Cryo-electron diffraction as a tool to study local variations in the lipid organization of human stratum corneum

The human skin provides the body with a barrier against transepidermal water loss and the penetration of harmful agents (e.g. microbes) from outside. This barrier function is produced mainly by the outermost, nonviable layer of the epidermis, the stratum corneum (s.c.). The s.c. consists of terminally differentiated corneocytes surrounded by a continuous intercellular lipid domain, which contains mostly ceramides, cholesterol and free fatty acids. Small- and wide-angle X-ray diffraction studies have elucidated the lamellar and lateral lipid organizations in these domains. However, these techniques require bulk quantities of SC, as a result of which local structure information on the lipids cannot be obtained. Insights to these local lipid arrangements are important when new transdermal drug delivery systems have to be developed. Therefore, the technique of electron diffraction arose as a tool to study the lateral packing of the lipids in the intercellular domains of SC, locally. In a previous study, the suitability of electron diffraction was demonstrated using a lipid model system that resembled the lipid composition of the SC. The spacings calculated from the electron diffraction patterns were in good agreement with the spacings revealed by wide-angle X-ray diffraction. The results presented here succeed this previous study. We improved the microscope settings and developed a new preparation method to study ex vivo human s.c. by cryo-electron diffraction. The method is based on the conventional tape-stripping method and offers the possibility to study depth-related changes in the lipid organization of human SC. Diffraction patterns of both hexagonal and orthorhombic lipid lattices have been recorded with spacings that resembled those found in human s.c. by wide-angle X-ray diffraction. After lipid extraction, such diffraction patterns could no longer be detected in the samples.