Franz type diffusion cells

Microemulsion penetration was investigated using Franz-type diffusion cells (PermeGear Inc., Hellertown, PA, USA) with a diffusion area of 1 cm² and an acceptor compartment of 8 mL containing fetal bovine serum and PBS (pH 7.4) (1 : 9, v/v). Skin was mounted on Franz-type diffusion cells, epidermal side up, and dermal side facing the receptor compartment. Diffusion cells were kept at 32°C. 100 μL of different treatments (the final curcumin concentration was 1%w/w (27.1 mM)) was applied to the mounted skin. Following 24 h incubation, skin was removed and washed three times using a cotton cloth containing ethanol and the viable epidermis was separated from the dermis. Separation of the full epidermis from the dermis was achieved by heat shock treatment; skin was placed for 30 seconds at 55–60°C followed by 1 min at 4°C, both in PBS. Curcumin was extracted from the separated layers with DMSO. The extraction was performed by incubation in a shaker (60 ×g) until all curcumin were released (24 h). Finally, 100 μL from the receptor fluids was collected. Curcumin existence in skin layers and in the acceptor compartment was determined by measuring fluorescence excitation at 485/40 nm and emission at 528/20 nm, using a BioTek microplate reader (BioTek Instruments Inc., Winooski, VT).

In vitro permeation experiments were performed by means of Franz-type diffusion cells (Perme Gear, Pensilvania USA) with an effective diffusion area of 0.75 cm², using ear skin excised from pigs provided by a local slaughterhouse (Granada, Spain). After careful removal of the subcutaneous fat, the skin was cut into squares of 3 × 3 cm and stored at −20 °C until use. To increase reproducibility, skin was pre-hydrated with PBS for 30 min before starting experiments. The skin was positioned with the stratum corneum facing the donor phase, between the donor and the receptor compartments of the Franz cell. The receptor chamber was filled with a mixture of PBS (pH 7.4) and ethanol (1:1 v/v) previously degassed and continuously stirred by a magnetic bar. Each hydrogel (100 mg) was placed onto the skin, thermostated at 37 °C to obtain the physiological skin temperature (32 °C). After 24 h, the skin surface was washed, and the stratum corneum (SC) was separated by stripping with adhesive tape (Strappal^®, Barcelona, Spain). The adhesive tape was firmly pressed on the SC and pulled off with a rapid and fluent stroke. The epidermis was separated from the dermis with a surgical scalpel. Tape strips, epidermis and dermis were put individually in methanol [26 (link)]. The resulting samples were sonicated to extract CUR, and the methanol extracts were analyzed by HPLC.