Direct visualization of lipid domains in human skin stratum corneum's lipid membranes: effect of pH and temperature

Abstract

The main function of skin is to serve as a physical barrier between the body and the environment. This barrier capacity is in turn a function of the physical state and structural organization of the stratum corneum extracellular lipid matrix. This lipid matrix is essentially composed of very long chain saturated ceramides, cholesterol, and free fatty acids. Three unsolved key questions are i), whether the stratum corneum extracellular lipid matrix is constituted by a single gel phase or by coexisting crystalline (solid) domains; ii), whether a separate liquid crystalline phase is present; and iii), whether pH has a direct effect on the lipid matrix phase behavior. In this work the lateral structure of membranes composed of lipids extracted from human skin stratum corneum was studied in a broad temperature range (10 degrees C-90 degrees C) using different techniques such as differential scanning calorimetry, fluorescence spectroscopy, and two-photon excitation and laser scanning confocal fluorescence microscopy. Here we show that hydrated bilayers of human skin stratum corneum lipids express a giant sponge-like morphology with dimensions corresponding to the global three-dimensional morphology of the stratum corneum extracellular space. These structures can be directly visualized using the aforementioned fluorescence microscopy techniques. At skin physiological temperatures (28 degrees C-32 degrees C), the phase state of these hydrated bilayers correspond microscopically (radial resolution limit 300 nm) to a single gel phase at pH 7, coexistence of different gel phases between pH 5 and 6, and no fluid phase at any pH. This observation suggests that the local pH in the stratum corneum may control the physical properties of the extracellular lipid matrix by regulating membrane lateral structure and stability.

FIGURE 1

DSC experiments and Laurdan GP temperature profile of _HSC(Cer/Chol)/FFA mixture (1:0.9:0.4 mol). The DSC temperature scans were collected from each sample between 10°C and 95°C, at a rate of 0.5°C/min. The GP values observed at the low temperature regime (below 40°C) indicate that the membrane is in a gel- (or solid)-like phase state (see text). For all samples two independent batches were used for the experiments. Four DSC scans and five GP measurements per temperature were performed per batch.

FIGURE 2

Confocal fluorescence 3D images (false color representation) of the _HSC(Cer/Chol)/FFA. (A) Sponge-like structure formed for the _HSC(Cer/Chol)/FFA lipid mixture after electroformation. Bar is 50 μm. (B) Effect of temperature on the _HSC(Cer/Chol)/FFA giant lipid structures. The images show the observed phase scenario for the skin lipids in MilliQ water (pH ∼5.7) at different temperatures. Bars are 20 μm. Lipid domains are observed below 70°C (arrows). The fluorescent probe used was DiI_C18. All measurements have been repeated at least twice from three independent preparations.

FIGURE 3

Laurdan GP images and Laurdan GP histograms of membranes composed of _HSC(Cer/Chol)/FFA (1:0.9:0.4) at three different temperatures in MilliQ water (pH ∼5.7). These pictures represent the behavior observed in the (A) 20°C–40°C, (B) 40°C–70°C, and (C) ≥70°C temperature range regimes. The different domains observed at the gel/fluid coexistence region are indicated by white arrows (orange/red areas: gel; greenish: fluid). The GP histograms reported in the figure correspond to a single area of the GP images (indicated by white circles). The bar corresponds to 20 μm.

FIGURE 4

Laurdan GP average values of _HSC(Cer/Chol)/FFA at different temperatures. These values were obtained by analyzing eight different regions of each Laurdan GP image at the desired temperature. At least four different Laurdan GP images were analyzed per temperature. The dotted lines in the plot indicate the observed phase transition events in the microscopy experiments.

FIGURE 5

pH effect in the _HSC(Cer/Chol)/FFA lipid membranes. (A) Laurdan GP versus temperature of independent samples prepared with _HSC(Cer/Chol)/FFA at different pH values (see Materials and Methods). (B) Confocal microscopy images of DiI_C18 labeled _HSC(Cer/Chol)/FFA membranes at different pH values. Bar is 20μm.

FIGURE 6

(A) DSC experiments and Laurdan GP temperature profile of bbCer/Chol/LA (1:1:1 mol). Two independent batches were used for this mixture. Four DSC scans and five GP measurements per temperature were performed per batch. (B) Confocal fluorescence 3D images (false color representation) of the bbCer/Chol/FFA in MilliQ water (pH ∼5.7). The images show the observed three different fluorescent areas (indicated by white arrows) at 25°C. Bars are 20 μm. The fluorescent probe used was DiI_C18. All measurements have been repeated at least twice from independent preparations.

FIGURE 7

The photoselection effect in the sponge-like structures is dictated by the relative orientation of the Laurdan electronic transition moment with respect to the polarization plane of the excitation light (see text in the Discussion and Conclusions Section). A photoselection effect is observed in the membranes oriented parallel to the polarization plane (black dotted border parallelepiped). This effect does not operate in the wall of the sponge-like structure (black solid border rectangle).

FIGURE 8

(A) Correlation between the structure of native stratum corneum (adapted from Simonetti et al. (79)) and (B) our lipid model system _HSC(Cer/Chol)/FFA. The size of keratinocytes in the stratum corneum is ∼30 μm (80) in close agreement with the size distribution observed in the lipid model system (the size of the cell unit is 20.6 μm ± 4.9).