Preparation of Model Intercellular Membrane Using Low-Toxic Solvent Ethanol

The stratum corneum, the outermost layer of the skin, is composed of keratinized cells known as corneocytes and intercellular lipids (IC lipids) such as ceramides (Cers), cholesterol (Chol), and free fatty acids (FFAs). These lipids form highly ordered multilamellar membranes in the intercellular spaces, serving as a key regulator of the skin barrier function. To investigate the mechanistic relation between the membrane structure and the skin barrier function, model membranes composed of Cers/Chol/FFA mixtures have been widely employed. In most of those studies, highly toxic solvents such as chloroform (CHCl₃) and methanol (MeOH) were used to dissolve and mix the IC lipids. However, growing concerns regarding safety and environmental hygiene have driven the selection of safer alternative solvents. In this study, we developed a new protocol for preparing model membranes using less toxic ethanol (EtOH). In the EtOH-based protocol, IC lipids were dissolved in near-boiling EtOH, diluted by a fivefold large volume of buffer, and cooled to 30°C. After 18 h of equilibration at 30°C, the model intercellular lamellae were collected by centrifugation. In addition, we demonstrated that the lamellar sample exhibited thermodynamic and structural properties comparable to those of the lamellae prepared by the conventional CHCl₃/MeOH-based protocols. Moreover, the developed EtOH-based protocol enhanced the miscibility of IC lipids to some extent. Therefore, the EtOH-based protocol would be a useful methodology for preparing model intercellular membranes, being well-suited for environmental hygiene.