ZnO impregnated MCM-41 nanocomposite hydrogel

Materials Science and Engineering C 73 (2017) 456–464

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Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec

A potential bioactive wound dressing based on carboxymethyl cellulose/ ZnO impregnated MCM-41 nanocomposite hydrogel Rasul Rakhshaei a, Hassan Namazi a,b,⁎ a b

Research Laboratory of Dendrimers and Nanopolymers, Faculty of Chemistry, University of Tabriz, P.O. Box 51666, Tabriz, Iran Research Center for Pharmaceutical Nanotechnology, Tabriz University of Medical Science, Tabriz, Iran

a r t i c l e

i n f o

Article history: Received 10 August 2016 Received in revised form 12 October 2016 Accepted 20 December 2016 Available online 21 December 2016 Keywords: Hydrogel nanocomposite Mesoporous silica Wound dressing Zinc oxide Drug delivery

a b s t r a c t Lack of antibacterial activity, deficient water vapor and oxygen permeability, and insufficient mechanical properties are disadvantages of existing wound dressings. Hydrogels could absorb wound exudates due to their strong swelling ratio and give a cooling sensation and a wet environment. To overcome these shortcomings, flexible nanocomposite hydrogel films was prepared through combination of zinc oxide impregnated mesoporous silica (ZnO-MCM-41) as a nano drug carrier with carboxymethyl cellulose (CMC) hydrogel. Citric acid was used as cross linker to avoid the cytotoxicity of conventional cross linkers. The prepared nanocomposite hydrogel was characterized using X-ray diffractometry (XRD), scanning electron microscopy (SEM), transmission electron microscopy (TEM), Zeta potential and UV–vis spectroscopy. Results of swelling and erosion tests showed CMC/ZnO nanocomposite hydrogel disintegrated during the first hours of the test. Using MCM-41 as a substrate for ZnO nanoparticles solved this problem and the CMC/ZnO-MCM-41 showed a great improvement in tensile strength (12%), swelling (100%), erosion (53%) and gas permeability (500%) properties. Drug delivery and antibacterial properties of the nanocomposite hydrogel films studied using tetracycline (TC) as a broad spectrum antibiotic and showed a sustained TC release. This could efficiently decrease bandage exchange. Cytocompatibility of the nanocomposite hydrogel films has been analyzed in adipose tissue-derived stem cells (ADSCs) and results showed cytocompatibility of CMC/ZnO-MCM-41. Based on these results the prepared CMC nanocomposite hydrogel containing ZnO impregnated MCM-41, could serve as a kind of promising wound dressing with sustained drug delivery properties. © 2016 Elsevier B.V. All rights reserved.

1. Introduction Skin is the largest organ in the human body and so cutaneous wounds severely affect human life and health. A wound dressing with perfect match to the demands of rapid wound closure has an important role in the entire management of wounds infection [1]. An ideal wound dressing should have appropriate mechanical properties, suitable oxygen and water vapor permeability, and keep the moisture in healing environment, and prevent wound from bacterial infection [2]. In addition, these dressings should be biocompatible and easily removed from wounds [3]. Hydrogels could absorb wound exudates due to their strong swelling ratio and give a cooling sensation and a wet environment [4,5]. Unique properties of natural polymers introduce them as very promising candidates for wound dressing materials [6]. Sodium carboxymethyl cellulose is a semi synthetic cellulose derivative that is biocompatible and biodegradable polymer and hence commonly used in wound dressings [7–9]. CMC alone has been used as ⁎ Corresponding author at: Research Laboratory of Dendrimers and Nanopolymers, Faculty of Chemistry, University of Tabriz, P.O. Box 51666, Tabriz, Iran. E-mail address: [email protected] (H. Namazi).

http://dx.doi.org/10.1016/j.msec.2016.12.097 0928-4931/© 2016 Elsevier B.V. All rights reserved.

dressing for burn wound dressing. It helps extracellular matrix formation and re-epithelialization due to its capability of maintaining an optimum moist environment in wound region [10–12]. Most of the available products are not clinically mature due to their characteristics and shortcomings [13]. For example, Ramli and Wong used noncrosslinked CMC films [14]. The membranes contain no antibacterial agent and have high degradation rate (films were changed every 6 h). Designing more effective dressing playing an active role in the wound healing process is in great demand. These bioactive dressings aim to deliver biomolecules including antibiotics and growth factors gradually [15,16]. Hence, combination of hydrogels with a drug delivery system is required to control and prolong the release of antibacterial agents to prevent wound infection during healing process. A wide variety of nanoparticles like Ag, ZnO and CuO nanoparticles are mixed with the polymeric network to obtain nanocomposite hydrogels with drug delivery and antibacterial properties [17–20]. ZnO is an eco-friendly and non-toxic material. It is widely used in drug delivery [21–23]. It has shown antibacterial activity and it is currently used in many cosmetic materials [24,25]. Furthermore, the Zn++ released from ZnO can enhance keratinocyte migration toward the wound site and promote healing [26,27]. In order to control the

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2.2. MCM-41 synthesis In a general synthesis, 2.74 mmol of (CTAB) were dissolved in 480 mL of NaOH aqueous solution (15.0 mM), and then 22.4 mmol of tetraethylorthosilicate (TEOS) was added dropwise to the solution. The mixture was vigorously stirred and heated to 80 °C for 2 h. Subsequently, the product was isolated by hot filtration, washed with distilled water and methanol, and dried. Finally, the resulted powder was calcinated at 600 °C in air for 6 h. To produce zinc impregnated MCM-41, a wet impregnation was performed. 0.33 g of calcinated MCM-41 was introduced into an aqueous solution of Zn(NO3)2·6H2O (0.20 g/12 mL). After 1 h, the resulting mixture was dried at 80 °C. The resulting precipitate was then heated in 400 °C for 3 h to oxidize the impregnated zinc. Fig. 1. SEM micrograph of the synthesized ZnO nanoparticles.

2.3. ZnO nanoparticles preparation stability of the ZnO nanoparticles, ZnO have been loaded on mesoporous silica materials through impregnation leading to surface developing, life extension, chemical and thermal stability (dissolution to yield Zn(OH)2) etc. [28,29]. MCM-41 (Mobil Composition of Matter No. 41, the number is added chronologically based on the date of discovery), one member of the mesoporous silica family, possess a highly ordered hexagonal array of one-dimensional cylindrical pores with changeable pore diameter between 1.5 and 30 nm [30]. Large surface area, large pore volume, and excellent biocompatibility make MCM-41 materials among the best candidates as hosts for many guest materials and have attracted significant interest as drug carriers [31,32]. Tian et al. prepared MCM-41 type mesoporous silica nanoparticles decorated with silver nanoparticles (Ag-MSNs). These Ag-MSNs possess an enhanced antibacterial effect [33]. Yu et al. used hyaluronic acid modified mesoporous silica nanoparticles for targeted drug delivery to cancer cells [34]. Buchtová et al. prepared nanocomposite hydrogels of mesoporous silica nano fibers interlinked with siloxane derived polysaccharide for cartilage tissue engineering. The prepared nanocomposite hydrogel showed enhanced mechanical properties and mesoporous silica acted as reservoirs for bioactive molecules [35]. Our group has shown previously that citric acid crosslinked carboxymethyl cellulose hydrogels could serve as potential wound dressings [36]. Here, for the first time, in order to design more effective dressing playing an active role in the wound healing process ZnO impregnated MCM-41 nanoparticles was added to the hydrogel. To maximize the antibacterial efficiency and reduce the chances of resistance development in the microbes by employing multiple targeting approaches TC was loaded to the nanoparticles. The prepared nanocomposite hydrogel was characterized using X-ray diffractometry (XRD), scanning electron microscopy (SEM), transmission electron microscopy (TEM), Zeta potential and UV–vis spectroscopy. Effect of the nanoparticles on the swelling, erosion, gas permeability and TC delivery was studied. Finally, microbiological assay were conducted to examine the antimicrobial activity and drug delivery properties of the prepared nanocomposite hydrogel films against Gram-positive S. aureus and Gram-negative E. coli.

ZnO nanoparticles were synthesized according to an approach reported elsewhere with modifications [37]. Briefly 1.5 g CMC was added to 250 mL distilled water. After complete dissolution of CMC, 7.44 g (0.025 mol) of Zinc nitrate hexahydrate was added to the solution, and then 250 mL of sodium hydroxide solution (0.2 mol/L) was added drop-wise with constant stirring. After the complete addition of sodium hydroxide the solution was centrifuged at 8000 rpm for 20 min, the settled precipitate washed several times, dried and calcinated by 7 h at 540 °C to complete the reaction and remove CMC. 2.4. Characterization and analysis UV–vis absorption spectra of the samples were recorded on a Shimadzu 1700 Model UV–vis spectrophotometer. The pattern of Xray diffraction of the samples was obtained by Siemens diffractometer with Cu-kα radiation at 35 kV in the scan range of 2θ from 2 to 10° and scan rate of 1°/min. The diameter of channels of the synthesized MCM-41 was calculated using Bragg's equation where λ was 0.154 nm. The morphology of the dried samples was observed using a scanning electron micro-scope (SEM) (LEO 1430VP) operated at 15 kV after coating the dried samples with gold and silver films. Transmission electron micrograph (TEM) was conducted by LEO 906E transmission electron microscope operating at 100 kV. Zeta potential of the sample is analyzed by a Zeta Sizer 2000 (Malvern Instruments Ltd., UK). An Analytik Jena flame atomic absorption spectrometer model Nov. 400 (Jena, Germany; www.analytik-jena.de) furnished with an air–acetylene flame and a cadmium hollow cathode lamp, operated at 3.0 mA, was used for Zn determination.

2. Experimental 2.1. Materials Sodium carboxymethyl cellulose (CMC), degree of substitution (DS) 0.55–1.0, and viscosity 15,000 mPas/s (1% in H2O, 25 °C) were obtained from Nippon Paper Chemicals Co., Ltd., Japan. Tetracycline hydrochloride was purchased from Sigma–Aldrich. Tetraethylorthosilicate (TEOS), cetyltrimethylammonium bromide (CTAB), Zn(NO3)2·6H2O, NaOH, citric acid, glycerol and all other materials were purchased from Merck.

Fig. 2. XRD patterns of native MCM-41 and ZnO impregnated MCM-41.

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Fig. 3. SEM micrographs of the MCM-41 (a) and ZnO-MCM-41 nanoparticles (b) and EDX of ZnO-MCM-41 nanoparticles(c).

2.5. Preparation of nanocomposite hydrogel films Using the data of our recently published paper the optimum crosslinking conditions of CMC hydrogel designated using 10% citric

acid along with curing at 60 °C for 24 h [36]. First, an aqueous solution (0.20 g/100 mL) of citric acid was prepared. Glycerol as plasticizer (1 g) was added to this solution. CMC/TC sample is the positive control as TC was directly added to the CMC hydrogel with the weight ratio of

Fig. 4. TEM micrographs of the native and ZnO impregnated MCM-41 nanoparticles.

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(Eq. (2)) were defined as: Swelling ratio ¼

Erosion ¼

Wt −Wi Wi

Wi −WtðdÞ  100% Wi

ð1Þ

ð2Þ

where, Wt = wet weight of sample at time t, Wt(d) = dry weight of sample collected at time t and Wi = initial dry weight. 2.8. Water vapor permeability

Fig. 5. Zeta potential of the native and ZnO impregnated MCM-41.

3%. In case of CMC nanocomposite hydrogels, drug loaded nanoparticles including MCM-41, ZnO-MCM-41 and ZnO powder with a weight ratio of 10% to CMC was added to this solution and sonicated for 10 min. Then 2.0 g of CMC was added to the resulted slurry with a continuous mechanical stirring to reach a homogeneous viscous mixture. The formed paste was transferred to a plastic container and then dried at 60 °C for 24 h. 2.6. Mechanical properties evaluation Tensile strength and elongation at break of the films were measured. The experimental procedure was as follows: the film specimens were prepared with dimensions of 6 cm × 0.5 cm. Thickness of the films using SEM micrographs were 0.1 ± 0.02 mm for CMC, 0.08 ± 0.04 mm for CMC/MCM-41, and 0.11 ± 0.02 mm for CMC/ZnO-MCM41 films. Both ends of tensile specimens were clipped with a special gripper. The tensile strength and percentage elongation at break of the films were measured by a universal testing machine (SANTAM, Model STM1, Iran) with the crosshead speed of 0.5 mm min−1 at room temperature. The average value out of six measurements was reported for each sample.

Water vapor permeability of films (g m−1 h−1 Pa−1) was gravimetrically determined at 20 °C. Prepared nanocomposite films were hermetically sealed with silicone grease in glass cups with an opening head of 2.5 cm diameter containing 15 mL distilled water. The cups were placed in a desiccator containing silicagel at 20 °C, thus obtaining an RH gradient equal to 100%. The water vapor transfer through the exposed film area (4.9 cm2) was measured from the cup weight loss. When steady-state conditions were reached (about 2 h), eight weight measurements were made over 7 h using a four-digit balance. At least three samples of each type of film were tested, and water vapor permeability (WVP) was calculated from the following equation: WVP ¼

Sd A  ΔP

ð3Þ

where S is the slope of the weight loss vs. time (g h−1), d is the film thickness (m), A is the area of exposed film (m2) and ΔP is the differential of water vapor pressure across the film (at 20 °C, ΔP = 2.33 × 103 Pa, assuming that the RH on the silicagel is negligible). 2.9. Oxygen permeability O2 permeation were measured at different pressure differences up to 0.1 MPa at 293 K. The samples were sealed in a stainless steel permeator module with the films facing the high-pressure side. Feed gas flows along outside of the membrane and permeated gas flow rate were measured on inner side of the membrane at pressure 1 bar. Pressure differences across the membrane were obtained by varying pressure on the upstream side and keeping the downstream pressure constant at 1 bar. Pressure in shell side of the membrane module was monitored via a pressure gauge.

2.7. In vitro swelling and erosion studies 2.10. In vitro drug loading In vitro swelling and erosion of the prepared films determined by immersing an accurately weighed sample with known size in 20 mL 1 × PBS (phosphate buffered saline) at physiological temperature and pH. At predetermined time intervals the samples were taken out and blotted onto filter paper to remove surface water. To determine erosion, the scaffold was then oven-dried at 50 °C for two days. The experiment was carried out in triplicates. The swelling ratio (Eq. (1)) and erosion

The adsorption of TC was studied by using UV–visible spectrophotometer. The TC was loaded via post-impregnation using a drug to nanoparticle carrier ratios of 1.5 and soaking 30 mg of carrier in 3 mL drug solution in distilled water. Finally, the drug loaded solid samples were centrifuged at 4500 rpm for 30 min and washed with distilled water followed by drying at room temperature. The loading percentage

Fig. 6. SEM micrographs of the CMC (a), CMC/MCM-41 (b), and CMC/ZnO-MCM-41 films (c).

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Fig. 7. Tensile strength (A) and elongation at break (B) of CMC-based films.

Fig. 9. Water vapor permeability (a) and oxygen permeability (b) of the prepared nanocomposite hydrogel films.

was achieved by measuring UV–vis absorbance at 276 nm [38]. For the original CMC sample, 3% TC was added directly to the CMC solution.

2.11. In vitro drug release To study the release of drugs, the nanocomposite hydrogels were immersed in 20 mL PBS (pH 7.4) at 37 °C under steady state condition. At appropriate time intervals, 5 mL aliquots were withdrawn and replaced with fresh PBS. The optical density of the aliquots was measured by UV–vis spectrophotometer. The cumulative release data were obtained using a predetermined calibration curve. The drug release from the hydrogels was determined by applying the amounts of released

Fig. 8. Erosion percentage (a) and swelling ratio (b) of the prepared nanocomposite hydrogels in PBS after 48 h.

Fig. 10. Tetracycline loading percent of the native and ZnO impregnated MCM-41.

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Fig. 11. Tetracycline cumulative release of the prepared nanocomposite hydrogel films in PBS.

and loaded drug to the following formula: Drug release ð%Þ ¼

ðthe amount of released drugÞ  100 ðthe amount of loaded drugÞ

ð4Þ

Fig. 13. Photographs of the inhibition zones of the prepared nanocomposite hydrogel films against S. aureus (films are allowed to release their loaded drug in PBS for 24 h, 48 h, and 72 h and then antimicrobial test was carried out).

2.12. Microbiological activity Microbiological tests were performed by zone inhibition methods. Five millimeter disks of samples after 24 h of TC release in PBS were placed on Petri dishes in contact with Mueller–Hinton agar previously inoculated with a Gram-positive bacterial strain S. aureus and a Gramnegative bacterial strain E. coli. The plates were incubated for 24 h at 37 °C. Bacteria developed on the agar medium, except in a clear zone around the disk samples. The radius of the clear zone around the disk

samples was measured visually using a ruler displaying their antibacterial activity.

2.13. Cell culturing For the cell culturing experiments, the samples (100 mg of each scaffold) were placed in siliconized 12-well cell culture plates and immersed three times in a 70% ethanol solution for 20–30 min each time, followed by evaporation of the ethanol in the air. Then the samples were rinsed three times with PBS solution to remove the residual of ethanol and incubated in culture medium DMEM at 37 °C for 24 h. ADSCs of passage 3 were used in the following studies. ADSCs were cultured in proliferation medium containing DMEM supplemented with 10% fetal bovine serum (FBS; Cambrex), 100 U mL−1 penicillin (Gibco), and 100 μg mL− 1 streptomycin (Gibco), 1 × Amphotricin B at 37 °C in a humid atmosphere with 5% CO2. When reaching 70–80% confluency cells were detached by trypsinization (0.05% trypsin containing 1 mM EDTA) from the cell culture flask and viable cells were counted by trypan blue assay and subsequently subcultured or seeded on the prepared nanocomposite films at a seeding density of 20,000–30,000 cells per scaffold. Cell numbers were determined by manual counting with a haemacytometer. The culturing period was 24 h, 72 h, and seven days. The medium was refreshed every other day. Culturing experiments were performed in triplicate for every type of hydrogel films.

Table 1 Inhibition zones (mm) of the prepared nanocomposite hydrogel films against E. coli (films are allowed to release their loaded drug in PBS for 24 h, 48 h, and 72 h and then antimicrobial test was carried out). E. coli CMC CMC/MCM-41 Fig. 12. Photographs of the inhibition zone of the prepared nanocomposite hydrogel films against E. coli (films are allowed to release their loaded drug in PBS for 24 h, 48 h, and 72 h and then antimicrobial test was carried out).

CMC/ZnO-MCM-41

Sample containing TC Control (without TC) Sample containing TC Control (without TC) Sample containing TC Control (without TC)

24 h

48 h

72 h

8±1 0 10 ± 1 0 25 ± 3 13 ± 1

9±1 0 10 ± 1 0 19 ± 2 13 ± 2

10 0 11 0 15 14

±1 ±1 ±2 ±2

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Table 2 Inhibition zones (mm) of the prepared nanocomposite hydrogel films against S. aureus (films are allowed to release their loaded drug in PBS for 24 h, 48 h, and 72 h and then antimicrobial test was carried out). S. aureus CMC CMC/MCM-41 CMC/ZnO-MCM-41

Sample containing TC Control (without TC) Sample containing TC Control (without TC) Sample containing TC Control (without TC)

24 h

48 h

72 h

7±1 0 11 ± 1 0 29 ± 3 10 ± 1

9±1 0 10 ± 1 0 18 ± 2 11 ± 2

9±1 0 11 ± 1 0 16 ± 2 10 ± 1

3. Results and discussion

Results proved ZnO nanoparticles changed the negative surficial charge of the native MCM-41 (− 20 ± 3.1 mV) to positive one (+ 9.3 ± 1.2 mV). 3.3. Morphology study of the films Fig. 6 depicts Scanning electron microscopy images of the pure CMC hydrogel and CMC/mesopore nanocomposite hydrogel films. In Fig. 6a, a clear and uniform surface morphology was observed for the pure CMC hydrogel. In the case of nanocomposite hydrogel films containing MCM-41 and ZnO-MCM-41 nanoparticles, the surface of the films is very rough and there are visible spherical particles (Fig. 6b and c). SEM results showed that the MCM-41 nanoparticles were well dispersed in the CMC matrix. However, some aggregations are visible.

3.1. Nanoparticles characterization 3.4. Evaluation of mechanical properties SEM micrograph of the synthesized ZnO nanoparticles is presented in Fig. 1. SEM image of ZnO shows aggregated particles with different sizes. XRD patterns of the native MCM-41 and ZnO modified MCM-41 nanoparticles are shown in Fig. 2. An intense diffraction peak at 2θ of 2.2° as well as the low intensity broad peaks at 2θ of 3.8 and 4.4° are the characteristic (1 0 0), (1 1 0), and (2 0 0) peaks of MCM-41, respectively. These peaks indicate the formation of hexagonal MCM-41 mesoporous material [39]. The pore diameter estimated from the (1 0 0) peak using Bragg's equation, was approximately 4 nm. For ZnO-MCM-41, diffraction peaks are appeared in 2θ 2.55° (1 0 0), 4.29° (1 1 0), and 4.91° (2 0 0). The pore diameter of ZnO-MCM-41 using Bragg's equation was approximately 3.5 nm. This shows ZnO impregnation of MCM-41 results in a decrease of MCM-41 pore diameter. SEM micrograph of the native (Fig. 3a) and ZnO impregnated MCM41nanoparticles (Fig. 3b) showed no especial difference between the two samples. There is not any sign of aggregated ZnO particles in ZnOMCM-41. Using EDX analysis (Fig. 3c), Zn content was estimated to be 14%. In addition to XRD for evaluating the microstructure of nanocomposites, TEM instrument as the best direct evidence to describe the morphology was used to visualize the channels of MCM-41 and ZnO nanoparticles. Fig. 4 illustrates the TEM micrographs for MCM-41 and ZnO-MCM-41 nanoparticles. Nano-metric channels of MCM-41 and spherical ZnO nanoparticle inside the MCM-41 channels are clearly visible in Fig. 4. 3.2. Zeta potential It was assumed positively charged ZnO nanoparticles could change the electric charges on MCM-41 surface. To probe this hypothesis we characterized the surficial charge of the native (MCM-41) and ZnO impregnated (ZnO-MCM-41) mesopores using Zeta potential test (Fig. 5).

The tensile strength of the CMC and CMC/mesopore nanocomposite hydrogel films was measured, and CMC showed a tensile strength of 50.5 MPa (Fig. 7A). The incorporation of MCM-41 and ZnO-MCM-41 enhanced the tensile strength by 24% and 12% respectively, which was due to the hydrogen bonding between MCM-41 and CMC. The obtained results showed that the nanocomposite hydrogel films had sufficient strength to bare the force applied on them. Elongation at break values indicates the flexibility of the material. CMC/MCM-41 showed an enhanced elongation (Fig. 7B). However ZnO-MCM-41 decreased the flexibility of the films. The obtained data were adequate to show the flexible nature of the nanocomposite hydrogel films. The flexible nature would be supportive for the application of these nanocomposite hydrogel films over any type of wound surface. 3.5. Swelling ratio and erosion Fig. 8a presents the results of erosion experiment. Erosion experiment showed the citric acid cross linked CMC is stable during 48 h but CMC/ZnO disintegrated during the first hours of the experiment and no crosslinking effect was observed. Failure to crosslinking in CMC/ ZnO could be due to the fact that zinc oxide nanoparticles react with protons under acidic conditions which lead to dissolution of ZnO nanoparticles and waste of crosslinker (citric acid) [40]. To overcome this problem ZnO nanoparticles stabilized within MCM-41 channels. Results showed this method was effective and CMC/ZnO-MCM-41 film was stable during 48 h. Fig. 8b illustrates the swelling ratio of the nanocomposite hydrogel films. Swelling ratio of CMC/ZnO-MCM-41 is higher than CMC and CMC/MCM-41. Addition of 10% ZnO-MCM-41 dramatically increases the swelling capacity of CMC films by 2.2 fold. The increase of the swelling ratio of the CMC/MCM-41 in comparison with CMC hydrogel is related to the hydrophilic surface of the MCM-41 nanoparticles [41]. The higher swelling ratio of CMC/ZnO-MCM-41 in comparison to CMC/MCM-41 is related to the electric charge of the ZnO nanoparticles which results in the penetration of more water molecules to balance the build-up ion osmotic pressure, which causes the hydrogel to swell [24, 42]. Due to insufficient erosion and swelling properties of CMC/ZnO films, this sample was eliminated and the experiments continued on the other samples. 3.6. Permeability

Fig. 14. Cell viability study using ADSC cells.

The influence of MCM-41 modification on the water vapor and O2 permeability of the CMC/MCM-41 nanocomposite films has been shown in Fig. 9a and b respectively. As it can be seen in the figures, addition of MCM-41 dramatically increased the WVP and O2 permeability values of the CMC films. It is proposed the dramatic increase in permeability of polymer/MCM-41 nanocomposite films is attributed to the high adsorption capacity of MCM-41 for gases due to its porosity and the formation of a permeable polymer corona around the MCMC-41

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particles which prepares a permeation pathway for gas molecules [43, 44]. However, the increase in gas permeability of nanocomposite films was smaller for ZnO modified MCM-41 containing CMC nanocomposite films. This could be attributed to the lower pore diameter of ZnO-MCM41 in comparison with MCM-41. 3.7. Drug loading and release Fig. 10, shows TC loading into native MCM-41 and ZnO impregnated nanoparticles. Charge of the Tetracycline molecules above pH 7.4 change to negative [45] and in this pH the surface of the MCM-41 has partially negative charge [46]. Accordingly there is a repulsive force between TC molecules and surface of the MCM-41 nanoparticles. It is visible that ZnO modification of the MCM-41 highly increased the TC loading. This could be attributed to the positive charge of the ZnO nanoparticles which attracts TC molecules to the MCM-41 surface. Tetracycline cumulative release experiments executed in PBS solution to evaluate the drug delivery properties of the prepared nanocomposite hydrogels (Fig. 11). It can be seen almost all of the drug released explosively during the first 3 h in pure CMC films. This could be explained in consideration that both TC molecules and carboxymethyl cellulose in pH 7.4 have negative charge causing a strong repulsive force between TC and CMC hydrogel [45,47]. In sample CMC/MCM-41, approximately TC release continued up to 6 h and then reached to its plateau state. This could be attributed to the possible hydrogen bonding between TC molecules and the available hydroxyl groups of the MCM41 surface. TC release of the sample containing ZnO-MCM-41showed a sustained release. The extended TC release in CMC/ZnO-MCM-41 could be attributed to the attractive force between positively charged ZnO nanoparticles and negatively charged TC molecules in pH 7.4 [45]. Moreover, the dissolution of the ZnO nanoparticles could be another reason for this sustained release of TC from CMC/ZnO-MCM-41. To compare the ZnO solubility of the free ZnO nanoparticles and ZnO-MCM-41, samples were dispersed in 0.01 M citric acid solution overnight. Then the concentration of dissolved Zn++ was obtained using atomic adsorption spectroscopy analysis. Knowing the Zn percentage in ZnO-MCM-41 (14%) from EDX analysis (Fig. 3c), it was calculated that the solubility of ZnO was decreased by 21.7% in ZnO-MCM-41 sample compared to free ZnO nanoparticles.

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3.9. Cell viability study Fig. 14 illustrates cell viability results. CMC hydrogel film showed 81%, 90% and 94% Cell viability after 24 h, 72 h and 7 days in contact with ADSCs. CMC/MCM-41 and CMC/ZnO-MCM-41 showed 72% and 63% viability after 24 h of incubation. All samples showed an increase in the viability after 72 h and 7 days of incubation. The reduced viability of CMC/ZnO-MCM-41 at 24 h was due to the interaction of nZnO with the cells. After 24 h, the remaining viable cells began to multiply and, hence, the viability increased. 4. Conclusions A flexible and tetracycline-eluting nanocomposite hydrogel film was prepared through combination of ZnO modified mesoporous silica MCM-41 as a nano drug carrier with CMC hydrogel. Results of swelling and erosion tests showed CMC/ZnO nanocomposite hydrogel disintegrated during the first h of test. Using MCM-41 as a substrate for ZnO nanoparticles, this problem was solved and the CMC/ZnOMCM-41showed a great improvement in swelling, erosion and gas permeability properties. TC loading and release studies showed ZnO impregnation of the MCM-41 highly increased the TC loading and addition of ZnO-MCM-41 into CMC films caused a prolonged and continued release of TC related to dissolution of ZnO. Zeta potential results showed this prolonged release could be as a result of surficial charge change of MCM-41. ZnO nanoparticle with positive charge change the negative charge of the MCM-41 resulting in an attractive force between TC molecules and ZnO-MCM-41 nanoparticles. The microbiological assay showed a powerful antibacterial effect in TC loaded CMC/ZnOMCM-41 after 24 h of release. The antimicrobial property of the CMC/ ZnO-MCM-41 is a result of intrinsic antibacterial properties of ZnO nanoparticles and confirmed the prolonged release of TC. Cytocompatibility of the nanocomposite hydrogel films has been analyzed in adipose tissue-derived stem cells (ADSCs) and results showed cytocompatibility of CMC/ZnO-MCM-41. These properties could be potentially beneficial for wound healing and dressing systems. Acknowledgments Authors gratefully acknowledge the financial supports from the University of Tabriz (grant number S/27/3243-29).

3.8. Antibacterial experiments References The in vitro drug delivery properties examined using antibacterial assay against Gram-negative E. coli (Fig. 12) and Gram-positive S. aureus (Fig. 13) bacteria by disk diffusion method. Samples allowed to release their loaded drug during 24 h, 48 h, and 72 h in PBS and then antibacterial test was carried out to determine the amount of drug remained in the samples and their antibacterial power after 24 h, 48 h, and 72 h drug release. Inhibition zone around the tested samples for bacterial growth was detected visually using a ruler and are summarized in Tables 1 and 2. In control series (samples without TC), inhibition zone was detectable just in CMC/ZnO-MCM-41 and in the other samples bacteria grew even on the surface of the films. This exceptional behavior is referred to the antibacterial properties of the ZnO particles. In samples containing TC, after 24 h of TC release, CMC/ZnO-MCM-41/TC showed an exceptional inhibition zone, while the inhibition zone of the CMC/ TC and CMC/MCM-41/TC films is just limited to the area of the films. The observed strong antibacterial property observed for CMC/ZnOMCM-41/TC in comparison with the other samples can be attributed to a synergistic effect. On the one hand as explained in previous section ZnO particles change the surficial charge of the MCM-41 resulting in a prolonged release of TC. On the other hand, Zinc Oxide nanoparticles itself show antibacterial properties. These results indicate that the ZnO nanoparticles have a synergistic effect with TC and result in more effective antibacterial property.

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