Permeability Barrier and Microstructure of Skin Lipid Membrane Models of Impaired Glucosylceramide Processing

Ceramide (Cer) release from glucosylceramides (GlcCer) is critical for the formation of the skin permeability barrier. Changes in β-glucocerebrosidase (GlcCer’ase) activity lead to diminished Cer, GlcCer accumulation and structural defects in SC lipid lamellae; however, the molecular basis for this impairment is not clear. We investigated impaired GlcCer-to-Cer processing in human Cer membranes to determine the physicochemical properties responsible for the barrier defects. Minor impairment (5–25%) of the Cer generation from GlcCer decreased the permeability of the model membrane to four markers and altered the membrane microstructure (studied by X-ray powder diffraction and infrared spectroscopy), in agreement with the effects of topical GlcCer in human skin. At these concentrations, the accumulation of GlcCer was a stronger contributor to this disturbance than the lack of human Cer. However, replacement of 50–100% human Cer by GlcCer led to the formation of a new lamellar phase and the maintenance of a rather good barrier to the four studied permeability markers. These findings suggest that the major cause of the impaired water permeability barrier in complete GlcCer’ase deficiency is not the accumulation of free GlcCer but other factors, possibly the retention of GlcCer bound in the corneocyte lipid envelope.

. Lamellar phases of selected hCer/FFA/Chol/CholS membranes studied using X-ray powder diffraction (XRPD). Roman numerals mark the short periodicity phase (SPP); Arabic numerals mark the long periodicity phase (LPP); and asterisks mark crystalline cholesterol (Chol). Figure S3. Repeat distances (d) of the lamellar phases in the hCer/GlcCer/FFA/Chol/CholS membranes studied by X-ray powder diffraction (XRPD). Panels a-d show the d values of SPP, LPP, SPP2, and Chol, respectively. Mean±SEM, n = 2-6. * Significant difference compared with control at p < 0.05; + significant difference between the membranes with and without GlcCer at p < 0.05. Figure S4. Infrared spectra of the GlcCer/FFA/Chol/CholS membrane. Panel A shows methylene asymmetric and symmetric stretching vibrations that suggest wellordered lipid chains. Panels B and C show methylene scissoring and rocking doublets, respectively, that indicate orthorhombic chain packing. All data are shown as second derivative spectra for clarity.

Human skin
The skin was obtained from female patients who had undergone abdominal plastic surgery in Sanus surgical center (Hradec Králové, Czech Republic). The procedure was approved by the Ethics Committee and conducted according to the principles of the Declaration of Helsinki. The residual subcutaneous fat was carefully removed from the skin fragment by a scalpel. The skin was washed in phosphate-buffered saline (PBS) at pH 7.4 with 50 mg/l gentamicin, blotted dry and stored at -20°C.

Isolation of human skin Cer (hCer)
The extracted human SC lipids (pooled from skin fragments from 6 subjects) were purified by column chromatography on Silica gel 60 (Merck, Darmstadt, Germany). The SC lipids classes were eluted sequentially with chloroform/acetic acid 99:1 (v/v) and then chloroform/methanol in 100:1, 50:1, 10:1, 3:1, 2:1, 1:1 and 1:2 ratios (v/v). The fractions containing the eluted Cer were collected, and the solvent was removed using a rotatory vacuum evaporator and then in high vacuum over P4O10 and solid paraffin. The isolated hCer were stored under nitrogen at -20°C. The composition of the isolated hCer was verified using high-performance thin layer chromatography (HPTLC) 1,2 .

Preparation of model SC membranes
First, mixture of FFA was prepared. Individual FFA were dissolved in hexane/96% ethanol 2:1 (v/v) and mixed in a molar % that corresponds to the composition of human skin FFA: 1.8 % hexadecanoic acid, 4.0 % octadecanoic acid, 7.6 % eicosanoic acid, 47.8 % docosanoic acid and 38.8 % tetracosanoic acid. 3 Chol, CerNS or hCer were dissolved in hexane/96 % ethanol 2:1 (v/v) (hCer created a fine suspension that was carefully homogenized before use 4 ), GlcCer in hexane/96% ethanol 1:1 (v/v) and CholS in 96% ethanol. The organic solutions/suspension were mixed in the desired ratio (FFA, Chol, CerNS or hCer in equimolar ratio with addition of 5 wt% of CholS = control sample). Lipid solutions for the preparation of model membranes with hCer replaced by GlcCer contained the same amount of FFA, Chol and CholS, only the "Cer fraction" (= CerNS or hCer with or without GlcCer) was modified (for details, see Figure 1). Lipid solutions for model membranes with reduced hCer content contained less lipids in Cer fractions, therefore total amounts of lipids were lower in these samples.
The solvents from sample mixtures were evaporated and samples were dried. Then the samples were redissolved in hexane/96% ethanol 2:1 (v/v) at 4.5 mg/ml. Nuclepore polycarbonate filters were washed in hexane/96% ethanol 2:1 (v/v), dried and mounted in steel holders with an opening of 1 cm diameter, which exposed 0.79 cm 2 of the filter. The lipid solutions were sprayed on the filters under a stream of nitrogen using Linomat V equipped with additional y-axis movement. On one filter (1 cm 2 ) were applied 300 µl of lipid solution (=1.35 mg lipids), divided in three steps with changing the direction of application after each step. Prepared lipid membranes were kept in the steel holders also during heating to 90 °C and subsequent cooling down. The membranes were directly transferred from the steel holders to Teflon holders used in permeation experiments.

High-performance liquid chromatography (HPLC)
The TH-and IND-containing samples of acceptor phase were measured by isocratic reverse-phase HPLC using s Shimadzu Prominence instrument (Shimadzu, Kyoto, Japan) consisting of LC-20AD pumps with a DGU-20A3 degasser, SIL-20A HT autosampler, CTO-20AC column oven, SPD-M20A diode array detector, and a CBM-20A communication module. Data were analyzed using the LCsolutions 1.22 software. Reverse phase separation of TH was achieved in a LiChroCART 250-4 column (LiChrospher 100 RP-18, 5 µm, Merck, Darmstadt, Germany) at 35°C using 4:6 methanol/0.1 M NaH2PO4 (v/v) as a mobile phase at a flow rate of 1.2 ml/min. Acceptor phase sample (20 µl) was injected into the column, and the UV absorption of the effluent was measured at 272 nm. The retention time of TH was 3.2±0.1 min. The IND samples were assayed on a LiChroCART 250-4 column (LiChrospher 100 RP-18, 5 µm, Merck) using a mobile phase containing 90:60:5 acetonitrile/water/acetic acid (v/v/v) at a flow rate of 2 ml/min. Next, 100 µl of acceptor phase sample was injected into the column, which was maintained at 40°C. The UV absorption was monitored at a wavelength of 260 nm, and the retention time of IND was 3.1±0.1 min. Both methods were previously validated according to EMA European Medicines Agency, Guideline on bioanalytical method validation (2011) 5 .

X-ray powder diffraction (XRPD)
The XRPD data were collected at ambient temperature with an X´Pert PRO θ-θ powder diffractometer (PANalytical B.V., Almelo, Netherlands) with parafocusing Bragg-Brentano geometry using CuKα radiation (λ = 1.5418 Å, U = 40 kV, I = 30 mA) in modified sample holders over the angular range of 0.6-30° (2θ). Data were scanned with an ultrafast detector X´Celerator with a step size of 0.0167° (2θ) and a counting time of 20.32 s step -1 . The data were evaluated using the software package HighScore Plus (PANalytical B.V., Almelo, Netherlands). The XRPD diffractograms show the scattered intensity as a function of the scattering vector Q [nm -1 ], which is proportional to the scattering angle 2θ according to the equation: Q = 4π sinθ/λ (λ = 0.15418 nm is a wavelength of the X-rays). The repeat distance d [nm] characterizes the regular spacing of parallel lipid bilayers arranged on a one-dimensional lattice, a lamellar phase (L). The diffractograms of lamellae phases exhibit a set of Bragg reflections whose reciprocal spacing are in characteristic ratios of Qn = 2πn/d (reflection´s order number n = 1, 2, 3…). The repeat distance d was obtained from the slope a of a regression function of the dependence Qn = a×n, according to the equation d = 2π/a.

Fourier transform infrared spectroscopy (FTIR)
Infrared spectra were collected on a Nicolet 6700 spectrometer (Thermo Scientific, USA) equipped with a single-reflection MIRacle ATR ZnSe crystal (PIKE technologies, Madison, USA). A clamping mechanism with constant pressure was used. The spectra were generated by the coaddition of 256 scans collected at a resolution of 2 cm -1 and analyzed using Bruker OPUS software.