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Chitosan and Low Molecular Weight Chitosan 189 density of the cell surface as well as changes in the characteristics of chito- san adsorption to the cell wall [41]. The interaction then leads to extensive surface alterations albeit via unknown mechanisms. This alters cell per- meability, further prevents the entry of material or causes the leakage of cytoplasmic constituents and eventually, cell death [42]. Several studies have illustrated the phenomenon by demonstrating leakage using hydrophobic membrane probes or electron microscopy technique. The leakage of glucose and lactate dehydrogenase from E. coli [43] as well as of nucleic acid and proteins [44, 45], of UV-absorbing and proteinaceous materials from A. niger [46], Glycine max and Phaseolus vulgaris [47], of electrolytes from Glycine max and Phaseolus vulgaris [48] have been observed. Electron microscopical examinations of various chitosan-treated micro- organisms do suggest that chitosan’s site of action is indeed at the microbial cell surface. The researchers observed a frayed cell wall [49], cell mem- brane detached from cell wall [50] and even the appearance of protoplasts upon chitosan treatment [51]. The morphological studies of Actinobacillus actinomycetemcomitans exposed to chitooligosaccharide (MW 2–30 kDa with DDA of 91.5 % by SEM, TEM revealed signs of cell membrane disrup- tion [52]. The extra- and intracellular changes included separation of the cytoplasmic membrane from the cell envelope, coagulation of the cytosolic components and disruption of the outer membrane structure with mem- brane sloughing, breaching and blebbing (Figure 6.1 & Figure 6.2). Kumar and colleagues claimed that the binding of chitosan to cell sur- face structures of Bacillus cereus resulted in pore formation[21]. An analo- gous hypothesis was put forward by Young, et al., who suggested that quite (A) (B) Figure 6.1 Scanning electron photomicrograph of A. actinomycetemcomitans cells after treatment with the chitooligosaccharides for 30 min (A) Non-treated cells and (B) treated cells [52].

190 Advanced Biomaterials and Biodevices (A) (B) Figure 6.2 Transmission electron photomicrograph of A. actinomycetemcomitans cells after treatment with the chitooligosaccharides for 30 min(A) Non-treated cells and(B) treated cells [52]. large ‘pores’ can be induced in the plant membrane by chitosan, through displacing cations (such as Ca2+) from complexes that stabilise the cell membrane of Glycine max cells [47, 48]. The nature of the surface components involved in interaction with chi- tosan was rarely accurately defined. While working with plants, Young, et al., suggested that chitosan might bind to polygalacturonate, a component of plant cell walls, thereby increasing the membrane permeability of plant cells [47]. Regarding bacteria, on the other hand, several research groups later hypothesised that an electrostatic interaction takes place between chi- tosan and either (i) negatively charged cell membrane components, i.e., phospholipids or proteins [53] (ii) amino acids in the Gram-positive bac- terial cell wall [24] or (iii) various lipopolysaccharides in the outer mem- brane of Gram-negative bacteria., thereby affecting membrane integrity and permeability. Morimoto, et al., reported the specific binding of a chitosan derivative to a receptor on the cell surface of P. aeruginosa [54]. Raafat, et al., showed that the initial contact between chitosan and the bacterial cell is most probably mediated through electrostatic interaction with the negatively charged teichoic acids of Gram-positive bacteria coupled with a potential extraction of membrane lipids (predominantly lipoteichoic acid) lead to cell death [50]. This finding is consistent with the much lower activ- ity of chitosan against Gram-negative bacteria, because a possible binding of chitosan to LPS would not significantly influence the dynamics of the cell envelope, since these molecules are confined to the outer membrane. It is believed that chitosan’s mode of action is not confined to a single target molecule but that killing results from a sequence of rather ‘untar- geted’ molecular events, taking place simultaneously or successively [44,  50]. Two-step sequential mechanism was reported by Chung, et al., indicating that the inactivation of E. coli by chitosan occured via an initial separation of the cell wall from its cell membrane, followed by destruction of the cell membrane[41]. The mechanism was verified by the similarity

Chitosan and Low Molecular Weight Chitosan 191 between the antibacterial profiles and patterns of chitosan and those of control substances, polymyxin and EDTA. The other proposed mechanism involves the binding of positively charged chitosan with DNA to inhibit its functioning [38, 55]. However, this mechanism is still controversial due to considerations of MW and size. The possibility of chitosan’s direct access to the intracellular targets is unlikely because of its high MW (upto 1000 kDa) and size. It would not normally be able to reach these targets unless it could overcome the bar- rier of the plasma membrane such as by getting hydrolysed to a lower MW polymer. Nakae and Nikaido have reported inablilty of chitosan to pass the outer membrane of Gram-negative bacteria [56]. The chelating activity of chitosan and deprivation of metals, trace ele- ments or essential nutrients limiting the production of toxins, growth of microorganisms have often been implicated as a possible modes of action [37, 57]. Otherwise chitosan draws ions as Ca2+ or Mg2+ from the cell wall by its chelating ability [44]. Yet, chelation of metals does not seem to be important for the antibiotic activity of chitosan; in contrast, the forma- tion of complexes with metal ions appears to abrogate this activity [50]. Chitosan is also able to interact with and flocculate proteins, but this action is highly pH-dependent. Moreover, growth inhibition through blockage of nutrient flow has been suggested by several researchers who attribute the antibacterial activity of chitosan to its deposition (stacking) onto the surface of bacteria, thereby impeding mass transfer and suppressing the metabolic activity of bacteria [24, 58, 59]. 6.4.2 Factors Affecting Antimicrobial Activity 6.4.2.1 Molecular Weight (MW) Tanigawa, et al., reported that D-glucosamine hydrochloride (chitosan monomer) did not show any growth inhibition against several bacteria, whereas chitosan was effective [60]. This suggests that the antimicrobial activity of chitosan is related not only to its cationic nature, but also to its chain length and MW. Kendra and Hadwiger examined the antifungal effect of chitosan oligomers on Fusarium solani f. sp. pisi and Fusarium solani f. sp. phaseoli [61]. In the assessment of minimum inhibitory con- centration, the antifungal activity was found to increase as the polymer size increased. Monomer and dimer units did not show any antifungal activ- ity at the concentration of 1000 μg/ml, whereas, heptamer (DP 7) showed maximal antifungal activity and the minimum concentrations were identi- cal for native chitosan and heptamers.

192 Advanced Biomaterials and Biodevices N-acetyl glucosamine oligomers of DP ≥ 30 possess antimicrobial activ- ity against number of Gram-neagtive, Gram-positive and lactic acid bac- teria and low DP oligomers are ineffective [62, 63]. When Tokura, et al., examined the antimicrobial action of chitosan with average MW of 2200, DDA of 0.54 and of 9300, DDA 0.51; they found the inhibition of E.coli growth with stacking of chitosan MW 9300 on the cell wall and the accel- eration of the E. coli. growth with permeation of MW 2200 into the cell wall [59] Apparantly, the smaller chitosan oligomers serve as nutrients for bacteria, whereas, the higher oligomers are toxic. To elucidate the relation- ship between MW of chitosan and its antimicrobial activity against E. coli, the existing experimental data are summarised in Table 6.3. The wide variability in reported data makes it difficult to find a clear correlation between MW and antimicrobial activity even for same tar- geted microorganisms. Generally, the antimicrobial activity against E.coli increases as the MW of chitosan increases. However, the activity decreases over a certain high MW. The discrepancies between data may result from the different DDA and molecular weight distributions (MWD) of chitosan. The evaluation of only the MW dependence of the antimicrobial activity requires a wide MW range of chitosan samples with the same DDA and MWD. It is almost impossible to obtain this because chitosan is a natural polymer. Batch to batch variation occurs and the properties of chitosan are very sensitive to DDA and MWD. Therefore, from the existing data, it is Table 6.3 Effect of chitosan MW on its antimicrobial activity against E. coli. Ref Effectiveness, MW in Da, (DDA) Hwang [45] 29,800 (0.93)>102,200 (0.93)>9,800 (0.96)>174,700 (0.94) Liu [38] 91,600 (0.86)>51,100 (0.88)>8,000 (0.75)>5,000 (0.73), 274,000 (0.74)>650,000 (0.85), 1,080,000 (0.85) Jeon [63] 685,000 (0.89) ≥24,000−7,000>6,000–1,500>~1,000 Ueno [64] 10,500, 9,300>8,000, 7,300>6,200>5,500>4,100, 2,200 Tanigawa [60] 80,000 (0.80)>166,000 (0.91)>190,000 (0.84)>2,000 (<0.80)>4,000 (<0.80)>12,000, 8,000 (<0.80) Chang [65] 35,000>29,000>32,000>97,000, 95,000, 68,000>293,000, 275,000>820,000, 11,000 Chen [66] 600,000 (0.9) ≥ 600,00 (0.9) >600,000 (0.7) ≥ 600,00(0.7) > 600,000 (0.5) ≥ 600,00 (0.5)

Chitosan and Low Molecular Weight Chitosan 193 difficult to determine the most optimal MW for the maximal antimicrobial activity. The antimicrobial activity of chitosan varies depending on the micro- organisms tested. The relationship between MW of chitosan and the anti- microbial activity too is affected by the test organisms. Shimojoh, et al., treated several oral bacteria with the same concentration of chitosans of different molecular weights [67]. It was found that the chitosan with MW 2,20,000 was most effective and MW 10,000 was the least effective in their bactericidal activities. The antimicrobial activity of chitosan with MW of 70,000 was better than MW of 4,26,000 for some bacteria, but for the oth- ers, the effectiveness was reversed. Yalpani, et al., reported that chitosans (medium and high MW) showed higher antimicrobial activities against Bacillus circulans than chitooligosaccharides (DP 2–30), whereas they were less effective against E. coli than chitooligosaccharides [68]. A posi- tive correlation between the MW of chitosan and its activity against the Gram-positive bacteria, Staph. aureus, and the negative correlation for Gram-negative bacteria has been reported by Zheng and Zhu also [69]. Similar trends were reflected in inspection of antimicrobial activity by Atomic Force Microscopy (AFM), where specifically, cell lysis, surface roughening and cell clustering were observed [70]. In the case of E. coli, AFM results displayed clustering due to the ionic interaction between chi- tosan oligosaccharide and cell wall (or due to the production of extracel- lular polymeric substance). This apparent response strategy presumably protected the bacilli in the interior of the clusters from the action of chito- san oligosaccharide and led to only a short-lived effect on the cell-viability, i.e., bacteriostatic action. However, for the high-MW chitosan, the poly- mer prevented this clustering mechanism (possibly due to comparatively lower ionic influence v/s the chitosan oligosaccharide); keeping bacilli iso- lated from each other and consequently was a more effective antibacterial agent. AFM images for Staph. aureus showed much less intense cellular morphological changes on chitosan and oligosaccharide treatment than for E. coli. This was in accordance to presence of much thicker peptidogly- can layer of the cell wall of Gram-positive bacteria; but nanoindentation studies revealed that even so, the cells were weakened by treatment with the chitooligosaccharides. It has been proven that lower MW leads to longer persistence length (PL) at the same deacetylation degree [71, 72] and the persistence length decides whether the chitosan molecule will penetrate into the bacteria cell or not. Scherrer and Gerhardt found that the minimum persistence length to pass the cell wall of Gram-positive bacteria is 11Ǻ [73]. Decad and Nikaido found that the minimum persistence length to pass the cell wall of Gram-negative

194 Advanced Biomaterials and Biodevices bacteria is only 5 Ǻ [74]. For the same purpose Chen et al., reported the persistence length range from 8 to 12 Ǻ [66]. This proves that the chitosan with higher MW (shorter persistence length) can easily pass through the cell wall of Gram-positive species, but it is blocked outside of the cell wall of the Gram-negative species. Hence, the effect of MW of chitosan is significant on the growth inhibition ability of Gram-positive species. The effect of MW and antibacterial activity is said to be dependent on the concentration range used [75]. Different MW chitosans (55 to 155 kDa) with the same degree of deacetylation (80 % ± 0.29), were investigated for antimicrobial activities against E. coli. All of the chitosan samples with MW from 55–155 kDa had antimicrobial activities at the concentrations higher than 200 ppm. The antibacterial activity of chitosan had relation- ship to the MW at the concentration range from 50–100 ppm. At a lower concentration (<0.2 mg/mL), the polycationic chitosan does probably bind to the negatively charged bacterial surface to cause agglutination, while at higher concentrations, the larger number of positive charges may have imparted a net positive charge to the bacterial surfaces to keep them in suspension [58, 76]. 6.4.2.2 Degree of Deacetylation (DDA) The antimicrobial activity of chitosan is directly proportional to the DDA of chitosan [38, 60, 65, 77]. Higher DDA of chitosan brings more positive charges and the positive charges will interact with the negatively charged bacteria. So, a higher DDA of chitosan causes higher growth inhibition activity. However, higher DDA also results in longer persistence length that hinders chitosan penetrating through the cell wall due to the more positive charges and increased intermolecular electric repulsion [78]. In vitro and in silico studies supplement the observations related to MW and DDA with respect to the mechanism of surface interference [79]. The in vitro studies of chitosan- lipopolysaccharide (LPS) interactions using ligand-enzyme solid phase assay were carried out for high MW chitosans (80 kD) of different degree of acetylation, low MW chitosan (15 kD), acyl- ated oligochitosan (5.5 kD) and chitooligosaccharides (biose, triose and tetraose). The LPS-binding activity of chitosans (80 kD) increased with increase in degree of deacetylation. Activity of N-monoacylated chitooli- gosaccharides was in the order- oligochitosan >tetra> tri> disaccharides. In silico studies of the three-dimensional structures of complexes of LPS and chitosans was performed by molecular modeling with MOE software pack- age (Molecular Operating Environment, http://www.chemcomp.com/) and its docking module (FlexX). The number of bonds stabilising the complexes

Chitosan and Low Molecular Weight Chitosan 195 and the energy of LPS binding with chitosans decreased with increase in acetate group content in chitosans and resulted in changing of binding sites. It was also observed that binding sites of chitooligosaccharides on LPS over- lapped and chitooligosaccharide binding energies increased with increase in number of monosaccharide residues in chitosan molecules. 6.4.2.3 pH The antimicrobial activity of chitosan is strongly affected by pH [47, 58, 71]. The antibacterial property is observed at acidic pH since at that pH, chitosan will be protonated [80]. Tsai and Su examined the antimicrobial activity of chitosan (DDA 0.98) against E. coli at different pH values of 5.0, 6.0, 7.0, 8.0 and 9.0 [43]. The greatest activity was observed at pH 5.0. The activity decreased as the pH increased, and chitosan had little antibacterial activity at pH 9.0. Other researchers reported that chitosan had no anti- microbial activity at pH 7.0 due to the deprotonation of amino groups and poor solubility in water at pH 7 [43, 58]. 6.4.2.4 Cations and Polyanions Results regarding the effect of ionic strength on chitosan’s antibacterial activ- ity are still contradictory. While Chung, et al. [41], proposed that higher ionic strength might enhance the solubility of chitosan and thus increase its anti- bacterial activity, regardless of the test strain, Tsai and Su [43] suggested that the presence of sodium ions (100 mM) reduced chitosan’s activity against E. coli. Raafat however, observed no detectable effect of NaCl (10 or 25 mM) on the antimicrobial activity of chitosan against several indicator strains [81]. The divalent cations at concentrations of 10 and 25 mM reduced the antibac- terial activity of shrimp chitosan against E. coli in the order of Ba2+, Ca2+ and Mg2+ [43]. Furthermore, the addition of Zn2+ ions inhibited the antibacterial activity of 0.2 M acetic acid-chitosan solution the most, compared to Ba2+, Ca2+ and Mg2+ ions [41]. Analogous findings have been made for plant cells (Glycine max), where chitosan-induced permeability changes were strongly inhibited by divalent cations in the order Ba2+>Ca2+>Sr2+>Mg2+>Na+>K+ [47]It was assumed that the cations displaced Ca2+ released from the cell sur- face, form complexes stabilising the cell membrane and consequently reduce the chitosan-induced leakage. However, Takanori, et al [80] and Chung, et al [41] claimed that the pH, rather than metal ion concentration, is more important in antibacterial activity. The polyanions like Na-polygalacturonate and Na-poly-L-aspartate, but not the monomeric galacturonate and aspartate, prevented the effect of chitosan on plant cell. The explanation of this provided was, that individual

196 Advanced Biomaterials and Biodevices ionic bonds between anionic monomers and polycations could dissociate, but the multiple bonds between polyanion and polycation would not dis- sociate at the same time [47]. 6.4.2.5 Temperature Tsai and Su examined the effect of temperature on the antibacterial activ- ity of chitosan against E. coli. The cell suspensions in phosphate buffer (pH 6.0) containing 150 ppm chitosan were incubated at 4, 15, 25, and 37 °C for various time intervals, and the surviving cells were counted. The antibacterial activity was found to be directly proportional to the temperature. At temper- atures of 25°C and 37°C, the E. coli cells were completely killed within 5 h and 1 h respectively. However, at lower temperatures (4°C and 15oC), the number of E. coli declined within the first 5 h and then got stabilised. The antibacte- rial activity was found to be directly proportional to the temperature [43]. The temperature and duration for which chitosan solutions are stored, affect the antibacterial activity [82]. Antibacterial activity of chitosan solutions (1 % (w/v) in 1 % (v/v) acetic acid) before and after 15-week storage differed, depending on the MW of chitosans, the storage temperature and the bacte- ria. In general, chitosan solutions before storage showed higher antibacterial activity than those after 15-week storage. Chitosan solutions stored at 25°C possessed similar or weaker antibacterial activity compared to those at 4°C. In sum, the findings, especially related to MD, DDA and pH, are con- sistent with the above-mentioned hypothesis of cell envelop disturbance. The test organism-related factors too boost this hypothesis. Mutants of Salmonella typhimurium, with strongly reduced negative cell surface charge, were found to be more resistant to chitosan than the parent strans [40]. The compatible data is provided with use of Staph. aureus mutants displaying different overall cell surface charges [50]. Various derivatives of chitosan have therefore been investigated as potential substitutes for chitosan with enhanced properties. The most pop- ular derivatives include those that comprise acidic (anionic) or quaternary ammonium (cationic) moieties on the polymer backbone [83]. 6.5 Chitosan as Haemostatic Agent Probably one of the most prominent commercial applications of chitosan is its use as a hemostatic and wound healing agent. In vitro, the hemostatic properties of chitosan have been observed even in severe anticoagulating conditions and in cases of abnormal activity of platelets [84, 85].

Chitosan and Low Molecular Weight Chitosan 197 Hemostasis depends on the successful balance between coagulation, com- plementary and fibrinolytic pathways, with complex interactions between plasma proteins, blood cells, blood vessel endothelium, as well as blood flow and viscosity [86]. The hemostatic activity of chitosan may not depend on any part of the normal blood coagulation cascade. Many investigators indi- cate that the red blood cells in blood are activated to form the coagulum, on being induced by chitosan. There appears to be an electrostatic interaction between the cell membrane of erythrocytes (negative charge) and chitosan (positive charge) [87–89]. The agglutination of red blood cells induced by chitosan may be thought of as a crosslinking of these cells, as they are bound together by chitosan polymer chains. Therefore, the hemostatic activity of chitosan is dependent on molecular weight, degree of deacetylation, and/or other characteristics of chitosan, particularly its polycationic properties inter- fering with negatively charged molecules at the cell surface. (Table 6.4). On Table 6.4 Characteristics of chitosan studied for hemostatic action. Chitosan physical form MW (Da), DDA (%) Ref Filament composite 150,000–200,000 (94) [95] Coating 240,000 (90) [96] Coating 800,000 (46) [97] Coating 50,000 (>90) [88] Coating 900,000 [ 84] Film 1,200,000 [98] Film 600,000 (85) [99] Film – (80) [100] Solution 800,000 –1,500,000 [101] Hydrogel 800,000 –1,000,000 (80) [102] Hydrogel 43,000 (85) [103] Sponge 10,500 (85) [104] Powder – (90) [105] Film (N-acyl chitosan) 125.7 × 104 to 66.9 × 104 (17.5 – 47.3 [21] % acylation)

198 Advanced Biomaterials and Biodevices the effect of weight of chitosan molecule on blood coagulation, the review by Whang et al., mentioned that low molecular weight chitosans were unable to initiate firm coagulum formation [90]. The in vivo studies carried out by Hirano, et al., showed that more blood coagulum is formed on suture surfaces coated with fully deacetylated chitosan rather than on sutures impregnated with acetylated chitosan [91]. The recent studies by Fischer by electrophoretic and Western blot analysis of red blood cell surface proteins demonstrated that chitin microfibers were bound to band three of the red blood cells. However, they stated that the interaction resulted in the activation of the intrinsic coag- ulation cascade associated with the presentation of phosphatidylserine on the outer layer of the surface membrane of nanofiber-bound red blood cells. The results demonstrated that red blood cells play a direct and important role in achieving surface hemostasis by accelerating the generation of thrombin [92]. Chitosan also mediates platelet aggregation [93]. Study of the mecha- nism for this phenomenon by Chou, et al., demonstrated that chitosan is an effective inducer of rabbit platelet adhesion and aggregation [88]. The potent platelet aggregation induced by chitosan was proportional to the concentration of platelets in the plasma [94]. The relationship was investigated by Fischer, et al., between confor- mation of chitins and activation of hemostasis, including SyvekPatch , whose chitin fibers are organised in a parallel tertiary structure that can be chemically modified to an antiparallel one; and hydrogels consisting of either partially or fully deacetylated daughter chitosans [106]. Several studies were performed on the said chitosans, including, (1) an analysis of the ability of chitosans to activate platelets and turnover of the intrinsic coagulation cascade, (2) an examination of the viscoelastic properties of mixtures of platelet-rich plasma and chitosans via thrombo-elastography and (3) scanning electron microscopy to examine the morphology of the chitosans. The haemostatic responses to the chitosans were highly depen- dent on their chemical nature and tertiary/quaternary structure, while the microalgal chitin fibers were found to have superior hemostatic activity compared to the other chitosans. The action of chitosan on blood could be modulated by formation of its sulfate esters which chemically behave as heparin-like substances. 6.6 Chitosan as Immunity Modulator The immunological activity of chitosan is particularly interesting and con- tributes to potentially very important applications of this polymer in the treatment of various tumoral afflictions and in the treatment of several

Chitosan and Low Molecular Weight Chitosan 199 pathologies of viral origin. Since chitosan can be degraded in living cell media, the question remains whether the biological activity is due to the monomer and oligomer or directly due to the polymer. It seems that both kinds of involvements must be considered. Immune-stimulating function of chitosan oligosaccharide is condi- tioned by the similarity of its molecular structure to that of cell membrane. On the basis of this hypothesis, the immune stimulatory activity of chi- tin and chitin derivatives was extensively explored in the middle to late 1980s especially with the oligomers. The water of soluble hexa-N-acetyl- chitohexaose, the hexamer of N-acetylglucosamine, and chitohexaose, the hexamer of glucosamine, at 100×5 mg/kg dosage, gave complete regres- sion of solid tumors in all mice observed [107]. Besides growth inhibitory effect against solid Meth-A tumors N-acetylchitohexose was also found to display antimetastatic effects against Lewis lung carcinoma transplanted into mice, giving rise to 40–50 % inhibition ratio of pulmonary metasta- sis when administered intravenously (1mg/g) on day 6 after implantation [108]. This oligosaccharide was also shown to enhance the tumorocidal effect of splenic T-lymphocytes against mastocytoma cells and to increase the NK activity of splenic T-lymphocytes [109]. Protection against infec- tion by Pseudomonas aeruginosa, Listeria monocytogenes, and Candida albicans was also noticed in tumor-bearing mice [110–112]. The triggering of the defense functions of macrophages, polymorphonuclear leukocytes, cytotoxic and NK cells activity was proposed and evidenced as mode of action [113]. Immunological activities of higher chitin derivatives were evaluated where, deacetylated chitin derivatives such as 70 % deacetylated chitin and 30 % deacetylated chitin among the test derivatives were potent immuno- logical activators of murine peritoneal macrophages and NK cells in vivo, suppression of Meth-A tumour cells in syngeneic BALB/c mice and stimu- lation of host resistance against Escherichia coli infection in mice [114]. The immunomodulating effect was mainly by stimulation of production of cytokines [115]. Shibata, et al., evaluated the immunological effects of chitin in vivo and in vitro using phagocytosable small (1–10 mm) chitin particles. These studies demonstrated that intravenous administration of fractionated chi- tin particles into the lung activated alveolar macrophages to express cyto- kines such as IL-12, tumor necrosis factor (TNF)-α, and IL-18, leading to INF-γ production mainly by NK cells [116]. Subsequent studies by the same group of investigators demonstrated that cytokine production was mediated by a mannose-receptor-dependent phagocytic process [117]. The mannose receptors also mediated the internalisation of the chitin particles

200 Advanced Biomaterials and Biodevices that were eventually degraded by macrophage lysozyme and N-acetyl-β- glucosaminidase [118] Reese, et al., addressed the in vivo effects of chitin on innate immune cells – macrophages, basophils and eosinophils – and modulation of adaptive type I or type 2 responses [119]. Suzuki, et al., have been investigating the mechanisms of accelerated wound healing by chitin and chitosan for over a decade focusing on com- plement activation by these materials as their major biological effect [120]. Chitin and chitosan both activated complement via the alternative path- way components C3 and C5, but not C4. Chitosan did so more intensely than chitin. The differences in the sensitivity to chitosan between species were found. Chitosan (10 mg/kg) induced an increase of the C3 level in dogs, but not in mice. To attain the same C3 level in mice as in dogs, the dose of chitosan had to be increased five-fold. Regarding the activation of complement chitosans, the team assessed the influence of MW and water solubility of the amino-polysaccharides [121]. A water-soluble mixture of a monomer (D-glucosamine, MW 216) and chitooligosaccharides (2–14 residues) did not activate complement C3. In addition, 50 % homogenously acetylated chitosan (MW 80000), which was soluble in water, also did not activate C3. But the insoluble 50 % heterogeneously acetylated chitosan (MW 80000) caused C3 activation. From these results, it was concluded that the most important characteristic of amino-saccharides-inducing complement activation seemed to be insolubility. The team further inves- tigated non-water soluble chitooligosaccharides (MW: 8800; 14200; 18200; and 33000) for complement activation by the single radial immuno-dif- fusion method and demonstrated that chitosan activates complement in an NH2 group dependent fashion [122]. After activation of C3, C3b was produced and effectively bound to chitosan, while stabilised C3b acted as a binder for factor B. The mechanism of complement activation by chitosan seems to closely resemble that for zymosan, a complement activator, via alternative pathway. The immunomodulatory activities of chitosan and chitosan oligomers envisage its use as possible antitumor and antiviral agent. Low molecular weight chitosan with MW 5–10 kDa and with a DDA 58 % inhibited the growth of Sacroma 180 tumor cells in the mice by intraperitoneal and oral administration [123]. Chitosans of MW 21-kDa, 46-kDa displayed antitu- mor activity in sarcoma 180-bearing mice in dose dependent manner when given by intragastric intubation; on the other hand, 130-kDa water-soluble chitosan had no effect on growth of tumor [124]. Chitosan nanoparticles of size 40, 70 and 100 nm and positive surface charge about 50 mV showed significant antitumor activity in vivo against Sarcoma-180 and mouse hep- atoma H22 and hepatocellular carcinoma cells BEL7402 [125, 126].

Chitosan and Low Molecular Weight Chitosan 201 Other mechanisms proposed to be involved in antitumor activity are apoptosis induction, inhibition of nitric oxide production. Apoptosis induction may occur by elevated caspase-3-like activity (observed in human bladder tumor cells 5637), by Fas signaling pathway (observed in macrophages), by up-regulation of proapoptotic protein Bax (observed in human hepatocellular carcinoma cells SMMC-7721) [127–130]. Activated macrophages by inflammatory agents such as interferon-γ (IFN-γ) and bacterial LPS are known to produce a large quantity of NO as major cytotoxic mediator and inhibit the growth of invading microor- ganisms and tumor cells. It seems chitosan affects nitric oxide production, the process dependent on MW of chitosan used. Chitin (MW 450,000) and chitosan (MW 150,000, DDA 95 % showed a significant inhibitory effect on NO production by the LPS activated murine macrophage cell line, RAW 264.7. Hexa-N-acetylchitohexaose and penta-N-acetylchito- pentaose also inhibited NO production but with less potency. However, N-acetylchitotetraose, N-acetylchitotriose, N-acetylchitobiose and mono- mer of chitin, N-acetylglucosamine and glucosamine had little effect on NO production by the activated cells [131]. The low molecular weight chi- tosan oligosaccharides (MW<1 kDa and MW<10kDa and DDA 90–95 %) inhibited LPS induced NO production, whereas, chitosan of MW 20 kDa inhibited interferon-g induced NO production in these cells [132, 133]. Chitosan of low MW (1 kDa<MW<3 kDa) inhibited cytokine induced NO production in HT-29 cells, chitosan oligosaccharides 10 kDa, 90 % DDA inhibited the glycerol-induced NO production in the proximal tubules and kidney tissue [132, 134]. However, the reports about NO production seem contradictory. Porporatto, et al., reported a moderate induction of NO production by chitosan (MW 50 kDa) in macrophages via activating the inducible nitric oxide synthase and arginase pathways [135]. The high MW chitosan (300 kDa DDA > 90 %) did not affect NO production by itself, but in combination with IFN-g, there was a marked cooperative induc- tion of NO synthesis in a dose-dependent manner in RAW 264.7. The syn- ergy between rIFN-γ and water soluble chitosan was ascribed mainly to chitosan-induced tumor necrosis factor-α and nuclear factor-kB activation [136]. Seo, et al., informed that treatment of macrophages with a com- bination of chitooligosaccharide DP 3–10 and interferon-γ synergically increased NO synthesis and enhanced killing of tumor cells [137]. Some investigators have reported that certain chemically modified chi- tin derivatives, including sulfated and carboxymethylated chitin, inhibited degradation of certain enzymes including collagenase, heparanase and this mechanism was thought to be responsible for the inhibition of tumor cell metastasis [138, 139]. Neutrophil emigration, as occurs in model of acute

202 Advanced Biomaterials and Biodevices respiratory distress syndrome, is comparable to the mechanism of tumor cell metastasis. The sulfated derivatives of chitosan also exhibit antiviral effects. Many plant viral pathogens are inhibited by chitosan [140, 141]. 6.7 Chitosan as Adjuvant This immunostimulating activity along with the structural similarities between chitin derivatives and glucans, an immunoadjuvant class of natu- ral polysaccharides, led several scientists to study the adjuvant capabilities of chitosan. Nishimura, et al., formulated various chitin derivatives with antigen and incomplete Freund’s adjuvant (IFA) to measure adaptive immune responses in guinea pigs and mice [142]. 30 and 70 % deacetylated chi- tins were active adjuvants for the circulating-antibody formation to bac- terial a-amylase and for the induction of delayed type hypersensitivity to azobenzenearsonate-N-acetyl-L-tyrosine. 70 % deacetylated chitin enhanced the helper T-cell functions, the generation of alloreactive cyto- toxic T-lymphocytes, and the activity of natural killer cells in mice. Other derivatives of chitin as carboxymethyl-, hydroxyethyl and dihydroxypro- pyl-chitin showed weaker or no adjuvant activity compared with 70 % deacetylated chitin. In other studies, this chitin derivative induced protection against the Sendai virus, when it was nasally administered prior to virus infection. Macrophages might have played an important role in the induction of this protection [143]. Marcinkiewicz, et al., found that intraperitoneal admin- istration of a water insoluble chitosan suspension enhanced humoral responses, but not cell-mediated immune responses in mice [144]. Subcutaneous administrations of chitosan suspensions were found to be ineffective. Seferian and Martinez found that chitosan particles, formu- lated in an emulsion with antigen, squalene and Pluronic L121, gave a prolonged, high antigen-specific antibody titer and sensitised animals for antigen-specific delayed hypersensitivity responses, following an intraperi- toneal injection [145]. Chitosan particles alone offered no enhancement of an adaptive immune response. Zaharoff, et al., explored chitosan solution as an adjuvant for subcutaneous vaccination of mice with a model protein antigen. Chitosan enhanced anti- gen specific antibody titers over five-fold and antigen-specific splenic CD4+ proliferation over six-fold. Strong increases in antibody titers, together with robust delayed hypersensitivity responses, revealed that chitosan induced both humoral and cell-mediated immune responses. When compared with

Chitosan and Low Molecular Weight Chitosan 203 traditional vaccine adjuvants, chitosan was equipotent to IFA and superior to aluminum hydroxide. Mechanistic studies revealed that chitosan exhibited at least two characteristics that may allow it to function as an immune adjuvant. First, the viscous chitosan solution created an antigen depot. More specifi- cally, less than 9 % of a protein antigen, when delivered in saline, remained at the injection site after 8 hrs. However, more than 60 % of a protein antigen delivered in chitosan remained at the injection site for 7 days. Second, chi- tosan induced a transient 67 % cellular expansion in draining lymph nodes. The expansion peaked between 14 and 21 days after chitosan injection and diminished as the polysaccharide was degraded. The results envisage chito- san as a promising and safe platform for parenteral vaccine delivery. Because of its mucoadhesive properties, chitosan has also been explored as an adjuvant for mucosal vaccination by oral and intranasal route [146, 147]. The mechanisms of vaccine action enhancement by chitosan are believed to be due to both, retention of vaccine in the mucosal passages via mucoadhesion, and opening of endothelial cell junctions for paracellu- lar transport of vaccine. Augmentation in immunogenicity of co-adminis- tered antigens and stimulation of immune system has been widely studied for a variety of antigens and for various routes (Table 6.5). 6.8 Chitosan as Wound Healing Accelerator The history of chitin and its derivatives as wound healing accelerators began with the studies of Prudden, et al [178]. They noticed that the shark cartilage accelerated wound healing and suggested that glucosamine, which is a com- ponent of shark cartilage, functioned mainly as a healing accelerator. Hence, chitosan, a copolymer of N-acetyl-D-glucosamine and D-glucosamine has been scrutinised for the wound healing activity [179–184]. Wound healing process comprises of five overlapping stages that involve complex biochemical and cellular processes. These are described as hae- mostasis, inflammation, migration (epithelial cells and fibroblasts, mac- rophages), proliferation (granulation tissue formation, collagen synthesis, vascular tissue formation, epithelisation) and maturation (remodelling) phases [185]. This involves the fine tuning of complex interactions among cells, extracellular matrix components and signaling compounds. Chitosan accelerates various activities in the wound healing process, as infiltration of inflammatory cells [186] chemoattraction [187–189], func- tions of polymorph neutrophils [phagocytosis, production of osteopontin [190], tumor necrosis factor-α, interleukin (IL)-1, IL-8, IL-12, leukotriene B4, macrophage inflammatory protein (MIP)-1a and MIP-1b [191–193]

Table 6.5 A few examples of use of chitosan and its derivatives as immune adjuvant. 204 Advanced Biomaterials and Biodevices Vaccine Route, in Chitosan or derivative Used as Ref Diptheria toxoid Oral Chitosan Microparticles [148] <10 mm Influenza Nasal, Human Chitosan glutamate Solution 149] Bordetella pertusis Nasal, Mice Chitosan glutamate Solution [150] (CRM)197 of diphtheria toxin Nasal, Human Chitosan Powder [151, 152] (CRM)197 of diphtheria toxin Intraperitoneal, Chitosan glutamate Solution, Powder [153] Nasal (booster), Ovalbumin BALB/c mice, Chitosan, Solution [154] Guinea pigs 270 kDa, DDA 93% Nasal, 500 kDa, DDA 96 % BALB/c mice Trimethyl chitosan DQ 20, 40 , 60 % Ovalbumin, cholera toxin Nasal, Nanoparticles [155] intraperitoneal, Rat Chitosan 700, 1300, 3000 nm & chitosan-coated [156, 157] Group C Meningococcal 10, 100, 500 kDa Conjugate Vaccine Nasal, BALB/c mice DDA 80 % emulsions Bordetella bronchiseptica In vitro, Chitosan, DDA 94.5% Microparticles dermonecrotoxin RAW264.7 cells Trimethyl chitosan Microspheres [158] Chitosan 4.39 ± 0.68 mm 10, 100 and 300 kDa

Vaccine Route, in Chitosan or derivative Used as Ref Chitosan and Low Molecular Weight Chitosan 205 Bordetella bronchiseptica Nasal, Microspheres [159] BALB/c mice Chitosan 5.23 ± 0.46 dermonecrotoxin 10,100, and 300 kDa, [160] Nasal, Mice DDA 90.8 %. Microspheres Bordetella bronchiseptica 5.73± 0.42 mm [161] dermonecrotoxin In vitro, Mannosylated chitosan RAW264.7 cells 100 kDa; DDA 95.4 % Microspheres [162] Bordetella bronchiseptica DS 5.9 mol %. 5.47mm dermonecrotoxin Nasal, Mice [163] Pegylated chitosan Nanoparticles 0.85±0.03mm [164] Influenza A subunit H3N2 10 kDa, DDA 80.4 % [165] DS 5–8 mol % Nanoparticles [166] Tat toxoid Nasal, Mice Solution Trimethyl chitosan [167] Helicobacter pylori urease and Nasal, intramuscular, 177 kDA, DDA 93 %, [168] muramyl di-peptide BALB/c mice DQ 25 % (Continued) GM-CSF Subcutaneous, Mice Chitosan PEI- DNA complex (hepatitis B) Intramuscular, Chitosan BALB/c mice Chitosan Solution DNA peanut allergen gene Oral, Mice Mannosylated chitosan Microspheres (pCMVArah2) Nasal, BALB/c mice 102 kDa; DDA 85 % 86.6±30.2 nm DS 0.3 Plasmid DNA (Respiratory Chitosan ~390 kDa Nanoparticles syncytial virus) 150–300 nm Chitosan Nanoparticles 300–330 nm

Table 6.5 (Cont.) 206 Advanced Biomaterials and Biodevices Vaccine Route, in Chitosan or derivative Used as Ref [169] Plasmid DNA hepatitis B Nasal, BALB/c mice Chitosan Nanoparticles [170] 400 kD, DDA 85 % 300–400 nm [171] DNA vaccine encoding Pulmonary, Chitosan Nanoparticles 376±59 nm [172] HLA-A*0201-restricted HLA-A2 trangenic mice DDA 93.2 % [173] T-cell epitopes of [174] Mycobacterium tuberculosis [175] [176] PEI- DNA complex (HBsAg) Intramuscular, Mannosylated chitosan Microspheres 200–300 nm BALB/c mice 12 kDa; DDA 85 % [177] Plasmid 11 CpG pig inter- Intramuscular, Chitosan Nanoparticles leukin-6 gene (Pasteurella BALB/c mice 150 kD, DDA 95 % multocida and hog cholera vaccine) Plasmid DNA encoding house Oral, Mice Chitosan Nanoparticles 506.9±9.7 nm dust mite allergen, Der p 1 390 kDa, DDA 83.5 % Plasmid pCR-X8-HBc-CETP Nasal, Rabbit Chitosan 173 kDa Nanoparticles 506.9±9.7 nm Plasmid AAV-tetO-CMV-mEpo Oral, Mice Chitosan 300 kDa Nanoparticles 70–150 nm Replication defective HAdV-5 Nasal, Calves 6O-Glycol chitosan Solution expressing BoHV-1 400 kDa, DS 0.18–0.12 glycoprotein DNA (pcDNA3-VP1) encoding Nasal, BALB/c mice Chitosan Nanoparticles VP1, of Coxsackievirus B3 390 kDa 80–100 nm Chitosan glutamate MW 150–300 kDa, DDA 84 %, DS=Degree of substitution, DQ= Degree of quaternisation

Chitosan and Low Molecular Weight Chitosan 207 activation of macrophages [194] (phagocytosis, production of IL-1 [195, 196], transforming growth factor-a1 and platelet derived growth fac- tor [197], N-acetyl-β-D-glucosaminidase), functions of fibroblasts (pro- duction of IL-8, cytokine-induced neutrophil chemoattractant) [196], complement activation [120, 121], integrin-mediated cell motility, integ- rin-dependent regulation of the pro-angiogenic transcription factor Ets1, angiogenesis [198, 199]. Chitosan also entraps growth factors for accelera- tion of the healing; limitation of scar formation and retraction [200, 201]. Recent evidence points to the DG42 protein, a chito oligomer synthase that during embryogenesis produces chito oligomers acting as primers in the synthesis of hyaluronan [202]. Most preparations of hyaluronan have chito oligomers at their reducing end, that act as templates for hyaluro- nan synthesis and the high concentration of hyaluronan helps in healing the wounds without scar formation [203]. In addition, chitosan’s intrinsic hemostatic and antimicrobial activity assist the wound healing function The inhibitory action of chitosan on enzymes as metalloproteinases may also be beneficial in wound healing, or diseases that involve exces- sive extracellular matrix degradation, mediated by family of these proteins. Chitosan with MW 500 kDa and DDA 0.30 was found to decrease the inva- siveness of human melanoma cells, via, not gene expression inhibition of MMP2, but probably by a post-transcriptional effect as direct non-com- petitive, highly specific molecular interaction [204]. Partially hydrolysed chitosans showed the inhibition of activation and expression of MMP2 in primary human dermal fibroblasts, the highest inhibitory effect being exerted by lower molecular weights (3–5 kDa). The inhibition is caused by the decrease of the gene expression and transcriptional activity of MMP2. It was speculated that the inhibitory effect might be explained by the effec- tive chelating capacity of chitosan for Zn2+ that would become unable to exert correctly its cofactor role in MMP2 [205, 206]. The hydrolysed chito- sans were found to be potent inhibitors of gene and of protein MMP9 also [207–209]. Therefore said chitosans may prevent and treat several health problems mediated by MMP2 (that can hydrolyse the basement membrane collagen IV), such as wound healing and wrinkle formation [210]. The inherent biological properties of chitosan can be potentially improved with derivitisation by carboxymethyl, carboxyethyl and car- boxybutyl groups [211–213]. Carboxymethyl chitosan promoted the pro- liferation of the normal skin fibroblasts significantly, but inhibited the proliferation of keloid fibroblasts, as it reduces the ratio of collagens I/III in keloid fibroblasts, by inhibiting the secretion of collagen type I, while bearing no effect on the secretion of collagens I and III in the normal skin fibroblasts [214–216].

Table 6.6 Some commercial chitin- and chitosan-based bandages and wound dressings. 208 Advanced Biomaterials and Biodevices Product Description Comment Chitin-based Beschitin Unitika Non-woven material manufactured from chitin. Available Favours early granulation, no retractive scar formation. in Japan since 1982. For traumatic wounds, surgical tissue defects SyvekPatch Made of chitin microfibrils from the centric diatom It is claimed to be 7 times faster in achieving haemostasis Marine Polymer Thalassiosira fluviatilis grown under aseptic conditions than fibrin glue, because it agglutinates red blood cells, Technologies activates platelets whose pseudopodia make a robust contact with chitin and promotes fibrin gel formation Chitipack S Sponge-like chitin from squid within the patch, thus acting in a redundant way even Eisai Co. on heparinised patients Dispersed and swollen chitin supported on poly(ethylene Chitipack P therephthalate). For traumatic wounds and surgical tissue defects. Favours Eisai Co early granulation, no retractive scar formation For the treatment of large skin defects. Favours early granulation. Suitable for defects difficult to suture Chitosan-based Chitosan powder with adsorbed elementary iodine For the disinfection and cleaning of wounded skin and for Chitodine IMS surgical dressing ChitoFlex Antibacterial, biocompatible wound dressing designed to be Foe controlling moderate to severe bleeding. It adheres HemCon stuffed into a wound track strongly to tissue surfaces forming a flexible barrier that seals and stabilises the wound surface. Easily removed Chitopack C Eisai with saline or water Cotton-like chitosan obtained by spinning chitosan acetate Complete reconstruction of body tissue, rebuilding of salt into a coagulating bath of ethylene glycol, ice and normal subcutaneous tissue and regular regeneration NaOH; fibers washed with water and methanol of skin

Product Description Comment Chitopoly Fuji Chitosan and polynosic Junlon poly(acrylate) for the manu- Suitable to prevent dermatitis spinning facture of antimicrobial wears Chitosan Skin Hainan Xinlong Non-Wovens A chitosan-based skin substitute Chitoseal Abbott Clo-Sur Scion. A Based on chitosan For bleeding wounds Chitosan and Low Molecular Weight Chitosan 209 Crabyon Non-Wovens Mainly for comfortable sportwear Made of cellulose viscose and chitosan Ohmikenshi HemCon Freeze-dried chitosan acetate salt For emergency use to stop bleeding. Used on battlefields since the time of the Desert Storm expedition in Iraq HemCon The dressing contains chitosan particles that swell while Tegasorb 3M absorbing exudate and producing a soft gel A layer For leg ulcers, sacral wounds, chronic wounds. Reportedly of waterproof Tegaderm film dressing covers the superior to Comfeel and Granuflex TraumaStat hydrocolloid. Ore-Medix Vulnosorb Freeze-dried chitosan containing highly porous silica Tesla-Pharma CELOX Freeze-dried sponge of collagen and chitosan On the European market since 1996 Medtrade Products TraumaStat Based on chitosan nonwovens For moderate to severe hemorrhage and currently used for Ore-Medix Porous silica and chitosan-based hemostatic dressing hemostasis in the emergency and military settings Reportedly superior to HemCon

210 Advanced Biomaterials and Biodevices Photocrosslinkable chitosan, bearing p-azidobenzoic acid and lactobi- onic acid, when crosslinked by UV-irradiation (250W lamp, 2 cm distance, 240–380 nm prevalent 340 nm), resulted in a rubber-like flexible and transparent hydrogel. The hydrogel showed a strong adhesion to tissues, significant induction of wound contraction and acceleration of wound clo- sure and healing [102, 217]. Experimental wound dressings derived from chitins and chitosans are available in the form of hydrogels, xerogels, powders, composites, films, non-wovens, nanofibrils, sponges and scaffolds: the latter are easily colo- nised by human cells in view of the restoration of tissue defects with the advantage of avoiding retractive scar formation. Chitosan’s positive surface charge provides effective ability for promotion of cell attachment, prolifer- ation and growth with retention of the normal cell morphology [218]. The occlusive, semi-permeable chitin film dressings are flexible, soft, transpar- ent and less distressing in removal from wound. These are generally non- absorbent, exhibiting a total weight gain of only 120–160 % in physiological fluid. Dry chitin films transpire water vapour at a rate of about 600 g/m2/24 hr, (similar to commercial polyurethane-based film dressings), that rises to 2400 g/m2/24 hr when wet (higher than the water vapour transmission rate of intact skin) [210] However, pure chitosan films, sponges, have a poor tensile strength and elasticity due to their brittleness. Hence, addition of other polymers is necessary to achieve films with improved strength and elasticity. Wound healing chitosan-based materials tested experimentally include, chitosan-chitin nanofibers [219], Chitosan glycolate-chitin nanofibers [182] chitin nonofibrils-alginate [220], chitosan-polyethyl- ene glycol [221, 222], chitosan-polyvinylpyrrolidone hydrogels [223], Chitosan-polyurethane/poly(N-isopropylacrylamide) thermosensitive membrane [224], semi-interpenetrating polymer networks of chitosan- poloxamer [225], chitosan-polysaccharide composites with corn starch, dextran [226] and polyelectrolyte complexes of chitosan with oppositely charged polyelectrolytes as its own anionic derivatives (sulfated chitosan or N-carboxybutyl chitosan). Chitin nanofibrils in addition to restructur- ing the medium, contribute significantly to effectivity by improving the overall susceptibility to lysozyme and release of chito oligomers. To improve the healing process, chitosan has been combined with a variety of modified materials such as growth factors [227, 228], extracel- lular matrix components as hyaluronic acid [229], heparin [230], collagen [231], gelatin [232, 233], taurine [234], and antibacterial agents as minocy- cline [235], chlorhexidine [182, 229], norfloxacin [236], silver sulfadiazine [237], preformed silver nanoparticles [238], etc.

Chitosan and Low Molecular Weight Chitosan 211 6.9 Chitosan as Lipid Lowering Agent & Dietary Supplement in Aid of Weight Loss Unlike inactive cellulose, chitin and chitosan, as dietary fibers, exhibit hypolipidemic activity. This is confirmed by the reduced cholesterol and triglyceride levels in serum and liver of rats, in a 4 week study in animals kept on diet of 1 % cholesterol + 0.1 % bile salts containing 4 % chito- san [239]. Chitosan was much more effective than chitin. A similar trend was observed by other investigators as well [240]. Chitosan has also been shown to reduce plasma cholesterol in cholesterol-fed broiler chickens at dietary concentrations of 1.5–3.0 % [241, 242]. Sugano, et al., studied the relationship between hypocholesterolemic efficacy and average molecular weight of chitosan in rats fed on cholesterol-enriched diet [243]. At a 5 % dietary level, chitosan almost completely prevented the rise of serum cholesterol level. However, at a 2 % level, chitosan, with viscosity at both the extremes, exerted a comparable cholesterol lowering action. Thus, the hypocholesterolemic action of chitosan was proposed to be independent of its molecular weight. Effect of various different grades of chitosan on faecal excretion of fat in rats fed on a high fat diet was investigated by Deuchi, et al [244]. Chitosan intake resulted in a higher level of fat being excreted by rats receiving corn oil than those receiving lard, although the effect was strong for both diet groups. A supplement of ascorbic acid to each chitosan diet resulted in a significant depression of fat digestion and absorption in the lumen. It was observed that an increase in the viscosity or the degree of deacetylation of chitosan resulted in a pronounced effect on the apparent digestibility of fats. Ormrod, et al., studied effect of chitosan in apolipoprotein E-defficient mice which had hypercholesterolemia, rapid development of atheroscle- rosis and the lesions histologically similar to those seen in humans was observed [245]. Mice were fed for 20 weeks on a diet containing 5 % chi- tosan or on a control diet. Blood cholesterol levels were found to be sig- nificantly lower in chitosan treated mice, and at 20 weeks, were 64 % of control levels. On comparing the aortic plaque in the two groups, a highly significant inhibition of atherogenesis in both the whole aorta (42 %) and the aortic arch (50 %) was observed in the chitosan fed mice, suggesting the possible use of chitosan in inhibiting the development of atheroscle- rosis in individuals with hypercholesterolemia. Kanauchi, et al., attributed chitosan’s ability to inhibit fat digestion to its dissolution in the acidic envi- ronment of the stomach, with a subsequent change to a gel form capable of entrapping fats and oils in the intestine [246, 247]. A synergistic effect

212 Advanced Biomaterials and Biodevices observed with sodium ascorbate was attributed to: (1) a viscosity reduction of the polymer combination in the stomach, thus implying that chitosan mixed with a lipid might be better than chitosan alone; (2) an increase in oil holding capacity of the chitosan gel; and (3) chitosan-fat gel being more flexible and less likely to leak the entrapped fat in the intestinal tract. Three-fold fecal bile acid excretion in acid micelles in toto, with con- sequent assimilation of bile acids, cholesterol, monoglycerides and fatty acids. In rats kept on diets containing 7.5 % chitosan, compared to cellu- lose-fed animals was observed [248]. But in several studies, rats on diets containing 5 % chitosan did not have increased fecal bile acid excretion [249, 250]. However, increased fecal bile acid excretion due to dietry chito- san, has been observed in humans and rabbits [251, 252]. It is reported that, in vitro bile acid binding capacity of chitosan was approximately one-half or equal to that of cholestyramine, a strong synthetic anion exchanger and a hypocholesterolemic agent [250, 253, 254]. Chitosan was compared with cholestyramine and oat gum for its lipid lowering effects and on intestinal morphology in rats. All the three agents lowered liver cholesterol signifi- cantly; chitosan did so without producing any deleterious changes in the intestinal mucosa [255]. Thus prevalent evidence supports a reduction in lipid absorption, by dearth of bile acid, since chitosan engages it by binding. Chitosan, upon reaction with bile acids (cholate, taurocholate, etc.), forms insoluble salts that collect lipids by hydrophobic interaction [256–258]. Yet another explanation for lipid lowering effect is provided by the finding that, in vitro chitosan inhibits pancreatic lipase activity by acting as an alterna- tive substrate [259–261]. Han et al. assessed the effect of chitosan treatment on the activity of pancreatic lipase, in vitro, and on the degree of fat storage induced in mice by oral administration of a high fat diet for 9 weeks [262]. Chitosan prevented the increase in body weight, hyperlipidemia and fatty liver induced by a high fat diet. However, the antiobesity effects of chito- san in high fat treated mice were attributed to the inhibition of intestinal absorption of dietary fat with no effect being observed on pancreatic lipase activity. LeHoux and Grondin investigated the effects of chitosan on plasma and liver cholesterol levels, liver weight and 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, a key regulatory enzyme of choles- terogenesis, in rats fed on a sterol diet (1 % cholesterol and 0.2 % cholic acid) [263]. Chitosan, at a level of 5 %, lowered plasma and liver cholesterol levels by 54 % and 64 %, respectively. High molecular weight chitosan (750 kDa) had less hypocholesterolemic potential than a 70 kDa preparation. Incorporation of chitosan (70 kDa, DA 7.5 %) for 3 weeks, in a sterol diet, completely prevented any decrease in plasma high-density lipoproteins

Chitosan and Low Molecular Weight Chitosan 213 cholesterol, or increase in plasma cholesterol level and liver weight. HMG- CoA reductase activity was 7.7-times more elevated in the sterol chitosan group, than in the sterol group. No significant difference was observed in results obtained at 1, 3 or 6 weeks. It was concluded that chitosan at 7.5 % was able to adequately maintain cholesterol homeostasis in rats despite a greatly increased intake of cholesterol. When discussing the role of chitosan as cholesterol lowering nutriceuti- cal, and considering possible mechanisms for its action, one should keep in mind that animal studies might not be predictive of results in humans because of the presence of chitinases in the digestive systems of many ani- mals, as a point of difference from humans [264]. Chitosan was first shown to reduce serum cholesterol in humans in 1993, when adult males, fed chi- tosan-containing biscuits for 2 weeks (3 g/day for week 1. 6 g/day for week 2), experienced a significant decrease of 6 % in total cholesterol [251]. The subjects also demonstrated a 10 % increase in HDL cholesterol. However, in a 28 day study in overweight subjects, given a daily dose of approxi- mately 0.6 g/day of chitosan, no reduction in total cholesterol was detected [265]. The failure to find any reduction in cholesterol in this study was due to the small quantity of chitosan used. Two studies have reported serum cholesterol reductions with chitosan treatment. A double blind study involving 51 obese women consuming 1.2 g of microcrystalline chitosan for 8 weeks demonstrated significant reductions in LDL, although not total serum cholesterol [266]. Eighty-four female subjects with mild to moder- ate hypercholesterolemia, receiving 1.2 g of chitosan per day, experienced a significant decrease in total serum cholesterol [267]. The results of the animal studies and human trials provide convincing evidence that chitosan is effective in lowering total and LDL cholesterol. Chitosan appears to be active in humans at rather low doses, with as little as 1.2 g per day, produc- ing significant reductions in serum cholesterol. Chitosan is widely promoted and available as dietary supplement to aid weight loss and lower blood cholesterol levels, since chitosan could theo- retically bind ingested fats and oils directly and/or prevent lipid absorption indirectly, via removal of bile acids and other acttions. Numerous clinical trials have been conducted to validate the claim. Jull, et al., reviewed published results of fifteen such studies which lasted for 4–24 weeks, including a total of 1219 participants [33]. Reviews suggest that there is some evidence for chitosan being more effective than placebo in the short-term treatment of overweight and obesity. However, many of the included trials had been of poor quality. Results obtained from high quality trials indicated that the effect of chito- san on body weight was minimal and unlikely to be of clinical significance.

214 Advanced Biomaterials and Biodevices 6.10 Chitosan as Antioxidant Oxidative stress, induced by oxygen radicals, is believed to be a primary factor in various degenerative diseases, as well as in the normal process of aging. Superoxide anion radicals (O2 –), hydroxyl radicals (HO ) and hydrogen peroxide, generated by metabolic processes or from exogenous factors and agents, initiate the oxidative reactions with lipids, proteins, amines, lipoproteins, carbohydrates and DNA resulting in cell damage and diseases [268]. Antioxidants scavenge these free radicals and protect the biological system from oxidative stress. Chitosan has been reported frequently as scavenger of O2 –, HO , peroxyl, carbon and nitrogen centered radicals, as well as metal chelator [269–272]. Different parameters used to report the antioxidant activity include scavenging of O2 –, HO , peroxyl carbon and nitrogen centered radicals AAPH (2,2’-azobis(2-amidinopropane) hydrochloride (C centerd radical), DPPH 1,1’-diphenyl-2-picrylhydrazyl (N-centered), ABTS 2,2’-azino bis(3-ethylbenzthiazoline-6-sulfonic acid (N-centered radical), Barton’s PTOC esters, N-Hydroxy-2-thiopyridone esters of aliphatic and aromatic acids (C- or O- centered radical), AMVN (2, 2’-azobis (2,4-dimethylvaleronitrile, peroxyl radical), H2O2; chelating ability on ferrous ion, copper ion; reducing power; linoleic acid oxidation, electron spin trapping studies of radicals. Water soluble chitosan, as well as lipid soluble chitosan derivatives inhibit lipid peroxidation [273]. Radical scavenging efficacies of different molecular weight bearing chito oligosaccharide (1–3 kDa) were assessed intracellularaly, employing murine melanoma cell B16F1, mouse macro- phages RAW 264.7 [274, 275]. The antioxidant activity of chitosan is impli- cated in preventing the oxidation of human serum albumin by peroxyl radical [276]. Treatment with chitosan for 4 weeks lowered the ratio of oxidised to reduced albumin and increased total plasma antioxidant activ- ity in human volunteers [277]. The radical scavenging effect of chitosan compounds are attributed to their ability to abstract hydrogen atoms easily from free radicals. This abil- ity directly correlates to the structural property that they have amino and hydroxyl groups attached to C2, C3 and C6 positions of the pyranose ring [278]. Scavenging of HO by chitosans of various molecular weights was consistently reported and is supported by results demonstrating depoly- merisation of the polysaccharide chain by HO . The scavenging activities of chitosan derivatives against HO may be derived from some or all of the following [279]

Chitosan and Low Molecular Weight Chitosan 215 i. The hydroxyl groups in the polysaccharide unit can react with HO by the typical H-abstraction reaction. ii. HO can react with the residual free amino groups NH2 to form stable macromolecule radicals. iii. The NH2 groups can form ammonium groups NH3+ by absorbing hydrion from the solution, then reacting with HO through addition reaction. The presence of more than two free amino groups in chitooligosaccha- rides may be mandatory for the antioxidant activity because glucosamine and N-acetylchitooligosaccharides were not effective at all in inhibition of H2O2-induced hydroxylation of benzoate. On the other hand, the oligosac- charide of glucosamine with DP 2–5 as chitobiose, chitotriose, etc., were active and shown to possess antioxidant properties against carcinogen induced oxygen radical species, as well as hydrogen peroxide released from activated polymorphonuclear leukocytes [280, 281]. Among the chitosans DDA 90, 75 and 50 %, the most potent antioxidant found was chitosan DDA 90 % [282]. This is due to accessibility of more number of free -NH2 groups for reaction with radicals. Variation in the MW of chitosan DDA 90 % influenced the antioxidant activity. Medium MW chitosan MW 1–5 kDa, DDA 90 %, exhibited high- est activity in comparison to high MW (10–5 kDa) and low MW (<1kDa) chitosans of same and lesser DDA [283]. Other studies also reported pro- nounced activity in low molecular weight chitosans [284 285]. Evaluation of chito oligomers of varied MW (2.3, 3.27, 6.12, and 15.250 kDa) obtained from high MW chitosan (850 kDa) also displayed the similar trends of anti- oxidant activity and MW relation and lack of activity in initial high MW chitosan [286]. A comparison of the antioxidant action of high MW chi- tosans (604, 931 kDa) with that of low MW chitosans (2.8, 17.0, 33.5, 62.6, 87.7 kDa), showed that low MW chitosans were more effective in prevent- ing the formation of carbonyl groups in human serum albumin exposed to peroxyl radicals and scavenging nitrogen centered radicals [287]. The plausible reason for such observation is short chain composition of low MW chitosans or oligomers as compared to high MW chitosan. In short chains, the ability to form intramolecular hydrogen bonds between N2-O6 and O3-O5 decline sharply, i.e., the hydroxyl and amino groups are acti- vated and help the radical scavenging process. The hydrolysates of chitosan for the same reason were good antioxidants [287, 289]. Many of the chitosan derivatives abide by these observations. The reported antioxidant derivatives of chitosan are shown in Figure 6.3. With screening of

216 Advanced Biomaterials and Biodevices fully water-soluble O6-aminoethyl chitosan (DS 1.01) obtained from chitin (MW ~310 kDa, DDA 10 %), Je and Kim, suggested that the additional free amino groups in the –CH2CH2NH2 enhanced the scavenging activity [290]. The charged derivatives, chitosan-N-2-hydroxypropyl trimethyl ammonium chloride, followed the same trend as that of native chitosan, with respect to molecular weight and radical scavenging ability [291]. The results obtained by Lin and Chou for water-soluble chitosans-N-alkylated with cellobiose, maltose and lactose units are in line with the previous ones in so far as the highest scavenging activity was observed for rarely substituted products (DS 0.20–0.30) indicative of the main role of the primary amino group [292]. Schiff bases obtained from chitosan and carboxymethyl chitosan exhibited softer scavenging activity [293]. The Schiff bases of chitosan destroyed part of the hydrogen bonds, but at the same time, formed new hydrogen bonds with change of -NH2 group to C=N, so the activities of antioxidant were not benefited from the modification. As a point of difference, N-alkyl qua- ternised chitosans were better HO scavengers, possibly due to the positive charge [294]. There is no report about the antioxidant activity of positive charge. However, Liu, et al., described that the strengthening of the charge density by electronegativity of the substituted groups used for quaternisation increased antioxidant activity [295]. The order of HO and H2O2 scaveng- ing activity in N,N-dimethyl N-alkyl chitosan was -CCl3 > -CBr3 > -CH3 > -CH2CH3 > -CH(CH3)2. The O2 – scavenging action among N,N,N-trimethyl chitosan and N,N-dimethyl N-aryl chitosan (aryl group = benzyl, phenyl- ethyl, furfuryl or salicyl) was highest for N,N,N-trimethyl chitosan and lowest for N,N-dimethyl N-salicyl chitosan. Comparing these quaternary ammonium salts of chitosan, trimethylchitosan has three strong electron- withdrawing methyl groups. The electron-withdrawing groups improve the energy level of the highest occupied molecular orbital (HOMO) and decline the dissociation energy of O-H [296]. Therefore, trimethylchitosan has the highest scavenging ability; conversely salicyl derivative has lowest scaveng- ing ability, for that, it has an electron releasing phenolic group [297]. The modification as the O-carboxymethylation is unfavorable because it drastically lowers the number of primary alcohol functions necessary for the scavenging action. N-carboxymethylation was shown to decrease N-centered radical scavenging and increase reducing power, with increase in DS of values 0.28, 0.41, 0.54. As for O2 – scavenging, the order was DS 0.41> DS 0.54> DS0.28. The difference may be related to the different radi- cal scavenging mechanisms and donating effect of substituting carboxy- methyl group [298]. Just as an electron-donating carboxymethyl group can enhance the elec- tron cloud density of active hydroxyl and amino groups in the polymer

Chitosan and Low Molecular Weight Chitosan 217 chain, so can the scavenging effect on superoxide anion increase when DS increases from 0.28 to 0.41. Although with further increase in DS to 0.54, the electron cloud density of active hydroxyl and amino groups increases, the content of active amino groups decreases, and thus the electron-donat- ing activity decreases. Nevertheless, carboxymethylated chitosan (no car- boxymethyl position specified) was used with the intention of protecting the chondrocytes from induced apoptosis: in fact it restored the level of mitochondrial membrane potential, down-regulated the NO synthase expression and scavenged reactive oxygen species in chondrocytes [299]. Another carboxylated derivative, succinyl chitosan, identified as inhibi- tor of free radical mediated oxidation of cellular biomolecules [300]. High MW O-carboxymethyl chitosan at the maximum concentration of 40 mg/ ml did not show any scavenging activity against the O2 –, but low MW O-carboxymethyl chitosan (1.1–4.35 kDa) showed very modest activity, indicative of the importance of the molecular size and free functions [301]. Quaternised carboxymethyl chitosan had better HO scavenging activity than that of carboxymethyl chitosan, as a result of the positive charge on the quaternised chitosan [302]. Huang, et al., synthesised two chitooligosaccharides derivatives, succi- nyl chitosan and chitosan-N-2-hydroxypropyl trimethyl ammonium chlo- ride by introducing carboxyl (-COCH2CH2COO¯) and quaternised amino -CH2CH(OH)CH2N(CH3)3+ groups to the amino position with different substitution degrees for the purpose of altering the total amount of hydro- gen atoms capable of reacting with radicals, and modifying their metal ion chelating ability [303]. Scavenging of carbon-centered and nitrogen-cen- tered radicals was directly affected by the amount of abstractable hydro- gen atoms in oligosaccharide molecules. In contrast, structure-activity relationships revealed that chelation of Fe2+ ions indirectly contributed for their observed HO scavenging activity apart from hydrogen abstraction. Xie, et al., reported that water-soluble chitosan derivatives prepared by graft copolymerisation of maleic acid sodium onto hydroxyporpyl chito- san and carboxymethyl chitosan sodium, showed radical scavenging activ- ity against HO and O2 – which could be related to the contents of active hydroxyl and amino groups in the polymer chains [278, 304]. The antioxi- dant activity of eugenol grafted or gallic acid conjugated, flavonoid grafted chitosan, 2-(4(or2)-hydroxyl-5-chloride-1,3-benzene-di-sulfanimide)- chitosan or sulfanilamide derivative of chitosan is accredited to additional -OH groups and disruption of intramolecular hydrogen bonds between amine and hydroxyl groups [305–309]. Chitosan substituted at N with 1,3,5-thiadiazine-2-thione residue dis- played superior activity than chitosan because the substituted group

OH OR 218 Advanced Biomaterials and Biodevices O OH O O O O O HO O O O O HO O HO HO O CH3 N N+ NH OH CH3 CH3 N COO– NH2 O R = –CH2CH2NH2 H3C CH3 CH2R R = CH2CH2NEt2 R= –CCl3, –CBr3, –CH3, –CH2CH3, –CH(CH3)2 OR O OH OR NH O O O SO2 O O R = SO2 NH2 O HO HO O H3C O Cl HO NH NH O SO2 N NR SS HO OH R = SO2 NHCOCH3 OH = SO3 OH OSO3H OH OH O O O O OH NH O NH O O HO C HO O O O O H2 NH2 O O H3OSO CH HO HO n NHSO3H R= NH R OMe SO2 H/OH OH SO2 H/OH Cl Figure 6.3 Chitosan derivatives evaluated for antioxidant activity (a) quaternizezd chitosan (b) chitosan-N-2-hydroxypropyl trimethyl ammonium chloride (c) succinyl chitosan) (d) gallic acid conjugated chitosan (e) sulfanilamide derivative of chitosan (f) 1,3,5-ththiadiazine-2-thione substituted chitosan (g) 2-(4(or2)-hydroxyl-5-chloride-1,3-benzene-di-sulfanimide)-chitosan (h) sulfated chitosan (i) eugenol grafted chitosan.

Chitosan and Low Molecular Weight Chitosan 219 hydrolyses in water solutions to generate free -SH group. Because -SH and -OH groups have so many same properties, more -SH groups in the mol- ecule may induce stronger radical scavenging ability on HO [310]. Chitosan sulfate obtained via modification of hydroxyl or amino groups has a strong negative charged nature. Therefore, it can be expected that antioxidant effects of chitosan sulfate might be different from other well- known antioxidants such as vitamins C and E that usually contain mul- tiple aromatic hydroxyl groups. Highly sulfated, high MW and low MW chitosans were also reported to be effective against O2 – and HO reduc- tion of N-centered radicals and chelation of Fe+2 [311–312]. The potency was in the sequence of chitosans sulfated at positions N,3,6 > 3,6 > 6 > 3 [313]. Although the study of hydroxyethyl chitosan sulfate led to the unlikely conclusion that sulfated chitosan did not react with OH radical but increased its generation. It could scavenge N-centered (33.78 %, 2.5 mg/mL) and carbon-centered radicals (67.74 %, 0.25 mg/mL) effectively. This was different from the published literature and was presumed due to the loss of chelating ability on Fe2+. However, there are no reports that have tested the relationship between OH scavenging of chitosan and their Fe2+ chelating ability to date [314]. Research is needed to affirm the mechanism of antioxidation by sulfated derivatives. It is suggested that radical scavenging properties of chitosan and its derivatives not only depend on the presence of free alcohol and amino groups as hydrogen atom donors, but also on the ability of these matri- ces to work as radical cages, entrapping and constraining free radicals to undergo copulation reactions. The study demonstrated that modified poly- saccharides, 5-methylpyrrolidinone chitosan bearing free –OH and –NH2 groups, and lipophilic O- and N-persubstituted chitin derivative dibutyryl chitin, behave as effective radical scavengers since they are able to prevent the propagation of chain reaction onto polymeric framework when free radical species are generated inside the matrix [315]. Anarku, et al. put forth that more plausible mechanism for chitosan’s ability to protect human serum albumin from oxidative damage in vivo as scavenge of secondary peroxyl radicals and not O2 –, HO scavenge [276]. Kinetic measurements have confirmed that the reaction of HO• with carbo- hydrates is close to diffusion control, with reported rate constants between 1.64×109M−1 s−1 for monosaccharides and 1.8×108M−1 s−1 for glycogen with 3000 glucose subunits [316]. The one value measured for chitosan of Mr =400 kDa was in that range, at 6.4×108M−1 s−1.[317] However, the com- mon assumption that chitosan performs much of its antioxidant role in vivo by scavenging HO• is incorrect, because achievable chitosan concen- trations in cells and tissues cannot compete successfully with alternative

220 Advanced Biomaterials and Biodevices HO targets such as proteins, DNA and lipids [318] It may not be due to scavenging of the O2 – by chitosan as this radical does not react with car- bohydrates. Any observations of apparent reaction with O2 – are due to the well-documented ability of chitosans to chelate metal ions, especially Fe3+, which after reduction, reacts with H2O2 derived from the dismuta- tion of the O2 – or from other sources (Fenton reaction) [319]. Antioxidant activity in cellular system may be mediated by other activities of chitosan and derivatives like inhibition of myeloperoxidase activity [300], induction of intracellular glutathione [273, 274] and protective effects on oxidative damage of DNA [320]. 6.11 Conclusion The cationic and polymeric nature of chitosan molecule bestows it a novel set of biological properties apart from being the biodegradable, nontoxic, biocompatible. It is marketed as a food additive or as a dietary supple- ment. Playing on the health card, chitosan is commercially projected as weight lowering agent however high quality clinical trials do not sub- stantiate this claim definitely. Noteworthy number of reports related to its 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 commercialized to some extent. The immunological and antioxidant activities of chitosan are par- ticularly interesting and contribute to potentially very important applica- tions of this polymer in the treatment of various tumoral afflictions and in the treatment of several pathologies of viral origin. The immunoadju- vant 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 monomer and oligomer or directly due to the polymer. There is need to understand the mechanisms underlying these activities and to exploit the actions by chemical derivitization. Chitosan-based oligomers and derivatives may assume significant biological role. The role of chitosan as a constituent of the composites for different biomedical applications based on its bioactivi- ties and physicochemical properties has not been addressed here. Taken that into account along with this overview, it can safely be said that, in the near future, biomedical products based on chitin and chitosan will be embarking on a commercialization program.

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