P1.13 Application of laser scanning microscopy for penetration studies of topically applied substances

J. Lademann, N. Otberg, H. Richter, U. Jacobi, U. Blume-Peytavi, H. Schaefer, W. Sterry

Center of Experimental and Applied Cutaneous Physiology, Department of Dermatology and Allergology, Charité - Universitätsmedizin, Berlin, Germany

The quantification of penetration processes and pathways of topically applied substances into and through the skin is the basis for the development and optimization of cosmetic products and drugs. Three different penetration pathways can be discussed: the intercellular, the transcellular and the follicular penetration routes. Intercellular penetration has been considered to be the predominant pathway in the past. Many studies were performed to investigate the influence of the structure of the lipid layers on this process. The dermatological laser scanning microscope (LSM), Stratum, OptiScan, was applied in-vivo to investigate skin physiology. The radiation of an Ar⁺ laser at 488 nm was used to excite the topically applied food dyes, curcumin and sodium fluoresceine. The penetration kinetics of a dye-containing formulation into the lipid layers were investigated with LSM by analyzing the penetration depth of the same skin area at different time points after application. The fluorescence images of the stratum corneum of the forearm of volunteers demonstrate that the fluorescent dye is mainly located in the lipid layers around the  corneocytes. The dye curcumin could only be located in the first layers of the stratum corneum 5 min after application. Later, deeper corneocyte layers could also be detected step by step, because the dye had reached these layers as a result of the penetration process. After 20 min, the shifted structure of 4 layers of the stratum corneum became visible. In this way, the penetration kinetics could be detected online and non-invasively. Follicles and sweat glands account only for approximately 0.1% of the skin surface area. Therefore, they were not considered to represent significant penetration routes. However, recent in vitro and in vivo studies on follicular penetration show a surprisingly high influence of this route on the penetration process. Laser scanning confocal microscopy also allows the online investigation of the follicular penetration process in-vivo. Penetration kinetics into the follicles could be detected by analyzing the distribution of the fluorescent dye at different time points after application. The measurements visualized the penetration of substances into the orifices of the hair follicles. The phenomena of active and passive follicles, concerning the penetration of topically applied substances were investigated.