Characterization of stratum corneum molecular dynamics by natural-abundance ¹³C solid-state NMR

Abstract

Despite the enormous potential for pharmaceutical applications, there is still a lack of understanding of the molecular details that can contribute to increased permeability of the stratum corneum (SC). To investigate the influence of hydration and heating on the SC, we record the natural-abundance (13)C signal of SC using polarization transfer solid-state NMR methods. Resonance lines from all major SC components are assigned. Comparison of the signal intensities obtained with the INEPT and CP pulse sequences gives information on the molecular dynamics of SC components. The majority of the lipids are rigid at 32°C, and those lipids co-exist with a small pool of mobile lipids. The ratio between mobile and rigid lipids increases with hydration. An abrupt change of keratin filament dynamics occurs at RH = 80-85%, from completely rigid to a structure with rigid backbone and mobile protruding terminals. Heating has a strong effect on the lipid mobility, but only a weak influence on the keratin filaments. The results provide novel molecular insight into how the SC constituents are affected by hydration and heating, and improve the understanding of enhanced SC permeability, which is associated with elevated temperatures and SC hydration.

Figure 1. Dynamic regimes and resulting signal intensities from PT ssNMR experiments.

Theoretical ¹H to ¹³C polarization transfer efficiency as a function of correlation time τ _c and order parameter |S _CH| for a CH₂ segment at the magnetic field 11.74 T and the magic-angle spinning frequency 5 kHz, calculated with input parameters equal to the present experimental settings (see Solid-state NMR). The map is color-coded according to the calculated intensities of the INEPT (red) and CP (blue) polarization transfer schemes. White represents inefficient polarization transfer for both INEPT and CP. Typical values of τ _c and S _CH, in the different dynamic regimes, and the expected intensities for the INEPT and CP polarization transfer schemes, are listed to the right of the figure. Adopted from ref. .

Figure 2. Peak assignment of molecular segments in the SC.

¹³C DP MAS NMR spectra of (A) intact SC, (B) isolated corneocytes, and (C) SC model lipids at 60°C. The schematics illustrate SC with corneocytes filled with keratin filaments, surrounded by a multilamellar lipid matrix. Peaks originating from the keratin and the lipids are assigned in (B) and (C), respectively, following the IUPAC nomenclature. For peaks assigned to several amino acid residues, the names are ordered according to the expected abundance. Spectra A and B are scaled to equal intensity at 172.8 ppm, while spectra A and C are scaled to give equal intensity at 30.4–30.6 ppm. See Figure S1 (E) for standard numbering of cholesterol carbons and labels of lipid carbons.

Figure 3. Water affects the molecular mobility of the SC components.

¹³C MAS NMR spectra of intact SC as a function of RH (water content) at 32°C using DP (grey), CP (blue), and INEPT (red) pulse sequences for preferential enhancement of the signals from molecular segments in either rigid (CP) or mobile (INEPT) microenvironments. Prominent resonance lines from keratin (Leu C_β, Lys C_ε, Gly C_α, Ser C_α, Ser C_β) and lipids (all-trans and trans/gauche (CH₂)_n, (ω−1)CH₂, ωCH₃) are labeled in the data obtained at 50 wt% water. The dots in the 30 wt% water spectra indicate peaks of cholesterol (C12/24, C4, C14/17 and C9, cf., Fig. 2 C).

Figure 4. Heating affects the SC molecular mobility differently than hydration.

¹³C MAS NMR spectra of intact SC as a function of temperature and water content using DP (grey), CP (blue), and INEPT (red) pulse sequences. The schematics illustrate the dynamic state of the SC components by color coding according to the observed INEPT (red, mobile) and CP (blue, rigid) intensities of the signature peaks for lipids (all-trans and trans/gauche (CH₂)_n, (ω−1)CH₂, ωCH₃), as well as the keratin core (C_α-region centered around 57 ppm, Leu C_β, Lys C_ε) and the protruding terminals of the keratin filaments (Gly C_α, Ser C_α, Ser C_β).