polyvinyl

Journal of Molecular Liquids 250 (2018) 335–343

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Nanocomposite framework of chitosan/polyvinyl alcohol/ZnO: Preparation, characterization, swelling and antimicrobial evaluation Zizi I. Abdeen a, Ahmed F. El Farargy b, Nabel A. Negm a,⁎ a b

Petrochemicals Department, Egyptian Petroleum Research Institute, Nasr City, Cairo, Egypt Chemistry Department, Faculty of Science, Zagazig University, Egypt

a r t i c l e

i n f o

Article history: Received 9 November 2017 Received in revised form 2 December 2017 Accepted 7 December 2017 Available online 09 December 2017 Keywords: Chitosan Chitosan-polyvinyl alcohol polymer ZnO nanocomposite Swelling properties Antimicrobial activity

a b s t r a c t Chitosan was prepared by deactylation of chitin. Chitosan (CH)/polyvinyl alcohol (PVA) hydrogel and CH/PVA/ ZnO nanocomposite were prepared using gluteraldehyde as crosslinking agent. The structures of CH, CH/PVA and CH/PVA/ZnO were investigated using Fourier transformer infrared spectroscopy (FTIR) and scanning electron microscopy (SEM). The factors affect the swelling properties of the prepared polymers were studied including: cross linker ratio, temperature, pH and immersion time. The properties of the prepared hydrogels were studied and discussed including the mechanical properties and the thermal stability. The evaluated biocidal activities of the different compounds were performed using inhibition zone diameter measurements. The antimicrobial activities of the prepared CH/PVA polymer were higher than the conventional biocides of Erythromycin and Metronidazole; while the activity was highly improved after ZnO nanocomposites formation. It was found that the increase of ZnO ratio in the formed nanocomposite increases their antimicrobial activities against gram positive and gram negative bacteria. © 2017 Published by Elsevier B.V.

1. Introduction Nanocomposites are new class of materials made by loading different nanoparticles such as metals and metal oxides nanoparticles on polymeric substrates. The metal-polymer nanocomposites are obtained by either in- or ex-situ techniques [1–4]. In in-situ technique, metal nanoparticles are created in the polymeric matrix via decomposition (e.g., thermolysis, photolysis, radiolysis), or reduction of metallic precursors dissolved in the solution. In ex-situ technique, metal nanoparticles are generated using soft-reduction route followed by dispersion in the polymeric solution [5–9]. The inorganic nanoparticle materials are dispersed in the polymer medium to enhance the materials performances such as structural, physical, chemical, swelling, optical, electrical, and mechanical characteristics. Among the various inorganic nanoparticles, ZnO nanoparticles (ZnO-NPs) have acquired wide considerations due to their wide employment in diversity of applications including: functional devices, catalysts, pigments, optical materials, cosmetics, UV-absorbers, and additives in many industrial products [10–12]. Recently, the biocidal efficacy of ZnO-NPs was extensively reported [13]. The dispersion of nanoparticles in polymeric framework has significant influences on the ultimate characteristics of nanocomposites. ⁎ Corresponding author. E-mail address: [email protected] (N.A. Negm).

https://doi.org/10.1016/j.molliq.2017.12.032 0167-7322/© 2017 Published by Elsevier B.V.

Hydrogels usually have well-defined structures that can be modified to yield functionality and realize profile. This has attracted the research trends toward its claim in antibacterial application either by direct use of the polymer for its antibacterial property or, by combining antibacterial agents on the polymeric hydrogel framework [14–16]. Chitosan is a naturally occurring polymer produced in a bulk quantity from the shells of crustaceans as by-product and showed promising characteristics in fabricating antibacterial hydrogels owing to its innate antibacterial property [14–16]. Other promising characteristics include: formation of gel substrates, ability to dissolve in acidic medium, and lack of toxicity, which make it suitable in different biomedical applications. However, the poor mechanical properties of chitosan considered the main drawback of chitosan hydrogel during its swelling which minimizes its application in disinfection purposes. Enhancing the mechanical and physical properties can be performed by physical blending, chemical modification, polymer network interpenetrating and crosslinking [14–16]. Chemical crosslinking of chitosan with high mechanical, chemical and hydrophilic polymers as polyvinyl alcohol (PVA) leads to assembly of hydrogel composites with good mechanical, chemical and physical properties [17]. Recently, a few studies have shown the use of chitosan/PVA hydrogels for antimicrobial and food packing application [17]. CH/PVA have attracted more attention as biodegradable polymers as a result of their excellent biocompatibility and suitable physical properties, which can be used in environmentally friendly materials such as packaging sheets, membrane filtration, dye

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adsorption and biomedical materials for controlled release, improved comfort, reduced irritation, and tissue engineering [14–16]. PVA polymer is widely applied due to it is not expensive, non-toxic, biocompatible, highly durable and chemically stable [18–19]. Several studies on the CH/PVA films have been reported [20–22]. Chitosan/PVA copolymers have higher tensile strengths than the individual components. In this study, chitosan-polyvinyl alcohol copolymer was prepared using gluteraldehyde as a cross linker. The prepared polymer was loaded by ZnO nanoparticles at different ratios and the properties of the obtained nanocomposites were investigated including mechanical and thermal properties. The prepared polymers were evaluated for their potential in the antimicrobial field against gram positive, gram negative bacteria and fungi. The prepared systems of hydrogels nanocomposites can be widely used in medical applications such as manufacturing of medical tissues which used as a covering for injuries or after the medical operations. In industrial applications, these compounds can be used as additives for paints or in covering the surfaces required continuous sterilizing as IC or surgical operations rooms.

nanocomposites of cross-linked CH/PVA polymers were prepared by adding different zinc oxide nanoparticles ratios of 0.5–3.5% to the homogenized CH/PVA. The prepared polymers and nanocomposites were poured in glass plates and kept for two days at room temperature. The obtained films were washed with water to eliminate unreacted glutaraldehyde and kept in de-ionized water at 25 °C. 2.3. Measurements 2.3.1. Degree of deactylation of chitosan FTIR spectroscopy of chitosan was used to determine its deacetylation degree by using (JASCO, Nicolet IS-10, Japan). Chitosan solution in 2% acetic acid was subjected to infrared spectroscopy and two absorption peaks were determined at 1660, 3450 cm−1 to calculate its deactylation degree (DD%) using Eq. (1) [21]: DD% ¼ 100−

A1660 100=1:33 A3450

ð1Þ

where: A: absorption bands intensities of amide group (at 1660 cm−1) and hydroxyl group (at 3450 cm−1).

2. Experimental 2.1. Materials PVA used in this study is analytical grade with average Mwt. of 127,000 and 89% degree of hydrolysis. Hydrochloric acid, oxalic acid, potassium permanganates, potassium hydroxide, gluteraldehyde, and sodium hydroxide were purchased from Merck (Germany) and used without purification. Zinc oxide nanoparticles (Merck, Germany) were 20 nm with a specific surface area of ≈ 60 m2/g. The shrimp shells were collected, washed thoroughly and dried at 65 °C for 1 day until complete dryness. 2.2. Reactions 2.2.1. Preparation of chitosan Shells of the shrimp were decalcificated using 1.0 M HCl (3.0% w/v) at 25 °C under stirring for 90 min; then filtered, washed, dried, and treated by 4% NaOH solution at 55 °C under stirring for 5 h to eliminate any contaminated proteins. The product was filtered and washed using deionized water until neutralization (pH = 7), followed by dehydration with methanol/acetone mixture, and finally dried in oven at 40 °C for 24 h. To remove its odor and color, the dried chitin was treated by 0.1% KMnO4 and 15% oxalic acid solutions, and then filtered, washed and dried. Chitosan was prepared by refluxing chitin and a solution of NaOH (50% w/v) in a three-necked flask under nitrogen flow at 135–140 °C for 2 h, and then filtered, washed with bi-distilled water, and dried (Scheme 1) [21–23]. 2.2.2. Preparation of CH/PVA and CH/PVA/ZnO Aqueous solution of polyvinyl alcohol (100 mL, 10% w/w) was mixed with chitosan solution (20 g chitosan dissolved in 100 mL acidified water by 2% acetic acid) in three-necked flask and stirred for 5 min, followed by addition of gluteraldehyde as a crosslinking agent (in a ratio of 1–5% compared to chitosan amount added). KOH solution (0.01 g/10 mL H2O) was drop wisely added and homogenized. The

2.3.2. Molecular weight of chitosan The molecular weight of the deacetylated chitosan was determined by measuring the viscosity of chitosan solution at 25 °C. The viscosityaverage molecular weight was considered using Mark-Houwink Eq. (2) [22]: ½η ¼ Km Mva

ð2Þ

where: Km = 8.93 × 10−4, a = 0.716, and 25 °C for chitosan polymer, solvent and temperature. 2.3.3. Degree of swelling (DS) A known weight of CH/PVA and CH/PVA/ZnO films were immersed in solutions of different pH (5–8) for different periods at temperatures of 5, 25, 50 °C till attaining the swelling equilibrium. Then, the films were dried with filter papers to get rid the excess water and weighed. The influences of swelling time, temperature and gluteraldehyde ratio on the swelling ability were calculated using Eq. (3) [23]: DS ¼ ðmw −md Þ=md

ð3Þ

where: mw and md: weights of wet and dry samples, respectively. It is noticeable that the polymers were soluble at pH lower than 5, and no change was observed at pH values higher than 8. 2.3.4. Scanning electron microscopy The surface morphologies of cross linked CH/PVA polymer sheets were examined using scanning electron microscopy (SEM) Model TOPCON ABT-150 S (Japan). The samples were hanged on metal grids and coated under vacuum with gold vapor before scanning. 2.3.5. Thermal analysis Thermogravimetric profiles of the prepared polymers in the temperature range of 25–700 °C were carried out under nitrogen

Scheme 1. Preparation of chitosan by deactylation of chitin.


atmosphere using Nietzsche DSC 204 (Germany) at heating rate of 10 °C/min.

2.3.6. Mechanical properties The mechanical properties of the prepared polymers (dry and wet polymers) were measured using Instron type universal testing machine (USA) with a tensile speed of 10 mm/min.

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2.3.7. Antimicrobial evaluation The antimicrobial efficacies of the prepared copolymers and their ZnO nanocomposites were measured against a wide range of microorganisms in concentration of 5 mg/mL using diffusion agar technique [24]. The tested microorganisms were: Gram positive bacteria (Bacillus subtilis, Staphylococcus aureus), Gram negative bacteria (Escherichia coli and Pseudomonas aeruginosa), Yeast (Candida albicans) and Filamentous Fungus (Aspergillus niger). The bacteria and yeast were

Fig. 1. a: FTIR spectra of chitosan polymer; b: Cross linked PVA/chitosan copolymer; c: PVA/chitosan/ZnO nanocomposite.

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grown on nutrient agar while the fungus was grown on Czapek's Dox agar medium [25]. DMF was used as a solvent and Erythromycin was used as antibacterial reference, while Metronidazole was used as antifungal and anti-yeast reference. The measurements were performed in triplicates and their average was considered.

3. Results and discussion 3.1. Compounds characterization 3.1.1. Infrared spectroscopy (FTIR) Chitosan is produced from deacetylation reaction of chitin using aqueous solution of sodium hydroxide. FTIR spectroscopic analysis is used to determine its structure and degree of deacetylation. Fig. 1a represents the FTIR spectra of the prepared chitosan. It is clear from Fig. 1 the appearance of broad absorption band at 3500–3300 cm−1 contributed to the stretching of the primary amine (NH2) and the hydroxyl groups (OH). The absorption bands at 2925 and 2882 cm−1 are attributed to symmetric and asymmetric stretching of CH2 groups of chitosan polymer [26]. While, the absorption bands at 1647 and 1078 cm−1 are ascribed to bending of\\NH group and C\\O stretching, respectively. The absorption bands at 1429 and 1427 cm−1 corresponded to the \\COOCH3 (deacetylated hydroxyl groups), and two medium intensity bands at 1652 and 1620 cm−1 referred to C_O stretching of acetyl groups. Fig. 1b shows the FTIR spectra of the cross linked CH/PVA copolymer. It represents a broad combined absorption band in the range of 3627–2600 cm− 1 assigned for stretching vibrations of the primary amine and hydroxyl groups (NH2 and OH) [27]. A broad absorption band was appeared in the range of 3125–3550 cm− 1 and ascribed to the hydrogen bonding occurred between the hydrogen atoms of \\OH and \\NH2 in chitosan skeleton and the oxygen atom of the carbonyl groups of the gluteraldehyde structure. A wide broad band was appeared at 1095 cm− 1 referred to the crosslinking of CH/PVA and gluteraldehyde [26], in addition to a narrow absorption band at 1150–1130 cm − 1 which prove the formation of acetal group as a result of the crosslinking between CH/PVA and gluteraldehyde. Fig. 1c represents the FTIR spectra of CH/PVA/ZnO nanocomposite. It is clear that the characteristic bands were shifted to lower wavenumbers and became stronger; the absorption band at 3427 cm−1 corresponded to the stretching vibration of hydroxyl, amino and amide groups were noticeably moved to lower wavenumbers of 3390 cm− 1, which indicates the strong interaction between these groups and ZnO nanoparticles. The absorption bands at 662 and 865 cm−1 are appeared due to the stretching mode of amide groups attached to ZnO nanoparticles [28].

3.1.2. Degree of deacetylation The deactylation degree (DD%) of the chitosan represents the extent deacetylation occurred to the acetyl groups in the chitin chains. The calculated deacetylation degree of the prepared chitosan using Eq. (1) was found to be 61.8%.

3.1.3. Molecular weight of chitosan The viscosity-average molecular weight (Mwtv) of the prepared chitosan was determined by measuring the intrinsic viscosities of chitosan solutions in 0.3 M acetic acid and 0.2 M sodium acetate at 25 °C using Ostwald viscometer and applying Eq. (2) [23]. Fig. 2 represents the linear variation of intrinsic viscosity for different concentrations of chitosan solutions at 25 °C. The obtained molecular weight of the deacetylated chitosan was 156,000 Da (based on viscosity measurements).

Fig. 2. Dependence of chitosan solution viscosity on its concentration at 25 °C.

3.2. Swelling characteristics of the prepared hydrogels The factors affect the swelling properties of the prepared hydrogels were determined including: cross linking agent ratio, pH of the medium, temperature and immersion time. Chitosan polymer contains amino groups (NH2) which can easily protonated to form ammonium group (NH+ 3 ) in the acidic solution (acetic acid). That enhances its solubility in the aqueous medium. On the other hand, PVA polymer has large number of hydroxyl groups which form hydrogen bonds by water molecules and consequently its solubility in aqueous medium is large. As a result, CH/PVA polymer has enhanced ability to swell in the aqueous medium. The swelling characteristics of the prepared hydrogels were determined under different conditions, and the factors which affect their swelling were studied including: the cross linking, pH, temperature and immersion time.

3.2.1. Effect of cross linking The solubility of water soluble polymers can be controlled by the cross linking using suitable cross linkers. The prepared CH/PVA polymer is completely water soluble, and to enhance its hydrogel property (swelling tendency), cross-linking takes place using gluteraldehyde as cross-linker. Fig. 3a represents the swelling degree of CH/PVA polymer cross linked by different ratios of gluteraldehyde (1, 2, 3, 4 and 5%). It is clear from Fig. 3 that the degree of swelling of CH/PVA cross linked polymer is increased by increasing the ratio of gluteraldehyde from 1% to reach the maximum at 2%, and then starts to decrease to reach the minimum at 5% of gluteraldehyde. That can be attributed to the validity of the hydroxyl groups and the amino groups in the polymeric segments of CH/PVA. At 1% content of gluteraldehyde, the solubility of the polymer being high and no swelling tendency can be determined due to the high validity of the hydroxyl and amino groups within the chains by the aqueous medium. At this low ratio, the polymer segments are flexible and can interact by large number of water molecules which increases their solubility in the aqueous medium. Increasing the cross linker ratio (gluteraldehyde) to 2% restricts the mobility of the chains and consequently decreases their solubility in the aqueous medium. But, the available hydroxyl and amino groups in the medium are still large. Hence, the interaction between these groups and the water molecules becomes in the maximum value, and consequently the degree of swelling increased considerably to reach to 24%. Increasing the cross linker ratio to/and over 3% has two functions. First, decreases the number of amino groups in chitosan segments as they react by the gluteraldehyde molecules. Second, decreases the flexibility of the polymer segments due to the cross linking, which decreases the availability and the interaction of the polar groups in the polymer segments with the aqueous medium. That decreases the amount of adsorbed water by the polymer chains and consequently, the degree of their swelling. The minimum degree of swelling was observed in the presence of 5% cross linker ratio at 10%.


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Fig. 3. Effect of cross linker (GA) ratio on the swelling of: a: CH/PVA polymer at 25 °C; b: CH/PVA/ZnO nanocomposite at 25 °C.

The degree of swelling of CH/PVA/ZnO nanocomposite (Fig. 3b) has similar trend as represented in case of CH/PVA polymer. The swelling of the nanocomposite was decreased than the parent polymer to reach the maximum value of 14% at 2% cross linker ratio. That can be attributed to the participation of the polar groups of the polymer segments in the formation of electrostatic bonds by ZnO nanoparticles. The electrostatic interaction between the polar groups and the nanoparticles (observed from the shifts of their wave numbers in IR spectra) decreases the number of active polar groups which are responsible for swelling of the prepared copolymers in the aqueous medium [29]. 3.2.2. Effect of pH The swelling tendency of the hydrogel is strongly related to the available hydrophilic (polar) groups in their segments and also depends on their nature. Strong interaction between the polar groups and aqueous medium increases the polymer swelling, and vice versa. Fig. 4 represents the effect of pH of the medium (pH = 5, 7, 8) on the degree of swelling of the prepared polymers. It is clear from Fig. 4a that the maximum degree of swelling was obtained at neutral medium, i.e., pH = 7 for all cross linker ratio. The swelling properties of CH/PVA polymer are strongly dependent on the pH of the medium. That can be attributed to the nature of the polar groups within the polymer

segments in the aqueous medium at the different pH values. In alkaline medium (pH = 8), the amino groups are strongly deactivated and the lone pairs of electrons on the nitrogen atoms became unavailable due to the mutual repulsion with the hydroxyl groups in the medium. As a result, the interaction between the amino groups and the water molecules is inhibited, which consequently decreases the water uptake and the swelling of the polymer chains. In acidic medium (pH = 5), the hydroxyl groups of PVA and chitosan chains are protonated and acquiring positive charges, which increases the mutual repulsion with the water molecules. That decreases the polymer swelling at the various cross linking ratios. The variation of cross linking ratio within CH/PVA polymer has similar trend. At neutral medium (pH = 7), the polar groups of OH and NH2 have high validity to interact by water molecules to form hydrogen bonds. The neutral medium does not change their polarity, and consequently their interaction by the water molecules. As a result, the swelling tendency of the polymer at pH = 7 in its maximum value compared to the swelling at different pH values for the same cross linking ratio. The effect of pH on the swelling properties of CH/PVA/ZnO nanocomposite is represented in Fig. 4b. Generally, the behavior of swelling properties of CH/PVA/ZnO nanocomposite is similar to that of the parent polymer (CH/PVA). It is clear that the formation of the nanocomposite

Fig. 4. Effect of pH on the swelling of: a: CH/PVA polymer at 25 °C; b: CH/PVA/ZnO nanocomposite at 25 °C.

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decreases the swelling properties compared to the parent polymer at the entire pH range of the medium, i.e., pH = 5, 7, 8. The decreases in the swelling properties of the nanocomposite at the different pH medium can be attributed to the participation of the polar groups of the polymer segments in formation of the nanocomposite. The decrease of the swelling by nanocomposite formation follows the same trend of CH/ PVA polymer but with lower extent. 3.2.3. Effect of temperature Fig. 5 represents the influence of increasing the temperature on the swelling properties of the prepared hydrogels. It is clear from Fig. 5a that the swelling is at the lowest extent at 5 °C. The gradual increase in the temperature gradually increases the swelling to reach its maximum at 25 °C. Then, it steeply decreased by rising the temperature to 50 °C. This behavior can be ascribed due to the effect of temperature on the hydrogen bond formation between the polar groups (OH, and NH2) in the prepared polymer chains and the water molecules in the medium; and also to the stability of the formed hydrogen bonds at the various temperatures. The formation of hydrogen bonds required energy of formation, which is absorbed from the medium. Increasing the temperature of the medium increased the activation energy of the medium and consequently the H-bonding accelerated. At low temperatures (5 °C), the hydrogen bonds formed in a very slow rate which reflected on the swelling of the polymer. Increasing the temperature increases the agitation of the medium and consequently the hydrogen bonds are easily formed between the polar groups and the water molecules. Continuous increasing in the temperature leads to breakdown of the formed hydrogen bonds as the free energy of the molecules increased, which gradually decreased the swelling of the polymers. Decreasing the number of the polar groups in the polymeric chains appears significantly in case of CH/PVA/ZnO nanocomposite, which leads to decrease its swelling at higher temperatures compared to the parent polymers, Fig. 5b. From Fig. 5a–b, the extent of cross linking has a vital influence on the swelling of the prepared hydrogels and their nanocomposites. That can be attributed to the effective number of polar groups in case of the polymer and its nanocomposite at each cross linker ratio. 3.2.4. Effect of immersion time Fig. 6 represents the variation of degree of swelling of the prepared polymer and nanocomposite by immersion in aqueous medium for different immersion time. It is clear that swelling of the prepared polymer and its nanocomposite is increased by increasing the immersion time to reach the maximum swelling after 100 and 60 min for CH/PVA and CH/

Fig. 6. Effect of immersion time on the swelling of CH/PVA and CH/PVA/ZnO nanocomposites at 25 °C.

PVA/ZnO, respectively. The degree of swelling of the polymer in the aqueous medium depends on the contact between the polar groups and the water molecules. Longer immersion time facilitates the diffusion of water molecules into the polymer segments and consequently large number of polar groups forms hydrogen bonds by the water molecules. The fast saturation of the nanocomposites is ascribed to the lower number of polar groups in the nanocomposite segments. 3.3. Thermal properties (Thermogravimetric analysis, TGA) TGA measurements were performed to determine the compatibility of the PVA, chitosan, and zinc oxide components. Fig. 7a represents the TGA of the prepared chitosan and it was comparable to the reported data [30–32]. The thermogram of chitosan showed two characteristic endothermic decomposition processes. The first is located around 60 °C accompanied by the evaporation of the water molecules adsorbed on the chitosan segments via hydrogen bonds. The second is located around 225 °C and reaches the maximum 260 °C ascribed the dehydration of chitosan segments via removal of hydroxyl groups along the polymer segments. The thermal degradation of chitosan is followed by a broad decomposition process around 360 °C accompanied by charring of the residue and the total loss reached to 39% at 450 °C. Fig. 7b, c showed TGA curves of CH/PVA and CH/PVA/ZnO nanocomposite hydrogels. All the samples showed a weight loss of more or less 10%

Fig. 5. Effect of temperature on the swelling of: a: CH/PVA copolymer; b: CH/PVA/ZnO nanocomposite.


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incorporation of the ZnO nanoparticles by CH/PVA, indicating that the thermal stability of CH/PVA polymer was enhanced by incorporation of ZnO nanoparticles in the CH/PVA segments. 3.4. Mechanical properties The swelling behavior of the polymer is intimately related to the structural and mechanical properties of the hydrogel; therefore, its elastic behavior is highly dependent on the quantity of liquid that can be absorbed. In hydrated phase, most hydrogels behave like elastomers; as a result, their mechanical behavior is nonlinear. This mainly depends on the polymer network architecture [22]. Fig. 8a–b represents the mechanical response in term of tensile strength of CH/PVA polymer and CH/PVA/ZnO nanocomposite hydrogels. The tensile strength profiles of the prepared polymers showed that the mechanical behaviors of the hydrogels are not similar. This may be due to changes in the crosslinking and polymerization processes, swelling conditions, or even the inherent mechanical anisotropy of these materials. It is found that an increasing in strength of CH/PVA film under stress can, in general, be attributed to either the strain-dependent increase in hydrogen bonding [21,33]. In addition, it was also reported for that cross-linking of hydrogels become less flexible and increases their tensile strength. The addition of (1.5%) zinc oxide nanoparticles improved the mechanical properties to PVA/CH/ZnO nanocomposite hydrogel. Increasing the zinc oxide nanoparticle content than (1.5%) showed a decrease in the tensile strength values of the hydrogels nanocomposite, i.e., the compound became more brittle. 3.5. Scanning electron microscopy (SEM)

Fig. 7. Thermogravimetric analysis of: a: chitosan; b: CH/PVA; c: CH/PVA/ZnO nanocomposite.

within 100 °C. The major weight losses were observed in the temperature range of 250–350 °C. It is also clear that the onset degradation temperature of the nanocomposites is slightly increased by the

Fig. 9a–d shows the SEM (scanning electron microscopy) micrograph of the prepared chitosan, CH/PVA polymer and CH/PVA/ZnO nanocomposite. It is visible from chitosan micrograph (Fig. 9a) its smooth surface, this is in accordance with the reported studies [31]. The SEM image of CH/PVA film (Fig. 9b) shows that the surface is quite rough and dense in nature, which provides the maximum surface area [32]. The SEM image of pure zinc oxide nanoparticles is shown in Fig. 9c, the average nanosize of the particles is ranged between 30 and 35 nm. Fig. 9c showed the agglomerations of ZnO particles. As shown in Fig. 9c, the larger irregular shape particles of several nanometer sizes were obtained without separating medium between particles to permit the as produced particles coalescence easily. Combination of zinc oxide nanoparticles and polymer network significantly decreased in particle size of polymer forming nanocomposites as shown in Fig. 9d.

Fig. 8. Tensile strength of: (a) CH/PVA polymer at different cross linking ratio; (b) CH/PVA/ZnO nanocomposite at 25 °C.

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Fig. 9. The SEM images of: a: chitosan, b: CH/PVA and c: zinc oxide nanoparticles.

3.6. Antimicrobial activity The samples were evaluated for antibacterial activity against Gram positive bacteria (Bacillus subtilis, Staphylococcus aureus), Gram negative bacteria (Escherichia coli and Pseudomonas aeruginosa), yeast (Candida albicans) and fungus (Aspergillus niger). Erythromycin was used as standard biocide, and metronidazole was used for fungi and yeast which showed 12 mm and 11 mm in inhibition zone diameter measurements. Table 1 lists the antimicrobial activities of the prepared chitosan, CH/PVA polymer, CH/PVA/ZnO nanocomposite, in addition to ZnO nanoparticles using inhibition zone diameter measurements, and the results were compared to the standards used. The cell membrane of microorganisms is composed of several lipids and protein layers arranged together in specific arrangement called the bi-layer (or multilayer lipoprotein structure) [34]. The presence of the

lipids as building units in the cell membrane gave them their hydrophobic characters [35]. It is clear from Table 1 that all the tested compounds exhibit antimicrobial activities higher than the standards used. The prepared chitosan has comparatively higher antimicrobial activities toward the tested microorganisms, which can be attributed to its natural structure. The natural source of the chitosan makes it easily to interact with the cellular membrane and eases its penetration into the cellular membrane. Modification of the chitosan structure by polyvinyl alcohol increases its polarity, which consequently decreases its penetration into the cellular membrane due to the increase of the chains polarity. That decreases the antimicrobial activity to lower extent than chitosan itself, as can be seen from Table 1. The action mode of the nanoparticles in antimicrobial action is quite differing from the ordinary biocides. The major contribution for the

Table 1 Antimicrobial activity of chitosan, CH/PVA polymer, zinc oxide nanoparticles, CH/PVA/ZnO (1.5%) nanocomposite, and CH/PVA/ZnO (3.5%) nanocomposite. Compounds

Chitosan CH/PVA CH/PVA/ZnO (1.5%) nanocomposite CH/PVA/ZnO (3.5%) nanocomposite ZnO nanoparticles Erythromycin Metronidazole

Tested microorganisms B. subtilis

S. aureus

E. coli

P. aeruginosa

C. albicans

A. niger

23 22 30 33 36 12 –

20 13 22 24 33 12 –

19 12 21 23 35 12 –

18 12 20 22 35 12 –

12 11 13 18 27 – 11

11 10 14 20 29 – 11


nanoparticles in the biocidal process is the interaction by the cellular membrane due to their extremely small size and their ability to its penetration, after that, the nanoparticles release reactive species by the interaction with the inert environment of the cells. These reactive species include ionic or free radical threats which attack the enzymes and DNA in the cellular nucleus, which leads to microbial growth inhibition of microbial death. The maximum antimicrobial activity was obtained in case of pure ZnO nanoparticles, due to its above discussed action on the microorganisms. Loading of ZnO nanoparticles on CH/PVA polymer with two loading ratios (1.5% and 3.5%) showed gradual decreases in the antimicrobial activity than the pure ZnO nanoparticles. Comparing the two loaded nanocomposites CH/PVA/ZnO (1.5%) and CH/PVA/ZnO (3.5%) revealed that the latter is more toxic than the former, which can be attributed to the higher ZnO nanoparticles content. 4. Conclusions The study of chitosan modification with polyvinyl alcohol and ZnO nanoparticles revealed several concluding points: 1. The cross linking of chitosan affect its swelling properties. 2. Increasing the temperature increases the swelling of chitosan hydrogel until 25 °C. 3. At pH 7, the maximum swelling of CH/PVA can be obtained. 4. Tensile strength of CH/PVA is varied depending on the cross linking ratio. 5. The antimicrobial activity of CH/PVA is extremely increased by loading ZnO nanoparticles on the polymer matrix. References [1] Z.H. Mbhele, M.G. Salemane, C.G.C. Sittert, J.M. Nedeljkovic, V. Djokovic, A.S. Luyt, Chem. Mater. 15 (2003) 5019.

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