The phase behavior of skin-barrier lipids: A combined approach of experiments and simulations

Abstract

Skin barrier function is localized in its outermost layer, the stratum corneum (SC), which is comprised of corneocyte cells embedded in an extracellular lipid matrix containing ceramides (CERs), cholesterol (CHOL), and free fatty acids (FFAs). The unique structure and composition of this lipid matrix are important for skin barrier function. In this study, experiments and molecular dynamics simulation were combined to investigate the structural properties and phase behavior of mixtures containing nonhydroxy sphingosine CER (CER NS), CHOL, and FFA. X-ray scattering for mixtures with varying CHOL levels revealed the presence of the 5.4 nm short periodicity phase in the presence of CHOL. Bilayers in coarse-grained multilayer simulations of the same compositions contained domains with thicknesses of approximately 5.3 and 5.8 nm that are associated with elevated levels, respectively, of CER sphingosine chains with CHOL, and CER acyl chains with FFA chains. The prevalence of the thicker domain increased with decreasing CHOL content. This might correspond to a phase with ∼5.8 nm spacing observed by x-rays (other details unknown) in mixtures with lower CHOL content. Scissoring and stretching frequencies from Fourier transform infrared spectroscopy (FTIR) also indicate interaction between FFA and CER acyl chains and little interaction between CER acyl and CER sphingosine chains, which requires CER molecules to adopt a predominantly extended conformation. In the simulated systems, neighbor preferences of extended CER chains align more closely with the FTIR observations than those of CERs with hairpin ceramide chains. Both FTIR and atomistic simulations of reverse mapped multilayer membranes detect a hexagonal to fluid phase transition between 65 and 80°C. These results demonstrate the utility of a collaborative experimental and simulation effort in gaining a more comprehensive understanding of SC lipid membranes.

Figure 1

Molecular structures and CG mapping schemes for (a) CHOL, (b) FFA C24, and (c) CER NS C24. The carbon atoms bound to the perdeuterated FFA C24 (DFFA C24) and CER NS C24 (CER NSd47) are highlighted in red in the respective skeletal structures. CG representations of CER NS C24 in the (d) hairpin conformation and (e) extended (linear) conformation. Adapted with permission from Shamaprasad et al. (34) Copyright 2022 American Chemical Society.

Figure 2

SAXD profiles of six lipid mixtures. The plot titles describe the composition (molar ratio) of each lipid system. The Roman numerals (I, II) indicate the diffraction orders attributed to the SPP; asterisk (^∗), crystalline CHOL with a reflection at q = 1.8 nm⁻¹; plus (+), the first and third diffraction order of a lamellar phase that has double the repeat distance of the SPP (∼10.8 nm); hash (#), the peaks corresponding to unknown phases. The phases are described in Table 1.

Figure 3

Plots of the local bilayer thickness distribution across the bilayer plane for the central bilayer from simulations of three stacked bilayers for the final frame of one simulation at each of four CER NS/CHOL/FFA molar ratios: (A) 1:0:1, (B) 1:0.2:1, (C) 1:0.5:1, and (D) 1:1:1.

Figure 4

Histograms of the local bilayer thickness for the central bilayer of the three-bilayer systems with varying molar ratios of CHOL in an equimolar mixture of CER NS and FFA. The CER NS/CHOL/FFA molar ratios are indicated in black. Results are aggregated over four independent simulations at each composition.

Figure 5

Histograms of the local excess fraction (positive number) or deficit fraction (negative number) of each tail type in the central bilayer of the three-bilayer system relative to the average fraction of each in lipid mixtures with equimolar CER NS and FFA and varying CHOL molar ratio: (A) CER NS acyl tails, (B) CER NS sphingosine tails, (C) CHOL, and (D) FFA. Acyl and sphingosine tails may have slightly different local fractions in the central bilayer because extended conformations of CER NS have only one tail in the central bilayer. The average tail fractions depend on the composition of the central bilayer. For example, the average fraction of each tail type is 0.25 in 1:1:1 CER/CHOL/FFA mixture, whereas for the 1:0.5:1 mixture, the average fraction of CHOL tails is 0.14, and 0.29 for each of the other tails. Results are aggregated over four independent simulations at each composition.

Figure 6

Pearson correlation coefficient (r) between the local fraction of each tail type and local bilayer thickness of the central bilayer in a three-bilayer system tabulated separately for CHOL, FFA C24, and the sphingosine and acyl tails of CER NS (x axis) for four mixtures at the indicated CER NS/CHOL/FFA molar ratios (y axis). Results are aggregated over four independent simulations at each composition. White text designates |r| > 0.5.

Figure 7

Thermotropic curves indicate the phase transitions of the lipids in the temperature interval 10–90°C for CER NS/CHOL/FFA at a 1:0.5:1 molar ratio with (A) perdeuterated FFA C24 chain (DFFA C24), (B) perdeuterated acyl chain of CER NS (NSd47), and (C) both CER NS acyl chain perdeuterated and the perdeuterated chain of the FFA C24. The wavenumber of the ν_sCH₂ peak position is shown on the left y axis (blue), the right y axis displays the wavenumber of the ν_sCD₂ peak position (red). The graphs represent an average of three measurements for each lipid model.

Figure 8

Temperature variation of structural properties calculated from atomistic simulations of the reverse mapped three-bilayer membrane containing CER NS C24/CHOL/FFA C24 with a 1:0.5:1 molar ratio: (A) nematic order parameter (S2) calculated separately for the FFA, and the acyl and sphingosine chains of CER NS, (B) normalized lipid area (NLA), and (C) total membrane thickness. The gray shading designates the estimated 65–80°C range of the hexagonal-fluid phase transition. Snapshots from simulations at temperatures below (D) and above (E) the hexagonal-fluid phase transition at the same scale. CER lipids are shown in silver, FFA in blue, and CHOL in yellow; oxygen atoms are rendered as red spheres to highlight layers; water is shown as a density isosurface. A movie constructed from simulation snapshots as a function of temperature is included in the supporting material (Video S1).

Figure 9

(A) δCD₂ and (B) δCH₂ vibrations for the CER NS/CHOL/FFA C24 (1:0.5:1) system with: deuterated NS acyl chain (black bottom line), deuterated FFA chains (blue middle line), and both deuterated NS acyl and FFA chains (orange top line). The vibrations shown are measured at 10°C. The peak splitting indicates the protiated and deuterated lipid chain interactions. The data presented are the average of three measurements for each model.