ZnO nanocomposite hydrogels

Accepted Manuscript Title: Synthesis and Characterization of Antibacterial Carboxymethyl Chitosan/ZnO Nanocomposite Hydrogels Author: Fazli Wahid Jun-Jiao Yin Dong-Dong Xue Han Xue Yu-Shi Lu Cheng Zhong Li-Qiang Chu PII: DOI: Reference:

S0141-8130(16)30268-9 http://dx.doi.org/doi:10.1016/j.ijbiomac.2016.03.044 BIOMAC 5938

To appear in:

International Journal of Biological Macromolecules

Received date: Revised date: Accepted date:

2-2-2016 21-3-2016 22-3-2016

Please cite this article as: Fazli Wahid, Jun-Jiao Yin, Dong-Dong Xue, Han Xue, YuShi Lu, Cheng Zhong, Li-Qiang Chu, Synthesis and Characterization of Antibacterial Carboxymethyl Chitosan/ZnO Nanocomposite Hydrogels, International Journal of Biological Macromolecules http://dx.doi.org/10.1016/j.ijbiomac.2016.03.044 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Synthesis and Characterization of Antibacterial Carboxymethyl Chitosan/ZnO Nanocomposite Hydrogels Fazli Wahid1, Jun-Jiao Yin1, Dong-Dong Xue2, Han Xue1, Yu-Shi Lu1, Cheng Zhong2, Li-Qiang Chu1* 1. College of Chemical Engineering and Materials Science, Tianjin University of Science and Technology, No.29, 13th Ave., TEDA, Tianjin 300457, China 2. Key Laboratory of Industrial Fermentation Microbiology (Ministry of Education), Tianjin University of Science and Technology, Tianjin 300457, China * To whom correspondence should be addressed: Tel: +86 22 60602476; Fax: +86 22 60602430; E-mail: [email protected]

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Abstract The antibacterial carboxymethyl chitosan/ZnO nanocomposite hydrogels were successfully prepared via in situ formation of ZnO nanorods in the crosslinked carboxymethyl chitosan (CMCh) matrix, by treating the CMCh hydrogel matrix with zinc nitrate solution followed by the oxidation of zinc ions with alkaline solution. The resulting CMCh/ZnO hydrogels were characterized by using FTIR spectroscopy, X-ray diffractormetry and scanning electron microscopy (SEM). SEM micrographs revealed the formation of ZnO nanorods in the hydrogel matrix with the size ranging from 190 nm to 600 nm. The swelling behavior of the prepared nanocomposite hydrogels was also investigated in different pH solutions. The CMCh/ZnO nanocomposite hydrogel showed rather higher swelling behavior in different pH solutions in comparison with neat CMCh hydrogel. Furthermore, the antibacterial activity of CMCh/ZnO hydrogel was studied against Escherichia coli and Staphylococcus aureus by CFU assay. The results demonstrated an excellent antibacterial activity of the nanocomposite hydrogel. Therefore, the developed CMCh/ZnO nanocomposite hydrogel can be used effectively in biomedical field.

Keywords: Hydrogel; Carboxymethyl chitosan; Antibacterial activity.

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1. Introduction Hydrogels are a class of materials with three dimensional polymeric networks which can bind high amount of water or biological fluids. Despite their high affinity towards water absorption, they show swelling behavior instead of dissolving in aqueous surrounding environment [1]. Hydrogels consist of critical crosslinks, which are provided by covalent bonds, hydrogen bondings, van der Waals interactions or physical entanglements. The excellent hydrophilicity of hydrogels, high swelling ratio and biocompatibility, promote their usage widely in biomedical areas such as tissue engineering, drug delivery, biosensor, and so on [1, 2]. Due to the superior biomedical relevance, there is an increasing interest to develop the antibacterial hydrogels [3, 4]. Among the antibacterial hydrogels, the inorganic-based nanocomposite hydrogels are particularly promising to inhibit the microbial growth, thus making them attractive in the fields of biomedical and biotechnology [5]. Silver-based materials are of special interest because of their broad-spectrum inhibitory and strong antimicrobial effect [6]. Therefore, much effort had been placed onto the development of silver nanocomposite hydrogels in the medical field during the last few years [7-10]. However, the use of silver-based materials has to face the restrictive applications attributed to color changing, expensive cost and strong influence on living systems (i.e., silver can accumulate in the human body and lead to severe toxicity) [11]. The antimicrobial property of ZnO nanostructures is well known and it is currently used as cosmetic materials and in food packaging applications [12]. The microbial inhibition property of ZnO may be due to the induction of oxidative stress causes the production of reactive oxygen species, which may degrade the membrane structure of the cells [13]. Recently, there is an increasing research interest on the fundamental studies of the antimicrobial activities of ZnO 3

nanostructures. The advantages of combining hydrogels with ZnO nanoparticles as an alternative to the widely used Ag nanoparticles resided in their lack of color, lower cost and UV-blocking properties [14, 15]. However, there are only a few reports on the preparation of ZnO nanocomposite hydrogels [16, 17]. Yadollahi et al.[17], have developed carboxymethyl cellulose/ZnO nanocomposite hydrogels which showed good swelling ability in various pH solutions. It has also showed enhanced antibacterial properties. Pati et al.[18], demonstrated the immunological and antibacterial mechanism of ZnO nanoparticles against human pathogens. They found that ZnO nanoparticles showed greater activity against Staphylococcus aureus while have lesser activity against Mycobacterium bovis. Moreover, ZnO nanoparticles significantly reduced the skin infection, bacterial load and inflammation in mice by intradermal administration, and also improved infected skin architecture [18]. Hashem et al.[19] prepared CMC/ZnO nanocomposite hydrogel and studied its swelling behavior and antibacterial activity against both gram-positive and gram-negative bacteria. The results showed excellent swelling properties and good antibacterial activity.

Other group of researchers also worked on the ZnO nanocomposite

hydrogels and studied antibacterial properties [20, 21]. Chitosan, an amino polysaccharide, is the deacetylated derivative of chitin, which is one of the renewable, nontoxic, biodegradable and abundant carbohydrate polymers and can be obtained in large amount from the exoskeletons of both shellfish and insects. Therefore, chitosan has received much attention in various fields as a functional biopolymer [22, 23]. However, due to low water solubility of chitosan, its hydrogels do not show high water binding capacities. Carboxymethyl chitosan (CMCh) is an important derivative of chitosan. It is an amphoteric material which shows hydrophilic characteristics [24, 25]. Thus CMCh has several advantages 4

over chitosan, such as increased water solubility, better biocompatibility, high moisture retention ability, excellent biodegradability [26], improved antioxidant property [27], as well as, enhanced antibacterial activity [28]. The preparation of O-CMCh is achieved simply by reacting chitosan with monochloroacetic acid in alkaline medium [29]. Recently, CMCh hydrogels have successfully prepared by crosslinking in aqueous media followed by drying the resultant gel, which have high water binding capacity [30]. In the light of the above results mentioned in the literature, in this work we explored the synthesis of a series of antibacterial CMCh/ZnO nanocomposite hydrogels, simply by in situ oxidation of the Zn2+ ions in the CMCh hydrogel matrix. The resulting CMCh/ZnO hydrogels were characterized by FTIR, XRD and SEM. The antibacterial activity of the nanocomposite hydrogels was studied against Escherichia coli and Staphylococcus aureus by CFU assay. 2. Materials and Methods 2.1 Materials Chitosan was purchased from Zhejiang Golden-shell Pharmaceutical Co., Ltd (Zhejiang, China), with a molecular weight of ca. 50,000 Da and the degree of deacetylation of ca. 90%. Epichlorohydrin (ECH, 99%) was obtained from Sigma-Aldrich. Monochloroactic acid (99%) was purchased from Xiya Chemical Industry Co., Ltd (Shandong, China). Yeast extract and Tryptone were obtained from Oxoid Ltd (U.K.). Agar powder was purchased from Solarbio Science and Technology Co. Ltd. All other reagents like zinc nitrate hexahydrate, sodium hydroxide, methanol and acetic acid were analytical grade. Deionized water was used throughout the experiments. Bacterial strains were kindly provided by the Key Laboratory of Industrial Fermentation Microbiology, Tianjin University of Science & Technology.

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2.2 Methods 2.2.1 Preparation of CMCh Carboxymethyl chitosan was prepared based on the procedure reported previously [31]. Briefly, 5 g of chitosan was stirred for 15 min in 20% NaOH solution (w/v, 100 mL). Then 15 g of monochloroactic acid was added portion wisely to the reaction mixture with constant stirring and the reaction was continued for 2 h at T=40 °C. The reaction mixture was then neutralized with 10% acetic acid and poured into an excess of 70% methanol. The product was filtered through a G2 sintered funnel and washed several times with methanol. Finally, the obtained CMCh powder was dried in a vacuum oven at T=55 °C for 8 h to give dry product. 2.2.2 Preparation of crosslinked CMCh hydrogel CMCh hydrogel was prepared by dissolving CMCh (3 g) in 3% NaOH solution (w/v, 100 mL) with continuous stirring until a homogeneous viscous mixture was obtained. Then 5 mL ECH was added dropwise with constant stirring for 2 h until a homogenous mixture was obtained. The obtained mixture was heated at T=80 °C in water bath for two hours. The insoluble crosslinked CMCh paste was collected and washed with distilled water for several times to remove the residual ECH and NaOH. Finally, the hydrogel was dried at T=50 °C for 24 h [32]. 2.2.3 Loading of ZnO to CMCh hydrogel ZnO nanorods were loaded to CMCh hydrogel according to the method described in the literature [17]. Briefly, 0.6 g of dry CMCh hydrogel was immersed in different concentrations of Zn(NO3)2 solutions (0.000, 0.005, 0.010, 0.020, 0.030 and 0.050 M) for 24 h, and then it was washed with copious deionized water. The resulting hydrogel was then placed in a NaOH solution of 0.2 M for 24 h. After the oxidation of bound Zn ions, the nanocomposite hydrogel 6

was washed with distilled water and finally dried in an oven at T=50 °C for 24 h. In the rest of the article, Z0, Z1, Z2, Z3, Z4 and Z5 represent CMCh/ZnO nanocomposite hydrogels, which have 0.000, 0.005, 0.010, 0.020, 0.030 and 0.050 M of Zn(NO3)2 contents, respectively. 2.3. Characterization and analysis 2.3.1. FTIR Spectroscopy Fourier transform infrared (FTIR) spectrum was measured in the wave number ranging from 4000 to 400 cm-1 at a resolution of 4 cm-1 using a Bruker Tensor 27 FTIR instrument. The samples were prepared in KBr and scanned against a blank KBr pellet. 2.3.2 SEM measurements Surface morphologies of pure CMCh hydrogel and nanocomposite hydrogel were examined on a SU8000 scanning electron microscope. The samples were mounted on a double sided carbon tape and then coated with a thin layer of platinum. 2.3.3 XRD analysis The pattern of X-ray diffraction of the samples was obtained by using an X-Ray diffractometer (General Instrument Co. Ltd., Beijing XD-3) at 40 kV with the scan range from 5 to 70◦ and a scan rate of 2 ◦/min. 2.3.5 Swelling test The swelling test of CMCh/ZnO nanocomposite hydrogel was performed by immersing 1 g of sample into a pre-weighed nonwoven tea bag in 50 mL solution of desired pH for 24 h to attain the maximum swelling equilibrium. Then it was hanged until the last drop of water was fallen down. The swelling ratio was calculated from the following equation. Swelling ratio = [

𝑊𝑤 − 𝑊𝑑 ] × 100 𝑊𝑑

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Where Ww and Wd are the wet and dry weights of hydrogels, respectively. To get the desired pH solutions, standard solutions of HCl (0.1 M) and NaOH (0.1 M) were used [33]. 2.3.5 Antibacterial assays The antibacterial experiments were performed against Staphylococcus aureus (gram-positive) and Escherichia coli (gram-negative) by the enumeration of viable organisms using a method described in the literature [34, 35]. Briefly, the bacterial cells were grown overnight in LuriaBertani (LB) broth at T=37 °C. The cells were harvested by centrifugation, washed and resuspended in a PBS buffer solution. The optical density (OD) was adjusted to 0.5, approximately 4 × 108 colony forming units (CFUs) per mL at λ=600 nm. All samples with concentration of 5 mg/mL were already swollen in a sterilized PBS. The samples, each of which contained test materials at a concentration of 5 mg/mL, were incubated with bacterial suspension in a shaker incubator at T=37 °C for 6 h. A PBS solution was used as negative control. All samples were diluted serial wisely, 100 μL of bacterial suspension was drawn from each sample tube, spread on the LB agar plate in triplicate and finally incubated at T=37 °C for 24 h for colony forming. The viable colonies were counted and the experiments were repeated three times. 4. Results and discussion 4.1 In situ formation of ZnO The schematic representation of the crosslinking of CMCh in alkaline medium is shown in Scheme 1, while ZnO formation in the CMCh hydrogel is shown in Scheme 2. Due to the negative charge on the CMCh, it can interact with the positively charged metal cations [36, 37]. Zn(NO3)2 produce positively charged zinc ions, and therefore it can be easily bound onto the negatively charged carboxylic group (COO-) of the CMCh hydrogel via electrostatic force. In the presence of suitable basic solution like NaOH, zinc ions can be easily oxidized giving rise to the 8

ZnO nanostructures. This is a facile and economical method for in situ preparation of ZnO nanostructures, not requiring heat or other tools for nanostructures synthesis.

Scheme 1. Schematic representation of the crosslinking of CMCh with epichlorohydrin in alkaline solution.

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Scheme 2. Schematic representation of the incorporation of ZnO into the crosslinked CMCh hydrogel. 4.2 FTIR analysis FTIR spectra of both the as-prepared CMCh and the crosslinked CMCh hydrogel are shown in Fig. 1. The bands in the as-prepared CMCh spectrum (Fig. 1a) at 3439 cm−1, 1598 cm−1 and 1414 cm−1 are assigned to stretching vibration of O−H, COO− (asymmetric) and COO− (symmetric), respectively [31]. The FTIR spectrum of the crosslinked CMCh (Fig. 1b) is similar to that of the as-prepared CMCh, but the new absorption peak appeared at 1377 cm−1, which could be assigned to C−N stretching vibration [38]. The peak at 1457 cm−1 belongs to stretching vibration of C−O−C of the reacted ECH with CMCh. Our results are similar to the literature [32].

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Compared to the FTIR spectrum of the as-prepared CMCh, the characteristic bands of COO− (symmetric) and –OH in the spectrum of crosslinked CMCh can be found to shift to slight higher wavenumber of 1421 cm-1 and 3443 cm-1 (as shown in Fig. 1b), which may be due to the decrease in the number of the formed hydrogen bonds because of the consumption of carboxylate ions in cross linking.

Transmittance / %

b 1457 1421

a 1597 1377

3443

1414 1598

3439

4000

3500

3000

2500

2000

1500

1000

500

Wavenumber / cm-1

Fig. 1. FTIR spectra of as-prepared CMCh (a) and crosslinked CMCh hrdrogel (b). 4.3 XRD analysis The XRD patterns of neat CMCh hydrogel and CMCh/ZnO nanocomposite hydrogel, in the 2θ range of 5-70° are shown in Fig. 2. The typical peaks in a CMCh hydrogel diffractogram (Fig. 2a) at 10° and 20° are assigned to the polymeric matrix of CMCh. There are additional peaks in a CMCh/ZnO nanocomposite hydrogel diffractogram (Fig. 2b) at 31°, 34°, 37°, 47°, 56°, 62° and 68°, which are attributed to the (100), (002), (101), (102), (110), (103), and (112) crystal planes of ZnO with hexagonal wurtzite structure. Our results are matched well with the standard values

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reported by JCPDS No. 36-1451 [39, 40]. None of other peaks can be observed, which indicate the pure ZnO formation. The results also revealed that ZnO nanostructures were successfully prepared in the polymeric CMCh hydrogel matrix.

Intensity / a.u

101 100 002 b

102

110

103 112

a

10

20

30

40 Angle / 2

50

60

70

Fig. 2. XRD patterns of neat CMCh hydrogel (a) and CMCh/ZnO nanocomposite hydrogel of Z3 (b). 4.4 SEM analysis SEM measurements were carried out to analyze the morphology of the as-prepared CMCh/ZnO nanocomposites and the size of ZnO nanostructures. Fig. 3 gives the SEM images of neat CMCh hydrogel and three CMCh/ZnO nanocomposite hydrogels prepared with different concentrations of Zn(NO3)2. A clear and uniform surface morphology can be seen for the neat hydrogel (Fig. 3a), whereas rod like crystalline shape ZnO nanostructures can be observed in the nanocomposite hydrogels (Fig. 3b, 3c and 3d). SEM results also shows that the ZnO nanorods are uniformly dispersed in the CMCh matrix. For lower concentration of Zn(NO3)2 (0.005 M, see Fig. 3b), the particle size range is about 190-450 nm. When the concentration of Zn(NO3)2 is 12

increased to 0.010 M, the particle size increases up to 600 nm (Fig. 3c). When further increasing the concentration of Zn(NO3)2 to 0.020 M, the nanorods form three dimensional structures. One also can observe the agglomeration of these three dimensional ZnO particles (Fig. 3d). However, the average particle size becomes smaller as compared to those in Fig. 3c. Similar three dimensional nanorods of ZnO nanorods have been reported in the literature [41].

Fig. 3. SEM images of neat CMCh hydrogel (a) and CMCh/ZnO nanocoposite hydrogels prepared with different concentrations of zinc nitrate: (b) 0.005 M; (c) 0.010 M; (d) 0.020 M.

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4.5 Swelling behavior In order to investigate the pH sensitivity of the prepared hydrogels, the swelling behavior was studied at pH = 2, 4, 6, 7, 8 and 10. As shown in Fig. 4, the swelling increased with the increase of pH from 2 to 7, and then decreased at pH = 8 and 10. With the increase of pH from 2 to 7, carboxyl groups on the CMCh chains convert to negatively charged carboxylate ions, causing higher electrostatic repulsion and water would be taken up [42]. A reducing pattern of swelling values was observed at pH > 7, although we were expecting rise in the swelling behavior at higher pH values. The reduction in swelling values may be due to the shielding of carboxylate ions by Na+ cations from NaOH, which may prevent the complete anion-anion repulsion and restrain the extending of the tangled molecular chain of the hydrogel. As the ionic strength of the medium increases, the swelling decreases. Furthermore, Fig. 4 suggests that the CMCh/ZnO nanocomposite hydrogels exhibit a higher swelling capability in the comparison to neat CMCh hydrogel. The increase of the swelling capacity of the CMCh/ZnO nanocomposite hydrogels may be attributed to the presence of ZnO nanorods with different sizes, morphologies and surface charges. Charged ZnO nanoparticles results in the penetration of more water molecules to balance the build-up ion osmotic pressure, which causes the swelling of hydrogels [43-45]. In addition, the formation of ZnO nanorods may cause the expansion of hydrogel network and thus can increase the pores and free spaces within the hydrogel, which would absorb more water accordingly [46, 47].

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900

Swelling ratio / %

800 700 600 500 Z5 Z4 Z3 Z2 Z1 Z0

400 300 200 100

2

4

6

8

10

pH

Fig. 4. Swelling behavior of CMCh/ZnO nanocomposite hydrogels at different pH values.

4.6 Antibacterial activity CFU assay was employed to investigate the antibacterial activity of both the neat CMCh hydrogel and the nanocomposite hydrogel against gram-positive bacteria S.aureus and gramnegative bacteria E.colli. The results obtained are given in Fig. 5. While in contrast to the results of Mohamed and Sabaa [32], we found that the crosslinked CMCh hydrogel showed poor antibacterial activity (Fig. 5A and 5B). Furthermore, the results suggested that the ZnO embedded nanocomposite hydrogel had a more toxic effect on bacteria than neat hydrogel under similar conditions. However, when increasing the concentration of ZnO nanorods in the nanocomposite hydrogel the bactericidal effect increased. It is clear from Fig. 5A and 5B, relative to control Z3 reduced the bacterial population by 99%, while no viable cells could be found on the agar plates when bacteria were incubated with Z4 and Z5.

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To study the timing effect, we incubated the bacteria with a Z3 nanocomposite hydrogel, and drawn the bacterial suspension after certain intervals of time, diluted and spread on the agar plate for counting the survival colonies. The results are depicted in Fig. 5C (S. aureus) and Fig. 5D (E.colli). It is apparent that Z3 reduced the bacterial population by 40% after half an hour of incubation, while 60% after 1 hour incubation. After that the viability percentage was decreased. No viable colony was found after 4 hours contact time in case of S. aureus, while in case of E.colli it took about 6 hours to reduce the bacterial population by 99%. These results suggest that S.aureus is more susceptible to nanocomposite hydrogel in comparison to E.colli. Several mechanisms have been postulated to elucidate the antimicrobial activity of chitosan. The most acceptable mechanism is mediated by the electrostatic forces between the protonated −NH3+ groups of chitosan and the negative charges on the microbial cell surface [48]. Since such mechanism is based on the electrostatic interaction, it means, the greater the positive charge on chitosan is, the higher the antibacterial activity will be. In comparison to chitosan, CMCh has higher positive charge density, in this case the −COOH groups may react with the −NH2 groups leading to an increase of the polycationic character of CMCh, so it showed higher antibacterial activity [49]. The CMCh pure hydrogel (Z0) showed poor antibacterial property (Fig. 5A and 5B), it may be due to the crosslinking of CMCh by the reaction of −NH2 group with ECH causing a decrease of positive charge on the resultant hydrogel, or might be due to the poor dispersion of hydrogel in PBS solution. Moreover, distinctive antibacterial mechanisms of ZnO nanoparticles have been discussed in literature including the direct contact of ZnO nanostructure with cell walls, resulting in the destruction of bacterial cell integrity. The production of reactive oxygen species like H2O2, hydroxyl radicals (•OH) and singlet oxygen (1O2), and the liberation of Zn2+, which may cause the killing of bacterial population [50].

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A

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Viability / %

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NB NB Control CMCh Z0

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Control CMCh Z0

Z1

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Fig. 5. Comparison of antibacterial activities of neat CMCh and CMCh/ZnO hydrogels of different concentration of ZnO against S.aureus (A) and E.colli (B); typical antibacterial activity of Z3 hydrogel at different time intervals against S.aureus (C) and against E.colli (D). 5. Conclusion In this study we demonstrated facile synthesis of CMCh/ZnO nanocomposite hydrogels via in situ incorporation of ZnO nanorods into the crosslinked CMCh hydrogels, which were prepared by reacting CMCh with ECH in an alkaline medium. XRD and SEM measurements clearly showed that ZnO nanorods were formed in the hydrogel matrix in the size range of 190 nm to 600 nm. The swelling capacity of the CMCh/ZnO nanocomposite hydrogels was 17

dependent on both the pH value and the abundance of ZnO nanorods in the CMCh hydrogels. Antimicrobial activity of the nanocomposite hydrogels was examined against E. coli and S. aureus according to CFU assay. The results showed an excellent antibacterial activity against both kinds of bacteria. The future study will be focused on the biocompatibility and biodegradability of CMCh/ZnO nanocomposite hydrogels.

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Scheme and figure captions Scheme 1. Schematic representation of the crosslinking of CMCh with epichlorohydrin in alkaline solution. Scheme 2. Schematic representation of the incorporation of ZnO into the crosslinked CMCh hydrogel. Fig. 1. FTIR spectra of as-prepared CMCh (a) and crosslinked CMCh hrdrogel (b). Fig. 2. XRD patterns of neat CMCh hydrogel (a) and CMCh/ZnO nanocomposite hydrogel of Z3 (b). Fig. 3. SEM images of neat CMCh hydrogel (a) and CMCh/ZnO nanocoposite hydrogels with prepared different concentrations of zinc nitrate: (b) 0.005 M; (c) 0.010 M; (d) 0.020 M. Fig. 4. Swelling behavior of CMCh/ZnO nanocomposite hydrogel in different pH solutions. Fig. 5. Comparison of antibacterial activities of neat CMCh and CMCh/ZnO hydrogels of different concentration of ZnO against S.aureus (A) and E.colli (B); typical antibacterial activity of Z3 hydrogel at different time intervals against S.aureus (C) and against E.colli (D).

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