burns constitute one of the most important and common injuries that can disrupt skin function . according to the area of the body surface affected by burns , it can be classified into three types ( mild , moderate , and severe ) ; moreover , based on the depth of the wounds , it can be classified into four degrees . in second- , third- , and fourth - degree burn injuries that exhibit heat - induced skin degradation , the protective effects of skin are lost , and a scar is formed , which is characterized by a wet , alkaline , non - vascular , and protein - rich environment . a scar is a type of open wound that lacks immune function - related cells ; thus , it is considered an appropriate environment for the growth of microorganisms ( 1 ) . microorganisms such as streptococcus , staphylococcus aureus , and pseudomonas aeruginosa can infect the wound surface within 24 to 48 hours , following which , in 5 - 7 days , gram - negative bacteria such as klebsiella and escherichia coli multiply on the wound surface , eventually leading to the spread of infection ( 2 , 3 ) . application of topical medication on the scar surface is the most convenient method to deliver antibiotic drugs . although topical antimicrobial treatment does not sterilize the wound , it reduces the number of bacteria to an extent that can be controlled by the host immune system . three common topical medicines used in this field are silver sulfadiazine , silver nitrate , and mafenide acetate . mafenide acetate , a sulfonamide - type antibiotic , is the only drug that is able to penetrate into the scar and inhibit bacterial proliferation in the tight tissue below the scar ( 4 ) . after debridement , the skin s surface must be covered with a suitable membrane to reduce trans - epidermal moisture loss and prevent microbial infection ; this would , in turn , lead to enhanced wound healing . one of the efficient choices for wound dressing is nanofibrous films , which offer many advantages over conventional methods ( 5 ) . electrospinning is a simple and cost - effective technique for fabricating nanofibrous films ( 6 ) . in this method , the polymer solution or melt is injected through a nozzle and charged by a dc electrode ; once the critical voltage between the polymer and the collector is reached , the charge imbalance overcomes the surface tension of the polymer , forming a fiber jet toward the collector , where the polymer solvent evaporates , generating nanofibers ( 7 ) . different polymer solutions that may be biodegradable , nondegradable , natural materials , or a mixture of these polymers are used in this technique ( 8) . chitosan is among the polymers considered in the production of wound dressings owing to its unique properties such as healing and bacteriostatic effects , biocompatibility , and biodegradability ( 9 ) . despite the desirable features of chitosan , obtaining pure chitosan nanofibers by electrospinning is difficult . to facilitate nanofiber formation , different blends of chitosan with other spinnable polymers such as polyvinyl alcohol ( pva ) pva is a water - soluble , completely biodegradable , non - toxic , and biocompatible polymer that has several biomedical applications such as wound or lesion dressing ( 11 ) . despite numerous studies on the efficiency of nanofibers as alternative wound dressing materials ( 12 ) , few studies have evaluated the effect of topical application of antibiotic - loaded nanofibers on the prevention of wound infections . in a recent animal study , it has been shown that pcl / peo nanofibers containing nanosilver and silversulfadiazine improve the antimicrobial effect in comparison to that achieved by traditional application of silversulfadiazine cream ( 13 ) . on surveying the available literature , we were unable to find studies on mafenide acetate - loaded nanofibers . the objective of this study was to fabricate nanofibrous films containing chitosan and pva by electrospinning , to investigate their antimicrobial effect , to evaluate the effect of incorporating different concentrations of mafenide acetate on their antimicrobial activity , and to evaluate their ability to prevent microbial penetration , with an aim to assess their applicability in burn care . pva of molecular weight 72000 and glacial acetic acid was purchased from merck ( germany ) . staphylococcus aureus ( atcc29213 ) and pseudomonas aeruginosa ( atcc27853 ) were purchased from the pasteur institute ( iran ) . to prepare polymer solutions for electrospinning the percentage of chitosan in chitosan - pva solution ( 0% , 10% , and 30% ) and the amount of mafenide acetate in the formulation ( 0% , 20% , and 40% ) were selected as independent variables . pva solution in water ( 10% w / v ) , chitosan solution in acetic acid ( 2% w / v ) , and 0.5 g / ml stock solution of mafenide acetate in water were used for the preparation of polymer solutions . finally , nine formulations containing different amounts of chitosan and drug were prepared ( table 1 ) . the polymer solution was filled in a 10-ml syringe with a metallic needle , which was fixed at a 15-cm distance from a rotary steel drum covered with aluminum foil . a voltage of 19 kv was applied to the polymer solution through an electrode connected to the needle . the speed of the rotating collector was 600 rpm and the feed rate of the solution was set at 1 ml / hour . surface morphology of the films was evaluated by scanning electron microscopy ( sem ) ( vp1455-leo , germany ) . the film samples were mounted on aluminum stubs , sputter - coated by a thin layer of platinum under an argon atmosphere by using a sputter - coater ( polaron , england ) , and then observed by sem . the average diameter of the electrospun nanofibers was measured from sem images by using microstructure measurement software ( nahamin pardazan asia co. , iran ) . vertical diffusion cells ( franz cells ) with acetate cellulose membrane between the receptor and donor areas were employed for determining drug release rate from nanofibrous films containing various concentrations of mafenide acetate . phosphate buffer ( 6.2 ml ; ph 7.4 ) at 37c was used as a receptor medium , and it was magnetically stirred at 300 rpm . film samples equivalent to 4 mg mafenide acetate were placed in the donor area on the cellulose acetate membrane , and they were moistened by adding 200 l phosphate buffer . samples were withdrawn from the receptor area at predetermined time intervals and analyzed by uv - spectrophotometry ( biochrom , uk ) at 267 nm . to investigate the films ability to inhibit microbial penetration , each film was tightly placed on an open test tube containing 5 ml autoclaved nutrient broth ( nb ) medium . an open test tube containing nb without any covering material was used as the positive control , while another tube that was closed with a tightly packed cotton ball was used as the negative control . the transmittance of nb was measured at 600 nm as an indicator of microbial contamination ( 14 ) . to determine the minimum inhibitory concentration ( mic ) and minimum bactericidal concentration ( mbc ) of mafenide acetate , a suspension of s. aureus and p. aeruginosa with turbidity equivalent to 1 mcfarland standard was prepared and then diluted 1:500 with trypticase soy broth ( tsb ) ( sigma - aldrich co. ; usa ) medium ( 15 ) . using the serial dilution method , 12 test tubes containing different drug concentrations were prepared , such that the concentration in each tube was half the concentration in the previous tube . then , 1 ml of p. aeruginosa suspension was added to each of these tubes . the tubes were incubated at 37c for 18 hours , following which their turbidity was examined against light according to the clinical and laboratory standard institute ( clsi ) guidelines ( 16 ) . the lowest concentration at which the tube did not show turbidity was considered the mic . to determine mbc , 20 l of medium was taken from the tube considered the mic and the next tubes and inoculated on mueller - hinton s agar plates . then , the plates were placed in a 37c incubator for 18 - 24 hours , following which the results were recorded ( 17 ) . the lowest concentration at which no colony growth was observed on the plate was considered the mbc . according to the mbc , 5 l of the microbial suspension ( 1:500 dilution of the 1 mcfarland standard ) inoculated on 10-mg cuts of the film was placed into the test tube . the films were sterilized under a uv lamp ( 200 - 280 nm ) for 12 hours . then , 500 l tsb was added to the films . immediately after adding tsb ( zero time ) and after 4 and 8 hours , 50 l of the medium was taken and inoculated into 20 ml mueller - hinton melt agar ( sigma ; usa ) medium by using the pour plate technique . after 18 - 24 hours of incubation at 37c , the colonies were counted ( 17 ) . the percentage of chitosan in chitosan - pva solution ( 0% , 10% , and 30% ) and the amount of mafenide acetate in the formulation ( 0% , 20% , and 40% ) were selected as independent variables . pva solution in water ( 10% w / v ) , chitosan solution in acetic acid ( 2% w / v ) , and 0.5 g / ml stock solution of mafenide acetate in water were used for the preparation of polymer solutions . finally , nine formulations containing different amounts of chitosan and drug were prepared ( table 1 ) . the polymer solution was filled in a 10-ml syringe with a metallic needle , which was fixed at a 15-cm distance from a rotary steel drum covered with aluminum foil . a voltage of 19 kv was applied to the polymer solution through an electrode connected to the needle . the speed of the rotating collector was 600 rpm and the feed rate of the solution was set at 1 ml / hour . surface morphology of the films was evaluated by scanning electron microscopy ( sem ) ( vp1455-leo , germany ) . the film samples were mounted on aluminum stubs , sputter - coated by a thin layer of platinum under an argon atmosphere by using a sputter - coater ( polaron , england ) , and then observed by sem . the average diameter of the electrospun nanofibers was measured from sem images by using microstructure measurement software ( nahamin pardazan asia co. , iran ) . vertical diffusion cells ( franz cells ) with acetate cellulose membrane between the receptor and donor areas were employed for determining drug release rate from nanofibrous films containing various concentrations of mafenide acetate . phosphate buffer ( 6.2 ml ; ph 7.4 ) at 37c was used as a receptor medium , and it was magnetically stirred at 300 rpm . film samples equivalent to 4 mg mafenide acetate were placed in the donor area on the cellulose acetate membrane , and they were moistened by adding 200 l phosphate buffer . samples were withdrawn from the receptor area at predetermined time intervals and analyzed by uv - spectrophotometry ( biochrom , uk ) at 267 nm . to investigate the films ability to inhibit microbial penetration , each film was tightly placed on an open test tube containing 5 ml autoclaved nutrient broth ( nb ) medium . an open test tube containing nb without any covering material was used as the positive control , while another tube that was closed with a tightly packed cotton ball was used as the negative control . the transmittance of nb was measured at 600 nm as an indicator of microbial contamination ( 14 ) . to determine the minimum inhibitory concentration ( mic ) and minimum bactericidal concentration ( mbc ) of mafenide acetate , a suspension of s. aureus and p. aeruginosa with turbidity equivalent to 1 mcfarland standard was prepared and then diluted 1:500 with trypticase soy broth ( tsb ) ( sigma - aldrich co. ; usa ) medium ( 15 ) . using the serial dilution method , 12 test tubes containing different drug concentrations were prepared , such that the concentration in each tube was half the concentration in the previous tube . then , 1 ml of p. aeruginosa suspension was added to each of these tubes . the tubes were incubated at 37c for 18 hours , following which their turbidity was examined against light according to the clinical and laboratory standard institute ( clsi ) guidelines ( 16 ) . the lowest concentration at which the tube did not show turbidity was considered the mic . to determine mbc , 20 l of medium was taken from the tube considered the mic and the next tubes and inoculated on mueller - hinton s agar plates . then , the plates were placed in a 37c incubator for 18 - 24 hours , following which the results were recorded ( 17 ) . the lowest concentration at which no colony growth was observed on the plate was considered the mbc . according to the mbc , 5 l of the microbial suspension ( 1:500 dilution of the 1 mcfarland standard ) inoculated on 10-mg cuts of the film was placed into the test tube . the films were sterilized under a uv lamp ( 200 - 280 nm ) for 12 hours . then , 500 l tsb was added to the films . immediately after adding tsb ( zero time ) and after 4 and 8 hours , 50 l of the medium was taken and inoculated into 20 ml mueller - hinton melt agar ( sigma ; usa ) medium by using the pour plate technique . after 18 - 24 hours of incubation at 37c , the colonies were counted ( 17 ) . morphological evaluation of the film formulations showed that pure pva generated smooth and uniform nanofibers with a mean diameter of 1064 nm , which upon addition of 20% and 40% mafenide acetate , decreased in diameter to 418 and 102 nm , respectively ( figure 1 ) .   in vitro release tests show that all film samples containing mafenide acetate released over 50% of their drug within 30 minutes , ( figure 2 ) . the results also showed that chitosan / pva blend nanofibers had a lower release rate than did pure pva nanofibers , which can be attributed to the lower solubility of chitosan compared to that of pva at ph 7.4 . higher transmittance of the medium represents a lower rate of microbial growth ( figure 3 ) .   to study the antibacterial effect of the films , the mic and mbc of mafenide acetate were first measured against both the microorganisms tested in this study . mic and mbc were 20 g / dl and 40 g / dl for s. aureus and 0.125 g / dl and 8 g / dl for p. aeruginosa , respectively . the results of the antibacterial effects of various films are shown in figures 4 and 5 . the results revealed that chitosan / pva nanofibrous films could inhibit the growth of both microorganisms , which is attributed to the antibacterial effects of chitosan ( 18 ) . with respect to p. aeruginosa , addition of mafenide acetate into nanofibers led to a considerable decrease in bacterial count after 4 hours and elimination of viable bacteria after 8 hours . as expected , the rate of reduction in microbial population was greater for nanofibers containing 40% drug . with respect to s. aureus , the formulation containing 20% drug reduced the microbial population remarkably , but could not eliminate it after 8 hours . this observation could be explained by the sensitivity of p. aeruginosa to mafenide acetate , which was confirmed by the mic and mbc . another reason could be the lower rate of drug release from nanofibers containing 20% drug , resulting in a lower concentration of drug being accessible to the bacteria ( figure 2 ) . the results also indicated that blending various amounts of chitosan with pva led to thinning of the nanofibers in the range of 441 - 652 nm , with bead formation being observed at high concentrations of mafenide acetate . since chitosan and mafenide acetate are ionized in solution , their addition to pva solution increased the charged density of the polymer blend , resulting in stronger stretch forces on the ejected fiber jet and formation of ultrafine nanofibers ( 19 ) . to evaluate the ability of different nanofibrous films to resist microbial penetration , the microbial penetration test was used . penetration of microorganisms through the film into the test tube containing medium causes microbial growth and increased the turbidity of the medium . intensity of the turbidity directly correlated to the microbial growth rate and consequently to the rate of microbial penetration . to determine the intensity of the opacity , transmittance of the medium was measured at 600 nm . in this test , intense turbidity of the positive control tubes indicated that nutrient broth medium is suitable for microbial growth . lack of turbidity in the negative control tube is indicative of the maintenance of sterile , microbe - free conditions during the experiment . the results show that almost all of the studied films were able to prevent the penetration of microbes into the medium ; as a result , the medium showed transmittance that was similar or close to that of the negative control tube . these results indicate that despite the films porous and non - compact structure , they do not allow the penetration of microorganisms and can be used to protect wounds from secondary bacterial infections ( 14 ) . the first reason for this observation could be the small size of the pores in the nanofibrous membrane , which is sufficiently smaller than the size of the microorganisms ; this feature ensures that only air and water vapor permeates the membrane , while airborne microorganisms are trapped in the pores . another reason may be the presence of nh3 groups on chitosan , which can bond with negatively charged bacteria and inhibit their penetration ( 20 ) . according to figures 4 and 5 , it is clear that all drug - loaded formulations could reduce the number of bacteria , whereas in control samples ( tubes without films ) , both microorganisms showed rapid growth , such that after 4 hours , the number of colonies was not countable ( > 400 ) . the results showed that compared to the pure pva films , drug - free formulations containing chitosan have shown antimicrobial effects , which can be attributed to the antimicrobial properties of chitosan . the antimicrobial effect of chitosan and its underlying mechanisms have been reviewed recently ( 21 ) . it seems that the main mechanisms underlying the antimicrobial effect of chitosan are alteration in cell permeability , structural damage of the cell membrane , and interactions between charged groups . it is also supposed that chitosan applies its antimicrobial effect through bonds with sialic acid and phospholipids , by the penetration of its oligomers into the cell , and by inhibition of transcription ( 18 ) . for both microorganisms , a comparison between drug - loaded formulations and drug - free formulations indicates that the release of the drug from the formulation resulted in a reduction in the viable bacterial count . these findings are in a close conformity with those of the in - vitro release tests . the higher the amount of drug in the film formulation , the stronger and faster is the antibacterial effect observed . overall , the results of this study indicate that both drug - free and drug - loaded nanofibrous chitosan / pva membranes have full functionality for protecting wounds from secondary bacterial infections . moreover , compared to drug - free films , films containing mafenide acetate exhibited a greater antibacterial effect on the studied microorganisms ; hence , these films can be considered for future studies evaluating their application as a wound dressing with protective , healing , and antimicrobial effects .
background : chitosan , an important biodegradable and biocompatible polymer , has demonstrated wound - healing and antimicrobial properties.objectives:this study aimed to evaluate the antimicrobial properties of mafenide acetate - loaded nanofibrous films , prepared by the electrospinning technique , using chitosan and polyvinyl alcohol ( pva).materials and methods : a 32 full factorial design was used for formulating electrospinning solutions . the chitosan percentage in chitosan / pva solutions ( 0% , 10% , and 30% ) and the drug content ( 0% , 20% , and 40% ) were chosen as independent variables . 
 the release rate of mafenide acetate from nanofibrous films and their microbial penetration were evaluated . the antimicrobial activity of different nanofibrous film formulations against staphylococcus aureus and pseudomonas aeruginosa was studied.results:the results indicated that all nanofibrous films , with and without drug , can prevent bacterial penetration . 
 incorporation of mafenide acetate into chitosan / pva nanofibers enhanced their antimicrobial activity against p. aeruginosa and s. aureus.conclusions:overall , the results showed that chitosan / polyvinyl alcohol ( pva ) nanofibrous films are applicable for use as a wound dressing with protective , healing , and antimicrobial effects .