The development and optimization of transdermal and topical formulations should ideally include the measurement of drug permeation through the skin as well as drug retention in the skin. Current methods have primarily focused on permeation rather than retention in the skin and, in addition, are limited by their low-throughput, time-consumption, and quantity of tissue. High-throughput screening methods have been investigated to increase the throughput of skin permeation studies. However, efforts to develop high-throughput screening methods have other limitations such as the use of synthetic membranes that do not mimic the structure of the skin, the use of indirect measurements of permeability, and the focus on permeation rather than retention. To address these limitations, our aim is to design a novel micro-well plated based screening method to enable direct screening of large numbers of topical/transdermal formulations simultaneously. We hypothesized that our high-throughput screening (HTS) method is able to assess both permeation and retention in the skin when compared to the Franz cells and that is able to detect changes in compositions in a high-throughput manner.
Fresh-frozen dermatomed human skin from the same donor and anatomical area was used for method validation. Sulforhodamine B (SRB) was used as a model drug to allow for direct quantification of drug permeated across and into the skin. The skin was tape stripped 15 times using a standardized procedure prior to each permeation study to assure permeation of the hydrophilic model drug across the skin and to allow validation of the method. The HTS system was developed using modified microwell plates.
In vitro permeation studies were conducted using the HTS method by applying 50μL of SRB (0.5mg/ml in PBS) in the donor compartment, on top of the skin. Franz cells were used in the same manner as the HTS system, and 85μL of SRB (0.5mg/ml in PBS) was applied in the donor compartment and samples were drawn from the receptor fluid at predetermined timepoints, up to 8 hours to determine steady-state.
A spectrophotometer was used to determine the amount of drug that permeated across the tissue and into the receptor compartment and 2-photon microscopy was used to determine the amount of drug retained inside the skin. Statistical analysis using a student’s t-test was performed to assess for differences between the methods. To evaluate the high-throughput of the HTS method, a 3x2x3 factorial design was used to analyze changes in permeation and retention between the solutions. 18 different solutions containing propylene glycol (PG), dimethyl sulfoxide (DMSO), oleic acid (OA), and Tween 80 (Tw80) at different concentrations were prepared and analyzed using the HTS method.
The results showed that the permeability of SRB across the skin was 1.23x10-6 ± 5.41x10-7 cm/sec for Franz cells and 1.26x10-6 ± 2.05x10-7 cm/sec for the HTS system, with no statistical differences (p-value >0.05). Additionally, the relative peak fluorescence intensity of SRB retained in the skin at 15 µm down the surface was 90.10 ± 2.20 % for the Franz cells and 85.12 ± 2.77 % for the HTS, respectively. Below the stratum corneum and into the viable epidermis (depth=50µm) the relative fluorescence intensity of SRB was 44.30 ± 3.34 % and 37.62 ± 2.28 % for the Franz cells and HTS system, respectively.