In Vitro Permeability Assessment of AA

A preliminary screen of the effects of individual components on the permeability of AA utilized an in vitro artificial skin permeability assay (Strat-M^®; EMD Millipore, Temecula, CA, USA). The artificial membrane served as a barrier between the donor and receptor compartment of a Franz diffusion cell system with a diffusion area of 0.785 cm² (Labfine, Gunpo-si, Republic of Korea). The receptor compartment was filled with 5 mL phosphate buffer saline (PBS, pH 7.4), which was assumed to maintain a sufficient sink condition, with a solubility of 1.10 ± 0.01 mg/mL. Indeed, the PBS solubility level was 11.0-fold higher than the maximum drug concentration (100 µg/mL) maintained in receptor compartments with 100% permeability. The receptor solution was stirred at 600 rpm using a magnetic stirrer and the membrane surface was maintained at 32 °C with a heating system during the experiments. After 30 min of equilibration, 50 µL of 1% (w/w) AA in ethanol or AA–TFs (all equivalent to 500 µg AA) was added to the donor chamber (0.785 cm² permeation area). The receptor solution (500 µL) was collected at predetermined time points (0, 1, 2, 3, 4, 5, 6, 8, 10, and 24 h) and the equivalent volume of fresh PBS (pH 7.4) was used to refill the receptor compartment. The collected samples were filtered with a polyvinylidene fluoride (PVDF) membrane (0.45-µm pore size). The cumulative amount of AA that permeated the artificial membrane was determined using an HPLC system with 254 nm detection, as previously described.