Study of the Nanofibers Fabrication Conditions from the Mixture of Poly(vinyl alcohol) and Chitosan by Electrospinning Method

Nanofiber fabrication is attracting great attention from scientists and technologists due to its applications in many fields of life. In order to design a nanosized polymer-based drug delivery system, we studied the conditions for the fabrication of electrospun nanofibers from poly (vinyl alcohol) (PVA) and chitosan (CS), which are well-known as biocompatible, biodegradable and non-toxic polymers that are widely used in the medical field. Aiming to develop nanofibers that can directly target diseased cells for treatment, such as cancerous cells, the ideal choice would be a system that contains the highest CS content as well as high quality fibers. In the present manuscript, it is expected to become the basis for improving the low bioavailability of medicinal drugs limited by poor solubility and low permeability. PVA–CS nanofibers were obtained by electrospinning at a PVA:CS ratio of 5:5 in a 60% (w/w) acetic acid solution under the following parameters: voltage 30 kV, feed rate 0.2 mL/h, needle-collector distance 14 cm. The obtained fibers were relatively uniform, with a diameter range of 77–292 nm and average diameter of 153 nm. The nanofiber system holds promise as a potential material for the integration of therapeutic drugs.


Introduction
In recent years, electrospinning technology has gained more and more attention along with the development of nanotechnologies, which have been used by scientists and technicians in a wide variety of scientific and technological fields. It is important to mention that healthcare has been one of the pioneers in the development of nanotechnologies, with applications in such areas as fluorescent biological labeling, drug and gene delivery, biodetection of pathogens, detection of proteins, probing of DNA structure, tissue engineering, tumor detection, separation and purification of biological molecules and cells, MRI contrast enhancement and pharmacokinetic studies [1]. Nano-sized drug capsules also have gained increasing attention due to the fact that they are believed to have outstanding advantages over conventional sized capsules. Given the same drug mass, nano-sized capsules have a significantly higher total surface area as well as a higher drug decomposition and absorption rates. Another nanomaterial that exhibits outstanding advantages is electrospun fibers, which are used as a drug carriers. Due to their size, nano-sized capsules allow otherwise difficult-to-absorb drugs to be absorbed and slowly distributed into the body, thus enhancing their absorption and providing better therapeutic effect. In addition, biodegradable materials used as drug carriers can be broken down into small molecules that are absorbed or excreted from the body; during this process, the drug can be easily released and assimilated, improving its therapeutic function [2]. The fabrication of nanofibers by the electrospinning technique follows the fundamental principle of fabricating nanofibers from molten polymers or polymer solutions under the effect of high-voltage current. In that process, the polymer solution is streamed under the action of a high-voltage electric sesses excellent biological properties such as biodegradability, lack effects, and the ability to accelerate wound healing and stimulate the Due to its ability to function in a variety of forms, chitosan has attr in the fields of orthopedics and periodontitis therapy [10,11], tissu wound healing [10,12], and drug transport [10]. In biomedical app used for artificial skin, surgical sutures, artificial blood vessels, co contact lenses, eye moisturizers, bandages, sponges, cholesterol co tory treatment, tumor inhibition, antiviral drugs, inhibition of plaq celerated wound healing; hemostatic, antibacterial, and antifun weight loss effects [10]. The reason for our preference for chitosan a material was the distribution of amino groups along the molecule, charide structure. The amino groups can be protonated, providing ferent properties such as passing through characteristic neutraliza line compounds [8] and ensuring solubility in dilute acidic aqueo [5,13]. It is known that pathological microenvironments are mostl inflammation-associated acidic pH of 7.2-6.5 for the extracellular pH inflamed tissue; pH 4.7 in fracture-related hematomas; and pH 5. [14,15]. Thus, the base structure of chitosan will help the formed n targeted to pathological cells and tissues. As a polycationic polyme of positive charges, it can adhere to both hard and soft tissues such cous tissues through hydration, hydrogen bonding and ionic inter extensively investigated as a drug carrier for targeted drug deliver cerous cells are characterized by an abnormal glucose metabolism the need for high glucose to rapidly generate energy for their surv has been shown to be suitable as a material to target cancerous cells ride structure. In addition, as a polysaccharide that contains degrad chitosan is biodegradable by several protease enzymes, and mainl Chitosan is a biopolymer that behaves like a hydrogel due to its thr ture, which can absorb and retain large amounts of water, allowing need for complete dissolution [7]. In solution, chitosan macromole and form physical networks due to the abundant intermolecular hy at low concentrations. This causes some difficulties in the fabricati only chitosan, requiring that it be combined with another polymer th properties for nanofiber fabrication; PVA was chosen for our invest  Poly (vinyl alcohol) (PVA) (Figure 2) has attracted considerable research interest and is recognized as one of the most extensively produced synthetic polymers worldwide [20]. PVA is a widely used thermoplastic polymer that is safe for living tissues, harmless and non-toxic. Orally administered poly (vinyl alcohol) has an LD 50 ≈ 15-20 g/kg [21,22]. PVA is a semicrystalline synthetic polymer, which is soluble in water, slightly soluble in ethanol and insoluble in other organic solvents [22][23][24][25]. It is also a biodegradable polymer, and its degradability is enhanced by hydrolysis due to the presence of hydroxyl groups. Under the action of the microbial community or some enzymes such as ß-diketone hydrolase and secondary alcohol oxidase, the PVA molecular chain can be partially cleaved at C-C bonds to form ketone and carboxylic compounds [26][27][28]. The final breakdown product during PVA degradation is acetic acid, which is transferred into the central metabolic pathway. Acetic acid is readily metabolized by most human and animal tissues. It is also involved in the formation of phospholipids, neutral lipids, sterols, and saturated and unsaturated fatty acids in many human and animal tissues [29,30].
Poly (vinyl alcohol) (PVA) (Figure 2) has attracted considerable researc is recognized as one of the most extensively produced synthetic polymers w PVA is a widely used thermoplastic polymer that is safe for living tissues, non-toxic. Orally administered poly (vinyl alcohol) has an LD50 ≈ 15-20 g/kg is a semicrystalline synthetic polymer, which is soluble in water, slightly so nol and insoluble in other organic solvents [22][23][24][25]. It is also a biodegradable its degradability is enhanced by hydrolysis due to the presence of hydroxyl g the action of the microbial community or some enzymes such as ß-diketone secondary alcohol oxidase, the PVA molecular chain can be partially cleaved to form ketone and carboxylic compounds [26][27][28]. The final breakdown p PVA degradation is acetic acid, which is transferred into the central metab Acetic acid is readily metabolized by most human and animal tissues. It is in the formation of phospholipids, neutral lipids, sterols, and saturated an fatty acids in many human and animal tissues [29,30]. PVA is commercially produced through the hydrolysis of poly (vinyl ace step process consisting of free radical polymerization of vinyl acetate follow drolysis [20,31]. Therefore, the structural properties of PVA are mainly dep molecular weight of the polymer and the degree of hydrolysis, i.e., the perce alcohol in the polymer [31,32]. As the degree of vinyl acetate hydrolysis into increases, the polymer structure becomes more crystalline, which results in a h PVA structure, which becomes chemically inert [31]. The degree of crystallini jor role in controlling the diffusion of drugs from PVA hydrogels [33], wh signed either as matrix or reservoir for drug delivery platforms [34]. In gen biocompatibility, drug compatibility, water solubility, film formation, mech ties and good swelling, PVA has been studied as a material for ocular inserts nanoparticles, microspheres, floating microspheres, mucoadhesives, transde and intramuscular drug delivery systems, as well as targeted drug delivery fo buccal, transdermal, pH-and temperature-sensitive drug delivery systems controlled drug delivery systems [20,31,35]. Due to its susceptibility to hydr and excessive crystallization, PVA is very sensitive to moisture; PVA hydro have low mechanical properties and offer very low swelling capacity, makin ble only for specific biomedical and pharmaceutical applications [31,36,37]. I prove its mechanical properties and stability, polyvinyl alcohol is often mix biopolymers. It has been reported that the inclusion of chitosan into a polyvin trix can improve its biocompatibility and mechanical properties [24,38,39]. To date, there have been many reports referring to the fabrication o PVA-chitosan nanofibers, from which the smallest fibers are reported to ha diameter of 20 to 100 nm [39]. However, such fibers were interspersed with e dle-like sections of about 500 ± 100 nm in width. The addition of PVA facil mation of chitosan nanofibers, but only when the chitosan content was equ PVA is commercially produced through the hydrolysis of poly (vinyl acetate) in a two-step process consisting of free radical polymerization of vinyl acetate followed by its hydrolysis [20,31]. Therefore, the structural properties of PVA are mainly dependent on the molecular weight of the polymer and the degree of hydrolysis, i.e., the percentage of vinyl alcohol in the polymer [31,32]. As the degree of vinyl acetate hydrolysis into vinyl alcohol increases, the polymer structure becomes more crystalline, which results in a highly durable PVA structure, which becomes chemically inert [31]. The degree of crystallinity plays a major role in controlling the diffusion of drugs from PVA hydrogels [33], which can be designed either as matrix or reservoir for drug delivery platforms [34]. In general, due to its biocompatibility, drug compatibility, water solubility, film formation, mechanical properties and good swelling, PVA has been studied as a material for ocular inserts, ocular films, nanoparticles, microspheres, floating microspheres, mucoadhesives, transdermal patches, and intramuscular drug delivery systems, as well as targeted drug delivery for colon, rectal, buccal, transdermal, pH-and temperature-sensitive drug delivery systems and swelling-controlled drug delivery systems [20,31,35]. Due to its susceptibility to hydrogen bonding and excessive crystallization, PVA is very sensitive to moisture; PVA hydrogels generally have low mechanical properties and offer very low swelling capacity, making them desirable only for specific biomedical and pharmaceutical applications [31,36,37]. In order to improve its mechanical properties and stability, polyvinyl alcohol is often mixed with other biopolymers. It has been reported that the inclusion of chitosan into a polyvinyl alcohol matrix can improve its biocompatibility and mechanical properties [24,38,39].
To date, there have been many reports referring to the fabrication of electrospun PVA-chitosan nanofibers, from which the smallest fibers are reported to have an average diameter of 20 to 100 nm [39]. However, such fibers were interspersed with enlarged spindle-like sections of about 500 ± 100 nm in width. The addition of PVA facilitated the formation of chitosan nanofibers, but only when the chitosan content was equal or less than 25% [39]. In another report on a morphological study of PVA-CS, nanofibers were prepared by electrospinning and collected on reticulated vitreous carbon; nanofibers with diameters from 132 to 212 nm were obtained from a mixture of 8% PVA and 2% chitosan in 2% acetic acid solution [40]. PVA-CS nanofibers produced by the electrospinning technique were also obtained from an acetic acid solution with up to 70% concentration and PVA: CS  [41]. There are obviously major differences in PVA-CS nanofiber fabrication methods and results, so more detailed and specific studies are needed. In this study, we conducted our experiments step-by-step to estimate the correlation between the influence of the solution conditions and the electrospinning parameters on the formation of PVA-CS nanofibers.

Materials
CS (M W 200 kDa) and PVA (M W 55 kDa) were used as received from the Russian Federation limited liability company Bioprogres.
Acetic acid (99.5%) and distilled water were used as components of the binary solvent system.

Electrospinning Technique
PVA-CS solutions were electrospun in a NANON-01A system (MECC CO., LTD., Fukuoka, Japan). The electrospinning process ( Figure 3) was performed at the temperature of 28.0 ± 1.5 • C and the relative humidity of 20 ± 3%. The technical parameters were changed in order to find the optimal conditions for the production of fibers: voltage range from 18 to 30 kV; feed rate range from 0.1 to 0.4 mL/h; needle-collector distance switched between 140 mm and 150 mm; traverse speed 10 mm/s, 16G steel needles; plate stainless steel collector 150 mm × 200 mm (L × W). fabrication methods and results, so more detailed and specific studies are needed. In study, we conducted our experiments step-by-step to estimate the correlation betwee influence of the solution conditions and the electrospinning parameters on the form of PVA-CS nanofibers.

Materials
CS (MW 200 kDa) and PVA (MW 55 kDa) were used as received from the Ru Federation limited liability company Bioprogres.
Acetic acid (99.5%) and distilled water were used as components of the binary so system.

Electrospinning Technique
PVA-CS solutions were electrospun in a NANON-01A system (MECC CO., L Fukuoka, Japan). The electrospinning process ( Figure 3) was performed at the tem ture of 28.0 ± 1.5 °C and the relative humidity of 20 ± 3%. The technical parameters changed in order to find the optimal conditions for the production of fibers: voltage r from 18 to 30 kV; feed rate range from 0.1 to 0.4 mL/h; needle-collector distance swit between 140 mm and 150 mm; traverse speed 10 mm/s, 16G steel needles; plate stai steel collector 150 mm × 200 mm (L × W). The study of nanofiber fabrication included studying the influence of diff PVA:CS ratios and an investigation of the solvent's influence on the formation of n fibers at different needle-collector distances, voltages, and feed rate speeds.

Rheological Properties of Polymer Solutions
An MCR 502 rheometer with a cylinder was utilized to obtain the kinematic visc of the solutions. Measurements were performed at a temperature of 25 °C and in a rate range from 0.1 to 500 s −1 .
The conductivity of the polymer solution was measured on a WTW inoLab C 7110 conductivity meter with a WTW TetraCon 325 sensor. Measurement error shoul exceed 0.5%. The study of nanofiber fabrication included studying the influence of different PVA:CS ratios and an investigation of the solvent's influence on the formation of nanofibers at different needle-collector distances, voltages, and feed rate speeds.

Rheological Properties of Polymer Solutions
An MCR 502 rheometer with a cylinder was utilized to obtain the kinematic viscosity of the solutions. Measurements were performed at a temperature of 25 • C and in a shear rate range from 0.1 to 500 s −1 .
The conductivity of the polymer solution was measured on a WTW inoLab Cond 7110 conductivity meter with a WTW TetraCon 325 sensor. Measurement error should not exceed 0.5%.

Morphology and Diameters of Nanofibers
For the preliminary characterization of the morphology and diameters of the electrospun PVA-CS fibers, the optical microscope Olympus STM6 (OLYMPUS Corporation, Tokyo, Japan) was used. Differentially interferential contrasting technique (DIC) was applied to emphasize the colorfulness and contrast of the obtained fibers. ImageJ (National Institutes of Health, Bethesda, MD, USA) was used for the analysis and measurement of the fiber diameters from the obtained microphotograph program.

Infrared Spectroscopy
A Bruker alpha Fourier transform Infrared (FTIR) spectrometer (Bruker, Germany) was used to obtain the infrared absorption spectra of the samples.

Statistical Analysis
The diameter distribution of the obtained nanofibers was estimated by OriginPro 2019b (OriginLab Corporation, Northampton, MA, USA). To measure the diameter distribution, several images were used.

Selection of PVA:CS Ratio to Investigate Nanofiber Fabrication Conditions
During the fabrication of PVA nanofibers from acetic acid solution, we found that the optimal parameters were 12% (w/w) PVA and 30% (w/w) CH 3 COOH with the rest as deionized water. In order to develop a nanofiber system that could integrate medicinal compounds, we added CS to PVA nanofibers. The first step for obtaining the PVA-CS fibers was to keep constant the CS concentration at 2% (w/w) and change the PVA concentration to find the optimal concentration for the electrospun fibers.
Polymer solutions were prepared by mixing 2% (w/w) CS with different concentrations of PVA in a 30% (w/w) acetic acid solution. All solutions were stirred at 90 • C until homogeneous. The electrospinning parameters used during the process were: feed rate range 0.1-0.4 mL/ h; voltage range 18-30 kV; and needle-collector distance of 140 and 150 mm. The results of nanofiber fabrication from the polymer solutions with different PVA:CS ratios are presented in Table 1. A PVA: CS ratio of 8:2 was chosen for the investigation of the solvents and electrospinning conditions used to fabricate the nanofiber system.

Influence of Acetic Acid Concentration on PVA-CS Nanofiber Fabrication
We studied the effect of CH 3 COOH concentration on fiber formation from the PVA:CS (8:2) polymer system, see Table 2   Table 2. Results of the investigation of fiber formation from the electrospinning experiment using PVA 8% (w/w) and CS 2% (w/w) with different concentrations of CH 3 COOH.

Needle-Collector
Distance ( 150 The solution is too viscous to conduct the electrospinning. (2) No solution is obtained because PVA polymer particles are insoluble in pure acetic acid while CS forms salts with it. (+) Nanofiber formation without drops.
When the acid concentration was increased to 50% (w/w) and 60% (w/w), the solution easily formed nanofibers. However, when the concentration of acetic acid was increased to 70% (w/w), the solution became too viscous, which inhibited its use for electrospinning.

Determination of Electrospinning Parameters for PVA-CS Nanofiber Fabrication
The use of acetic acid at a concentration of 50% or 60% allows the formation of fibers with a PVA:CS ratio of 8:2. Therefore, the study on the formation of nanofibers from different PVA:CS ratios was carried out at both acetic acid concentrations.
Solutions with different PVA:CS ratios were dissolved in 50% (w/w) and 60% (w/w) CH 3 COOH. The solutions were prepared by stirring at 90 • C until a homogeneous solution was obtained. The electrospinning parameters were fixed as follows: needle-collector distance 140 mm, feed rate range 0.1-0.4 mL/h and voltage range 18-30 kV. The results of nanofiber fabrication from the polymer solutions at different PVA:CS ratios are presented in Table 3.
Solutions with the concentration of acetic acid at 60% (w/w) proved to be the most favorable for the fabrication of nanofibers. From the obtained results, it was possible to observe that the solution containing a higher concentration of PVA needed a lower feed rate and voltage, which eased the fabrication of fibers. However, lower feed rates and higher voltages resulted in polymer solutions that dried faster, causing clogging at the needle tip, which easily occurred especially with solutions with high chitosan concentrations above 4% (w/w) and 5% (w/w). Additionally, from visual observation, it could be seen that the solutions with a higher concentration of PVA resulted in easier fiber formation. The solution with a concentration of chitosan greater than 5% (w/w) was too viscous and could not be used for electrospinning. In order to choose the proper ratio of polymers for fabrication of nanofibers, the morphology of the fibers must be analyzed using microscopy techniques.
For further analysis, we selected all the samples obtained under the following parameters: needle-collector distance of 140 mm, voltage of 30 kV and feed rate of 0.2 mL/h.

Morphology and Size Distribution of PVA-CS Nanofibers
In order to select the proper polymer ratio, the influence that different polymer concentrations have on the formation and morphology of the fibers must be taken into consideration. PVA-CS solutions with ratios of 8:2; 7:3; 6:4 and 5:5 (%, w/w) and acetic acid concentrations at 50% (w/w) and 60% (w/w) were electrospun under the same parameters: voltage 30 kV, needle-collector distance 140 mm, and feed rate 0.2 mL/h. The morphological characteristics and diameter distribution of the obtained PVA-CS fibers are presented in Figures 4-6, and Table 4. -------------- Solutions with the concentration of acetic acid at 60% (w/w) proved to be the most favorable for the fabrication of nanofibers. From the obtained results, it was possible to observe that the solution containing a higher concentration of PVA needed a lower feed rate and voltage, which eased the fabrication of fibers. However, lower feed rates and higher voltages resulted in polymer solutions that dried faster, causing clogging at the needle tip, which easily occurred especially with solutions with high chitosan concentrations above 4% (w/w) and 5% (w/w). Additionally, from visual observation, it could be seen that the solutions with a higher concentration of PVA resulted in easier fiber formation. The solution with a concentration of chitosan greater than 5% (w/w) was too viscous and could not be used for electrospinning. In order to choose the proper ratio of polymers for fabrication of nanofibers, the morphology of the fibers must be analyzed using microscopy techniques.
For further analysis, we selected all the samples obtained under the following parameters: needle-collector distance of 140 mm, voltage of 30 kV and feed rate of 0.2 mL/h.

Morphology and Size Distribution of PVA-CS Nanofibers
In order to select the proper polymer ratio, the influence that different polymer concentrations have on the formation and morphology of the fibers must be taken into consideration. PVA-CS solutions with ratios of 8:2; 7:3; 6:4 and 5:5 (%, w/w) and acetic acid concentrations at 50% (w/w) and 60% (w/w) were electrospun under the same parameters: voltage 30 kV, needle-collector distance 140 mm, and feed rate 0.2 mL/h. The morphological characteristics and diameter distribution of the obtained PVA-CS fibers are presented in Figures 4-6, and Table 4.     Presence of individual small spots From the nanofiber morphology investigation, it could be seen that by increasing t CS concentration to the point where PVA and CS concentrations were equal, the diame    From the nanofiber morphology investigation, it could be seen that by increasing the CS concentration to the point where PVA and CS concentrations were equal, the diameter of the fibers decreased; moreover, it was possible to observe an improvement in the fibers' morphology. Therefore, for the fabrication of high-quality fibers, it is necessary to find the optimal polymer ratios as well as the optimal electrospinning parameters.
In summary, increasing the concentration of acetic acid not only improved the fabrication of the fibers, but also improved the morphology and decreased the diameters of the PVA-CS nanofibers. The higher the concentration of PVA used, the more easily that fibers were obtained, but also the more defects that appeared; on the other hand, higher CS concentrations required more precise electrospinning parameters but resulted in fibers with better morphology and increased nanoscale. When the CS concentration was higher than the PVA concentration (higher than 5% (w/w)), the solution became too viscous and could not form nanofibers. However, the ratio of 5% (w/w) CS together with 5% (w/w) PVA was higher if compared to previously published reports on the PVA:CS ratio for nanofiber fabrication, that is, in the range from 8:2 to 7:3 [39][40][41]. With the expectation of including as much CS in the PVA-CS nanofiber as possible, in order to increase the ability to integrate drugs and increase the targeting ability to treat diseased cells in the body, the 5:5 ratio is ideal. Thus, it was determined that the optimal parameters for the production of highquality nanofibers are: for the polymer solution, the optimal concentrations of polymers are 5% (w/w) PVA, 5% (w/w) CS, and 60% (w/w) acetic acid. For the electrospinning, the optimal parameters are: needle-collector distance-140 mm; feed rate-0.2 mL/h, and voltage-30 kV.

Investigation of Rheological Properties of PVA-CS Solutions
The nanofiber fabrication ability as well as the nanofiber morphology are closely related to the rheological properties of the polymer solutions used. To understand this correlation, we studied the change in the conductivity and viscosity values of the polymer solutions at different PVA:CS ratios.

Electrical Conductivity
The conductivity of the PVA-CS solutions was considered in two cases: first, the CS concentration was kept at 2% (w/w) while the PVA concentration was changed in 30% (w/w) acetic acid; in the second case, the concentrations of both PVA and CS were changed in 60% (w/w) acetic acid. The results are presented in Tables 5 and 6 and Figure 7.  By comparing the results presented in Figure 7A,B it is easy to conclude that whi increasing the concentration of PVA decreased the conductivity of the solution, a high concentration of CS led to an increase in solution conductivity. It can be concluded that th CS molecules have an extremely large charge carrier release, which overcomes the enta glement of the polymer system and the abundant hydrogen bonding in the solution.

Viscosity
The viscosity of the PVA-CS solutions was studied. PVA-CS solutions were consi ered in two cases: first, the CS concentration was kept at 2% (w/w) while the PVA conce tration was changed in 30% (w/w) acetic acid; in the second case, the concentrations both PVA and CS were changed in 60% (w/w) acetic acid. Measurements were made at °C and in a range of shear rates from 0.1 to 500 s −1 ; the results were obtained at a shear ra of 75.3 s −1 and are presented in Tables 7 and 8 and Figure 8. The viscosity values of the PVA:CS solutions with the ratio of 9:2 in the above tw cases changed from 4275.6 mPa.s to 7016.4 mPa.s along with the increase in the conce tration of acetic acid from 30% (w/w) to 60% (w/w). Similarly to the effect of acid conce tration on electrical conductivity, the increase in viscosity in this case can also be explaine by the increasing number of hydrogen bonds between the acetic acid and the polymers the solution, thereby increasing the viscosity of solutions with higher concentrations acetic acid. Considering the conductivity values of PVA:CS solutions with the ratio 9:2 in the above two cases, it was found that the increase in acetic acid concentration from 30% (w/w) to 60% (w/w) caused the conductivity to decrease from 1777 µS/cm to 802 µS/cm. The cause of the decrease in conductivity with the increased acetic acid concentration was due to the increase in the density and strength of the hydrogen bonds between the acetic acid and polymer molecules. It is these connections that increase the degree of entanglement of the polymers in solution and hinder the movement of charge carriers and free ions present in the solution.
By comparing the results presented in Figure 7A,B it is easy to conclude that while increasing the concentration of PVA decreased the conductivity of the solution, a higher concentration of CS led to an increase in solution conductivity. It can be concluded that the CS molecules have an extremely large charge carrier release, which overcomes the entanglement of the polymer system and the abundant hydrogen bonding in the solution.

Viscosity
The viscosity of the PVA-CS solutions was studied. PVA-CS solutions were considered in two cases: first, the CS concentration was kept at 2% (w/w) while the PVA concentration was changed in 30% (w/w) acetic acid; in the second case, the concentrations of both PVA and CS were changed in 60% (w/w) acetic acid. Measurements were made at 25 • C and in a range of shear rates from 0.1 to 500 s −1 ; the results were obtained at a shear rate of 75.3 s −1 and are presented in Tables 7 and 8 and Figure 8.  By comparing the results presented in Figure 7A,B it is easy to conclude that while increasing the concentration of PVA decreased the conductivity of the solution, a higher concentration of CS led to an increase in solution conductivity. It can be concluded that the CS molecules have an extremely large charge carrier release, which overcomes the entanglement of the polymer system and the abundant hydrogen bonding in the solution.

Viscosity
The viscosity of the PVA-CS solutions was studied. PVA-CS solutions were considered in two cases: first, the CS concentration was kept at 2% (w/w) while the PVA concentration was changed in 30% (w/w) acetic acid; in the second case, the concentrations of both PVA and CS were changed in 60% (w/w) acetic acid. Measurements were made at 25 °C and in a range of shear rates from 0.1 to 500 s −1 ; the results were obtained at a shear rate of 75.3 s −1 and are presented in Tables 7 and 8 and Figure 8. The viscosity values of the PVA:CS solutions with the ratio of 9:2 in the above two cases changed from 4275.6 mPa.s to 7016.4 mPa.s along with the increase in the concentration of acetic acid from 30% (w/w) to 60% (w/w). Similarly to the effect of acid concentration on electrical conductivity, the increase in viscosity in this case can also be explained by the increasing number of hydrogen bonds between the acetic acid and the polymers in the solution, thereby increasing the viscosity of solutions with higher concentrations of acetic acid.  By comparing the results presented in Figure 7A,B it is easy to conclude that while increasing the concentration of PVA decreased the conductivity of the solution, a higher concentration of CS led to an increase in solution conductivity. It can be concluded that the CS molecules have an extremely large charge carrier release, which overcomes the entanglement of the polymer system and the abundant hydrogen bonding in the solution.

Viscosity
The viscosity of the PVA-CS solutions was studied. PVA-CS solutions were considered in two cases: first, the CS concentration was kept at 2% (w/w) while the PVA concentration was changed in 30% (w/w) acetic acid; in the second case, the concentrations of both PVA and CS were changed in 60% (w/w) acetic acid. Measurements were made at 25 °C and in a range of shear rates from 0.1 to 500 s −1 ; the results were obtained at a shear rate of 75.3 s −1 and are presented in Tables 7 and 8 and Figure 8.  An increase in the concentration of any polymer had the effect of increasing th lution's viscosity. As illustrated in Figure 8A, when the concentration of CS was fix 2% (w/w) and the concentration of PVA was changed, it was observed that the viscosi the solution was proportional to the increase in PVA concentration. When the conce tion of CS was increased and the concentration of PVA decreased, the viscosity o solution still increased very quickly, by up to about four-fold compared to case (Fi 8A, 8B). In addition, considering the difference in molecular weight between the two ymers, the increase in viscosity caused by one mole of CS was 53 times higher than of PVA.
Thus, in the PVA-CS solution, CS was the polymer with the main effect that dr cally changed the rheological properties of the solution, exerting a direct influence o fabrication of nanofibers as well as on their morphology and diameter distribution increase in the viscosity and electrical conductivity of the solution resulted in fibers were more nano-sized, but the electrospinning performance was reduced. Therefore the fabrication of nanofibers with a higher ratio of CS to PVA, the electrospinning pa eters need to be more strictly controlled.

FTIR spectroscopy
To investigate the characteristics of bonds formed in the PVA-CS fiber matrix, i red spectroscopy of PVA-CS fibers, PVA-CS film (obtained by drying PVA-CS solut pure PVA powder and pure CS powder was performed (Figure 9).
From Figure 9, it can be easily seen that the IR spectrum of the PVA-CS film conta the peaks with highest intensity. The broad peak with strong intensity in the regio 3000-3600 cm −1 showed the abundance of stretching motion of OH and NH bonds pre in the PVA-CS film. The simplest spectra were those of the PVA-CS nanofibers, in w the strong peak at position 1705 cm −1 corresponded to the stretching of the C=O bond and the peak at 1264 cm −1 corresponded to the C-O [43] bond in the carboxyl grou acetic acid; the strong peaks at 1546 cm −1 and 1408 cm −1 corresponding to the fluctua of the carboxylate group of the salt [42] completely disappeared.
In general, the spectrum of PVA-CS nanofibers was quite similar to that of pure P The viscosity values of the PVA:CS solutions with the ratio of 9:2 in the above two cases changed from 4275.6 mPa.s to 7016.4 mPa.s along with the increase in the concentration of acetic acid from 30% (w/w) to 60% (w/w). Similarly to the effect of acid concentration on electrical conductivity, the increase in viscosity in this case can also be explained by the increasing number of hydrogen bonds between the acetic acid and the polymers in the solution, thereby increasing the viscosity of solutions with higher concentrations of acetic acid.
An increase in the concentration of any polymer had the effect of increasing the solution's viscosity. As illustrated in Figure 8A, when the concentration of CS was fixed at 2% (w/w) and the concentration of PVA was changed, it was observed that the viscosity of the solution was proportional to the increase in PVA concentration. When the concentration of CS was increased and the concentration of PVA decreased, the viscosity of the solution still increased very quickly, by up to about four-fold compared to case ( Figure 8A,B). In addition, considering the difference in molecular weight between the two polymers, the increase in viscosity caused by one mole of CS was 53 times higher than that of PVA.
Thus, in the PVA-CS solution, CS was the polymer with the main effect that drastically changed the rheological properties of the solution, exerting a direct influence on the fabrication of nanofibers as well as on their morphology and diameter distribution. The increase in the viscosity and electrical conductivity of the solution resulted in fibers that were more nano-sized, but the electrospinning performance was reduced. Therefore, for the fabrication of nanofibers with a higher ratio of CS to PVA, the electrospinning parameters need to be more strictly controlled.

FTIR Spectroscopy
To investigate the characteristics of bonds formed in the PVA-CS fiber matrix, infrared spectroscopy of PVA-CS fibers, PVA-CS film (obtained by drying PVA-CS solution), pure PVA powder and pure CS powder was performed (Figure 9).
The peaks at 2940 cm −1 and 2910 cm −1 were related to the asymmetric and symm vibrations of CH2 stretching, respectively [43,45]. The vibration of the C-H alkyl bon the chitosan molecule at the position 2869 cm −1 was mixed and transformed into a sh der in the spectrum of the polymer blend; this transition has been confirmed by a prev publication [42]. Peaks 1709 cm −1 and 1659 cm −1 were related to the stretching vibrations of the and C-O bonds present in the remaining acetate units in the PVA molecule [45,46] w were still visible in the spectrum of PVA-CS fibers. The peak at 1425 cm −1 referred to vibrations of the C-H bond of the methyl group (-CH3). The peak at 1089 cm −1 was ca by the asymmetric stretching vibration of the C-O bond of the acetate group. The pe 844 cm −1 was associated with the bending vibrations of C-H bonds in the molecule. T peaks are in agreement with previous statements about PVA [20,25,47,48].
A band at 1589 cm −1 in the spectrum of chitosan corresponded to the N-H bond b ing of the basic amine. The C-H bending and C-N symmetry deformations of chit were confirmed by the presence of bands around 1423 cm −1 , 1375 cm −1 and 1322 cm − spectively. The absorption band at 1153 cm −1 may have been due to asymmetric stretc of the C-O-C bridge in the glucose group. The bands at 1066 cm −1 and 1028 cm −1 c sponded to the stretching of the C-O bonds. Ring stretching corresponded to the 896 peak of OH out-of-plane and the 690 cm −1 peak of N-H twist vibrations. These peak in accordance with previous statements about PVA [49][50][51].
The peaks in the 1590-890 cm −1 region of the individual polymers were more com than those obtained from the PVA-CS nanofibers. In general, the peak position of PVA a slight shift, the wavenumber decreased, and the peaks of chitosan became shou shaped. The OH out-of-plane vibrations and the torsional vibrations of the NH grou From Figure 9, it can be easily seen that the IR spectrum of the PVA-CS film contained the peaks with highest intensity. The broad peak with strong intensity in the region of 3000-3600 cm −1 showed the abundance of stretching motion of OH and NH bonds present in the PVA-CS film. The simplest spectra were those of the PVA-CS nanofibers, in which the strong peak at position 1705 cm −1 corresponded to the stretching of the C=O bond [42] and the peak at 1264 cm −1 corresponded to the C-O [43] bond in the carboxyl group of acetic acid; the strong peaks at 1546 cm −1 and 1408 cm −1 corresponding to the fluctuations of the carboxylate group of the salt [42] completely disappeared.
In general, the spectrum of PVA-CS nanofibers was quite similar to that of pure PVA, but the peaks shifted and changed in intensity depending on the concentrations of the polymers in the system. The higher PVA component in the fiber led to stronger peaks. The two peaks associated with the stretching vibrations of the NH bond and OH bond in the chitosan molecule at position 3362 cm −1 and position 3294 cm −1 were associated with the wide peak at position 3299 cm −1 of the stretching vibration of the OH bond in the PVA molecule into a wide and high intensity peak in the region of 3000-3600 cm −1 common to all OH and NH groups in the polymer system. This is similar to observations reported in previous publications [42][43][44][45].
The peaks at 2940 cm −1 and 2910 cm −1 were related to the asymmetric and symmetric vibrations of CH 2 stretching, respectively [43,45]. The vibration of the C-H alkyl bond in the chitosan molecule at the position 2869 cm −1 was mixed and transformed into a shoulder in the spectrum of the polymer blend; this transition has been confirmed by a previous publication [42].
Peaks 1709 cm −1 and 1659 cm −1 were related to the stretching vibrations of the C=O and C-O bonds present in the remaining acetate units in the PVA molecule [45,46] which were still visible in the spectrum of PVA-CS fibers. The peak at 1425 cm −1 referred to the vibrations of the C-H bond of the methyl group (-CH 3 ). The peak at 1089 cm −1 was caused by the asymmetric stretching vibration of the C-O bond of the acetate group. The peak at 844 cm −1 was associated with the bending vibrations of C-H bonds in the molecule. These peaks are in agreement with previous statements about PVA [20,25,47,48].
A band at 1589 cm −1 in the spectrum of chitosan corresponded to the N-H bond bending of the basic amine. The C-H bending and C-N symmetry deformations of chitosan were confirmed by the presence of bands around 1423 cm −1 , 1375 cm −1 and 1322 cm −1 , respectively. The absorption band at 1153 cm −1 may have been due to asymmetric stretching of the C-O-C bridge in the glucose group. The bands at 1066 cm −1 and 1028 cm −1 corresponded to the stretching of the C-O bonds. Ring stretching corresponded to the 896 cm −1 peak of OH out-of-plane and the 690 cm −1 peak of N-H twist vibrations. These peaks are in accordance with previous statements about PVA [49][50][51].
The peaks in the 1590-890 cm −1 region of the individual polymers were more complex than those obtained from the PVA-CS nanofibers. In general, the peak position of PVA had a slight shift, the wavenumber decreased, and the peaks of chitosan became shoulder shaped. The OH out-of-plane vibrations and the torsional vibrations of the NH group of chitosan also disappeared. This showed that the mobility of the OH and NH 2 groups of chitosan were no longer available. These changes together with the broadening of the absorption band in the region of 3000-3600 cm −1 indicated that many hydrogen bonds had formed between PVA and CS molecules. Previous publications also reached similar conclusions [44,45].

Conclusions
The investigation of nanofiber fabrication and rheological properties of the solutions containing PVA and CS polymers together with acetic acid as solvent showed that these factors were closely related to each other. Any increase in the concentration of either the acetic acid or the polymers led to an increase in the solution's viscosity. While PVA and acetic acid reduced the conductivity of the solution, CS greatly increased the solution's conductivity. For this reason, it is easier to obtain fibers from solutions with a high concentration of PVA; however, the fibers obtained from these solutions have larger sizes and more defects. In contrast, although requiring more precise electrospinning parameters, solutions with high CS content resulted in more nanoscale fibers.
Infrared spectroscopy confirmed the complete separation of the acetic acid from the nanofibers, ensuring its safety for future drug integration purposes. The results of infrared spectrum analysis also showed that hydrogen bonds were the only type of bond formed between the polymers in the nanofibers.
PVA-CS nanofibers were successfully fabricated from a solution containing 5% (w/w) PVA; 5% (w/w) CS; and 60% (w/w) CH 3 COOH. The following electrospinning parameters were used: voltage 30 kV, feed rate 0.2 mL/h, needle-collector distance 140 mm. The obtained fibers exhibited diameters that were in the range of 77-292 nm, with an average diameter of 153 nm. These nanofiber systems had relatively few defects and could be used for the integration of drugs.
The formed PVA-CS nanofiber system holds promise as a potential material for the integration of therapeutic molecules.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.

Conflicts of Interest:
The authors declare no conflict of interest.