2.12. In Vitro Permeation Testing

Cryopreserved, dermatomed human skin was obtained from a skin bank (New York FireFighters, New York, NY, USA). Prior to the experiment, the skin was thawed and cut into sections large enough to mount on vertical Franz diffusion cells with a diffusion area of 0.64 cm² (PermeGear, Philadelphia, PA, USA). The receptor chamber was filled with 10 mM PBS (pH 7.4), and the diffusion apparatus was temperature-controlled in order to maintain the surface skin temperature at around 32 °C. The thickness of dermatomed human skin used for IVPT studies was 400–500 µm.

The barrier integrity of the skin was evaluated by measuring the electrical resistance before the application of the formulations at a frequency of 100 Hz and a low voltage of 100 mV. Skin specimens in which the resistance was found to be acceptable were used for further permeation studies [23]. A finite dose of 10 mg/cm² of the formulation was added to the donor compartment. Sink conditions were maintained throughout the experiment. After 24 h, the excess formulation was wiped off with a cotton swab. Any formulation remaining on the skin surface was also removed by tape stripping (CuDerm Corp., Dallas, TX, USA) by using up to 2 consecutive strips. The remaining stripped skin, including the epidermis and dermis, was collected separately for analysis. The cotton swabs, the tape strips, and stripped skin were placed in a vial containing 10 mM PBS pH 7.4, and the mixtures were stirred overnight in order to ensure adequate drug extraction.