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

Abstract

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).

Keywords: PMVEMA; TRPM8; electrospinning; menthol; microfibers; nanofibers; viability.

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 non-loaded and menthol-loaded fibers, respectively). Best-fit adjustments to a Gaussian distribution are also correspondingly indicated with a line.

Figure 2

Fourier transform infrared spectroscopy (FTIR) spectra of menthol (red line), F₁₃₉s (blue line), and menthol-loaded F₁₃₉s (green line) in KBr pellets. Evidence of the encapsulated menthol, including the new IR bands, is indicated in circles.

Figure 3

Stability over time of the menthol content in loaded F₁₁₉s and F₁₃₉s. The amount of menthol in acetone-dissolved fibers was analyzed by gas chromatography and mass spectrometry (GC-MS) at 0-, 3- and 10-days post fiber preparation. The data are represented in percentages relative to the amount of menthol initially added. Results are shown as the mean with standard deviation from three independent experiments performed in triplicate. Statistical analysis comprised two-way ANOVA corrected with Tukey’s test. ***, p < 0.001.

Figure 4

Viability of HEK293-hTRPM8 and HaCaT cells treated with F₁₁₉s experimental samples. Treatments include a gradient of menthol concentrations and equivalent dilutions of F₁₁₉s, menthol-loaded F₁₁₉s, and acetone. Cell viability is calculated in percentage relative to non-treated control cells from the optical densities (570–620 nm) obtained at 24 h post treatment by the Thiazolyl Blue Tetrazolium Bromide (MTT) method. 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.

Figure 5

Activation of TRPM8 by the menthol contained in electrospun F₁₁₉s and F₁₃₉s in HEK293-hTRPM8 cells. All data correspond to intracellular Ca²⁺ measurements and are represented as mean percentages with standard deviation of the maximum response observed for each treatment to that of 1000 µM menthol (n = 3). (a) Representative dynamics of the intracellular Ca²⁺ in response to different menthol concentrations. (b) Dose–response curve to menthol. (c,d) Representative dynamics of the intracellular Ca²⁺ in response to different concentrations of menthol encapsulated in F₁₁₉s and F₁₃₉s, respectively. (e) Intracellular Ca²⁺ increases recorded in response to 250, 83.3, and 25 µM of free and encapsulated menthol, including as controls: samples without menthol and 0.05% acetone (Acet.), and the specific TRPM8 blocker AMTB at 10 µM against 83.3 µM menthol samples. Statistical analysis calculated from the datasets in (e) 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.