2.5. In Vitro Drug Release and Skin Permeation

The in vitro drug release and skin permeation were determined using a Franz diffusion cell (PermeGear, Hellertown, PA, USA), as previously described [33]. The final total drug concentration in the DST-NLCs was 0.29 mg/mL. The total dutasteride content of 250 uL of DST-NLCs or DST-NLCs coated with CSO-LA in 5 mL of 2% SDS in PBS at pH 7.4 was measured using HPLC and found to be approximately 67 and 61 µg, respectively. For the release study, a 0.45 µm HA nitrocellulose membrane (MF™ Membrane Filters, Merck Millipore, Cork, Ireland) and 0.29 mg/mL of dutasteride in 70% ETOH were applied. For the permeation study, pig ear skin (freshly slaughtered pigs for food consumption from Farnborough, UK) was used as shown in Figure 1. The ears were washed with deionised water, and the hair was carefully cut with scissors. The subcutaneous tissue was removed. The average thickness of the skin was ~0.5 mm, which was cut and frozen (−20 °C) for later use. The Franz diffusion cell (surface area = 0.64 cm²) was maintained at 37 °C and stirred using a magnetic stirrer (600 rpm). Due to the very low solubility of dutasteride in water (0.038 ng/mL), for the release study 2% sodium dodecyl sulfate (SDS; required to ensure sink conditions as determined in preliminary experiments, data not shown) in phosphate-buffered saline (PBS 7.4) with the addition of 0.02% sodium azide for the permeation study was used. DST-NLCs (250 µL), uncoated and coated with 5% CSO-LA, were pipetted in the donor chamber. Samples (200 µL) were taken at 0, 0.25, 0.5, 1, 2, 3, 6, 8, 12, 24, 30, 36, and 48 h from the receptor chamber, with the medium replaced with 200 µL of fresh buffer. The samples were injected into the HPLC to quantify the drug released.

Diagram of the Franz diffusion cell used for the in vitro drug release and permeation studies.

The skin was placed between the donor and receptor chambers to assess the in vitro permeation and was allowed to rest for 1 h. Then, 200 μL samples were taken from the receptor chamber at 0, 0.25, 0.5, 1, 2, 3, 6, 8, 12, 24, 30, 36, and 48 h, and replaced with 200 μL of fresh buffer. The formulation in the donor compartment was carefully collected using a pipette tip and a tissue paper. A total of 2 mL of ethanol was used to collect the remaining formulation in the donor compartment and to rinse the upper part of the skin. The stratum corneum was removed after 48 h using adhesive tape (25 mm × 20 mm; 3 M Scotch magic™ Tape, Saint Paul, MN, USA) 10 times. The tapes were collected and 1 mL of ethanol was applied, following bath sonication for 1 h. Using a scalpel, the epidermis/dermis was cut into small pieces and added to 0.5 mL ethanol for 24 h and sonicated for 1 h. A 0.22 μM polyethersulfone (PES) membrane syringe filter (Millex^® GP, Merck Millipore, Cork, Ireland) was used to filter the solutions before injection into the HPLC for drug quantification.