Poly(vinyl alcohol) (PVA) was supplied by Vetec Química Fina (Rio de Janeiro, RJ, Brazil) with molecular weight of 85,300 g/mol and 88% degree of hydrolysis. Medium molecular weight chitosan (CS), degree of acetylation (DA = 15%) and the viscosity-average molecular weight ( M ¯ v = 296772 ), and glutaraldehyde were provided by Sigma Aldrich (São Paulo, SP, Brazil). Analytical grade acetic acid was purchased from Vetec Química Fina Ltda. (Rio de Janeiro, RJ, Brazil). All materials were used without previous purification.

The nanofibers were soaked in distilled water and after 1 h they were carefully removed and dried with filter paper to remove surface water and then weighed. The materials were dried under vacuum to constant weight.

The morphology of PVA/CS nanofibers was observed by scanning electron microscopy (SEM), using a Tescan model 212 Vega 3LMU (Brno-Kohoutovice, Czech Republic) equipment. The surfaces were vacuum coated with gold prior to measurements. SEM images were used to measure the average fiber diameters with the Size Meter Software. At least 50 nanofibers were randomly selected from each of the SEM images.

Structural characterization was performed by X-ray diffraction (XRD) operated with CuKα radiation (λ = 0.15418 nm). XRD analyses were performed in an Ultima IV diffractometer (Rigaku Corporation, Osaka, Japan) operating at the CuKα wavelength of 1.5418 Å, at 40 kV and 20 mA. The scattered radiation was detected at ambient temperature in the 0.6˚ to 80˚ (2θ) angular region at 0.5˚ (2θ)/min in the reflection mode. For a better visualization of the characteristic reflections, the diffractograms were smoothed using the software OriginPro 8.0 (Savitsky-Golay, polynome: 2, points of window: 10). The free software Fityk, downloaded at http://www.unipress.waw.pl/fityk, was used to determine the degree of crystallinity.

Infrared spectroscopy (FTIR) analyses were carried from 4000 cm⁻¹ to 600 cm⁻¹, at ambient temperature, using a Perkin Elmer Frontier spectrometer (Waltham, MA, USA), equipped with an attenuated total reflection accessory (ATR), by averaging 60 scans with a resolution of 4 cm⁻¹ in transmission mode.

Dynamic mechanical analyses (DMA) were performed in a Q800 DMA from TA Instruments, equipped with an accessory to measure tensile properties of mats with (13.0 ± 0.2) mm × (6.0 ± 0.02) mm × (0.09 ± 0.006) mm. The dynamic experiments were carried out at 1 Hz in the tensile mode, from −60˚C to 310˚C, at a heating rate of 3˚C/min, after conditioning the samples at 25˚C and 50% relative humidity, for a period of at least 48 h. Uniaxial tensile tests were performed in the same Q800 equipment, at 25˚C, by the controlled force method under a force ramp of 1 N/min to 18 N up to the break of the sample. The average value was taken from a total of three measurements.

The combined effects of the independent variables (applied voltage, polymeric solution flow rate, chitosan concentration in the mixture and distance between plate and needle) on nanofiber diameter and homogeneity were evaluated by Box-Behnken design. According to statistical theory, a Box-Behnken design with four independent factors comprises 27 experiments as shown in Table 2, which also included coded variables and experimental and predicted values. The observed mean diameter (Y₁) and standard deviation (Y₂) values for PVA/CS nanofibers were in the range 186.6 - 354.2 nm and 27 - 139.7 nm, respectively, depending on the variation of conditions. The results were then evaluated by an ANOVA test to verify the significance of the independent variables, as well as their possible interactions, in order to obtain a mathematical model. The effect of each of the responses as well as their respective estimated coefficients and p values were compiled in Table 3. The p-value was used as a measure of statistical significance to assess the impact of each factor on the response, where p values greater than 0.05 do not significantly affect response within a confidence interval greater than 95%. Also, the lower the p-value, the more important that factor would be. For both responses, voltage was the factor with the greatest impact, followed by the distance between the plates and the chitosan concentration.

The insignificant coefficients (p > 0.05) were taken from the complete quadratic model in order to obtain a more refined mathematical model, and again through an ANOVA test, the coefficients were recalculated. The reduced response surface model for the mean diameter (Y₁) containing only statistically

significant terms within a 95% confidence interval (p ≤ 0.05) is presented in Equation (3).

Y 1 = 254.61 − 36.37 X 1 − 9.67 X 1 2 − 24.04 X 3 − 22.76 X 3 2             + 24.73 X 4 − 20.95 X 1 X 3 − 29.1 X 1 X 4 (3)

where X₁ is the applied voltage (kV), X₃ is the concentration of chitosan solution in the mixture (%) and X₄ is the distance between the collecting plate and the needle.

The mathematical model for nanofiber standard deviation (Y₂) after the elimination of insignificant terms is presented in Equation (4).

Y 2 = 62.85 − 25.55 X 1 − 9.30 X 1 2 + 8.27 X 3 − 9.03 X 3 2     + 11.3 X 4 − 17.5 X 1 X 3 − 20.3 X 1 X 4 (4)

The p-value was also used to measure the statistical significance related to estimated coefficients of the presented models. It was observed that the equations obtained for the responses Y₁ (mean diameter) and Y₂ (standard deviation) were considered significant, with no lack of fit in reduced models. The p-value observed for mean diameter and standard deviation in reduced models was 0.1014 and 0.1256, respectively.

The adequacy of the predicted values by the model with the experimental data was also confirmed by the analysis of the coefficient of determination (R²), which represents the proportion of the total variability that can be explained by a regression model. After excluding non-significant terms, the calculated value of R² was 0.84002 for the average diameter model, indicating that the model can explain 84% of the average diameter variation of nanofibers. The standard deviation model can explain 83% of the variation in the experimental observations. The adjusted R² value for the mean diameter (0.781) and standard deviation (0.7677), after withdrawal of insignificant terms also showed a good correlation between the observed and predicted values, showing that the quadratic models obtained are significant and favorable for the representation of the relationship between the response and the independent variables.

Figures 1(a)-(d) show the estimated three-dimensional (3D) surface plots for the evaluated responses as a function of the selected independent parameters (two-factors-at-a-time) illustrating their relationships. The impact of the electrospinning parameters on fiber diameter and their homogeneity was assessed by 3D surface plots.

The influence of applied voltage on fiber diameter and their standard deviation was illustrated in Figures 1(a)-(d). It may be observed that, in relation to mean diameter and standard deviation, keeping one or more factors constant, the increase in applied voltage led to a decrease in the responses, up to a threshold limit in some regions. High voltage created an electrically charged polymeric jet out of the needle, and the electric field formed with the collecting plate, reducing the drop. Overcoming the surface tension by voltage led to the formation of the Taylor cone at the capillary outlet, initiating the electrospinning process. Increasing the applied voltage causes an increase on strength of electric field and

electrostatic stretching force leading to an accelerated jet, forming thinner nanofibers [2] [40] [41]. Also, it was possible to observe the curvature on the plots, which confirms the need for a quadratic regression model. The models show that applied voltage has significant interactions with tip-to-collector distance (X₁X₃) and chitosan content on blend (X₁X₄).

Figure 1(a) and Figure 1(c) shows the impact of tip-to-collector distance and applied voltage in nanofibers with flow rate and chitosan content kept constant at center point. A minimum distance was required to give the fibers enough time to evaporate the solvent before reaching the collecting plate, thus avoiding the appearance of failures if they are too close or too far away [42]. The impact of tip-to-plate distance is given by competition between long- and short-range effects. While shorter distances led to the formation of thinner fibers, due to increased electric field, longer distances provided longer jet stretching, due to longer flight times [43] [44].

Figure 1(b) and Figure 1(d) shows the effect of increasing chitosan concentration on PVA/CS mixture. As shown in Figure 2(a), because of increased entanglements and intermolecular interactions between polymer molecules the solution viscosity was enhanced.

The liquid-like behavior of the solutions is shown in Figure 2(b), in which the loss modulus (G’’) is larger than the storage modulus (G’) for all studied solutions. Thus, a higher voltage was required to charge the solutions and start the electrospinning process as shown in Figure 1(b). It is possible to observe that in low voltage regions, regardless of the chitosan concentration, the average fiber diameter increased. However, the increase of the two factors caused a decrease in the responses, as already observed in the model, probably because of the significance of the term (X₁X₄).

The desirability function was used to optimize the influence of the production variables on the process of obtaining PVA/CS nanofibers, seeking to minimize the mean diameter and standard deviation. Figure 3 shows the conditions

obtained after the test application, in which the smallest diameter and standard deviation nanofibers were obtained by optimizing all independent variables. The conditions for lower mean fiber diameter and lower standard deviation by desirability function were Applied Voltage = 13.1 kV, Chitosan concentration = 30% and distance tip-to-plate = 10 cm. As the flow rate was not statistically significant, it was kept at the center point value (0.5 mL/h). The predicted values for mean fiber diameter and the standard deviation were calculated based on the obtained model. The observed values for mean fiber diameter and standard deviation were (196.5 ± 28.3) nm, compared to the predicted values of (185.9 ± 26.8) nm. The error was less than 5% and acceptable.

Since PVA is water soluble, a simple contact with water can immediately destroy PVA/CS mats (Figure 4(a)) compromising their wound dressing application as shown in Figure 4(b). A crosslink treatment was performed to guarantee water resistance, maintaining the nanofibrous structure with contact with water. After a 24 h treatment with GTA, water stability was increased. Glutaraldehyde treatment was able to increase stability in water, regardless of exposure time, as shown in Figure 4(c) and Figure 4(e). After a 24 h treatment, it was possible to observe a decrease in nanofiber diameter to 126.5 ± 25.7 nm. A 35.5% reduction compared to fiber diameter without crosslinking. Regarding morphology, it can be observed that after 1 h of immersion, the fibrous morphology was not lost; however, there was swelling of the nanofibers with visible coalescence that prevented the correct measurement of the average diameter (Figure 4(d)). The 48 h treatment (Figure 4(e) and Figure 4(f)) showed an even larger diameter reduction, 44.8% (108.4 ± 15.4) nm, with fibers slightly altered after exposure to water,

with an average diameter of (128.4 ± 22.4) nm, maintaining its homogeneous appearance, confirming the efficiency of the vapor-phase crosslinking reaction by GTA.

FTIR spectra were used to assess structural changes that occurred after crosslinking with GTA. Figure 5(a) shows the spectra before (trace I) and after crosslinking (trace II—24 h and trace III—48 h), and it is possible to observe that the position of most bands remained the same; only their intensities were changed. In trace I, the broad band observed in the range 3650 cm⁻¹ to 3000 cm⁻¹ resulted from O−H stretching of PVA and O–H and N–H stretching from CS. The two bands in the 3000 cm⁻¹ to 2750 cm⁻¹ range were attributed to asymmetrical and symmetrical C−H stretching. The band at ~1740 cm⁻¹ was assigned to C=O stretching of nonhydrolyzed vinyl acetate groups [45]. The absorptions at 1658 cm⁻¹ and 1590 cm⁻¹ may be attributed, respectively, to the amide I (C=O stretching) and amide II (C‒N stretching and C‒N‒H bending vibrations) from CS. After exposure to GTA (trace II and trace III), competitive and parallel reactions may occur between GTA and hydroxyl and amine groups present in PVA and chitosan. The appearance of a doublet at the C−H region, as well as the increase in intensity of the band associated to C−H bending at ~1459 cm⁻¹ may be attributed to the introduced crosslinking agent in the chitosan phase, when a vapor phase GTA crosslinking procedure was applied [46] [47] [48] [49].

The crystallinity of uncrosslinked and crosslinked 70:30 PVA/CS mats were evaluated by X-ray diffraction (Figure 5(b)). According to the literature, the characteristic chitosan crystalline peaks are not visualized when the PVA composition is high in PVA/CS mats [46]. For the uncrosslinked mat (Figure 5(b), trace I), the only maximum is in the 19˚ - 20˚ (2θ) region, and was related to the combined 101 and 200 reflections of semicrystalline PVA [50]. The intensity of this characteristic PVA reflection decreased significantly for the crosslinked mat (Figure 5(b), trace II and III). Therefore, the degrees of crystallinity found were 22.5% for the uncrosslinked and 9.5% and 4.7% for the 24 h and 48 h crosslinked materials, respectively. This behavior is in accordance with the formation of crosslinks, which hinder the mobility of PVA molecules, and this phenomenon depends on the exposure time to GTA.

Dynamic mechanical analyses were carried out for uncrosslinked and crosslinked optimized PVA/CS mats. The variation of the loss factor (tanδ), given by the ratio between loss modulus (E’’) and storage modulus (E’) values, as a function of temperature are shown in Figure 6(a), within the –60˚C to 310˚C range. This factor is used as an indicator of stiffness or flexibility. In Figure 6(a), the peak at 10.5˚C, observed for the uncrosslinked sample, was attributed to the γ relaxation of CS. No significant change was observed for the 24 h crosslinked mat. The peak at 110.3˚C, observed for the uncrosslinked mat may be attributed to the PVA α relaxation, associated with its dynamic glass transition temperature (T_g), and to encompass the β relaxation peak for CS [51]. At a higher temperature, the peak with its maximum at 201.8˚C may be attributed to the α transition of CS. The presence of two α relaxations for the PVA/CS uncrosslinked mat revealed lack of miscibility between PVA and CS. On the other hand, for the crosslinked samples only one broad peak was observed in the ~60˚C to 235˚C temperature range for both crosslinked products. However, it is possible to observe a shift of the tanδ peak with increasing exposure time to GTA. The increase in tanδ observed for the product obtained after 48 h indicates a higher crosslinking density. In the crosslinked structure, a higher crosslinking density means a shorter distance between molecular chains. Such an arrangement forces the segments of the chains to be in a more intimate contact improving energy dissipation [52] [53].

Figure 6(b) shows the Tensile tests results for the mats. For the uncrosslinked mat, the lowest Young’s modulus, E = (153.5 ± 12.8) MPa, and tensile stress at break, σ_max = (6.6 ± 0.3) MPa are attributed to the brittle character of chitosan. However, the highest strain at break, ε_max = (10.1 ± 1.3)%, is assigned to the flexibility of PVA chains of this PVA-richest composition. For the 24 h and 48 h crosslinked mats, enhanced properties were observed in both treatments and the results are summarized in Table 4. The decrease in elongation at break was assigned to the reduction in stretchiness after crosslinking reactions, which involved –OH and −NH₂ groups of PVA and CS.