# In Situ Preparation and Properties of Poly(vinyl alcohol)/Carboxymethyl Chitosan/Cyanidin Hydrogel Films

Authors: Xiaojuan Zhang; Wenbin Cai; Lingyun Hao; Wei Zhang
Journal: Journal of Nanomaterials
 (2019)

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## Abstract

This study synthesized poly(vinyl alcohol)/carboxymethyl chitosan/cyanidin (PVA/CMCS/CY) hydrogel films. First, PVA and CMCS were used to synthesize hydrogel films by ultraviolet irradiation. Meanwhile, CY was in situ combined into the hydrogel films through the electrostatic attraction between CMCS and CY. Next, the products were analyzed using Fourier-transform infrared spectroscopy, scanning electron microscopy, contact angle test, swelling analysis, and mechanical property test. The results revealed that compared with PVA/CMCS hydrogel films, the PVA/CMCS/CY hydrogel films had an interporous structure and good swelling and mechanical properties. Moreover, the drug release experiments demonstrated that the PVA/CMCS/CY hydrogel films had a CY encapsulation efficiency of 33.5% with a sustained CY release of up to 60 h. Furthermore, the examined antibacterial activities againstStaphylococcus aureus and Escherichia coli showed that the porous PVA/CMCS/CY hydrogel films exhibited a certain inhibition. Cell viability experiments demonstrated that the PVA/CMCS/CY films displayed no obvious cytotoxicity to human umbilical vein endothelial cells.


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## Body

## 1. Introduction

Cyanidin (CY) is an anthocyanin (ATC) that belongs to flavonoids and is found in fruits, vegetables, and flowers [1]. In recent years, CY has been extensively studied because of its α-glucosidase inhibitory, antioxidative, anti-inflammatory, antibacterial, and antitumor properties [2–6]. However, due to its insufficient absorption and short half-life in vivo metabolism, its clinical application is limited [7, 8]. To improve its absorption, CY has been loaded into different carriers, such as gelling systems [9], poly(lactide-co-glycolide)@polyethylene glycol (PEG) nanoparticles [10], liposomes [11], chitosan-cellulose microcapsules [12], cyclodextrin [13], ferritin nanocage [14], chitosan nanoparticles [15], and chitosan/PVA films [16]. The combination of CY into different materials will support the development of multifunctional drug delivery. For instance, Jeong et al. [17] prepared a chondroitin sulfate- (CS-) ATC nanocomplex and loaded doxorubicin (DOX) into the nanocomplex through an intermolecular stacking interaction. Their results showed that the CS-ATC2-DOX nanocomplex exhibited a reactive-oxygen-species-sensitive therapeutic effect and inhibited tumor growth. Kim et al. [18] synthesized ATC-loaded PEG-gold nanoparticles, which achieved an enhanced delivery of ATC in the mouse brain and could prevent neurodegenerative diseases.Hydrogel is a three-dimensional network polymeric material containing water or biological substances, which can be applied in drug delivery, regenerative medicine, and wound dressing [19]. There are various types of hydrogel wound dressing materials, including poly(vinyl alcohol) (PVA), chitosan, alginate, nanofibrillar cellulose, and gelatin [20, 21]. Sung et al. used the freeze-thaw method to prepare a minocycline-loaded PVA/CS hydrogel wound dressing, which displayed an excellent swelling ability, elasticity, form, and enhanced wound healing [22]. Swaroop et al. [23] used gamma irradiation to synthesize a PVA/Ag hydrogel with good swelling rate (SR) and antibiotic property for wound dressing. Chitosan hydrogel films and membranes have been widely applied in wound dressing. Chitosan does not exhibit any antibacterial activity at neutral pH. To impart the antibacterial properties of hydrogels, drugs were loaded into hydrogel films. For instance, Kumar et al. [24] prepared flexible and microporous CS/ZnO composite hydrogel films with enhanced swelling, blood clotting, antibacterial activity, and good biocompatibility. Singh and Pal [25] synthesized PVA/poly(AAm) hydrogel films loaded with tetracycline and gentamicin by sterculia crosslinking, which significantly improved the wound healing effect.In this study, the antibacterial CY was introduced into PVA/CMCS hydrogel films through in situ ultraviolet (UV) irradiation, and the properties of PVA/CMCS/CY were examined. Furthermore, the biocompatibility of PVA/CMCS/CY on human umbilical vein endothelial cells (HUVECs) was investigated.


## 2. Experimental

### 2.1. Materials

PVA was procured from Aladdin. Carboxymethyl chitosan (CMCS), acrylic acid (AA), phosphate buffer (PBS), cyanidin chloride (CY),Escherichia coli (E. coli), Staphylococcus aureus (S. aureus), Luria–Bertani (LB) agar, and drug-sensitive papers (streptomycin and cefixime) were purchased from Sinopharm Chemical Reagent Co. Ltd. Distilled water from a Millipore-Q system was used as the solvent.


### 2.2. Synthesis of PVA/CMCS/CY Hydrogel Films

The synthesis of the PVA/CMCS/CY hydrogel films is shown in Figure1. CY was in situ incorporated into the PVA/CMCS hydrogel network through an electrostatic attraction between CMCS and CY. First, 0.4 g of CMCS and 0.0022 g of CY were dispersed into 38.6 mL of H2O. Then, 1.0 g of PVA was added into the solution at 90°C for 2 h with stirring. After the mixture was cooled to 50°C, 2.5 mL of AA was added. Subsequently, the solution containing PVA, CMCS, CY, and AA was cast onto glass slides and exposed to a UV lamp at room temperature for 10 h under N2. The resultant hydrogel films were collected from the slides and extracted in deionized water at 50°C for 48 h. Finally, the PVA/CMCS/CY hydrogel films were vacuum-dried at 40°C for 24 h. For comparison, we synthesized PVA/CMCS hydrogel films without adding CY.Figure 1
Schematic of the formation processes of PVA/CMCS/CY hydrogel films.


### 2.3. Determination of the Infrared (IR) Spectrum, Scanning Electron Microscopy (SEM) Morphology, and Contact Angles of the Hydrogel Films

The Bruker Vector 22 spectrometer was used to measure the IR spectrum of hydrogel films. The films’ morphology was measured by SEM (Model S-4800). The contact angles of the hydrogel films were tested using a contact angle meter (Kruss DSA25, Germany).


### 2.4. Swelling Properties of the Hydrogel Films

The SR was measured in PBS (pH 7.4) at different temperatures: (a) 25°C, (b) 37°C, and (c) 40°C. The PVA/CMCS and PVA/CMCS/CY hydrogel films (2cm×2cm) were dispersed in 100 mL of PBS solution at different temperatures for 24h to reach the maximum swelling equilibrium. The SR of the hydrogel films was determined using the following [26]:
(1)SR=w2−w1w1×100%,where w1 is the initial mass of the sample and w2 is the mass of the sample after swelling for 24 h.


### 2.5. Mechanical Properties of the Hydrogel Films

The tensile strength and elongation at break of the PVA/CMCS and PVA/CMCS/CY hydrogel films were tested using a universal testing machine (Instron, Model 5943), and the crosshead speed was 20 mm/min at room temperature. The PVA/CMCS hydrogel films were prepared with dimensions of15mm×9.5mm×0.1285mm, and the PVA/CMCS/CY hydrogel films were cut with dimensions of 15mm×9.9mm×0.077mm.


### 2.6. Drug Release of the PVA/CMCS/CY Hydrogel Films

The release of PVA/CMCS/CY was tested inpH=5.5 (mimicking the endosome pH) and pH=7.4 (simulated body fluid) so as to speculate whether acidic conditions could influence the release behaviors or not. Approximately 10 mg of PVA/CMCS/CY was suspended in a conical flask containing 50 mL buffer and was kept in a shaking incubator at 37°C and 95 rpm. The incubated solution (3 mL) was collected and replaced with an equal volume of buffer. The CY release was characterized by an UV-visible spectrophotometer (UV1101M054) at 282 nm. The encapsulation and drug loading efficiencies were calculated using equations (2) and (3), respectively, as follows:
(2)Encapsulationefficiency=mdm0×100%,(3)Drugloadingefficiency=mdmt×100%,where md is the mass of CY in the PVA/CMCS/CY hydrogel films, mt is the total mass of the PVA/CMCS/CY hydrogel films, and m0 is the total mass of CY.


### 2.7. Antibacterial Study of the PVA/CMCS/CY Hydrogel Films

The antibacterial activity of the prepared PVA/CMCS/CY hydrogel films was evaluated againstE. coli and S. aureus. LB agar was used as the culture medium. The PVA/CMCS/CY hydrogel films, the PVA/CMCS hydrogel films, and the drug-sensitive papers (streptomycin and cefixime) were spread on the culture medium, which was evenly coated with bacteria. After 24 h of culture, the antibacterial effect was measured based on the inhibition zone surrounding each sample.


### 2.8. In Vitro Cytotoxicity of the PVA/CMCS/CY Hydrogel Films

HUVECs were selected to measure the cytotoxicity of PVA/CMCS/CY hydrogel films by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay [27]. Approximately 200 μL of HUVEC was seeded into a 96-well plate. Different concentrations of PVA/CMCS/CY (0.5, 1, 10, and 50 μg/mL) were added to each group (six wells) for 24 h. The absorbance was measured at a wavelength of 490 nm by using the Multiskan FC enzyme-labeled instrument (Thermo Scientific, USA). In addition, the number of viable cells was measured on day 3 by fluorescence microscopy (Olympus CKX41).


## 2.1. Materials

PVA was procured from Aladdin. Carboxymethyl chitosan (CMCS), acrylic acid (AA), phosphate buffer (PBS), cyanidin chloride (CY),Escherichia coli (E. coli), Staphylococcus aureus (S. aureus), Luria–Bertani (LB) agar, and drug-sensitive papers (streptomycin and cefixime) were purchased from Sinopharm Chemical Reagent Co. Ltd. Distilled water from a Millipore-Q system was used as the solvent.


## 2.2. Synthesis of PVA/CMCS/CY Hydrogel Films

The synthesis of the PVA/CMCS/CY hydrogel films is shown in Figure1. CY was in situ incorporated into the PVA/CMCS hydrogel network through an electrostatic attraction between CMCS and CY. First, 0.4 g of CMCS and 0.0022 g of CY were dispersed into 38.6 mL of H2O. Then, 1.0 g of PVA was added into the solution at 90°C for 2 h with stirring. After the mixture was cooled to 50°C, 2.5 mL of AA was added. Subsequently, the solution containing PVA, CMCS, CY, and AA was cast onto glass slides and exposed to a UV lamp at room temperature for 10 h under N2. The resultant hydrogel films were collected from the slides and extracted in deionized water at 50°C for 48 h. Finally, the PVA/CMCS/CY hydrogel films were vacuum-dried at 40°C for 24 h. For comparison, we synthesized PVA/CMCS hydrogel films without adding CY.Figure 1
Schematic of the formation processes of PVA/CMCS/CY hydrogel films.


## 2.3. Determination of the Infrared (IR) Spectrum, Scanning Electron Microscopy (SEM) Morphology, and Contact Angles of the Hydrogel Films

The Bruker Vector 22 spectrometer was used to measure the IR spectrum of hydrogel films. The films’ morphology was measured by SEM (Model S-4800). The contact angles of the hydrogel films were tested using a contact angle meter (Kruss DSA25, Germany).


## 2.4. Swelling Properties of the Hydrogel Films

The SR was measured in PBS (pH 7.4) at different temperatures: (a) 25°C, (b) 37°C, and (c) 40°C. The PVA/CMCS and PVA/CMCS/CY hydrogel films (2cm×2cm) were dispersed in 100 mL of PBS solution at different temperatures for 24h to reach the maximum swelling equilibrium. The SR of the hydrogel films was determined using the following [26]:
(1)SR=w2−w1w1×100%,where w1 is the initial mass of the sample and w2 is the mass of the sample after swelling for 24 h.


## 2.5. Mechanical Properties of the Hydrogel Films

The tensile strength and elongation at break of the PVA/CMCS and PVA/CMCS/CY hydrogel films were tested using a universal testing machine (Instron, Model 5943), and the crosshead speed was 20 mm/min at room temperature. The PVA/CMCS hydrogel films were prepared with dimensions of15mm×9.5mm×0.1285mm, and the PVA/CMCS/CY hydrogel films were cut with dimensions of 15mm×9.9mm×0.077mm.


## 2.6. Drug Release of the PVA/CMCS/CY Hydrogel Films

The release of PVA/CMCS/CY was tested inpH=5.5 (mimicking the endosome pH) and pH=7.4 (simulated body fluid) so as to speculate whether acidic conditions could influence the release behaviors or not. Approximately 10 mg of PVA/CMCS/CY was suspended in a conical flask containing 50 mL buffer and was kept in a shaking incubator at 37°C and 95 rpm. The incubated solution (3 mL) was collected and replaced with an equal volume of buffer. The CY release was characterized by an UV-visible spectrophotometer (UV1101M054) at 282 nm. The encapsulation and drug loading efficiencies were calculated using equations (2) and (3), respectively, as follows:
(2)Encapsulationefficiency=mdm0×100%,(3)Drugloadingefficiency=mdmt×100%,where md is the mass of CY in the PVA/CMCS/CY hydrogel films, mt is the total mass of the PVA/CMCS/CY hydrogel films, and m0 is the total mass of CY.


## 2.7. Antibacterial Study of the PVA/CMCS/CY Hydrogel Films

The antibacterial activity of the prepared PVA/CMCS/CY hydrogel films was evaluated againstE. coli and S. aureus. LB agar was used as the culture medium. The PVA/CMCS/CY hydrogel films, the PVA/CMCS hydrogel films, and the drug-sensitive papers (streptomycin and cefixime) were spread on the culture medium, which was evenly coated with bacteria. After 24 h of culture, the antibacterial effect was measured based on the inhibition zone surrounding each sample.


## 2.8. In Vitro Cytotoxicity of the PVA/CMCS/CY Hydrogel Films

HUVECs were selected to measure the cytotoxicity of PVA/CMCS/CY hydrogel films by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay [27]. Approximately 200 μL of HUVEC was seeded into a 96-well plate. Different concentrations of PVA/CMCS/CY (0.5, 1, 10, and 50 μg/mL) were added to each group (six wells) for 24 h. The absorbance was measured at a wavelength of 490 nm by using the Multiskan FC enzyme-labeled instrument (Thermo Scientific, USA). In addition, the number of viable cells was measured on day 3 by fluorescence microscopy (Olympus CKX41).


## 3. Results and Discussion

### 3.1. Fourier-Transform Infrared (FT-IR) Analysis

The FT-IR spectra for PVA/CMCS and PVA/CMCS/CY hydrogel films are shown in Figure2, a and b, respectively. In Figure 2(a), C=O amide stretching vibration peaks appeared at 1650 cm-1. The peaks at 1580, 1423, 1324, and 1100 cm-1 could be ascribed to the absorption of the amide group, CH–OH bending, C–H bond, and C–C backbone, respectively [28]. This result indicated that PVA and CMCS were successfully cross-linked to form a hydrogel. In Figure 2(b), the peaks at 3290, 1539, 1400, and 1050 cm-1 could be attributed to the O–H, benzene ring, C–O bending, and C–O–C of CY, which indicated that CY was loaded into PVA/CMCS hydrogel films.Figure 2
FT-IR spectra of (a) PVA/CMCS and (b) PVA/CMCS/CY hydrogel films.


### 3.2. SEM Analysis

The SEM images of PVA/CMCS and PVA/CMCS/CY hydrogel films are shown in Figure3. As shown in Figure 3(a), the surface of PVA/CMCS hydrogel films displayed a honeycomb-like structure with interconnecting pores. As shown in Figure 3(b), the pore sizes of the network were in the range of several micrometers. Figures 3(c) and 3(d) show that the PVA/CMCS/CY hydrogel films exhibited a 3D porous network structure [29], and the addition of CY obviously affected the interior morphology of the hydrogel. The addition of CY led to a compact interior structure and small pores of the PVA/CMCS/CY hydrogel films. The pore size decreased correspondingly with the concentration of CY increasing due to the decrease in the distance between the polymer chains and the increase of crosslink density. Besides, the decrement of the pore size may be attributed to the CY solution entering into the inner side of the sample through these interconnected pores by capillary force [30].Figure 3
SEM images of (a, b) PVA/CMCS and (c, d) PVA/CMCS/CY hydrogel films.
(a)
(b)
(c)
(d)


### 3.3. Contact Angle Analysis

The contact angles of the PVA/CMCS and PVA/CMCS/CY hydrogel films were48.2°±0.9° and 55.0°±1.2°, respectively. The contact angle of the PVA/CMCS/CY hydrogel films was higher than that of the PVA/CMCS films, indicating that the PVA/CMCS/CY hydrogel films were more hydrophobic than the PVA/CMCS films because of the presence of CY [30].


### 3.4. Analysis of Swelling Properties

Figures4(a) and 4(b) show the SRs of the PVA/CMCS and PVA/CMCS/CY hydrogel films, respectively, in PBS (pH 7.4) and at different temperatures: (a) 25°C, (b) 37°C, and (c) 40°C. All of the hydrogel films had a high swelling ratio due to CMCS and PVA having many hydrophilic groups. In Figure 4(a), the equilibrium SR of PVA/CMCS at 37°C reached up to 716% at 250 min, which was higher than that of PVA/CMCS at 25°C (645%) and 40°C (680%). Therefore, the swelling properties of the PVA/CMCS hydrogel films were beneficial for wound dressing application [31]. As shown in Figure 4(b), the SRs of the PVA/CMCS/CY hydrogel films were 467% (a, 25°C), 380% (b, 37°C), and 415% (c, 40°C). The decrease in the PVA/CMCS/CY hydrogel films may be attributed to the binding between CY and CMCS, which are responsible for higher cross-links. Compared with the PVA/CMCS hydrogel films, the higher cross-links restricted the water penetration for swelling. Moreover, the swelling ratio obviously decreased with the addition of CY, because the electrostatic attraction between CMCS and CY could limit the swelling force. Besides, the swelling ratio of the PVA/CMCS/CY hydrogel films was limited, which was attributed to that there were a lot of hydrogen bonds formed from CY and PVA (–OH and –OH) and CY and CMCS (–OH and –COOH).Figure 4
SRs of PVA/CMCS (a) and PVA/CMCS/CY hydrogel films in PBS (pH 7.4) and at different temperatures: (a) 25°C, (b) 37°C, and (c) 40°C.
(a)
(b)


### 3.5. Analysis of Mechanical Properties

The tensile tests provided an indication of the strength and elasticity of the hydrogel films, and the results suggested that the hydrogel films applied in wound dressing were strong yet flexible [32]. The mechanical properties of the PVA/CMCS and PVA/CMCS/CY hydrogel films are presented in Figure 5. As shown in Figure 5(a), PVA/CMCS had a tensile strength of 31.13 MPa and an elongation at break of 27.36%. As shown in Figure 5(b), the PVA/CMCS/CY hydrogel films exhibited a higher elongation at break (167.04%) but lower tensile strength (15.82 MPa) than the PVA/CMCS hydrogel films. These results indicated that PVA/CMCS/CY was softer and more flexible than the PVA/CMCS hydrogel films. Therefore, the addition of CY into the polymeric network could facilitate the rearrangement of molecular chains in composite films [28].Figure 5
Typical tensile stress-strain curves of (a) PVA/CMCS and (b) PVA/CMCS/CY hydrogel films determined at room temperature.


### 3.6. UV Analysis

The release profiles of CY from PVA/CMCS/CY are shown in Figure6. The standard curve of CY can be derived using equation (4). The drug loading and encapsulation efficiencies of PVA/CMCS/CY were 17.3% and 33.5%, respectively.
(4)A=56.4972·C+0.1695,R2=0.981.Figure 6
Release behavior of CY from PVA/CMCS/CY at (a) pH 7.4 and (b) pH 5.5.In Figure6, the PVA/CMCS/CY hydrogel films exhibited a severe burst release, and more than 22% CY was released in the first 2 h, which could be due to the attachment of the drugs to the surface of the hydrogel films. Approximately 33.5% (Figure 6(a)) and 27.6% (Figure 6(b)) of CY were released at pH 7.4 and 5.5 after 60 h, respectively. Therefore, the addition of CY resulted in the compact network structure of the PVA/CMCS/CY hydrogel, so the drug stored in the hydrogel films needed to be diffused by more polymer layers. Under a weak alkaline condition (pH=7.4), the carboxylic acid groups in PVA/CMCS/CY hydrogel films were gradually ionized into carboxylate ions (–COO-), which generate electrostatic repulsion to make the hydrogel films release fast. However, at an acidic condition (pH=5.5), the electrostatic attraction from the protonation of amino groups (–NH3+) and CY could limit the drug release of hydrogel films. Moreover, in pH=5.5, the protonation of amino groups on CMCS caused the breakage of the dynamic Schiff base linkage and thus increased the connectivity of internal pores of hydrogel films and limited the drug release.


### 3.7. Antibacterial Properties

The in vitro antibacterial potentials of (A) streptomycin (S10), (B) cefotaxime (CTX30), (C) PVA/CMCS, and (D) PVA/CMCS/CY hydrogel films were investigated for positive S. aureus (Figure 7(a)) and negative E. coli (Figure 7(b)) through the disc diffusion method. Streptomycin, cefotaxime, and PVA/CMCS/CY (Figure 7(a) (A, B, and D) and Figure 7(b) (A, B, and D)) expressed an inhibition zone, whereas the PVA/CMCS hydrogel films (Figures 7(a) (C) and Figure 7(b) (C)) did not possess an antibacterial activity. The control cefotaxime displayed an inhibition ring size of 9.3 and 14.3 mm for S. aureus and E. coli, respectively. The control streptomycin exhibited an inhibition ring size of 12.4 and 11.2 mm for S. aureus and E. coli, respectively. The PVA/CMCS/CY hydrogel films showed a growth inhibition ring size of 10.3 and 10.2 mm for S. aureus and E. coli, respectively. The PVA/CMCS/CY hydrogel films showed good antibacterial activities on both positive S. aureus and negative E. coli. The good antibacterial activity of the PVA/CMCS/CY hydrogel films could be attributed to the efficient release of CY in the media.Figure 7
Antibacterial activity screening images of samples against (a)S. aureus and (b) E. coli: (A) streptomycin (S10), (B) cefotaxime (CTX30), (C) PVA/CMCS hydrogel films, and (D) PVA/CMCS/CY hydrogel films.
(a)
(b)


### 3.8. In Vitro Cytotoxicity Assay

Cell viability data (Figure8) indicated that the PVA/CMCS/CY hydrogel films had no considerable cytotoxicity to HUVECs. The PVA/CMCS/CY hydrogel films with different concentrations achieved 90%–95% viability after 24 h of incubation. The cell viability results of the products were further confirmed by fluorescence microscopy. The control group contained the largest number of viable cells (Figure 9(a)). With the addition of different concentrations of PVA/CMCS/CY (0.5, 1, 10, and 50 μg/mL, Figures 9(b)–9(e), respectively), HUVECs displayed no obvious morphological change. The PVA/CMCS/CY hydrogel films showed good biocompatibility for HUVECs, indicating their suitability for wound healing.Figure 8
Histogram of cell viability as a function of PVA/CMCS/CY concentration in culture media.Figure 9
HUVECs observed by fluorescence microscopy on (a) the control and (b–e) the PVA/CMCS/CY hydrogel films of (b) 0.5μg/mL, (c) 1 μg/mL, (d) 10 μg/mL, and (d) 50 μg/mL.
(a)
(b)
(c)
(d)
(e)


## 3.1. Fourier-Transform Infrared (FT-IR) Analysis

The FT-IR spectra for PVA/CMCS and PVA/CMCS/CY hydrogel films are shown in Figure2, a and b, respectively. In Figure 2(a), C=O amide stretching vibration peaks appeared at 1650 cm-1. The peaks at 1580, 1423, 1324, and 1100 cm-1 could be ascribed to the absorption of the amide group, CH–OH bending, C–H bond, and C–C backbone, respectively [28]. This result indicated that PVA and CMCS were successfully cross-linked to form a hydrogel. In Figure 2(b), the peaks at 3290, 1539, 1400, and 1050 cm-1 could be attributed to the O–H, benzene ring, C–O bending, and C–O–C of CY, which indicated that CY was loaded into PVA/CMCS hydrogel films.Figure 2
FT-IR spectra of (a) PVA/CMCS and (b) PVA/CMCS/CY hydrogel films.


## 3.2. SEM Analysis

The SEM images of PVA/CMCS and PVA/CMCS/CY hydrogel films are shown in Figure3. As shown in Figure 3(a), the surface of PVA/CMCS hydrogel films displayed a honeycomb-like structure with interconnecting pores. As shown in Figure 3(b), the pore sizes of the network were in the range of several micrometers. Figures 3(c) and 3(d) show that the PVA/CMCS/CY hydrogel films exhibited a 3D porous network structure [29], and the addition of CY obviously affected the interior morphology of the hydrogel. The addition of CY led to a compact interior structure and small pores of the PVA/CMCS/CY hydrogel films. The pore size decreased correspondingly with the concentration of CY increasing due to the decrease in the distance between the polymer chains and the increase of crosslink density. Besides, the decrement of the pore size may be attributed to the CY solution entering into the inner side of the sample through these interconnected pores by capillary force [30].Figure 3
SEM images of (a, b) PVA/CMCS and (c, d) PVA/CMCS/CY hydrogel films.
(a)
(b)
(c)
(d)


## 3.3. Contact Angle Analysis

The contact angles of the PVA/CMCS and PVA/CMCS/CY hydrogel films were48.2°±0.9° and 55.0°±1.2°, respectively. The contact angle of the PVA/CMCS/CY hydrogel films was higher than that of the PVA/CMCS films, indicating that the PVA/CMCS/CY hydrogel films were more hydrophobic than the PVA/CMCS films because of the presence of CY [30].


## 3.4. Analysis of Swelling Properties

Figures4(a) and 4(b) show the SRs of the PVA/CMCS and PVA/CMCS/CY hydrogel films, respectively, in PBS (pH 7.4) and at different temperatures: (a) 25°C, (b) 37°C, and (c) 40°C. All of the hydrogel films had a high swelling ratio due to CMCS and PVA having many hydrophilic groups. In Figure 4(a), the equilibrium SR of PVA/CMCS at 37°C reached up to 716% at 250 min, which was higher than that of PVA/CMCS at 25°C (645%) and 40°C (680%). Therefore, the swelling properties of the PVA/CMCS hydrogel films were beneficial for wound dressing application [31]. As shown in Figure 4(b), the SRs of the PVA/CMCS/CY hydrogel films were 467% (a, 25°C), 380% (b, 37°C), and 415% (c, 40°C). The decrease in the PVA/CMCS/CY hydrogel films may be attributed to the binding between CY and CMCS, which are responsible for higher cross-links. Compared with the PVA/CMCS hydrogel films, the higher cross-links restricted the water penetration for swelling. Moreover, the swelling ratio obviously decreased with the addition of CY, because the electrostatic attraction between CMCS and CY could limit the swelling force. Besides, the swelling ratio of the PVA/CMCS/CY hydrogel films was limited, which was attributed to that there were a lot of hydrogen bonds formed from CY and PVA (–OH and –OH) and CY and CMCS (–OH and –COOH).Figure 4
SRs of PVA/CMCS (a) and PVA/CMCS/CY hydrogel films in PBS (pH 7.4) and at different temperatures: (a) 25°C, (b) 37°C, and (c) 40°C.
(a)
(b)


## 3.5. Analysis of Mechanical Properties

The tensile tests provided an indication of the strength and elasticity of the hydrogel films, and the results suggested that the hydrogel films applied in wound dressing were strong yet flexible [32]. The mechanical properties of the PVA/CMCS and PVA/CMCS/CY hydrogel films are presented in Figure 5. As shown in Figure 5(a), PVA/CMCS had a tensile strength of 31.13 MPa and an elongation at break of 27.36%. As shown in Figure 5(b), the PVA/CMCS/CY hydrogel films exhibited a higher elongation at break (167.04%) but lower tensile strength (15.82 MPa) than the PVA/CMCS hydrogel films. These results indicated that PVA/CMCS/CY was softer and more flexible than the PVA/CMCS hydrogel films. Therefore, the addition of CY into the polymeric network could facilitate the rearrangement of molecular chains in composite films [28].Figure 5
Typical tensile stress-strain curves of (a) PVA/CMCS and (b) PVA/CMCS/CY hydrogel films determined at room temperature.


## 3.6. UV Analysis

The release profiles of CY from PVA/CMCS/CY are shown in Figure6. The standard curve of CY can be derived using equation (4). The drug loading and encapsulation efficiencies of PVA/CMCS/CY were 17.3% and 33.5%, respectively.
(4)A=56.4972·C+0.1695,R2=0.981.Figure 6
Release behavior of CY from PVA/CMCS/CY at (a) pH 7.4 and (b) pH 5.5.In Figure6, the PVA/CMCS/CY hydrogel films exhibited a severe burst release, and more than 22% CY was released in the first 2 h, which could be due to the attachment of the drugs to the surface of the hydrogel films. Approximately 33.5% (Figure 6(a)) and 27.6% (Figure 6(b)) of CY were released at pH 7.4 and 5.5 after 60 h, respectively. Therefore, the addition of CY resulted in the compact network structure of the PVA/CMCS/CY hydrogel, so the drug stored in the hydrogel films needed to be diffused by more polymer layers. Under a weak alkaline condition (pH=7.4), the carboxylic acid groups in PVA/CMCS/CY hydrogel films were gradually ionized into carboxylate ions (–COO-), which generate electrostatic repulsion to make the hydrogel films release fast. However, at an acidic condition (pH=5.5), the electrostatic attraction from the protonation of amino groups (–NH3+) and CY could limit the drug release of hydrogel films. Moreover, in pH=5.5, the protonation of amino groups on CMCS caused the breakage of the dynamic Schiff base linkage and thus increased the connectivity of internal pores of hydrogel films and limited the drug release.


## 3.7. Antibacterial Properties

The in vitro antibacterial potentials of (A) streptomycin (S10), (B) cefotaxime (CTX30), (C) PVA/CMCS, and (D) PVA/CMCS/CY hydrogel films were investigated for positive S. aureus (Figure 7(a)) and negative E. coli (Figure 7(b)) through the disc diffusion method. Streptomycin, cefotaxime, and PVA/CMCS/CY (Figure 7(a) (A, B, and D) and Figure 7(b) (A, B, and D)) expressed an inhibition zone, whereas the PVA/CMCS hydrogel films (Figures 7(a) (C) and Figure 7(b) (C)) did not possess an antibacterial activity. The control cefotaxime displayed an inhibition ring size of 9.3 and 14.3 mm for S. aureus and E. coli, respectively. The control streptomycin exhibited an inhibition ring size of 12.4 and 11.2 mm for S. aureus and E. coli, respectively. The PVA/CMCS/CY hydrogel films showed a growth inhibition ring size of 10.3 and 10.2 mm for S. aureus and E. coli, respectively. The PVA/CMCS/CY hydrogel films showed good antibacterial activities on both positive S. aureus and negative E. coli. The good antibacterial activity of the PVA/CMCS/CY hydrogel films could be attributed to the efficient release of CY in the media.Figure 7
Antibacterial activity screening images of samples against (a)S. aureus and (b) E. coli: (A) streptomycin (S10), (B) cefotaxime (CTX30), (C) PVA/CMCS hydrogel films, and (D) PVA/CMCS/CY hydrogel films.
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## 3.8. In Vitro Cytotoxicity Assay

Cell viability data (Figure8) indicated that the PVA/CMCS/CY hydrogel films had no considerable cytotoxicity to HUVECs. The PVA/CMCS/CY hydrogel films with different concentrations achieved 90%–95% viability after 24 h of incubation. The cell viability results of the products were further confirmed by fluorescence microscopy. The control group contained the largest number of viable cells (Figure 9(a)). With the addition of different concentrations of PVA/CMCS/CY (0.5, 1, 10, and 50 μg/mL, Figures 9(b)–9(e), respectively), HUVECs displayed no obvious morphological change. The PVA/CMCS/CY hydrogel films showed good biocompatibility for HUVECs, indicating their suitability for wound healing.Figure 8
Histogram of cell viability as a function of PVA/CMCS/CY concentration in culture media.Figure 9
HUVECs observed by fluorescence microscopy on (a) the control and (b–e) the PVA/CMCS/CY hydrogel films of (b) 0.5μg/mL, (c) 1 μg/mL, (d) 10 μg/mL, and (d) 50 μg/mL.
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## 4. Conclusions

CY was successfully introduced into PVA/CMCS hydrogel films by in situ UV irradiation, and the effects of CY on the structure and properties were studied. The SEM images illustrated that the addition of CY resulted in the compact interior structure and small pores of the PVA/CMCS/CY hydrogel films. The tensile test indicated that the inclusion of CY within PVA/CMCS hydrogel networks improved the elasticity of the composite films. Moreover, the developed PVA/CMCS/CY hydrogel films showed good antibacterial activities againstS. aureus and E. coli. Therefore, PVA/CMCS/CY nanocomposites have a promising application potential in wound dressing.



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*Source: 7510573-2019-03-27.xml*