Effects of 6-Hydroxyceramides on the Thermotropic Phase Behavior

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Effects of 6-hydroxyceramides on the thermotropic phase behavior and permeability of model skin lipid membranes Andrej Kovacik, Michaela Silarova, Petra Pullmannová, Jaroslav Maixner, and Katerina Vavrova Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.7b00184 • Publication Date (Web): 23 Feb 2017 Downloaded from http://pubs.acs.org on February 24, 2017

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Effects of 6-hydroxyceramides on the thermotropic phase behavior and permeability of model skin lipid membranes Andrej Kováčik 1, Michaela Šilarová 1, Petra Pullmannová 1, Jaroslav Maixner 2 and Kateřina Vávrová 1,* 1

Charles University, Faculty of Pharmacy in Hradec Králové, Czech Republic 2

KEYWORDS:

University of Chemistry and Technology, Prague, Czech Republic

6-hydroxysphingosine,

ceramide,

stratum

corneum,

lipid

membranes,

permeability, X-ray powder diffraction, infrared spectroscopy

ACS Paragon Plus Environment

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ABSTRACT: Ceramides (Cer) based on 6-hydroxysphingosine are important components of the human skin barrier, the stratum corneum. Although diminished concentrations of 6-hydroxyCer have been detected in skin diseases such as atopic dermatitis, our knowledge of these unusual sphingolipids, which have only been found in the skin, is limited. In this work, we investigate the biophysical behavior of N-lignoceroyl 6-hydroxysphingosine (Cer NH) in multilamellar lipid membranes composed of Cer/free fatty acids (C16-C24)/cholesterol/cholesteryl sulfate. To probe the Cer structure-activity relationships, we compared Cer NH membranes with membranes containing Cer with sphingosine (Cer NS), dihydrosphingosine (Cer NdS) and phytosphingosine (Cer NP), all with the same acyl chain length (C24). Compared with Cer NS, 6-hydroxylation of Cer increased membrane water loss and permeability in a lipophilic model compound but also dramatically increased the membrane opposition to electrical current, which is proportional to the flux of ions. Infrared spectroscopy revealed that Cer hydroxylation (in either Cer NH or Cer NP) increased the main transition temperature of the membrane but prevented good Cer mixing with free fatty acids. X-ray powder diffraction showed lamellar phases with shorter periodicity upon Cer hydroxylation but also the formation of an unusually long periodicity phase (d = 10.6 nm) in Cer NH-containing membranes. Thus, 6-hydroxyCer behaves differently from sphingosine- and phytosphingosine-based Cer. In particular, the ability to form a long periodicity lamellar phase and highly limited permeability to ions indicate the manner in which 6hydroxylated Cer contribute to the skin barrier function.


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INTRODUCTION Ceramides (Cer) are the dominant lipids of the mammalian stratum corneum (SC), the uppermost epidermal layer, which acts as the main barrier that prevents water loss and protects the body from external insults.1-4 Cer are simple sphingolipids composed of a sphingoid base (sphingosine (S, according to Motta nomenclature5), dihydrosphingosine (dS), phytosphingosine (P) or 6-hydroxysphingosine (H)), to which a fatty acid is attached by an amide bond. The acyl chain in skin Cer might be a non-substituted (N) or α-hydroxylated (A) or contain linoleic acid ester-linked to the ω-hydroxy group (EO).5 A combination of the letters in parentheses defines the major skin Cer subclasses, e.g., N-acyl 6-hydroxysphingosine is denoted by Cer NH, and N2-hydroxyacyl 6-hydroxysphingosine is denoted by Cer AH.3-5 Cer based on 6-hydroxysphingosine, i.e., 6-hydroxyCer,6 are probably the most unusual human Cer. In 1989, Hamanaka et al., reported sphingolipids with a double bond and three hydroxyls in the sphingoid base in human epidermis.7 The additional hydroxyl was assigned to carbon 6 in 19948, and the absolute stereochemistry at carbon 6 was confirmed only in 2003.9 In human SC, 6-hydroxyCer (Cer NH, AH and EOH) account for up to 30% of Cer10 but have not been detected in other tissues. Differences in the levels of 6-hydroxyCer and free 6-hydroxysphingsine have been described in skin diseases such as atopic dermatitis11-15 and psoriasis,16 respectively (for a review, see Ref.6). However, neither the biosynthesis of these lipids nor their role in the human body has been established. In general, the skin barrier lipids are highly hydrophobic because of their central role in preventing desiccation. Therefore, it is unclear why there is need for an additional hydroxyl group in Cer (beyond those produced by phytoCer such as Cer NP and α-hydroxylated Cer such as Cer AS). Several studies have shown that an additional hydroxyl in Cer NP changes


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its thermotropic behavior, chain order, hydrogen bonding, and mixing with other lipids compared with Cer NS.17-24 The major limitation to a better understanding of the role of 6-hydroxyCer is their unavailability. We recently reported a synthesis method for Cer NH using a Ru-catalyzed hydrosilylation/protodesilylation strategy, and by applying X-ray powder diffraction (XRPD) on model lipid membranes, we found that Cer NH, in contrast to Cer NS, promotes the formation of an unusual lamellar phase with 10.6 nm periodicity.25 This unexpected finding led us to further study the behavior of Cer NH compared with that of Cer that contain other sphingoid bases. In this study, we investigate the manner in which the 6-hydroxy group in Cer NH influences the basic biophysical properties of such Cer in lipid membranes. To describe the structureactivity relationships in skin Cer, we compared Cer NH (with a C24 acyl chain, i.e., Nlignoceroyl 6-hydroxysphingosine or t18:1;24:0) with Cer NS, Cer NdS and Cer NP of the same acyl chain lengths (Figure 1). We described how Cer NH assembles with other skin barrier lipids, including free fatty acids (FFA), cholesterol (Chol) and cholesteryl sulfate (CholS) using XRPD and attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) with unlabeled and deuterated FFA (d-FFA). Furthermore, we studied how these lipid assemblies resist water loss, electrical current and permeation of model substances with varying degrees of lipophilicity.


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Figure 1. Structures of the studied Cer (Cer NS, Cer NdS, Cer NP and Cer NH) and other SC lipids in the model membranes, including free fatty acids (FFA), cholesterol (Chol) and cholesteryl sulfate (CholS). The FFA mixture was composed of 1.8% hexadecanoic (C16:0), 4.0% octadecanoic (C18:0), 7.6% eicosanoic (C20:0), 47.8% docosanoic (C22:0) and 38.8% lignoceric (C24:0) acids (molar %)26 or their perdeuterated counterparts (d-FFA).

EXPERIMENTAL SECTION Chemicals Cer NH was prepared, and its structure and purity were determined as described.25 Cer NS (synthetic, over 99% stereochemically pure, i.e., (2S,3R,4E)), Cer NdS (synthetic, over 99% stereochemically pure, i.e., (2S,3R)), and Cer NP (synthetic, over 99% stereochemically pure, i.e., (2S,3S,4R)) were purchased from Avanti Polar Lipids (Alabaster, AL). Perdeuterated FFA (d-FFA, over 98.9% D) were purchased from CDN Isotopes (Pointe-Claire, Canada). All other chemicals, including Chol, CholS and unlabeled FFA, were purchased from Sigma-Aldrich (Schnelldorf, Germany). Water was deionized, distilled, and filtered through a Millipore Q purification system. Preparation of Lipid Membranes The model SC lipid membranes were prepared in a manner similar to that described previously27 as equimolar mixtures of Cer (Cer NS, Cer NdS, Cer NP or Cer NH), Chol, and FFA with the addition of 5 wt% of CholS.28 FFA (or d-FFA) were mixed in a molar percentage that corresponds to the native composition of human skin FFA26 (Figure 1). The lipids were dissolved in 2:1 hexane/96% EtOH (v/v) at 4.5 mg/mL (note: the use of 96% aq. EtOH is necessary to dissolve CholS). These lipid solutions (3 × 100 µL per cm2) were slowly sprayed on


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Nuclepore polycarbonate filters with 15 nm pores (Whatman, Kent, UK) under a stream of nitrogen using a Linomat V (Camag, Muttenz, Switzerland) instrument equipped with additional y-axis movement. These lipid films were heated to 90°C, a temperature that is above the main phase transitions of all of the studied membranes (57-81°C), equilibrated for 10 min, and slowly (~3 h) cooled to room temperature. The membranes were equilibrated at 32°C and 30% air humidity for 24 h. The lipid films for ATR-FTIR experiments were prepared in the same manner. The homogeneity of the membranes was previously validated.29 Permeation Experiments The lipid membranes were mounted into Franz diffusion cells with an available diffusion area of 0.5 cm2 with the lipid film facing the donor compartment. The acceptor compartment of the cell (6.5 mL) was filled with phosphate-buffered saline (PBS, containing 10 mM phosphate buffer, 137 mM NaCl and 2.7 mM KCl) at pH 7.4 with 50 mg/L gentamicin. The acceptor phase was stirred at 32°C throughout the experiment. After a 12-h equilibration, the electrical impedance and water loss through the model membranes were measured (see below). An amount of 100 µL of the model permeant (either 5% theophylline (TH) or 2% indomethacin (IND) suspensions in 60% propylene glycol) was applied into the membrane. Propylene glycol had no adverse effects on the membranes.29-31 This setup ensured sink conditions for the selected compounds. Samples of the acceptor phase (300 µL) were withdrawn every 2 h over a period of 10 h to reach steady-state situation and were replaced with the same volume of PBS. TH and IND were analyzed by HPLC on a LiChroCART 250-4 column (LiChrospher 100 RP-18, 5 µm, Merck, Darmstadt, Germany) using 4:6 methanol/0.1 M NaH2PO4 (v/v) at 1.2 mL/min or 90:60:5 acetonitrile/water/acetic acid (v/v/v) at 2 mL/min, respectively, as the mobile phases. TH


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and IND were detected at 272 nm and 260 nm, respectively, using a Shimadzu Prominence instrument (Kyoto, Japan). Both methods were validated.31 Water Loss through the Lipid Membranes The water loss through the model membranes [g/h/m2] was measured using a Tewameter® TM 300 probe and Multi Probe Adapter Cutometer® MPA 580 (CK electronic GmbH, Kӧln, Germany). The upper portion of the Franz diffusion cell was removed, and the probe was placed on a membrane holder containing a cylindrical hole with a diameter of 0.8 cm (0.5 cm2) located 0.6 cm from the membrane surface. The measuring time was usually between 80 and 100 s, and the average steady-state value was recorded. Because the use of the membrane holder affects the measured values, we performed calibration measurements over the various water/propylene glycol mixtures using an empty Nuclepore filter with and without the membrane holder.29 The environmental conditions were comparable during all measurements, i.e., ambient air temperature of 23-25°C and relative air humidity of 39-41%. Electrical Impedance of the Lipid Membranes The electrical impedance (normalized to an area of 1 cm2; [kΩ×cm2]) of the model SC lipid membranes was recorded using an LCR 4080 meter (Conrad Electronic, Hirschau, Germany) operated in parallel mode with an alternating frequency of 120 Hz. This setup yields the best sensitivity to small impedance changes. To record the membrane impedance, the donor compartment of the Franz diffusion cell was filled with 0.5 mL of PBS, and the tips of the stainless-steel probes were carefully immersed in PBS in the donor and acceptor compartments of the diffusion cell.32 Attenuated Total Reflectance Fourier Transform Infrared Spectroscopy (ATR-FTIR)


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Infrared spectra of the model SC lipid membranes 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 co-addition of 256 scans collected at a resolution of 2 cm-1. The temperature dependence of the FTIR spectra was studied over the range of 28-100°C in 2°C steps using a temperature control module (PIKE technologies, Madison, USA). After each temperature increment, the sample was allowed to stabilize for 6 min before the spectrum was measured. The spectra were analyzed using Bruker OPUS software. The exact peak positions were determined from the second derivative spectra. X-ray Powder Diffraction (XRPD) The lipid mixtures for the XRPD measurements were prepared in the same manner as those for the permeation experiment, but the lipids were sprayed onto a 22 mm × 22 mm cover glass instead of the polycarbonate filters. The membranes were hydrated either at ambient humidity (30%) or at 100% relative humidity. 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 an angular range of 0.6-30° (2θ). Data were scanned with an ultrafast position-sensitive linear (1D) detector X’Celerator with a step size of 0.0167° (2θ) and a counting time of 20.32 s/step. 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 the wavelength of the X-rays). The data were evaluated using the X'Pert Data Viewer software (PANalytical B.V., Almelo, Netherlands). The repeat distance d [nm] characterizes the regular spacing of parallel lipid layers


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arranged on a one-dimensional lattice, i.e., a lamellar phase (L). The diffractograms of the lamellar phases exhibit a set of Bragg reflections whose reciprocal spacing appear in the characteristic ratios of Qn = 2πn/d (reflection order number n = 1, 2, 3…). The repeat distance d was obtained from the slope of a linear regression function of the dependence Qn = a × n, according to the equation d = 2π/a. Data Analysis The cumulative amount of the drug that penetrated across the lipid membrane (corrected for the acceptor phase replacement; [µg/cm2]) was plotted against time [h], and the steady-state flux [µg/h/cm2] was calculated from the linear region of the plot. Data are presented as the mean ± standard error of the mean (SEM), and the number of replicates is given in the pertinent figure. One-way analysis of variance (ANOVA) with Dunnettʹs post-hoc test method was applied for statistical analysis, and p < 0.05 was considered significant.

RESULTS Relationships between Cer Structure and Membrane Permeability To evaluate the structure-permeability relationships in Cer with different sphingoid bases, we prepared model lipid membranes containing equimolar Cer/FFA/Chol and 5 wt% of CholS, where the Cer was Cer NS, Cer NdS, Cer NP or Cer NH. These model membranes that mimic certain of the important features of the SC lipid barrier (major lipid classes and their proportions) were prepared as thin lipid films on supporting filter via a previously validated method.30,33,34 The supporting filters did not significantly contribute to the barrier properties of the model lipid membranes. We used four complementary permeability markers: (a) water loss through the


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membrane; (b) electrical impedance, which is proportional to the movement of ions through the membrane; (c) TH, which is a small molecule (Mw = 180.2 g/mol) with balanced lipophilicity (log P = 0) that is likely to cross the membrane via free-volume diffusion;35 and (d) IND, which is a large (Mw = 357.8 g/mol) and lipophilic (log P = 4.3) permeant that prefers lateral diffusion along lipid layers.35 The water loss through the membrane that contained Cer NS/FFA/Chol/CholS was 1.4 ± 0.1 g/h/m2 (Figure 2A). The membranes with Cer NdS, Cer NP and Cer NH achieved water loss values of 2.7 ± 0.1 g/h/m2, 2.1 ± 0.5 g/h/m2 and 2.1 ± 0.2 g/h/m2, respectively, which were significantly higher than that of the Cer NS-containing membrane. The electrical impedances of the model membranes containing Cer NS, Cer NdS and Cer NH were 58 ± 17 kΩ × cm2, 43 ± 16 kΩ × cm2 and 69 ± 16 kΩ × cm2 (Figure 2B), respectively, with no significant differences among them. Interestingly, the electrical impedance of the model membrane based on Cer NH reached a notably high value of 353 ± 48 kΩ × cm2, which indicates a 6 times lower permeability to electrical current than the membrane containing Cer NS (p < 0.05). The flux of TH (Figure 2C) through the control model membrane containing Cer NS was 0.20 ± 0.04 µg/cm2/h, which is comparable to previous studies.28,30 The membranes based on Cer NdS and Cer NP were almost three times more permeable to TH (p < 0.05) than the Cer NS membrane, reaching flux values of 0.65 ± 0.09 and 0.57 ± 0.13 µg/cm2/h, respectively. In contrast, the membrane with Cer NH showed permeability to TH similar to that of the Cer NScontaining membrane at 0.26 ± 0.02 µg/cm2/h. The permeabilities of the model membranes composed of Cer NS, Cer NdS and Cer NP to the lipophilic IND were similar, with flux values from 0.07 ± 0.02 to 0.12 ± 0.02 µg/cm2/h (Figure


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2D). Interestingly, the Cer NH-containing membrane showed significantly higher permeability to IND (0.31 ± 0.04 µg/cm2/h) compared with all other membranes.

Figure 2. Permeabilities of the studied model membranes composed of Cer NS (black), Cer NdS (blue), Cer NP (red), or Cer NH (green)/FFA/Chol/CholS. (A) Water loss through the membrane; (B) Electrical impedance of the membrane; (C) Permeation profiles for theophylline (TH); (D) Permeation profile for indomethacin (IND). The flux values of TH and IND are shown in the table below the permeation profiles. Data are presented as the mean ± standard error of the mean (SEM), n = 8-37. The asterisks show statistically significant differences against the Cer NScontaining membrane (p < 0.05).

Lipid Chain Conformations at 32°C


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Infrared spectroscopy is a powerful and non-perturbing technique used to study lipid membranes.36 Lipid conformation order and phase transitions can be deduced from the methylene symmetric stretching vibrations – the higher the wavenumber, the lower the conformational order. The methylene symmetric stretching wavenumbers of Cer NS-, Cer NdS-, Cer NP- and Cer NH-containing membranes are shown in Figure 3. At 32°C, i.e., skin temperature, the lipid chains in all membranes contained high proportions of all-trans conformers because all methylene symmetric stretching wavenumbers were below 2850 cm-1, Ref.36 The highest lipid chain order, deduced from the methylene symmetric stretching wavenumbers, was found in the Cer NS membrane followed by Cer NH, Cer NP and Cer NdS, with wavenumbers of 2848.4 ± 0.1 cm-1, 2848.4 ± 0.0 cm-1, 2848.9 ± 0.1 cm-1 and 2849.0 ± 0.0 cm-1, respectively. Interestingly, this lipid chain order approximately followed the water loss through the respective membranes.

Figure 3. ATR-FTIR spectra of the model SC lipid membranes containing the studied Cer, i.e., Cer NS (black circles in panel A), Cer NdS (blue circles in panel B), Cer NP (red circles in panel


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C), or Cer NH (green circles in panel D), (d)-FFA, Chol, and CholS. Data are presented as the temperature dependence of methylene symmetric stretching, either CH2 (filled circles) or CD2 (d-FFA, open circles). The first graphs in each panel show the behavior of the membrane with unlabeled lipids, i.e., the circles describe the behavior of both Cer and FFA chains. The second and third graphs show the behavior of the membrane with d-FFA, where the second graph shows the CH2 vibrations of Cer chains, and the third graph shows the CD2 vibrations.

Phase Transitions of the Model Membranes In general, the lipid chain order decreased with increasing temperature, as indicated by the shift of the methylene stretching to higher wavenumbers because of the increased proportion of gauche conformers in the lipid chains. Figure 3 shows the temperature dependence of the methylene symmetric stretching of either all lipid chains in the unlabeled membrane (first graph) or the unlabeled (Cer) and deuterated chains (d-FFA) separately in the second and third graph, respectively. The transition temperatures are also given in Figure 3. The Cer NS/FFA/Chol/CholS-containing membrane (Figure 3A) first showed an approximately 1 cm-1 gradual increase in the methylene stretching wavenumber with increasing temperature. This increase in wavenumber suggests gradual disordering with increased rotational movements (as can be seen from the evolution of the scissoring bands – Supporting Figure S1) of the lipid chains with temperature. This disordering is likely connected with the sphingosine chain in Cer NS, not the acyl, as indicated by experiments using deuterated Cer NS,34 and/or with Chol that is partly separated and also contributes to the behavior of the CH2 vibrations. At 57°C, the membrane underwent an order-to-disorder transition, as indicated by a sharp shift of the wavenumber to over 2850 cm-1, which is in good agreement with previous studies.30,37 In the


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sample with d-FFA, both Cer (CH2) and d-FFA (CD2) became disordered at the same temperature of 55°C, which suggests their good miscibility. The thermotropic behavior of the lipid chains in the Cer NdS/FFA/Chol/CholS membrane (Figure 3B) did not show any disordering up to 70°C. Subsequently, a sharp phase transition occurred centered at 71°C, and thus, the saturation of the double bond in the sphingosine chain increased the phase transition of such a membrane, which is in agreement with previous results.37 With the replacement of FFA by d-FFA, two distinct transitions were observed. Cer NdS chains became disordered at 83°C, preceded by selected chain rearrangement, as suggested by the decrease in wavenumber immediately before the transition, whereas d-FFA became disordered at 59°C (Figure 3B). These different phase transition temperatures indicate phase separation. The Cer NH/FFA/Chol/CholS membrane underwent an order-to-disorder transition at 71°C (Figure 3C). Thus, hydroxylation of the sphingosine chain in position 6 increased the phase transition temperature by 14°C compared with the Cer NS membrane. Deuteration of FFA revealed that the phase transition temperature of the CH2 chains (mostly Cer NH) was the same as the overall transition of all lipid chains, i.e., 71°C. The CD2 chains in d-FFA underwent disorder in two steps, the first at 57°C and second at 79°C. In the model membranes composed of Cer NP/FFA/Chol/CholS, (Figure 3D), the methylene symmetric stretching wavenumbers increased slowly with increasing temperature. The main phase transition was rather broad. We estimated the transition temperature at 81°C when the wavenumbers reached values greater than 2850 cm-1. Thus, a combination of saturation of the double bond and an additional hydroxyl in the sphingoid base led to the highest transition temperature in this membrane. In the pertinent membrane with d-FFA, the individual phase transitions of the CH2 and CD2 chains were well separated, similar to the membrane with Cer


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NH. The d-FFA CD2 chain became disordered at 58°C, whereas the Cer NP chains showed a thermotropic behavior similar to that of the unlabeled membrane. Interestingly, the CD2 chains disordered only partially, with wavenumbers of approximately 2090 cm-1 after the transition. Relative Proportion of Orthorhombic Chain Packing The methylene rocking and scissoring bands are sensitive to lateral packing of the lipid chains. The splitting of these bands into doublets is indicative of orthorhombic chain packing because the vibrations in this tight lateral packing are coupled.36 Figure 4 shows the methylene rocking (Figure 4A) and scissoring vibrations (Figures 4B-E) at 32°C, and the thermal evolution of the main bands is given in Supporting Figure S1. The contours of the methylene rocking bands suggested different relative proportions of orthorhombic lipid chains in the model membranes (Figure 4A). The Cer NS-containing membrane clearly contained a doublet (at 719.3 cm-1 and 729.6 cm-1). The relative intensity of the 729.6 cm-1 component of the doublet decreased with saturation of the double bond in the Cer NdS-containing membrane compared with the Cer NS membrane. 6-Hydroxylation of the sphingosine chain in the membrane led to a broad peak at 719.2 cm-1 but with a clearly visible shoulder at 729.3 cm-1, indicating that certain orthorhombic lipids were present in the Cer NHcontaining membrane. This effect of hydroxylation was confirmed in the Cer NP-containing membrane, for which only a small shoulder at 730.2 cm-1 was visible. These effects of saturation and hydroxylation of the sphingosine chain in Cer were confirmed by the scissoring contours at 32°C (Figures 4B-E, first graphs in each panel). Although all membranes showed orthorhombic doublets at approximately 1462 cm-1 and 1472 cm-1, a central band at approximately 1469 cm-1 was also present, and its relative intensity increased in the membranes with hydroxylated Cer, i.e., Cer NH and Cer NP. The orthorhombic doublet merged


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into a singlet with increasing temperature, either before the main phase transition (at 37°C in the Cer NS membrane and at 58°C in the Cer NP membrane) or at the onset of the main transition (at 65°C in the Cer NdS and Cer NH membranes) (Supporting Figure S1).

Figure 4. ATR-FTIR spectra (methylene rocking – panel A and methylene scissoring vibrations – panels B-E) of the model SC lipid membranes containing the studied Cer: Cer NS (black lines in panels A and B), Cer NdS (blue lines in panels A and C), Cer NH (green lines in panels A and D) and Cer NP (red lines in panels A and E), (d)-FFA, Chol, and CholS. The first graphs in panels B-E show the behavior of the membrane with unlabeled lipids, i.e., of both Cer and FFA chains. The second and third graphs show the behavior of the membrane with d-FFA, where the second graph shows the CH2 vibrations of Cer chains, and the third graph shows the CD2 vibrations. The splitting of the rocking or scissoring band into a doublet is indicative of orthorhombic chain packing. The thermal evolution of the main bands is given in the Supporting Information.


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Lipid Mixing Deuteration of the lipid chain shifts the methylene scissoring wavenumbers to approximately 1090 cm-1. Thus, the behaviors of the CH2 and CD2 chains could be individually examined. In addition, when the CH2 scissoring doublet (at approximately 1462 cm-1 and 1472 cm-1) changes into CH2 and CD2 singlets (at approximately 1468 cm-1 and 1088 cm-1, respectively) upon deuteration of one component of the lipid mixture, it indicates mixing of the deuterated and unlabeled components because the vibrational coupling does not occur between different isotopes.36 This behavior was observed in the membrane with Cer NS (Figure 4B), which together with the coordinate melting of Cer NS and d-FFA indicates good mixing of Cer NS and FFA in this membrane. This result is in agreement with previous results using either the same FFA mixture37 or lignoceric acid.30 Both singlets further indicated an amount of rearrangement at the main transition temperature (Supporting Figure S1). Upon saturation of the double bond in the sphingoid base, Cer NdS still mixes with d-FFA, although shoulders indicating separation of a subset of molecules of Cer NdS into small orthorhombically packed domains were present (Figure 4C, second graph). This result is in agreement with separate phase transitions of these lipids. Both CH2 and CD2 singlets indicate a degree of lipid chain rearrangement before melting (Supporting Figure S1) that is consistent with the changes in the methylene symmetric stretching. Both Cer with additional hydroxyls (either Cer NH or Cer NP) showed separated d-FFA, as indicated by clear CD2 doublets in the third graphs in Figures 4D-E. Both Cer NH and Cer NP displayed broad CH2 scissoring singlets with visible shoulders. The CD2 doublets in both Cer NH and Cer NP membranes were approximately 7 cm-1 wide, suggesting formation of large domains


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of d-FFA. This observation is consistent with the disappearance of these doublets at approximately the melting temperature of this d-FFA mixture (Supporting Figure S1).

Membrane Microstructure (XRPD) In all XRPD diffractograms, (Figure 5), reflections that give d = 3.42 ± 0.01 nm (n ≥ 3; marked by asterisks) were found and assigned to separated mixture of Chol and CholS. The Cer NS/FFA/Chol/CholS membrane (Figure 5A; black line) further contained a set of peaks marked by Roman numerals that gave a repeat distance d = 5.34 ± 0.01 nm. This La phase is similar to the short periodicity lamellar phase (SPP) that occurs in human SC (d = 5.3 – 6.5 nm).38,39 The diffractogram of the Cer NdS-based membrane (Figure 5B; blue line) was similar to that of the Cer NS membrane and contained peaks with d = 5.38 ± 0.02 nm assigned to the La phase. Cer

hydroxylation

dramatically

changed

the

membrane

microstructure.

The

Cer

NP/FFA/Chol/CholS membranes (Figure 5D; red line) showed two lamellar phases (in addition to separated Chol). A La phase with d = 5.56 ± 0.01 nm was barely visible, and the dominant lamellar phase was Lb phase with d = 3.72 ± 0.01 nm (marked by letters). In the Cer NH/FFA/Chol/CholS membranes (Figures 5C, E; green lines), two lamellar phases and Chol were also found. The Lb with d = 3.78 ± 0.01 nm was preserved, but the La phase was lost and replaced by the Lc phase with periodicity d = 10.60 ± 0.01 nm, which was approximately twice as long (marked by Arabic numerals). Figure 5F shows a sample without Cer, i.e., FFA/Chol/CholS. In this figure, the La phase (d = 4.99 ± 0.01 nm with separated Chol were dominant. In addition, a weak phase with d = 4.26 ± 0.01 nm (Ld phase) was observed.


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The wide-angle XRPD region (at the scattering vector Q = 14-18 nm-1) contains information on short-range molecular arrangement, and in our case, the lateral lipid packing. In all of the studied membranes, regardless of Cer structure, two peaks were found with distances between lattices of 0.41-0.42 nm and 0.37-0.38 nm. These peaks were assigned to the orthorhombic chain packing,40 which is in agreement with the FTIR spectroscopy results.

Figure 5. XRPD diffractograms of Cer/FFA/Chol/CholS lipid membranes containing Cer NS (black; A), Cer NdS (blue; B), Cer NH (green; at 30% and 100% relative humidity, panels C and E, respectively), or Cer NP (red; D). Roman numerals mark the La phase (d = 5.3-5.6 nm), letters mark the Lb phase (d = 3.7-3.8 nm); Arabic numerals mark the Lc phase (d = 10.6 nm) and asterisks mark separate Chol (d = 3.4 nm). Panel F shows the sample without Cer (i.e., FFA/Chol/CholS); La phase (d = 5.0 nm) and letters mark the Ld phase (d = 4.3 nm). The intensity is given in arbitrary units (a.u.). In the wide-angle region (14-18 nm-1), arrows indicate reflections that were assigned to the orthorhombic lipid packing.


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DISCUSSION Cer play an important role in skin barrier homeostasis.3 This highly heterogeneous collection of simple sphingolipids is indispensable as a protective barrier between the external environment and the living organism. However, the role of the individual sphingoid bases and their diversity in the SC has not been fully clarified. In particular, 6-hydroxyCer, including Cer NH, Cer AH and Cer EOH, have not been investigated yet because they are not commercially available. We recently developed an efficient synthesis of the 6-hydroxysphingosine base.25 In this work, we use the synthesized Cer NH to investigate the biophysical and permeability consequences of Cer 6-hydroxylation in model systems composed of major SC lipids. To establish the basic structure-activity relationships, we compare Cer based on four main sphingoid bases: sphingosine (namely, Cer NS), dihydrosphingosine (Cer NdS), phytosphingosine (Cer NP) and 6-hydroxysphingosine (Cer NH), all with 24-carbon acyl chain lengths. These Cer were incorporated in model lipid membranes composed of Cer/FFA/Chol/CholS, where the FFA were 16 to 24 carbons long,26 to mimic the main components and their ratios in the skin barrier. The lipid films were annealed at 90°C because the Cer NP-based membrane has a high transition temperature (approximately 81°C). These models were simpler than the real SC, e.g., they lacked corneocytes, corneocyte lipid envelope and diversity in Cer subclasses. The latter simplification was used in this work on purpose to enable direct comparison of the effects of the studied Cer. Thus, it is appropriate to ask whether our model systems are relevant to the SC lipid barrier. Such models reproduce many of the important features of the SC lipids, e.g., their lateral packing, lamellar phases (either a short periodicity phase or both lamellar phases depending on the lipid composition) and permeability. 28,30,32-34,38,41 Of course the findings from simple models


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cannot be translated to real human tissue, but they can point to relationships and mechanisms that could not be found using the complex native SC. First, the permeabilities of the prepared lipid membranes were investigated using four complementary markers: water loss, electrical impedance, and the TH and IND flux values. Water loss is a common dermatological technique for assessment of the skin barrier function in vivo42,43 and also in vitro, including model membranes.44,45 The water loss through the studied lipid membranes ranged from 1.4 ± 0.1 g/h/m2 to 2.7 ± 0.1 g/h/m2, which is highly similar to a membrane prepared in the same manner from isolated human SC lipids (1.8 g/h/m2; Sochorová et al., unpublished data). The membrane based on Cer NS offered the best resistance to the movement of water. Hydroxylation of the sphingoid base, either in position 6 in Cer NH or in position 4 (together with saturation of the double bond) in Cer NP, increased water loss through these membranes by 50%. Saturation of the sphingosine trans-double bond without hydroxylation in Cer NdS was apparently the least advantageous structural change from this point of view because it increased water loss by nearly a factor of two compared with the Cer NS-based membrane. Electrical impedance, i.e., the membrane opposition to alternating current, which is inversely proportional to the permeability to ions, was our second marker. In contrast to water loss, no significant differences among Cer NS, Cer NdS and Cer NP were found. The impedance values were in the tens of kΩ × cm2 under these conditions, which is similar to the values found in human skin (22.1 ± 0.8 kΩ × cm2).46 However, 6-hydroxylation of sphingosine in Cer NH dramatically increased the impedance, which means highly limited permeability of the Cer NHcontaining membrane to ions compared with all other membranes. To confirm this finding, we repeated this experiment using three independently prepared batches of membranes (total n =


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24). However, the same high values were obtained. Interestingly, the membrane with Cer NP, which also has three hydroxyls, had impedance values similar to those of membranes with Cer NS and Cer NdS. Such high impedance values in the Cer NH membrane suggest certain profound changes in lipid organization, probably connected to differences in lipid mixing/domain formation compared with the other Cer. Notably, the impedance values are likely the most sensitive of our markers to small changes in the membrane composition and structure, e.g., using lignoceric acid (24 carbons, the most abundant FFA in SC47) instead of a FFA mixture in membranes with Cer NS30 or Cer NdS34 yielded impedance values in the hundreds of kΩ × cm2. We studied the membrane permeabilities using the flux of TH, which represents a small molecule with balanced lipophilicity, and the flux of IND, which is a large lipophilic compound. It should be noted that TH and IND were selected purely as model compounds and not for their therapeutic potential. The results further emphasized the highly specific effects of Cer NH on the studied permeants and the likely different permeation pathways. Although 6-hydroxylation in Cer NH did not significantly change the flux of TH, it increased the flux of IND by a factor of four compared with the membrane with Cer NS. No significant differences were found among the membranes with saturated Cer, Cer NdS and Cer NP using either TH or IND. In general, the flux of TH was 3-5 times higher than that of IND in the membranes with Cer NS, Cer NdS and Cer NP, which is in agreement with the physicochemical properties of these compounds and also with their permeabilities through the human epidermis (the flux values of TH and IND were 0.15 ± 0.05 µg/cm2/h and 0.05 ± 0.02 µg/cm2/h, respectively; Kováčik et al., unpublished data). In contrast, the Cer NH-containing membrane yielded similar fluxes of TH and IND.


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Thus, it appears that the effects of introducing further hydroxyls in position 6 of Cer NH are highly different from introducing hydroxyls in position 4 (in the absence of double bond) in Cer NP. The reason might be connected with the fact that both secondary hydroxyls in Cer NH are actually allylic hydroxyls with distinct properties compared with the diol structure in Cer NP. In addition, the distance of the 6-OH group from the other hydrogen-bonding groups in the Cer polar head might lead to a rather different hydrogen-bonding network and/or seriously change the spatial arrangement of the entire molecule. These structural features in the Cer NH molecule led to a high resistance of their assemblies with FFA, Chol and CholS to the permeation of ions but low opposition to the flux of lipophilic IND. To obtain a link on the molecular level between the structure of these Cer and the membrane permeability, we studied multilamellar lipid films that were prepared in the same manner as those for the permeation experiments using ATR-FTIR and XRPD. At 32°C, i.e., skin temperature, the lipid chains in all membranes were well ordered with prevailing trans conformers, and the most ordered lipids were found in the Cer NS membrane. Introduction of further hydroxyls or saturation of the double bond slightly decreased the chain order. Although the differences in wavenumbers were rather small, the membranes with the most ordered chains were those with the lowest water loss and vice versa. These results are consistent with the work of Stahlberg et al., who found a less crystalline phase in Cer NdS than in Cer NS (both with 24 carbon acyl chains)48 and more fluid phases in Cer NP than in Cer NS (both with 18 carbon acyl chain lengths) using deuterium solid-state NMR spectroscopy of equimolar Cer/FFA/Chol mixtures.49 The least permeable membrane (with Cer NS) also showed the highest relative proportion of orthorhombic lipid packing. This notably tight lipid packing was also found in native skin in vivo


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and is a good indicator of the correct barrier function.50,51 All of the studied structural alterations of the sphingosine backbone led to a lipid arrangement with apparent less orthorhombic packing, which might explain the lower resistance of such membranes to water loss. In addition, the presence of a third hydroxyl group in Cer (either 6-hydroxylation of the sphingosine chain in Cer NH or hydration of the double bond leading to Cer NP) strongly decreased the mixing of such Cer with FFA. Previous study of native skin using very highresolution cryo-electron microscopy suggested that Cer prefer the extended conformation, i.e., with chains pointing in opposite directions, and FFA associates with the Cer acyl chain and Chol associates with the Cer sphingoid base chain.52 We confirmed this finding in membrane models composed of Cer NS/FFA (either lignoceric acid or a mixture of FFA with 16 to 24 carbons)/Chol, either with or without CholS using selectively deuterated lipids and FTIR spectroscopy.37 However, it appears that Cer hydroxylation either prevents this particular arrangement or diminishes it such that it cannot be detected. Considering the thermotropic behavior of the membranes, both saturation of the double bond and hydroxylation of the sphingoid backbone in Cer increased the phase transition temperatures compared with the membrane with Cer NS. This result means that additional energy was needed to disrupt the cohesive forces between the lipids of the membranes with Cer NP, followed by Cer NH and Cer NdS, than for the membranes with Cer NS. In addition, the orthorhombic packing in the membranes with Cer NH and Cer NdS persisted to higher temperatures than in the Cer NScontaining membrane. In the membrane with Cer NS, the short periodicity lamellar phase La and separated Chol were found by XRPD, which is consistent with previous studies.28,30,38,53 A long periodicity phase, which is also present in human SC and has d = 11-13 nm,38,54-56,52 was not formed because we


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did not include acylCer (e.g., Cer EOS) in our model. Separated Chol was observed in all of the model membranes studied in this work, in accordance with previous results from isolated human SC.38 The relative intensities of the reflections of the short periodicity phase relative to the reflections of Chol decreased in the following order of Cer NS > Cer NdS > Cer NP and disappeared in the Cer NH membrane. Hydroxylation of the sphingoid base chain (in Cer NH and Cer NP) induced formation of a lamellar phase Lb with even shorter periodicity (3.7-3.8 nm). For pure Cer NP with the same acyl chain length (24 carbons), a complex polymorphism was described by Dahlén and Pascher.57,58 One of the six solid phases of Cer NP, i.e., phase F, was described as a crystalline triclinic unit cell with a repeat distance d = 3.73 nm. No such phase was found in a membrane without Cer, i.e., composed of FFA/Chol/CholS. Thus, a separated phase rich in such V-shaped Cer NP might correspond to the Lb phase found in our membrane with Cer NP. This observation is consistent with the decreased mixing between Cer NP and FFA found by FTIR. However, the separated FFA probably formed a phase without a periodically repeated arrangement because no such phase was found in the diffractograms. This Lb lamellar phase was also observed in the Cer NH-containing membrane, suggesting a similar behavior of Cer NH to Cer NP. The most interesting finding was the formation of an additional lamellar phase Lc with 10.6 nm periodicity in the Cer NH-based membrane. To exclude any possible artifacts, we repeated this experiment three times, and this Lc phase was consistently found in all the diffractograms. Hydration of the membrane further increased the intensity of this Lc phase over Lb and Chol. Thus, Cer NH promotes the formation of a long periodicity phase, although the repeat distance is slightly shorter than the conventional long periodicity phases induced by acylCer or found in human SC.38,53,59 However, Iwai et al.52 suggested that only a single lamellar phase with a


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periodicity of approximately 11 nm is actually present in native SC examined by cryo-electron microscopy, which is similar to the periodicity of our Cer NH-based lamellar phase. It would be interesting to study this 6-hydroxyceramide-based long periodicity phase further, e.g., to examine how much FFA and Chol is actually incorporated in this phase, how it behaves when other Cer subclasses are added and, in particular, its interactions with conventional long periodicity phases formed by acylCer. It might be possible that 6-hydroxyCer aids the acylCer in creating or stabilizing the long periodicity phase.

SUMMARY AND CONCLUSIONS Cer with a 6-hydroxysphingosine backbone, an epidermal-specific sphingolipid subclass, was studied for the first time in this work. The behavior of Cer NH, 6-hydroxyCer with a 24-carbon acyl chain length, was investigated in model systems composed of the major skin barrier lipids and compared with ceramides with sphingosine, dihydrosphingosine and phytosphingosine bases. Permeability experiments revealed highly permeant-specific effects of Cer 6hydroxylation that were detrimental, neutral and beneficial. FTIR suggested that 6-hydroxylation negatively influenced the lipid chain order, lateral packing and miscibility between the ceramide and fatty acids but increased the thermal stability of the ordered domains. XRPD showed that Cer NH promoted formation of a long periodicity phase even in the absence of acylCer. Thus, the Cer polar head structure specifically influences the permeability and organization of the model lipid membranes. It appears that all of these diverse mechanisms contribute to the unique barrier properties of the SC lipids and their resistance to the heterogeneous external stressors that are constantly faced by the skin barrier. Although it is not possible to directly translate our data obtained from simple model lipid membranes to real SC tissue, this special behavior of ceramide


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NH warrants further study because it might be connected with the reason why skin is the only human tissue that synthesizes Cer with a 6-hydroxylated sphingoid base.


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SUPPORTING INFORMATION

The Supporting Information (ATR-FTIR spectra of the model SC lipid membranes – temperature dependence of the scissoring wavenumbers) is available free of charge on the ACS Publications website at DOI:

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] Author Contributions The manuscript was written with contributions from all authors. All authors have approved the final version of the manuscript. ACKNOWLEDGMENT

This work was supported by The Czech Science Foundation (13-23891S) and Charles University (SVV 260 291). The authors thank Mrs. Iva Vencovská for technical assistance. ABBREVIATIONS SC, stratum corneum; Cer, ceramide(s); Chol, cholesterol; FFA, free fatty acids; CholS, cholesteryl sulfate; TH, theophylline; IND, indomethacin; XRPD, X-ray powder diffraction; ATR-FTIR, attenuated total reflectance Fourier-transform infrared spectroscopy.


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(18) Glombitza, B.; Müller-Goymann, C. C. Influence of different ceramides on the structure of in vitro model lipid systems of the stratum corneum lipid matrix. Chemistry and Physics of Lipids 2002, 117 (1–2), 29. (19) Janssens, M.; Gooris, G. S.; Bouwstra, J. A. Infrared spectroscopy studies of mixtures prepared with synthetic ceramides varying in head group architecture: coexistence of liquid and crystalline phases. Biochim Biophys Acta 2009, 1788 (3), 732. (20) Ohta, N.; Hatta, I. Interaction among molecules in mixtures of ceramide/stearic acid, ceramide/cholesterol and ceramide/stearic acid/cholesterol. Chem Phys Lipids 2002, 115 (1-2), 93. (21) Rerek, M. E.; Chen; Markovic, B.; Van Wyck, D.; Garidel, P.; Mendelsohn, R.; Moore, D. J. Phytosphingosine and Sphingosine Ceramide Headgroup Hydrogen Bonding: Structural Insights through Thermotropic Hydrogen/Deuterium Exchange. J Phys Chem B 2001, 105 (38), 9355. (22) Raudenkolb, S.; Hubner, W.; Rettig, W.; Wartewig, S.; Neubert, R. H. Polymorphism of ceramide 3. Part 1: an investigation focused on the head group of Noctadecanoylphytosphingosine. Chem Phys Lipids 2003, 123 (1), 9. (23) Raudenkolb, S.; Wartewig, S.; Neubert, R. H. Polymorphism of ceramide 3. Part 2: a vibrational spectroscopic and X-ray powder diffraction investigation of N-octadecanoyl phytosphingosine and the analogous specifically deuterated d(35) derivative. Chem Phys Lipids 2003, 124 (2), 89. (24) Rerek, M. E.; Van Wyck, D.; Mendelsohn, R.; Moore, D. J. FTIR spectroscopic studies of lipid dynamics in phytosphingosine ceramide models of the stratum corneum lipid matrix. Chemistry and Physics of Lipids 2005, 134 (1), 51. (25) Kovacik, A.; Opalka, L.; Silarova, M.; Roh, J.; Vavrova, K. Synthesis of 6hydroxyceramide using ruthenium-catalyzed hydrosilylation-protodesilylation. Unexpected formation of a long periodicity lamellar phase in skin lipid membranes. RSC Advances 2016, 6 (77), 73343. (26) Groen, D.; Gooris, G. S.; Bouwstra, J. A. Model membranes prepared with ceramide EOS, cholesterol and free fatty acids form a unique lamellar phase. Langmuir 2010, 26 (6), 4168. (27) de Jager, M.; Gooris, G.; Ponec, M.; Bouwstra, J. Acylceramide head group architecture affects lipid organization in synthetic ceramide mixtures. J. Invest. Dermatol. 2004, 123 (5), 911. (28) Pullmannová, P.; Staňková, K.; Pospíšilová, M.; Školová, B.; Zbytovská, J.; Vávrová, K. Effects of sphingomyelin/ceramide ratio on the permeability and microstructure of model stratum corneum lipid membranes. Biochim Biophys Acta 2014, 1838 (8), 2115. (29) Pullmannová, P.; Pavlíková, L.; Kováčik, A.; Sochorová, M.; Školová, B.; Slepička, P.; Maixner, J.; Zbytovská, J.; Vávrová, K. Permeability and microstructure of model stratum corneum lipid membranes containing ceramides with long (C16) and very long (C24) acyl chains. under review. (30) Skolova, B.; Janusova, B.; Zbytovska, J.; Gooris, G.; Bouwstra, J.; Slepicka, P.; Berka, P.; Roh, J.; Palat, K.; Hrabalek, A.; Vavrova, K. Ceramides in the skin lipid membranes: length matters. Langmuir 2013, 29 (50), 15624. (31) Školová, B.; Kováčik, A.; Tesař, O.; Opálka, L.; Vávrová, K. Phytosphingosine, sphingosine and dihydrosphingosine ceramides in model skin lipid membranes: permeability and biophysics. Biochim. Biophys. Acta Biomembranes 2017.


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(32) Janusova, B.; Zbytovska, J.; Lorenc, P.; Vavrysova, H.; Palat, K.; Hrabalek, A.; Vavrova, K. Effect of ceramide acyl chain length on skin permeability and thermotropic phase behavior of model stratum corneum lipid membranes. Biochim Biophys Acta 2011, 1811 (3), 129. (33) de Jager, M.; Groenink, W.; Bielsa i Guivernau, R.; Andersson, E.; Angelova, N.; Ponec, M.; Bouwstra, J. A novel in vitro percutaneous penetration model: evaluation of barrier properties with p-aminobenzoic acid and two of its derivatives. Pharm Res 2006, 23 (5), 951. (34) Skolova, B.; Jandovska, K.; Pullmannova, P.; Tesar, O.; Roh, J.; Hrabalek, A.; Vavrova, K. The role of the trans double bond in skin barrier sphingolipids: permeability and infrared spectroscopic study of model ceramide and dihydroceramide membranes. Langmuir 2014, 30 (19), 5527. (35) Mitragotri, S. Modeling skin permeability to hydrophilic and hydrophobic solutes based on four permeation pathways. J Control Release 2003, 86 (1), 69. (36) Mendelsohn, R.; Moore, D. J. Vibrational spectroscopic studies of lipid domains in biomembranes and model systems. Chem Phys Lipids 1998, 96 (1-2), 141. (37) Školová, B.; Hudská, K.; Pullmannová, P.; Kováčik, A.; Palát, K.; Roh, J.; Fleddermann, J.; Estrela-Lopis, I.; Vávrová, K. Different Phase Behavior and Packing of Ceramides with Long (C16) and Very Long (C24) Acyls in Model Membranes: Infrared Spectroscopy Using Deuterated Lipids. J Phys Chem B 2014, 118 (35), 10460. (38) Bouwstra, J. A.; Gooris, G. S.; van der Spek, J. A.; Bras, W. Structural investigations of human stratum corneum by small-angle X-ray scattering. J. Invest. Dermatol. 1991, 97 (6), 1005. (39) White, S. H.; Mirejovsky, D.; King, G. I. Structure of lamellar lipid domains and corneocyte envelopes of murine stratum corneum. An X-ray diffraction study. Biochemistry 1988, 27 (10), 3725. (40) Bouwstra, J. A.; Ponec, M. The skin barrier in healthy and diseased state. Biochim Biophys Acta 2006, 1758 (12), 2080. (41) Opalka, L.; Kovacik, A.; Maixner, J.; Vavrova, K. Omega-O-Acylceramides in Skin Lipid Membranes: Effects of Concentration, Sphingoid Base, and Model Complexity on Microstructure and Permeability. Langmuir 2016, 32 (48), 12894. (42) Motta, S.; Monti, M.; Sesana, S.; Mellesi, L.; Ghidoni, R.; Caputo, R. Abnormality of water barrier function in psoriasis. Role of ceramide fractions. Arch Dermatol 1994, 130 (4), 452. (43) De Paepe, K.; Houben, E.; Adam, R.; Wiesemann, F.; Rogiers, V. Validation of the VapoMeter, a closed unventilated chamber system to assess transepidermal water loss vs. the open chamber Tewameter. Skin Res Technol 2005, 11 (1), 61. (44) Netzlaff, F.; Kostka, K.-H.; Lehr, C.-M.; Schaefer, U. F. TEWL measurements as a routine method for evaluating the integrity of epidermis sheets in static Franz type diffusion cells in vitro. Limitations shown by transport data testing. European Journal of Pharmaceutics and Biopharmaceutics 2006, 63 (1), 44. (45) Vávrová, K.; Hrabálek, A.; Mac-Mary, S.; Humbert, P.; Muret, P. Ceramide analogue 14S24 selectively recovers perturbed human skin barrier. British Journal of Dermatology 2007, 157 (4), 704. (46) Kopečná, M.; Macháček, M.; Prchalová, E.; Štěpánek, P.; Drašar, P.; Kotora, M.; Vávrová, K. Dodecyl amino glucoside enhances transdermal and topical drug delivery via reversible interaction with skin barrier lipids. Pharm Res 2017, in press.


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Effects of 6-Hydroxyceramides on the Thermotropic Phase Behavior

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