Ex vivo porcine skin permeation study

The ability of DOCTOR to deliver DOX and CPT inside the skin was assessed via ex vivo porcine skin permeation studies using protocols reported previously (39). Briefly, full-thickness porcine skin samples (Lampire Biological Laboratories, Pipersville, PA) stored at −80°C were defrosted, and hair was trimmed before use. The pieces were washed with PBS (pH 7.4), and resistivity was measured to ensure that samples with an intact barrier were used for the study. The permeation rates were assessed in Franz diffusion cells (penetration area, 1.77 cm²; receptor volume, 12.0 ml) as described here. The receptor compartment was filled with PBS (pH 7.4), and skin samples were mounted with the stratum corneum (SC) facing up. Care was taken to ensure that the donor compartment was dry and left open to air for 30 min. Extra caution was taken to remove air bubbles between the skin’s base and the receptor solution. Lyophilized DOCTOR was dissolved in DI water (pH 5.0) and then diluted in the same solvent to obtain a dose of 0.5 mg/ml or 920 μM DOX in DOCTOR. This formulation (200 μl) was applied topically on the skin surface inside the donor compartment and incubated for 24 hours at 37°C under occlusive conditions with moderate stirring (200 rpm) in the acceptor compartment. The controls and treatments for the study were assessed in triplicates. At 24 hours, the skin surface was washed at least five times with PBS (pH 7.4) to remove any excess drugs. The skin samples were then cryosectioned for confocal microscopy analysis or subjected to tape-stripping studies for drug quantification purposes.

For quantifying skin permeation of the applied DOCTOR, the skin sections were retrieved after incubation for 24 hours and rinsed with PBS. Each section was then subjected to the tape-stripping method as described. For this, an adhesive tape (Scotch Transparent Tape, 3M Corporate, St. Paul, MN) was used to separate the upper stratum corneum from the lower stratum corneum and the epidermis. Ten consecutive tape strips were performed to remove the upper stratum corneum. The lower stratum corneum combined with the epidermis of each skin section was then separated from the dermis with a sterile surgical scalpel and placed in the same glass vial. The dermis obtained from each section was then cut into small pieces and placed in different glass vials. For drug extraction, each separated skin layer was incubated with 3 ml of 50% methanol/PBS mixture. To determine the amount of drug that passed completely through the length of the skin, 3 ml of the acceptor chamber solution was mixed with 3 ml of methanol/PBS. All vials were shaken overnight at room temperature. The solutions were then centrifuged to remove the skin remnants. The supernatants collected were used to measure the concentration of DOX and CPT using their distinct fluorescence spectra at Ex/Em 470/590 and 370/448 nm, respectively.