Folic acid-containing nanobers by simultaneous process for transdermal drug delivery: preparation, characterization, and in vitro biocompatibility

Nanobers with bioactive agents are good candidates for skin-care applications due to high spesic surface area, low density and highly porous structure. In this study, hydrophilic based bioactive nanobers were produced via an electrospinning and electrospraying simultaneous process. Polyvinyl alcohol (PVA), polyvinyl alcohol-gelatin (PVA-Gel) and polyvinyl alcohol-alginate (PVA-Alg) polymers were used as the matrix material and folic acid (FA) particles were dispersed simultaneously on the surface of these hydrophilic nanobers. The morphology of the nanobers (NFs) was uniform and dispersed folic acid particles incorporated into the structure of nanobers as conrmed by scanning electron microscopy (SEM). Thermal behavior, chemical structure of the composite nanobers were analyzed/investigated by thermogravimetric analysis (TGA) and Attenuated Total Reectance-Fourier Transform Infrared Spectroscopy (ATR-FTIR) which showed that no chemical bonding between vitamin and polymers. A controlled release of FA-loaded electrospun bers were carried out by UV-Vis in vitro study within the 8 hour-period in articial sweat solutions (acidic media, pH 5,44). The obtained PVA/FA, PVA-Gel/FA and PVA-Alg/FA bers released 49.6 %, 69.55 % and 50.88 % of the sprayed FA in 8 h, indicating the inuence of polymer matrix and polymer-drug interactions, on its release from the polymer matrix. Moreover, biocompatibility of all developed novel NFs was assessed by two different cytotoxicity tests,3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and neutral red uptake (NRU) assay in L929 cell lines. In all cases, it is clearly concluded that these new electrospun bers had fast-release of the vitamin and the hybrid process is suitable for transdermal patch applications, especially for skin-care products. Moreover, it has been proposed nanober with folic acid as a patch may prevent the COVID-19.


Introduction
Human skin, the largest organ in the body, has vital functional role by protecting from pollution, ultraviolet radiation and other damage, which cause to skin aging and disorders [1][2][3]. Skin has well-organized morphological structure and self-renewing barrier property. It comprises of three multilayers structure with different levels of cellular and epidermal differentiations that serves as a protective barrier [4,5]. The outmost layer of the skin is epidermis known as the stratum corneum (SC) contains dead cells (corneocytes) interdispersed within a greasy matrix [1,2,6]. Therefore, SC provides selective permeability and limits hydrophilic rich compounds to penetrate in to the inner layers. Transportation process of many bioactive materials in skin is mainly resulted by passive diffusion [7] in related to Fick's Law [8]. (Ficks Law summarized the absorption model in skin) Transdermal drug delivery system (TDDS) is de ned as pharmaceutical ingredients are transported locally or systemically via skin [1,9,10]. The approach is alternative to oral, intravascular, subcutaneous, and transmucosal routes due to loss of effectiveness and toxicity of drugs (degradation) [11,12]. Besides, TDDS have not cause painful administration and other unwanted side effects on the gastrointestinal tract compared with oral or injection administration, especially. Therefore, this route has not damage to liver. However, the penetration of drugs via SC has many limitations. It is known that drug molecules penetrate/pass through/ via SC by two routes including intercellular (hydrophilic drugs) and intracellular (hydrophobic drugs) mechanisms. A drug molecule needs some physicochemical properties such as su cient hydrophobicity, high and su cient partition coe cient (Log P 1-3), low molecular weight (<500 Da) and the short half life for better penetration [1,13,14].
During the last decades, nano bers have become excellent candidate for transdermal drug release applications due to high porosity, large spesi c surface area and small size of electrospun bers [10,15].
There have been many attemptions about pharmaceutical ingredients incorporated into nano bers for usage of transdermal applications. In this regard, Galkina et al. (2013) reported cellulose NFs -titania nanocomposites grafted with three different drugs can be potential transdermal patch [16]. Gencturk et al. (2017) fabricated polyurethane/hydroxypropyl cellulose electrospun nano bers with Donepezil (DNP) hydrochloride and in their study, controlled release of DNP from the bers within 6 h was observed [17].  [20]. These NFs showed cumulative drug release reached to about 99 % after 24 h. In another study, Tran et al. (2015) investigated release behavior of Ibuprofen from NFs. PCL, pNIPAM and pNIPAM/PCL were used as carriers of the drug for thermoresponsive transdermal delivery systems [21]. The release behavior of the bers signi cantly changed with 4-hour period depending on the temperature. Song et al (2016) developed an alternative patch for the use of Daidzein in oral delivery applications [22]. The Daidzein-loaded PLGA bers showed around 65.38% with sustained released behavior after 72 h. Furthermore, vitamin loaded NFs have received increasing attention as transdermal patches. Within this aim, Madhaiyan et al. (2013) synthesized vitamin B12-loaded polycaprolactone NFs and the resulting bers indicated gradually vitamin release behavior in 48-hour period [23]. A slow release of the vitamin E was observed from silk broin NFs over 72 h to utilize for skin care applications [24]. In another study, electrospun bers made of gelatin was loaded with the vitamin A and E and investigated as wound healing patch [25]. The in vitro test results displayed the bers have sustained release behavior for more than 60 hours.
Previous studies mainly focused on drug-loaded NFs fabricated by conventional electrospinning. Apart from electrospinning, there is one approach used in drug release applications which is called electrospraying. Electrospraying is sister technology of electrospinning to fabricate nanomaterials with < 1 μm diameter [26]. In electrospinning, polymer solution concentration is su cient to develop chain formation in the capillary by using high voltage potential while the polymer solution concentration is too low and droplets is spraying from the capillary in electrospraying [27]. However, nanoparticles micro/nanoparticle dispersions are processed with electrospraying due to low viscosity. There are many studies on nano bers produced together with electrospinning and electrospraying process. However, to the best of our knowledge, there is no report on the transdermal drug delivery of nano bers produced with simultaneous electrospinning and electrospraying process.
In the current study, electrospinning and electrospinning were performed simultaneously to fabricate the electrospun PVA, PVA-Gel and PVA-Alg brous mats. Folic acid utilized as model drug. By varying the polymer matrix type, the properties of as-spun/FA NFs were investigated in terms of morphology, thermal and chemical structure (polymer-polymer and polymer-drug compatibility). In vitro drug release from the NFs was assessed. The biocompatibility of the nano bers was also evaluated through by 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and neutral red uptake (NRU) assayson the L929 cell lines (mouse broblasts) as recommended in ISO10993-5 standard. water was used in the experiments and all of the reagents were used without any puri cation.

Fabrication of Hybrid Nano bers
The hydrophilic based polymer/FA nano bers were fabricated by combining electrospinning and electrospraying process (INOVENSO Nanospinner24, Turkey). PVA powders were dissolved in pure water at 90°C till a 10 % (w/w) homogeneous PVA solution was obtained. Gelatin was dissolved in pure water and acetic acid binary-solvent systems (7:3 w/w) solution to obtain a 20 % (w/w) gelatine solution by stirring for 2 hours at ambient conditions. Alginate powders were also dissolved in water to obtain a 2 % (w/w) alginate solution. PVA-alginate (4:1 v/v) and PVA-gelatin solutions (3:2 v/v) were mixed at room temperature for overnight. In our previous experiments, it was determined optimum folic acid (FA) concentration in electrospraying system. 22 mg FA was dispersed in 10 ml (2:1 v/v) pure water and alcohol solution for each polymer system via ultrasonic homogenizer (Bandelin/Sonoplus HD3200). Each polymer solution (PVA, PVA-gelatin and PVA-alginate) and FA particle dispersion were transferred into two seperate plastic syringes (10 mL) and put side to side on two syringe pumps. The plastic syringes were attached to two stainless steel nozzle. The PVA, PVA-gelatin and PVA-alginate solutions and FA dispersion were dispensed via nozzle which was vertical to the collecting plate. The feed rates were performed at 2 mL/h and 1.5 mL/h for polymers and FA dispersion, respectively. Neat PVA, PVA-gelatine and PVA-alginate solutions were prepared as a control (Fig. 1).

Scanning electron microscopy (SEM)
Morphology of all nano bers was observed with Carl Zeiss/Gemini 300 Scanning Electron Microscope (SEM) (ZEISS Ltd.,Germany). All samples were coated with gold for 20 minutes before analysis. Fiber diameters were measured using Image J, version 1.520 software.

Thermal Analysis (TGA)
TA /SDT650 TGA(USA) were used for the thermal analysis. TGA analyses were performed under nitrogen atmosphere with 20˚C min -1 heating rate and 30˚C -600˚C temperature range and applied oxygen atmosphere with 20˚C min -1 heating rate 600˚C-900˚C temperature.

Fourier transform infrared spectroscopy (FT-IR)
FTIR data were obtained with a Thermo Nicolet iS50 FTIR (USA) spectrometer with a ATR (Attenuated Total Re ectance) adapter (Pike, USA) in the range of 4000-500 cm −1 recorded with 16 scans at 4 cm -1 resolutions.

In vitro Release Study
The pH of skin surface ranges from 4.2 and 5.6 [28]. Furthermore, solubility of folic acid as a drug is maximum at pH 5 to 6 [29]. Therefore, arti cial sweat solutions were prepared according to ISO 105-E04:2013 method [30]. The vitamin-release behavior of FA sprayed resulting bers were studied in acidic sweat solutions at pH 5.44 by total immersion method [23]. 12-30 cm 2 of the nano bers were put into sealed glass tubes with each containing 50 mL of acidic sweat solution, separately. Then, they were placed in shaking incubator at 37.5 °C with stirring 120 rpm in order to apply the release pro le of the folic acid. Samples of 3 ml was removed at the speci ed time intervals with the sweat solution and the corresponding absorbance value was determined in a UV spectrophotometer (Scinco/NEOYSY 2000) at λ max = 282 nm, which was the characteristic peak of folic acid. The drug concentration was obtained from the calibration curve of the model vitamin prepared with a folic acid solution of known concentrations in acidic solutions (pH 5.44). The calibration curve was found to be Y = 0,0486X + (-0,0402) (R 2 = 0,99992), where X is the concentration of FA (mg/L) and Y is the solution absorbance at 282 nm (linear range of 0.5-25 mg/L) (Fig. 2). The amount of released drug was determined using UV-Vis spectroscopy.

In vitro biocompatibility
Preparation of nano ber extract solutions for cytotoxicity assays In this study in order to determine the cytotoxic effects of novel NFs prepared in different compositions, extracts of all samples were prepared among the methods recommended by the UNI EN ISO 10993-12 : 2009 regulation [31] and also suitable for the nature and shape of NFs as biomaterials. Before performing the extraction procedure, equal sizes of NF patch samples (3 cm 2 ) were cut and each sample was sterilized by UV light for 1 hour in order to keep the structural properties of the nano bers intact. The extraction procedure was carried out in sterile tubes containing 5ml of culture medium (99% RPMI 1640 and 1% Penicillin-Streptomycin, without serum in order to prevent protein interaction). All samples were kept in this medium at 37 °C for 30 minutes and it was observed that they were completely dissolved during this time period. Equal sample of an aluminum foil was also used simultaneously in the experiments as a reference base support material of NFs and treated as a sample and also extraction medium without sample was used as control and culture medium containing 1% Triton X-100 was used as a positive control. Bioengineering) were seeded in 75cm 2 culture asks containing RPMI 1640 supplemented with 10% FBS and 1% penicillin streptomycin. Cells were grown in an incubator at 37°C in an atmosphere supplemented with 5% CO 2 and monitored daily by using an inverted microscope with phase contrast attachment (Olympus CKX41). Subcultures were performed when an 80% of con uence was observed. Following disaggregation with trypsin/EDTA and resuspension of cells in medium, a total of 5x10 4 cells/well were plated in 96 well tissue-culture plates.
Determination of the cytotoxicity of nano bers by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay MTT assay was performed by the method of Mosmann (1983) [34] with the modi cations of Hansen et al. (1989) [35] and Kuz´ma et al. (2012) [36]. After 24 h incubation of cells seeded in 96 well tissue-culture plates, cells were exposed to the different concentrations of nano ber extract solutions in medium for 24 h at 37 °C in 5% CO2 in air. After exposure, the medium was aspirated and MTT (5 mg/ml of stock in PBS) was added (10 μl/well in 100 μl of cell suspension), and cells were incubated for an additional 4 h with MTT dye. At the end of incubation period, the dye was carefully taken out and 100 μl of DMSO was added to each well. The absorbance of the solution in each well was measured in a microplate reader at 570 nm. Results were expressed as the mean percentage of cell growth from three independent experiments.
Determination of the cytotoxicity of nano bers by neutral red uptake (NRU) assay The cytotoxicity of nano bers was performed in L929 cells by NRU assay following the protocols

Morphology of electrospun nano bers
The morphology of the resulting electrospun bers was investigated with SEM and presented in Fig. 3, respectively. When comparing with SEM images of PVA and PVA/FA bers, neat PVA sample has intensive beads on a string structure in the ber (Fig. 3A&B). However, applying to electrospray procedure of FA on the bers enhanced the formation of beadless morphology. With the adding of FA, the average diameter of ber with beads decreased from about 532 nm (neat PVA) to about 291 nm (PVA/FA) (Fig.   43A&B). Folic acid molecules connected PVA bers showed a little rough, randomly interconnected structure with FA clusters formed. Unfortunately, folic acid clusters were not seen too much on surface of the PVA bers and this situation can be explained by using the same solvent in both electrohydrodynamic process and thus folic acid molecules attached into PVA bers. The addition of gelatin into PVA solution also provided thin and homogenous ber structure (Fig. 3C). PVA/Gel bers with thin and regular structure were also obtained previously by Lihn et al. [39]. Furthermore, PVA-Gel NFs had a predominant ber diameter of 80-120 nm; whereas, PVA-Gel/FA bers had a fairly even distribution ranging from 60 to 220 nm. (Fig. 4 C&D). Folic acid molecules caused to form of thicker ber structure and destroyed the homogenous ber morphology. There were 2D at ber forms in some regions. In addition, folic acid clusters on the gelatin bers were seen clearly. Fig. 3E displays the blend of PVA and alginate bers. The diameter of ber increases by adding alginate into PVA solution. Therefore the resulting blend sample has shown slightly interconnected ber morphology. Although there are FA beads on the PVA-alginate bers, the structure destroyed seriously during electrospraying of FA. The reason of this is similar to PVA/FA bers related to same solvent for two hydrodynamic process. As shown in Fig. 4E and F, nanoscale PVA-Alg and PVA-Alg/FA bers were produced with diameters ranging from 150 to 550 nm and 50 to 350 nm, respectively.
As a result, SEM images revealed FA clusters have both partially deposited on the surfaces of hydrophilic bers and integrated into the ber structure. Moreover, morphology PVA bers with beads enhanced with their blends. The amount of weight loss was different for all bers. The maximum weight loss was seen at the PVA-Gel/FA mixture and the minimum was at the PVA/FA mixture. The amount of the weight losses were 9.28 % and 4.15 % for PVA-Gel/FA and PVA/FA , respectively. Apart from the weight loss related to moisture out three steps of weight loss were observed for the neat PVA ber. The major weight losses of 43% taken place in the temperature range from 210°C to 350°C due to dehydration of PVA [40,41]. The second step is dominated by chain scission about 450°C to 600°C [42] and the amount of weight loss was 8.18 %. The last step was related to burning of the pyrolysis product which was formed during analysis in the N 2 atmosphere. The weight loss of 3.77 % from the last step. It is found that FA loaded PVA bers started to decompose at about 150°C with 45% weight loss and continue to 300°C. The following weight loss of 13% from 350°C to 460°C was attributed to both elimination reaction of PVA and loss of pterin and then p-aminobenzoic acid units in folic acid. The last step was related to burning of the pyrolysis product which was formed during analysis in the N 2 atmosphere. The amount of weight loss was 8.83 % in this step.

Thermal Properties
The rst weight loss of almost 67 % apart from moisture out was seen between 280-450°C for PVA-Gel bers. The second weight loss in the amount of 6.49 % was occurred in the region of 450-550 °C. The last weight loss was about 11.68 % related to pyrolysis product which was formed during analysis in the N 2 atmosphere. The weight loss of blend ber had a decrease at 280°C and continue to 500°C with 78% weight loss which was mainly due to the cleavage of C=N bond is concerned with the presence of protein molecules in gelatin [43].
Thermal behavior of the PVA-Gel/FA was slightly difference from the thermogram of the PVA-Gel. The difference was only at the region of the 150-250°C temperature. There was slightly weight loss of the PVA-Gel/FA (about 3.34 %). The other difference was occurred after the temperature of the 450°C due to the FA content. The decomposition speed was decrease as a consequence of C=N group.
The lowest decomposition temperature was occurred for PVA/Alg blend nano bers about 170°C [44].
The rst decomposition weight loss of 68 % from the ber is related to dehydration of PVA. The second decomposition step temperature range of about 350-450°C, corresponding to the degradation of alginate with weight loss of 7.52. The third decomposition step the temperature ranges of about 450-600°C with weight loss of 6.21 % from the ber was related to PVA chain scission as the other PVA samples. The loss of the moisture from PVA-Alg/FA was less than the PVA-Alg. Furthermore, as seen in Fig. 5, PVA-Alg bers showed faster decomposition than PVA-Alg/FA after 450°C. There is no residue for all NFs.

FT-IR Analysis
The chemical composition of the all electrospun bers and folic acid were determined through FT-IR analysis. As seen in Fig. 6, the spectrum of folic acid has a number of characteristic peaks at 3590, 3496,   3330, 2925, 2840, 1694, 1650, 1605, 1487 and 1405 cm -1 . The band between 3600-3300 cm −1 is associated with (-OH) stretching bands of glutamic acid moiety and -NH group of pterin ring. The band at 1650 cm -1 belongs to the (-C=O) stretching of (-CONH 2 ). The another characteristic IR absorption peaks at 1605, 1694 and 1487 cm −1 is due to the N-H bending vibration of (-CONH) group, (-C=O) amide stretching of the α-carboxyl group and absorption band of phenyl ring respectively [45][46][47]. The frequencies for the neat PVA are indicated as follows: 3284 cm -1 for the stretching vibration peak of its (-OH) groups, 2943 and 2910 cm -1 for the stretching vibration of -CH 2 group, 1242, 1088, 1023 and 945 cm -1 (C-O) stretching vibration, respectively [41,48,49]. On the other hand, for the PVA-Gel bers the band appears at 3290 cm -1 for the (-OH) group. The bands appear at 2938 and 2913 cm -1 belongs to -CH 2 stretching vibration. The characteristic absorption of gelatin peaks show mainly to the peptide bonds (-CONH) with the amide I-III vibrations. The peak is 1643 cm -1 (amide-I) is related to -C=O stretching vibration whereas the peak at 1535 cm -1 (amide-II) is due to N-H bending and C-H stretching vibration. 1243 cm −1 for the (amide-III) peak was occurred. In addition, 1435 cm -1 (-CH 2 bending), 1374 cm -1 (C-H wagging), 1088 cm -1 (-C-O-C) and 837 cm -1 (C-C) stretching [50,51].
It is worth mentioning that the spectrums of NFs with drug ( Fig. 6) were similar the spectrum to neat NFs, indicating the absence of any chemical reaction between polymer and drug. This is explained by -OH groups in polymer structures shielded the characteristic peaks of drug molecules between 3300-3600 cm -1 , corresponding to penetration of drug into the bers. Additionally, the characteristic peaks of drug molecules could not seen after 1700 cm -1 due to the low intensity.

UV-Vis Spectroscopy-in vitro Study
UV-Vis spectrometry was used to evaluate the release pro le of FA from the nano bers, which helped to reveal the structure-function relationship of the electrospun FA-loaded bers in arti cial sweat solution (pH 5.44, 37°C) during the time course of 480 min. The cumulative FA release rate pro les, in the sweat solution FA from the ber samples, are plotted in Fig. 7.
The release pro le shows that the percentage cumulative FA releases are 49.6 %, 69.55 % and 50.88 % for PVA/FA, PVA-Gel/FA and PVA-Alg/FA, respectively after 8 h. As seen in Fig. 7, all bers exhibited initial burst release pro le at an early stage of the analysis. Especially, in PVA/FA ber at an early stage of the analysis within rst 5 min, more than half of the FA was released from the ber. In PVA-Gel/FA ber within rst 30 min about 65 % FA release whereas in PVA-Alg/FA composite bers 49 % FA release. The rapid release of FA from the composite bers is related to many factors, such as hydrophilic nature of matrix (PVA, gel and alg), high surface area of bers that supports to increase in wettability [55,56]. Illangakoon et al. have reported hydrophilic PVP with paracetamol/caffeine NFs release almost all caffein and paracetamol within the rst 6 min [57]. In this study, the burst release behavior of all bers might be related to the existence of hydrophilic based polymer matrix and drug as well. Furthermore, drug molecules depositted on the ber surface also cause the initial release due to the hybrid process. On the other hand, burst release is a phenomenon which may be preferred to obtain quick results in dermal applications [58]. Kataria et al. also prepared cipro oxacin loaded transdermal patches with fast release. In this study, the resulting PVA and PVA-Alg with cipro oxacin bers have sustained and controlled release pro le and reached maximum drug release curve in the rst 7 hours [20].
It is clearly seen PVA-Gel/FA had the slowest behavior in bers for 30 min. From the SEM images, the average ber diameter of PVA/FA, PVA-Gel/FA and PVA-Alg/FA are 291, 145 and 158 nm. Moreover, the sample has the highest drug release rate compared with the other samples in total time. This is explained as the thickness of the bers increase, the pathway drug diffusion of will increase [59].
The maximum drug was released from the bers within sustained behaviour untill 45 min. From the Fig. 7 showed diffusion based drug release occured for 30 min, then followed by a constant drug release till 8 h due to erosion of polymer matrix. Similar UV-Vis results were reported by Arthani et al. [53]. However, the release rate in PVA-Gel/FA ber is partially slower than others for the rst 30 min. due to the interaction hydrogen bonding between PVA, gelatin and folic acid. PVA-Alg/FA ber also indicated similar situation, but unfortunately the low amount of alginate in the ber limit the formation of too many interaction of hydrogen bonding (Fig. 6). Consequently, PVA-Gel/FA could be used as an e cient drug delivery system for beauty mask purpose with 8 h period.
Recently, there have been a variety approaches about COVID-19 pandemia. In this regard, inhibition of furin enzyme activity provide to limit of viral and bacterial growth [60]. Sheybani et al. have claimed that folic acid prevents furin activation. It has been simulated folid acid could be a potential drug in early respiratory diseases caused by COVID- 19 [61]. Results of the study indicates folic acid molecules interacted with active sites of furin due to formation of hydrogen bonds. Therefore, we also proposed nano ber with folic acid as a patch may prevent the COVID-19.

Cytotoxic effects of nano bers by MTT assay
The cytotoxic effects of PVA/FA, PVA-Gel/FA and PVA-Alg/FA nano bers extract solutions were evaluated by MTT assay in L929 cells. To determine and compare the cytotoxicity, extract solutions of all NFs and alsoa negative control group without any chemicals, aluminum foil as a planch material and a positive control group containing 1% Triton x-100 were prepared. In the NRU method, which is based on the measurement principle of lysosomal activity, cell viability is shown by a different mechanism than MTT assay (based on the measurement of mitochondrial activity as metabolic activity). It is thought that the nano ber extract solutions may cause toxic effects by interacting with the cell membrane or enter the cell through the membrane and accumulating in organelles and cytoplasm.

Conclusions
In this study, we successfully produced fast-dissolving drug delivery systems derived from hydrophilic composite bers by simultaneous electrospraying and electrospinning method. FA was used as model drug and the resulting composite bers consists of FA clusters entrapped in PVA, PVA-Gel and PVA-Alg NFs. The SEM images showed the composite bers possessed relatively uniform with average ber diameters between 145-291 nm and most of the FA clusters have been integrated into the bers rather than deposit on the ber surface due to polymers and folic acid solutions are hydrophilic based nature. Moreover, as FA is added ber morphology is formed with bead-free for PVA/FA ber. The FTIR spectra results demonstrated the apparent slight shifts of some peaks, corresponding to physical interactions of FA with bers. The TGA results pointed out that by the incorporation of FA into NFs, the degradation rate was increased slightly. However, FA was stable at high temperature. The in vitro release test clearly con rmed that the obtained PVA/FA, PVA-Gel/FA and PVA-Alg/FA NFs could release the FA in a sustained manner with initial burst release for the 8 hour-period. The observation of fast dissolving of all brous structures with FA in 30 min is directly related to two reasons : the strong hydrophilic nature of PVA, PVA-Gel and PVA-Alg NFs and FA clusters deposited on ber surfaces due to electrospraying process.
The biocompatibility test results based on the cytoxicity methods adapted from the ISO10993-5 standards indicatednovel PVA/FA, PVA-Gel/FA and PVA-Alg/FA electrospun nano brous patches revealed no cell toxicity on cultured broblasts in MTT assay but slight cell toxicity in NRU assay. In this study L929 cells were treated with a high concentrations of nano ber extracts to investigate exact cytotoxicity due to the nature of certain biodegradable/ leachable and extractable biomaterials. As indicated in biocompatibility test protocols the quantity exposed to used cell lines is dependent to the interface area, the volume of extraction, pH, temperature, time and many other factors. Results of this study and similar studies in the literature also indicated that the cytotoxic effect the different PVA nano bers are based on the components and content amounts. Our results reveal that PVA and PVA-Gel with/without FA nano bers seems more biocompatible than PVA-Alg nano bers. However, further biocompatibility tests should be carried out in different conditions, concentrations and different cell lines according to potential use for dermal or other biomedical applications as scaffolds. Moreover, the new nano bers can also be used as an effective patch against the COVID-19 thanks to folic acid molecules.
Declarations Figure 8 Effects of PVA nano ber extract solutions on cell viability of L929 cells by MTT assay. Results were expressed as themean percentage of cell growth inhibition from 3 independent experiments. Cell viability was plotted as percent of control (assuming data obtained from the absence of nano ber as 100%).