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CuO bio-nanocomposite hydrogel beads
Accepted Manuscript Title: Facile synthesis of antibacterial chitosan/CuO bio-nanocomposite hydrogel beads Author: Sana Farhoudian Mehdi Yadollahi Hassan Namazi PII: DOI: Reference:
S0141-8130(15)30023-4 http://dx.doi.org/doi:10.1016/j.ijbiomac.2015.10.018 BIOMAC 5432
To appear in:
International Journal of Biological Macromolecules
Received date: Revised date: Accepted date:
23-8-2015 2-10-2015 6-10-2015
Please cite this article as:http://dx.doi.org/10.1016/j.ijbiomac.2015.10.018 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.
Facile synthesis of antibacterial chitosan/CuO bio-nanocomposite hydrogel beads Sana Farhoudiana, Mehdi Yadollahia*[email protected], Hassan Namazia,b*[email protected] a
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Research Laboratory of Dendrimers and Nanopolymers, Faculty of Chemistry, University of Tabriz, P.O. Box 51666-16471, Tabriz, Iran b Research Center for Pharmaceutical Nanonotechnology, Tabriz University of Medical Science, Tabriz, Iran
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Tel.: +98413339321
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Abstract CuO nanoparticles were synthesized in situ during the formation of physically cross-linked chitosan hydrogel beads using sodium tripolyphosphate as the cross-linker. The aim of the study was to investigate whether these nanocomposite beads have the potential to be used in drug delivery applications. The formation of CuO nanoparticles (CuONPs) in the hydrogels was confirmed by X-ray diffraction and scanning electron microscopy studies. SEM micrographs revealed the formation of CuONPs with size range of 10–25 nm within the hydrogel matrix. Furthermore, the antibacterial and swelling properties of the beads were studied. The prepared nanocomposite hydrogels showed a pH sensitive swelling behavior. The CuO nanocomposite hydrogels have rather higher swelling in different aqueous solutions in comparison with neat hydrogel. The nanocomposite hydrogels demonstrated good antibacterial effects against Escherichia coli and Staphylococcus aureus bacteria.
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Keywords Chitosan bead, Antibacterial, Bio-nanocomposite
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1. Introduction During recent decades, biopolymers have been widely used as raw materials for the preparation of hydrogels owing to their excellent properties, such as non-toxicity, biocompatibility, biodegradability and environmental sensitivity, etc [1-3]. Chitosan is one of the most abundant natural biopolymers derived by the deacetylation of chitin. Chitosan has gained many attractions in the pharmaceutical and medical applications [4, 5]. On the other hand, in the presence of polyanionic molecules, chitosan can form spherical gel beads. Chitosan in the form of the gel beads has attracted much attention as a drug controlled release formulation owing to the simplicity and mildness of this process and formation of uniform and small size shape as compared with the conventional hydrogels [6-8]. Recently, there has been a great interest to generate antibacterial hydrogels because of their superior biomedical relevance [9]. Among antibacterial hydrogels, inorganic-based nanocomposite hydrogels are particularly promising for bacterial inactivation applications in materials and engineering science. These antibacterial agents possess a great potential to inhibit microbial growth. Because of they are easily functionalized with inorganic materials and biocompatible, this characteristic makes them attractive in the biomedical and biotechnological fields [10, 11]. At neutral pH, chitosan does not show any antibacterial activity; in order to impart that activity, it is necessary to incorporate some antibacterial agents into it [12]. Chitosan has previously been 1
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investigated as matrix to incorporate antibacterial nanoparticles like ZnO, Ag or Au nanoparticles [13-15]. It is well-known that copper oxide nanoparticles (CuONPs) possesses antibacterial activity. CuONPs are attractive antibacterial agents because in addition to their lowcost and easy release out of human body, they are known to have significant antibacterial properties. On the other hand, an added advantage of copper nanoparticles is that they oxidize and form copper oxide nanoparticles, which can easily mix with polymers or macromolecules and are relatively stable in terms of both chemical and physical properties [16]. Therefore, Copper-based nanocomposites are of great notice. Recently, nanocomposites of copper with several polymers such as chitosan [17, 18], polyacrylic acid [19], polypropylene [20] and cellulose [21], have been successfully prepared. In consideration of the mentioned concerns, a combination of chitosan hydrogel with CuONPs is quite attractive and, to the best of our knowledge, there is still no related report. In this study, novel chitosan/CuO nanocomposite hydrogels (CH/CuONPs) were successfully prepared by in situ formation of CuONPs in the chitosan hydrogel matrix. The structures of the nanocomposite hydrogels were characterized using X-ray diffraction (XRD) and scanning electron microscopy (SEM). The effect of the concentration of the CuONPs on the swelling behavior and antibacterial effect for the gram-negative E. coli and gram-positive S. aureus bacteria was investigated. 2. Materials and methods 2.1. Materials Chitosan, medium molecular weight, and viscosity 200-800 cP, 1 wt. % in 1% acetic acid (25 °C, Brookfield) and Sodium tripolyphosphate (STPP, technical grade 85%) was obtained from Aldrich. CuCl2.2H2O, NaOH were purchased from Merck and used as received. Distilled water was used throughout this study.
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2.2. Preparation of chitosan/CuONPs nanocomposite hydrogel beads A series of chitosan/CuONPs nanocomposite hydrogel beads (CH/CuO) were prepared according to a previously reported technique [8], using sodium tripolyphosphate (STPP) as the crosslinking agent and NaOH as oxidizing agent. Typically, 1 g of chitosan and desired amount of CuCl2.2H2O (0.0, 0.5, 1.0 and 1.5 mmol) were added to 35 ml distilled water. Then, 2.5 ml acetic acid was added to the above-mentioned mixture and stirred until a clear homogenous viscose solution obtained. Thereafter, the solution was extruded in the form of droplets, using a syringe (2 mm diameter), into an aqueous solution (400 ml) containing STPP (4 g) and NaOH (3.2 g). The beads were stayed in the solution for 24 h in order to crosslink with STPP and also converting Cu ions into CuONPs. The formation of CuONPs was confirmed by changing the beads' color from pale brown to black. After that, the beads were filtered and washed several times with distilled water to remove un-reacted STPP and NaOH on the surface of beads and dried under vacuum for 24 h. In remaining of the manuscript, CH/CuO0, CH/CuO1, CH/CuO2 and CH/CuO3, means chitosan/CuONPs nanocomposite hydrogel beads, which have 0.0, 0.5, 1.0 and 1.5 mmol of CuCl2 content, respectively. 2.3. Characterization and analysis Infrared spectra was obtained on an FTIR spectrometer (Bruker Instruments, model Aquinox 55, Germany) in the 4000-400 cm-1 range at a resolution of 0.5 cm-1as KBr pellets. The pattern of Xray diffraction of the samples was obtained by Siemens diffractometer with Cu-Ka radiation at 35 kV in the scan range of 2θ from 2 to 70o and scan rate of 1o/min. All of analyzed samples were in the powdery form. The d-spacing was calculated by Bragg’s equation where λ was 0.154
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nm. The morphology of the dried samples was examined using a scanning electron microscope (SEM) (TESCAN MIRA3) operated at 5 kV after coating the samples with gold and silver films. 2.4. Antibacterial activity Antibacterial activity of the CH/CuO nanocomposite hydrogels beads was tested against both E. coli (gram-negative) and S. aureus (gram-positive) according to agar diffusion test. For agar diffusion method, samples were exposed to bacteria on solid media (nutrient agar), and the inhibition zone around each sample was measured and recorded as the antibacterial effect of CuONPs. The agar plates were inoculated with 100 µL spore suspensions of bacteria. Swelled hydrogels placed on the agar plate and incubated at 37 °C for 24 h. Inhibition zone for bacterial growth was detected visually. 2.5. Swelling behavior Swelling ratio of nanocomposite beads was measured according to the previously reported methods [3] at buffer solutions (pH of 2.1 and 7.4) at room temperature. 0.1 g of CH/CuO nanocomposite beads was immersed in 50 ml of buffer solutions with desired pH at room temperature for 500 minutes to reach swelling equilibrium. The swelling ratio of nanocomposite beads was determined according to Equation 1. Eq. (1) Where W1 is initial weight of sample, and W2 is the weight of the sample after swelling for 500 min. 3. Results and discussions 3.1. Preparations of chitosan nanocomposite hydrogel beads Chitosan is one of the appropriate bio-polymers, which are capable of crosslinking by ionic interactions. Chitosan is cross-linked in the presence of poly-anions like sodium tripolyphosphate in the aqueous mediumsw. When the chitosan solution was dropped into STPP solution, gelled spheres formed instantaneously due to the electrostatic interaction between positively charged chitosan chains and negatively charged STPP anions. In the acidic conditions, amine moieties of chitosan chains convert to ammonium cations, which can electrostatically interact with STPP anions. In this way, the water-soluble chitosan is transformed into an insoluble gel, and chitosan droplets precipitated in the bead form in aqueous STPP solution. Scheme 1 schematically represents the formation of CuONPs in chitosan hydrogel network. Chitosan interacts with many metal cations, including Ag+, Cu2+, Fe3+ and Zn2+ [22]. Due to the existence of amine (-NH2) and hydroxyl (-OH) groups, the chitosan chains easily bind to the Ag+ cations. In this way, an almost uniformly distributed array of CuO nanoparticles obtained within the chitosan matrix. This procedure is facile and economically not requiring heat or any other tools for nanoparticle synthesis. 3.2. FT-IR analysis FT-IR spectra of pure chitosan, chitosan hydrogel beads and chitosan-CuO nanocomposite beads are shown in Fig 1. The FTIR spectra of the neat chitosan exhibited a broad band of multiple peaks between 3400-3600 cm−1, which attributed to the stretching of –OH and –NH2 groups and intermolecular and intramolecular hydrogen bonds. The peak about C–H stretching associated with methane hydrogen atoms appeared at 2924 cm−1. The peaks at 1640 and 1616 cm−1 related to the absorption band of the carbonyl stretching of the secondary amide and the bending vibrations of the N-H (N-acetylated residues, amide), respectively. The absorption bands between 1300-1460 cm-1 and 1000-1200 cm−1 were ascribed to the –C–N– and –C–O– stretching on the polysaccharide skeleton, respectively [23, 24]. Compared to the FTIR spectra of neat chitosan, chitosan beads reveals a single and narrower peak in the 3400-3600 cm−1 regions. Also,
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the intense characteristic bands of chitosan at 1640 and 1616 cm−1 shifted to lower wave numbers after the formation of chitosan beads. The FTIR results confirmed that chitosan polymeric chains was cross-linked with STPP in the beads. Compared with the FTIR spectra of chitosan beads, chitosan-CuO nanocomposite beads indicates the new peaks in the 400-800 cm−1 regions. These peaks were attributed to the vibration bands of metal-oxygen (Cu-O) bonds [25]. The interaction of CuONPs with chitosan hydrogels results in a reduction in the intensity of broad band of N–H and O–H bonds (3400-3600 cm−1). These results imply that the interaction also exists between the CuO nanoparticles and chitosan molecules. This difference arises from the participation of hydroxyl and amine groups of chitosan to the metal ions of CuO nanoparticles in order to form chelating structure and consequent decrease in hydrogen bonding which affords narrower and weaker band in chitosan-CuO nanocomposite beads [26]. 3.3. XRD analysis The XRD pattern of the pure chitosan hydrogel and CH/CuO3 nanocomposite hydrogel in the 2θ range of 2-70o is shown in Fig. 2. The pristine chitosan hydrogel without CuONP demonstrates the characteristic peaks at 2θ values of about 12o and 20o,which are typical finger prints of semicrystallinity of chitosan [27]. After the formation of CuONPs, the characteristic peaks of pure chitosan hydrogel were broadening a result of presence of CuONPs. In addition, the diffractogram of CH/CuO3 nanocomposite hydrogel is assigned to new diffractions at 2θ values of about 36o, 39o, 45o and 54o, which assigned to the (002), (111), (112) and (020) diffractions of CuO crystals, respectively. All the peaks match well with those of CuO crystals and confirm the formation of CuO particles in the chitosan hydrogel matrix [28, 29]. No peaks of impurity are observed in the XRD pattern, indicating the high purity of obtained CuO particles.
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3.3. Morphology of CH/CuO nanocomposite hydrogel beads Generally, the wet beads were spherical with a diameter of about 3-4 mm and possessed a smooth surface. After air drying, the diameter of test beads decreased to about 1 mm but still kept the spherical shape. For CH/CuO nanocomposite beads, there was no obvious variation with the bead size (Fig. 3). Fig. 4 a, b and c exhibit the SEM images of the hydrogel bead surface at low magnification. As it can be seen in Fig. 4a, the surface morphology of the neat chitosan hydrogel beads showed severe wrinkles and many cavities, which was caused by partial collapsing of the polymer network during drying. The CH/CuO beads show a smooth and tight surface which becomes smoother with the increase of CuONPs content. This could be due to the interfacial interactions between chitosan chains and CuONPs (as shown in Scheme 1) which could possibly act as intermolecular cross-linkers. Consequently, the CuONPs could contract and restrict the movability of the chitosan chains, and then change the surface morphology.
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With further magnification of the beads (Fig. 4 d, e and f), CuO nanoparticles appeared on the surface of the CH/CuO hydrogel beads. SEM results showed that the CuONPs were well dispersed in the chitosan matrix for sample with 5 wt% CuONPs content and have particle size less than 25 nm (Fig. 4e). However, some aggregation and bigger particle sizes (marked with circle) can be seen on the sample with the highest nano CuO content (sample CH/ CuO 3), Fig. 4f). These results indicate a close interaction of the CuONPs with the chitosan. 3.4. Antibacterial properties The in vitro antibacterial properties of CH/CuO nanocomposite beads tested against gramnegative E. coli and gram-positive S. aureus bacteria by disk diffusion test. Inhibition zone around the tested samples for bacterial growth was detected visually and summarized in Table 1. 4
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The inhibition zones are presented in Fig. 5. The results suggest that the CuONPs embedded hydrogel beads revealed a more toxic effect on bacteria than pure chitosan hydrogel under similar conditions as evidenced by higher inhibition zone. The results in the Table 1 show that the antibacterial efficiency of the nanocomposite hydrogels is influenced by the concentration of the CuONPs regardless of the kind of bacterial used. Hydrogels with more CuONPs demonstrate greater antibacterial properties. The antibacterial effect of CH/CuO nanocomposite hydrogels could be associated to the attachment of CuO nanoparticles to the cell wall of bactericides which damages the cell wall and causing leakage of proteins and other intracellular constituents and ultimately causes cell death [16, 28, 30]. 3.5. Swelling behavior The swelling behavior of the hydrogel beads was studied in the pH of 2.1 and 7.4 in order to investigate the pH sensitivity of the prepared hydrogels. As shown in the Fig. 6, swelling increases with time, first rapidly and then slowly, reaching a maximum constant swelling. It is obvious that the swelling ratio of test beads at pH 2.1 was higher than that of pH 7.4. The swelling mechanism at pH 2.1 involves the protonation of chitosan amine groups, which leads to chain repulsion that result in diffusion of proton and counter-ions together with water inside the beads [4].
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In addition, the results in Fig. 6 shows that CH/CuO nanocomposite hydrogel beads revealed a higher swelling capacity in comparison to that of the neat chitosan hydrogel. The enhancement of the swelling capacity of the nanocomposite hydrogels may be due to the presence of CuO nanoparticles with different sizes, morphologies and surface charges. The charged CuO nanoparticles results in penetration of more water molecules in order to neutralize the build-up ion osmotic pressure [31, 32]. Furthermore, formation of CuONPs in the hydrogel could expand the hydrogel network and increases the pores and free spaces within the network and as consequence CH/CuO samples adsorb more water [33]. However, CH/CuO2 and CH/CuO3 samples revealed less swelling capacity in comparison to that of the CH/CuO1 sample. This could be attributed to the role of CuONPs as the knot tying functions, which restricts the expanding of polymer chains. The knot tying function of CuONPs may be due to the chelation of some hydroxyl and amine groups of the hydrogel networks with CuO nanoparticles [34]. 4. Conclusions In this study, antibacterial CH/CuO nanocomposite hydrogel beads were successfully prepared by in situ generation of CuONPs during the formation of chitosan beads. Structural details were provided by XRD and SEM analysis. In addition, the influence of CuONPs on the swelling, drug release and antibacterial behavior of the chitosan beads was studied. The CuO nanoparticles were successfully generated in the beads and had a clear influence on the surface morphology of the beads as evidenced by XRD and SEM analysis. CH/CuO nanocomposite hydrogel beads revealed a higher swelling capacity in comparison to that of the neat chitosan hydrogel which was dependent upon the CuONPs content. Antimicrobial activity of the hydrogels was examined on E. coli (gram-negative) and S. aureus (gram-positive) according to agar diffusion test. The CH/CuO hydrogels has shown good antibacterial activity against gram-positive and gramnegative bactericides. Hydrogels with more CuONPs demonstrate greater antibacterial properties. Based on these findings, the prepared CH/CuO nanocomposite hydrogels can be used in different medical fields. Acknowledgments
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Authors also gratefully acknowledge the University of Tabriz (grant number S/27/3243-29) for the financial supports for this research.
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Scheme1. The schematic representation of the interactions of chitosan with Cu ions and CuONPs Fig. 1. FTIR spectra of pure chitosan, chitosan hydrogel bead and CH/CuO nanocomposite hydrogel bead Fig. 2. XRD pattern of pure chitosan hydrogel bead (a) and CH/CuO nanocomposite hydrogel bead Fig. 3. Digital photo of pure chitosan hydrogel bead (a) and CH/CuO nanocomposite hydrogel bead; Fig. 4. SEM micrographs of pure chitosan hydrogel bead (a) CH/CuO1 (b) CH/CuO3 (c) at low magnification (×1000) and micrographs of pure chitosan hydrogel bead (d) CH/CuO1 (e) CH/CuO3 (f) at high magnification (×100000) Fig. 5. Photographs of the inhibition zones of CH/CuO nanocomposite hydrogel beads against (a) S. aureus (b) E. coli. Fig. 6. Swelling behavior of CH/CuO nanocomposite beads at pH values of 2.1 and 7.4 Table 1. Zones of inhibition (mm) against the tested bacteria for CH/CuO nanocomposite hydrogel beads Sample Bacteria Zone of inhibition (mm) 1 day 3 days CH/CuO0 S. aureus 0 0 E. coli 0 0 CH/CuO1 S. aureus 6 0 E. coli 6 6 CH/CuO2 S. aureus 6 0 E. coli 9 9 CH/CuO3 S. aureus 8 7 E. coli 11 10
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S0141-8130(15)30023-4 http://dx.doi.org/doi:10.1016/j.ijbiomac.2015.10.018 BIOMAC 5432
To appear in:
International Journal of Biological Macromolecules
Received date: Revised date: Accepted date:
23-8-2015 2-10-2015 6-10-2015
Please cite this article as:
Facile synthesis of antibacterial chitosan/CuO bio-nanocomposite hydrogel beads Sana Farhoudiana, Mehdi Yadollahia*[email protected], Hassan Namazia,b*[email protected] a
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Research Laboratory of Dendrimers and Nanopolymers, Faculty of Chemistry, University of Tabriz, P.O. Box 51666-16471, Tabriz, Iran b Research Center for Pharmaceutical Nanonotechnology, Tabriz University of Medical Science, Tabriz, Iran
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Tel.: +98413339321
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Abstract CuO nanoparticles were synthesized in situ during the formation of physically cross-linked chitosan hydrogel beads using sodium tripolyphosphate as the cross-linker. The aim of the study was to investigate whether these nanocomposite beads have the potential to be used in drug delivery applications. The formation of CuO nanoparticles (CuONPs) in the hydrogels was confirmed by X-ray diffraction and scanning electron microscopy studies. SEM micrographs revealed the formation of CuONPs with size range of 10–25 nm within the hydrogel matrix. Furthermore, the antibacterial and swelling properties of the beads were studied. The prepared nanocomposite hydrogels showed a pH sensitive swelling behavior. The CuO nanocomposite hydrogels have rather higher swelling in different aqueous solutions in comparison with neat hydrogel. The nanocomposite hydrogels demonstrated good antibacterial effects against Escherichia coli and Staphylococcus aureus bacteria.
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Keywords Chitosan bead, Antibacterial, Bio-nanocomposite
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1. Introduction During recent decades, biopolymers have been widely used as raw materials for the preparation of hydrogels owing to their excellent properties, such as non-toxicity, biocompatibility, biodegradability and environmental sensitivity, etc [1-3]. Chitosan is one of the most abundant natural biopolymers derived by the deacetylation of chitin. Chitosan has gained many attractions in the pharmaceutical and medical applications [4, 5]. On the other hand, in the presence of polyanionic molecules, chitosan can form spherical gel beads. Chitosan in the form of the gel beads has attracted much attention as a drug controlled release formulation owing to the simplicity and mildness of this process and formation of uniform and small size shape as compared with the conventional hydrogels [6-8]. Recently, there has been a great interest to generate antibacterial hydrogels because of their superior biomedical relevance [9]. Among antibacterial hydrogels, inorganic-based nanocomposite hydrogels are particularly promising for bacterial inactivation applications in materials and engineering science. These antibacterial agents possess a great potential to inhibit microbial growth. Because of they are easily functionalized with inorganic materials and biocompatible, this characteristic makes them attractive in the biomedical and biotechnological fields [10, 11]. At neutral pH, chitosan does not show any antibacterial activity; in order to impart that activity, it is necessary to incorporate some antibacterial agents into it [12]. Chitosan has previously been 1
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investigated as matrix to incorporate antibacterial nanoparticles like ZnO, Ag or Au nanoparticles [13-15]. It is well-known that copper oxide nanoparticles (CuONPs) possesses antibacterial activity. CuONPs are attractive antibacterial agents because in addition to their lowcost and easy release out of human body, they are known to have significant antibacterial properties. On the other hand, an added advantage of copper nanoparticles is that they oxidize and form copper oxide nanoparticles, which can easily mix with polymers or macromolecules and are relatively stable in terms of both chemical and physical properties [16]. Therefore, Copper-based nanocomposites are of great notice. Recently, nanocomposites of copper with several polymers such as chitosan [17, 18], polyacrylic acid [19], polypropylene [20] and cellulose [21], have been successfully prepared. In consideration of the mentioned concerns, a combination of chitosan hydrogel with CuONPs is quite attractive and, to the best of our knowledge, there is still no related report. In this study, novel chitosan/CuO nanocomposite hydrogels (CH/CuONPs) were successfully prepared by in situ formation of CuONPs in the chitosan hydrogel matrix. The structures of the nanocomposite hydrogels were characterized using X-ray diffraction (XRD) and scanning electron microscopy (SEM). The effect of the concentration of the CuONPs on the swelling behavior and antibacterial effect for the gram-negative E. coli and gram-positive S. aureus bacteria was investigated. 2. Materials and methods 2.1. Materials Chitosan, medium molecular weight, and viscosity 200-800 cP, 1 wt. % in 1% acetic acid (25 °C, Brookfield) and Sodium tripolyphosphate (STPP, technical grade 85%) was obtained from Aldrich. CuCl2.2H2O, NaOH were purchased from Merck and used as received. Distilled water was used throughout this study.
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2.2. Preparation of chitosan/CuONPs nanocomposite hydrogel beads A series of chitosan/CuONPs nanocomposite hydrogel beads (CH/CuO) were prepared according to a previously reported technique [8], using sodium tripolyphosphate (STPP) as the crosslinking agent and NaOH as oxidizing agent. Typically, 1 g of chitosan and desired amount of CuCl2.2H2O (0.0, 0.5, 1.0 and 1.5 mmol) were added to 35 ml distilled water. Then, 2.5 ml acetic acid was added to the above-mentioned mixture and stirred until a clear homogenous viscose solution obtained. Thereafter, the solution was extruded in the form of droplets, using a syringe (2 mm diameter), into an aqueous solution (400 ml) containing STPP (4 g) and NaOH (3.2 g). The beads were stayed in the solution for 24 h in order to crosslink with STPP and also converting Cu ions into CuONPs. The formation of CuONPs was confirmed by changing the beads' color from pale brown to black. After that, the beads were filtered and washed several times with distilled water to remove un-reacted STPP and NaOH on the surface of beads and dried under vacuum for 24 h. In remaining of the manuscript, CH/CuO0, CH/CuO1, CH/CuO2 and CH/CuO3, means chitosan/CuONPs nanocomposite hydrogel beads, which have 0.0, 0.5, 1.0 and 1.5 mmol of CuCl2 content, respectively. 2.3. Characterization and analysis Infrared spectra was obtained on an FTIR spectrometer (Bruker Instruments, model Aquinox 55, Germany) in the 4000-400 cm-1 range at a resolution of 0.5 cm-1as KBr pellets. The pattern of Xray diffraction of the samples was obtained by Siemens diffractometer with Cu-Ka radiation at 35 kV in the scan range of 2θ from 2 to 70o and scan rate of 1o/min. All of analyzed samples were in the powdery form. The d-spacing was calculated by Bragg’s equation where λ was 0.154
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nm. The morphology of the dried samples was examined using a scanning electron microscope (SEM) (TESCAN MIRA3) operated at 5 kV after coating the samples with gold and silver films. 2.4. Antibacterial activity Antibacterial activity of the CH/CuO nanocomposite hydrogels beads was tested against both E. coli (gram-negative) and S. aureus (gram-positive) according to agar diffusion test. For agar diffusion method, samples were exposed to bacteria on solid media (nutrient agar), and the inhibition zone around each sample was measured and recorded as the antibacterial effect of CuONPs. The agar plates were inoculated with 100 µL spore suspensions of bacteria. Swelled hydrogels placed on the agar plate and incubated at 37 °C for 24 h. Inhibition zone for bacterial growth was detected visually. 2.5. Swelling behavior Swelling ratio of nanocomposite beads was measured according to the previously reported methods [3] at buffer solutions (pH of 2.1 and 7.4) at room temperature. 0.1 g of CH/CuO nanocomposite beads was immersed in 50 ml of buffer solutions with desired pH at room temperature for 500 minutes to reach swelling equilibrium. The swelling ratio of nanocomposite beads was determined according to Equation 1. Eq. (1) Where W1 is initial weight of sample, and W2 is the weight of the sample after swelling for 500 min. 3. Results and discussions 3.1. Preparations of chitosan nanocomposite hydrogel beads Chitosan is one of the appropriate bio-polymers, which are capable of crosslinking by ionic interactions. Chitosan is cross-linked in the presence of poly-anions like sodium tripolyphosphate in the aqueous mediumsw. When the chitosan solution was dropped into STPP solution, gelled spheres formed instantaneously due to the electrostatic interaction between positively charged chitosan chains and negatively charged STPP anions. In the acidic conditions, amine moieties of chitosan chains convert to ammonium cations, which can electrostatically interact with STPP anions. In this way, the water-soluble chitosan is transformed into an insoluble gel, and chitosan droplets precipitated in the bead form in aqueous STPP solution. Scheme 1 schematically represents the formation of CuONPs in chitosan hydrogel network. Chitosan interacts with many metal cations, including Ag+, Cu2+, Fe3+ and Zn2+ [22]. Due to the existence of amine (-NH2) and hydroxyl (-OH) groups, the chitosan chains easily bind to the Ag+ cations. In this way, an almost uniformly distributed array of CuO nanoparticles obtained within the chitosan matrix. This procedure is facile and economically not requiring heat or any other tools for nanoparticle synthesis. 3.2. FT-IR analysis FT-IR spectra of pure chitosan, chitosan hydrogel beads and chitosan-CuO nanocomposite beads are shown in Fig 1. The FTIR spectra of the neat chitosan exhibited a broad band of multiple peaks between 3400-3600 cm−1, which attributed to the stretching of –OH and –NH2 groups and intermolecular and intramolecular hydrogen bonds. The peak about C–H stretching associated with methane hydrogen atoms appeared at 2924 cm−1. The peaks at 1640 and 1616 cm−1 related to the absorption band of the carbonyl stretching of the secondary amide and the bending vibrations of the N-H (N-acetylated residues, amide), respectively. The absorption bands between 1300-1460 cm-1 and 1000-1200 cm−1 were ascribed to the –C–N– and –C–O– stretching on the polysaccharide skeleton, respectively [23, 24]. Compared to the FTIR spectra of neat chitosan, chitosan beads reveals a single and narrower peak in the 3400-3600 cm−1 regions. Also,
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the intense characteristic bands of chitosan at 1640 and 1616 cm−1 shifted to lower wave numbers after the formation of chitosan beads. The FTIR results confirmed that chitosan polymeric chains was cross-linked with STPP in the beads. Compared with the FTIR spectra of chitosan beads, chitosan-CuO nanocomposite beads indicates the new peaks in the 400-800 cm−1 regions. These peaks were attributed to the vibration bands of metal-oxygen (Cu-O) bonds [25]. The interaction of CuONPs with chitosan hydrogels results in a reduction in the intensity of broad band of N–H and O–H bonds (3400-3600 cm−1). These results imply that the interaction also exists between the CuO nanoparticles and chitosan molecules. This difference arises from the participation of hydroxyl and amine groups of chitosan to the metal ions of CuO nanoparticles in order to form chelating structure and consequent decrease in hydrogen bonding which affords narrower and weaker band in chitosan-CuO nanocomposite beads [26]. 3.3. XRD analysis The XRD pattern of the pure chitosan hydrogel and CH/CuO3 nanocomposite hydrogel in the 2θ range of 2-70o is shown in Fig. 2. The pristine chitosan hydrogel without CuONP demonstrates the characteristic peaks at 2θ values of about 12o and 20o,which are typical finger prints of semicrystallinity of chitosan [27]. After the formation of CuONPs, the characteristic peaks of pure chitosan hydrogel were broadening a result of presence of CuONPs. In addition, the diffractogram of CH/CuO3 nanocomposite hydrogel is assigned to new diffractions at 2θ values of about 36o, 39o, 45o and 54o, which assigned to the (002), (111), (112) and (020) diffractions of CuO crystals, respectively. All the peaks match well with those of CuO crystals and confirm the formation of CuO particles in the chitosan hydrogel matrix [28, 29]. No peaks of impurity are observed in the XRD pattern, indicating the high purity of obtained CuO particles.
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3.3. Morphology of CH/CuO nanocomposite hydrogel beads Generally, the wet beads were spherical with a diameter of about 3-4 mm and possessed a smooth surface. After air drying, the diameter of test beads decreased to about 1 mm but still kept the spherical shape. For CH/CuO nanocomposite beads, there was no obvious variation with the bead size (Fig. 3). Fig. 4 a, b and c exhibit the SEM images of the hydrogel bead surface at low magnification. As it can be seen in Fig. 4a, the surface morphology of the neat chitosan hydrogel beads showed severe wrinkles and many cavities, which was caused by partial collapsing of the polymer network during drying. The CH/CuO beads show a smooth and tight surface which becomes smoother with the increase of CuONPs content. This could be due to the interfacial interactions between chitosan chains and CuONPs (as shown in Scheme 1) which could possibly act as intermolecular cross-linkers. Consequently, the CuONPs could contract and restrict the movability of the chitosan chains, and then change the surface morphology.
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With further magnification of the beads (Fig. 4 d, e and f), CuO nanoparticles appeared on the surface of the CH/CuO hydrogel beads. SEM results showed that the CuONPs were well dispersed in the chitosan matrix for sample with 5 wt% CuONPs content and have particle size less than 25 nm (Fig. 4e). However, some aggregation and bigger particle sizes (marked with circle) can be seen on the sample with the highest nano CuO content (sample CH/ CuO 3), Fig. 4f). These results indicate a close interaction of the CuONPs with the chitosan. 3.4. Antibacterial properties The in vitro antibacterial properties of CH/CuO nanocomposite beads tested against gramnegative E. coli and gram-positive S. aureus bacteria by disk diffusion test. Inhibition zone around the tested samples for bacterial growth was detected visually and summarized in Table 1. 4
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The inhibition zones are presented in Fig. 5. The results suggest that the CuONPs embedded hydrogel beads revealed a more toxic effect on bacteria than pure chitosan hydrogel under similar conditions as evidenced by higher inhibition zone. The results in the Table 1 show that the antibacterial efficiency of the nanocomposite hydrogels is influenced by the concentration of the CuONPs regardless of the kind of bacterial used. Hydrogels with more CuONPs demonstrate greater antibacterial properties. The antibacterial effect of CH/CuO nanocomposite hydrogels could be associated to the attachment of CuO nanoparticles to the cell wall of bactericides which damages the cell wall and causing leakage of proteins and other intracellular constituents and ultimately causes cell death [16, 28, 30]. 3.5. Swelling behavior The swelling behavior of the hydrogel beads was studied in the pH of 2.1 and 7.4 in order to investigate the pH sensitivity of the prepared hydrogels. As shown in the Fig. 6, swelling increases with time, first rapidly and then slowly, reaching a maximum constant swelling. It is obvious that the swelling ratio of test beads at pH 2.1 was higher than that of pH 7.4. The swelling mechanism at pH 2.1 involves the protonation of chitosan amine groups, which leads to chain repulsion that result in diffusion of proton and counter-ions together with water inside the beads [4].
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In addition, the results in Fig. 6 shows that CH/CuO nanocomposite hydrogel beads revealed a higher swelling capacity in comparison to that of the neat chitosan hydrogel. The enhancement of the swelling capacity of the nanocomposite hydrogels may be due to the presence of CuO nanoparticles with different sizes, morphologies and surface charges. The charged CuO nanoparticles results in penetration of more water molecules in order to neutralize the build-up ion osmotic pressure [31, 32]. Furthermore, formation of CuONPs in the hydrogel could expand the hydrogel network and increases the pores and free spaces within the network and as consequence CH/CuO samples adsorb more water [33]. However, CH/CuO2 and CH/CuO3 samples revealed less swelling capacity in comparison to that of the CH/CuO1 sample. This could be attributed to the role of CuONPs as the knot tying functions, which restricts the expanding of polymer chains. The knot tying function of CuONPs may be due to the chelation of some hydroxyl and amine groups of the hydrogel networks with CuO nanoparticles [34]. 4. Conclusions In this study, antibacterial CH/CuO nanocomposite hydrogel beads were successfully prepared by in situ generation of CuONPs during the formation of chitosan beads. Structural details were provided by XRD and SEM analysis. In addition, the influence of CuONPs on the swelling, drug release and antibacterial behavior of the chitosan beads was studied. The CuO nanoparticles were successfully generated in the beads and had a clear influence on the surface morphology of the beads as evidenced by XRD and SEM analysis. CH/CuO nanocomposite hydrogel beads revealed a higher swelling capacity in comparison to that of the neat chitosan hydrogel which was dependent upon the CuONPs content. Antimicrobial activity of the hydrogels was examined on E. coli (gram-negative) and S. aureus (gram-positive) according to agar diffusion test. The CH/CuO hydrogels has shown good antibacterial activity against gram-positive and gramnegative bactericides. Hydrogels with more CuONPs demonstrate greater antibacterial properties. Based on these findings, the prepared CH/CuO nanocomposite hydrogels can be used in different medical fields. Acknowledgments
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Authors also gratefully acknowledge the University of Tabriz (grant number S/27/3243-29) for the financial supports for this research.
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Scheme1. The schematic representation of the interactions of chitosan with Cu ions and CuONPs Fig. 1. FTIR spectra of pure chitosan, chitosan hydrogel bead and CH/CuO nanocomposite hydrogel bead Fig. 2. XRD pattern of pure chitosan hydrogel bead (a) and CH/CuO nanocomposite hydrogel bead Fig. 3. Digital photo of pure chitosan hydrogel bead (a) and CH/CuO nanocomposite hydrogel bead; Fig. 4. SEM micrographs of pure chitosan hydrogel bead (a) CH/CuO1 (b) CH/CuO3 (c) at low magnification (×1000) and micrographs of pure chitosan hydrogel bead (d) CH/CuO1 (e) CH/CuO3 (f) at high magnification (×100000) Fig. 5. Photographs of the inhibition zones of CH/CuO nanocomposite hydrogel beads against (a) S. aureus (b) E. coli. Fig. 6. Swelling behavior of CH/CuO nanocomposite beads at pH values of 2.1 and 7.4 Table 1. Zones of inhibition (mm) against the tested bacteria for CH/CuO nanocomposite hydrogel beads Sample Bacteria Zone of inhibition (mm) 1 day 3 days CH/CuO0 S. aureus 0 0 E. coli 0 0 CH/CuO1 S. aureus 6 0 E. coli 6 6 CH/CuO2 S. aureus 6 0 E. coli 9 9 CH/CuO3 S. aureus 8 7 E. coli 11 10
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