Interactions between vesicle formulations and human skin were studied, in vitro, in relation to their composition and elasticity. The skin ultrastructure was investigated using transmission electron microscopy (TEM), freeze-fracture electron microscopy (FFEM) and two-photon fluorescence microscopy (TPE). The main difference between the vesicle formulations was their elasticity. Elastic vesicle formulations contained bilayer forming surfactants/lipids and single-chain surfactant octaoxyethylenelaurate-ester (PEG-8-L), whereas rigid vesicles contained bilayer surfactants in combination with cholesterol. TEM results showed three types of interactions after non-occlusive application of elastic PEG-8-L containing vesicle formulations on human skin: (1) the presence of spherical lipid structures containing or surrounded by electron dense spots; (2) oligolamellar vesicles were observed between the corneocytes in the upper part of the stratum corneum; and (3) large areas containing lipids, surfactants and electron dense spots were observed deeper down into the stratum corneum. Furthermore, after treatment with vesicles containing PEG-8-L and a saturated C12-chain surfactant, small stacks of bilayers were found in intercellular spaces of the stratum corneum. Rigid vesicles affected only the most apical corneocytes to some extent. FFEM observations supported the TEM findings. Major morphological changes in the intercellular lipid bilayer structure were only observed after treatment with PEG-8-L containing elastic vesicles. TPE showed a distinctdifference in penetration pathways after non-occlusive application of elastic or rigid vesicles. After treatment with elastic vesicles, thread-like channels were formed within the entire stratum corneum and the polygonal cell shape of corneocytes could not be distinguished. Fluorescent label incorporated in rigid vesicles was confined to the intercellular spaces of the upper 2-5 micrometer of the stratum corneum and the cell contours could still be distinguished.
The intercellular lipid bilayers of the stratum corneum provide the permeability barrier of the skin. To perform an electron microscopical examination of the ultrastructure of these bilayers, ruthenium tetroxide fixation is required. In this study an optimal fixation protocol was developed and selected upon comparing seven different fixation procedures, using glutaraldehyde (GA) and the postfixatives ruthenium red, osmium tetroxide (OsO4) and ruthenium tetroxide (RuO4) in combination with potassium ferrocyanide (K4Fe(CN)6) and potassium ferricyanide (K3Fe(CN)6). Instead of fixing skin with either OsO4 or RuO4, these two fixatives were combined in one protocol. In addition, the use of RuRed was introduced and the influence of both K4Fe(CN)6 and K3Fe(CN)6 in combination with RuO4 were examined. Furthermore, we compared two dehydration solvents, methanol and acetone.
The most satisfying results were obtained when the skin was prefixed in GA and postfixed in OsO4 and RuO4 with K3Fe(CN)6, i.e. with Fe in its highest oxidation state (Fe3+). No differences were observed between dehydration in methanol and acetone.
In this paper we describe a preparative procedure which allows maximal visualization of all lipid bilayers in the human stratum corneum (SC). We used 50-microns-thick vibratome sections of paraformaldehyde/glutaraldehyde-fixed human skin. The sections were post-fixed in 1% osmium tetroxide and 0.5% ruthenium tetroxide. The vibratome sections were dehydrated only in 70% ethanol in order to prevent dissolution of the lipids. Lipid bilayers, including the alternating electron-dense and electron-lucent lamellae, were visible between all cell layers of the SC. In addition, this preparative procedure also appeared to be excellent for the ultrastructural preservation of lamellar bodies.
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