Nuclepore polycarbonate filters

The skin barrier models were prepared as equimolar mixtures of Cer or its unnatural analogs, Chol, and LIG with the addition of 5 wt% of CholS^{9 (link),46 (link)}. First, the lipids were dissolved in 2:1 hexane/96% EtOH (or 96% EtOH for CholS) with sonication and mixed to yield the desired composition. The lipid solutions were evaporated under a stream of nitrogen, dried under vacuum, and then redissolved in 2:1 hexane/96% ethanol (v/v) at 4.5 mg/mL. These lipid solutions (3 × 100 µL per cm²) were slowly sprayed on Nuclepore polycarbonate filters with 15 nm pores (Whatman, Kent, UK) or on 22 mm × 22 mm supporting glass cover slides under a stream of nitrogen using a Linomat V (Camag, Muttenz, Switzerland) equipped with additional y-axis movement. This fast drying suppressed artefactual lipid unmixing during solvent evaporation. The supporting filters did not significantly contribute to membrane barrier properties^{8 (link)}. The lipid films were heated to 90 °C, a temperature that is above the main lipid phase transitions in our samples, equilibrated for 10 min, and then slowly (~3 h) cooled to room temperature. All samples were equilibrated at 32 °C for at least 3 days before the experiments.

FFA were mixed in a molar % that corresponds to the composition of human skin FFA^{36 (link)} (see Supplementary information). Then the FFA mixture was combined with equimolar amounts of Chol and sphingolipids (CerNS, hCer or GlcCer, for details, see Fig. 1 and Supplementary information) and 5 wt% of CholS. The lipid mixtures were dissolved in hexane/96% ethanol 2:1 (v/v) at concentration 4.5 mg/ml. Mixtures containing hCer created fine suspensions, which were homogenized by ultrasound. Then, the lipid solutions/suspensions (3 × 100 µl; 1.35 mg/cm²) were sprayed to Nuclepore polycarbonate filters with 15 nm porosity (Whatman, Kent, UK) under nitrogen using a Linomat V (Camag, Muttenz, Switzerland) equipped with additional y-axis movement^{36 (link)}. Thus, each membrane (0.79 cm²) contained 1 mg of lipids. The prepared lipid membranes were dried in vacuum over P₄O₁₀ and solid paraffin and stored at −20 °C. The day before the permeation experiments, the lipid membranes were annealed at 90 °C for 10 min and then slowly (3–4 h) cooled to 32 °C. During this process, a lamellar structure was created. Afterward, the membranes were equilibrated at 32 °C and 45 ± 5% relative humidity for at least 24 h. The lipid films for FTIR experiments were prepared in the same manner. The homogeneity of the membranes was previously validated³⁷ .

Esterified omega-hydroxyacyl-sphingosine (CER EOS), as well as shorter CERs including: N-(tetracosanoyl)-sphingosine (CER NS), N-(tetracosanoyl)-phytosphingosine (CER NP); N-(2R-hydroxy-tetracosanoyl)-sphingosine (CER AS), and N-(2R-hydroxy-tetracosanoyl)-phytosphingosine (CER AP), were all kindly donated by Evonik, Essen, Germany. CER nomenclature was used based on the definitions from Motta et al. [22 (link)]. The sphingoid chain length for all the CERs was C18, while the acyl chain was C24, except for CER EOS with C30 chain, and an additional shorter CER NP with C16 acyl chain. Palmitic acid (C16), stearic acid (C18), arachidic acid (C20), behenic acid (C22), tricosylic acid (C23), lignoceric acid (C24), cerotic acid (C26), octacosanoic acid (C28), and cholesterol (CHOL) were purchased from Sigma-Aldrich Chemie GmbH, Schnelldorf, Germany. The deuterated FFA C16 and C24, which were deuterated along the entire acyl chain (C16-d31 and C24-d47), were obtained from Larodan (Malmö, Sweden) and Arc Laboratories B.V. (Apeldoorn, The Netherlands), respectively. All solvents used were of analytical grade and supplied by Labscan, Dublin, Ireland. The water was of Millipore quality produced by a Milli-Q water filtration system with a resistivity of 18 MΩ cm at 25 °C. Nuclepore polycarbonate filters, with 0.05 μm pore size were purchased from Whatman, (Kent, UK).