Bioactive Materials Based on Biopolymers Grafted on Conducting Polymers

Chapter

10

Bioactive Materials Based on Biopolymers Grafted on Conducting Polymers: Recent Trends in Biomedical Field and Sensing Salma Khan and Anudeep K. Narula Guru Gobind Singh Indraprastha University, Delhi, India

1.

INTRODUCTION

In 1977, the seminal research of Shirakawa et al. discovered conjugated semiconducting polyacetylene via a simple chemical oxidation method. The key revolutionary change in the field of conducting polymers comes when Kanazawa et al. (1979) and Diaz et al. (1981), all reported the electrochemical polymerization method of a highly conductive, stable, and processed polypyrrole film. From that time onward, researches explored the various properties of the conducting polymers profoundly. Conducting polymers exhibited unique properties like electrical conductivity (Groenendaal et al., 2000), optical activity (Kim et al., 2013), high electron affinity (Ma et al., 2008), and redox activity (Mathiyarasu et al., 2008) due to which these conducting plastics have become the integral part of the various field such as supercapacitors, light emitting diode, field effect transistors, solar cells, actuators, and sensors. Moreover, with the discovery of biological compatibility, they also have established their niche in biosensing and in biomedical field (Rivers et al., 2002) like tissue engineering (Hardy et al., 2013), artificial organs (Baughman, 1996), drug delivery scaffolds (Pernaut and Reynolds, 2000), etc. However, individual conducting polymer can not be used efficiently due to many problems such as lack of specificity, poor biodegradability, poor stability at definite pH, poor mechanical properties, and lesser flexibility (Kitani et al., 1993). Therefore, biofunctionalization of conducting polymers is achieved through the combination with biopolymers and gives rise to a new category of materials known as smart advance bioactive Biopolymer Grafting: Synthesis and Properties. http://dx.doi.org/10.1016/B978-0-323-48104-5.00010-X Copyright © 2018 Elsevier Inc. All rights reserved.

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442 CHAPTER 10 Bioactive Materials Based on Biopolymers Grafted on Conducting Polymers

materials which in turn allow the direct delivery of the electrical simulation to the cells. The presence of two different polymers in these fabricated hybrid smart bioactive materials may result in sensing and biomedical field with more efficient manner and improved characteristics. It can be understood that intermolecular force of attractions or affinity between conducting polymers and biopolymers in the (Van Krevelen and Te Nijenhuis, 2009) hybrid materials may help to eliminate their individual drawbacks due to synergistic effects, leading to the fabrication of new class of smart bioactive materials. Moreover, efforts have been made to discuss the biosensing mechanism of these fabricated materials. Surface modification of the fabricated biomaterials is achieved via molecular engineering (Ma et al., 2007), which is a new widespread strategy used to control the developed interfacial interactions in the materials effectively; therefore a desired specific biological response can be identified. Henceforth, the better control on the properties of the produced biomaterials leads to the commercialization of the smart bioactive materials.

2.

CONDUCTINGeNATURAL POLYMERS COMPOSITE: SMART ADVANCED FUNCTIONALIZED MATERIAL IN BIOMEDICAL FIELD AND BIOSENSING

The field of conductive biohybrid composites is a rapidly growing area of research in advanced functionalized material science (Fig. 10.1). The smart bioactive materials consist of conducting and natural polymer parts, wherein the strong biological interactions like van der Waal force and hydrogen bonding etc. at the molecular level generate unique properties at the interface. Since 1995, Schmidt et al. has been exploring the origin, fundamentals, advancement, prospectus, and possible application of bioactive materials. This research group, for the first time, has explored the usage of the conducting polymer polypyrrole in biomedical field viz. tissue engineering. Design of the bioactive material using conducting polymer and natural polymer and studies of their properties, like antibacterial, electrical stimulus and mechanical properties along with their sensing application, have been explained by Tasteta et al. (2011) and Kristi et al. (2013). The controlled and improved properties, generated at the interface through molecular interactions, were explored by Ebrahimiasl et al. (2014) and Kuralay et al. (2014). The necessity of the molecular and surface engineering of the functionalized bioactive materials regarding their aspect in biomedical field has been highlighted by Ma et al. (2007) and Molino et al. (2013).

2. ConductingeNatural Polymers Composite 443

n FIGURE 10.1 Yearly publications of biohybrid polymer composites use in biomedical field. PubMed.

According to Nickels and Schmidt (2013) and Tiefenauer and Ros (2002), the characteristic properties of the hybrid bioactive material is a function of the morphological, interfacial, and characteristic properties of the each individual component present in the material. Comprehensive studies of supramolecular chemistry make us understand about the developed interactions between two macromolecules moieties and therefore generate unique properties at the interface. Furthermore, the heterogeneous or homogeneous mixing, suspension, and cross-linking of conducting and natural polymer components generate interesting composite materials with commercial prospectus. Significant efforts (Kumar and Geckeler, 2012) have also been made to give nanodimension to the biohybrid material with the desired shape, size, and properties using innovative synthesis techniques. The surface modification and functionalization of (Rashidi et al., 2014) natural polymer or conducting polymer, covalent attachment, self-assembly, and way of organization on the surface provide a means to produce smart biofunctionalized bioactive material with tunable surface properties. There is a strong interest in adding metal nanoparticles as additives to the bioactive materials to be used in various device applications. A promising and demanding area of research relates to the utilization of bioactive material as biosensor and in tissue engineering, bioimagining, and organ transplantation. Many reports (Cen et al., 2004; Ding et al., 2014) pertaining to the

444 CHAPTER 10 Bioactive Materials Based on Biopolymers Grafted on Conducting Polymers

favorable interaction between biological moiety/analyte and functionalized bioactive material framework have been explored. To achieve selective and sensitive detection (Huang et al., 2013), optimization of biological parameters such as pH, temperature, surface area, biocompatibility, and effect of pore size on the properties of the bioactive material are a matter of considerable interest till today (Thakur et al., 2013). Other than interfacial interactions, control of surface morphology is an important aspect that influences the properties of the bioactive material and creates appropriate pore size for imbibing of enzyme through which they become functionalized toward target biomolecule and therefore transfer the electrical signal to the analyzer or detector. Some of the applications of biohybrid material are shown in the flow chart.

The adopted methods to fabricate smart functionalized bioactive material of desired properties and applications are discussed below.

2.1 Preparation Methods of Smart Functionalized Bioactive Material Depending on the nature of natural polymer and conducting polymer, the different synthesis techniques are likely to result in the fabrication of nanowires or films of smart bioactive materials via different electrostatic interactions. The bioactive hybrid composites exhibit flexible and porous structure (Ulbricht, 2006; Khan and Narula, 2016). This enables biomolecules or enzymes to penetrate into the polymer matrix and be adsorbed onto the surface, resulting in the strong sensing behavior (Hoa et al., 1992).

2. ConductingeNatural Polymers Composite 445

Many techniques such as grafting, blending, and surface modification which is achieved either electrochemically or chemically are used to fabricate these smart bioactive materials (Thakur and Thakur, 2014a,b). Moreno et al. (2009), studied the effect of the synthesis parameters on the composites of polypyrrole doped with polysaccharide and also observed the effect of the surface roughness of the fabricated composites on cell adhesion and proliferation. They reported that the smooth surfaces facilitate the best adhesion of osteoblasts (stem cell) on electropolymerized films. Their study also revealed about the cell interactions with fabricated composites; according to them, cell interactions depend on the explicit chemistry of the dopant type and also on other physical surfaces properties (modulus, surface potential, and morphology) which are influenced by the presence of the dopant and other parameters (e.g., polymerization charge of polymer growth) used to fabricate the biohybrid polymer. The electrochemical doping of the conducting polymer with the polyelectrolyte dopants of biopolymer is shown in Fig. 10.2. The technique provides a trajectory for further chemical modification of coating on the surface and gives unambiguous biochemical characteristics. Higgins et al. (2011) have studied the fabrication of biohybrid and electroactive electrode comprising conducting polymer polypyrrole doped with gellan

n FIGURE 10.2 Pictorial representation of electrochemical polymerization.

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gum (a biologically derived polysaccharide) electrochemically to be used as a neural prosthetic probe. Pelto et al. (2010) also prepared the bioactive films consisting of polypyrrole and a biopolymer hyaluronic acid using an electrochemical method. In this work they also studied the surface roughness, elasticity, and net surface charge distribution of the fabricated bioactive material. The charge density on the surface of the bioactive/biohybrid materials allows the facile adhesion of the biomolecules on them, and it can be estimated using atomic force microscopy technique. Hong et al. (2011), reported that elasticity or flexibility of the smart bioactive polymer is of prime interest in tissue engineering as it enables the bone/nerve to attain a physiological acceptable stress. Therefore, to combat the challenge of modulus-mismatch between available implant materials and bone/nerve, the fabricated flexible bioactive material stimulates the development of a safe bond between the fabricated implant material and host tissue; Ravichandran et al. (2010) also introduced the concept of other analogous biomaterials. Since then, number of biohybrid materials have been produced and investigated. Electrochemical-based synthesis and doping of conducting polymer with biopolymer electrolyte has been achieved by specific functionalization of the polyfunctionalized natural biopolymer hydrogels like chitosan, gellan gun etc. Electrochemical polymerization occurs by applying an appropriate voltage across the electrodes placed in the polyelectrolyte solution containing the monomer of the conducting polymer and the doping agent (natural polymer) (Gerard et al., 2002). The method allows the deposition of a film of biohybrid polymer composites with a well-controlled thickness (down to 20 nm) and surface morphology (Baolin et al., 2011). The applied potential across the electrodes causes the monomer to oxidize and polymerize along with the biopolymer and result in the deposition of films of bioactive polymer composite on the positively charged working electrode. The properties of the prepared biohybrid composite film will be specified by the deposition charge and time, temperature, pH of the solvent, doping agent, and the electrode system (Wallace et al., 1999). Electrochemical polymerization allows the synthesis of the biohybrid polymer composites, the monomer undergoes oxidation and the biopolymer gets cross-linked in between the chains of the conducting polymer in the presence of an appropriate applied electrical potential (Cosnier et al., 1997). All of the main biocompatible conducting polymers (Guimard et al., 2007) like polypyrrole, polyaniline, and poly(3, 4-ethylene dioxythiophene) (PEDOT) along with natural biopolymers such as dextran and cellulose fulfill this criterion. The electrochemical method of synthesis of biohybrid polymer composites can be accomplished using three techniques, namely the

2. ConductingeNatural Polymers Composite 447

galvanostate, the potentiostate, and potentiodynamic (Otero et al., 2012; Otero and Abadias, 2008). In the galvanostatic method of polymerization, the electric current of the electrodes is controlled, but the potential is allowed to vary. Using this technique, the rate of deposition of biohybrid polymer composites can be controlled accurately; henceforth the thickness of the fabricated film can also be controlled easily. In the potentiostatic method of polymerization, the potential across the auxiliary electrode is controlled, against the working electrode, therefore the potential difference between working electrode and reference electrode is well stated, but current is allowed to vary (monitor by coulometer). Therefore the integrity of the prepared biohybrid composites remains protected, making this method suitable for the fabrication of biosensors (Weng et al., 2015). In the potentiodynamic method, the polymerization potential is swept between low- and highpotential limits in cycles; using this technique, the polymer is deposited in the layer structural morphology. It should be noted that all the abovementioned electrochemical techniques produce different structural and surface morphology along with varied thickness of the film, fibrous to rodlike structure. So depending upon the course of application of the biohybrid polymer composites, they could be fabricated using appropriate electrochemical method. Molino et al. (2013) stated the fabrication of the biohybrid composite of polypyrrole doped with dextran sulfate electrochemically. They also studied the physicochemical properties like polymer film mass, thickness, interfacial roughness, wettability, electroactivity, and porosity, and make the biohybrid material to be appropriate for biomimetic physiochemical and electrochemical stimuli on cells and tissues. The chemical method of polymerization involves the use of appropriate oxidizing agent to polymerize the given monomer. During the polymerization of monomer biopolymer is placed. The fabrication of nanosize biohybrid composites has also been reported by chemical polymerization, depending upon the amount of oxidizing agent and monomer ratio the surface morphology of the prepared composites can be varied from chain-like structure to porous honeycomb structure (Khan and Narula, 2016). Chemical polymerization is relatively easier and a more economic method and many conducting biohybrid composites of polypyrrole and PEDOT combine with poly(D, L-lactide), dextran, chitosan, cellulose, protein, and silk has been reported (Severt et al., 2015; Cabuk et al., 2014). Biohybrid composites can be prepared by chemical or electrochemical polymerization. The electrochemical method of polymerization allows the direct deposition of thin films of conducting polymer with improved control of thickness and surface morphology, which are more appropriate for direct application.

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Takano et al. (2014) reported the fabrication of film of biohybrid composite of polypyrrole with cellulose acetate. Table 10.1 showed the methods of synthesis of biohybrid composites, type of dopant used, and its biomedical application. Rubber-toughened amorphous glasses have been synthesized using blending technique since 1940, but from the last decade the physical blending technique of two polymers to generate bioactive materials is gaining popularity. The physical approach of fabrication of biomaterials is anticipated to be a more efficient, rapid, and a less expensive route to meet the anxieties of the marketplace. Blending of preexisting polymers offers a means of engineering with certain physical forces into one material, by which certain combinations of desired properties exhibited individually by the individual polymer component present in the composite. Biomolecules or biopolymers are incorporated into the conducting polymer matrix

Table 10.1 Representing Synthesis Method of Biohybrid Composites Polymers, Type of Dopant Used, and Its Biomedical Application So. No.

Name of Conducting Polymer

Name of Dopant

1

PEDOT

Chitosan

2

Polypyrrole/PEDOT

Silk fibroin

3

Polypyrrole

Dextran sulfate

4

Polypyrrole

5

Polyaniline

Poly(L-lactide) (PLLA) or poly (e-caprolactone) (PCL) Dextrin

6

Polyaniline

Cellulose

7

PEDOT

Bacterial cellulose

PEDOT, poly(3, 4-ethylene dioxythiophene).

Method of Synthesis

Application

References

Chemical oxidative polymerization Chemical oxidative polymerization/ electrochemical polymerization Electrochemical polymerization Chemical oxidative polymerization

Antibacterial

Khan and Narula (2016) Severt et al. (2015)

Chemical oxidative polymerization

Antioxidant, antibacterial, and removal of heavy metals Electroactive material Biointerface materials used in tissue engineering

Chemical oxidative polymerization Chemical oxidative polymerization

Biomimetic material in tissue engineering Cell proliferation Biocompatibility with human fibroblast cell

Molino et al. (2015) Boutry et al. (2013)

Zare and Lakouraj (2014)

Namazi et al. (2016) Chen et al. (2015)

2. ConductingeNatural Polymers Composite 449

n FIGURE 10.3 Types of chemical/physical interactions during the fabrication of biohybrid polymer

composites.

by means of various physical interactions like covalent, coordinate, ionic, hydrogen bonding, and van der Waal forces of interactions (Fig. 10.3; Table 10.2). Kristi et al. (2013) blended the plasma-modified chitosan and PEDOT together and developed the conducting biohybrid nanofibers with polyvinyl Table 10.2 Representing Characteristics and Strength of Different Developed Interactions S. No.

Interactions

1 2

Covalent Coordinate covalent bond Ionic van der Waal Hydrogen

3 5 6

Strength (kJ/mol)

Range

Characteristics

350 50e250

Short Short

Irreversible Directly

50e250 50 5e65

Long Short Short

Nonselective Nondirectional Semidirectional

450 CHAPTER 10 Bioactive Materials Based on Biopolymers Grafted on Conducting Polymers

alcohol as a supporting polymer in different volumetric ratio using an electrospinning method. The electrospinning method is used to give nanodimensions to the prepared biohybrid composites (Piskin et al., 2007). Doshi and Reneker (1995) were the first researchers to report the fabrication of nanofibers by the electrospinning method. The electrospinning method requires a high-voltage power source (30 kV), syringe pump, and conducting collectors (target). The method involves the passage of high voltage through the droplet polymeric solution followed by extrusion from the tip of the needle and a grounded target. On applying an appropriate amount of electrical voltage, electrostatic charge develops on the surface of the droplets of the polymer, the intensity of the charge increases, till it suppresses the surface tension of the droplet, and then only charged polymer fluid jet is ejected toward the target. The jet shows blending instabilities due to the repulsion of mutual surface charges. The repulsion causes the elongation of the jet thousand times so that it becomes extremely thin, the solvent allowed to evaporate, and long nanofibers are collected on the grounded target (Huang et al., 2003). Kristi et al. (2013) also reported that the blend of PEDOT with chitosan exhibited good antibacterial and electrochemical properties and are the potential material to be used in biomedical field. Although blending of polymers is an efficient and beneficial technique to produce advantageous combination of properties, the components of polymers are thermodynamically immiscible and held by weak physical forces with each other and the nature of the interface generated between the two phases exhibit complications in both the melt and the solid state. Hence, the resultant biohybrid composite shows poor mechanical properties. Therefore, surface modification is done to facilitate the intermixing of two immiscible polymers. The surface modification is achieved through the grafting technique (Bhattacharya and Misra, 2004; Thakur and Thakur, 2014a,b). Graft copolymer is a branched molecule wherein the main chain or the backbone chain consists of entirely of one type of the repeating unit with short side chains of the second type of the repeating unit attached at random points. Grafting of polymer (Thakur et al., 2016) can be achieved by means of either free radical mechanism or by anionic polymerization. In grafting technique (Chergui et al., 2011), block or grafted copolymers are chosen carefully which act as a coupling agents between the different phases of composites and possess the capability of producing single-phase composite. Chen et al. (2013) modified the surface of the polyaniline and allowed copolymerization with the macromolecule called methoxy-poly(ethylene glycol) monomethacrylate and reported that the fabricated biohybrid composites could be used as a blood compatible material. In another study, Chen et al. modified the surface of polyaniline by copolymerization with acrylic

3. Biosensing Principle of Biohybrid Polymer Composite 451

monomer. They used the prepared biohybrid polymer matrix for the covalent immobilization of enzyme invertase and used polymeric matrix as a biosensor for the sensing of enzyme activity. The fabrication of biohybrid composite materials with the desired properties depends upon the selection of the synthesis method (Fig. 10.4). It was observed that the conducting properties of the fabricated biohybrid composites depend on the number of parameters, such as type of counterion, type of electrolyte and its concentration, synthesis temperature, electrochemical voltage, and pH of the electrolyte. Therefore, the selection of suitable technique is crucial for the preparation of a stable, selective, sensitive, and flexible biohybrid composite. It has been found that electrical, optical, and molecular properties of biohybrid composite can be altered and tuned using an appropriate synthetic route and varying precursor/operational parameter. Biosensing and tissue engineering has been focused in this chapter.

3.

BIOSENSING PRINCIPLE OF BIOHYBRID POLYMER COMPOSITE

Biosensors are the analytical tools using which the nature and concentration of the analyte is analyzed. Biosensors are the diagnostic devices and is comprised of a layered structure, consisting of biological recognition element (analogous to the analyte) attached to a biohybrid polymer matrix which is further joined to a transducer (Fig. 10.5). The biological recognition element interacts with the analyte; this interaction produces a biochemical signal and the biochemical signal is

n FIGURE 10.4 Schematic representation of fabrication techniques of biohybrid polymer composites.

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n FIGURE 10.5 Schematic representation of working of biosensor.

transformed by the transducer into a readable output amplified by an amplifier and all the data is displayed on the computer screen (Malhotra et al., 2006). The father of biosensors, Clark, first invented the biosensor by imbibing the enzyme onto the surface of electrode, since then considerable development has been done in the direction of this analytical tool. Conducting polymers, like polyaniline, polypyrrole, and PEDOT, have been extensively used as transducers, which transmitted the electrochemical signal generated from the interaction of biomolecules. Depending on the basis of biological recognition site, type of transducer used, and nature of interactions developed, the biosensors can be classified (Table 10.3).

Table 10.3 Classification of Biosensors on the Basis of Transducer (Thévenot et al., 2001) S. NO.

Transducer

Biosensor

1

Electrochemical

2 3 4

Electrical Heat/thermal Ion-selective

Amperometric Potentiometric Conductometric/Impedimetric Calorimetric Biosensors based on ion-selective field-effect transistors (ISFETs)

3. Biosensing Principle of Biohybrid Polymer Composite 453

In biosensors, generally two common types of transducers viz. amperometric and potentiometric are used (Monosík et al., 2012). An amperometric biosensor measures the produced current when the analyte is oxidized or reduced at a constant applied potential. The biopolymer generates the active imbibing site for the enzyme, which interacts with the analyte, and the conducting polymer mediates the electron transfer from an enzyme to the working electrode; however, the exact mechanism is yet to be explored more. Since the pioneering work of Rew and Hill (1988), it has been proved that there should be direct attachment of the redox group to the surface of the fabricated biosensor which would facilitate the transfer of electrons between the transducer and biomolecule. Nanoparticles such as gold and silver facilitate the electron transfer from the biochemical reaction to the biohybrid polymer matrix and therefore increase the sensitivity and selectivity of the sensor. The nanoparticles get entrapped on incorporating them as dopants, or they can be chemically conjugated to the monomer of polymer (Mazeiko et al., 2013). Gambhir et al. (2001) prepared a potentiometric biosensor for the detection of urea by coimmobilization of enzymes namely urease and glutamate dehydrogenase onto a polypyrrole polyvinyl sulfonate matrix. They reported that the sensing of urea by ureases is accomplished by the production of NH3, which interacts with polypyrrole to generate an electrical signal. The produced signal would be directly influenced by the concentration of the NH3 ions which in turn is measured by the pH of the solution. Therefore, potentiometric biosensors correlate the electrical potential with the concentration of analyte by means of an ion-selective electrode or gas-sensing electrode as the transducers Therefore, it is concluded that unlike amperometric biosensors (electron based transducer), potentiometric biosensors are based on the ion-selective transducer electrode. Being an electron transfer-based transducer, amperometric biosensors exhibit fast response times and good sensitivities. An efficient biosensor should be reusable, reproducible, quick response time, selective, and sensitive. To achieve all the qualities, immobilization of biomolecule to the biohybrid polymer composite is a vital part and has been explored thoroughly by Murugaiyan et al. (2014). A biosensor involves stable and reproducible immobilization of a biological entity (e.g., vitamins, coenzymes, proteins, DNA, polypeptides, cells, microorganisms) onto the surface of transducer, with complete retention of its biological activity. The precarious part of the immobilization is to maintain the activity, selectivity, and stability of the imbibed biomolecule. The immobilization of the biomolecule can be done either covalently or noncovalently to the biohybrid polymer matrix. Noncovalent interactions comprises adsorption, physical entrapment, and affinity binding while in covalent interactions a

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covalent bond (Chen et al., 2006) is generated between the imbibed biomolecule and conducting biohybrid polymer matrix by their respective complementary functional groups (Fig. 10.6). Fu et al. (2005) fabricated the DNA biosensor using polypyrrole-gold-silver hybrid composite polymer matrix, the DNA was allowed to adsorb onto the surface of hybrid polymer matrix by mercapto-oligonucleotide immobilization probe. They also reported that adsorption; a noncovalent technique of immobilization is very simple which does not offer selectivity and stability to the immobilized biomolecule. Physical entrapment is also a kind of primitive adsorption technique. Chen et al. (2013) made the physical entrapment of the enzyme horseradish peroxidase into a conducting polymer matrix of PEDOT film. They fabricated the amperometric glucose biosensor for the detection of hydrogen peroxide. The affinity-binding techniques of immobilization is based on the weak van der Waal force of interactions arises between the enzymes with the transducer; this method of immobilization is relatively offered more specificity and stability to the biosensor. Moy et al. (1994) reported

n FIGURE 10.6 Schematic representation of interactions developed between biomolecules (enzyme) and

matrix. (A) Physical adsorption. (B) Entrapment. (C) Affinity.

3. Biosensing Principle of Biohybrid Polymer Composite 455

another kind of noncovalent interactions known as ligandereceptor pairing found in biotine-avidin, the strongest reported noncovalent interaction, with an unbinding force of up to 250 pN. Henry et al. (2000) functionalized the surface of the polymer matrix (PMMA) with the covalent attachment of the biomolecules (DNA) to the surface of polymer and ensures the immobilization without leaching of the biomolecule from the surface of the biohybrid polymer matrix. To achieve more specificity and longer life time to the fabricated biosensor, covalent immobilization is preferred. Covalent immobilization involves the functionalization of the surface of the polymer matrix or generation of active binding site; e.g., Xia et al. (2013) functionalized the conducting polymer matrix with N-hydroxysuccinimide/1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (NHS/EDC) group on its surface so that carboxylic acid (eCOOH) present on the matrix can efficiently attach to the amine (eNH2) groups (condensation reaction) present on the biomolecule. After polymerization, the assembly (biomolecule and conducting polymer) is finalized. The fabricated biohybrid composite exhibited the multipurpose activity and noninvasiveness and can be modified according to the properties of the probe molecules. The fabricated assembly offers strong binding (lock and key type) between the biomolecule and conducting polymer and henceforth enhances the target accessibility. As the immobilization occurs at the conducting polymer surface, the coupling procedure is performed under mild aqueous conditions. Fang et al. (2012) prepared a high-sensitive glucose biosensor by immobilizing the glucose oxidase’s (GOD) enzyme onto the surface of biocompatible chitosanepolypyrrole (CSepolypyrrole) polymer composites followed by deposition of the whole biohybrid film assembly onto the surface of glassy carbon electrodes. The CSepolypyrrole biohybrid polymer composite provides the 3D micro-nano topological surface morphology which enhances the electron transfer between the enzyme and electrode surface significantly. They also showed that the electrode coated with the biohybrid CSe polypyrrole composites exhibited a fast response time of 5 s, with a linear range of concentration of 5.00  10 4 Me1.47  10 1 M and a limit of detection (LOD) of 1.55  10 5 M. Mehmet Senel (2015) also prepared the glucose biosensor via an in situ chemical reaction. The involved biohybrid composite transducer was comprised of CSepolypyrrole-gold nanoparticles. The prepared biosensor represented a high and reproducible sensitivity of 0.58 mA/mM, a response time of about 4 s, the linear range of concentration from 1 to 20 mM, and an LOD of 0.068 mM.

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4.

BIOMEDICAL APPLICATION OF BIOHYBRID POLYMER COMPOSITES 4.1 Tissue Engineering The damage or miscarriage of an organ or tissue is one of the most regular, demoralizing, and expensive snags in human health care. Tissue engineering technology depends on those biomaterials which can provide physical support for the growth of tissue and also stimulate the specific cell functions. The objective of tissue engineering is to fabricate a biohybrid material that could syndicate the intrinsic biological properties which can specifically trigger the desired cellular responses (e.g., angiogenesis) with electrical properties which are indicated to have the marked effect on the regeneration of several tissues including bone and nerve. Langer and Vacanti (1993a,b) carried out the pioneering work in the direction of tissue engineering. According to their report, regeneration of new tissue should consist of the following three strategies: 1. Isolated cell or cell substituted: To avoid the complications of surgery, this strategy is applied in which it replaces only those cells that reconcile the required function and allows the manipulation of cells before infusion. Its major limitation is the rejection of the infused cell by the immunological system. 2. Tissue-inducing substances. The implantation and success of the method depends on the involved technical aspect in it, like purification, growth factors, and the involved strategy of delivering these molecules to their targets. 3. Cells placed on or within matrices: the strategy consists of the isolation of the cells from the body using a selective permeable membrane that allows the nutrients and wastes but prevents large entities such as antibodies or immune cells which can destroy the transplantation. After successful growth, the systems can be implanted or used as extracorporeal. The matrixes used in that strategy are derived from natural or biocompatible materials. One key aspect and challenge to the tissue engineers is to fabricate a biocompatible material. Biocompatible conducting polymers like polypyrrole and PEDOT have attracted the attention of the engineers. The conducting polymers have been considered as a suitable biomaterial for multiple applications within biotechnology due their electroactive properties that can be used for the trigger of any specific cellular response like cell adhesion, migration, DNA synthesis, or in ion exchange process through a semipermeable membrane under external electrical stimulus. Biotechnologists combined conducting polymers with natural polymers to

4. Biomedical Application of Biohybrid Polymer Composites 457

fabricate a new class of biohybrid material, taking benefit of their synergistic properties like flexibility, biocompatibility, and biodegradability in tissue engineering (Wang, 2003). Biotechnologists till date have almost investigated every part of the mammalian tissue. Human tissue can be categorized as epithelial, connective, muscular, and nervous tissue. Nervous tissue: Parkinson’s disease in which loss of dopamine production occurs slowly; this causes damage to the neural growth of the nervous system. Presently, grafting technique is used for the regeneration of damaged nerve. The technique of grafting involves autografting in which the grafting of the nerve is done from the same nerve. Allografting includes grafting of nerve tissues with the same individual’ s cells but from a different healthy individual’s nerve. Heterografting means grafting of nerve tissues from different may be artificial or from the cells of different species (Naumann et al., 2004). To minimize the chances of rejection from the immune system of the body, autografting methodology is preferred. However, the process of autograft repairing has been reported to have many problems like difficulties with isolation which involved the second surgical step for the elimination of the donor nerve function and the mismatch between nerve and graft dimensions (Wang, 2003). To avoid all these difficulties various natural, synthetic polymers and biohybrid composites are used as scaffolds. Scaffolds are the biocompatible materials constructed for the cell attachments by which cellular interactions can be tailored to generate new functional tissues for clinical purpose. Scaffolds are the mimicry biomaterials that serve as the native extracellular matrix (ECM) of the preferred innate tissues. Cells are seeded in vitro and allowed to grow onto these structures which in turn give favorable conditions for the formation of three-dimensional tissues (Chan and Leong, 2008). Moreover, growth factors are also implanted within the porous ECM, which in turn allowed their sustained released, to boost cell growth and morphogenesis and also cause the functionality to the engineered tissue (Amini et al., 2012). Scaffolds are characterized by some key properties like (1) porous structure which allow penetration of cell and transportation of nutrients and waste; (2) biodegradability and biocompatibility; (3) appreciable mechanical properties to meet the desired specific function (specially for bone tissue engineering); (4) surface modification able to attach cell and its proliferation. Biohybrid polymeric scaffolds are fabricated in various forms varied from the form of solid foam, nanofibrous matrix, microsphere, to hydrogel, depending on the nature of target regenerated tissue. Scaffolds are used for various aspects of tissue engineering such as bone, cartilage, ligament, skin, vascular tissues, neural tissues, muscle, and as a medium for the sustained release of drugs, proteins, and DNA. Nanofibrous scaffolds have more interesting properties, due to their high

458 CHAPTER 10 Bioactive Materials Based on Biopolymers Grafted on Conducting Polymers

surface area and surface roughness which enhance neural cell adhesion (Ravi and Chaikof, 2010). Li et al. (2006) fabricated the nanofibrous conductive scaffold consisting of conducting polymer polyaniline and natural polymer gelatin by blending technique. They used the fabricated nanofibrous matrix for supporting the cell growth of H9c2 rat cardiac myoblast cells for the cardiac tissue engineering. They cultured the cells on the surface of the conductive fibrous biohybrid matrix and were observed for cell proliferation and morphology (Fig. 10.7). They concluded that fabricated biohybrid (polyanilineegelatin) blend fibers supported the H9c2 cell attachment and proliferation efficiently. Schmidt et al. (1997) did pioneer work for the very first time; they used polypyrrole for the purposes of tissue engineering and demonstrated that electrical stimulation boosted neural growth factors uniformly and also encouraged neuronal differentiation of PC 12 (an immortalized cell line resulting from rat pheochromocytoma cells). In further studies, along with Collier, they prepared biohybrid bilayer films of polypyrroleehyaluronic acid. The prepared film was biocompatible, conducting, noncytotoxic, with angiogenic properties, with PC 12 cells. Stewart et al. (2013) synthesized the conducting biohybrids of PEDOTe glycol and studied the cell attachment and proliferation of fibroblast cell and keratinocyte cell. They also observe the effect of different oxidizing and reducing potential on the structural morphology of fabricated biohybrid which in turn affects significant cell adsorption at the surface. Bone is a natural composite. In case of any injury to bone, it can be repaired by the classical medical approach, i.e., bone grafting but the grafting technique is not a choice of engineers due to its severe limitations like rejection by immune system or transfer of disease. The use of pure ceramics and polymer scaffolds is also not favorable due to their poor mechanical and flexibility problems. Hayati et al. (2011) fabricated hydroxyapatite nanoparticle embedded with poly(3-hydroxybutyrate) scaffolds which served as noble material for the regeneration of bone tissue due to their excellent physicochemical properties with good mechanical strength (Fig. 10.8).

4.2 Drug Release The famous microelectrode microelectromechanical systems technology of drug delivery has serious limitations due to its slow delivery rate and high power consumption. Langer and Folkman (1976) introduced the concept of sustained release of proteins and macromolecule, since then polymer systems have been investigated for the controlled release of biomolecules. Zinger and Miller (1984) for the first time studied the release of dopamine and glutamate through the actuation of polypyrrole polymer film on applying suitable potential values. Drug is loaded into the polymeric matrix and then

4. Biomedical Application of Biohybrid Polymer Composites 459

(A)

(B)

(C)

(D)

(E)

n FIGURE 10.7 Morphology of H9c2 myoblast cells at 20 h of postseeding on: (A) gelatin fiber; (B) 15:85 PANiegelatin blend fiber; (C) 30:70 PANiegelatin blend fibers; (D) 45:55 PANiegelatin blend fibers; and (E) glass matrices. Staining for nuclei-bisbenzimide and actin cytoskeleton-phalloidin, fibers autofluorescence. Reprinted with permission from Li, M., Guo, Y., Wei, Y., MacDiarmid, A.G., Lelkes, P.I., 2006. Electrospinning polyaniline-contained gelatin nanofibers for tissue engineering applications. Biomaterials 27, 2705e2715, Copyright, 2016, Elsevier.

the sustainable release of drug is monitored under applied potential (Fig. 10.9). The pKa value of the drug is considered as the charge on the drug which influences the drug loading, film forming, and releasing capabilities. Lin and Wallace (1994) studied the release of 2,6-anthraquinone disulfonic acid (ASQA) from polypyrrole polymeric film on applying appropriate reduction potential to polymeric film. They also optimized the physiochemical factors like pH, ionic strength, polarity, and hydrophobicity and

n FIGURE 10.8 Schematic representation of bone regeneration using scaffolds.

n FIGURE 10.9 Schematic representation of drug delivery using hydrogels of biohybrid composites polymer.

5. Conclusion 461

observed their effect on the release of ASQA from polymer directly. They concluded the rate of release of drug increases with increasing values of reduction potentials, ionic strength, and pH of the solution. However, a lone conductive polymeric film after some time losses its actuation power due to which release of drug becomes slower. Conducting biohybrid hydrophilic hydrogel scaffolds have become the choice of material for the purpose of smart drug delivery for the investigators due to their lucrative properties like biocompatibility, cell-controlled degradability, and intrinsic cellular interactions. Freed et al. (1994) said that hydrogels are biocompatible due to their exhibition of structural similarity with biomolecules based components in the body. Freed and Novakovic (1998) reported that hydrogels can be produced by using synthetic or natural polymers, which are cross-linked through either covalent or noncovalent bonds. Abidian et al. (2006) prepared the conducting biodegradable nanofibers by the polymerization of PEDOT on electrospun poly(L-lactide) or poly (lactide-co-glycolide) (PLGA) for the controlled release of drug dexamethasone in the presence of external electrical stimulation of PEDOT nanotubes. On the degradation of PLGA, dexamethasone penetrates into PEDOT polymeric film. On applying appropriate potential control, sustainable release of drug takes place.

5.

CONCLUSION

This chapter reviews the recent developments in the fabrication methods of biohybrid polymer composites and their applications in biosensing and biomedical field. The biohybrid materials were found to be an excellent alternative to conventional sensing material and biomedical props due to their exclusive properties like biocompatibility, biodegradability, noncytotoxicity, electroactivity, and topography. The properties of biohybrid materials can be tailored by using appropriate synthetic route and dopant. The review clearly demonstrates the working principle and mechanism of the biohybrid materials. However, commercialization of immobilized enzyme biosensors and scaffolds in tissue engineering is still a challenge because of their costs and maintenance problems. Research should be focused to overcome the present confines related to immobilization techniques in biosensors and implantation techniques in tissue engineering so as to meet the expectations of marketing and commercialization of the probes.

462 CHAPTER 10 Bioactive Materials Based on Biopolymers Grafted on Conducting Polymers

REFERENCES Abidian, M.R., Hwan, K.D., Martin, D.C., 2006. Conducting-polymer nanotubes for controlled drug release. Advanced Materials 18, 405e409. Amini, A.R., Laurencin, C.T., Nukavarapu, S.P., 2012. Bone tissue engineering: recent advances and challenges. Critical Reviews in Biomedical Engineering 40, 363e408. Baolin, G., Anna, F.W., Christine, A.A., 2011. Facile synthesis of degradable and electrically conductive polysaccharide hydrogels. Biomacromolecules 12, 2601e2609. Baughman, R.H., 1996. Conducting polymer artificial muscles. Synthetic Metals 78, 339e353. Bhattacharya, A., Misra, B.N., 2004. Grafting: a versatile means to modify polymers techniques, factors and applications. Progress in Polymer Science 29, 767e814. Boutry, C.M., Hörler, I.G., Hierold, C., 2013. Electrically conducting biodegradable polymer composites (polylactide-polypyrrole and polycaprolactone-polypyrrole) for passive resonant circuits. Polymer Engineering and Science 53, 1196e1208. Cabuk, M., Alan, Y., Yavuz, M., Unal, H.I., 2014. Synthesis, characterization and antimicrobial activity of biodegradable conducting polypyrrole-graft-chitosan copolymer. Applied Surface Science 318, 168e175. Cen, L., Neoh, K.G., Li, Y., Kang, E.T., 2004. Assessment of in vitro bioactivity of hyaluronic acid and sulfated hyaluronic acid functionalized electroactive polymer. Biomacromolecules 5, 2238e2246. Chan, B.P., Leong, K.W., 2008. Scaffolding in tissue engineering: general approaches and tissue-specific considerations. European Spine Journal 17, 467e479. Chen, C., Zhang, T., Zhang, Q., Feng, Z., Zhu, C., Yu, Y., Li, K., Zhao, M., Yang, J., Liu, J., Sun, D., 2015. Three-dimensional bc/pedot composite nanofibers with high performance for electrodeecell interface. ACS Applied Materials and Interfaces 51, 28244e28253. Chen, J., Winther-Jensen, B., Lynam, C., 2006. A simple means to immobilize enzyme into conducting polymers via entrapment. Electrochemical and Solid-State Letters 9, H68eH70. Chen, C., Xie, Q., Yang, D., Xiao, H., Fu, Y., Tan, Y., Yao, S., 2013. Recent advances in electrochemical glucose biosensors: a review. RSC Advances 3, 4473e4491. Chergui, S.M., Derouich, S.G., Mangeney, C., Chehimi, M.M., 2011. Aryl diazonium salts: a new class of coupling agents for bonding polymers biomacromolecules and nanoparticles to surfaces. Chemical Society Reviews 40, 4143e4166. Cosnier, S., Innocent, C., Allien, L., Poitry, S., Tsacopoulos, M., 1997. An electrochemical method for making enzyme microsensors. Application to the detection of dopamine and glutamate. Analytical Chemistry 69, 968e971. Diaz, A.F., Castillo, J.I., Logan, J.A., Lee, W.-Y., 1981. Electrochemistry of conducting polypyrrole films. Journal of Electroanalytical Chemistry and Interfacial Electrochemistry 129, 115e132. Ding, H., Zhong, M., Kim, Y.J., Pholpabu, P., Aditya Balasubramanian, A., Hui, C.M., He, H., Yang, H., Matyjaszewski, Y., Bettinger, C.J., 2014. Biologically derived soft conducting hydrogels using heparin-doped polymer networks. ACS Nano 8, 4348e4357. Doshi, J., Reneker, D.H., 1995. Electrospinning process and applications of electrospun fibers. Journal of Electrostatics 35, 151e160. Ebrahimiasl, S., Zakaria, A., Kassim, A., Basria, N., 2014. Novel conductive polypyrrole/ zinc oxide/chitosan bionanocomposite: synthesis, characterization, antioxidant, and antibacterial activities. Polymer, International Journal of Nanomedicine 10, 217e227.

References 463

Fang, Y., Ni, Y., Zhang, G., Mao, C., Huang, X., Shen, J., 2012. Biocompatibility of CSepolypyrrole nanocomposites and their application to glucose biosensor. Bioelectrochemistry 88, 1e7. Freed, L.E., Novakovic, G.V., 1998. Culture of organized cell communities. Advanced Drug Delivery Reviews 33, 15e30. Freed, L.E., Vunjak-Novakovic, G., Biron, R.J., Eagles, D.B., Lesnoy, D.C., Barlow, S.K., Langer, R., 1994. Biodegradable polymer scaffolds for tissue engineering. Biotechnology (NY) 12, 689e693. Fu, Y.Z., Yuan, R., Xu, L., Chai, Y.Q., Zhong, X., Tang, D.P., 2005. Indicator free DNA hybridization detection via EIS based on self-assembled gold nanoparticles and bilayer two-dimensional 3-mercaptopropyltrimethoxysilane onto a gold substrate. Biochemical Engineering Journal 23, 37e44. Gambhir, A., Gerard, M., Mulchandani, A.K., Malhotra, B.D., 2001. Coimmobilization of urease and glutamate dehydrogenase in electrochemically prepared polypyrrolepolyvinyl sulfonate films. Applied Biochemistry and Biotechnology 96, 249e257. Gerard, M., Chaubey, A., Malhotra, B.D., 2002. Application of conducting polymers to biosensors. Biosensors and Bioelectronics 17, 345e359. Groenendaal, L.B., Jonas, F., Freitag, D., Pielartzik, H., Reynolds, J.R., 2000. Poly (3, 4ethylenedioxythiophene) and its derivatives: past, present, and future. Advanced Materials 12, 481e494. Guimard, N.K., Gomez, N., Schmidt, C.E., 2007. Conducting polymers in biomedical engineering. Progress in Polymer Science 32, 876e921. Hardy, J.G., Lee, J.Y., Schmidt, C.E., 2013. Biomimetic conducting polymer-based tissue scaffolds. Current Opinion in Biotechnology 24, 847e854. Hayati, A.N., Rezaie, H.R., Hosseinalipour, S.M., 2011. Preparation of poly(3hydroxybutyrate)/nano-hydroxyapatite composite scaffolds for bone tissue engineering. Materials Letters 65, 736e739. Henry, A.C., Tutt, T.J., Galloway, M., Davidson, Y.Y., McWhorter, C.S., Soper, S.A., McCarley, R.L., 2000. Surface modification of poly(methyl methacrylate) used in the fabrication of microanalytical devices. Analytical Chemistry 72, 5331e5337. Higgins, T.M., Moulton, S.E., Gilmore, K.J., Wallace, G.G., Panhuis, M.I.H.P., 2011. Gellan gum doped polypyrrole neural prosthetic electrode coatings. Soft Matter 7, 4690e4695. Hoa, D.T., Suresh, T.N.K., Punekar, N.S., Srinivasa, R.S., Lal, R., Contractor, A.Q., 1992. A biosensor based on conducting polymers. Analytical Chemistry 64, 2645e2646. Hong, Y., Huber, A., Takanari, K., Amoroso, N.J., Hashizume, R., Badylak, S.F., Wagner, W.R., 2011. Mechanical properties and in vivo behavior of a biodegradable synthetic polymer microfiber - extracellular matrix hydrogel biohybrid scaffold. Biomaterials 32, 3387e3394. Huang, Z.M., Zhang, Y.-Z., Ramakrishna, S., 2003. A review on polymer nanofibers by electrospinning and their applications in nanocomposites. Composites Science and Technology 63, 2223e2253. Huang, H., Wu, J., Lin, X., Li, L., Shang, S., Yuen, M.C.W., Yan, Y., 2013. Self assembly of polypyrrole/chitosan composite hydrogels. Carbohydrate Polymers 95, 72e76. Kanazawa, K.K., Diaz, A.F., Geiss, R.H., Gill, W.D., Kwak, J.F., Logan, J.A., Rabolt, J.F., Street, G.B., 1979. Organic metals polypyrrole a stable synthetic metallic polymer. Journal of the Chemical Society, Chemical Communications 19, 854e855. Khan, S., Narula, A.K., 2016. Bio-hybrid blended transparent and conductive films PEDOT: PSS: chitosan exhibiting electro-active and antibacterial properties. European Polymer Journal 81, 161e172.

464 CHAPTER 10 Bioactive Materials Based on Biopolymers Grafted on Conducting Polymers

Kim, Y.H., Lee, J., Hofmann, S., Gather, M.C., Meskamp, L.M., Leo, K., 2013. Achieving high efficiency and improved stability in ITO-free transparent organic light-emitting diodes with conductive polymer electrodes. Advanced Functional Materials 23, 3763e3769. Kitani, A., Yoshioka, K., Sasaki, K., 1993. Improvement of mechanical properties of electropolymerized polyaniline. Synthetic Metals 57, 3566e3570. Kristi, M., Oksuz, A.U., Oksuz, L., Ulusoy, S., 2013. Electrospun chitosan/PEDOT nanofibers. Materials Science and Engineering 33, 3845e3850. Kumar, P.T., Geckeler, K.E., 2012. GrapheneeDNA hybrid materials: assembly, applications, and prospects. Progress in Polymer Science 37, 515e529. Kuralay, F., Demirci, S., Kiristi, M., Oksuz, L., Oksuz, A.U., 2014. Poly(3, 4-ethylenedioxythiophene) coated chitosan modified disposable electrodes for DNA and DNAedrug interaction sensing. Colloids and Surfaces B: Biointerfaces 123, 825e830. Langer, R., Folkman, J., 1976. Polymers for the sustained release of proteins and other macromolecules. Nature 263, 797e800. Langer, R., Vacanti, J.P., 1993a. Tissue engineering. Science 260, 921e936. Langer, R., Vacanti, J.P., 1993b. Tissue engineering. Science 260, 920e928. Li, M., Guo, Y., Wei, Y., MacDiarmid, A.G., Lelkes, P.I., 2006. Electrospinning polyaniline-contained gelatin nanofibers for tissue engineering applications. Biomaterials 27, 2705e2715. Lin, Y., Wallace, G.G., 1994. Factors influencing electrochemical release of 2, 6-anthraquinone disulphonic acid from polypyrrole. Journal of Controlled Release 30, 137e142. Ma, Z., Mao, Z., Gao, C., 2007. Surface modification and property analysis of biomedical polymers used for tissue engineering. Colloids and Surfaces B: Biointerfaces 60, 137e157. Ma, L., Li, Y., Yu, X., Yang, Q., Noh, C.H., 2008. Using room temperature ionic liquid to fabricate pedot/tio2 nanocomposite electrode-based electrochromic devices with enhanced long-term stability. Solar Energy Materials and Solar Cells 92, 1253e1259. Malhotra, B.D., Chaubey, A., Singh, S.P., 2006. Prospects of conducting polymers in biosensors. Analytica Chimica Acta 578, 59e74. Mathiyarasu, J., Senthil, K.S., Phani, K.L.N., Yegnaraman, V., 2008. Pedot-au nanocomposite film for electrochemical sensing. Materials Letters 571e573. Mazeiko, V., Minkstimiene, A.K., Ramanaviciene, A., Balevicius, Z., Ramanavicius, A., 2013. Sensor. Actuat. B: Chemical 189, 187e193. Molino, P.J., Innis, P.C., Higgins, M.J., Kapsa, R.M.I., Wallace, G.G., 2015. Influence of biopolymer loading on the physiochemical and electrochemical properties of inherently conducting polymer biomaterials. Synthetic Metals 200, 40e47. Molino, P.J., Zhang, B., Wallace, G.G., Hanks, T., 2013. Surface modification of polypyrrole/biopolymer composites for controlled protein and cellular adhesion. Biofouling 29, 1155e1167.  Monosík, R., Stredanský, M., Sturdík, E., 2012. Biosensors d classification, characterization and new trends. Acta Chimica Slovaca 5, 109e120. Moreno, J.S., Panero, S., Materazzi, S., Martinelli, A., Sabbieti, M.G., Agas, D., Materazzi, G., 2009. Polypyrrole-polysaccharide thin films characteristics: electrosynthesis and biological properties. Journal of Biomedical Materials Research 88A, 832e840.

References 465

Moy, B.V., Florin, E., Gaub, H.E., 1994. Intermolecular forces and energies between ligands and receptors. Science 226, 257e259. Murugaiyan, S.B., Ramasamy, R., Gopal, N., Kuzhandaivelu, V., 2014. Biosensors in clinical chemistry: an overview. Advanced Biomedical Research 3, 67. Namazi, H., Baghershiroudi, M., Kabiri, R., 2016. Preparation of electrically conductive biocompatible nanocomposites of natural polymer nanocrystals with polyaniline via in situ chemical oxidative polymerization. Polymer Composites. http://dx.doi.org/ 10.1002/pc.23943. Naumann, A., Dennis, J.E., Aigner, J., Oticchia, J., Arnold, J., Berghaus, A., Kastenbauer, E.R., Caplan, A.I., 2004. Tissue engineering of autologous cartilage grafts in three-dimensional in vitro macroaggregate culture system. Tissue Engineering 10, 1695e1706. Nickels, J.D., Schmidt, C.E., 2013. Surface modification of the conducting polymer, polypyrrole, via affinity peptide. Journal of Biomedical Materials Research A 10, 1464e1471. Otero, T.F., Abadias, R., 2008. Potentiostatic oxidation of poly(3-methylthiophene): influence of the prepolarization time at cathodic potentials on the kinetics. Journal of Electroanalytical Chemistry 618, 39e44. Otero, T.F., Sanchez, J.J., Martinez, J.G., 2012. Biomimetic dual sensing-actuators based on conducting polymers: galvanostatic theoretical model for actuators sensing temperature. Journal of Physical Chemistry B 116, 5279e5290. Pelto, J., Haimi, S., Puukilainen, E., Whitten, P.G., Spinks, G.M., Bahrami, S.M., Ritala, M., Vuorinen, T., 2010. Electroactivity and biocompatibility of polypyrrolehyaluronic acid multi-walled carbon nanotube composite. Journal of Biomedical Research 93, 1056e1067. Pernaut, J.M., Reynolds, J.R., 2000. Use of conducting electroactive polymers for drug delivery and sensing of bioactive molecules. A redox chemistry approach. Journal of Physical Chemistry B 104, 4080e4090. Piskin, E., Bölgen, N., Egri, S., Isoglu, I.A., 2007. Electrospun matrices made of poly(!hydroxy acids) for medical use. Nanomedicine 2, 441e457. Rashidi, H., Yang, G., Shakesheff, K.M., 2014. Surface engineering of synthetic polymer materials for tissue engineering and regenerative medicine applications. Biomaterials Science 2, 1318e1331. Ravi, S., Chaikof, E.L., 2010. Biomaterials for vascular tissue engineering. Regenerative Medicine 5, 107. Ravichandran, R., Sundarrajan, S., Venugopal, J.R., Mukherjee, S., Ramakrishna, S., 2010. Applications of conducting polymers and their issues in biomedical engineering. Journal of The Royal Society Interface 7, 559e579. Rew, J.E., Hill, H.A.O., 1988. Direct and indirect electron transfer between electrodes and redox proteins. European Journal of Biochemistry 172, 261e269. Rivers, T.J., Hudson, T.W., Schmidt, C.E., 2002. Synthesis of a novel, biodegradable electrically conducting polymer for biomedical applications. Advanced Functional Materials 12, 33e37. Schmidt, C.E., Shastri, V.R., Vacanti, J.P., Langer, R., 1997. Applied biological sciences stimulation of neurite outgrowth using an electrically conducting polymer. Proceedings of the National Academy of Sciences 94, 8948e8953.

466 CHAPTER 10 Bioactive Materials Based on Biopolymers Grafted on Conducting Polymers

Senel, M., 2015. Simple method for preparing glucose biosensor based on in-situ polypyrrole cross-linked chitosan/glucose oxidase/gold bionanocomposite film. Materials Science and Engineering C 48, 287e293. Severt, S.Y., Snider, N.A.O., Leger, J.M., Murphy, A.R., 2015. Versatile method for producing 2D and 3D conductive biomaterial composites using sequential chemical and electrochemical polymerization. ACS Applied Materials and Interfaces 7, 25281e25288. Stewart, E.M., Fabretto, M., Mueller, M., Molino, P.J., Griesser, H.J., Short, R.D., Wallace, G.G., 2013. Cell attachment and proliferation on high conductivity PEDOT-glycol composites produced by vapour phase polymerisation. Biomaterials Science 1, 368e378. Takano, T., Mikazuki, A., Kobayashi, T., 2014. Conductive polypyrrole composite films prepared using wet cast technique with a pyrroleecellulose acetate solution. Polymer Engineering and Science 54, 78e84. Tasteta, D., Savea, M., Fatima, C., Charrier, B., Ledeuila, B., Dupina, J.D., Billona, L., 2011. Functional biohybrid materials synthesized via surface-initiated MADIX/ RAFT polymerization from renewable natural wood fiber: grafting of polymer as non leaching preservative functional biohybrid materials synthesized via surfaceinitiated MADIX/RAFT polymerization from renewable natural wood fiber: grafting of polymer as nonleaching preservative. Polymer 52, 606e616. Thakur, M.K., Thakur, V.K., 2014a. Recent trends in hydrogels based on psyllium polysaccharide: a review. Journal of Cleaner Production 82, 1e15. Thakur, V.K., Thakur, M.K., 2014b. Recent advances in graft copolymerization and applications of chitosan: a review. ACS Sustainable Chemistry and Engineering 2, 2637e2652. Thakur, M.K., Thakur, V.K., Gupta, R.K., 2013. Rapid synthesis of graft copolymers from natural cellulose fibers. Carbohydrate Polymers 98, 820e828. Thakur, M.K., Thakur, V.K., Gupta, R.K., Pappu, A., 2016. Synthesis and applications of biodegradable soy based graft copolymers: a review. ACS Sustainable Chemistry and Engineering Journal. http://dx.doi.org/10.1021/acssuschemeng.5b01327. Thévenot, D.R., Toth, K., Durst, R.A., Wilson, G.S., 2001. Electrochemical biosensors: recommended definitions and classification. Biosensors Bioelectronics 16, 121e131. Tiefenauer, L., Ros, R., 2002. Biointerface analysis on a molecular level new tools for biosensor research. Colloids and Surfaces B: Biointerfaces 23, 95e114. Ulbricht, M., 2006. Advanced functional polymer membranes. Polymer 47, 2217e2262. Van Krevelen, D.W., Te Nijenhuis, K., 2009. Properties of Polymers: Their Correlation with Chemical Structure; Their Numerical Estimation and Prediction from Additive Group Contributions. Elsevier. Wallace, G.G., Smyth, M., Zhao, H., 1999. Conducting electroactive polymer- based biosensors trends. Analytical Chemistry 18, 245e251. Wang, M., 2003. Developing bioactive composite materials for tissue replacement. Biomaterials 24, 2133e2151. Weng, B., Diao, J., Xu, Q., Liu, Y., Li, C., Ding, A., Chen, J., 2015. Bio-interface of conducting polymer-based materials for neuroregeneration. Advanced Materials Interfaces 2, 1500059-1e1500059-23.

References 467

Xia, N., Xing, Y., Wang, G., Feng, Q., Chen, Q., Feng, H., Sun, X., Liu, L., 2013. Probing of edc/nhss-mediated covalent coupling reaction by the immobilization of electrochemically active biomolecules. International Journal of Electrochemical Science 8, 2459e2467. Zare, E.N., Lakouraj, M.M., 2014. Biodegradable polyaniline/dextrin conductive nanocomposites: synthesis, characterization, and study of antioxidant activity and sorption of heavy metal ions. Iranian Polymer Journal 23, 257e266. Zinger, B., Miller, L.L., 1984. Timed release of chemicals from polypyrrole films. Journal of the American Chemical Society 106, 6861e6863.