Abstract
Acne vulgaris is a common chronic skin disease in young adults caused by infection of the pilosebaceous unit, resulting in pimples and possibly permanent scarring on the skin. Minocycline, a common antibiotic, has been widely utilized as a systemic antimicrobial treatment for acne via oral administration. Recently, a topical minocycline gel (BPX-01) was developed to directly deliver minocycline through the epidermis and into the pilosebaceous unit to achieve localized treatment with lower doses of drug. As the effectiveness of the drug is directly related to its successful delivery, there is a need to evaluate the pharmacokinetics at the cellular level within tissue. Advantageously, minocycline is naturally fluorescent and can be directly visualized using microscopy-based approaches. Due to high endogenous autofluorescence, however, imaging of weakly emitting fluorescent molecules such as minocycline in skin tissue can be challenging. Here, we demonstrate a method for the selective visualization of minocycline within human skin tissue by utilizing two-photon excitation fluorescence (TPEF) microscopy and fluorescence lifetime imaging microscopy (FLIM). To demonstrate the feasibility of this approach, ex vivo human facial skin samples treated with various concentrations of BPX-01 were investigated. From the TPEF analysis, we were able to visualize relatively high levels of drug uptake within facial skin. However, minocycline fluorescence could be overwhelmed by endogenous fluorescence that complicates TPEF quantitative analysis, making FLIM more advantageous for visualizing drug uptake. Importantly, we found a unique signature of minocycline uptake via FLIM analysis that enabled the successful differentiation of the drug and enabled the extraction of drug local distribution from the endogenous fluorescence using a non-Euclidean phasor analysis method. Based on these results, we believe that the drug local distribution visualization method using TPEF and FLIM with phasor analysis can play an important role in studying the pharmacokinetics and pharmacodynamics of a topically applicable drug.