Chitosan nanofiber biocomposites for potential wound healing applications: Antioxidant activity with synergic antibacterial effect

Abstract Bacterial wound infection is one of the most common nosocomial infections. The unnecessary employment of antibiotics led to raising the growth of antibiotic‐resistant bacteria. Accordingly, alternative armaments capable of accelerating wound healing along with bactericidal effects are urgently needed. Considering this, we fabricated chitosan (CS)/polyethylene oxide (PEO) nanofibers armed with antibacterial silver and zinc oxide nanoparticles. The nanocomposites exhibited a high antioxidant effect and antibacterial activity against Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa. Besides, based on the results of the cell viability assays, the optimum concentration of ZnONPs and AgNPs in the nanofibrous mats is 0.2% w/v and 0.08% w/v respectively and had no cytotoxicity on fibroblast cells. The scaffold also showed good blood compatibility according to the effects of coagulation time. As well as significant fibroblast migration and proliferation on the wound margin, according to wound‐healing assay. All in all, the developed biocompatible, antioxidant, and antibacterial Ag‐ZnO NPs incorporated CS/PEO nanofibrous mats showed their potential as an effective wound dressing.


| INTRODUCTION
Wound infection is a common and unsolved issue in preventing wound healing. Any kind of wound on the skin is susceptible to the accumulation of infectious bacteria. 1 With the spread of antibiotic-resistant bacteria, employing antibiotics to prevent and treat wound infections is inefficient.
In addition, it imposes extra costs on the patient and society. Consequently, alternative materials are pivotal to combat pathogens. 2,3 Advanced wound dressing is the barrier to care for sores and should mainly target bacterial infection by sufficient sealing of the injury microenvironment from peripheral contaminants. Simultaneously, they can eradicate excessive exudate, enhance autolysis debridement, and maintain adequate water for healing. In addition to barrier property, they would also have specific flexibility, applicability, constancy, biodegradability and be able to remove, pace the healing process, and decrease the risk of infection. 4,5 Today, the use of advanced mats comprising antibacterial properties is essential in preventing and treating wound infections. Despite the diversity of wound dressings in the market, highly exuding wounds are difficult to heal as dressings cannot efficiently inhibit the wound's microbial invasion. 6 Antimicrobial dressings involve incorporating an antiseptic agent comprising zinc oxide, silver, titanium oxide, and iodine, adding to the dressings to avoid microbial contamination. 7 Electrospinning is one of the most common methods to produce electrospun wound dressing mats. 8,9 By fabricating nanofibers with the appropriate surface nanotopography, density, and two-dimensional structure, electrospinning is an effective way to fabricate substrates targeted for tissue engineering. 10 These nanofibrous mats can be employed as a wound dressing because of their high porosity, making them suitable for ventilation and air exchange. Since the skin is repaired in a moist environment, the porous structure of mats can provide an environment ideal for healing through the exchange of gas. 11 Chitosan is a cationic biopolymer with a linear structure derived from hydrolysis of the natural polymer chitin. 12 Due to its biocompatibility, biodegradability, cell transplantation ability, along with antibacterial and antifungal properties, it is widely deployed in scaffolds for regenerative medicine applications and wound healing. 13,14 Due to its natural origin and wound healing capability, chitosan wound management products, for example, ChitoSorb (ChitoTech), KytoCel (Aspen Medical), ChitoGauze Pro (HemCon), and Opticell (Medline), used to treat severe cases of burned skin, open and deep wounds. 15 Besides, nanofibrous chitosan scaffolds play a significant role in wound healing due to their positive influence on the re-epithelialization and regeneration of the granular layer of the wounds. 15,16 Nonetheless its application for wound dressing has some limitations, for example, poor mechanical strength and low antibacterial activity, which is inadequate for effective wound dressing. 17 Recently, there has been a growing interest in research on nanofibrous scaffolds developed by electrospinning of natural and synthetic polymers solely or as blend with bioactive plant extracts and metallic nanoparticles for tissue regeneration. 18,19 Electrospinning of chitosan is very difficult because of the cationic nature of the solution, its rigid chemical structure, and its specific intermolecular interactions. 14,20 The simplest and most effective way to enhance the electrospinning capabilities of chitosan is blending with another polymer (e.g., polyethylene oxide) with high electrospinning potential. 21,22 Polyethylene oxide (PEO) is a biocompatible, noncytotoxic, and hydrophilic polymer and is widely used in biomedical applications. Moreover, PEO is employed in combination with chitosan to form uniform nanofibers with suitable mechanical properties for wound healing and tissue engineering applications. 23,24 Silver nanoparticles are the most prevalent nanostructures for antimicrobial therapy due to their potent microbicide properties. 25 Ag nanoparticles have been employed in numerous approaches for wound treatment, such as impregnation of silver nanoparticles in polymeric bandages for upgrading of the existing polymer-based moist wound dressings or adding of silver ions in hydrocolloids, foams (PolyMem Silver), hydrogel (SilvaSorb gel), alginate dressings and silver sulfadiazine (SSD) creams and gauze dressings (Urgotul ® SSD, Askina Calgitrol Ag [silver alginate], Acticoat™). For instance, Argovit™ is a silver nanoparticle formulation with antimicrobial activity, showing neither cytotoxic nor genotoxic effects in human lymphocytes, and promotes diabetic wound healing. 6,26 Porous and nanofibrous antibacterial materials of the nanosilver/cellulose composite showed excellent antibacterial properties as a result of the Ag nanoparticles. The composite alsoimproved infected wound healing owing to the absorbing capability for wound exudate besides promoted keratinocyte proliferation and growth, thus providing an appropriate environment for cell growth. 25,27 Other antimicrobial nanocomposite scaffolds containing Ag NPs were blended with polymers such as α-chitin/β-chitin hydrogel, alginate/hydroxyapatite, poly(lactic acid), poly(ϵ-caprolactone), and poly(3-droxybutyrate-co-3-hydroxyvalerate) 25 for tissue engineering applications.
Antimicrobial properties of low concentration of ZnO NPs and their role in fibroblast proliferation, angiogenesis, and increased reepithelialization properties make them an active ingredient in wound dressings. 7,16,[27][28][29] It was reported that the bactericidal activity of zinc oxide NPs against Streptococcus mutans is significantly higher than that of Ag NPs at both 1% w/w. 25 Blending ZnO NPs with chitosan increases microbicidal capability and increases collagen deposition in the wound area. 16 Altogether, the purpose of this study was to design antibacterial and hemocompatible chitosan/PEO-based wound dressings using the electrospinning technique. In this regard, silver and zinc oxide nanoparticles were employed to enhance the antibacterial capability of the nanofibers. We then evaluated the wound healing capability, cytotoxicity, and hemocompatibility of the nanofibrous mats. As the T A B L E 1 MIC determination of AgNPs, ZnONPs, and control antibiotics against Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa medical applications of metal-based nanomaterials are associated with their cytotoxicity, many efforts have been committed for diminishing the cytotoxicity of nanometals. Different strategies can be deployed to decrease the cytotoxicity of metallic nanoparticles, such as immobilization of nanoparticles in polymeric matrices/nanocarriers or surface coatings. For instance, chitosan, dextran, chitosan/alginate beads, polyethylene glycol, silica, or titania prepare unique materials with new characteristics to both components, e.g., tensile strength, flexibility, and biodegradability. 7,25 Combining biopolymers with nanofillers is an alternative for achieving materials with improved antibacterial properties. 27 Antioxidant are suggested to assist the management of wound oxidative stress and, thus, accelerate wound healing. 30

| Characterization
The surface morphology of the nanofibrous mats assessed using FESEM is shown in Figure 1. Uniform fibers 100-300 nm in diameter were obtained for nanofibrous mats containing silver and zinc oxide nanoparticles suitable for fibroblast cell attachment. The optimal diameter of chitosan/PEO nanofibers has been reported between 120 and 230 nm. 32 In the present study, based on FESEM results, the average diameter of electrospun nanofibers containing AgNPs, ZnONPs, and AgNPs-ZnONPs was 185 ± 15 nm, 184 ± 26 nm, and 193 ± 15 nm, respectively.
FTIR test was performed to characterize the specific chemical bonds in the prepared nanofibrous mats, as shown in Figure 2a.
The antibacterial effect of the prepared nanofibrous mats against Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli. Inhibition zone of the various contents of CS, AgNPs, ZnONPs, and AgNPs-ZnONPs mats compared with selected antibiotic(Ampicillin, Gentamicin, and Penicillin), (b) schematic antibacterial mechanisms of silver and zinc oxide nanoparticles (****p < 0.0001; *p < 0.05) chitosan amine groups. As is well known, chitosan is a fragile natural polymer, whereas PEO is a flexible polymer. 18 Accordingly, as expected, the blend of chitosan and PEO showed sufficient flexibility.
Based on the tensile test results, the incorporation of nanoparticles into this blend of nanofibers and nanocomposite fabrication leads to a significant increase in nanofibrous mats' tensile properties and strength.

| Antibacterial assessment
An antibacterial assessment was performed using a disk diffusion method for each bacterium. The diameter of the inhibition zone was measured after 24 h of incubation by a caliper. As shown in Figure 4a, prepared nanofibrous mats have antibacterial properties compared to the selected antibiotic. In the disk diffusion test, the inhibition zone diameter was employed as an indicator of antibacterial activity of scaffolds; According to the inhibition zone diameter of mats containing silver, zinc oxide, and mixtures of them against Gram-positive S. aureus was more significant than the other two Gram-negative bacteria. As expected, the Gram-positive bacterium is more sensitive to these mats. By adding silver nanoparticles, the inhibition zone was formed for all three bacteria. The sensitivity of S. aureus was more than the other two bacteria by measuring and comparing the inhibi- ascorbic acid (p < 0.005), and scaffold contain zinc oxide nanoparticles demonstrated the lowest scavenging activities (p < 0.001). When the skin is damaged, large amounts of ROS are produced in the inflammatory phase of wound healing, resulting in biological damage, including degradation of lipids, proteins, nucleic acids, and ultimately cell death, which disrupts the wound healing process. The use of antioxidants can effectively help with enzymatic repair and improve metabolism. 41 Many studies have shown that nanomaterials are a reliable source of antioxidant activity. 3,42 The DPPH free radical-scavenging activity of the scaffolds at different concentrations was evaluated and compared to ascorbic acid and BHT used as a standard. Results indicated that AgNPs/CS/PEO exhibited antioxidant ability against DPPH in a dose-dependent manner ( Figure 5). The highest activity was recorded with AgNPs/CS/PEO and Ag-ZnONPs/CS/PEO, with the lowest SC 50 value in scavenging the DPPH (0.47 mg/ml).

| Cell viability and hemocompatibility
To assess the cell viability of the fibroblast cells against the nanofibrous mats, two different analysis including MTT and CCK-8 assay were employed. 43 According to GB/T 16886. 5-20035- (ISO 10993-5: 1999), samples with cell viability higher than 75% can be counted as noncytotoxic. 44 According to the OD of samples and standard curve ( Figure S1), cell proliferation and cell viability have similar pattern, confirming that the MTT assay results (Figure 6a   Our results suggested wound healing process can be accelerated through antioxidant, antimicrobial of prepared nanofibrous mats. 56 The results showed the prepared nanofibrous mats adorned with silver and zinc nanoparticles possess the considerable antioxidant ability and synergic antimicrobial activity. Consequently, these characteristics can synergistically prompt wound healing processes in acute and chronic forms. Figure 8 shows a schematic illustration for the nanofibrous mats with antioxidant activity and synergic antibacterial properties to stimulate wound healing.
As reported, zinc oxide and silver nanoparticles cause bacterial death by acting on the bacterial cell wall and membrane, inhibiting protein and DNA synthesis, and disturbing the antioxidant system. 25,38,[57][58][59] In our study, adding these two nanoparticles to the chitosan scaffold showed a synergistic effect, indicating that scaffolds observed that the antibacterial potential of zinc nanoparticles was significantly higher than that of zinc powder, which could be due to the higher surface-to-volume ratio of smaller particles than larger particles, which increased their efficiency. It was also reported that Gram-positive bacteria are more sensitive to these nanoparticles than Gram-negative bacteria. In this study, the inhibition zone diameter nanoparticles for E. coli, P. aeruginosa, and S. aureus were 21, 17, and 31 mm, respectively. The MICs of these nanoparticles were also announced as 16, 26, and 10 μg/ml, respectively. 63 The results of the present study, which include an inhibition zone diameter for the stud-  66 Also, in other studies, the increase in antibacterial effect following the activity of both silver nanoparticles and zinc oxide has been well evident. 67,68 In antibacterial studies, high sensitivity of S. aureus to composite mats containing the nanoparticles was observed compared to

| Suspension preparation of nanoparticles
Based on similar studies, 100 μg/ml of silver nanoparticle was selected to prepare the suspension of Ag NP. 69 The stock solution was prepared as follows: 200 μg of the nanoparticle powder was dispersed in 1 ml of Müller Hinton broth and prepared as the primary stock for MIC testing for the bacteria studied.
The solution comprising zinc oxide nanoparticle prepared as following; a specific concentration was considered by default for each studied bacteria based on previous studies. For S. aureus, the concentration of initial stock was 1.98 mg/ml: for E. coli, 64.8 mg/ml and for P. aeruginosa, 20 mg/ml. 63,70

| Broth-microdilution method
There was no particular antibacterial guideline for nanoparticles.

| Preparation of chitosan/PEO solution containing silver nanoparticle for electrospinning
To prepare chitosan/PEO solution, chitosan and PEO solutions separately in 80% V/V and 0.5molar acetic acid, prepared respectively.
Then, the prepared solutions are mixed to a certain proportion.
To prepare a 3 wt% chitosan solution, first, 0.3 g of chitosan powder was poured into a beaker, then 80% acetic acid was added. The solution was stirred for 16 h at room temperature on a magnetic stirrer at 700 rpm. A 3 wt% PEO solution was prepared similar to chitosan. After adding 0.5 molar acetic acid to 0.3 g of a PEO powder to give a uniform solution, it was stirred for 8 h at ambient temperature at 400 rpm; after separate preparation of these solutions, the chitosan solution and the PEO were mixed at a weight ratio of 9:1 and stirred at room temperature at 400 rpm for 12 h. Then, proper concentrations of silver nanoparticles according to MIC were added to the chitosan solution for each bacterium. Thus, a solution containing 0.16% silver nanoparticle was prepared for S. aureus, a solution containing 0.08% silver nanoparticle for E. coli, and a solution containing 0.16% silver nanoparticle for P. aeruginosa. The prepared solution was then filled into the syringe and put into an electrospinning device with the following parameters: voltage 20 KV, a distance of needle from collector 14 cm, rotation of collector 700 rpm. To achieve nanofibrous mats adorned with silver nanoparticles with appropriate thickness, electrospinning was performed using the obtained parameters for 6 h.

| Preparation of chitosan/PEO solution containing zinc oxide nanoparticle for electrospinning
For fabrication of the nanofibers containing zinc nanoparticles, concentrations obtained from MIC zinc nanoparticles for each bacterium were added to the chitosan solution. Thus, a scaffold containing 0.2% zinc nanoparticles was produced for S. aureus. A scaffold containing 27% zinc nanoparticles was fabricated for E. coli. A scaffold containing 8.3% zinc nanoparticles was fabricated for P. aeruginosa. The same settings obtained from the electrospinning of silver scaffold were used for electrospinning, the polymeric solution containing zinc oxide nanoparticles.

| Preparation of chitosan/PEO solution containing silver and zinc oxide nanoparticle
To preparing the nanofibrous mats containing the silver and zinc nanoparticles, concentrations obtained from MIC nanoparticles for each bacterium were added to the chitosan solution. Thus, Mix1 scaffold contains 0.28% zinc and silver nanoparticles (0.08% silver nanoparticles and 0.2% zinc nanoparticles), and Mix2 scaffold containing 8.4% zinc and silver nanoparticles (0.08% silver nanoparticles and 8.3% zinc nanoparticles) were obtained.
A 1% TPP solution was used as a cross-linker to improve the strength of the nanofibrous mats. Specimens were prepared from scaffolds measuring 3 Â 2 cm and immersed in TPP solution for 5-15 min, washed with PBS after each step, and dried at room temperature.

| Characterization
The morphology and diameter of the nanofibers were examined by scanning electron microscopy (FESEM; Philips XL30) after coating with gold by gold sputtering apparatus. ImageJ software was used to measure the average diameter of nanofibers. X-ray energy dispersive spectroscopy coupled to FESEM apparatus was used for elemental analysis of prepared nanofibrous mats to confirm nanoparticles' presence in the nanofibrous mats. The FTIR was performed in the 4000 to 400 cm À1 region using Bruker Tensor

| In vitro antioxidant activities
The antioxidant activities of the prepared scaffolds were evaluated using DPPH radical-scavenging activity. The DPPH radical-scavenging activities of CS/PEO, AgNPs/CS/PEO, ZnONPs/CS/PEO, and AgNPs/ ZnONPs/CS/PEO were determined as described as reported in 71 with slight modification. Briefly, a volume of 1 ml of methanolic dilutions equivalent volumes of prepared scaffolds was mixed with 1 ml of 80 μg/ml of DPPH as a free radical source. The mixtures were then kept for 30 min in the dark at room temperature (25 C ± 1). The lower absorbance of the reaction mixture indicated a higher DPPH radicalscavenging activity. Neutralization of DPPH was measured against the NC at 517 nm by a Shimadzu UV-1800 spectrophotometer according to the following equation: NCs consisted of all the reagents except the antioxidant components.

| Cell viability
The MTT assay was done to determine the fibroblast viability on the prepared nanofibrous mats. The cells were cultured in DMEM medium supplemented with 10% fetal bovine serum and 1% penicillin/ streptomycin antibiotic under standard conditions of 37 C and 5% CO 2 . Each experiment was performed three times at the specified culture durations of 1, 3, and 7 days in a 96-well plate (3 Â 10 3 cells/well) as reported previously. 21,22 Finally, the microplate reader (ChroMate-4300, FL, USA) at 570 nm was used to measure the absorbance of the samples. For better assessment and evaluating biocompatibility, after assessing the cytotoxicity with MTT assay, we evaluated the viability as well as cell proliferation using cell counting kit-8 (CCK-8) using fibroblast (L929) cells, respectively. CCK-8 assay is a more sensitive assay than any other tetrazolium salts (e.g., MTT, MTS, or XTT). Such analysis allows more sensitive colorimetric assays for the indication of the number of viable cells in the proliferation assays and cytotoxicity. 43 Cells were seeded on nanofibrous mats and well as controll in 96-well plates at 5 Â 10 3 cells/well. Cell proliferation was investigated after 1, 3, and 7 days using CCK-8 assay. To create a calibration curve, after preparing the wells with known numbers of viable cells, 10 μL of the CCK-8 solution was added to each well of the plate. The plates were incubated for 1 to 4 h in the incubator, and the absorbance were measured at 450 nm using a microplate reader. The calibration curve prepared using the data obtained from the wells that contain known numbers of viable cells ( Figure S1). 43 The one-way analysis of variance (ANOVA) with a statistical significance level of less than 5% (p-value < 0.05) was used to compare the cytotoxicity data. Hemolysis % ð Þ¼ OD s À OD n OD p À OD n Â 100 OD s , OD n , and OD p were the absorbances of sample, negative, and positive controls. All the hemolysis experiments were performed in triplicate.

| In vitro wound-healing assay
The wound healing potential of the realized formulations was assessed by in vitro wound-healing assay. 73 To this aim, fibroblast cells (L929) were seeded at a density of 5 Â 10 4 cells/ml on six-wells to obtain a monolayer of cells. Then, a scratch was made across the middle of each well using a sterile 1000 μL pipet tip, and the plates were washed twice with PBS to remove the detached cells. A new medium was added, and approximately 1 Â 1 cm sized of prepared scaffolds were placed over the scratched area. A control without any samples was also included. Scratches were observed and imaged under the microscope (Nikon Inverted Fluorescent Microscope) immediately after the wounding procedure and after 24 h of incubation.
The wound area was calculated using the Image J public domain software. The percentage of wound area reduction or wound closure, expression of the cell migration rate, can be expressed as: A 0 is the area of the wound measured immediately after scratching, and A t is the area of the wound measured 24 h after the scratch is performed. The closure percentage increases as cells migrate into the scratch over time.

| CONCLUSION
In the presented work, the results showed the chitosan nanofibrous mats adorned with silver and zinc nanoparticles possess considerable antioxidant ability and synergic antimicrobial activity. Moreover, the enhanced activity of the Zn/Ag combination with chitosan is also attractive as it improves the overall performance, bringing out the potential of single-device antibiotic combinations with synergic antibacterial and antioxidant properties. In antibacterial studies, high sensitivity of S. aureus to composite mats containing the nanoparticles was observed compared to P. aeruginosa and E. coli. Further investigation of cytotoxicity assessment revealed no toxicity for fibroblast cells, while the viability of fibroblast on composite mats was significantly increased, indicating that not only the composite mats had no toxic effect, but also is a promising scaffold for cell growth. This study showed the simultaneous use of these two nanoparticles in the combination of chitosan nanofibers, accelerating the migration and proliferation of fibroblast cells. Scratches after 24 h of treatment showed significant repair. These results indicated that the chitosan/PEO nanofibers scaffolds containing 8.3% AgNPs-ZnONPs could be a suitable choice for a wound dressing that facilitates the wound healing process; also, composite dressings, in addition to being more effective in treating wounds, can reduce the cost of treatment.

CONFLICT OF INTEREST
The authors declare that they have no conflict of interest.