2.5. In Vitro Skin Permeation

Human cadaver skin samples used in the study were via a donation, from a 57-year-old Caucasian female, obtained from Science Care (Phoenix, AZ, USA), without an identifier and, hence, exempted from ethical review. Dermatomed thigh skin (epidermis and partial dermis) with thickness of 250–300 µm was used for the skin permeation study. Vertical Franz diffusion cell was used for the study, with an effective diffusion area of 0.25 cm². Skin was placed between the donor and receptor compartment, with the SC side facing the donor compartment. A dose of 50 µL of proposome solution was added to the donor compartment and 4 mL of PBS (pH 7.4) was placed inside the receptor compartment to maintain sink condition. A brief discussion considering sink conditions can be found in Supplementary Materials S1, with the solubility data of each drug found in literature [30,31,32,33]. The system was placed under constant stirring of 250 rpm at 32 °C to mimic in vivo skin environment. Parafilm was used to cover both the donor compartment and sampling port to prevent evaporation throughout the experiment. Samples of 1 mL were collected from the receptor compartment at 1, 2, 4, 8, 12, and 24 h. After each sampling, 1 mL of fresh PBS was added to the receptor compartment to maintain constant volume. Samples were then centrifuged at 10,000 rpm for 5 min, and the supernatant was analyzed with an LC-20AD HPLC machine (Shimadzu, Kyoto, Japan). Control was also prepared by dissolving the same amount of drug in 30% PG solution without SPC.

After 24 h of skin permeation, the skin samples were removed and washed with PBS to remove the free drug on the skin surface. The skin sample was processed and analyzed using the method we reported previously [7,34]. Briefly, the washed skin samples were digested by proteinase K solution. Then the digested skin samples were processed and analyzed using HPLC.

The skin permeation results were processed to generate the following parameters: cumulative permeation (CP), and area under the curve (AUC) from proposomes. The AUC was employed to represent the total drug permeation through skin across time. The formulation process and drug molar concentrations were the same for all proposomes. Further, enhancement and enhancement ratio were calculated for CP and AUC of proposomes compared to CP and AUC of the control (free drug in 30% PG). The CP enhancement (EN_CP) (Equation (3)), CP enhancement ratio (ER_CP) (Equation (4)), AUC (Equation (5)), AUC enhancement (EN_AUC) (Equation (6)), and AUC enhancement ratio (ER_AUC) (Equation (7)) were calculated using the following equations:

where, Aproposome and AControl were the cumulative amounts permeated in 24 h for proposomes and control, respectively.

where, X was time and Y was the cumulative permeation at the time.

where, AUCproposome and AUCcontrol were AUC_0→24h for proposome and control respectively for 24 h.

The EN_CP presents the increase in absolute drug amount permeated through the skin from proposomes compared to the drug amount permeated from the free drug in 30% PG (control). The ER_CP is the ratio that shows the factor by which drug permeation is enhanced in proposomes compared to the control. The AUC presents the overall drug permeation profile over 24 h. The EN_AUC presents the increased AUC from proposomes compared to the control. The ER_AUC is the ratio that shows the factor by which the AUC is enhanced.