percentage by weight (called mass fraction) of the nanoparticulates introduced can remain very low (on the order of 0.5% to 5%) due to the low filler percolation threshold, especially for the most commonly used non-spherical, high aspect ratio fillers (e.g. nanometer-thin platelets, such as clays, or nanometer-diameter cylinders, such as carbon nanotubes). The general class of nanocomposite organic/inorganic materials is a fast growing area of research. Significant effort is focused on the ability to obtain control on the nanoscale structures via innovative synthetic approaches. The properties of nano-composite materials depend not only on the properties of their individual parents but also on their morphology and interfacial characteristics. This rapidly expanding field is generating many exciting new materials with novel properties. The latter can derive by combining properties from the parent constituents into a single material. There is also the possibility of new properties which are unknown in the parent constituent materials. The inorganic components can be threedimensional framework systems such as zeolites, two-dimensional layered materials such as clays, metal oxides, metal phosphates, chalcogenides, and even one-dimensional and zerodimensional materials such as (Mo3Se3-)n chains and clusters. Experimental work has generally shown that virtually all types and classes of nanocomposite materials lead to new and improved properties when compared to their macrocomposite counterparts. Therefore, nanocomposites promise new applications in many fields such as mechanically reinforced lightweight components, non-linear optics, battery cathodes and ionics, nano-wires, sensors and now in biomedical field. The general class of organic/inorganic nanocomposites may also be of relevance to issues of bio-ceramics and biomineralization in which in-situ growth and polymerization of biopolymer and inorganic matrix is occurring. Finally, lamellar nanocomposites represent an extreme case of a composite in which interface interactions between the two phases are maximized. Since the remarkable properties of conventional composites are mainly due to interface interactions, the materials dealt with here could provide good model systems in which such interactions can be studied in detail using conventional bulk sample (as opposed to surface) techniques. By judiciously engineering the polymer-host interactions, nanocomposites may be produced with a broad range of properties. Inorganic layered materials exist in great variety. They possess well defined, ordered intralamellar space potentially accessible by foreign species. This ability enables them to act as matrices or hosts for polymers, yielding interesting hybrid nano-composite materials. Lamellar nano-composites can be divided into two distinct classes, intercalated and exfoliated. In the former, the polymer chains alternate with the inorganic layers in a fixed compositional ratio and have a well defined number of polymer layers in the intralamellar space. In exfoliated nano-composites the number of polymer chains between the layers is almost continuously variable and the layers stand >100 Å apart. The intercalated nanocomposites are also more compound-like because of the fixed polymer/layer ratio, and they

Recent Advances in Biomedical Applications of Multifunctional Nanocomposites 411 are interesting for their electronic and charge transport properties. On the other hand, exfoliated nano-composites are more interesting for their superior mechanical properties.

2. BACKGROUND OF DIFFERENT MULTIFUNCTIONAL NANOCOMPOSITES FOR BIO-MEDICAL APPLICATIONS In the past decades, colloidal II-VI semiconductor nanocrystals, often referred to as “quantum dots (QDs)” or, have gained increasing attention in technological applications and fundamental studies due to their unique optical properties [2-5]. Among them most importantly, ZnO, embedded/capped/core-shell structures of ZnO in different inorganic (SiO2) or organic (PVA, PVP, biotin, citric acid, CTAB etc.) polymers [6-9], bare and capped CdSe [10], CdTe, CdS, ZnS have made significant advances towards bio-medical applications. Magnetic nanoparticles, e.g., Fe3O4, are additional important materials due to their interesting magnetic properties and have been sophisticatedly employed in many advanced technology areas, including biology, pharmacy, and diagnostics [11, 12].

Figure 1. Colloidal nanocrystals of different materials. Colloidal nanocrystals can be synthesized from metallic, semiconductor or insulating materials. Low-resolution TEM images from nanocrystalsmade out of Co, CdSe and Fe3O4 arranged on a TEM grid are shown. The images were recorded by V Puntes, L Manna and M Casula. Each sphere corresponds to one single nanocrystal. Figure adopted from Nanotechnology 14 (2003) R15–R27.

Magnetic nanoparticles combined with semiconductor nanocrystals would lead to a special functionalized luminescent magnetic nanocomposite that enjoys both the advantages of magnetic nanoparticles and semiconductor QDs and offers higher potential applications. To reduce the unwanted crystallite coarsening and particles aggregation, attempts have been made to synthesize nanocomposites by embedding nanoparticles in a suitable matrix such as silica. Incorporation of the inorganic nanoparticles into a polymer matrix has extended their practical applications, for example, as high-sensitivity chemical gas sensors [13]. Size and shape-controllable synthesis of monodisperse inorganic nanocrystals has attracted a great deal of attention, owing to their unique size and shape-dependent properties. Drug-carrying magnetic nanocomposite spheres were synthesized using magnetite nanoparticles and poly (D, L-lactide-coglycolide) (PLGA) for the purpose of magnetic targeted drug delivery [14].

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Figure 2. TEM images of drug-carrying nanocomposite spheres showing the distribution of magnetite nanoparticles in PLGA matrix at low (a) and high (b) magnifications. Figure adopted from Journal of Nanotechnology, Volume 2009, Article ID 238536, 6 pages doi:10.1155/2009/238536.

Wang et al. used dimercapto-succinimid acid modified ç-Fe2O3 nanoparticles reacted with CdSe/ZnS QDs to prepare water soluble magnetic luminescent composites in an organic/water two-phase mixture [15]. Gaponik et al. encapsulated both CdTe QDs and Fe3O4 simultaneously in polymer microcapsules to prepare a magnetic luminescent composite [16]. The Fe3O4 nanoparticles were used as a template for the deposition of polyelectrolyte multilayers/CdTe QDs (i.e., Fe3O4/PEn/CdTe) or alternative adsorption of three polyelectrolyte layers/CdTe QDs multilayers (i.e., Fe3O4/(PE3/CdTe)n) [17, 18]. One-pot syntheses of size- and shape-controlled spinel ferrite, MFe2O4 (M = Fe, Co, and Mn), nanocrystals (NCs) larger than 20 nm in size were achieved by the thermolysis of mixedvalence tri-iron complexes and heterotrimetallic complexes as a precursor using benzilic acid (BA) as a capping ligand [19]. Magnetic/fluorescent nanocomposites of Fe3O4/CdTe and their applications in immuno-labeling and fluorescent imaging of cancer cells have been shown by Pan Sun et al. [20]. Preparation of Fe3O4/CdS nanocomposites via a sonochemical route in an aqueous solution as effective and convenient recyclable photocatalysts has been shown by Xiaowang Liu et al. [21]. Pd nanoparticle immobilized Fe3O4 and NiFe2O4 nanoparticles have been used for hydrogenation reactions and Suzuki and Heck reactions as facile recoverable catalysts [22, 23]. Among the various magnetic materials, the cubic spinel structured MFe2O4 represents an important class of magnetic transition metal oxide materials. Nanometer-sized MFe2O4 materials and their dispersions have been widely used in many important technological applications ranging from information storage [24], electronics [25, 27], catalysis [28], magnetic resonance image (MRI), to biomedicine and drug delivery [29-30]. In particular, they have been considered to be the most promising magnetic materials in biological applications. NiFe2O4 nanocrystal has shown its advantage in hyperthermia application [31].

Recent Advances in Biomedical Applications of Multifunctional Nanocomposites 413 Synthesis and applications of magnetic nanocomposite catalysts Pd/SiO2/Fe2O3 system was successfully applied to hydrogenation reaction [32].

Figure 3. Schematic illustration of immuno-labeling using Fe3O4/CdTe nanocomposites which are formed by linking multiple TGA stabilized CdTe QDs with the thiol-functionalized silica-coated iron oxide nanoparticles. Figure adopted from Langmuir 2010, 26(2), 1278–1284.

Figure 4. Photographs of aqueous solutions of the as-synthesized Fe3O4/CdTe nanocomposites without applying a magnetic field (left), with a magnetic field (middle), and after removing the magnetic field and stirring (right) under normal light (a) and 365 nm excitation (b). Figure adopted from Langmuir 2010, 26(2), 1278–1284.

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Figure 5. TEM image of Co/CdSe core/shell nanocomposites. (b) High-resolution TEM image of a composite nanocrystal revealing the polycrystalline nature of the shell. Figure adopted from J. AM. CHEM. SOC. 2005, 127, 544-546.

Some nanocrystals are widely used as fluorescence probes for visualizing biological processes in vitro and in vivo, the magnetic nanocrystals are frequently served as magnetic resonance image (MRI) agents for the diagnosis of many diseases, and noble metal nanoparticles are usually exploited as catalysts in organic synthesis or fuel cells [33]. Synthetic biodegradable aliphatic polyester/montmorillonite nanocomposites were synthesized by Sung T. Lim et al. Polymer or SiO2 coated magnetic nanoparticles have been widely investigated due to their superparamagnetic properties and biocompatibility [34-37]. For many applications, these magnetic nanoparticles would benefit from having SiO2 or polymer shells to impart ease of functionalization and biocompatibility. Poly (ethylene glycol) conjugation onto the SiO2 layer has increased cell uptake efficiency in silica coated magnetic nanoparticles and made them feasible for cell separation applications. Selfassembled block copolypeptides on magnetic nanoparticles could be used as a smart drug delivery system controlled by magnetic field. Magnetic nanoparticles can also be processed with quantum dots within a silica matrix to yield magnetic and fluorescent nanocomposites polymeric matrixes that enhance stability and biocompatibility are frequently used to disperse magnetic nanoparticles. It may help to understand the magnetic behavior of nanoparticles due

Recent Advances in Biomedical Applications of Multifunctional Nanocomposites 415 to new possible surface, interparticles, and exchange interactions in magnetic/nonmagnetic matrix.

Figure 6. Magnetic hysteresis loops of Fe3O4 microspheres (black) and Fe3O4/CdS nanocomposites (red) at room temperature; Figure adopted from Xiaowang Liu et al., Crystal Growth and Design, 2009, 9 (1), 197–202.

Figure 7. Schematic Illustration of the LbL Process Forming the Magnetic Luminescent Nanocomposites. Figure adopted from Chem. Mater., Vol. 16, No. 21, 2004.

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Besides these nanocomposites, Sharma et al. [8] reported the nanoparticles of ZnO embedded in SiO2 matrix and proposed its use in bio-imaging purpose. In their another work Dutta et al. [38] have designed and surface modified the potential luminomagnetic nanocarriers of ZnO:Fe for biomedical applications. They suggested the use of ZnO:Fe nanoparticles in targeted drug delivery and localized hyperthermia applications.

Figure 8. Shows prepared ZnO quantum dots embedded in SiO2 matrix under (a) ordinary and (b) UV (365nm excitation) lamps. The letters A, B and C correspond to solid samples, samples dissolved in ethanol and samples dissolved in water. Photographs were taken with a digital camera just after completion of synthesis. Figure adopted from Sharma et al. J. Lumin. 129, 605, 2009,

Figure 9. TEM image of (A) bare ZnO quantum dots with spherical particles and (B) symmetrically dispersed ZnO quantum dots embedded in SiO2 matrix. Figure adopted from Sharma et al. J. Lumin. 129, 605, 2009.

Thus multifunctional nanocomposites have been prepared since decades according to their utilities. These nanocomposites have higher magnetism, luminescence, resistive properties, biocompatible, thermal, and optical stability. Magnetic nanocomposites were synthesized to circumvent the shortcomings associated with conventional magnetic materials. Polymers and surfactants are used to control the size and obtain composites of monodisperse uninform size, shape and properties which is not possible in nanoparticles. Composite materials have been instilled with the desired modifications to obtain multifunctional

Recent Advances in Biomedical Applications of Multifunctional Nanocomposites 417 nanocomposites for various applications in opto-electronic devices and bio-medical applications. In the present book chapter the main focus is on the recent advances made by the multifunctional nanocomposite materials in bio-medical applications such as bio-imaging, targeted drug delivery, nanosensors, magnetic chips, hyperthermia agents, contrast agents for magnetic resonance imaging (MRI) and thermostable devices.

3. BIOMEDICAL APPLICATIONS OF MULTIFUNCTIONAL NANOCOMPOSITES 3.1. Nanocomposites for Magnetic Resonance Imaging (MRI) MRI is as a powerful imaging tool due to its non-invasive nature, high spatial resolution and tomographic capabilities. The applications of MRI have steadily increased over the past decade, offering the advantage of high spatial resolution of contrast differences between tissues. In comparison with conventional gadolinium chelates, nanoparticle-based contrast agents offer enhanced cellular internalization and slower clearance from the tumour site [39, 40]. For MRI purposes iron oxide cores are commonly used as so-called T2 contrast agents and can be divided into either superparamagnetic iron oxides (SPIOs), with diameters of >50 nm, or ultrasmall SPIOs (USPIOs) with diameters of