Advanced Biomaterials and Biodevicess

Fabrication, Properties of Nanoshells 137 stable for long periods of time when dry and can be repeatedly isolated and redissolved in common organic solvents without irreversible aggre- gation or decomposition. In synthesis, the gold salt is first transferred to the organic phase using a suitable surfactant. Then sodium borohydride (NaBH4) is added to the aqueous phase. The formation of nanoparticles is monitored by the generation of change of color in the organic phase. Biological methods: Microorganisms have been used with a certain suc- cess in nanoparticle synthesis has been observed. Among the microorgan- isms, prokaryotic bacteria have received the most attention in the area of metal nanoparticle biosynthesis. The formation of extracellular and intra- cellular metal nanoparticles by bacteria like Escherichia coli, Pseudomonas stutzeri, Pseudomonas aeruginosa, Plectonema boryanum, Salmonells typlus, Staphylococcus currens, Vibrio cholerae, etc., have been reported [52,53]. The Au NPs were synthesized adopting similar procedure using two Pseudomonas aeruginosa isolate [55]. The reduction of Au3+ complexes by C. metallidurans to AuNPs occurred through fast accumulation leading to the formation of intermediate Au+-S complexes followed by a slow biochemically-driven reduction and intra- and extracellular deposition of Au NPs. Here, gold colloid was prepared by using citrate reduction method [56]. 5 mL of 0.03 M trisodium citrate (Na3C6H5O7) was added to 10 mL of 5 mM boiling tetrachloroauric acid (HAuCl4·2H2O) aqueous solution while stirring. The mixture was kept boiling for about 30 min until a deep wine red colour was observed, indicating the formation of gold nanoparticles and then cooled down to room temperature. Gold nanoparticles were iso- lated from the colloidal solution by centrifugation at 14000 rpm for 20 min and then washed 3–4 times with water. 4.9 SiO2@Ag and SiO2@Au Core-shell Nanocomposites Any core-shell nanostructures may be fabricated by combining various types of dielectric materials, metals, semiconductors and pigments, where one material is a core and another is shell. Another type of core-shell nanostruc- tures are where the core is a small (10–50 nm) metal nanoparticle such as gold or silver, and the shell is made of silica. These nanostructures are of great interest for improving the chemical stability of colloids and for enhancing luminescent properties of various systems. The method chosen for core-shell fabrication depends on the desired type and properties of the chemicals used. For preparation of SiO2@Ag core-shell nanocomposites silver nitrate (AgNO3) (50 mL, 2◊10–3 M) was reduced in presence of silica particles

138 Advanced Biomaterials and Biodevices Figure 4.11 TEM image of prepared SiO2@Ag nanocomposites. (0.05 gm dispersed in 10 mL of water) using trisodium citrate (50 mL, 2◊10–2 M). These particles were centrifuged and washed with water. Yellow colour precipitate was again redispersed in water. To enhance the thick- ness of coating the second step was repeated again till the desired thick- ness was obtained. Coating thickness can also be controlled by modifying the reaction conditions such as amount of silica particles added. SiO2@Ag coreshell particles were isolated from the colloidal solution by centrifuga- tion at 4000 rpm for 20 min and then washed 3–4 times with water. The prepared nanocomposites were observed using TEM as is clearly shown in Figure 4.11. In order to prepare SiO2@Au particles first the silica particles was func- tionalized using 3-Aminopropyltriethoxysilane (APTES). APTES mol- ecules have one −OH end and the other end has −NH4. So it was bonded to silica through oxygen and gold via nitrogen atom. Functionalization of silica particles was performed by using 12 mM APTES in C2H5OH:H2O (3:1volume ratio) and adding silica particles to it with APTES: silica ratio as 4.3:1 by weight. The resulting solution was vigorously stirred at 75°C for 4 h. The solution was centrifuged and washed with water. Functionalized particles were then re-dispersed in water. Speckled SiO2@Au core shell particles were synthesized in a three step procedure [57–58] as follows 1) 20 mL (6.25 mM) of gold solution, 4.5 mL (0.1 M) of NaOH solution (pH = 7.0) and 1 mL functionalized silica particles in water were stirred at 75 °C for 10 min. These form the gold seeds with silica particles. Above solution was centrifuged, washed with water and re-dispersed in 40 mL water. (2) 28 mg K2CO3, 100 mL Milli-Q water and 1.5 mL of 25 mM gold sol solution were poured in 250 mL round

Fabrication, Properties of Nanoshells 139 Figure 4.12 FE-SEM image of prepared SiO2@Au nanocomposites. bottomed flask, stirred and aged in the dark for 12 h. (3) Finally silica - gold seed solution in the step (1) and gold hydroxide solution in the step (2) were mixed and stirred for 10 min with (5.3 mM) NaBH4. A variety of linker molecules with different functional groups, a number of materials can be anchored on dielectric cores like MPTMS (3-mercap- topropyltrimethoxysilane) which makes surface terminated with thiols, DPPETES (2-(diphenylphosphino)ethyltriethoxysilane) leaves the surface terminated by diphenylphosphine group and PTMS (propyltrimethoxysi- lane) giving surface terminated with methyl group. The prepared nano- composites were observed using FE-SEM as is clearly shown in Figure 4.14. 4.10 Surface Enhanced Raman Scattering Surface-enhanced Raman scattering (SERS) is a powerful analytical tech- nique for ultrasensitive chemical or biochemical analysis. When a light beam interacts with the surrounding electrons of the metal, they begin to oscillate as a collective group across the surface. These oscillations are known as “Surface Plasmons”. SERS gives an enhancement of up to about 106 in scattering efficiency over normal Raman scattering. When a molecule with a chromophore which fluoresces is adsorbed on the SERS active metal surface, the fluorescence is almost completely quenched. On a smooth surface, the oscillation occurs along the plane of the surface. Absorption can occur but no light will be scattered. To get scattering, there needs to be an oscillation perpendicular to the surface plane and this is achieved by roughening the surface or preparing colloid metal particles.

140 Advanced Biomaterials and Biodevices Silver and gold plasmons oscillate at frequencies in the visible region and therefore, they are suitable for use with the visible and NIR laser systems commonly used in Raman scattering. For analytical applications, it is important to distinguish two types of SERS signals when using colloidal nanoparticles [59]. First, the “aver- age SERS” enhanced spectra, [60] i.e. the SERS spectrum of a given ana- lyte obtained from an ensemble of colloidal particles and aggregates and characterized by a stable intensity pattern, with well-defined and reproduc- ible frequencies and bandwidths. Second, SERS intensities obtained from silver or gold nanostructures sustaining a “hot spot” (large enhancement factors), which permits the detection of a few molecules with fluctuating spectral characteristics [61]. Although large enhancement factors have been reported, attempts are still underway to improve the SERS signals by using metal nanoparticles, aligned carbon nanotubes etc. along with the theoreti- cal efforts to understand the huge enhancements. SERS can mainly take place either due to chemical, electromagnetic or combined effect [62–63]. Speckled SiO2@Au core-shell particles with speckled particle shells i.e. shells constituting the nanoparticles should be ideal for enhancing the Raman signal as there would be natural surface roughness due to small particles, increasing the area for molecular adsorption and the nanogaps between the particles to enhance the electric field between the particles. Here the Raman enhancement due to speckled nanoparticle shells in case of Crystal Violet (CV) molecules as well as Single Wall Carbon Nanotubes (SWNT) is demonstrated. CV molecules also known as methyl violet are known since nineteenth century. They are water soluble and their 18.0k λLaser = 532 nm 1590 16.0k 14.0k Intensity (a.u.) 12.0k 10.0k 8.0k 6.0k 4.0k 1350 2.0k B A 0.0 500 1000 1500 2000 Raman shift (cm–1) Figure 4.13 Raman spectra of Single Wall Carbon Nanotubes (SWNT) without (A) and with (B) SiO2@Au particles.

Fabrication, Properties of Nanoshells 141 color can change with pH of the solution. They are used in histological stain as well as antiseptic and antibacterial application. CV molecules also have applications in fertilizers and ball pen inks. There are also previous reports on SERS due to CV molecules on different substrates [64]. Excitation by laser near the localized surface plasmon resonance (SPR) of gold core-shell particles is better than simply the closeness to the SPR of gold nanopar- ticles deposited on core-shell particles. They have the advantage that they provide large surface area due to their large area providing platform for accommodating large number of nanoparticles. Nanoparticles themselves provide surface roughness desired for enhancing the Raman signal due to increased localized electric field. A spectrum was observed for CNT prepared in lab using Chemical vapor deposition method and enhancement in signal was observed as they interacted with SiO2@Au core-shell nanocomposites. 4.11 Conclusions It has been shown that consistent with previous reports the enhancement of signals can depend on particular vibrations as well as overall closeness of the laser excitation wavelength to the SPR wavelength of the substrate. We find in general that speckled SiO2@Au core-shell particles are promis- ing SERS material. Acknowledgements We are thankful to Prof. Aditya Shastri, Vice Chancellor of Banasthali Vidyapith for kindly extending the facilities of ‘‘Banasthali Centre for Education and Research in Basic Sciences” sanctioned under CURIE pro- gramme of the Department of Science and Technology, New Delhi. References 1. A. S. Tawfik, A Strategy for Integrating Basic Concepts of Nanotechnology to Enhance Undergraduate Nano-Education: Statistical Evaluation of an Application Study, A journal of nanoeducation, 4, 1, 2013. 2. M. C. Roco, C. A. Mirkin, and M. C. Hersam, Nanotechnology research directions for societal needs in 2020: summary of international study, Journal of nanoparticle research, 2011, doi: 10.1007/s11051-011-0275-5.

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5 Chitosan as an Advanced Healthcare Material M.A. Jardine* and S. Sayed Department of Chemistry, University of Cape Town, Cape Town, South Africa Abstract Chitin and chitosan are renewable biopolymers with inherently favourable proper- ties allowing diverse chemical modification that generates novel materials ideal for biomedical applications. Chitin itself has very poor aqueous solubility and hence found limited utility. For this reason the use of chitosan, in particular, modified chitosan, has increased exponentially in biomedical applications in recent years. The review of chitosan as a health care material includes wound healing or tissue regeneration, drug delivery and antimicrobial studies. Specialist applications are vast and due to the shortened publication process, biomedical applications of chi- tosan may warrant more frequent reviews. In this review, highlights of recent research on the synthesis and analysis of chitin and chitosan based nanomaterials and nanofibres will be covered. A detailed review of the synthetic transformation of chitosan that further added value by enabling diverse application has been discussed. The value of co- polymerization and grafting of biodegradable synthetic polymers with chitosan in order to improve stability and control of the functional biomaterial has been a major driver in the biotechnological innovation. Keywords: Chitosan, biomedical, polymers, nano, wound healing, drug delivery, antimicrobial 5.1 Introduction Chitosan is a linear semi-crystalline polysaccharide which has recently been receiving significant scientific interest, owing to its unique properties including its biocompatibility, chemical versatility, biodegradability and *Corresponding author: [email protected] Ashutosh Tiwari and Anis N. Nordin (eds.) Advanced Biomaterials and Biodevices, (147–182) 2014 © Scrivener Publishing LLC 147

148 Advanced Biomaterials and Biodevices low toxicity. For this reason the utility of chitosan, in particular, modified chitosan in biomedical applications increased exponentially in recent years [1]. Regular reviews have been reported, more so in a general sense rather than special applications in the biomedical field [1, 2]. 5.1.1 Chitosan Chitosan may be obtained from the partial deacetylation of chitin, the sec- ond most abundant biopolymer in nature, after cellulose (Figure 5.1) [2]. Sources of chitin include the exoskeletons of arthropods such as crus- taceans, fungi, insects, annelids, etc. Approximately 1010 tons of chitin is produced annually in nature, where it is the largest source of nitrogen available to living organisms [3, 4]. Chitin is converted to chitosan by thermochemical deacetylation in the presence of an aqueous base whereas chitosan only occurs naturally in certain fungi e.g. Mucoraceae. Chitosan is relatively inexpensive and is a cost effective alternative to expensive syn- thetic polymers which can perform a similar function [1]. It is considered to be an environmentally friendly ‘green’ polymer which has been clas- sified as a Generally Regarded As Safe (GRAS) material [5, 6]. Chitosan is composed of randomly distributed β-(1–4)-linked D-glucosamine and N-acetyl-D-glucosamine units (Figure 5.1). As a result, the polymer does not have a single well-defined molecular structure and may have differ- ent molecular weights and sequences [3, 4]. The degree of deacetylation Chitin N-deacetylation Chitosan Figure 5.1 The deacetylation of chitin.

Chitosan as an Advanced Healthcare Material 149 (DDA) and depolymerization, determines the molecular weight. The DDA ranges from between 40 to 98 % with a variation in the molecular weight from 5 × 104 to 2 × 106 Daltons. As a result of polymers of different molec- ular weights, different properties of chitosan can be exploited. There are currently four grades of chitosan available, depending on their application these are, agricultural (DDA ≥ 85%), industrial (DDA > 75%), food & cos- metics (DDA 65–90 %, 78–82%) and pharmaceutical grade chitosan (DDA 90–95%) [2]. However, the global standardization of chitosan grades is currently in progress [7]. Chitosan contains two reactive hydroxyl groups (C-3 & C-6) and an amino group at the C-2 position of the glucosamine residue which is responsible for the unique properties of chitosan. The reactivity of chitosan is largely dependent on pH which affects its charged state and properties. Chitosan is protonated and thus positively charged at a low pH where it is also partially water soluble. In contrast, at a neutral to high pH chitosan is insoluble. Chitosan has an almost neutral pKa where the soluble-insoluble transition occurs at a pH of ~ 6.0 - 6.5, a range which is favourable for bio- logical applications [8, 9]. Due to the presence of strong intra- and intermolecular hydrogen bonds, the polymer does not dissolve in most organic or aqueous solvents. This poor solubility restricts the possible applications of the polymer. In order to increase polymer solubility, derivatives of chitosan have been syn- thesized by attaching hydrophilic or hydrophobic groups to the polymer backbone. One particular route to increase solubility, involves the conver- sion of the C-6 hydroxy group into a carboxy or amino group, thereby increasing solubility in organic and aqueous solvents [10, 11]. 5.1.2 General Applications At present a wide range of industrial applications of chitosan exist. These include water treatment, agriculture, biotechnology, food/health supple- ments, cosmetic, biomedical, textile and paper [4, 12]. Most of these appli- cations require chitosan to be aqueous soluble therefore modifications which enhance solubility are favourable. Chitin and chitosan are biopoly- mers that can offer structural versatility for chemical modifications to gen- erate novel materials with interesting properties. The antimicrobial, antifungal and haemostatic properties of chitosan have found numerous biomedical applications. Since chitosan is fully bio- degradable in addition to being non-toxic, the utilization of this polymer in various products will not have a negative effect on humans or the envi- ronment [1, 2, 4]. 

150 Advanced Biomaterials and Biodevices 5.2 Chemical Modification and Analysis Chitosan has been modified by a variety of methods which include alkyl- ation, acylation, Schiff base formation, nitration, phosphorylation, sul- fation, xanthation, hydroxyalkylation, and graft co-polymerization[13]. These modifications have chemical, biological and functional advan- tages compared to native chitosan. Some enhanced properties have been reviewed by Sarmento et al. and Inamdor et al [14, 2]. These include an increase in solubility, gelling properties and reversion of the net charge from polycationic to polyanionic. In addition, designs for hydrophobic derivatives with amphiphilic character and the capacity to harness self- assembling nanostructures and chemical conjugates with an assortment of bioactive and therapeutic molecules have been evaluated with modi- fied chitosan. Improved biocompatibility (e.g., haemocompatibility) can also be observed as well as an enhancement of properties for complex- ing and transfection of plasmid DNA or messenger RNA (siRNA) [14]. A variety of chemical transformations enable chitosan to be an attrac- tive material for the preparation of many functional polymer products. Selected examples are shown below (Figure 5.2) [2]. N-Carboxyacyl-chitosan Click chemistry Reductive with metal N-alkylation Metal free click chemistry R1 = H or trifluoromethylated oxanorbornadienes Sugar-linked chitosan derivatives Schiff base Reductive Reductive methylation animation R2 = azido-modified substrates Quaternized N-Alkyl chitosan chitosan Figure 5.2 Chemical modification of chitosan has been possible in mostly polar protic and aprotic solvents. Some of the many possibilities are represented here.

Chitosan as an Advanced Healthcare Material 151 The chemistry involved in the synthesis of chitosan derivatives includes a range of reaction conditions and purification methods. In the Schiff base reaction between chitosan and aldehydes or ketones, the product obtained is an aldimine or ketamine which is subsequently converted to the N-alkyl derivatives by hydride reduction with borohydride, generally known as reductive amination. It is also common to utilize bifunction- alized aldehydes such as glutaraldehyde as chitosan cross-linking agents. Water soluble carboxymethyl chitosan is obtained by chitosan’s reaction with glyoxylic acid while cationic derivative N,N,N-trimethyl chitosan is synthesized via reductive methylation under alkaline conditions. A cross- linked chitosan marketed as Chitopearl is produced by the reaction of chi- tosan with excess 1,6-diisocyanatohexane which is later exposed to water vapour. This polyurethane-type chitosan is used in chromatography and as an enzyme support. To produce alternative sugar linked chitosan, the poly- mer undergoes reductive N-alkylation with sodium cyanoborohydride and a sugar/sugar-aldehyde derivative useful in targeted drug delivery. Thiolated chitosan derivatives are produced by reacting chitosan with reagents bearing thiol moieties. Thiolation with cysteine via coupling through its carboxyl group have been well studied. Many other methods exist for the introduction of a thiol group. Once incorporated in chitosan, the thiol group permits redox mediated polymer gelation which is attrac- tive in drug delivery technology [15]. An important reaction used in the synthesis of chitosan derivatives is ‘click chemistry’. Click chemistry is essentially a azide-alkyne Huisgen 1,3- polar cycloaddition catalysed by in situ generated Cu(I). This chemistry allows for the synthesis of complex polymers by rapidly and consistently linking small units at room temperature in polar solvents. This method has to date produced a variety of chitosan-based derivatives which can have several different functional groups present. These modifications improve the utility of these polymers for various applications [16, 17]. However, limitations to this reaction due to oxidative instability of Cu(I) and sub- sequent difficulty of metal removal from the complexing polymer, led to the development of a metal free coupling method. Thus, the coupling of chitosan-oxanorbornadiene derivatives to azides provided an ideal means of coupling water soluble chitosan derivatives with either small polar mol- ecules or other polymers [18]. The latter reaction has not yet been exploited in the coupling of bioactive molecules for biomedical application. 5.2.1 Characterization Due to the variability in chitosan structure and physical properties, it is challenging to obtain consistent batch-to-batch analytical data. Synthetic

152 Advanced Biomaterials and Biodevices polymers can be made under strict experimental control or specifications, whereas chitosan requires careful selection as a starting material. Successful commercialisation depends on reliable quality control and quality assess- ment protocols, especially in the biomedical filed. Thus, standardisation of native chitosan on a global scale is warranted in order to promote its utility. Chitosan polymers are typically analysed using a variety of spectroscopic and analytical techniques. In the analysis of chitosan and its derivatives, determining the degree of deacetylation (DDA) is the main priority. The DDA influences proper- ties such as solubility and reactivity and the molecular weight affects the physico-chemical and biological properties of the compounds [19, 20]. Many different techniques are available to determine the DDA where all techniques having both advantages and disadvantages. There is no solitary technique to determine DDA, a combination of techniques can be used to confirm the value obtained. These techniques also provide additional information on the polymer such as incorporation of different functional groups onto the polymer backbone. Techniques available to determine structural and physical properties include: Size exclusion chromatography (SEC) - offers the determination of the number average molecular weight (Mn) and the weight average molecu- lar weight (Mw) which can be used to calculate the polydispersity index (Mw/Mn). American Standard Test Method (ASTM) Organisation utilizes ASTM F2602–08e1 as the standard test method for determining the molar mass of chitosan and chitosan salts by virtue of SEC coupled to Multi-angle Light Scattering Detection (SEC-MALS). Nuclear Magnetic Resonance Spectroscopy (NMR) - 1H and 15N-NMR are used for samples in solution and 13C NMR is used for solid samples. Solution NMR is ideal when a suitable NMR solvent is available. However, solid state NMR provides an opportunity to assign structure to insoluble samples. This quantitative analysis is preferred as it is simple, quick and accurate. The ASTM utilizes NMR as the method of choice to determine the DDA of chitosan samples [20]. Kasaai et al. reviewed the various NMR spectroscopy techniques in determining DDA and found that solution 1H NMR is by far the best method available. NMR can also be used to find the degree of substitution of a chitosan derivative [21]. Viscometry – viscosity is related to the DDA where viscosity is a widely used method to determine DDA [20]. The intrinsic viscosity of a poly- mer solution is related to the polymer molecular weight according to the Mark-Houwink-Sakurada (MHS) equation, [η] = K(Mv)a. Where [η] is the intrinsic viscosity, Mv the viscosity-average molecular weight, and K and a, are the constants for a given solute-solvent system [22].

Chitosan as an Advanced Healthcare Material 153 Thermogravimetric Analysis (TGA) and Differential Scanning Calorimetry (DSC) – Thermal methods, such as TGA and DSC are pow- erful thermoanalytical techniques to monitor characteristic physical and chemical changes in both natural and synthetic polymers. Modified chito- san would yield curves that are unique for a particular composition where slight changes in chemical structure will bring about discrete and repro- ducible variations in the thermograms. The loss of structural water in vola- tilization and subsequent decomposition can be quantitatively measures by TGA. Thus, TGA is a useful method to compare the thermal stability of chitosan vs. chitosan derivatives. For chitosan, there are two characteristic degradations, at 50–100 °C attributed to the loss of water from the polysac- charide chains and 250–400 °C resulting from the depolymerization of the polysaccharide with the loss of volatile compounds [20]. DSC is generally used to confirm results obtained from TGA and NMR. The heights of the peaks obtained can be used to approximate the DDA [20]. Ultraviolet Spectroscopy (UV) – this technique is generally used to anal- yse chitosan derivatives since chitosan itself does not have any character- istic absorption in the range 200–400 nm. However, after introduction of elements that permit surface plasmon resonance e.g. silver nanoparticles, UV-analysis can provide some structural support data [20]. Infra-red spectroscopy (IR) – this technique can be utilized as a qualita- tive or quantitative method in the determination of DDA. It is one of the preferred methods when characterizing chitosan derivatives due to its sim- plicity of sample preparation, promptness and it is non-destructive nature. Several groups have used different absorption band ratios e.g. A1560/A2875, A1655/A2875, A1655/A3450 to determine the DDA of chitosan, where these bands are characteristic of chitosan [19]. IR is also useful for chitosan deriva- tives as the introduction of new bands or the disappearance or change in existing bands can be used as an indicator of degree of substitution. This technique does have certain disadvantages such as, peak broadening and overlapping which can lead to incorrect results [20]. Elemental Analysis (EA) – the elemental composition of chitosan deriv- atives is obtained through the use of this technique [20]. The degree of substitution for modified chitosan can be determined from the C/N ratio [23]. Disadvantages associated with this method, is the destruction of the sample during analysis and inaccurate estimations due to varying molecu- lar weights and solvent inclusion. X-ray diffraction – this technique provides information on the crys- talline nature of the polymer. The crystallinity depends on the DDA of the polymer since a higher DDA leads to greater flexibility of the polymer where fewer bulky acetyl groups are present. When metals are incorporated

154 Advanced Biomaterials and Biodevices into the polymer, the presence and identity of the metal can be confirmed by the changes observed in the X-ray pattern. Disadvantages associated with this technique are the high cost and samples need to be in the solid state [24]. This is by no means an exhaustive list of the techniques available to characterize all properties of chitosan and its derivatives. Other common techniques include: light scattering (dynamic and static), zeta potential (measures charge which directly affects antimicrobial activity of the poly- mer), titrations (conductimetric, colloidal, isothermal titration calorim- etry, turbidimetric, acid-base, potentiometric), High Performance Liquid Chromatography, gas chromatography (can only be performed after deg- radation and derivitization of sample) – MALDI-TOF mass spectrometry, gel-permeation chromatography, membrane osmometry, etc [20, 23, 25, 26]. These analysis techniques provide a clear understanding of the composition and nature of the compound being studied allowing for the prediction of various properties and applications of the samples analyzed. 5.3 Chitosan Co-polymers A co-polymer is defined as a polymer chain consisting of more than one type of polymer. This combination leads to the synthesis of polymers with a specific set of desired properties. There are different types of co-polymers depending on the monomers present and the synthetic method used. A random co-polymer is defined as a polymer with a relatively random dis- tribution of the constituent monomers. Alternating co-polymers have their polymers alternate regularly along the polymer chain. A block co- polymer has long sequences of each polymer whereas a graft co-polymer is a polymer that has a backbone consisting of one polymer and branches of the other polymer [27]. In the case of chitosan, co-polymerization offers the introduction of new advantageous properties without the loss of the original properties of the poly- mer as only a selected number of amino/hydroxyl groups are functionalized (Table 5.1) [88]. The common synthetic methods available for co-polymer- ization include free radical polymerization used for graft co-polymers where free radicals are generated on the polymer backbone; these radicals react with vinyl or acrylic monomers. Chemical methods used to initiate this co- polymerization include the use of ceric ions, Fenton’s reagent, microwaves, ammonium persulfate, gamma and UV radiation. Disadvantages associated with free radical polymerization include the generation of homopolymers and thereby undermining the stability of the polymer backbone [28, 29].

Chitosan as an Advanced Healthcare Material 155 Other methods utilize controlled/living free radical polymerization e.g. living free radical nitroxide-mediated polymerization, atom transfer radi- cal polymerization and reversible addition-fragmentation chain transfer (RAFT) polymerization [30]. Atom transfer radical polymerization (ATRP) refers to a system where equilibrium between dormant species and radi- cals is established [31]. Reversible addition fragmentation chain transfer (RAFT) is a reversible deactivation of radical polymerization. These radical polymerisation methods of generating co-polymers normally yield com- plex polymers which have a predetermined MW with a narrow distribution and a known composition. Nonradical methods include the ring opening polymerization of chitosan followed by a nucleophilic attack on the amino groups present on the polymer backbone [28]. Table 5.1: Selected co-polymers and the associated improved properties. Polymer Grafted onto chitosan Property Reference Methyl methacrylate Improved biocompatibility [32, 28] Polyethylene glycol (PEG) Improved solubility, [33, 28] stability and blood residence time Vinyl acetate (VAc) Improved swelling and [34] antifungal activity Polyethylenimine (PEI) Increased transfection [35] efficiency and reduced toxicity of PEI polyplexes Poly(N- Temperature sensitivity [28] isopropylacrylamide(PNIPAM) Increased solubility [36] Polystyrene Enhanced hydrophobicity [37] Polyvinylpyrrolidone pH sensitivity [38] Poly(Lactic acid (LA)) Conductivity and improved [39] Polyaniline stability [40] Polyamidoamine (PAMAM) Improved solubility and dendrimer antibacterial activity

156 Advanced Biomaterials and Biodevices Amongst the chitosan-grafted polymers, the more important polymers in the biomedical field are chitosan-grafted-PEG and chitosan-grafted- PEI. PEG is commonly used in the biomedical sector due to its hydrophi- licity and biocompatibility. It is non-toxic, does not cause irritation and is odourless, making it an ideal polymer which has been used in pharmaceu- ticals as a solvent, ointment and tablet excipient [27]. The grafting of PEG onto chitosan promotes solubility at physiological pH and the stability of the polymer in vivo. In addition, PEG delays the host immune system from degrading the polymers leading to a longer blood residence time. As a result of these advantageous properties, this co-polymer has been applied in the development of carriers for transmucosal drug delivery [41, 42]. PEG has been approved by the Food and Drug Administration for human intravenous, oral, and dermal application which is favourable for the development of biomedical applications of the chitosan-PEG co- polymer [43]. These chitosan-PEG co-polymers are typically synthesized via reductive amination or through the use of activated esters. PEGylation can proceed at the amino (C2) or hydroxyl groups (C3/C6) present on chi- tosan depending on the reaction conditions used. These chitosan-PEG co- polymers can be further functionalized to incorporate functional groups which offer advantageous properties [28]. Grafting of PEI onto chitosan lowers the cytotoxicity associated with PEI polyplexes while increasing transfection efficiency associated with chitosan polyplexes. These co-poly- mers can be synthesized via many different routes which include peri- odate oxidation of the chitosan backbone together with imine formation and Michael addition [28]. 5.4 Nanoparticles When moving to the nanoscale, certain properties are improved due to the higher surface area to volume ratio [44]. Nanoparticles are defined as ultrafine particles which range from 1 to 100 nm in size. Polymeric nanoparticles (NPs) are of great interest in many areas although much effort has been focused on the application of these par- ticles in the biomedical sector. Biodegradable polymeric NPs are the main focus of scientists which have been studied mainly as carriers of drugs or bioactive compound agents due to their favourable properties [45]. These include a longer half-life (sustained release), stability in vivo, higher drug loading, enhanced permeation (due to small size), targeted delivery, pro- tection of the drug and improvement of pharmacokinetics [45, 46]. In addition, release of the bioactive compounds in response to a trigger such

Chitosan as an Advanced Healthcare Material 157 as pH or heat could be possible. Polymeric nanocarriers have been success- fully used to transport drugs, DNA, peptides, imaging agents and proteins [46, 47]. However, the use of NPs is not without disadvantages, there have been concerns related to the use of NPs and their potential cytotoxicity [48]. Therefore, when applying NPs for any specific application, the toxic- ity must be thoroughly studied. Nanoparticles are synthesized using meth- ods such as ionic gelation, microemulsion, self-assembly, reverse micelle formation, coacervation/precipitation and the emulsion-droplet coales- cence technique [47, 49]. Chitosan nanoparticles are particularly attractive since the polymer is biocompatible, biodegradable, non-toxic and can be loaded with hydropho- bic molecules [50]. Chitosan and its derivatives are applied regularly in the area of nanotechnology. They are used as drug, antigen, protein and gene carriers as well as surfactants in the production of nanoparticles [51]. In addition, chitosan is mucoadhesive and can open tight junctions of epithelial cells leading to an increase in the delivery of drugs or bioactive compounds to the desired sites [52]. Chitosan nanoparticles are synthesized primarily by ionic crosslinking or gelation in the presence of a cross-linking agent e.g. sodium tripolyphosphate (TPP) [53]. Chen et al. recently reviewed chitosan based nanoparticles synthesized with the aim of using these NPs as carri- ers in the oral delivery of macromolecules [54]. The derivatives synthesized displayed enhanced properties such as increased mucoadhesivity, increased residence time, increased absorption, etc. The more popular derivatives are those that have been quaternized, thiolated and carboxylated [54]. Recently, chitosan based NPs have been loaded with hydrocortisone, the antimalarial curcumin, insulin, siRNA, ovalbumin, tumor-hypoxia activated phototrig- ger (TAP), etc [45, 55–59]. Chitosan has also been used in the production of metal nanoparticles where the polymer serves as a surfactant, keeping the NP size to a mini- mum. Silver (Ag) nanoparticles have been synthesized using chitosan by numerous groups for a variety of applications. Chitosan-Ag complexes have been reported by Zhan et al. and the complexes exhibited antibacte- rial activity [60]. Due to the enhanced antimicrobial properties of both Ag nanoparticles and chitosan, the evaluation of silver loaded chitosan deriva- tives as antimicrobial agents has been explored. Sanpui et al. investigated the efficacy of a chitosan−Ag nanoparticle composite against Escherichia coli (E. coli) and results indicated that the composite had a higher antimi- crobial activity compared to the parent polymers [61]. Chen et al. synthe- sized a thiourea chitosan−Ag+ complex which displayed a wide spectrum of antimicrobial activities against Staphylococcus aureus (S. aureus), E. coli, Bacillus subtilis (B. subtilis), Aspergillus flavus (A. flavus), Mucor

158 Advanced Biomaterials and Biodevices (A) (B) Figure 5.3 Ag (A) and Fe (B) nanoparticles synthesized in the presence of chitosan derivatives. bacilliformis and Paecilomyces variotii. The minimum inhibitory concen- tration range was found to be 20 times lower than that reported for chi- tosan [62]. Other metal NPs which have been synthesized with chitosan include iron, gold, palladium, platinum and copper to name a few [63]. Bae et al. have synthesized chitosan oligosaccharide stabilised ferrimagnetic iron oxide nanoparticles which were utilized in magnetically modulated cancer hyperthermia [64]. This study showed promising antitumour effi- cacy in an animal tumour model [64]. Previously, Ag and Fe nanoparticles have been synthesized in the presence of chitosan based polymers. The TEM images below show the typical morphologies obtained for these par- ticles [Figure 5.3, A (Ag) & B (Fe) at a resolution of 200 nm]. 5.5 Nanofibres (Electrospinning) Electrospinning is a method used for the fabrication of ultrafine fibres, where macromolecules are spun into fibres as thin as a few nanometres. Almost any soluble polymer with a sufficiently high molecular weight can be electrospinned to provide nanofibres with diameters in the range of 20–200nm. The principle of electrospinning is the application of an elec- tric field to a polymer fluid where the polymer is introduced to the field via a capillary needle. The resultant non-woven fibre mat is collected on a collector plate (Figure 1.4) [65, 29]. Adjusting the parameters of the electrospinning process produces fibres with variable structures [66]. Nanofibres produced have favourable proper- ties such as large surface area to volume ratios, high porosity, high gas per- meability and small pore sizes, all of which are advantageous in biomedical

Chitosan as an Advanced Healthcare Material 159 High voltage supplier Polymer solution Fibre formation Syringe pump Collector Figure 5.4 The typical set-up for electrospinning. applications. Nanofibres have been applied in wound dressing, drug deliv- ery, as tissue engineering scaffolds and in filtration [67]. Biopolymers are more favourable compared to synthetic polymers due to their biodegrad- ability and biocompatibility however, these polymers require polar sol- vents which are harder to spin due to unfavourable dissolved polymer desolvation characteristics. Examples of biomacromolecules which have been successfully electrospun include: silk, collagen, fibrinogen and chi- tosan [68]. Electrospun fibres have been used in a variety of commercially available products such as air filters, liquid filters, performance apparel fabrics, acoustic insulation, medicine (wound healing, tissue engineer- ing, barrier textiles and membranes for drug delivery & release), battery separators and inorganic nanofibres (catalysts, sensors, etc.). Chitosan has been successfully spun into nanofibres using dry and wet spinning meth- ods. However, the bulk scale production of these nanofibres is challenging, fibres have poor mechanical strength and hydrolysis may occur in water or tissue fluid. In addition, toxic solvents are sometimes used in the produc- tion of these fibres which may still be present in the final product. To over- come these shortcomings certain methods have been proposed. Chitosan may be spun together with a water-soluble polymer, neutralized using alkaline compounds, or a cross-linking reagent (e.g. epichlorohydrin) can be employed, etc. Elmarco s.r.o (Czech Rep. Eur.) pioneered the scale-up of electrospin- ning of polymers. The company offers research support to scaling up the production of experimental nanofibres with the use of their Nanospider™ technology. This method allows for the production of nanofibres from polymers dissolved in water, acids or bipolar solvents and from melted polymers. This technology can produce organic and inorganic fibres where the process parameters may be altered to obtain the desired effect

160 Advanced Biomaterials and Biodevices [69]. Chitosan has been successfully electrospun together with alginate, poly(ethylenoxide) (PEO), poly(vinyl alcohol) (PVA), silk fibroin and many other synthetic and natural polymers [70]. The co-spinning of chi- tosan with these bioactive substances may confer favourable properties to the nanofibres. One such example is the synthesis of hydroxyapatite (HA) containing chitosan nanofibres. The presence of HA promotes cell attach- ment, osteoblast proliferation and the production of bone extracellular cell matrix when used as scaffolds for wound healing [71]. Commercially avail- able nanofibres are increasing, thus paving the way for advanced health- care materials R & D. One of the most successful products which are based on chitosan electrospun nanofibres is the haemostatic wound dressings manufactured by Hemcon Inc [72]. 5.6 Visualising Nanostructures Structures at the nano level are typically visualised using high powered microscopes. With the help of these microscopes, the morphology and surface & characteristics of nanomaterials can be observed. The renewed interest in nanotechnology in recent years brought attention to visuali- sation techniques. Selected imaging devices can be categorized starting with those that image materials from the surface profile, right down to the nanoscale or atomic level. The atomic force microscope (AFM) is commonly used to observe the surface topology of nanomaterials. This technique utilizes a cantilever with a tip made of either a ceramic or a semiconducting material which moves on the surface of the sample. A laser measures the deflection of the beam from the surface producing a profile of the surface. The laser beam operates at a wavelength of 1300 nm. The AFM produces three-dimensional images of the surface. Advantages of this technique include minimal sample prep- aration and sample analysis can be carried out in different environments such as ambient conditions, in liquids, and in ultra-high vacuum over a large temperature range [73]. A disadvantage with this technique is that the widths of nano-objects may be overestimated due to tip sharpness. Object resolution depends on the sharpness of the tip. This problem can be solved by using deconvolution algorithms to correct for the overestimation [74]. AFM can also be used to manipulate objects at the nanoscale [75]. This technique has been successfully utilized to visualise chitosan [76]. AFM studies on chitosan are relatively new and only a few studies investigate chitosan properties on a molecular level in relation to its interaction with surfaces. The application is yet to be exploited with biomedical materials. A

Chitosan as an Advanced Healthcare Material 161 single molecule study of chitosan using AFM as well as AFM-based single molecular force spectroscopy allowed the imaging of a positively charged single strand of chitosan on negatively charged mica [77]. Scanning electron microscopes (SEM) are commonly used to get a bet- ter picture of the surface of a nanomaterial. SEM can be used to collect information on the topography, morphology, crystallographic arrange- ment and composition of the sample being analysed. The principle upon which this microscope works, involves directing a beam of electrons (0.2 – 40 KeV) at the sample which dislodges the sample’s electrons, thus gen- erating signals. These signals are used to generate an image of the surface of the sample. The sample needs to be electrically conductive to be exam- ined by SEM. Thus, the sample needs to be coated with a thin layer of an electrically conducting material such as gold or palladium. This technique can be used at low vacuum, high vacuum and in wet conditions. An advan- tage of SEM analysis is that a resolution better than 1 nanometre can be achieved [75, 78]. Polymer size distribution can easily be visualised with SEM as well as the surface morphology of nanofibres and nanoparticles. Numerous publications display chitosan surface features using SEM, it is certainly a key tool in polymer surface analysis. Transmission electron microscopy (TEM) involves the focusing of a thin beam of electrons on a sample with a wavelength of < 200 nm. The electrons are scattered when they interact with matter and an image of the interacting electrons is magnified and focused onto an imaging device. The electrons provide a picture of the sample that is being studied. The micro- scope has a resolution of up to 0.2 nm enabling the determination of parti- cle size, arrangement of atoms in a sample and composition of the sample. An improved version of this microscope is the scanning-transmission elec- tron microscope (STEM) which combines attractive features of both TEM and SEM. Samples are typically analysed at low pressures. The images gen- erated can be used to build a three dimensional picture of the sample. A disadvantage associated with this technique is the higher cost compared to other characterization techniques [75, 79]. This technique has previously been utilized to view amongst others, a chitosan-clay nanocomposite [80]. The scanning tunnelling microscope (STM) is intended to image surfaces at the atomic level. This technique can also provide information on the elec- tronic structure of the sample at a specified point. The samples analysed are required to conduct electricity. During analysis a current hovers over the surface of the material where changes in surface height and density of states alter the current. These changes are recorded and used to produce images [81]. Therefore the sample resolution is independent of radiation wave- length and is based only on the size of the probe which is the size of a single

162 Advanced Biomaterials and Biodevices atom. Samples can be analysed in air or liquids however; a vacuum is typi- cally used to prevent sample contamination. This technique is not limited to information gathering, the STM can also be used to manipulate atoms at the nanolevel [75]. This technique has been used for the observation of DNA deposited on graphene, but not yet on chitosan derived materials [82]. Figure 5.5 shows examples of images obtained using SEM, TEM and AFM. Magnetic Resonance Force Microscopy (MRFM) is a technique which uses the three dimensional imaging abilities of magnetic resonance imag- ing (MRI) together with AFM. This leads to a technique which is non- destructive and chemical-specific which can produce high resolution images of many different materials potentially at the atomic scale [85]. Low-voltage electron microscopes (LVEM) have also been used to image samples at the nanoscale. These microscopes can operate in TEM, STEM, SEM and electron diffraction modes. This instrument produces images with better spatial resolution by reducing the beam/sample interac- tion volume and the lower voltage allows for a longer collecting time espe- cially in cases where samples are beam sensitive. This instrument is also Figure 5.5 Representative images generated using SEM (A), TEM (B) and AFM (C & D). The SEM and TEM images show Ag nanoparticles which have been stabilized using chitosan [83]. The AFM images show chitosan/ halloysite nanotube nanocomposites produced by Liu et al. where image (C) and (D) show the height and three dimensional topography of the sample [84].

Chitosan as an Advanced Healthcare Material 163 less costly compared to other electron microscopes and a table top version is available [86, 87]. No reference has been found in literature relating to the use of STM and MRFM on chitosan-based samples. Most visualisation techniques have been applied to chitosan or chitosan derivatives and the more modern techniques can potentially be used in characterizing chitosan. However, as accessibility to some of the technologically advanced instrumentation increases, it’s utility in the visualisation of chitosan and chitosan deriva- tives will also increase. The most powerful nanotech applications have been briefly reviewed, but these are not the only options available for visu- alising nanostructures. Other methods such as confocal microscopy and many other florescence based techniques for often real-time live biologi- cal cell observations are well established [88]. The choice of the appropri- ate technique is dependent on the sample being analysed and the kind of information that is required from the analysis. 5.7 Biomedical Applications of Chitosan There has been a significant amount of interest in the utilization of chitosan in the biomedical sector. A recent review of the literature which covered an extensive range of biomedical applications of chitosan was conducted by Sarmento et al [89]. The diverse uses of this polymer are due to its inherent properties such as biocompatibility, biodegradability, bacteriostatic nature and permeation enhancing properties [89]. The natural abundance of this renewable resource, the ecological safety, low toxicity and low immuno- genicity also promote the use of chitosan [49]. The common topics under review include wound healing or tissue regeneration and antimicrobial studies. The widespread use of chitosan as a pharmaceutical excipient and in cosmetic applications is not within the scope of this review. However, chitosan modifications that enable targeted or triggered delivery of func- tional actives are of particular interest. Many biomedical applications of chitosan have resulted in the market- ing of products which make use of the favourable properties of this poly- mer. A variety of chitosan based products have been marketed for uses ranging from antibacterial fibre to dietary supplements and bandages [90]. Figure 5.6 highlights selected biomedical applications of chitosan. The util- ity of a chitosan derivative depends on the physical properties, in particu- lar the rheology of the polymer. In a recent review by Castro et al., it was mentioned that in solution or as a gel, chitosan can be used as a bacterio- static, fungistatic and as a coating agent [90]. Gels and suspensions may act

164 Advanced Biomaterials and Biodevices Biomedical applications of chitosan Delivery Wound healing/ Antimicrobial Other agents Tissue Wound dressings Dietary Gene regeneration Fibres supplement Drugs Surface coatings Slimming Peptides Scaffold for cell growth agent Protein Resorbable sutures Bioimaging Biopharmaceuticals Promotes healing Hypocholest- Vaccine Haemostatic agent erolemic Nucleic acids Growth factors effect Figure 5.6 Biomedical applications of chitosan. as an immobilising medium or an encapsulation material for the transport and controlled release of drugs. Films and membranes of chitosan have been applied in wound dressings, cell cultures, contact lenses as well as in dialysis. Sponges of the polymer are used to stop bleeding in mucous membranes and as wound dressings. Fibres of the polymers have found utility as drug carriers, non-woven materials for wound dressings and as resorbable sutures. 5.7.1 Current Technology Status Patented technologies are a good indicator of market potential and future projections in biomedical applications of chitosan. Enabling technologies that are on the critical path for expeditious development of new biomate- rials and applications increased rapidly in recent years. This is evident as applied research relating to the biomedical applications of chitosan often yields innovations which can be used in commercial applications warrant- ing intellectual property protection through patenting. In recent years, an exponential increase in patents relating to the use of chitosan in numer- ous areas such as wound healing, tissue reconstruction, delivery of drugs, bioactive materials & genes, coating of medical devices and many more applications appeared. The translation of patented proof of concept to commercialisation is a long process. However, a few companies have estab- lished themselves using chitosan as a core technology. A selected number

Chitosan as an Advanced Healthcare Material 165 of patents that have been awarded recently are highlighted here. These pat- ents form the basis of technology that is currently being developed in the hope of commercialisation. The utility of chitosan as a final coating has found widespread biomedi- cal application. A chitosan-based coating process for ophthalmic lenses has recently been reported. This coating allows the lenses to move freely with- out adhering to the eye and has antimicrobial properties making this an appealing product [91]. Halada and co-workers reported on a method for the electrochemical deposition of metal NPs (ruthenium, rhodium, pal- ladium, silver, osmium, iridium, platinum and gold) together with a chito- san coating on a stainless steel surface [92]. This coating has antimicrobial properties and can potentially be used to confer antimicrobial activity to the surface of medical implants [92]. A patent filed by Filee and colleagues described the preparation of chitosan-based biomimetic scaffolds consist- ing of two layers, a nanofibre scaffold membrane and a porous support layer (sponge) [93]. These scaffolds can potentially be utilized in wound dressing, tissue engineering and other biomedical applications [93]. Thixotropic hydrogels have found widespread application in the cos- metic market, thus allowing the delivery of water soluble actives. However, it is the ability to control gelation using external stimuli or chemical change on demand that is particularly useful in biomedical applications. Yu et al. described the application of a dextran-chitosan based in-situ gelling hydro- gel in the biomedical sector [94]. Potential uses of this hydrogel include controlled drug release, biofilm prevention, tissue in-growth prevention (tissue engineering) and as a matrix for cell proliferation [94]. Wang et al. used gelatin, fibroin, chitosan, collagen or sodium alginate in the prepara- tion of electrospun natural material nanofibres which were loaded with inorganic nanoparticles (mesoporous silica or dye, growth factor, nucleic acid, bioenzyme or drug-coated mesoporous silica) [95]. These nanofibres are biocompatible, simple to prepare and relatively cheap for potential applications in tissue engineering (growth factor and gene release) and other biomedical fields such as cancer treatment, drug delivery, etc [95]. Arab merchants brought cotton cloth to Europe about 800 A.D. Cotton, which comprise of cellulose, a structurally related polymer to chitosan his- torically has a very long lead in technology improvements. Over the past decades, much effort has gone into making cotton fabrics more resistant to microbial degradation. It is only in recent times that the inherent antimi- crobial nature of chitosan has drawn attention. An example of successful technology commercialisation is that of Crabyon® fibre, is a blend of chito- san and viscose manufactured by the textile company Swicofil. This fibre is based on the patent filed by the company Omikenshi which developed a

166 Advanced Biomaterials and Biodevices method to produce this fibre without the use of organic solvents [96]. The production of chitosan-based textiles is important for the manufacture of antimicrobial apparel. During the period 2011 - 2013, there were a large number of patents filed, at least 58 were attributed to the biomedical applications of this ver- satile polymer. [97]. The majority of these patents were filed by the Chinese since China is the largest producer of chitosan worldwide. In addition, the Chinese government has employed an incentive policy which privileges inventors and applicants [98]. With a polymer as diverse as chitosan, the number of potential applications increases as new technology is developed. The patented technologies can be divided into the following sections: bone composite, platforms for cell growth, scaffolds for delivery of cell growth factors, delivery agents and antimicrobial studies. 5.7.2 Wound Healing/Tissue Regeneration Tissue engineering or wound healing comprise of three main components, biomaterials, cells and growth factors [99]. One of chitosans most widely studied applications is that of wound healing or tissue generation. It has been shown that chitosan can contribute to wound healing in different ways such as enhancing the filtration of polymorphonuclear neutrophils and macrophages into the injured tissue, activating the complement and normal fibrolasts, increasing granulation, vascularisation and promot- ing re-growth of the epithelium. In the market there are currently quite a few examples of chitosan-based wound dressings, examples include: HemCon , ChitoFlex , Chitopack C and Chitodine . These dressings range from bandages to sponges and films of chitosan [100]. Wound heal- ing or tissue regeneration generally comprises platforms for cell growth (tissue engineering), scaffolds for delivery of cell growth factors or bone composites. Chitosan-based scaffolds can be produced using a variety of techniques these include, solvent casting and particulate leaching, phase separation and freeze drying, solution spinning, microsphere sintering, hydrogel formation and other less commonly used techniques such as solid free-form fabrication and rapid prototyping integrated with computer- aided design. When designing a scaffold for tissue regeneration, certain requirements should be met; the scaffold should ideally be biodegradable and mimic the native tissue extracellular matrix thereby providing an ideal environment for cellular activities and tissue regeneration. Chitosan meets these requirements and it has functional groups present on the backbone which can be modified with a variety of biological agents with advanta- geous properties. To date, chitosan has been used in the regeneration of

Chitosan as an Advanced Healthcare Material 167 hard and soft tissues (bone, skin, cartilage, intervertebral discs, ligaments & tendons, nerves, vascular and liver tissue) [100]. Numerous co-polymers have been recently reported as promising potential candidates for application in tissue engineering. Balaji et al. has synthesized a keratin-chitosan three dimensional (3-D) scaffold which was found to have antibacterial properties and degrade slowly [101]. Gu and co-workers fabricated a chitosan-poly(ε-caprolactone-co-2-oxepane- 1,5-dione) blend which demonstrated a slower degradation rate compared to native chitosan [102]. Compact rods consisting of chitosan and apatite were prepared by Pu et al [103]. These rods were found to promote cell proliferation and maintain their integrity for much longer compared to native chitosan rods [103]. Kwon et al. utilized a chitosan hydrogel in the presence of valproic acid as a 3-D substrate for the attachment, prolifera- tion and differentiation of rat muscle-derived stem cells [104]. The study concluded that the hydrogel in the presence of valproic acid can differenti- ate the stem cells into cells which have a neural-like phenotype [104]. A 3-D silk fibroin/chitosan composite sponge was prepared by Sionkowska et al. and an investigation of the sponges properties suggested that it could potentially be used as a scaffold to temporarily support the formation of new tissue [105]. Yu and colleagues produced a novel injectable biode- gradable glycol chitosan hydrogel which is an appealing candidate in the development of cell-specific bioactive extracellular matrices [106]. Zhang and colleagues investigated the effect of cold plasma treatment on chitosan films and chitosan-nanoliposome blends for applications in tissue engi- neering [107]. Ideal candidates for wound dressings should be biocompatible, not trigger an allergic or unwanted immune response, have minimal wound adhesion and provide thermal insulation [108]. Recent reports of chitosan- based compounds applied as wound dressings have been reported. These reports are based on chitosan’s effect on the wound healing process. It has been shown that chitosan positively affects immune cells (neutrophils and macrophages), osteoblasts, keratinocytes and fibroblasts all of which are crucial in the healing process. So far, chitosan has been tested in the treat- ment of incisional skin, surgical & subcutaneous wounds, burns, mucosal wounds, wounds of urogenital tissue, liver tissue and spinal tissue [108]. Bellini et al. applied a membrane composed of chitosan and the polysac- charide xanthan in the treatment of skin lesions. These membranes showed potential for use as scaffolds in tissue regeneration and as wound dressings [109]. Kang and co-workers prepared chitosan dressings which had been treated with sodium hydroxide and or sodium tripolyphosphate [110]. The dressings were compared to commercially available chitosan-based

168 Advanced Biomaterials and Biodevices products. Results showed that the dressing absorbed blood quickly, accel- erated clotting and enhanced red blood cell adhesion. This bandage showed great potential as a haemostatic dressing [110]. Lin et al. combined chito- san with bacterial cellulose to produce membranes on a large scale [111]. Testing of these membranes showed that they inhibited the growth of com- mon pathogens E. coli and S. aureus and promoted wound healing [111]. These wound dressings may also contain other bioactive molecules such as the semi-interpenetration hydrogel based on polyacrylamide and chitosan. This hydrogel was loaded with growth factors and the antibiotic piperacil- lin–tazobactam allowing for effective wound healing management [112]. In bone tissue engineering, Tanase and colleagues report the syn- thesis of a chitosan-calcium phosphate matrix via a novel biomimetic co-precipitation method [113]. It was found that these scaffolds were com- parable to the controls tested making these biomaterials strong candidates for use in tissue regeneration [113]. Le et al. investigated chitosan com- posites containing precipitated hydroxyapatite particles where the mineral increased the durability of the compound [114]. These composites can potentially be applied as bone tissue scaffolds [114]. In another study, Lee and co-workers investigated the effects of varying concentrations of chi- tosan and β-tricalcium phosphate on a collagen matrix [115]. The study found that the composite membranes were good candidates for guided bone regeneration membranes [115]. Moving into the nano-scale, Dorj and co-workers produced a nanocomposite scaffold of chitosan and nano- bioactive glass through the method of robocasting [116]. The study sug- gested that these composites may find utility in bone tissue engineering as matrices [116]. Liu et al. incorporated halloysite nanotubes into chitosan forming bionanocomposite films [88]. These films have the potential to act as scaffold materials in tissue engineering. Overall, chitosan is an excellent compound for use in wound healing/ tissue regeneration [88]. This is due to the favourable characteristics of this polymer as evidenced by the many commercially available dressings which are superior to most wound care products. 5.7.3 Targeted Delivery Agents The application of biopharmaceuticals is hampered by the delivery of bio- active agents to the intended target. This could be a result of solubility, stability, charge and size issues which restricts the choice of an appropriate delivery method. To date, chitosan and its derivatives have been studied as peptide, protein, gene, drug (oral, ocular, nasal and buccal), biopharmaceu- tical, nucleic acid and anticancer biopharmaceutical delivery systems. This

Chitosan as an Advanced Healthcare Material 169 polymer is a non-viral vector which has enhanced absorption, controlled release and bioadhesive properties. The modified derivatives of chitosan possess added advantages such as increased solubility, mucoadhesivity and many other functional advantages [15]. Each route of administration depends on the biopharmaceutical being administered and the intended target [117]. As a result of chitosan’s mucoad- hesivity, the polymer can possibly remain in contact with the gastrointestinal tract for a longer period of time. This may result in an increase in the bioavail- ability of the biopharmaceutical being administered. Modifications of chito- san and its derivatives can be made to overcome some of the disadvantages associated with the various administration routes [15]. Vectors can be conju- gated to the polymer to allow for targeted delivery of the biopharmaceutical. These vectors can be biologically active substances (BAS) where the method of conjugation depends on several factors such as the mechanism of thera- peutic effect, the ideal final structure of the biopolymer and the nature of the BAS [118]. Chitosan-based nanoparticles have been used in the transport of peptides, antigens and plasmid DNA among others. Studies have shown that these nanoparticles enhance drug absorption, promote mucosal immunisa- tion and gene expression. Chitosan-hybrids NPs have been used as carriers as these particles protect the entrapped biopharmaceuticals from degradation, improve cellular uptake and can be delivered via different administration routes (oral, nasal and pulmonary) [119]. Triggered release is also an impor- tant point to consider when designing delivery agents. This refers to the release of a BAS in response to stimuli such as pH or temperature changes. Other factors which also affect release of active substances are exposure to redox species, certain biomolecules, ions, electric fields or light [120]. Zhang et al. synthesized a dibenzaldehyde-functionalized chitosan-based hydrogel whose response to various stimuli was investigated [121]. This hydrogel was also used to encapsulate small molecules and proteins whose release could be controlled by exposure to various stimuli [121]. A dual responsive chitosan terpolymer (poly[(2-dimethylamino)ethyl methacrylate] and poly(N-iso- propylacrylamide)) was recently synthesized using a combination of Atom transfer radical polymerization (ATRP) and click chemistry. This polymer reacted to pH and temperature changes making it an ideal candidate for gene/drug delivery and triggered release applications [122]. Targeted drug delivery may also involve bio-imaging. There is a great need for non-invasive imaging methods for the study of pathological con- ditions within the body. Chitosan has been studied as a delivery agent, which can also be utilized for bio-imaging applications. Potara et al. syn- thesized chitosan-silver nanotriangles labelled with p-aminothiophenol. These nanoparticles were utilized as a multi-response contrast agent for

170 Advanced Biomaterials and Biodevices surface-enhanced Raman scattering (SERS) imaging of living cancer cells [123]. Raveendran and colleagues synthesized mauran/chitosan nanopar- ticles which were loaded with the anti-cancer drug 5-fluorouracil and tagged with the fluorescein isothiocyanate. These NPs were non-toxic to normal cells and delivered sustained release of the drug under different pH conditions. Live cellular imaging was possible due to the fluorescent tag present [124]. Lien et al. synthesized a range of O-substituted alkylglyceryl chitosan nanoparticles to be used in transporting biologically active compounds across the blood-brain barrier [125]. A tracer molecule was attached to the nanoparticles and its transport across the barrier was monitored [125]. A novel thermo-gelling injectable nanogel based on chitosan was loaded with ethosuximide for the treatment of epilepsy. The gel was found to have a sustained in vivo release profile which was desirable for this application [126]. Berezin et al. reviewed the utility of chitosan and its derivatives in targeted drug delivery [118]. This review reported on the use of chitosan based compounds such as carboxymethylchitosan, hydroxypropylchito- san, histidine-chitosan and PEG-chitosan for the delivery of conjugated BAS which included doxorubicin, oligodeoxynucleotides and pDNA [118]. Ramesan et al. looked at gene delivery using modified chitosan nanoparti- cles [127]. These modified derivatives which included quaternized, amino acid-conjugated, thiolated, glycosylated, pegylated and phospholipid-con- jugated chitosans were designed to overcome the shortcomings of native chitosan [127]. Modified chitosan in drug delivery was reviewed by Riva et al. this review noted that quaternization and thiolation improves solubility and stability of ionic complexes while the mucoadhesive properties of the polymer increased [128]. The microencapsulation of cells and cell-based drug delivery involving chitosan was reviewed by Wan [129]. Patel and colleagues published an extensive review related to chitosan mediated tar- geted drug delivery systems [130]. Organ-specific delivery systems based on chitosan were discussed together with advances in drug delivery car- riers for cancer therapy through various targeting strategies. The review noted that chitosan is an excellent carrier due to properties such as bio- compatibility, biodegradability and low production costs associated with a renewable resource. Chitosan based carriers can be designed in different dosage forms such as beads, fibres and matrix type tablets. A noteworthy carrier mentioned was chitosan polyelectrolyte complexes utilized for drug delivery [130]. Currently, few chitosan-based delivery systems are in clinical use even though chitosan offers greater advantages compared to the currently used delivery agents such as increased solubility, enhanced absorption,

Chitosan as an Advanced Healthcare Material 171 controlled release and bioadhesive properties. Chitosan offers reactive functional groups, gel-forming properties, high adsorption capacity, bio- degradability and can be prepared as tablets, capsules, gels, membrane films, micro- & nanoparticles, sponges, etc [131]. The lack of technology transfer from academia to industry is an area that requires special focus in order to expedite chitosan innovations through the value chain and into the market. 5.7.4 Antimicrobial Studies Numerous papers have been published reviewing the utility of chitosan and its derivatives in the biomedical sector. These polymers have been applied in a range of products. Chitosan itself possesses inherent antimi- crobial properties where this property is dependent on the DDA, molecu- lar weight, concentration, the species tested, length of the test period, etc. A higher DDA results in a greater number of potentially cationic sites available on the polymer backbone making chitosan more active against bacteria at a lower pH. The mechanism of chitosans antimicrobial activity is yet to be proven. Common theories postulate that the positively charged chitosan binds to the negatively charged components present in the cell membrane of the bacteria (phospholipids, proteins, amino acids) via an electrostatic attrac- tion. Chitosan essentially affects cell membrane permeability leading to the loss of essential intracellular components of the bacterial cell (e.g. glucose and lactate dehydrogenase) which in turn leads to cell death. In addition, it was found that the cytoplasmic membrane of the bacterial cell detached from the cell wall when exposed to chitosan. As a result of chitosan’s metal chelating ability, the polymer is able to bind to metal ions from the bacte- rial intracellular fluid. Certain of these metals are essential for fungi and bacterial growth. This in turn leads to disruption of proper cell function resulting in cell death [132, 133]. Chitosan also interacts with DNA in the cell where it is thought that the polymer may inhibit synthesis of messen- ger RNA and proteins [133]. Selected chitosan derivatives have been tabulated (Table 5.2) along with organisms which they are active against. Micro- and nanoparticles of chitosan have also been proven to have antibacterial activity. To further improve this activity, certain metal ions with known antibacterial action (e.g. Ag+, Zn2+, Mg2+, Cu2+) have been used in conjunction with polymers. Nanoparticles loaded with these metal ions showed higher activity against E. coli, Salmonella choleraesuis, Salmonella typhimurium and S. aureus which suggests that the antibacterial activity is

172 Advanced Biomaterials and Biodevices Table 5.2 Selected chitosan derivatives and their associated antimicrobial activity. Polymer Structure Active against Chitosan Gram-negative bacteria (e.g. E. coli, P aeruginosa, S typhimunium, P fluorescens and Vibrio parahaemolyticus, Gram-positive bacteria S. aureus, S simulans, Listeria monocytogens, Bacillus megaterium, Lactobacillus plantarum, Lactobacillus bulgaricus and Bacillus cereus, fungi (Ramularia cercosporelloides, Aspergillus niger, Aspergillus parasiticus, Altemaria altenata, Botrytis cinerea, Colletrotichum gloeosporioides, Rhizopus stolonifer, Sclerotinia sclerotiorum, Rhizoctenia Solana) and has antiviral activity against certain viruses [132] 6-Deoxy-amino S. aureus, E. coli, P. aeruginosa, chitosan A. niger [134] N-acetylcysteinyl E. coli, Staphylococcus epider- chitosan midis, Streptococcus pneumonia, Haemophilus influenza, Moraxella catarrhalis Staphylococcus wamen, B. cereus, Acinetobacter cloacae, klebsiella pneumoniae. [135, 136–139]

Chitosan as an Advanced Healthcare Material 173 Polymer Structure Active against 3-trimethylammo- E. coli, S. aureus, P. aeruginosa, nium-2- B. subtilis, Aspergillus niveus hydroxypropyl- [140, 141] N-chitosan chloride Carboxymethyl E. coli, S. aureus, Fusanium chitosan solani and colletotrichum Lindemuthianum, A. flavus, Aspergillus parasiticus, Saprolegnia parasitica [142] related to the zeta potential of the compound. The most commonly studied metal used together with chitosan, is Ag. Silver nanoparticles coated with chitosan are a well-studied topic with applications in the biomedical sector being the main focus [133]. It has been shown that bacterial DNA is unable to replicate after exposure to Ag and changes have occurred in the cell membrane of the bacteria. In addition, the bactericidal effect of Ag leads to cell death [143]. Dutta et al. reviewed the antimicrobial activities of chitin, chitosan and chitosan oligosaccharides with the aim of applying these polymers in food applications [144]. Islam and colleagues reviewed green chemistry approaches in the development of antimicrobial textiles based on biopoly- mers [145]. This review noted that chitosan had great potential in a broad range of scientific areas due to its favourable properties [145]. Cota-Arriola et al. reviewed controlled release matrices based on micro/nanoparticles of chitosan with antimicrobial potential [133]. This review focused on the development of approaches for microbial control in agriculture [133]. Upadhyaya and co-workers looked at the water soluble carboxymethyl chitosan and its biomedical applications [146]. This polymer has a greater

174 Advanced Biomaterials and Biodevices antibacterial activity against E. coli compared to native chitosan and has been applied in drug delivery, bioimaging, biosensors and gene therapy [146]. The review by de Britto et al. focused on quaternary ammonium salts of chitosan, these salts are particularly useful since they are water soluble and have increased antimicrobial effects [147]. Common quater- nary ammonium salts are trimethyl chitosan chloride (TMC) and 3-tri- methylammonium-2-hydroxypropyl-N-chitosan chloride. These polymers have been applied in a variety of applications and have proven antibac- terial and antifungal activity [147]. Li and co-workers modified chitosan films with antimicrobial N-halamine, these films were subsequently chlori- nated to confer biocidal properties [148]. The films were found to be effec- tive against S. aureus and E. coli within 5–10 minutes after exposure. The authors suggest that these films have the potential to be applied as wound dressings, coatings for medical devices and food packaging [148]. A chito- san ethylene co-polymer (methyl acrylate and vinyl acetate) synthesized by Massouda et al. was found to be effective against E. coli, Salmonella enterica and L. monocytogenes making it a strong candidate for antimicrobial pack- aging [149]. Dilamian et al. produced electrospun membranes of chito- san/poly(ethylene oxide) in the presence of the broad spectrum antiseptic poly(hexamethylene biguanide) hydrochloride [150]. These nanofibres inhibited the growth of E. coli and S. aureus making these fibres good can- didates for biomaterials [150]. Similarly, Fouda and co-workers synthesized carboxymethyl chitosan/poly(ethylene oxide) nanofibres embedded with Ag nanoparticles [151]. These fibres were tested against S. aureus, P. aeru- ginosa, E. coli and Candida albicans and results showed that higher anti- microbial activity was obtained when nanofibres with silver nanoparticles (AgNPs) was tested [151]. There are currently a wide variety of bandages based on chitosan which claim antimicrobial effects one such example are the wound care products marketed by Hemcon. The company recently published a report on the antibacterial properties of their products where a wider range of bacteria was tested and shown to be inhibited by chitosan. A wide variety of products are on offer which includes haemostatic dressings, gels, nasal plugs and dental dressings to mention a few [152]. Other prod- ucts being marketed include the antimicrobial textile fibre Crabyon® [96]. Patents and applications of chitosan and its derivatives are based on the above research. New applications are being reported on a regular basis making chitosan a highly sought after commodity. The future of antimi- crobial chitosan technology looks promising. Based on the amount and continued increase in patented innovations, chitosan and its derivatives are poised to deliver on its untapped commercial potential.

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6 Chitosan and Low Molecular Weight Chitosan: Biological and Biomedical Applications Nazma N. Inamdar1 and Vishnukant Mourya2,* 1Government College of Pharmacy, Osmanpura, Aurangabad, India 2Government College of Pharmacy, Kathora Naka, Amravati, India Abstract The biorenewable polysaccharides chitin and chitosan are currently being explored intensively for their applications, especially in pharmaceutical, bio- medical, and biotechnological field. These are aminoglucopyrans composed of N-acetylglucosamine and glucosamine residues. The insolubility of chitin in most of the commonly used solvents has led to use of chitosan. The insolubility of chito- san, except in the aqueous acidic media, has prompted its derivitaztion and depo- lymerization to low molecular weight chitosans and chitooligosachharide. These polymers have emerged as a new class of physiological materials of highly sophisti- cated functions due to their versatile biological activity, excellent biocompatibility and complete biodegradability in combination with low toxicity. Noteworthy num- ber of reports related to variety of other biological activities is available. The most important of the array of biological activities are the antimicrobial, hemostatic and wound healing actions which have already been commercialised to some extent. The immunological and antioxidant activities of chitosan are particularly interest- ing and contribute to potentially very important applications of this polymer in the treatment of various tumoral afflictions and in the treatment of several pathologies of viral origin. The immunoadjuvant properties of chitosan are keenly observed for their possible use in vaccination. Since the chitosan can be degraded in living cell media, the question remains whether the biological activities are due to the mono- mer and oligomer or directly due to the polymer. There is a need to understand the mechanisms underlying these activities and to exploit the actions by chemical *Corresponding author: [email protected] Ashutosh Tiwari and Anis N. Nordin (eds.) Advanced Biomaterials and Biodevices, (183–242) 2014 © Scrivener Publishing LLC 183

184 Advanced Biomaterials and Biodevices derivatization. Chitosan-based oligomers and derivatives may assume significant biological roles. The role of chitosan as a constituent of the composites for different biomedical applications, based on its bioactivities and physicochemical proper- ties are too getting noticed and appreciated. These include non-viral gene vectors, carriers of drugs, biospecific sorbents, scaffold for tissue engineering, as enzyme immobilizing system etc. With this review, it can safely be said that, in the near future, biomedical products based on chitomaterial will get their due respect. Keywords: Chitosan, low molecular weight chitosan, chitooligosaccharide, biological action, biomedical applications, antimicrobial, immune modulator, adjuvant, antioxidant, haemostatic, lipid lowering 6.1 Introduction Carbohydrate polymers exert a variety of biological actions in modulat- ing the intra and extracellular environment. The look over of the literature related to chitin/chitosan is proof enough to substantiate the claim. Chitin is a linear cationic heteropolymer of randomly distributed 2-N-acetyl- 2-deoxy-glucose (N-acetylglucosamine) and 2-amino-2-deoxy-glucose (glucosamine) residues with β-1,4-linkage, mostly derived from the exo- skeleton of crustaceans [1]. Depending on the source and preparation procedure the charecteristics of chitn varies. In chitin, the number of glu- cosamine residues present in a molecule, denoted as the degree of deacety- lation (DDA), ranges from 5–10 % and the molecular weight (MW) of this linear polysaccharide can be as high as 1–2 × 106 Da, corresponding to a degree of polymerization (DP) of ca. 5000–10,000. Chitosan is a product derived from alkaline N-deacetylation of chitin. In chitosan, DDA is above 60 % and the MW ranges from 2000 Da (oligomers) to 104–2 × 106 Da [2, 3]. Chitin is insoluble in water and almost all commonly used organic solvents. Chitosan, in its crystalline form, is insoluble in aqueous solutions above pH 7; however, in dilute acids (pH<6.0), the protonated free amino groups on glucosamine facilitate solubility of the molecule. These unique polymers display a set of biological properties as biodegradability, biocom- patibility, non-toxicity and an array of pharmacological properties. 6.2 Biodegradability of Chitin and Chitosan The vast amount of chitin released into the seas by molting processes and dead chitinous organisms (so called marine snow) does not accumulate in this biosphere because of its degradation by microorganisms [4]. The

Chitosan and Low Molecular Weight Chitosan 185 wide variety of enzymes is involved in chitin and chitosan degradation, including endo- and exo-chitinases, chitosanases, chitodextrinases, chi- totriosidases, chitobiosidases, N-acetylhexosaminidases and enzymes of the terminal metabolism of N-acetyl glucosamine and glucosamine [5]. The occurrence of enzymes catalysing degradation of chitin chitosan has been found not only in microorganisms, but also in plants, arthropods and mammals. Chitinase activity has been discovered in human serum and the enzyme, as well as its production by cloning, is patented [6, 7]. Evidences collected suggests that significant portions of chitin-based dressings are depolymerised and that oligomers are further hydrolysed to N-acetylglucosamine, a common aminosugar in the body which either enters the innate metabolic pathway to be incorporated into glycoproteins or is excreted as carbon dioxide [8]. Lysozyme is the primary enzyme responsible for in vivo degradation of chitosan through hydrolysis of acetylated residues. Lysozyme may be mainly responsible for chitosan degradation in serum, but may not be the only enzyme involved in vivo degradation [9, 10]. The mechanism of deg- radation by these enzymes is not clearly elucidated. The biodegradation of chitosan is a phenomenon dependent on the several factors, especially DDA, MW and degree of crystallinity. The lower MW chitosans exert faster degradation in comparison to high MW chito- sans [11]. The in vitro and in vivo degradation rate of chitosan is inversely related to the DDA [12, 13]., In vitro studies showed that the chitosan with a DDA of 97 % was not degraded at all, while that with a 50 % degraded with maximum rate [14]. Another study found that the chitosans with DDA from 30–70 % were degraded well (above 50 %) within 4 weeks, whereas, the degradation of samples with very low or high deacetylation was minimal over this period [15]. In addition, it seems that at least three consecutive N-acetylated residues are necessary to be recognised by the enzyme [16]. The crystallinity of chitosan depends on DDA; so the rate of degradation, in turn, depends on crystallinity [14, 17]. Susceptibility of β-chitin to lysozyme was found to be much more than that of α-chitin due to weak intermolecular forces. In addition, the water content, the shape and the state of surface of the material, also influences biodegrada- tion [18, 19]. Chemically modified chitosans are also endowed with sus- ceptibility to lysozyme in vivo. It depends on the substitution on nitrogen of glucosamine unit and/or on various substituents at the 3-O, 6-O- posi- tion of the glucosamine and N-acetylglucosamine residues [20, 21]. Chitosan is susceptible to enzymatic degradation by other non-specific enzymes from a variety of sources such as cellulases [22], hemicellulases [23], proteases [24], lipases [25] and collagenases [26].

186 Advanced Biomaterials and Biodevices 6.3 Biocomapatibility and Toxicology of Chitin and Chitosan The low toxicity profile of chitosan compared with other natural polysac- charides is another of its many attractive features. Chitin is a normal con- stituent of sea food and mushrooms, both of which are considered to be non-toxic and safe for human consumption, except for a very small per- centage of the population that is allergic to seafood products, due to the presence of proteinaceous impurities [27]. High quality chitin and chito- san, after complete removal of toxic and contaminating bioburden as pro- teins, heavy metals and pyrogens, are biocompatible and safe for use. US FDA has proclaimed chitosan to be Generally Recognised As Safe (GRAS), a designation used to indicate that a chemical or substance added to foods and beverages is considered safe. The safety in terms of inertness and low or no toxicity has been dem- onstrated by in vivo toxicity studies. Its oral LD50 (median lethal dose) in mice was found to be in excess of 16 g/kg/day, which is higher than that of sucrose [28]. In the acute toxicity test, the LD50 was reported as over 15 g/ kg orally in rats, over 10 g/kg subcutaneously in mice, 5.2 g/kg intraperito- neally in mice and 3.0 g/kg in rats [29]. An extensive toxicological study with PROTASANTM UP (chitosan glu- tamate, ultrapure DDA 83 %) (Pronova Biomedical, Oslo, Norway) where administration was done by oral and parenteral routes for assays of hyper- sensitisation, mutagenicity and cytotoxicity, showed its essentially non- toxic nature in rats (see Table 6.1) [30]. Intravenous injections of chitosan oligomers (DP 2–8) in rabbits at low dose (4.5 mg/kg) or higher dose (7.1–8.6 mg/kg/day) did not cause any noticeable physiological symptoms, except increased hexosamine value and lysozyme activity in blood, returning to normal on cessation of treat- ment [31] At relatively high doses, deleterious effects were found in dogs Table 6.1 Toxicology of chitosan glutamate (protasantm up) in rats [30]. Application No toxic effect seen at Application for Oral Up to 600 mg/kg /day 13 weeks Intravenous Up to 25 mg/kg Once Intraperitoneal Bolus injection, up to 500 mg/kg 7 days Nasal mucosa Up to 3 mg per rat per day 7 days

Chitosan and Low Molecular Weight Chitosan 187 on subcutaneous administration (10–200 mg/kg) [32]. Anorexia and mortality were observed in dogs given doses above 50 mg/kg and above 150 mg/kg, respectively. From the findings of autopsy, severe hemorrhagic pneumonia was observed in all dead dogs. In hematologic findings, leu- kocytosis and increase of serum LDH-2 and LDH-3 isoenzymes were characteristic. With clinical trials of dietetic applications of chitosan, its effects on oral administration have been widely reported. To review effects of inter- vention on outcome of adverse effects, Jull, et al., reviewed combined data from the 13 trials [33]. It showed that there were no clear differ- ences between intervention and control groups in terms of frequency of adverse events. Common side effects reported in most trials included constipation, nausea, bloating, indigestion and abdominal pain, but only two studies found that these were significantly increased in participants taking chitosan. In addition to general side effects, some trials monitored changes in blood chemistry or fat-soluble vitamin levels, but none found any significant effect of chitosan on these parameters. However, Tanaka and colleagues cautioned that special care should be taken in the clinical use of chitosan over a long period of time, due to possible disturbances in intestinal microbial flora [34]. Concerns have also been raised that chitosan could cause the loss of fat-soluble vitamins; decrease mineral absorption and bone mineral content and block absorption of certain medicines [35]. The testing of cosmetic preparations of Hydagen CMF on the skin of volunteers revealed favorable properties of high MW chitosan when ery- thema and squamations were evaluated [36]. 6.4 Chitosan as Antimicrobial Agent One of the most unique biological properties of chitosan is its antibac- terial activity. The spectrum of antimicrobial activity of chitosan and its derivatives extends to include filamentous fungi, yeasts and bacte- ria. Many representative examples are given in Table 6.2. The minimum growth inhibitory concentrations of chitosan against Escherichia. coli and Staphylococcus aureus have been 20 ppm, while those against various bac- teria are 10–1000  ppm. But concentrations reported to be required for activity against the same specific target organism can vary up to 500-fold [37]. The vast variability in reported data, relying on in vitro investigations is due to intrinsic factors related to chitosan itself as molecular weight (MW), degree of polymerisation (DP), degree of deacetylation (DDA) and