Visualizing drug transport across stratum corneum: cryotechniques, vapour fixation, autoradiography

Abstract

The potentials of a method for visualizing the transport of drugs across human stratum corneum at electron microscopic level were investigated. Possible extraction and/or dislocation of the drug during sample preparation for electron microscopy was reduced as much as possible. In this “dry” sample preparation drug-loaded human skin was rapidly frozen, freeze-dried, osmium tetroxide vapour fixed, and embedded in Epon under vacuum. Visualization of the drug was achieved by using electron microscope autoradiography (general applicability). The model compound in the autoradiography studies was tritiated hydrocortisone. No detectable extraction of drug was measured during the “dry” sample preparation. Furthermore, this method preserved the ultrastructure of stratum corneum very well. The combination of the “dry” sample preparation and electron microscope autoradiography was feasible and there were no problems regarding the detection limit; the only remaining problem was an unsatisfactory resolution. However, considering the dimensions of the inter- and intracellular domains and the resolution attainable for electron microscope autoradiography, it should be possible to discriminate between intercellular and transcellular drug transport across stratum corneum.