L-Menthol-Loadable Electrospun Fibers of PMVEMA Anhydride for Topical Administration

Poly(methyl vinyl ether-alt-maleic anhydride) (PMVEMA) of 119 and 139 molecular weights (P119 and P139, respectively) were electrospun to evaluate the resulting fibers as a topical delivery vehicle for (L-)menthol. Thus, electrospinning parameters were optimized for the production of uniform bead-free fibers from 12% w/w PMVEMA (±2.3% w/w menthol) solutions, and their morphology and size were characterized by field emission scanning electron microscopy (FESEM). The fibers of P119 (F119s) and P139 (F139s) showed average diameter sizes of approximately 534 and 664 nm, respectively, when unloaded, and 837 and 1369 nm when loaded with menthol. The morphology of all types of fibers was cylindrical except for F139s, which mostly displayed a double-ribbon-like shape. Gas chromatography-mass spectrometry (GC-MS) analysis determined that not only was the menthol encapsulation efficiency higher in F139s (92% versus 68% in F119s) but also that its stability over time was higher, given that in contrast with F119s, no significant losses in encapsulated menthol were detected in the F139s after 10 days post-production. Finally, in vitro biological assays showed no significant induction of cytotoxicity for any of the experimental fibers or in the full functionality of the encapsulated menthol, as it achieved equivalent free-menthol levels of activation of its specific receptor, the (human) transient receptor potential cation channel subfamily M (melastatin) member 8 (TRPM8).


Introduction
The efficacy of any topical treatment depends on the ability of the pharmaceutical formulation to deliver the required drug to the specific target location and thus to penetrate the different skin layers. Many studies describe the difficulty of crossing these barriers by diffusion, which usually reduces the final bioavailability of the drug [1][2][3]. Different carriers have been tested to overcome this limitation, as well as to stabilize compounds of hydrophobic and/or thermolabile nature that may volatilize or degrade by isomerization or oxidation, such as terpenes. In the particular case of this family of compounds, their topical administration for localized skin disorders also pursues avoidance of their unnecessary, and sometimes problematic, systemic distribution resulting from other administration routes. For all of these reasons, several topical delivery systems of terpenes for skin healthcare applications based on nanosystems have been tested, such as nanocapsules, nanoemulsions, nanogels, nanofibers, and liposomes, most of time complexed with cyclodextrins (CD) [4].
The cyclic monoterpene alcohol L-menthol (hereinafter referred to as menthol) is the major constituent of the essential oils of peppermint and corn mint, and it is well known for its beneficial health properties among other characteristics [5,6]. As a result, menthol can be found in a wide range of commercial products, such as pharmaceuticals, cosmetics, pesticides, and oral hygiene products and as a flavoring agent [7,8]. Additionally, by Merck, Sigma-Aldrich (Saint Louise, MO, USA). Double recrystallized menthol (CAS: 2216-51-5) (≥99%) was also purchased from Merck, Sigma-Aldrich. The solvents employed were high-performance-liquid-chromatography (HPLC) grade and consisted of dichloromethane, acetone, ethanol (Merck KGaA, Darmstadt, Germany), and methanol (VWR International, Radnor, PA, USA).

Electrospinning
The preparation of nanofibers was performed by using the methodology previously described by Mira et al. (2017) [40]. Briefly, the electrospinning process was carried out in a horizontal set-up, including a 2 mL Discardit II syringe (Becton Dickinson, Franklin Lakes, NJ, USA) from which the polymer solution was pumped through a blunt end stainless steel hypodermic needle 316 of 20 gauge (Merck, Sigma-Aldrich) at a flow rate controlled by a KDS 100 infusion pump (KD Scientific, Holliston, MA, USA). The needle and the aluminum foil collector located at a settled distance were connected to a Series FC high voltage source (Glassman High Voltage Inc., High Bridge, NJ, USA). After a refinement process, an electrospinnable solution containing 12% of PMVEMA in acetone, together with 2.3% w/w menthol for drug-loaded fibers, was selected. Subsequently, the rest of operational parameters were optimized to achieve the most uniform and smallest fibers. It is of note that hereinafter all concentration percentages are given in w/w.

Optical Microscopy
Samples were electrospun directly onto the aluminum foil collector on which a microscope slide (Deltalab, Barcelona, Spain) was previously placed. The structure of the electrospun nanofibers was observed by using an optical microscope (Microsystems DMI3000B: Leica, Bensheim, Germany) provided with a compact light supply (Leica EL6000), as well as with a digital camera (Leica DFC 3000G). The 40× objective was used to take the images in phase contrast. As for imaging processing, it was performed manually by using the Leica Application Suite AF6000 Module Systems software. By this methodology, preliminary screening of the electrospun nanofibers was carried out.

Scanning Electronic Microscopy
After the optimization of the electrospinning parameters, samples of selected nanofibers arranged onto a microscope slide covered with aluminum foil, were analyzed (US English) in a Shottky type field emission scanning electron microscope (FESEM) Sigma 300 VP model (Carl Zeiss Microscopy GmbH, Oberkochen, Germany) at low kV without coating. From the images obtained for each sample, 100 measurements of nanofiber diameters were taken and analyzed by using ImageJ software (National Institutes of Health, NIH, Bethesda, MD, USA).

Fourier Transform Infrared Spectroscopy (FTIR)
FTIR spectra were obtained by using a Bruker IFS66s spectrometer (Karlsruhe, Germany). Samples were prepared as KBr pellets at room temperature. For each spectrum, there were recorded 256 scans (sample and background) in the range of 4000-400 cm −1 and at a resolution of 2 cm −1 .

Gas Chromatography and Mass Spectrometry (GC-MS)
The menthol content in the fibers was estimated with a gas chromatography-mass spectrometer (GCMS-QP2010 SE) equipped with a quadrupole detector and an automatic sample injector (AOC-20i/s; Shimadzu, Kyoto, Japan). The GC column employed for the assays was an Agilent J&W capillary HP-5MS UI (30 m × 0.25 mm id, 5% diphenyl-95% dimethylpolysiloxane, film thickness 0.25 µm) (Agilent Technologies Inc., Santa Clara, CA, USA). The protocol employed for these analytical assays was based on a previous work described in the literature [43]. In brief, a temperature of 250 • C and 210 • C was Pharmaceutics 2021, 13, 1845 4 of 12 used for the injector and detector, respectively. The carrier gas used was helium at a flow rate of 1.5 mL·min −1 . As for the program, the initial temperature was 40 • C for 2 min. Then, this temperature was increased up to 240 • C at a rate of 10 • C·min −1 . Finally, the temperature was raised at a rate of 5 • C·min −1 and maintained for 5 min at 270 • C. Menthol calibration curves were created for each batch of experiments, with a concentration range of 50-800 µg·mL −1 . All values corresponded to the area under the curve obtained for each concentration of menthol. In this way, all the curves were preferably adjusted to a quadratic equation correlating the area with concentration, and R 2 coefficients were greater than 0.99 in all cases ( Figure S1).
The analyzed samples (5-10 mg) corresponded to three independent production batches of nanofibers loaded with menthol. Each sample was analyzed just after its preparation and at 3-and 10-days post-production. They were stored uncovered, but protected from light, at room temperature. Immediately prior to their analysis, samples were dissolved in acetone, filtered with 0.2 µm nylon membranes (Millipore, Bedford, MA, USA) and injected at a concentration between the quantifiable ranges of the corresponding calibration curves. In order to check the concentration of each sample before performing the electrospinning process, an aliquot of the initial solution was also analyzed.

Cell Culture
For the in vitro cell assays of this work, human embryonic kidney cells stably expressing hTRPM8 (HEK293-hTRPM8) [44] and the well-known immortalized human keratinocyte cell line HaCaT were used. Both cell lines were maintained in high glucose (4.5 g·L −1 ) Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% v/v fetal bovine serum (FBS), 100 U·mL −1 penicillin-streptomycin, and 2 mM L-glutamine (Gibco, Waltham, MA, USA). Cells were grown in 25 cm 2 flasks at 37 • C in a humidified 5% CO 2 air atmosphere.

Cytotoxicity Assay
The potential viability changes in HEK293-hTRPM8 and HaCaT cells induced by the electrospun nanofibers were determined by the Thiazolyl Blue Tetrazolium Bromide (MTT) assay, similar to our previous study [40]. For this task, cells at 90-100% confluence, seeded in 96-well plates 48 h before, were treated with different concentrations of each type of nanofiber (i.e., menthol-loaded and unloaded F 119 s and F 139 s) in cell culture media (100 µL per well). Previously, all the fiber samples were dissolved at 6.8% in acetone (Merck, Sigma-Aldrich) to create 75× concentrated sample stocks, which were stored at −80 • C until use. A 75 mM menthol stock solution in acetone was also included. Testing samples consisted of 2-fold dilutions in cell culture media starting from 1× sample stocks. Thus, the menthol calibration curve encompassed a concentration range spanning from 1000 to 7.8 µM, as for the samples of menthol-containing nanofibers, considering full encapsulation efficiencies. There were also included solvent control samples consisting of acetone concentrations equivalent to those in the corresponding sample dilutions. The acetone-dissolved F 139 s partially precipitated in aqueous solvent, i.e., cell culture media, and therefore their cytotoxic effects were only evaluated at the highest concentration of the planned range.
After 24 h of incubation, treatments were replaced with 0.5 mg·mL −1 of MTT (from previously prepared 5 mg·mL −1 stock solutions in phosphate-buffered saline (PBS) stored at −20 • C) (Merck, Sigma-Aldrich) in fresh cell culture media (100 µL per well) for 2 h. Then, the media was carefully removed, and the colored product formazan was, subsequently, dissolved in an aliquot of 100 µL of dimethyl sulfoxide (DMSO). Absorbance measurements were acquired at 570 nm in an absorbance microplate reader SPECTROstar ® Omega (BMG Labtech, Offenburg, Germany). The resulting values were correlated with cell concentration and expressed in percentage relative to the untreated cells (control group). Experiments were carried out in quadruplicate, and the results are expressed as means with standard deviation (SD) of three independent experiments.

TRPM8 Channel Activity Assays
Functional assays were performed in HEK293-hTRPM8 cells. TRPM8 activity was estimated by measuring the variation of intracellular Ca 2+ concentration elicited by menthol and by using the fluorescent Ca 2+ indicator Fluo4-NW (Molecular Probes, Invitrogen, Milan, Italy), as described in Bonache et al. (2020) [45]. Briefly, two days before treatment, cells were seeded in opaque 96-well plates with transparent bottom ( Subsequently, plates were placed into a POLARStar (BMG Labtech) plate reader for fluorescence detection to record 16 measurements (excitation at 494 nm and emission at 516 nm). After a 3-cycle baseline recording prior to stimulation, the experimental samples (prepared as described before for the cytotoxicity assays to reach the desired concentrations by adding 1 µL per well) were inoculated between the third and fourth cycle using a multichannel pipette. The fluorescence increase induced by the treatments was quantified by subtracting the fluorescence value at the third cycle to the maximum fluorescence value obtained after the addition of the experimental compounds. All data were normalized to the highest response elicited by 1000 mM menthol. The specificity of the assay was assessed by blocking the TRPM8 activity with 10 µM AMTB, a well-known TRPM8 antagonist [46]. All experiments were performed in triplicate, and results are shown as means with SD (n = 3).

Statistical Analysis and Graphics
Data were statistically analyzed by two-way ANOVA corrected with Tukey's test for multiple comparisons using Prism 7 (GraphPad software, San Diego, CA, USA). Prism 7 was also used for general data analysis and plotting.

PMVEMA Fibers Encapsulating Menthol
Due to the low solubility in water and the high volatility of menthol [47], we first proceeded by confirming its solubilization in organic solvents, particularly, dichloromethane, ethanol, and acetone. The menthol in these solutions was then identified and quantified by GC-MS ( Figure S1). Among the different PMVEMA-derived polymers to employ in electrospinning, due to our previous knowledge, our first attempts focused on the ester derivative, which is soluble and commercially available in ethanol. In this way, menthol-loaded nanofibers were obtained. However, their residual content in ethanol, as is shown in the GC-MS chromatograms ( Figure S2), could interact with the menthol receptor TRPM8 [48] and then interfere with the functional biological assays planned in this study. For this reason, we finally opted for the anhydride PMVEMA of two different molecular weights (P119 and P139), which are soluble in acetone. After testing several polymer concentrations, both of these PMVEMA forms yielded uniform linear fibers at 12%-a percentage that was subsequently selected for the further optimization of the electrospinning procedure. Along this line, a concentration of 2.3% of menthol was also chosen in order to ideally reach 16% of this compound in the resulting solvent-free fibers, a moderately high bioactive concentration with no harmful effects [20].
Thus, using the selected concentrations and after testing several experimental conditions, the optimized parameters to produce reproducible uniform fibers without beads were 15.5 kV of voltage, 10 cm of distance between electrodes, and a flow rate of 0.25 mL·h −1 (Figure 1). spinning procedure. Along this line, a concentration of 2.3% of menthol was also chosen in order to ideally reach 16% of this compound in the resulting solvent-free fibers, a moderately high bioactive concentration with no harmful effects [20].
Thus, using the selected concentrations and after testing several experimental conditions, the optimized parameters to produce reproducible uniform fibers without beads were 15.5 kV of voltage, 10 cm of distance between electrodes, and a flow rate of 0.25 mL·h −1 (Figure 1).

Figure 1.
Representative field emission scanning electron microscopy (FESEM) images (left) and frequency distribution histograms of the diameter (right) of electrospinning-optimized F119s and F139s. Non-loaded fibers in main images and menthol-loaded ones inside the green-circle insets (scale bars: 5 μm). Each histogram was performed with the data obtained from several images (at least seven) until reaching 100 measurements (white and green bars correspond to nonloaded and menthol-loaded fibers, respectively). Best-fit adjustments to a Gaussian distribution are also correspondingly indicated with a line.
Assuming cylindrical morphologies, the histogram of the diameter measurements obtained by FESEFM for each type of fiber (100 measurements each) was best-adjusted to a Gaussian distribution with the following parameters (average size ± SD in nm, amplitude, and R 2 , respectively): 524 ± 189, 20.8, and 0.976 for (empty) F119s; 837 ± 275, 14.4, and 0.765 for menthol-loaded F119s; 664 ± 111, 36.4, and 0.977 for (empty) F139s; and 1369 ± 339, 11.8, and 0.788 for menthol-loaded F139s. In general, it was observed that the presence of menthol increased the diameter of the fibers, but a further analysis of these histograms also revealed that the non-loaded fibers presented a typical monomodal distribution, while the loaded ones were distributed in two populations. The most obvious explanation for this observation is a heterogenous distribution of the loaded menthol, but in the case Each histogram was performed with the data obtained from several images (at least seven) until reaching 100 measurements (white and green bars correspond to non-loaded and menthol-loaded fibers, respectively). Best-fit adjustments to a Gaussian distribution are also correspondingly indicated with a line.
Assuming cylindrical morphologies, the histogram of the diameter measurements obtained by FESEFM for each type of fiber (100 measurements each) was best-adjusted to a Gaussian distribution with the following parameters (average size ± SD in nm, amplitude, and R 2 , respectively): 524 ± 189, 20.8, and 0.976 for (empty) F 119 s; 837 ± 275, 14.4, and 0.765 for menthol-loaded F 119 s; 664 ± 111, 36.4, and 0.977 for (empty) F 139 s; and 1369 ± 339, 11.8 and 0.788 for menthol-loaded F 139 s. In general, it was observed that the presence of menthol increased the diameter of the fibers, but a further analysis of these histograms also revealed that the non-loaded fibers presented a typical monomodal distribution, while the loaded ones were distributed in two populations. The most obvious explanation for this observation is a heterogenous distribution of the loaded menthol, but in the case of F 139 s, the size of which was the most variable, the addition of menthol showed to profoundly affect their morphology by shaping double ribbon-like structures with distinct sizes depending on the observed dimension (Table S1).
Such an effect on the morphology of the loaded fibers may have been due to powerful Van der Waals interactions, mainly hydrogen bonds between the hydroxide group of menthol and the carbonyl groups of the anhydride. This effect appears to be reflected in the FTIR spectrum of the loaded F 139 s (Figure 2), which shows a slight shift (5 cm −1 ) of the two original anhydride bands (1851 and 1777 cm −1 ), as well as a correspondence with the changes observed between 3300-2500 cm −1 (typical OH stretch). The presence of the encapsulated menthol was confirmed by new IR bands in the fingerprint region (1000-500 cm −1 ) and in the saturated alkyl region (less than 3000 cm −1 ).
of F139s, the size of which was the most variable, the addition of menthol showed to profoundly affect their morphology by shaping double ribbon-like structures with distinct sizes depending on the observed dimension (Table S1).
Such an effect on the morphology of the loaded fibers may have been due to powerful Van der Waals interactions, mainly hydrogen bonds between the hydroxide group of menthol and the carbonyl groups of the anhydride. This effect appears to be reflected in the FTIR spectrum of the loaded F139s (Figure 2), which shows a slight shift (5 cm −1 ) of the two original anhydride bands (1851 and 1777 cm −1 ), as well as a correspondence with the changes observed between 3300-2500 cm −1 (typical OH stretch). The presence of the encapsulated menthol was confirmed by new IR bands in the fingerprint region (1000-500 cm −1 ) and in the saturated alkyl region (less than 3000 cm −1 ).

Determination of the Loaded Content and Its Stability over Time
For determining both the encapsulation efficiency and stability over time of the menthol loaded into the experimental fibers, the integrating compounds were separated, and the menthol was identified and quantified by GC-MS (see Figures S1 and S3 for further details on the calibration data). Thus, the menthol content in both F119s and F139s was analyzed right after their preparation 0, 3, and 10 days after that (Figure 3). The amount of menthol in the polymeric solutions prior to their electrospinning was also quantified to confirm its presence and as reference value (i.e., 100% of menthol content). By following this procedure, the resulting encapsulation efficiencies were 68 ± 2% and 92 ± 2% for F119s and F139s, respectively. Regarding the content stability over time, F119s showed a significant menthol decrease of about 20% at 10 days post preparation. In contrast, the menthol content in F139s remained stable throughout the testing period.

Determination of the Loaded Content and Its Stability over Time
For determining both the encapsulation efficiency and stability over time of the menthol loaded into the experimental fibers, the integrating compounds were separated, and the menthol was identified and quantified by GC-MS (see Figures S1 and S3 for further details on the calibration data). Thus, the menthol content in both F 119 s and F 139 s was analyzed right after their preparation 0, 3, and 10 days after that (Figure 3). The amount of menthol in the polymeric solutions prior to their electrospinning was also quantified to confirm its presence and as reference value (i.e., 100% of menthol content). By following this procedure, the resulting encapsulation efficiencies were 68 ± 2% and 92 ± 2% for F 119 s and F 139 s, respectively. Regarding the content stability over time, F 119 s showed a significant menthol decrease of about 20% at 10 days post preparation. In contrast, the menthol content in F 139 s remained stable throughout the testing period.
The loss of menthol appeared to be mostly related to the different molecular weights of the polymers employed rather than to differences in the relative surface area of the materials, which was notably higher in F 139 s (Figure 1). The containment stability of F 139 s ribbons could also be due to the above-mentioned molecular interactions between menthol and the maleic anhydride groups of P 139 (Figure 2), which were more numerous than in P 119 . In addition, the volatile nature of the encapsulated compound contributed negatively to its retention if other forces did not intervene in the other way, as we already observed in a previous work when encapsulating methyl salicylate [41].

Cytotoxicity Induced by Experimental Electrospun Nanofibers In Vitro
The potential toxicity induced in cells when treated with the electrospun fibers of this work was also evaluated. For these assays, HEK293-hTRPM8 and HaCaT cells were incubated for 24 h with different concentrations of the experimental samples previously dissolved in acetone. The concentrations used ranged from 7.8 to 1000 µM of menthol (for its calibration curve and menthol-loaded F 119 samples), testing equivalent amounts of non-loaded F 119S samples.  The loss of menthol appeared to be mostly related to the different molecular weights of the polymers employed rather than to differences in the relative surface area of the materials, which was notably higher in F139s (Figure 1). The containment stability of F139s ribbons could also be due to the above-mentioned molecular interactions between menthol and the maleic anhydride groups of P139 (Figure 2), which were more numerous than in P119. In addition, the volatile nature of the encapsulated compound contributed negatively to its retention if other forces did not intervene in the other way, as we already observed in a previous work when encapsulating methyl salicylate [41].

Cytotoxicity Induced by Experimental Electrospun Nanofibers In Vitro
The potential toxicity induced in cells when treated with the electrospun fibers of this work was also evaluated. For these assays, HEK293-hTRPM8 and HaCaT cells were incubated for 24 h with different concentrations of the experimental samples previously dissolved in acetone. The concentrations used ranged from 7.8 to 1000 μM of menthol (for its calibration curve and menthol-loaded F119 samples), testing equivalent amounts of nonloaded F119S samples.
As shown in Figure 4, the increase in sample concentration implied a decrease in cell viability in both cell lines. However, no significant differences were found in any of the cell lines between the treatments used, including the solvent control with acetone, suggesting that the observed toxicity was primarily induced by the vehicle (two-way ANOVA, p = 0.8763 and p = 0.9383 for HEK293-hTRPM8 and HaCaT cells, respectively). Such effect was more obvious in the HEK293-hTRPM8 cell line, which exhibited the lowest cell viability levels for all sample types at the highest dose used (1000 μM, 37.8-67.2%). HaCaT cells were much less sensitive to the cytotoxic effect caused by the treatments, and in none of the cases did the viability levels drop below ~80%. Regarding F139s, they were only tested at the highest concentration of the range, displaying comparable results with those of the F119s (data not shown). As shown in Figure 4, the increase in sample concentration implied a decrease in cell viability in both cell lines. However, no significant differences were found in any of the cell lines between the treatments used, including the solvent control with acetone, suggesting that the observed toxicity was primarily induced by the vehicle (two-way ANOVA, p = 0.8763 and p = 0.9383 for HEK293-hTRPM8 and HaCaT cells, respectively). Such effect was more obvious in the HEK293-hTRPM8 cell line, which exhibited the lowest cell viability levels for all sample types at the highest dose used (1000 µM, 37.8-67.2%). HaCaT cells were much less sensitive to the cytotoxic effect caused by the treatments, and in none of the cases did the viability levels drop below~80%. Regarding F 139 s, they were only tested at the highest concentration of the range, displaying comparable results with those of the F 119 s (data not shown).  Results are shown as the mean with standard deviation from three independent experiments performed in quadruplicate. Statistical analysis comprised two-way ANOVA corrected with Tukey's test for multiple comparisons, but no significant differences were found between the treatment groups at each concentration.

Assessment of the Activation Capacity of TRPM8 by the Encapsulated Menthol
The menthol encapsulated into the experimental fibers after being dissolved in acetone, as for the cytotoxicity assays, induced intracellular Ca 2+ increases in a concentrationdependent fashion in treated HEK293-hTRPM8 cells ( Figure 5). First, TRPM8 activation Results are shown as the mean with standard deviation from three independent experiments performed in quadruplicate. Statistical analysis comprised two-way ANOVA corrected with Tukey's test for multiple comparisons, but no significant differences were found between the treatment groups at each concentration.

Assessment of the Activation Capacity of TRPM8 by the Encapsulated Menthol
The menthol encapsulated into the experimental fibers after being dissolved in acetone, as for the cytotoxicity assays, induced intracellular Ca 2+ increases in a concentrationdependent fashion in treated HEK293-hTRPM8 cells ( Figure 5). First, TRPM8 activation dynamics were determined for our experimental model using free menthol (Figure 5a,b), describing a dose-response curve as in previous works [49].  Follow-up studies showed that the menthol contained in both types of fibers was able to activate TRPM8 similarly, and also in a concentration-dependent manner (Figure 5c,d for F 119S and F 139S samples, respectively). The vehicle (0.05% acetone) did not induce Ca 2+ internalization (Figure 5c-e), nor did the fiber samples without menthol (data not shown). TRPM8 activation was specific, as the observed Ca 2+ increase in cells was abolished with the TRPM8 blocker AMTB (Figure 5e). In Figure 5e, the representation of the highest Ca 2+ internalization values induced by different concentrations of menthol shows equivalent levels of TRPM8 activation between the free and the encapsulated compound, indicating no significant modifications in the loaded menthol after performing the electrospinning process. The concentrations chosen for this assay were 250, 83.3, and 25 µM, which were all comprised within the linear region of the dose-response menthol calibration curve.

Conclusions
In this work is described a procedure for obtaining uniform, beadless electrospun nano/microfibers loadable with 16% of the bioactive model terpene menthol without using CD, but with two PMVEMA derivates differing only in their molecular weights (F 119 s and F 139 s). All the resulting non-loaded fibers showed uniform, beadles, and cylindrical morphologies. However, when loaded with menthol, such difference in polymer molecular weight affected the obtained fibers by shaping ribbon-like F 139 s endowed with much higher encapsulation efficiency and content stability over time than the cylindrical-loaded F 119 s. Regarding their interaction with cells, none of the two types of fibers showed significant levels of toxicity in HaCaT and HEK293-hTRPM8 cells treated for 24 h in the range of concentrations used. Finally, the full functionality of the menthol encapsulated into both types of fibers was also verified in vitro, as it achieved TRPM8 activation levels equivalent to those of the free (i.e., non-manipulated) menthol.

Supplementary Materials:
The following are available online at https://www.mdpi.com/article/10 .3390/pharmaceutics13111845/s1, Figure S1: Procedure to quantify menthol by GC-MS, Figure S2: Representative GC-MS spectra of PMVEMA-ES 25% w/w and PMVEMA-ES 25% w/w with L-menthol 16% w/w fibers, Figure S3: GC-MS chromatograms of the samples corresponding to the stability experiment, Table S1: Data of histograms obtained for optimized fibers with and without menthol.

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