Polyacrylonitrile Nanofibers Containing Viroblock as Promising Material for Protective Clothing

Antimicrobial viroblock/polyacrylonitrile nanofiber webs fabricated using the electrospinning method were assessed in terms of the antimicrobial activity against infectious agents as a potential material used in mask production. Viroblock (VB) is an amalgam of lipid vesicle and silver. Lipid vesicle depletes the virus outer membrane, which contains cholesterol, while silver ions penetrate the virus, interact with sulfur-bearing moieties, and possess the virus bactericidal property. VB, having anti-coronavirus and anti-influenza properties, was prepared in four different concentrations, 0.5 wt%, 1 wt%, 1.5 wt%, and 2 wt%, in regard to nanofiber weight. The resultant nanofibers were characterized by scanning electron microscope (SEM), Fourier transform infrared (FTIR), thermogravimetric analysis (TGA), water contact angle, water content, and thermogravimetric analysis (TGA). Moreover, all nanofibrous samples were evaluated for cell proliferation assay and ATCC antibacterial tests. Based on characterization results and cytotoxicity, the developed composite nanofibers-based webs showed good promise for utilization in anti-viral masks. Particularly, 2 wt% VB/PAN nanofibers have the highest antibacterial properties against negative and positive bacteria along with excellent cell viability.


Introduction
The outbreak of the severe acute respiratory syndrome coronavirus 2 (SARS-COV-2 or COVID- 19) in Wuhan in 2019 is a global health emergency with many cases of death and huge economic losses [1]. The worldwide health crisis has affected all sectors of life and spread at a rapid pace. An unprecedented challenge is upon the scientific world to find effective medical treatments for the disease. COVID-19 infections are transmitted mainly through the inhalation of droplets produced by coughing and sneezing [2]. The droplets of variable sizes are dispersed through the air or rest on varied material surfaces. Droplets more than 20 µm easily fall onto objects, and viruses from these droplets have been reported to last anywhere from a few hours up to several days on plastic, glass, stainless steel, cardboard, and clothing surfaces. Smaller droplets of less than 10 µm are

Preparation of VB/PAN Nanofibers
In DMF, 10 wt% PAN was the constant concentration used for all solutions. The polymer was dissolved in DMF and the solution was gently stirred for 3 h. The VB was used in the PAN prepared solution in four different concentrations, such as neat Pan, 0.5% w/w VB/PAN, 1% w/w VB/PAN, 1.5% w/w VB/PAN, and 2% w/w VB/PAN. The spinning solution was injected into a needle syringe that had a metal needle attached with an inner diameter of 0.6 mm. There was a 140 mm distance between the collector and the tip of the needle. A total of 13 kV was applied on the positive electrode and the nanofibers were collected on the negative one. The resultant VB/PAN nanofiber webs were used for fabricating antiviral and antibacterial protective clothing such as face masks.

Characterization 2.3.1. Morphology Analysis
The morphology of PAN nanofibers and VB/PAN nanofibers was investigated by scanning electron microscopy (SEM, S-3000N, Hitachi Co., Tokyo, Japan) at 10 kV accelerating voltage. The average diameter of the PAN nanofibers and VB/PAN nanofibers was determined by analyzing the SEM images, using the Image J software version 1.49, measuring the diameter of 50 nanofiber strands.

Chemical State Analysis
Fourier transform infrared (FTIR-ATR) spectra were recorded between the wavelengths of 400-4000 cm −1 using an IR Prestige-21 (Shimadzu Co., Ltd., Kyoto, Japan) and studied for the chemical interactions between PAN and VB, with a resolution of 4 cm −1 and a cumulative addition of 128 scans.

Water Contact Angle and Water Content
The water contact angle was calculated to reflect the hydrophilicity of the composite nanofibers. The values of water contact angle were calculated statically using a contact angle meter (Digidrop, GBX, Romans-sur-Isère, France). After 5 measurements, the values were expressed as an average with standard deviation.
For the water content, nanofibrous specimens were cut into 1 × 1 cm pieces. They were weighed before being immersed in deionized water for 1 h. After that, the specimens were removed and placed on adsorptive papers to discard all dripping water. The water content was calculated as follows: where W w and W d were the weight of the wet nanofibers and dried ones, respectively.

TGA Study
The thermal behavior of PAN nanofibers, VB/PAN nanofibers, and the confirmed presence of VB in the VB/PAN nanofibers were investigated by Thermo Gravimetric Analysis. It was performed on the thermo-plus TG-8120 (Rigaku Corporation, Osaka, Japan). The measurement was carried out in a static mode under air temperature at a heating rate of 10 • C/min, with the temperature range of 0-600 • C.

In Vitro Assessment
To examine the cell attachment performance of PAN and VB/PAN nanofibers, an MTT study was performed for nine days in mouse fibroblast line L929, and was repeated in triplicate for each sample. After being sterilized, each nanofibrous specimen was immersed in a well containing DMEM-F12 medium with 10% FBS. The L929 cells were then seeded on each sample with a density of 16,000 cells/cm 2 . On the day of the test, 50 µL of medium culture containing Thiazolyl Blue Tetrazolium Bromide (MTT; 5 mg/mL) was added to each well. The specimens with cells were incubated at 37 • C with 5% CO 2 for a further 12 h.
After that, the samples were removed and washed with PBS. Formosan crystals of live cells were dissolved completely in 400 µL DMSO. Subsequently, 200 µL of supernatant solution was transferred into the 96-well plate and the absorbance was measured at 570 nm. To calibrate the dye absorption, control samples without cells were soaked in a medium culture with the addition of MTT, then washed with PBS, and soaked in DMSO before the absorbance of the control was acquired at 570 nm. For the evaluation of the cell viability, the absorbance of each well was subtracted by the control.

Air Permeability Analysis
To investigate the air permeability of the PAN and VB/PAN nanofibers, AirPerm model M021A, manufactured by SDL Atlas (Rock Hill, SC, USA), was implemented following ASTM D737.

Antibacterial Activity
To investigate the antibacterial properties of PAN nanofibers and VB/PAN nanofibers against Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) bacteria, the antibacterial activities were determined using the Kirby-Bauer method. The nanofiber samples of 10 mm diameter were placed for investigation on agar plates seeded with bacterial cells in the log phase and incubated at 37 • C for 16 h. All the samples were repeated three times using different specimens. The diameter of the inhibition zone was examined using Image J 1.50i software.

Morphology Analysis
SEM analysis was used to investigate the surface morphology of PAN electrospun nanofibers loaded with four different concentrations of VB. The prepared fibers were analyzed at a magnification of 3000 with a working voltage of 10 kV. It can be seen in Figure 1 that there is a smooth formation of electrospun nanofibers in all cases without the presence of any beads in all prepared samples.
Appl. Sci. 2021, 11, x FOR PEER REVIEW medium culture containing Thiazolyl Blue Tetrazolium Bromide (MTT; 5 mg/mL) was a to each well. The specimens with cells were incubated at 37 °C with 5% CO2 for a fu 12 h. After that, the samples were removed and washed with PBS. Formosan crys live cells were dissolved completely in 400 µL DMSO. Subsequently, 200 µL of sup tant solution was transferred into the 96-well plate and the absorbance was measu 570 nm. To calibrate the dye absorption, control samples without cells were soake medium culture with the addition of MTT, then washed with PBS, and soaked in D before the absorbance of the control was acquired at 570 nm. For the evaluation of th viability, the absorbance of each well was subtracted by the control.

Air Permeability Analysis
To investigate the air permeability of the PAN and VB/PAN nanofibers, Ai model M021A, manufactured by SDL Atlas (Rock Hill, SC, USA), was implemente lowing ASTM D737.

Antibacterial Activity
To investigate the antibacterial properties of PAN nanofibers and VB/PAN fibers against Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) bacteria, t tibacterial activities were determined using the Kirby-Bauer method. The nanofiber ples of 10 mm diameter were placed for investigation on agar plates seeded with ba cells in the log phase and incubated at 37 °C for 16 h. All the samples were repeated times using different specimens. The diameter of the inhibition zone was examined Image J 1.50i software.

Morphology Analysis
SEM analysis was used to investigate the surface morphology of PAN electr nanofibers loaded with four different concentrations of VB. The prepared fibers we alyzed at a magnification of 3000 with a working voltage of 10 kV. It can be seen in F 1 that there is a smooth formation of electrospun nanofibers in all cases without the ence of any beads in all prepared samples.  To determine the average diameter of electrospun nanofibers, Image J software was used to measure 50 different values from different areas of each prepared sample and the histogram was plotted to determine the average diameter of the prepared samples. In Figure 2, the horizontal axis indicated the diameter of electrospun nanofibers, while the vertical axis expressed the diameter distribution percentage. It was found that in the case of pure PAN electrospun nanofibers, the distribution curve showed that the average diameter of the nanofibers was 373.40 ± 49.80 nm. In Figure 2B, the PAN electrospun nanofibers loaded with 0.5% VB had an average diameter of 366.55 nm, with a standard deviation value of 49.55 nm. Similarly, the histograms in the cases of 1%, 1.5%, and 2% VBloaded PAN electrospun nanofibers indicated that the average diameters of the nanofibers were 381.52 ± 63.53, 341.17 ± 62.10, and 361.76 ± 55.67 nm, respectively, as shown in Figure 2C-E.
Appl. Sci. 2021, 11, x FOR PEER REVIEW 5 of 12 To determine the average diameter of electrospun nanofibers, Image J software was used to measure 50 different values from different areas of each prepared sample and the histogram was plotted to determine the average diameter of the prepared samples. In Figure 2, the horizontal axis indicated the diameter of electrospun nanofibers, while the vertical axis expressed the diameter distribution percentage. It was found that in the case of pure PAN electrospun nanofibers, the distribution curve showed that the average diameter of the nanofibers was 373.40 ± 49.80 nm. In Figure 2B, the PAN electrospun nanofibers loaded with 0.5% VB had an average diameter of 366.55 nm, with a standard deviation value of 49.55 nm. Similarly, the histograms in the cases of 1%, 1.5%, and 2% VB-loaded PAN electrospun nanofibers indicated that the average diameters of the nanofibers were 381.52 ± 63.53, 341.17 ± 62.10, and 361.76 ± 55.67 nm, respectively, as shown in Figure 2C-E.

Chemical Interactions Analysis
FTIR spectra were used to analyze the chemical groups of the prepared electrospun nanofibers. Figure 3 shows the FTIR spectra of pure PAN nanofibers, 0.5% VB-loaded PAN nanofibers, 1% VB-loaded PAN nanofibers, 1.5% VB-loaded PAN nanofibers, and 2% VB-loaded PAN nanofibers. The spectral range varies from 1000 cm −1 to 3500 cm −1 . In all spectra, there was broadband appearing in the range of 3000 cm −1 to 3500 cm −1 due to the presence of -OH group, belonging to moisture absorbed. In all spectra, the vibrational peak appeared at 2242 cm −1 ascribed to the presence of the nitrile (-C≡N) functional group. In the case of pure PAN electrospun nanofibers, the peaks that appeared at 2884 cm −1 and 2943 cm −1 indicated the stretching and bending of the methyl functional group [17,18]. The peaks present at 1732 cm −1 and 1453 cm −1 corresponded to the carboxylic functional group of PAN. The peak that emerged at 1170 cm −1 was attributed to (-C-O) stretching due to the formation of aliphatic ether. The peaks that appeared at 1224 cm −1 , 1360 cm −1 , and 1593 cm −1 were ascribed to the (-C-O) stretching and -OH bending, respectively [14,19]. The other four spectra were similar to that of the PAN spectrum with little variation in peak position. This is because the silver nanoparticles and other compounds that were present

Chemical Interactions Analysis
FTIR spectra were used to analyze the chemical groups of the prepared electrospun nanofibers. Figure 3 shows the FTIR spectra of pure PAN nanofibers, 0.5% VB-loaded PAN nanofibers, 1% VB-loaded PAN nanofibers, 1.5% VB-loaded PAN nanofibers, and 2% VB-loaded PAN nanofibers. The spectral range varies from 1000 cm −1 to 3500 cm −1 . In all spectra, there was broadband appearing in the range of 3000 cm −1 to 3500 cm −1 due to the presence of -OH group, belonging to moisture absorbed. In all spectra, the vibrational peak appeared at 2242 cm −1 ascribed to the presence of the nitrile (-C≡N) functional group. In the case of pure PAN electrospun nanofibers, the peaks that appeared at 2884 cm −1 and 2943 cm −1 indicated the stretching and bending of the methyl functional group [17,18]. The peaks present at 1732 cm −1 and 1453 cm −1 corresponded to the carboxylic functional group of PAN. The peak that emerged at 1170 cm −1 was attributed to (-C-O) stretching due to the formation of aliphatic ether. The peaks that appeared at 1224 cm −1 , 1360 cm −1 , and 1593 cm −1 were ascribed to the (-C-O) stretching and -OH bending, respectively [14,19]. The other four spectra were similar to that of the PAN spectrum with little variation in peak position. This is because the silver nanoparticles and other compounds that were present in the VB integrated into PAN, forming the composite material. In the case of 0.5% VB-loaded PAN electrospun nanofibers, a characteristic peak appeared at 1181 cm −1 corresponding to C-O stretching, the medium peak appeared at 1422 cm −1 representing the -O-H bending, and the peak at 1450 cm −1 was due to the -C-H bending. The peak at 1597 cm −1 was attributed to -N-H bending due to the formation of the amine group [20]. The stretching of 1656 cm −1 C=C was due to the conjugated alkene. The peak at 1728 cm −1 represented the carboxylic functional group as a result of the oxidation of PAN reacting with VB. In the case of 1% VB-loaded PAN electrospun nanofibers, the characteristic peaks appear at 1174 cm −1 and 1197 cm −1 , corresponding to -C-O stretching. The peaks that appeared at 1452 cm −1 , 2882 cm −1 , and 2914 cm −1 represented -C-H stretching of methyl functional group from VB. In the spectrum of 1% VB-loaded PAN, the medium peak emerged at 1391 cm −1 , corresponding to -C-N stretching, which was due to aromatic amine of VB. The peaks at 1653 cm −1 and 1731 cm −1 were ascribed to the carboxylic functional groups [21]. In the case of 1.5% VB-loaded PAN electrospun nanofibers, the peaks present at 1073 cm −1 and 1172 cm −1 corresponded to -C-O stretching of VB. The peak that emerged at 1453 cm −1 was due to the -CH methyl functional group. The peak at 1733 cm −1 represented the carboxylic functional group of oxidized PAN. A similar spectrum was obtained in the case of 2% VB-loaded PAN electrospun nanofibers and a shift in characteristic peaks was also observed. The peaks that appeared at 1042 cm −1 , 1122 cm −1 , and 1170 cm −1 were caused by the -C-O stretching of the secondary alcohol and aliphatic ether, respectively. The formation of phenol was demonstrated by the -OH bending, observed in the spectrum at 1316 cm −1 and 1415 cm −1 . -CH bending was observed at 1455 cm −1 , 2852 cm −1 , and 2922 cm −1 due to the methyl functional group of VB. The peak at 1738 cm −1 was ascribed to the carboxylic functional group of oxidized PAN [22,23].
Appl. Sci. 2021, 11, x FOR PEER REVIEW 6 in the VB integrated into PAN, forming the composite material. In the case of 0.5% loaded PAN electrospun nanofibers, a characteristic peak appeared at 1181 cm −1 co sponding to C-O stretching, the medium peak appeared at 1422 cm −1 representing the H bending, and the peak at 1450 cm −1 was due to the -C-H bending. The peak at 1597 was attributed to -N-H bending due to the formation of the amine group [20]. The stre ing of 1656 cm −1 C=C was due to the conjugated alkene. The peak at 1728 cm −1 represen the carboxylic functional group as a result of the oxidation of PAN reacting with VB the case of 1% VB-loaded PAN electrospun nanofibers, the characteristic peaks appea 1174 cm −1 and 1197 cm −1 , corresponding to -C-O stretching. The peaks that appeare 1452 cm −1 , 2882 cm −1 , and 2914 cm −1 represented -C-H stretching of methyl functi group from VB. In the spectrum of 1% VB-loaded PAN, the medium peak emerged at 1 cm −1 , corresponding to -C-N stretching, which was due to aromatic amine of VB.

Water Content and Water Contact Angle Study
COVID-19 is a highly infectious disease with the symptoms of fever, dry cough, weakness, and even respiratory failure in some severe cases. Masks are still the main protection to block water spatter and droplets from breath, cough, and sneezing of COVID-19 patients, and thus avoid virus transmission. Hydrophobic and anti-droplet properties are important characteristics in face masks; these attributes could be achieved by a coating or chemical modification of mask materials [24]. The water repellency or hydrophilicity was affirmed by water content and water contact angle, which are key parameters influencing the fouling propensity of a membrane. It was observed that the water content values were not affected by the incorporation of VB in the PAN nanofibers, as shown in Figure 4a. In addition to water content, the nanofiber-based web was characterized by the water contact angle. The static water contact angle measurement was performed to investigate the wetting properties of membrane surfaces. The water contact angle θ of all samples was less than 90 • and all prepared membranes were considered hydrophilic [25]. Conversely, the material surface was accepted as hydrophobic when the contact angle was larger than 90 • [26,27]. As shown in Figure 4b, the water contact angles of PAN and VB/PAN were 73.9 • , 77.4 • , 72.6 • , 78.3 • , and 74.2 • for PAN, 0.5% VB/PAN, 1% VB/PAN, 1.5% VB/PAN, and 2% VB/PAN, respectively, exhibiting no recognizable difference, which again confirmed that there was no significant influence of adding VB into the surface wettability of all membranes.
19 patients, and thus avoid virus transmission. Hydrophobic and anti-droplet properties are important characteristics in face masks; these attributes could be achieved by a coating or chemical modification of mask materials [24]. The water repellency or hydrophilicity was affirmed by water content and water contact angle, which are key parameters influencing the fouling propensity of a membrane. It was observed that the water content values were not affected by the incorporation of VB in the PAN nanofibers, as shown in Figure 4a. In addition to water content, the nanofiber-based web was characterized by the water contact angle. The static water contact angle measurement was performed to investigate the wetting properties of membrane surfaces. The water contact angle θ of all samples was less than 90° and all prepared membranes were considered hydrophilic [25]. Conversely, the material surface was accepted as hydrophobic when the contact angle was larger than 90° [26,27]. As shown in Figure 4b, the water contact angles of PAN and VB/PAN were 73.9°, 77.4°, 72.6°, 78.3°, and 74.2° for PAN, 0.5% VB/PAN, 1% VB/PAN, 1.5% VB/PAN, and 2% VB/PAN, respectively, exhibiting no recognizable difference, which again confirmed that there was no significant influence of adding VB into the surface wettability of all membranes.

TGA Study
The thermal degradation behaviors of PAN nanofibers and VB/PAN nanofibers were recorded using TGA. To ensure good reproducibility, all specimens were prepared with a weight of 10.0 ± 1.0 mg. It was operated in a static mode under normal air temperature with a heating rate of 10° C/min and a temperature range from 0 to 500° C. Figure 5 shows the dependence of weight retaining of PAN nanofibers with varied VB loading concentrations and temperature. It was observed that the weight residue percentages of VB/PAN nanofibers increased as the higher concentration of VB were loaded in the VB/PAN nanofibers. The trend was explained by the amounts of Ag presented in VB and Ag was thermally stable in this range of temperature. TGA spectra also confirmed the presence of VB in the VB/PAN nanofiber-based web because 2% VB/PAN nanofibers had 32% weight residue compared to neat PAN nanofibers, whose weight residue was much lower at 18%.

TGA Study
The thermal degradation behaviors of PAN nanofibers and VB/PAN nanofibers were recorded using TGA. To ensure good reproducibility, all specimens were prepared with a weight of 10.0 ± 1.0 mg. It was operated in a static mode under normal air temperature with a heating rate of 10 • C/min and a temperature range from 0 to 500 • C. Figure 5 shows the dependence of weight retaining of PAN nanofibers with varied VB loading concentrations and temperature. It was observed that the weight residue percentages of VB/PAN nanofibers increased as the higher concentration of VB were loaded in the VB/PAN nanofibers. The trend was explained by the amounts of Ag presented in VB and Ag was thermally stable in this range of temperature. TGA spectra also confirmed the presence of VB in the VB/PAN nanofiber-based web because 2% VB/PAN nanofibers had 32% weight residue compared to neat PAN nanofibers, whose weight residue was much lower at 18%.

In Vitro Assessment
In order to examine the toxicity of PAN nanofibers and VB/PAN nanofibers in tissue engineering applications, an MTT analysis was implemented, as shown in Figure 6. An MTT study was performed up to 9 days using mouse fibroblast L929 and was repeated in triplicate for each sample. With the increasing concentrations of VB, the cells were observed to be more viable with higher absorbent values of accumulated formazan in DMSO. The MTT reduced formazan crystals in living cells and the formazan amount was directly proportional to the living cell quantity. Cell viability of all nanofibers gradually increased with time progression, revealing the suitability of the scaffolds for metabolic activity and cell proliferation. It was also confirmed that when the concentration of VB was increased, the cell viability was increased, as shown in Figure 6, demonstrating that VB-loaded PAN nanofibers were non-toxic and had potential in soft tissue engineering. The MTT analysis confirmed that the resultant nanofiber webs can be used as protective clothing for COVID-19 due to their non-toxicity for fibroblast cells.

In Vitro Assessment
In order to examine the toxicity of PAN nanofibers and VB/PAN nanofibers in tissue engineering applications, an MTT analysis was implemented, as shown in Figure 6. An MTT study was performed up to 9 days using mouse fibroblast L929 and was repeated in triplicate for each sample. With the increasing concentrations of VB, the cells were observed to be more viable with higher absorbent values of accumulated formazan in DMSO. The MTT reduced formazan crystals in living cells and the formazan amount was directly proportional to the living cell quantity. Cell viability of all nanofibers gradually increased with time progression, revealing the suitability of the scaffolds for metabolic activity and cell proliferation. It was also confirmed that when the concentration of VB was increased, the cell viability was increased, as shown in Figure 6, demonstrating that VB-loaded PAN nanofibers were non-toxic and had potential in soft tissue engineering. The MTT analysis confirmed that the resultant nanofiber webs can be used as protective clothing for COVID-19 due to their non-toxicity for fibroblast cells.

In Vitro Assessment
In order to examine the toxicity of PAN nanofibers and VB/PAN nanofibers in tissue engineering applications, an MTT analysis was implemented, as shown in Figure 6. An MTT study was performed up to 9 days using mouse fibroblast L929 and was repeated in triplicate for each sample. With the increasing concentrations of VB, the cells were observed to be more viable with higher absorbent values of accumulated formazan in DMSO. The MTT reduced formazan crystals in living cells and the formazan amount was directly proportional to the living cell quantity. Cell viability of all nanofibers gradually increased with time progression, revealing the suitability of the scaffolds for metabolic activity and cell proliferation. It was also confirmed that when the concentration of VB was increased, the cell viability was increased, as shown in Figure 6, demonstrating that VB-loaded PAN nanofibers were non-toxic and had potential in soft tissue engineering. The MTT analysis confirmed that the resultant nanofiber webs can be used as protective clothing for COVID-19 due to their non-toxicity for fibroblast cells.

Antibacterial Activity of VB-Loaded PAN Nanofibers
The antibacterial activity of PAN nanofibers loaded with four different dosages of VB was analyzed for antibacterial activity against Gram-positive and Gram-negative bacterial strains by using the disc diffusion method. Figure 7 shows the antibacterial images of VBloaded PAN nanofibers. The activity against S. aureus was more pronounced in comparison to E. coli. The possible reason could be that the cell membrane of E. coli was relatively thicker than that of S. aureus.

Antibacterial Activity of VB-Loaded PAN Nanofibers
The antibacterial activity of PAN nanofibers loaded with four different dosages of VB was analyzed for antibacterial activity against Gram-positive and Gram-negative bacterial strains by using the disc diffusion method. Figure 7 shows the antibacterial images of VB-loaded PAN nanofibers. The activity against S. aureus was more pronounced in comparison to E. coli. The possible reason could be that the cell membrane of E. coli was relatively thicker than that of S. aureus. The zone of inhibition was calculated using Image J software by measuring the clear area around the nanofiber specimens. Figure 8 shows the histogram of the results. The values of the inhibition zone against S. aureus bacteria were 15.3, 15.7, 16.0, and 16.8 mm corresponding to 0.5%, 1%, 1.5%, and 2% VB-loaded PAN nanofibers, respectively. Similarly, in the case of E. coli bacteria, the values of the halo zone were 13.5, 14.85, 15.15, and 15.65 mm. VB mechanisms of E. coli and S. aureus termination were bacterial membrane perturbation and damage inflicted by vesicle and the ROS generated by nano silver intracellularly disturbing the functionality of DNA, enzymes, and protein. After the disruption of the bacterial membranes, silver penetrated the bacterial cells, interacting with sulfurcontaining proteins and thiol groups of enzymes, causing impaired functionality and DNA damage [28]. The zone of inhibition was calculated using Image J software by measuring the clear area around the nanofiber specimens. Figure 8 shows the histogram of the results. The values of the inhibition zone against S. aureus bacteria were 15.3, 15.7, 16.0, and 16.8 mm corresponding to 0.5%, 1%, 1.5%, and 2% VB-loaded PAN nanofibers, respectively. Similarly, in the case of E. coli bacteria, the values of the halo zone were 13.5, 14.85, 15.15, and 15.65 mm. VB mechanisms of E. coli and S. aureus termination were bacterial membrane perturbation and damage inflicted by vesicle and the ROS generated by nano silver intracellularly disturbing the functionality of DNA, enzymes, and protein. After the disruption of the bacterial membranes, silver penetrated the bacterial cells, interacting with sulfur-containing proteins and thiol groups of enzymes, causing impaired functionality and DNA damage [28].

Air Permeability Analysis
In order to investigate the air permeability of the PAN and VB/PAN nanofibers, an air permeability tester was used. It was confirmed that the air permeability of the VB/PAN nanofibers was slightly reduced, as shown in Figure 9. By increasing the concentration of VB, the air permeability of the nanofibers showed a slight decrease. This was because by increasing the amount of the loading, the diameter of the nanofibers increased, which re-

Air Permeability Analysis
In order to investigate the air permeability of the PAN and VB/PAN nanofibers, an air permeability tester was used. It was confirmed that the air permeability of the VB/PAN nanofibers was slightly reduced, as shown in Figure 9. By increasing the concentration of VB, the air permeability of the nanofibers showed a slight decrease. This was because by increasing the amount of the loading, the diameter of the nanofibers increased, which reduced the air permeability. The advantages of nanofiber masks are the thinness of their fibers, which promotes air permeability whilst maintaining a high rejection of pathogens such as bacteria and viruses due to their small interfiber spaces. The values of air permeability of the resultant nanofibrous membranes with the thickness of 0.35-0.45 mm were between 74.5-84 mm s −1 . For the commercial Nano-N95 or the surgical mask, the values were reported to be less than 20 mm s −1 ; thus, the fabricated membranes were much more breathable [29].

Air Permeability Analysis
In order to investigate the air permeability of the PAN and VB/PAN nanofibers, an air permeability tester was used. It was confirmed that the air permeability of the VB/PAN nanofibers was slightly reduced, as shown in Figure 9. By increasing the concentration of VB, the air permeability of the nanofibers showed a slight decrease. This was because by increasing the amount of the loading, the diameter of the nanofibers increased, which reduced the air permeability. The advantages of nanofiber masks are the thinness of their fibers, which promotes air permeability whilst maintaining a high rejection of pathogens such as bacteria and viruses due to their small interfiber spaces. The values of air permeability of the resultant nanofibrous membranes with the thickness of 0.35-0.45 mm were between 74.5-84 mm s −1 . For the commercial Nano-N95 or the surgical mask, the values were reported to be less than 20 mm s −1 ; thus, the fabricated membranes were much more breathable [29].

Conclusions
In this study, VB/PAN nanofibers were successfully fabricated with antiviral properties by the electrospinning method. The antimicrobial properties against E. coli and S. aureus were significantly increased with the higher loading of VB, demonstrating the antibacterial effects of this compound. The four different concentrations of VB material were proved to be successfully incorporated in the PAN nanofibers, confirmed by FTIR and TGA. The resultant nanofiber-based webs that were developed had excellent cell viability and showed great potential for antiviral-mask-based composite nanofibers. It was found that 2 wt% VB/PAN nanofibers had the strongest antibacterial and cell viability properties in comparison to the other concentrations of VB used in the PAN solution for electrospinning.