Coexistence of Lipid Phases Stabilizes Interstitial Water in the Outer Layer of Mammalian Skin

Abstract

The lipid matrix in the outer layer of mammalian skin, the stratum corneum, has been previously investigated by multiple biophysical techniques aimed at identifying hydrophilic and lipophilic pathways of permeation. Although consensus is developing over the microscopic structure of the lipid matrix, no molecular-resolution model describes the permeability of all chemical species simultaneously. Using molecular dynamics simulations of a model mixture of skin lipids, the self-assembly of the lipid matrix lamellae has been studied. At higher humidity, the resulting lamellar phase is maintained by partitioning excess water into isolated droplets of controlled size and spatial distribution. The droplets may fuse together to form intralamellar water channels, thereby providing a pathway for the permeation of hydrophilic species. These results reconcile competing data on the outer skin's structure and broaden the scope of molecular-based methods to improve the safety of topical products and to advance transdermal drug delivery.

Figure 1

Structure and permeability of skin lipid model bilayers. (A)–(D) show a simulation snapshot of a skin lipid bilayer in atomistic detail; individual lipid species of the same snapshot are shown separately, with hydrogen atoms hidden. CER[EOS] (A), CER[NS] (B), cholesterol (C), and behenic acid (D) are colored in gray, blue, pink, and cyan, respectively; to the right of each snapshot is one molecule of the same species, with a schematic of its coarse-grained (CG) representation. (E) shows the electron density profiles of the bilayer (black) and of terminal methyl groups (blue); the orange line is the boundary between ordered and disordered regions. (F) shows the computed skin permeability k_P (blue) and empirical estimates from the Potts-Guy equation (red) plotted against experimental values; squares indicate values for mannitol. Root mean-squared errors on log(k_P) are 0.73 and 0.72 for the computed and empirical values, respectively. To see this figure in color, go online.

Figure 2

Skin lipid bilayers subject to heat reach a hemifused state with interstitial water confined into droplets or channels. (A) shows the initial snapshot of the 5:1 water/lipid 16 × 16 × 32 nm³ atomistic model and (B) the same system after heating at 95°C for 0.2 μs, followed by annealing 30°C for 1.8 μs; small crystalline domains are formed during the latter stage. (C) shows the final snapshot of the 2:1 water/lipid model after heating. Lipid molecules are colored as in Fig. 1, water in white and Na⁺ ions in yellow. (D) shows the final snapshot of the 16 × 16 × 32 nm³ 2:1 water/lipid model, showing the distribution of interstitial water as clusters shown in distinct colors. To see this figure in color, go online.

Figure 3

Self-assembled ∼13 nm lamellae with and without interstitial water droplets. Initially randomized lipids (A) reorganize into dehydrated lamellae with homogeneous thickness (B) or hydrated lamellae with inhomogeneous thickness (C). Hydrated lamellae contain water droplets (D) formed by nucleation and growth of water molecules (E); (F) and (G) show the final distributions of droplets (seen from above the lamellar plane) for two independent runs. Multilamellar models were also simulated (H). For the hydrated lamellae, the number and the median, maximum, and minimum of the droplets’ radii are shown in (E) as a function of simulated time. To see this figure in color, go online.

Figure 4

Distributions of individual lipids in the ∼13 nm lamellae. (A)–(D) show the distribution of lipid headgroups for the dehydrated lamella shown in Fig. 3B (dotted lines) and the hydrated lamellae shown in Fig. 3, C and D (dashed lines). Profiles for CER[EOS], CER[NS], cholesterol, and behenic acid electron density profiles are colored in gray, blue, pink, and cyan, respectively. To see this figure in color, go online.

Figure 5

Water droplets are metastable within a lamellar core. (A) shows a CG simulation snapshot after 25 μs and (B) a snapshot from a 2 μs atomistic simulation. (C) shows the estimated thicknesses of the dehydrated CG lamella (orange circle), the hydrated CG lamellae (blue circles), and the hydrated atomistic lamella (cyan diamond). (D) shows the energy as a function of the droplet radius from Eq. 3; the shaded area reflects the uncertainty in the interfacial tension estimate. (E) shows the estimated rate of shrinkage of the larger droplets as a function of the gradient of Eq. 3 with respect to the droplet’s radius. (F) shows the energy landscape for a droplet shaped as a cylindrical capsule; dots indicate the radii and lengths distribution for self-assembled droplets (Fig. 3E). Error bars are standard deviations. To see this figure in color, go online.

Figure 6

Relative movements of the lipid lamellae promote droplet coalescence. (A) shows the initial condition and (B) the final snapshot after 5 μs. (C) shows the total number of droplets and radii distribution in the two lamellae; the solid line is the median and the shaded area the size range. Note the sudden jump at ∼0.3 μs, reflecting the first merging event. To see this figure in color, go online.

Figure 7

Summary of the stable and metastable skin lipid phases relevant to interstitial water. The structures simulated in this study (A–D) are compared to the LPP (E). Lipids are drawn schematically and colored in gray where the local composition equals the global composition, purple where locally enriched in ceramides, and orange where locally depleted in ceramides. Ordered water-lipid or lipid-lipid interfaces are highlighted with black lines. Splayed ceramides, although important to the cohesion of the dry lamellar phases, and chemical differences between the lipids are not drawn for clarity. Transitions from bilayers (A) to inverse-hexagonal and inverse-micellar phases (B and C) were observed as a result of heat (Fig. 2). Thick lamellae were also obtained by self-assembly (Fig. 3) in the hydrated state (C) and dehydrated state (D), with partial transitions between the two as a result of water diffusion. Permeability of hydrophilic molecules through the skin is thought to be nonvanishing only in the presence of metastable water channels (B). To see this figure in color, go online.