Comprendre l’imperméabilité cutanée : étude spectroscopique de mélanges modèles de la phase lipidique du stratum corneum

The stratum corneum (SC), the outermost layer of mammal epidermis, acts as a barrier dictating the rate of absorption of exogenous molecules through the skin and preventing water loss from the body. SC is mainly composed of flattened and fully keratinized dead cells (corneocytes), embedded in a lipid matrix, which is mostly constituted of ceramides, free fatty acids, and cholesterol. The transdermal diffusion of molecules is mainly related to the intercellular lipids, which form multilamellar membranes in the solid-crystalline phase. The main goal of this thesis is to better understand the relationship between the structure of the lipid matrix and the skin impermeability. SC lipid phase includes more than 400 different lipid species. Due to this very complex composition, model membranes are often used for the study of the physicochemical properties of membranes, the lipid behavior, and of the permeability of different chemicals. First, we determined how the length of the free fatty acid acyl chains influences the phase behavior of a lipid matrix composed of ceramide NS24, lignoceric (FFA24) or palmitic (FFA16) acid, and cholesterol. The structural properties of membranes were examined by 2H NMR and infrared spectroscopy. This study revealed that the phase behavior of these ternary mixtures is strongly affected by the length of the FFA. We found that lignoceric acid led to the formation of a more homogeneous mixture with ceramide NS24 and cholesterol, than the palmitic acid/ceramide NS24/cholesterol mixture. Also, the tertiary mixture containing lignoceric acid showed a transition from solid to gel phase when heated above 37 oC, an unusual feature for this type of model membranes. The combination of ordered lipids and homogeneous membranes is proposed as a critical element for SC impermeability. Second, we studied a more complex mixture containing cholesterol, a series of free fatty acids varying from 16 to 24 carbon atoms, and two types of ceramides: ceramide NS24 and EOS. The latter is considered a key component for the formation of the long periodicity phase in native and model SC membranes. The 2H NMR, infrared and Raman spectroscopy results showed that both the free fatty acid and the ceramide NS24 acyl chain remained in the solid-crystalline phase at physiological temperature while the oleate chain in ceramide EOS led to the formation of highly disordered domains. These liquid nanodrops remained in the liquid state down to -30 °C. The steric constraint imposed by the crystalline lipid matrix is proposed to prevent the crystallization of ceramide EOS oleate chains. This finding modifies the structural description of the SC substantially and proposes a novel physiological role of ceramide EOS as this lipid is a strong modulator of SC solid/liquid balance. The work leads to a re-examination of the mechanism currently proposed to explain the permeability of SC, as well as the effect of transdermal agents. Finally, we studied the interaction of very long-chain fatty acids with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) membranes to determine how these fatty acids adapt to spatial constraints. Three different FFAs with acyl chain varying from 16 to 24 carbon atoms were used: palmitic (FFA16), arachidic (FFA20) and lignoceric (FFA24) acid. The leaflet thickness of a POPC bilayer corresponds to the length of the hydrophobic part of FFA16, and therefore is smaller than the length of FFA20 and FFA24. The way in which these fatty acids structurally adapt in POPC bilayers was study by 2H NMR and molecular dynamics simulations. We found that the lower part of the protonated FFA24 acyl chain was disordered in a manner similar to that observed for the oleate chain of the EOS ceramide, its acyl chain interacts with the more fluid part of the opposite leaflet. This interdigitation of the end of the acyl chain caused a second plateau observed in the order profiles (SC−D), an unusual feature in lipid systems. In this case, the protonated carboxyl group of FFA24 was located slightly below the polar head of the POPC. The deprotonation of the FFA24 shifted the molecule toward the aqueous interphase. This movement reduces the spatial constraints, increases the order of the acyl chain and causes the disappearance of the plateau at the end of the chain.  The results presented in this thesis contributed to better understand how the structure of the SC lipid matrix dictates the skin impermeability. We showed spectroscopic evidences of the distinct phase behavior of some of the most important SC lipids. Furthermore, we suggested a novel mechanism for the regulation of transdermal diffusion of molecules.

Keyphrases: stratum corneum, phospholipides, model membranes, phase behavior, lipids, skin, very-long chain fatty acids, phospholipids.