2.7. In Vitro Skin Permeation Study

Skin permeation was measured using stillborn porcine skin via the Franz diffusion technique [20]. Full-thickness skin from the dorsal area of stillborn porcine skin was used in this study. The subcutaneous layer was then carefully trimmed off and rinsed with PBS (pH 7.4). The thickness of the porcine skin was 1.55 ± 0.14 mm. Franz diffusion cells with a diffusion area of 2.00 ± 0.42 cm² were used to study in vitro skin permeation. A receptor chamber (12 mL) was filled with the mixture of PBS (pH 7.4) and absolute ethanol at a volume ratio of 1:1 (v/v) [10,14]. The Franz cells were controlled in a water jacket at 32 ± 2 °C. A 1.0 mL aliquot containing 0.25% capsaicin from freshly prepared chili extract-loaded nanoparticles compared with chili extract solution dissolved in 10% ethanol (as the control) was transferred to the donor chamber. Samples (0.8 mL) from the receptor fluid were collected and immediately replaced with fresh solution at 0, 1, 2, 3, 4, 6, 12, and 24 h. All samples from the receptor fluid were filtered through 0.45 µm pore size membrane filter and assayed using the HPLC system at 280 nm with 80 µL injection volume. The experiments were carried out in triplicate.

The results of permeation through porcine skin are displayed as the cumulative amount of capsaicin permeated (Q_n) per area (µg/cm²) versus time (hour), steady state flux (J_ss; µg/cm² h), and permeability coefficient (K_p; cm/h). The cumulative amount of capsaicin was calculated using Equation (4) [20].

where n is sample time point; V_r and V_s are the volume in the receiver chamber (mL) and the volume of the sample, respectively, collected at the n^th time point (mL); and C_r(n) is the concentration of the drug in the receiver chamber medium at the n^th time point (µg/mL).

The rate of capsaicin permeated through the porcine skin was calculated using the slope of the cumulative amount of capsaicin permeated versus time (h) plot. The lag time is the time intercept of the linear portion of the graph. The steady state flux of capsaicin was evaluated using Equation (5) [20].

where Q is the cumulative amount of capsaicin permeated and A is the surface area of porcine skin (2 cm²).

The permeability coefficient (K_p) of capsaicin and enhancement ratio (E_r) were calculated following Equations (6) and (7), respectively [16,20].

After a 24 h permeation study, skin was washed with deionized water and allowed to dry. Capsaicin concentrations in the stratum corneum (SC) were determined from repeated tape stripping with 20 pieces of adhesive tape using 3M Scotch Magic^TM tape (1 × 1 cm). The capsaicin concentration retained in the skin was calculated. After stripping the skin, the sample was cut into small pieces and analyzed for the capsaicin content. The mixture of PBS (pH 7.4) and absolute ethanol at 1:1 (v/v) was placed in 5 and 2 mL vials for the tape strips and the cut skin, respectively. Then, all samples were sonicated for 30 min and filtered. The filtrate of tape stripping and skin retention was analyzed using the HPLC system at 280 nm with 20 µL injection volume. For both the in vitro release kinetic and the in vitro skin permeation studies, 0.25% capsaicin in chili extract-loaded SLN and NLC formulations were prepared and evaluated in comparison with the chili extract solution containing 0.25% capsaicin in 10% ethanol in water and standard 0.05% capsaicin solution in ethanol.