Supplemental Material for: Imaging Active Drug Transport in Viable Human Skin: A Functional ex vivo skin Model for Mechanistic and Regulatory Applications

ImageJ and MATLAB were used for quantitative image analysis.</p><p dir="ltr">Results</p><p dir="ltr">RD123 diffusion was faster and more extensive in verapamil-treated DSE, with increased fluorescence intensity in the basal and suprabasal layers at early time points compared to controls. HSE exhibited significantly reduced fluorescence and compromised cellular architecture. MPT–FLIM analysis confirmed that the average NAD(P)H fluorescence lifetime and a₁/a₂ metabolic ratios in DSE were comparable to control DTM, indicating preservation of metabolic activity. </p><p dir="ltr">Conclusion</p><p dir="ltr">This study establishes a viable, structurally intact epidermal model suitable for evaluating both passive and active transport in human skin.

Functional imaging confirmed the role of P-gp in modulating drug diffusion across the VE. This platform supports the generation of mechanistic data for improving dermal pharmacokinetic models and has translational relevance for formulation design and regulatory assessment of topical therapies.</p>