Radiation Synthesis and Characterization of N,O-Carboxymethyl Chitosan/poly(vinylpyrrolidone) Copolymer Hydrogel

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1 Radiation Synthesis and Characterization of N,O-Carboxymethyl Chitosan/poly(vinylpyrrolidone) Copolymer Hydrogel E.M.El-Nesr 1, A. I. Raafat 1, Sh. M.Nasef 1, E. A. Soliman 2 and El-Sayed.A.Hegazy 1. 1 Polymer Chemistry Department, National Center for Radiation Research and Technology, P.O. Box 29 Nasr City, Cairo, Egypt 2 Chemistry Department. Faculty of Science. Ain Shams University, Cairo, Egypt. Received:1/10/2013 Accepted: 2/11/2013 ABSTRACT In this study, water soluble N,O-carboxymethyl chitosan (N,O-CMCs) was obtained by chemical treatment of chitosan with chloroacetic acid in alkaline condition. The structure of N,O-carboxymethyl chitosan was confirmed by nuclear magnetic resonance ( 1 H.NMR) and Fourier transform infrared spectroscopy (FT-IR) techniques. The weight average molecular weight (MW) of the obtained (N,O-CMCs) was determined by a viscometric method. The degree of substitution (DS%) was calculated from 1 H.NMR spectra and by using potentiometric titration method. The degree of substitution calculated from 1 H.NMR spectrum was found to be close to that obtained using potentiometric titration method. (N,O-carboxymethylchitosan/polyvinyl pyrrolidone) (N,O-CMCs/PVP) copolymer hydrogels were synthesized using gamma irradiation as clean source of initiation and crosslinking. Preparation conditions such as effect of copolymer composition, concentration and irradiation dose on the gel content were investigated. The prepared (N,O-CMCs/PVP) copolymer hydrogels were characterized using Fourier transform infrared spectroscopy (FT-IR), thermogravimetric analysis (TGA), X-ray diffraction (XRD) as well as studying the swelling behavior. Keywords: Carboxymethyl chitosan, degree of substitution, potentiometric titration, hydrogel. INTRODUCTION Hydrogels are a polymeric material (synthetic or natural) have three-dimensional networked structures. Hydrogels swell when placed in a compatible aqueous medium but is not soluble and has the ability to retain the volume of the adsorbed aqueous medium in their three-dimensional swollen network (1,2). Natural polysaccharides such as chitin, chitosan, and cellulose, and their derivatives have a wide range of industrial and medical applications because of their unique structure, distinctive properties, safety, and biodegradability (3). Among the derivatives, N,O-Carboxymethyl chitosan (N,O-CMCs) is the N-deacetylated, carboxymethylated derivative of chitin and the second most abundant natural polysaccharide after cellulose (4). N,O-CMCs an amphoteric polymer having carboxymethyl substituents on some of the amino and primary hydroxyl sites of the glucosamine units of the chitosan structure (5, 6). Since there are three reactive sites of the C2-amino and C3-,C6-hydroxyl groups in molecular chains available for the carboxymethylation of chitosan, the choice of the appropriate reaction conditions and reagents allows the preparation of N-, O-, N,O- or N,N-carboxymethyl chitosan (7). Attention has been on carboxymethylated chitosan because of its good water solubility (it soluble in a wide range of ph), and it 41

2 (8, 9, is more convenient to be applied in medicine because it fits the neutral environment of the human body 10). However, the lack of bone-bonding bioactivity, low mechanical strength and loosening of structural integrity under wet conditions limits its biomedical application (11, 12). Blends with other synthetic polymers, such as poly(n-vinyl pyrrolidone) (PVP) as a nontoxic polymer has good biocompatibility and considerable biostability can get obviously increased properties, and play a significant role in a series of hydrogels as biomedical materials. Ionizing radiation as a method to prepare hydrogels has many advantages such as simultaneous sterilization, easy control of physical properties by combining dose with polymer composition, crossslinked via free radical polymerization on exposure to gamma radiation, without the use of a crosslinking agent (13) and safe for human being and the environment, and can lead to formation of human-friendly products (2). The aim of this work is to synthesis the water soluble N,O-carboxymethyl chitosan with high degree of substitution to enhance the quality and chemical properties of N,O-CMCs products. (N,O-CMCs/PVP) hydrogels will be prepared using -radiation as clean source of copolymeization and crosslinking. Characterization of the prepared (N,O-CMCs) and (N,O-CMCs/PVP) copolymer hydrogel was carried out by TGA, FTIR and XRD. MATERIALS AND METHODS Materials: EXPERIMENTAL Chitosan (MW; 7.7x10 4 g/mole, DDA; 95%) prepared as described in literature (14). Poly(vinylpyrrolidone) (PVP) has average M.W 1300,000 (Acros, Belgium). Chloroacetic acid, 99% (ClCH 2COOH) (Sigma-Aldrich Laboratory Rasayan). Sodium hydroxide (NaOH), Isopropyl alcohol, 98% [(CH 3) 2CHOH], Methanol, 96% (CH 3OH) were purchased from (El-Nasr for pharmaceuticals and chemicals company, Egypt). Methods: Synthesis of N,O-carboxymethyl Chitosan (N,O-CMCs) N,O-CMCs was synthesized according to the literature (15). In brief, chitosan (5 g) was suspended in 50 ml of isopropyl alcohol and the resulting slurry was stirred in a 200 ml flask at room temperature. A volume of 13 ml of 10 N aqueous NaOH solution, divided into five equal portions, was then added to the stirred slurry over a period of 25 min. The alkaline slurry was stirred for additional 30 min. Subsequently, monochloroacetic acid (30 g) was added, in five equal portions, at 1 min intervals. Heat was then applied to bring the reaction mixture to a temperature of 60 C and stirring at this temperature was continued for 3 h. Finally, the reaction mixture was filtered and the solid product (N,O-CMCs) was washed with methanol. The N,O-CMCs was dried at room temperature. Synthesis of (N,O-CMCs/PVP) copolymer hydrogels (N,O-CMCs/PVP) copolymer hydrogels were obtained using gamma irradiation of 10, 20, 30 wt% aqueous solutions of (N,O-CMCs/PVP). The solutions were mixed well, transferred into small glass vials then irradiated at (10, 20, 30, 40, 50 kgy) at room temperature using 60 Co gamma rays at fixed dose rate of 3.32 kgy/h. After copolymerization, the vials were broken; the formed hydrogel cylinders were 41

3 removed and cut into disks of 2 mm thickness and 5 mm diameter. The samples were then washed with excess water to remove the unreacted materials, and air dried at room temperature up to a constant weight. Determination of degree of substitution for (N,O-CMCs) Two methods were used to determine the degree of substitution; potentiometric titration and nuclear magnetic resonance (NMR). 1. Potentiometric titration: The degree of substitution of N,O-CMCs was determined by using potentiometric titration according to a method described elsewhere (16). In brief, N,O-CMCs (0.1 g) was dissolved in distilled water (20 ml). The solution was adjusted to ph < 2 by adding 0.1 M standard hydrochloric acid. Then, the N,O-CMCs solution was titrated with 0.05 M standard sodium hydroxide. The ph value of the solution was simultaneously recorded under continuous stirring. The obtained data were drawn between solution ph and volume of alkali added, which produced an integral curve with two inflection points and allows the determination of degree of substitution (DS%) of the N,O-CMCs using the following equation:- 161 A DS % (1) mcmcs A where A V NaOH C NaOH, V NaOH is the volume difference of NaOH solution between the two inflection points (in liter) and C NaOH is the molarity of aqueous NaOH (0.05 mol/l) and m CMCs is the mass of N,O-CMCs (g), and 161 and 58 are the molecular weights of glucosamine (chitosan skeleton unit) and a carboxymethyl group, respectively. 2. Nuclear magnetic resonance ( 1 H.NMR) The NMR spectra were recorded on a Varian Mercury VX-300 NMR spectrometer. 1 H spectra were run at 300 MHz in deuterium oxide. Chemical shifts are quoted in δ and were related to that of the solvents. The degrees of substitution (DS) of carboxymethyl groups on amino and primary hydroxyl sites of the modified chitosan (N,O-CMCs) were estimated by the relative peak intensities between the H on the carboxymethyl groups and the H at C2 of monosaccharide residue in the 1 H.NMR spectrum of N,O- CMCs, as a method reported in the literature (15). The equations used for the calculation are as follows: DS on amino = 1/2(I b/ I 2) 100 (2) DS on primary hydroxyl=1/2(i c/i 2) 100 (3) where I b, I c are the intensity of hydrogen atom in carboxymethyl group at N-position on C2 and the O- position on C6, respectively. I 2 is the intensity of proton on C2 of monosaccharide residue. Determination the average molecular weight of (N,O-CMCs) N,O-CMCs samples were dissolved in a solvent mixture (0.3 M acetic acid and 0.2 M sodium acetate). An Ubbelhode capillary viscometer was used to measure the passage time of the solutions flowing through a capillary at room temperature. The intrinsic viscosity of the solution η was obtained by extrapolating the reduced and inherent viscosity versus concentration data to zero concentration and the viscosity average 41

4 molecular weight of N,O-CMCs (M) was determined by Mark Houwink Sakurada s empirical equation, reported by (17), that relates the intrinsic viscosity to the polymer s molecular weight as follows: η = km α (4) where M is the average molecular weight, k and α, are the constants that depend on the solvent polymer system (k = ml/g & α = 0.76) (18). Determination gel fraction of (N,O-CMCs/PVP) copolymer hydrogel The gel fraction of the (N,O-CMCs/PVP) copolymer hydrogel was estimated by measuring its insoluble part after extraction. A dry weight of hydrogels (Wo) was immersed in distilled water at 60 o C for 24 h in order to remove the soluble part. The remaining gel was then dried at 25 o C to a constant weight (Wg). The gel fraction was calculated using eq.(5): Gel fraction % = [W g/w o] X 100 (5) Swelling behavior Evaluation A dry weight of (N,O-CMCs/PVP) copolymer hydrogel sample (Wo) was immersed in distilled water for different times at room temperature until equilibrium. After the excessive surface water was removed with filler paper, the weight of swollen gel was determined (Ws). The equilibrium degree of swelling was determined according to the following equation: Degree of swelling % = [Ws Wo /Wo] X 100 (6) APPARATUS Fourier Transform Infrared (FT-IR) spectroscopy FTIR studies of the prepared N,O-CMCs and hydrogel specimens were recorded on (JASCO FT- IR- 6300, Japan in the range of Cm -1 ). Thermogravimetric analysis (TGA) Shimadzu TGA system of Type TGA-50 under nitrogen atmosphere was used to determine the thermal stability and weight loss of the prepared N,O-CMCs and hydrogels as a function of temperature from ambient to 600 C at a heating rate of 10 C/ min. X Ray Diffraction (XRD) Analysis: The XRD patterns of the prepared N,O-CMCs and (NOMCs/PVP) copolymer hydrogel were measured using Shimadzu XRD 6000 diffractometer with Cu target. The XRD runs were carried out over the 2θ ranging from 10 to 40 at a scan speed of 8 /min. N,O-Carboxymethyl Chitosan (N,O-CMCs) RESULTS AND DISCUSSION The reactive sites for the carboxymethylation of chitosan are the amino groups bonded to C2 which can introduce two carboxymethyl groups and hydroxyl groups bonded to C3 and C6 of the glucopyranose unit (19). The amino groups of chitosan are weak bases which are predominantly protonated at ph < 6.5, leading to the solubilization of the polymer only in dilute acid solutions. However, the poor solubility of chitosan when ph > 6.5 is a serious drawback in many of its potential applications. Thus, the preparation of chitosan derivatives has been investigated to overcome its limited solubility in aqueous media. Such an 41

5 adequate chemical modification results, for instance, when the carboxymethylation of chitosan is carried out since N,O-carboxymethylchitosan is soluble in a wide range of ph (20). Additionally, the carboxymethylation reaction is not generally a complete one, and thus some hydroxyl and amino groups remain unsubstituted. Finally, one must consider that if the chitosan is not completely deacetylated there are also some units of glucosamine as well as acetylglucosamine units coming from the partial deacetylation of the parent chitin (21). Conversion yield of (N,O-CMCs) Conversion of chitosan into N,O-CMCs was determined as the dry weight of N,O-CMCs obtained from 29 g of parent chitosan. The obtained N,O-CMCs yield was 50 g which is higher than that of parent chitosan due to increase of sample mass or weight from excessive removal of amino groups from the chitosan and the addition of carboxymethyl groups. Factors affecting gel fraction of (N,O-CMCs/PVP) copolymer hydrogel Radiation copolymerization reaction of polymers in aqueous solution utilizing gamma rays would result in a crosslinked network structure (gel-like materials). The radiation crosslinking can be easily adjusted by controlling the copolymer concentration, composition as well as irradiation dose. 1. Effect of copolymer concentration The polymer concentration has a significant role during gel preparation by radiation copolymerization method. Figure (1) shows the dependence of the gel fraction of the (N,O-CMCs/PVP) hydrogel on the copolymer feed solution concentration. It is clear that, as the copolymer concentration increases, the gel fraction increases. At high copolymer concentration, a great number of molecules are available to react with the free radical on backbone of the polymer chain. Also, the production of large number of growing chains would increase the possibility of H-abstraction via chain transfer to give rise to the substrate macroradicals. This leads to the increased extent of chain propagation resulting in a greater degree of gelation add on Gel fraction (%) Copolymer Concentration (wt%) Fig. (1): Effect of copolymer concentration on the gel fraction (%) of (N,O-CMCs/PVP). Copolymer composition (50:50) wt% in d.h 2O and irradiation dose (30 kgy). 41

6 2- Effect of copolymer composition The gel fraction of (N,O-CMCs/PVP) hydrogel as a function of copolymer composition is shown in Fig. (2). It is clear that the gel fraction of the obtained (N,O-CMCs/PVP) hydrogels is dependent on the copolymer composition; the gel fraction increases by increasing the PVP ratio in the feed solution. These results suggested that the presence of PVP with N,O-CMCs enhanced the gelation process of the copolymers. This may be due to the difference in the diffusion coefficient of both polymers. Also, PVP has a higher sensitivity towards ionizing radiation than that of NOCMC. This is because under ionizing radiation, simultaneous crosslinking of synthetic polymer, degradation of polysaccharides and grafting of polysaccharides to crosslinked polymer chains occurs and leading to the formation of hydrogel (22) Gel fraction (%) PVP NOCMCs Fig. (2): Effect of copolymer composition on the gel fraction (%) of (N,O-CMCs/PVP) copolymer concentration 30 wt% in d.h 2O and irradiation dose 30 kgy. 3- Effect of irradiation doses Copolymer Composition (%) The gel fraction of (N,O-CMCs/PVP) hydrogel as a function of absorbed irradiation dose is shown in Fig. (3). It is clear that the gel fraction of (N,O-CMCs/PVP) hydrogels increases with the increase of the dose up to 30 kgy and then tends to slightly decrease at a higher absorbed dose. On the base of the fact that, the radiation crosslinking efficiency of PVP is much higher than that of N,O-CMCs which is a water soluble natural modified polysaccharide that degrades upon irradiation by the breakdown of the main chains (23). Irradiation of an aqueous concentrated solution of (N,O-CMCs/ PVP) (named as paste-like status ) would results in simultaneous crosslinking of PVP, degradation of N,O-CMCs and grafting of NOCMC fragments onto the crosslinked PVP chains, or physical crosslinking of N,O-CMCs but eventually lead to the formation of insoluble copolymer network (gel fraction) ( 22, 24). The formation of hydrogels via radiation crosslinking depends initially on the amount of free radicals generated. If this amount was not high enough for recombination, a physically stable hydrogel is 41

7 formed. However, when the absorbed dose reached a critical absorbed dose (gelation dose), the intermolecular recombination of free radicals readily occurred led to the formation of a three-dimensional network. Further increase of the absorbed dose would cause increased crosslinking density, while the fraction of the insoluble part did not change significantly in a certain dose range. At a sufficient high absorbed dose, the intramolecular free radical recombination tended to dominate over the intermolecular radical recombination, which would terminate the crosslinking and begin to degrade the network. Therefore, the gel fraction was slightly decreased (25, 26) Gel fraction (%) Irradiation Dose (kgy) Fig. (3): Effect of irradiation dose on the gel fraction (%) of (N,O-CMCs/PVP). Copolymer concentration 30 wt% and copolymer compositions ( ) 15:85, ( ) 25:75 (wt/wt). Characterization of N,O-CMCs and (N,O-CMCs/PVP) copolymer hydrogel 1. Determination of the average molecular weight In order to determine the molecular weight of polymeric chains, viscometry is selected as one of the simplest and most rapid methods (27). It was found that, the average molecular weight of the prepared N,O- CMCs determined using eq.(4) was approximately 13.1x10 4 (g/mole). It was also noticed that, the average molecular weight of N,O-CMCs is nearly two times greater than molecular weight of parent chitosan. These results confirmed the conversion of the amino and hydroxyl groups of chitosan into carboxymethyl groups in N,O-CMCs. 2. Determination of the degree of substitution 2.1. Using potentiometric titration method Typical ph-potentiometric titration curves are shown in Figure (4). At around 70% of titration process, precipitation occurred in the solutions and the response of the glass-electrode became sluggish. However, after the second equivalence point, a reasonably fast electrode response was achieved again, indicating that proton-exchange processes including N,O-CMCs are already over at this ph-range. The titration process results in a titration curve with two inflexion points: the first is the volume of sodium hydroxide consumed by excessive hydrochloric acid (ml), while the second corresponds to the volumes of sodium hydroxide corresponding to titration terminal of COOH (28, 7). The DS% of N,O-CMCs can be 02

8 calculated using Eq. (1) and was found to be 0.9. The obtained N,O-CMCs were completely soluble in distilled water at room temperature. Such phenomena could be interpreted in terms of the random distribution of N,O-CMCs residue which disturbs the formation of the ordered structure and the hydrogen bonding among the amino groups of chitosan. Furthermore, the presence of N,O-CMCs residues which are hydrophilic groups changes the solubility of chitosan in water significantly (29) ph Volume of NaOH (ml) Fig. (4): Potentiometric titration curve of the prepared N,O-CMCs Using Proton Nuclear magnetic resonance ( 1 H.NMR) Proton nuclear magnetic resonance spectroscopy ( 1 H.NMR) was used to confirm substitutions of amino and primary hydroxyl groups of the parent chitosan by carboxymethyl groups in the modified carboxymethychitosan (N,O-CMCs). ( 1 H NMR) spectrum of N,O-CMCs in D 2O is shown in Fig. (5). As shown, the chemical shift at 4.4 and 4.6 ppm represents the protons of -CH 2-COO- at N-position on C2 and the O-position on C6 of the carboxymethylated chitosan (N,O-CMCs), respectively (15). This indicated that carboxymethyl substitution reaction was occurred on some of the amino and primary hydroxyl sites of the chitosan structure. The degree of substitution (DS) of carboxymethyl groups on the amino and primary hydroxyl sites were determined using eqs.(2,3) and the resulting data were found to be approximately 63.36% and 23.60%, respectively. 04

9 3. FT-IR spectroscopy Analysis Fig. (5): 1 H.NMR spectra of N,O-carboxymethyl chitosan (N,O-CMCs). The occurrence of carboxymethylation reaction onto chitosan was confirmed by comparing the FT IR spectrum of chitosan with that of the prepared N,O-CMCs. As shown in Fig. 6 (a), the FT-IR spectrum of the parent chitosan showed a broad absorption band between 3500 and 3100 cm -1, centred at 3400 cm -1, which may be due to stretching O H and N H bond. The bands observed at 2894 cm -1 correspond to the CH stretching of chitosan. Another absorption band appears at 1656 cm -1 which is attributed to the axial stretching of C=O bonds of the acetamide groups, named as (amide I) band which indicated that the sample was not fully acetylated. A band observed at 1590 cm -1 is attributed to the angular deformation of the N H bonds of the amino group (amine II) (30). A band at 1370 cm -1 due to the symmetrical angular deformation of CH 3 and the (amide III) band at 1316 cm -1 were observed. The bands corresponding to the polysaccharide skeleton, including the vibrations of the glycoside bonds, C O and C O C stretching in the range cm -1 were observed (31). On comparing the FT IR spectra of the parent chitosan with that of N,O-CMCs, additional peaks appeared in the spectra which provoke structural changes. Thus, the broadening of the band between 3500 and 3100 cm -1, centred at 3400 cm -1 revealed more hydrophilic character of carboxymethyl chitosan as compared to the parent chitosan (Fig. 6b). The characteristic absorption bands of carboxyl group COOH appeared at 1737 cm -1. The occurrence of an intense band in 1588 cm -1 and a moderate band at 1423 cm -1 were attributed to the symmetric COO (which overlaps with N H bend) and asymmetric axial deformations of COO, respectively, confirmed the introduction of the carboxymethyl groups (19, 21, 32). The graft copolymerization process of PVP and N,O-CMCs was confirmed by comparing, the FT- IR spectra of, N,O-CMCs, with that of (N,O-CMCs/PVP) copolymer hydrogel. Figure 6(c) shows a characteristic bands for PVP appeared at 1654, 1286 and 962 cm -1 were assigned to the C=O stretching vibration (pyridine ring), C N stretching vibration and the out of plane rings C H bending, respectively (33). 00

10 a b Transmittance c Wavenumbers (cm -1 ) Fig. (6): FTIR spectrum of (a) pure chitosan, (b) pure N,O-CMCs and (c) (N,O-CMCs/PVP) hydrogel. 4. X-ray diffraction analysis (XRD) The X-ray patterns of chitosan and carboxymethyl chitosan is shown in Fig.7(a,b). It is clear that, the carboxymethylation of chitosan provoked important changes in the arrangement of the polymer chains in the solid state. Indeed, the spectrum of the N,O-CMCs is exhibiting poorly defined and much less intense peaks as compared to the parent chitosan. This is attributed to the presence of the carboxymethyl moieties which substitute the hydrogen atoms of the hydroxyl and amino groups of chitosan. Thus, as the carboxymethyl groups are much more voluminous than the hydrogen atoms, an important excluded volume effect occurs and a polyelectrolyte effect must also be considered due to the presence of charged groups in the chains of carboxymethylchitosan. The carboxymethylation of chitosan also affected the establishment of hydrogen bonds involving its hydroxyl and amino groups, these interactions being responsible for the adoption of a more ordered arrangement by the parent chitosan (21). On comparing the XRD spectra of the N,O-CMCs with that of (N,O-CMCs/PVP) copolymer hydrogel, additional peaks appeared in the spectra which provoke structural changes. Figure 7(c) showed that, there was two more intense peaks appeared at 2θ =13.9 and These results indicate that after grafting with PVP, the original structure of carboxymethyl chitosan has been altered leading to strong interaction between PVP and N,O-CMCs (34). 02

11 c Intensity b a Theta (degree) Fig. (7): XRD spectra of (a) Chitosan (b) N,O-CMCs. and (c) (N,O-CMCs/PVP) hydrogel. 5. Thermogravimetric analysis (TGA) TGA analysis was carried out to study the thermal stability of chitosan, N,O-CMCs and (N,O- CMCs/PVP) copolymer hydrogels. Figure 8(a,b) shows TGA curves of chitosan and N,O-CMCs. It can be seen that the degradation process of chitosan and N,O-CMCs occurs in tow steps: The first stage starts around 90 C for N,O-CMCs and around 110 C for chitosan. The second stage starts around 251 C for chitosan and 202 C for N,O-CMCs. The first stage is assigned to the loss of water because polysaccharides usually have a strong affinity for water and therefore may be easily hydrated. The second one corresponds to the thermal decomposition of main chain of polysaccharide (including saccharide rings and breaking of the C-O-C glycodidic bonds), vaporization and elimination of volatile product (34, 35, 36). Figure 8(c) shows TGA curves of (N,O-CMCs/PVP) copolymer hydrogel. It can be seen that, (N,O-CMCs/PVP) hydrogel started loss of water stage in the range of o C and started to degrade at about 205 C. The weight loss increases with increase in temperature from 205 to 404 C. The degradation step in the temperature range C is the main degradation step of poly(vinylpyrrolidone), leading to the formation of esters as a consequence of the scission of the N C O bonds at 485 C (37). It is observed that, the decomposition temperature of chitosan is higher than that of N,O-CMCs. The obtained results indicate that chitosan exists in a stable structure toward thermal decomposition than that of N,O-CMCs. Also, (N,O-CMCs/PVP) copolymer hydrogel is more stable than N,O-CMCs and chitosan. 01

12 Weight remained (%) a b 20 c Temperature ( C) Fig. (8): TGA curves of (): a chitosan, b:(..) N,O-CMCs. and c:( ) (N,O-CMCs/PVP) hydrogel. 6. Swelling Behavior The most characteristic feature of hydrogels is its ability to imbibe and hold an adequate amount of water in its network structure. Such water content is the prime factor that contributes to the biocompatible nature of the synthetic biomaterials. The high water content imparts several unique physiochemical properties to the synthetic biomaterials that shows living tissue like matrix, physiological stability, permeability to biomolecules that ease cell growth. The equilibrium swelling of a hydrogel is a result of the balance of osmotic forces determined by the affinity to the solvent and the network elasticity. The extent of hydrogel swelling depends strongly on the degree of cross-linking density, the hydrophilicity of hydrogel chains, the chemical composition, and the interactions of the network and surrounding liquid. Figure (9) shows the water swelling behavior of the (N,O-CMCs/PVP) hydrogels of different compositions. It is clear that, the swelling process occurred rapidly, reaching equilibrium of water uptake in about 5 h. Also as can be seen, the swelling degree is a function of the hydrogel composition. It increases by the increase of the N,O-CMCs ratio within the hydrogel due to its hydrophilic nature. While, PVP rich compositions possess lower degree of swelling than that of N,O-CMCs rich compositions. In spite of its hydrophilic nature, such decrease may be due to the increase in the crosslinking density by the increase of PVP ratio that reduces the swelling ability of the produced hydrogels. 01

13 Swelling degree (%) Time (hours) Fig. (9): Effect of time on the degree of swelling (%) of (N,O-CMCs/PVP) hydrogels. Copolymer compositions ( ) 75:25, ( ) 65:35, ( ) 35:65, ( ) 15:85, ( ) 0:100 (wt/wt). and irradiation dose 30 kgy. CONCLUSION In the current investigation, chitosan were successfully modified to N,O-carboxymethyl chitosan and its structure was confirmed by FTIR and 1 H.NMR. The degree of substitution of the obtained N,O- CMCs was determined using potentiometric titration and 1 H.NMR spectrum method. The obtained values of substitution degree were found to be close. The molecular weight was determined viscometry and found approximately 13.1x10 4 (g/mole). XRD analysis showed that chitosan is more crystalline than N,O- CMCs. TGA studies showed that chitosan exists in a stable structure toward thermal decomposition than that of N,O-CMCs. REFRENCES (1) B. D. Ratner, and A. S. Hoffman; (1976): Hydrogels for Medical and Related Applications, Andrade, J. D., ed., American Chemical Society, Washington, D.C., pp (2) F. Yoshii, K. Makuuchi, D. Darwis, T. Iriawan, M. T. Razzak, and J. M. Rosiak; Radiat. Phys. Chem.; 46, 169 (1995). (3) A. Hiroki, H.T. Tran, N. Nagasawa, T. Yagi, and M Tamad; Radiat. Phys. Chem.78, 1076 (2009). (4) M. Wang, X. Ling, J. Xuecheng,P. Jing, Z. Maolin, L. Jiuqiang, and W. Genshuan; Polymer Degradation and Stability; 93, 1807 (2008). (5) M. N. V. Ravi Kumar, R. A. A. Muzzarelli, C. Muzzarelli., H. Sashiwa, and A. J. Domb; Chem. Rev.; 104, 6017 (2004). (6) Y. H. Lin, F. L. Hsiang, C. K. Chung, M. C. Chen, and H. W. Sung; Biomaterials; 26, 2105( 2005). (7) X. Kong; Carbohydrate Polymers; 88, 336 (2012). (8) H. P. Ramesh, S. Viswanatha, and R.N. Tharanathan; Carbohydrate Polymers; 58, 435(2004). 01

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