2.6. In Vitro Skin Permeation Studies

Pig ears were gently cleaned and excised upon reception. The skin was separated from the porcine ear by carefully removing the connecting tissues with a surgical scalpel. After removing most of the subcutaneous fat, the skin was placed between two even plates, which were clamped together and cooled to −20 °C in a freezer. The frozen skin was removed from the freezer and the plate covering the stratum corneum taken off. A dermatome was used to cut skin strips with a thickness of 1 mm, the obtained strips were cut to circular disks using a die punch [19,20]. Subsequently, the integrity of the samples was assessed by visual inspection and by checking the tightness of the skins mounted onto jacketed franz diffusion cells (SES Analysensysteme, Bechenheim, Germany). Skin samples were stored at −20 °C and thawed at room temperature before use.

The prepared skin was placed between the donor and acceptor compartment of the cells, a pinch clamp secured both parts of the cells [19,21]. The acceptor compartment was filled entirely with PBS containing 0.5% γ -cyclodextrin. Initially, 2 mL of the acceptor solution were placed on top of the skin. The system was maintained at 32 ± 0.5 °C [19] and continuously stirred with a magnetic bead for 1 h to equilibrate the skin. Stirring was performed to maintain a constant hydrodynamic flow of the acceptor solution. After equilibration, the top side of the skin was dried and a DIA was placed upon the skin surface. After 1, 2, 4, 6, 9, 24, and 28 h, 2 mL of acceptor fluid were withdrawn through the sampling port and replaced with fresh acceptor solution, avoiding any air bubble under the skin.

Drug concentrations were determined by HPLC, and the amount of DMSO was determined by GC. Experiments were performed in triplicate. Flux J_SS [µg/h·cm²] is constant within the steady-state period and was calculated by linear regression analysis from the slope of the plot of cumulative E2/DMSO permeated per cm² of skin (dQ) against the time (dt). The lag-time is the intercept of J_SS with the time axis.

Following the permeation experiments, skin samples were cut into thin horizontal slices with a cryo-microtome (SLEE medical, Mainz, Germany). Acetone and methanol were added to the slices to extract the penetrated drug amount. The mixture was homogenized and centrifuged; the supernatant was assayed via HPLC.