Omega-O-Acylceramides in Skin Lipid Membranes: Effects of

This is an open access article published under an ACS AuthorChoice License, which permits copying and redistribution of the article or any adaptations for non-commercial purposes.

Article pubs.acs.org/Langmuir

Omega‑O‑Acylceramides in Skin Lipid Membranes: Effects of Concentration, Sphingoid Base, and Model Complexity on Microstructure and Permeability Lukás ̌ Opálka,† Andrej Kovácǐ k,† Jaroslav Maixner,‡ and Kateřina Vávrová*,† †

Faculty of Pharmacy, Charles University, Hradec Králové 500 05, Czech Republic University of Chemistry and Technology Prague, Prague 166 28, Czech Republic

‡

ABSTRACT: Omega-O-acylceramides (acylCer), a subclass of sphingolipids with an ultralong N-acyl chain (from 20 to 38 carbons, most usually 30 and 32 carbons), are crucial components of the skin permeability barrier. AcylCer are involved in the formation of the long periodicity lamellar phase (LPP, 12−13 nm), which is essential for preventing water loss from the body. Lower levels of acylCer and LPP accompany skin diseases, such as atopic dermatitis, lamellar ichthyosis, and psoriasis. We studied how the concentration and structure of acylCer influence the organization and permeability barrier properties of model lipid membranes. For simple model membranes composed of the sphingosine-containing acylCer (EOS), N-lignoceroyl sphingosine, lignoceric acid, cholesterol (Chol), and cholesteryl sulfate (CholS), the LPP formed at 10% Cer EOS (of the total Cer) and the short periodicity phase disappeared at 30% Cer EOS. Surprisingly, membranes with the LPP had higher permeabilities than the control membrane without acylCer. In the complex models consisting of acylCer (EOS, phytosphingosine EOP, dihydrosphingosine EOdS, or their mixture; at 10% of the total Cer), a six-component Cer mixture, a free fatty acid mixture, cholesterol (Chol), and cholesteryl sulfate (CholS), acylCer decreased the membrane permeability to model permeants (with the strongest effects for acylCer EOP and EOdS) when compared with the permeability of the control membrane without acylCer. However, in the complex model, only a mixture of acylCer EOS, EOdS, and EOP and not the individual acylCer formed both the LPP and orthorhombic chain packing at the 10% level. Thus, the relationships between acylCer, LPP formation, and permeability barrier function are not trivial. Lipid heterogeneity is essentialonly the most complex model with nine Cer subclasses mimicked both the organization and permeability of stratum corneum lipid membranes.

■

CholS.18 SC Cer are a structurally heterogeneous lipid group: 15 subclasses of SC Cer have been identified in humans.19 Human Cer can be based on the amino alcohols sphingosine, phytosphingosine, dihydrosphingosine, or 6-hydroxysphingosine or a tetrahydroxylated sphingoid base, which has not yet been fully characterized.17 The chain lengths of the sphingoid bases are usually 18 carbons, but other chain lengths are possible. It should be noted that not all mammals contain 6hydroxysphingolipids.20 These amino alcohols are acylated by three different types of FFAs: nonhydroxylated, α-hydroxylated, and ω-hydroxylated. Cer can further be esterified by linoleic acid at the ω-hydroxyl group21 or by a nonhydroxylated FFA at the hydroxyl group at position 1 of the sphingoid base.22 For the nomenclature, see Figure 1.23 Omega-O-acylceramides (acylCer; also known as Cer of the EO class) that have ultralong N-acyl chains (up to 38 carbons, most usually 30 and 32 carbons24,25) with an ω-hydroxyl group

INTRODUCTION Ceramides (Cer) belong to a family of sphingolipids. Cer are important regulators of cellular processes1 and essential components of the skin permeability barrier.2 This barrier resides in the uppermost epidermal layer, the stratum corneum (SC), which consists of corneocytes surrounded by a lamellar lipid matrix.3 To prevent excess water loss and the entry of undesired compounds from the environment, organization of the SC lipids is highly specialized. Unlike other biological membranes, this SC lipid matrix contains two coexisting lamellar periodicity phases: a short periodicity phase (SPP) with a repeat distance of 5.3−6.4 nm4−8 and a long periodicity phase (LPP) with a repeat distance of 11.9−13.1 nm.5,7,9−12 On the other hand, very high resolution cryo-electron microscopy on native skin showed that only a single LPP with a repeat distance of approximately 11 nm exists in the SC intercellular space.13 Cer are the dominant permeability barrier lipids, constituting 50% of the SC lipids by weight.14−17 Apart from Cer, the SC lipids include free fatty acids (FFAs) and Chol in an approximately 1:1:1 molar ratio, with a minor amount of © XXXX American Chemical Society

Received: August 18, 2016 Revised: November 9, 2016 Published: November 9, 2016 A

DOI: 10.1021/acs.langmuir.6b03082 Langmuir XXXX, XXX, XXX−XXX

Article

Langmuir

Figure 1. Panel A: structures of lipids in the model membranes and the compositions of Cer EO-mix, Cer-mix, and FFA-mix. Panel B: composition of the simple and complex models.

method for the efficient and scalable synthesis of acylCer that opens the possibility of further exploring their properties.36 The aim of this study was to investigate the relationships between the acylCer concentration, the acylCer polar head structure, the formation of lamellar phases, and the permeability of model skin lipid membranes using two levels of complexity. Such SC lipid membrane models show a high level of similarity to human SC.37−40 First, we studied the effects of different concentrations of Cer EOS in a simple lipid membrane model composed of Cer EOS, N-lignoceroyl-sphingosine (Cer NS), lignoceric acid (LA), Chol, and CholS (Figure 1). Formation of the LPP (studied using X-ray powder diffraction, XRPD) upon the addition of Cer EOS resulted in a higher permeability of these membranes compared with that of the control with SPP only. Thus, we constructed a more complex model that closely mimicked the composition of human SC lipids (Figure 1). In this experiment, we studied the contribution of the individual acylCer (namely, Cer EOS, EOdS, and EOP) and their mixture to the function and organization of the membrane permeability barrier. The membrane permeability was evaluated using four markers: transmembrane water loss, electrical impedance, and steady-state flux values of two model permeants with different lipophilicities.

esterified by linoleic acid are unique to the human epidermis.26,27 Different authors have indicated that acylCer constitute 7−12% (mean 9%) of the total SC Cer. Most acylCer contain sphingosine (Cer EOS; 4.6%), with the next most common being 6-hydroxysphingosine (Cer EOH; 3.1%), phytosphingosine (Cer EOP; 1.0%), and dihydrosphingosine (Cer EOdS; 0.3%) (Figure 1).14−17,20 Major defects in acylCer biosynthesis lead to neonatal death through massive transepidermal water loss (TEWL),28,29 likely because acylCer are necessary for the development of the LPP and also for the corneocyte lipid envelope.30−32 In addition, alterations in the LPP, such as shortening of the repeat distance or a complete lack of this phase, were found in skin diseases, such as atopic dermatitis,33 lamellar ichthyosis,34 psoriasis,23 and Netherton syndrome.35 The common denominator of these diseases is an altered level of SC lipids, mostly a decrease in the level of acylCer, and disturbed skin permeability barrier homeostasis. Despite the considerable effort that has gone into research on the roles of acylCer in the permeability barrier,5,7,9,10,23,30−34 the contribution of each acylCer subclass to the development of the LPP and the direct links between the LPP and the skin/ membrane permeability are not completely understood because acylCer are not commercially available and are extremely difficult to isolate from the skin. We have recently reported a

■

EXPERIMENTAL SECTION

Chemicals and Materials. AcylCer [namely, Cer EOS(d18:1/ h32:0/18:2), Cer EOP(t18:0/h32:0/18:2), and Cer EOdS(d18:0/ B


Article

Langmuir h32:0/18:2)] were prepared according to a published procedure.36 Cer NS(d18:1/24:0) (N-lignoceroyl sphingosine), NP(t18:0/24:0) (Nlignoceroyl phytosphingosine), AS(d18:1/h24:0) (N-((R)-2-hydroxylignoceroyl sphingosine)), and AP(t18:0/h24:0) (N-((R)-2-hydroxylignoceroyl phytosphingosine)) were purchased from Avanti Polar Lipids (Alabaster, AL, USA). Cer NdS(d18:0/24:0) (N-lignoceroyl dihydrosphingosine) and Cer AdS(d18:0/h24:0) (N-((R)-2-hydroxylignoceroyl dihydrosphingosine)) were prepared by the acylation of dihydrosphingosine by LA or 2-hydroxylignoceric acid using N-(3dimethylaminopropyl)-N′-ethylcarbodiimide (WSC) in the presence of N-hydroxysuccinimide. LA, docosanoic acid, eicosanoic acid, octadecanoic acid, hexadecanoic acid, Chol from lanolin, CholS, theophylline (TH), indomethacin (IND), gentamicin sulfate, sodium phosphate dibasic dodecahydrate, propylene glycol, and other solvents were purchased from Sigma-Aldrich Chemie Gmbh (Schnelldorf, Germany). All solvents were of analytical or high-performance liquid chromatography (HPLC) grade. Nuclepore track-etched polycarbonate membranes of 0.015 μm pore size were purchased from Whatman (Kent, Maidstone, UK). Water was purified using a Milli-Q system (Merck Millipore, Billerica, MA, USA). Preparation of Model SC Membranes. Two different SC lipid membrane models, containing equimolar mixtures of Cer, FFAs, and Chol with a 5 wt % addition of CholS, were prepared. This amount of CholS mimics the CholS/sphingolipids ratio in healthy skin, which is approximately 1:10.41 The simple models consisted of Cer EOS, Cer NS, LA, Chol, and CholS. As a simple control model, a membrane without Cer EOS was used. The molar ratios of the individual Cer are given in Figure 1. The complex models were constructed from acylCer, a sixcomponent mixture of Cer (Cer-mix), a mixture of FFAs (FFAmix), Chol, and CholS in the same molar ratios as described above. The compositions of Cer-mix14−17 and FFA-mix42 were selected to mimic the composition of these lipid classes in the SC as closely as possible and are specified in Figure 1. Because of the unavailability of acylCer and Cer based on 6-hydroxysphingosine, that is, Cer EOH, Cer NH, and Cer AH, these Cer were not included in the models; Cer AS and Cer AP were used instead of Cer NH and Cer AH, respectively. In this model, the studied acylCer were either sphingosine Cer EOS, phytosphingosine Cer EOP, dihydrosphingosine Cer EOdS, or their mixture (Cer EO-mix). As a negative control, a membrane without acylCer was used, and it contained Cer-mix, FFA-mix, Chol, and a 5 wt % addition of CholS (Figure 1). The lipid mixture (1.35 mg) was dissolved in 400 μL of hexane/ 96% ethanol 2:1 (v/v) and was sprayed on a cover glass (22 × 22 mm2) for XRPD or on a nuclepore polycarbonate filter for permeability experiments. The lipid solution was sprayed in four portions (4 × 100 μL) under a stream of nitrogen on the glass/filter using a Linomat V (Camag, Muttenz, Switzerland) equipped with an additional y-axis movement.42 The flow rate was 10.2 μL/min, and the sprayed area was 1 cm2. The lipid films were heated at 90 °C for 10 min and then slowly (3−4 h) cooled down to 32 °C. The membranes were equilibrated at 32 °C at 40−50% humidity for at least 24 h before experiments.39,40 To verify the uniform lipid composition in the membrane, the center of the membrane on the polycarbonate filter was punched out. The lipids from the center and from the remaining membrane periphery were each extracted using a chloroform/ methanol 2:1 (v/v) mixture (Pullmannová, unpublished results) and quantified using high-performance thin-layer chromatography.43 The lipid distribution was homogeneous, and the ratio of the individual lipid classes did not differ between the central area and the membrane periphery. X-ray Powder Diffraction. XRPD of the studied lipid membranes was performed on an X’Pert Pro θ−θ powder diffractometer (PANalytical B.V., Almelo, the Netherlands) with the parafocusing Bragg−Brentano geometry using Cu Kα 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 using an ultrafast detector X’Celerator with a step size of 0.0167° (2θ) and a counting time of 20.32 s/step. The data were evaluated using an X’Pert Data Viewer (PANalytical B.V., Almelo, the Netherlands). The XRPD diffracto-

grams 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 sin θ/λ (λ = 0.15418 nm is the wavelength of the X-rays). The repeat distance d (nm) characterizes the regular spacing of parallel lipid bilayers arranged on a onedimensional lattice. This lipid arrangement is called a lamellar phase (L). The diffractograms of lamellar phases exhibit a set of Bragg reflections whose reciprocal spacings are in the 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 linear regression fit of the dependence Qn = a × n, according to the equation d = 2π/a. Permeation Experiments. Franz-type diffusion cells with an acceptor volume of 6.5 ± 0.1 mL (the precise volume was measured for each cell and was included in the flux calculation) were used for the permeability experiments. Lipid membranes were fixed in Teflon holders with a diffusion area of 0.5 cm2, with the lipid membrane facing the donor compartment and mounted on the Franz cells. The acceptor compartment was filled with phosphate-buffered saline at pH 7.4 with 50 mg/L of gentamicin, and the membranes were equilibrated at 32 °C for 12 h. After 12 h, the transmembrane water loss and electric impedance were measured (see below). Next, 100 μL of the donor suspension (5% TH or 2% IND in 60% propylene glycol, prepared according to refs 39 and 40) was applied to the membrane. Samples of the acceptor phase (300 μL) were collected every 2 h over 10 h and replaced with the same amount of the acceptor solution.40 Typical lag times were less than 0.5 h; thus, 10 h was sufficient to reach a steady state. In all experiments, sink conditions were maintained, and the polycarbonate filter had no effect on the membrane permeability.39 Furthermore, 100 μL of 60% propylene glycol did not extract any lipids from the membranes (Pullmannová, unpublished results), which was verified using high-performance thinlayer chromatography. Water Loss through the Membranes. The water loss through the membranes was measured using a Tewameter TM 300 probe and a multiprobe adapter Cutometer MPA 580 (CK electronic GmbH, Köln, Germany). The probe was placed on top of the Teflon holder (after the donor part of the cell was temporarily removed) with an effective area of 0.5 cm2 and with a distance of 0.6 cm to the probe. The measuring time was 80 s, and the average steady state value (g/h/ m2) was recorded. The measurements were carried out at 26−29 °C and 40−46% relative humidity in a box that precluded ambient air flow. Electrical Impedance. The electrical impedance was measured using a 4080 LCR meter (Conrad Electronic, Hirschau, Germany) at an alternating frequency of 120 Hz. The measuring range of the instrument was 20 Ω to 10 MΩ, with an error at kΩ values less than 0.5%. For our experiments, 500 μL of phosphate-buffered saline was applied to the donor compartment of the diffusion cell, and the impedance (in kΩ × cm2) was measured using stainless steel probes placed in the donor and acceptor phases. At the end of the measurements, the buffer was carefully removed. HPLC. The collected samples from the permeation experiments with TH or IND were analyzed using isocratic reverse-phase HPLC using a Shimadzu Prominence instrument (Shimadzu, Kyoto, Japan) consisting of LC-20AD pumps with a DGU-20A3 degasser, SIL-20A HT autosampler, CTO-20AC column oven, SPDM20A diode array detector, and CBM-20A communication module. Data were analyzed using the LCsolution 1.22 software. Reverse-phase separation of TH was achieved on a LiChroCART 250-4 column (LiChrospher 100 RP18, 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. An acceptor-phase sample (20 μL) was injected into the column, and the UV absorption of the effluent was measured at 272 nm, with a bandwidth of 4 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. Then, 100 μL of the acceptor-phase sample was injected into the column, which was maintained at 40 °C. The UV absorption C


Article

Langmuir 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.40,44 Fourier-Transform Infrared Spectroscopy. Infrared spectra of selected lipid membranes were recorded on a Nicolet Impact 400 spectrometer (Thermo Scientific, USA) equipped with a singlereflection 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 recorded at a 2 cm−1 resolution. The temperature dependence of the IR spectra was studied over the range of 28−100 °C with 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. Data Treatment. The cumulative amounts of TH and IND that penetrated through the membrane were calculated from the concentration measured using HPLC and the Franz cell volume and were corrected for the acceptor-phase replacement. The cumulative amounts were plotted against time, and the steady-state flux of TH or IND (μg/cm2/h) was calculated as the slope obtained using linear regression to fit the linear region of the plot using Microsoft Excel. Water loss data were normalized by dividing the water loss measured (g/h/m2) by the average water loss of the control membranes (g/h/ m2) and multiplying by 100%. The electrical impedance values were normalized to the control in the same manner. All data are presented as mean ± standard error of mean (SEM). One-way analysis of variance (ANOVA) with Dunnett’s post-test or t-test as indicated in the pertinent figures was used for statistical analysis, and p < 0.05 was considered to be significant.

■

RESULTS LPP Starts to Form at 10% Cer EOS in the Simple Model. First, the lipid membranes of the simple model were studied using XRPD to establish how the concentration of Cer EOS influences the microstructure of these model membranes. Thus, four models with increasing amounts of Cer EOS (from 5 to 30%) were compared with those of a control without Cer EOS (Figure 2). It should be noted that the concentration of Cer EOS refers to its percentage relative to the sphingolipid fraction, not to the total lipids (Figure 1). The control sample (0% Cer EOS) and the sample with 5% Cer EOS contained a series of reflections with repeat distances of d = 5.40 ± 0.00 and 5.50 ± 0.06 nm, respectively (Figure 2A). This phase (La) has a repeat distance very similar to that of the SPP found in the human SC, which has a repeat distance of 5.3−6.4 nm.4−8,45 Furthermore, the diffractograms contained two reflections of the separated Chol, which is consistent with the findings in the pig and human SC.39,45,46 Separated Chol was found in all simple membranes, which had Cer EOS contents from 0 to 30% and d ranging from 3.39 to 3.44 nm (Figure 2B). Interestingly, the relative intensities of the Chol reflections compared with those of the SPP sharply decreased upon the incorporation of 5% Cer EOS. The membrane with 10% Cer EOS contained an La phase similar to that of the SPP, with a repeat distance d = 5.42 ± 0.02 nm. In addition, another lamellar phase (Lb) started to form at 10% Cer EOS. This Lb phase had a repeat distance larger than the La phase, with d = 12.81 nm, which is consistent with the LPP.45 When the amount of Cer EOS increased from 10 to 20%, the intensity of the Lb phase (d = 12.35 ± 0.25 nm, seven reflections) when compared with that of the La phase increased. At 20% Cer EOS, only two weak reflections of the La phase with d = 5.39 ± 0.03 nm were found. As the level of Cer EOS increased even further, the La phase completely disappeared from the diffractogram, and only the Lb phase

Figure 2. Microstructure of the simple skin lipid membranes composed of Cer EOS, Cer NS, LA, Chol, and CholS with the proportions of Cer EOS in the Cer fraction ranging from 0 (control) to 30%. Panel A: X-ray powder diffractograms of the simple membranes. Arabic numerals refer to the La phase (SPP), roman numerals refer to the Lb phase (LPP), and asterisks refer to separated Chol. Panel B: repeat distances of the lamellar phases La (SPP), Lb (LPP), and Chol as a function of Cer EOS concentration. Panel C: ratio of the full width at the half-maximum of the second and first orders of the lamellar phase La (SPP) as a function of the Cer EOS concentration (mean ± SEM).

with the repeat distance d = 12.22 ± 0.01 nm was found together with Chol (Figure 2B). The extent of bilayer fluctuations in the membrane (Figure 2C) could be estimated using the ratio of the full width at the half-maximum of the second (Δs2)- and first (Δs1)-order reflections. In our membranes, this value could be calculated only for the La phase because the first reflection of the Lb phase was absent or very weak. The Δs2/Δs1 ratio of the La phase in the control membrane was 0.86 ± 0.00. When the concentration of Cer EOS increased, the ratio dropped to 0.75 ± 0.17 for 5% EOS and then increased to 0.89 ± 0.06 for 10% EOS and 1.37 ± 0.23 for 20% Cer EOS. Thus, the fluctuations in the La phase decreased upon the incorporation of 5% Cer EOS but increased when the LPP formed. Cer EOS Increases the Permeability of the Simple Model. Then, we investigated how the percentage of Cer EOS and, consequently, the formation of lamellar phases in the simple skin lipid membrane models influence their permeability. Membrane permeability was assessed using four markers: the steady-state flux of TH, the steady-state flux of IND, the transmembrane water loss, and the electrical impedance. D


Article

Langmuir The TH flux through the simple control membrane without Cer EOS was 0.07 ± 0.01 μg/cm2/h (Figure 3A). The presence

impedance (not significant). The impedance of the membrane with 30% Cer EOS was three times higher than the control impedance (p < 0.01), which indicates less movement of ions through the membrane.48 Incorporation of 5% Cer EOS into the Simple Lipid Model Decreases the Lipid Chain Order and the Proportion of Orthorhombic Chain Packing. To gain insights into the molecular basis for the increased permeability of the simple lipid model with 5% Cer EOS when compared with that of the control (which corresponds to a 1.67% change in the total lipid compositions), we used infrared spectroscopy. Five percent Cer EOS caused a shift of the methylene stretching vibrations at 32 °C to higher wavenumbers when compared with the control (2848.6 ± 0.1 and 2847.9 ± 0.0 cm−1; Figure 4A). This suggests lipid disordering, that is,

Figure 3. Permeability markers of the simple skin lipid membranes composed of Cer EOS, Cer NS, LA, Chol, and CholS with the proportions of Cer EOS in the Cer fraction ranging from 0 (control) to 30%. Panel A: steady-state flux of TH, panel B: steady-state flux of IND, panel C: transmembrane water loss relative to the control, and panel D: electrical impedance relative to the control. Data are presented as the mean ± SEM. Asterisks indicate statistically significant differences against the control at p < 0.01 (**) and p < 0.05 (*).

Figure 4. Infrared spectroscopy of the simple model membranes composed of Cer EOS (0 and 5%), Cer NS, LA, Chol, and CholS. The thermal evolution of the symmetric methylene stretching vibration (panel A; means ± SEM) and methylene rocking vibration at 32 °C (panel B; representative spectra).

of 5 and 10% Cer EOS more than doubled the flux of TH (significant at p < 0.01) to values of 0.15 ± 0.02 and 0.15 ± 0.01 μg/cm2 /h, respectively. Further increases in the proportion of Cer EOS to 20 and 30% led to a decrease in the flux to 0.12 ± 0.01 and 0.08 ± 0.01 μg/cm2/h, respectively (not significantly different from the control). A similar bell-shaped trend in permeability was found for the steady-state flux of IND through the simple model membranes (Figure 3B). The flux of IND through the control membrane was 0.05 ± 0.01 μg/cm2/h. The presence of Cer EOS increased the flux of IND with maxima at 10 and 20% Cer EOS (0.11 ± 0.01 and 0.12 ± 0.01 μg/cm2/h, respectively; p < 0.01 when compared with the control). In the membrane with 30% Cer EOS, the flux decreased to 0.08 ± 0.01 μg/cm2/h, which is still 60% higher than the flux in the control membrane, although the difference was not significant. The third permeability marker was the transmembrane water loss (Figure 3C), which was measured using an evaporimeter that is commonly used for the measurement of TEWL in vivo, but it can also be used for in vitro experiments.47 The water loss of the control membrane was set at 100%, and the other values were normalized to the control. The presence of Cer EOS in the membranes led to higher water loss through these membranes when compared with the control with a maximum at 10% Cer EOS (37% higher than the control; the difference was not significant). The last parameter of the membrane permeability was the electrical impedance (Figure 3D), which is the opposition of the membrane to an alternating current. Transmembrane/skin current is mediated by the movement of charge-carrying ions and is related to the membrane/skin permeability. Membranes with 5, 10, and 20% Cer EOS had impedance values that were 50, 64, and 52%, respectively, which are higher than the control

incorporation of some gauche defects into the chains.49 The phase-transition temperature was broader with 5% Cer EOS when compared with that of the control, which is consistent with altered lipid mixing. The arrows in Figure 4B show the decreased relative intensity of the higher wavenumber peak of the methylene rocking doublet upon the incorporation of 5% Cer EOS into the membrane, which indicates that the proportion of orthorhombic packing decreased. Complex Model Better Mimics the Effect of AcylCer on the Permeability. The above-mentioned effects of Cer EOS and LPP formation on the membrane permeability were highly unexpected. We hypothesized that the reason for this behavior was that the above-mentioned models were too simple, that is, they contained only five lipid subclasses. Thus, we prepared a more complex model with six to nine Cer subclasses and five FFA subclasses.42 AcylCer were incorporated into the models at 10% (of the Cer fraction), which should be sufficient for the formation of LPP and is close to the physiological value.14−17 The permeability of the complex model was studied using the same four permeability markers as for the simple model (Figure 5). First, the permeability of the complex models to TH was studied. Figure 5A shows the permeation profiles (cumulative amounts of TH that permeated through 1 cm2 in time), and Figure 5B shows the flux of TH calculated from these profiles. The flux of TH through the control membrane was 0.36 ± 0.05 μg/cm2/h. Compared with that of the control, the incorporation of 10% acylCer decreased the TH flux: membranes with Cer EO-mix, Cer EOS, Cer EOP, and Cer EOdS had TH flux values of 0.24 ± 0.02 (not significant), 0.26 ± 0.07 (not E


Article

Langmuir

Only Cer EO-Mix, Not the Individual AcylCer, Forms the LPP in the Complex Model. To confirm that acylCerinduced decreases in the permeabilities of the complex model membranes to TH, IND, and water were consistent with the LPP formation, we performed XRPD measurements for these membranes (Figure 6). In all diffractograms, first- and second-

Figure 5. Permeability markers for the complex skin lipid membranes composed of Cer EO-mix, Cer-mix, FFA-mix, Chol, and CholS with 10% Cer EO-mix in the Cer fraction. Panel A: permeation profiles of TH, panel B: steady-state flux of TH, panel C: permeation profiles of IND, panel D: steady-state flux of IND, panel E: transmembrane water loss relative to the control, and panel F: electrical impedance relative to the control. Data are presented as mean ± SEM. Asterisks indicate statistically significant differences when compared with the controls at p < 0.01 (**) and p < 0.05 (*), ANOVA. The symbol # indicates a statistically significant difference when compared with the control at p < 0.05, t-test.

significant), 0.17 ± 0.02 (p < 0.01), and 0.14 ± 0.02 (p < 0.01) μg/cm2/h, respectively. When IND was used as a model permeant (Figure 5C,D), the effects of 10% acylCer were similar to those for the TH measurements: the control membrane without acylCer had the highest flux (0.25 ± 0.04 μg/cm2/h), whereas Cer EO-mix, Cer EOS, Cer EOP, and Cer EOdS decreased the IND flux to 0.12 ± 0.02 μg/cm2/h (p < 0.05), 0.17 ± 0.05 μg/cm2/h (not significant), 0.07 ± 0.01 μg/cm2/h (p < 0.01), and 0.08 ± 0.01 μg/cm2/h (p < 0.01), respectively. Similar trends were observed for the relative water loss (Figure 5E). The minima (by 16 and 20% less water loss than the control) were found in the membranes containing Cer EOmix and Cer EOdS, respectively. The electric impedance (Figure 5F) gave a different result: when compared with the control, all acylCer membranes had markedly (by 65−82%) decreased impedance, which means less opposition to electrical current, which indicates less opposition to the movement of ions through this complex membrane with 10% Cer EOS when compared with the control membrane.48 The reasons for these effects of acylCer on the membrane impedance are unknown, and we can only speculate that they might be related to domain formation.

Figure 6. Microstructure of the complex skin lipid membranes composed of Cer EO-mix, Cer-mix, FFA-mix, Chol, and CholS with 10% Cer EO-mix in the Cer fraction. Panel A: X-ray powder diffractograms of the complex membranes. Arabic numerals refer to the La phase (SPP), roman numerals refer to the Lb phase (LPP), and asterisks refer to separated Chol. The arrows show weak peaks that indicate the presence of orthorhombic packing. Panel B: repeat distances of lamellar phases La (SPP), Lb (LPP), and Chol of individual acylCer or their mixture. Panel C: ratio of the full width at the half-maximum of the second and first orders of the lamellar phase La (SPP) of individual acylCer or their mixture (mean ± SEM).

order reflections of separated Chol were found. The repeat distances of this Chol phase were d = 3.41−3.42 nm (Figure 6B). This result is similar to the above findings in the simple model and is consistent with crystalline Chol monohydrate.50 In the control membrane, only one series of reflections that corresponds to the La phase was found (in addition to Chol). The repeat distance of this phase (La) was d = 5.38 ± 0.01 nm, which is consistent with the SPP (d = 5.3−6.4 nm).4−8,45 The diffractograms of the membranes with a single acylCer, Cer EOS, Cer EOP, or Cer EOdS were surprisingly similar to the control diffractogram: only the SPP phase (La) with repeat F


Article

Langmuir distances d = 5.36 ± 0.02, 5.37 ± 0.00, and 5.37 ± 0.00 nm, respectively, and separated Chol were found. At Q = 6.75 nm−1, an unknown reflection was present. We also measured a set of membranes heated to 70 °C instead of 90 °C, but we found essentially the same results as in Figure 6. The only complex model that formed LPP was the membrane containing 10% Cer EO-mix. The first series of peaks (La) with a repeat distance d = 5.36 ± 0.01 nm was assigned to the SPP. The second phase, Lb, with a repeat distance d = 12.71 ± 0.00 nm was assigned to the LPP (Figure 6A,B). In addition to these two phases and Chol, unidentified peaks were found at Q = 1.36 and 6.75 nm−1. The fluctuations of the La phase were estimated using the complex model as described above (Figure 6C). The Δs2/Δs1 ratio in the control membrane was 0.83 ± 0.08, which is similar to the value for the simple model. These ratios were slightly decreased in the presence of Cer EO-mix and Cer EOS (0.70 ± 0.27 and 0.67 ± 0.17, respectively) and increased in membranes with Cer EOP and Cer EOdS (0.92 ± 0.08 and 0.98 ± 0.23, respectively). All complex membranes had Δs2/Δs1 ratios below 1, which indicated a well-ordered lamellar arrangement. Orthorhombic Lipid Packing in All Simple Skin Lipid Membranes, but Only in Cer EOS-Containing Complex Membranes. The wide-angle regions of the diffractograms (with Q ranging from 14 to 18 nm−1) are shown in Figure 2A and on the right of Figure 6A. This part of the diffractogram provides information on the short-range molecular arrangement, in this case, on the lateral packing of the lipid hydrocarbon chains. In all simple models with or without Cer EOS, two peaks were found at Q = 15.13−15.24 and 16.75− 16.84 nm−1. The corresponding distances between lattices (0.41−0.42 and 0.37−0.38 nm, respectively) were assigned to the orthorhombic chain packing of the lipid chains.51 In the complex model, the situation changed. The peaks indicating the orthorhombic chain packing were clearly detected only in the membrane containing 10% Cer EOS and Cer EO-mix (marked by an arrow in Figure 6A). The relative intensity of the peak at Q = 16.79 nm−1 was very low. The control membrane and the membranes containing 10% Cer EOP or Cer EOdS showed only one peak at Q = 15.13−15.24 nm−1, which might indicate less tight hexagonal chain packing5,52,53 or be caused by the detection limit of our measurement. Nevertheless, only those complex skin lipid models that contained Cer EOS (either alone or in Cer EOmix) clearly form some orthorhombic lateral chain packing at 10% acylCer.

AS24, Cer NP16, Cer AP24, FFA mixture, and Chol and found similar permeabilities of this model and isolated SC to paminobenzoic acid and its esters.37 We compared the effects of Cer with shortened chains (both acyl and sphingosine) in skin and simple lipid membranes composed of Cer NS24, LA, Chol, and CholS and found similar trends in permeabilities to TH and IND.38,39,54 Furthermore, a lipid model composed of Cer NS24, FFA, and Chol (with or without CholS) confirmed the existence of fully extended Cer with Chol molecules associated with the Cer sphingoid moiety and FFA with the acyl chains55 suggested using very high resolution cryo-electron microscopy of native skin.13 Simple Model Membranes Closely Mimic the SC Lipid Microstructure but Not the Permeability. First, we used XRPD on a simple SC membrane to verify that the incorporation of Cer EOS promoted the formation of the LPP. The purpose of this experiment was also to determine the concentration of acylCer (Cer EOS) that is necessary for the formation of LPP in our simple model. In the control membrane without acylCer, only SPP was found, which is in agreement with the central role of acylCer in the LPP arrangement in the SC.32 In addition, the control (and all other membranes) contained separated Chol, which was found in model SC lipid membranes50,56 and isolated SC57 using XRPD. Although some authors suggest the separation of Chol into crystals, no such crystals were found by using very high resolution cryo-electron microscopy on real skin.13 This separation of some Chol molecules might not be a formation of Chol crystals but only a formation of Chol domains within the lipid membranes. The lower acylCer content in skin diseases was associated with the lack of the LPP.58 In our membranes with 5% Cer EOS, which is approximately half of the physiological acylCer content, only the SPP lamellar phase was found. Thus, Cer EOS was likely incorporated into the SPP; this assumption is supported by the increased repeat distance and slightly increased order of this phase compared to the control. Another possible explanation is that 5% Cer EOS formed a structure that was not periodically organized and was therefore not detectable using XRPD. Our result is in contrast with that of de Jager et al., who constructed a model containing Cer EOS, Cer NP, bovine brain Cer (in a 1:7:2 molar ratio), an FFA mixture, and Chol and found that a small fraction of the LPP starts to form at 5% Cer EOS.6 This discrepancy may be caused by the different Cer used in that model and our model. When the concentration of Cer EOS was increased to 10 and 20%, the LPP appeared in the diffractograms together with the SPP and Chol. The formation of the LPP was accompanied by a decrease in the repeat distance of SPP and increased fluctuations in this phase, in particular, at 20% Cer EOS. The relative measure of the lipid fluctuations in the SPP at 10% Cer EOS was still less than 1, which indicates good membrane order (ratios greater than 2 that were typically reported for phospholipid membranes).59 Similar values were previously found for model skin lipid membranes that mimicked inadequate sphingomyelin processing.40 The coexistence of the SPP and LPP at 10−20% Cer EOS is consistent with the physiological skin lipid arrangement40,60 and with published model membranes. de Jager et al. studied a more complex model using a Cer mixture (Cer EOS, NS, NP, AS, NP16, and AP) containing 15% Cer EOS and found the LPP together with the SPP.30 For other skin lipid membrane models with 15% acylCer, the presence of both the SPP and

■

DISCUSSION AcylCer (Cer of the EO class, e.g., Cer EOS) are essential for proper permeability barrier function.23,33−35 Our understanding of the behavior of acylCer in the permeability barrier is limited by two drawbacks: first, the commercial unavailability of acylCer and, second, the highly complex lipid mixture in the permeability barrier that complicates detailed studies at the molecular level. We have recently developed a method for an efficient and scalable synthesis of acylCer to overcome the limited availability of these lipids.36 The second drawback can be (partially) overcome by using carefully selected lipid membrane models. Although all experimental models include a certain level of simplification, several models of the SC lipid membranes were shown to be useful in structure−permeability studies.39,40,52 For example, de Jager et al. studied a model lipid membrane composed of Cer EOS, Cer NS24, Cer NP24, Cer G


Article

Langmuir

Cer subclasses and five FFAs according to recent analyses of skin Cer14−17 and FFAs.42 This model simulated the composition of human SC more closely than did the simple model. On the basis of the physiological levels of acylCer and the microstructure of our simple model, the concentration of acylCer was set to 10% to include both the SPP and LPP. The increase in the complexity of the model membranes dramatically influenced their permeability. The permeabilities of our complex model (the flux values of TH and IND were 0.24 ± 0.02 and 0.12 ± 0.02 μg/cm2/h, respectively) are in very good agreement with those of a membrane model constructed from isolated human skin Cer (the flux values of TH and IND were 0.40 ± 0.05 and 0.23 ± 0.02 μg/cm2/h, respectively)40 and also with the permeabilities of isolated human epidermis under the same conditions (the flux values of TH and IND were 0.15 ± 0.05 and 0.05 ± 0.02 μg/cm2/h, respectively; unpublished data). However, the permeabilities of the control complex membranes were higher than those of the simple model. This finding suggests that our simple model indeed had a highly impermeable lipid organization, which, however, was prone to serious disturbances caused by minor changes in its lipid composition as suggested by the infrared spectroscopy of the 5% Cer EOS membrane. The apparent reinforcement of both the inward permeability barrier (against the flux of TH and IND) and outward permeability barrier (against the loss of water) by acylCer compared with that of the control membrane correlates well with the role of acylCer in SC. Imokawa et al. found a decrease in both the total levels of SC lipids and Cer (largest decrease in Cer EOS) in nonlesional and lesional skin of atopic patients.66 Further examination of atopic skin showed that a decrease in Cer EOS (and also Cer NP) was accompanied by a significantly increased TEWL.15,33,67 A significant decrease in the levels of Cer EOS23 and 2- to 3-fold higher TEWL47 were also found in psoriasis. Paige et al. found a statistically decreased Cer EOS in lamellar ichthyosis.34 Patients with lamellar ichthyosis also had significantly (2- to 3-fold) higher TEWL when compared with healthy volunteers.68 Interestingly, the effect of the most common acylCer with sphingosine as the base, Cer EOS, was the weakest of the studied acylCer. To probe the structure−activity relationships in acylCer, we replaced Cer EOS with Cer EOP and Cer EOdS. Both of these acylCer lack the trans double bond at carbon 4 of the sphingoid base, and Cer EOP has an additional hydroxyl at position 4. Both the phytosphingosine-based Cer EOP and the dihydrosphingosine-based Cer EOdS resulted in stronger permeability barrier reinforcement to TH and IND. By contrast, the effects of these acylCer on the water loss and impedance were similar to the Cer EOS effects. The reason for the difference in permeability between the membranes with various acylCer subclasses seems to be connected to the missing C4 trans double bond in Cer EOP and Cer EOdS. The double bond in sphingosine Cer decreases the phase transition temperature, which indicates lower cohesive forces in the lipid membrane.69 For acylCer, structure−activity relationships are scarce. In 2010, Kessner et al. compared pure Cer EOS with Cer EOP and found that Cer EOP was able to form the LPP even in the dry state in contrast to Cer EOS, which needed hydration for the formation of the LPP.10 de Jager et al. found that Cer EOS promotes the LPP formation more efficiently than does Cer EOP. However, their model membranes had a higher amount (15%) of acylCer than our membranes.30

LPP was reported.4,52,53 The repeat distance of the LPP was the largest at 10% Cer EOS and then slowly decreased. These repeat distances are consistent with the literature30 and are biologically relevant; for example, in reconstructed skin models supplemented with cytokines, periodicities ranging from 12.1 to 12.7 nm were found.61 In addition, orthorhombic chain packing, which is considered to be an indicator of good barrier function,53,62,63 was found in all simple membranes. At 30% Cer EOS, the SPP lamellar phase disappeared. The presence of the LPP without the SPP is in agreement with published studies (using 30−40% acylCer) that benefited from this phenomenon to better describe the LPP without interferences from the SPP reflections.5,9,64 Models with even larger amounts of acylCer were also constructed; Schröter et al. used approximately 70% Cer EOS together with Cer AP for a neutron diffraction study and proposed a different model of the SC lipid matrix with the SPP only and with Cer EOS incorporated in the SPP.65 In skin diseases, diminished acylCer levels are accompanied by increased skin permeability. This effect was also reproduced in vitro: lipid membranes lacking acylCer showed almost 2-fold larger permeability to model compounds (p-aminobenzoic acid and its derivatives) than did the membranes containing 15% acylCer.37 Thus, we expected similar results. In particular, we were interested in the permeability in relation to the presence of the SPP, LPP, or their mixture in the membranes. However, when compared with the control without Cer EOS, membranes with 5−20% Cer EOS displayed increased permeability to TH, IND, and water. Interestingly, even the incorporation of 5% Cer EOS, which corresponds to only a 1.67% change in the total lipid composition and did not yet induce the LPP formation, had a strong permeabilizing effect in this model. At 30% Cer EOS in the membrane, where only the LPP and Chol were detected, the permeabilities decreased to values similar to or slightly higher than for the control. These results are in a sharp contrast to in vivo findings and with the well-established importance of the LPP. Such behavior might be caused by the lack of heterogeneity in the simple model. In particular, LA is highly miscible with the Cer NS with a 24carbon acyl chain, and this Cer can be present in the extended conformation,55 which can cause the low permeability of the control membrane composed of Cer NS/LA/Chol/CholS. Using infrared spectroscopy, we found that the incorporation of 5% Cer EOS decreased lipid chain order and the proportion of orthorhombic chain packing. This behavior might explain the increased permeability of the 5% Cer EOS membrane over the control. Another possible explanation is a lower miscibility of Cer EOS with the lipids of the simple model than with those in the real SC and a formation of the LPP from separated Cer EOS. Such a separated Cer EOS or Cer EOS-rich phase would yield a large grain border with increased local permeability. However, a disturbing fact remains that this model mimicked both SC lamellar phases and orthorhombic lipid packing very well. Thus, the relationships between the SC lipid composition, microstructure, and permeability are not straightforward: apparently, the presence of neither the LPP nor the orthorhombic lipid packing is sufficient for a proper lipid barrier. Permeability of the Complex Model Membranes: Stronger Permeability Barrier Reinforcement by Cer EOP and Cer EOdS than by Cer EOS. The counterintuitive results from the permeability experiments using the simple model led us to construct a more complex model with six−nine H


Article

Langmuir

corneocyte likely plays an important role in the organization and permeability of the epidermal lipid barrier.

Finally, the single acylCer were replaced in the complex membranes by their mixture (Cer EO-mix), which contained 7% Cer EOS, 1.5% Cer EOP, and 0.5% Cer EOdS (of the Cer part) to approximately mimic their proportions in the skin. The effects of the Cer EO-mix on permeability seem to be consistent with the composition of this mixture and the effects of the individual acylCer. In general, the permeability of the membranes with Cer EO-mix was closer to the permeability of the membrane with only Cer EOS, likely because Cer EOS was the dominant acylCer in this mixture. Only a Mixture of Three AcylCer Leads to LPP Formation in the Complex Model Membranes. To understand the differences in membrane permeabilities in the presence of the different acylCer at 10%, we studied the microstructure of the complex model membranes using XRPD. We expected the coexistence of the SPP, LPP, and Chol; however, the membranes with individual acylCer, Cer EOS, Cer EOP, and Cer EOdS did not form the LPP, not even when we changed the heating temperature. The observed decrease in permeability in all of these membranes with acylCer was apparently not related to the formation of the LPP, or the long lamellae were not periodically organized (to be detected using XRPD). The only complex membrane in which the LPP (together with the SPP and Chol) was found was the model with Cer EO-mix. Thus, the heterogeneity of the acylCer polar head structure is very important for the formation of the LPP lamellar phase in the complex model. For the lateral lipid organization of the complex model, the orthorhombic packing was clearly detected only in the diffractograms of the membranes containing Cer EO-mix and Cer EOS. In the complex model without acylCer or with Cer EOP or Cer EOdS, the orthorhombic packing was not detected. This difference also suggests that Cer EOS (either alone or in Cer EO-mix) promotes the orthorhombic lipid packing more than other acylCer. This finding is consistent with previous data on a lipid membrane model containing Cer EOS and Cer EOP.30 Thus, there is no straightforward correlation between the lipid lamellar and lateral organization in the model membranes and permeability experiments. In the least permeable membranes with Cer EOdS, neither the LPP nor the orthorhombic packing was detected. Although previous studies indicated that the orthorhombic packing is not crucial for the barrier function of the SC model53 and that this packing may be confined to some segregated domains,39 this lack of correlation between orthorhombic lipid packing and permeability seems to contradict in vivo findings.62 Further studies will be directed toward the concentration-dependent effects of acylCer in the complex models to better understand this phenomenon. Nevertheless, this report is the first to show that the presence or absence of the LPP may not be a reliable indicator of better or worse permeability barrier functionat least in the in vitro membrane models. Although diminished acylCer and LPP were repeatedly found in skin diseases, the exact contribution of these alterations to the increased permeability in such diseases and the real structure of the LPP need further studies. The apparent contradiction between this result and the in vivo findings discussed above may be related to the lack of lipid heterogeneity in the experimental membrane models and the absence of the corneocyte lipid envelope in our model (and in all published models of the skin lipid membranes). This monolayer of covalently bound lipids on the surface of the

■

SUMMARY AND CONCLUSIONS AcylCer have long been recognized as essential components of the lipid permeability barrier, particularly because they promote the formation of the LPP. In this study, we investigated how the concentration and structure of acylCer influence the permeability and lipid organization of model skin lipid membranes. Our simple model was composed of one Cer, one acylCer, one fatty acid, Chol, and CholS, and it closely mimicked the lamellar and lateral lipid organization: the LPP started to form at 10% Cer EOS. However, the permeabilities of these membranes with Cer EOS were higher than those of the control. Next, a complex model that mimicked the composition of the human SC more closely was constructed. In the complex model, incorporation of 10% acylCer expectedly decreased the membrane permeability compared with that of the control without acylCer. The membranes with the phytosphingosinebased Cer EOP and the dihydrosphingosine-based Cer EOdS were less permeable than those with Cer EOS or a mixture of three acylCer. However, X-ray diffraction experiments showed that the least permeable membranes had neither the LPP nor the orthorhombic lipid packing. The orthorhombic packing was induced by Cer EOS, whereas the LPP was formed only in the membrane with Cer EO-mix. We conclude that the lipid heterogeneity is highly importantonly the most complex model with nine Cer subclasses mimicked both the organization and the permeability of SC lipid membranes.

■

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Kateřina Vávrová: 0000-0002-8502-4372 Author Contributions

The manuscript was written with contributions from all authors. All authors have given approval for the final version of the manuscript. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS This work was supported by the Czech Science Foundation (16-25687J) and Charles University in Prague (SVV 260 291).

■

ABBREVIATIONS Cer, ceramide; acylCer, acylceramide; LA, lignoceric acid; FFA, free fatty acid; Chol, cholesterol; CholS, sodium cholesteryl sulfate; TH, theophylline; IND, indomethacin; XRPD, X-ray powder diffraction

■

REFERENCES

(1) Hannun, Y. A.; Obeid, L. M. Principles of bioactive lipid signalling: Lessons from sphingolipids. Nat. Rev. Mol. Cell Biol. 2008, 9, 139−150. (2) Madison, K. C. Barrier function of the skin: “La Raison d’Ê tre” of the epidermis. J. Invest. Dermatol. 2003, 121, 231−241. (3) Elias, P. M.; Goerke, J.; Friend, D. S. Mammalian epidermal barrier layer lipids: Composition and influence on structure. J. Invest. Dermatol. 1977, 69, 535−546. I


Article

Langmuir

(23) Motta, S.; Monti, M.; Sesana, S.; Caputo, R.; Carelli, S.; Ghidoni, R. Ceramide composition of the psoriatic scale. Biochim. Biophys. Acta, Mol. Basis Dis. 1993, 1182, 147−151. (24) Masukawa, Y.; Narita, H.; Shimizu, E.; Kondo, N.; Sugai, Y.; Oba, T.; Homma, R.; Ishikawa, J.; Takagi, Y.; Kitahara, T.; Takema, Y.; Kita, K. Characterization of overall ceramide species in human stratum corneum. J. Lipid Res. 2008, 49, 1466−1476. (25) van Smeden, J.; Boiten, W. A.; Hankemeier, T.; Rissmann, R.; Bouwstra, J. A.; Vreeken, R. J. Combined LC/MS-platform for analysis of all major stratum corneum lipids, and the profiling of skin substitutes. Biochim. Biophys. Acta, Mol. Cell Biol. Lipids 2014, 1841, 70−79. (26) van Smeden, J.; Hoppel, L.; van der Heijden, R.; Hankemeier, T.; Vreeken, R. J.; Bouwstra, J. A. LC/MS analysis of stratum corneum lipids: Ceramide profiling and discovery. J. Lipid Res. 2011, 52, 1211− 1221. (27) Breiden, B.; Sandhoff, K. The role of sphingolipid metabolism in cutaneous permeabilitybarrier formation. Biochim. Biophys. Acta, Mol. Cell Biol. Lipids 2014, 1841, 441−452. (28) Jennemann, R.; Rabionet, M.; Gorgas, K.; Epstein, S.; Dalpke, A.; Rothermel, U.; Bayerle, A.; van der Hoeven, F.; Imgrund, S.; Kirsch, J.; Nickel, W.; Willecke, K.; Riezman, H.; Grone, H.-J.; Sandhoff, R. Loss of ceramide synthase 3 causes lethal skin barrier disruption. Hum. Mol. Genet. 2011, 21, 586. (29) Vasireddy, V.; Uchida, Y.; Salem, N.; Kim, S. Y.; Mandal, M. N. A.; Reddy, G. B.; Bodepudi, R.; Alderson, N. L.; Brown, J. C.; Hama, H.; Dlugosz, A.; Elias, P. M.; Holleran, W. M.; Ayyagari, R. Loss of functional ELOVL4 depletes very long-chain fatty acids (≥ C28) and the unique ω-O-acylceramides in skin leading to neonatal death. Hum. Mol. Genet. 2007, 16, 471−482. (30) de Jager, M. W.; Gooris, G. S.; Ponec, M.; Bouwstra, J. A. Lipid mixtures prepared with well-defined synthetic ceramides closely mimic the unique stratum corneum lipid phase behavior. J. Lipid Res. 2005, 46, 2649−2656. (31) McIntosh, T. J.; Stewart, M. E.; Downing, D. T. X-ray diffraction analysis of isolated skin lipids: Reconstitution of intercellular lipid domains. Biochemistry 1996, 35, 3649−3653. (32) Bouwstra, J. A.; Gooris, G. S.; Dubbelaar, F. E.; Weerheim, A. M.; Ijzerman, A. P.; Ponec, M. Role of ceramide 1 in the molecular organization of the stratum corneum lipids. J. Lipid Res. 1998, 39, 186−196. (33) Di Nardo, A.; Wertz, P.; Giannetti, A.; Seidenari, S. Ceramide and cholesterol composition of the skin of patients with atopic dermatitis. Acta Derm.-Venereol. 1998, 78, 27−30. (34) Paige, D. G.; Morse-Fisher, N.; Harper, J. I. Quantification of stratum corneum ceramides and lipid envelope ceramides in the hereditary ichthyoses. Br. J. Dermatol. 1994, 131, 23−27. (35) van Smeden, J.; Janssens, M.; Boiten, W. A.; van Drongelen, V.; Furio, L.; Vreeken, R. J.; Hovnanian, A.; Bouwstra, J. A. Intercellular skin barrier lipid composition and organization in Netherton syndrome patients. J. Invest. Dermatol. 2014, 134, 1238−1245. (36) Opálka, L.; Kovácǐ k, A.; Sochorová, M.; Roh, J.; Kuneš, J.; Lenčo, J.; Vávrová, K. Scalable Synthesis of Human Ultralong Chain Ceramides. Org. Lett. 2015, 17, 5456−5459. (37) de Jager, M.; Groenink, W.; Guivernau, R. B. i.; 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, 951−960. (38) Janůsǒ vá, B.; Zbytovská, J.; Lorenc, P.; Vavrysová, H.; Palát, K.; Hrabálek, A.; Vávrová, K. Effect of ceramide acyl chain length on skin permeability and thermotropic phase behavior of model stratum corneum lipid membranes. Biochim. Biophys. Acta, Mol. Cell Biol. Lipids 2011, 1811, 129−137. (39) Školová, B.; Janůsǒ vá, B.; Zbytovská, J.; Gooris, G.; Bouwstra, J.; Slepička, P.; Berka, P.; Roh, J.; Palát, K.; Hrabálek, A.; Vávrová, K. Ceramides in the skin lipid membranes: Length matters. Langmuir 2013, 29, 15624−15633. (40) Pullmannová, P.; Staňková, K.; Pospíšilová, M.; Školová, B.; Zbytovská, J.; Vávrová, K. Effects of sphingomyelin/ceramide ratio on

(4) Mojumdar, E. H.; Kariman, Z.; van Kerckhove, L.; Gooris, G. S.; Bouwstra, J. A. The role of ceramide chain length distribution on the barrier properties of the skin lipid membranes. Biochim. Biophys. Acta, Biomembr. 2014, 1838, 2473−2483. (5) de Sousa Neto, D.; Gooris, G.; Bouwstra, J. Effect of the ωacylceramides on the lipid organization of stratum corneum model membranes evaluated by X-ray diffraction and FTIR studies (Part I). Chem. Phys. Lipids 2011, 164, 184−195. (6) 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, 911−916. (7) Bouwstra, J. A.; Gooris, G. S.; van der Spek, J. A.; Bras, W. Structural investigations of human stratum corneum by small-angle Xray-scattering. J. Invest. Dermatol. 1991, 97, 1005−1012. (8) Bouwstra, J. A.; Gooris, G. S.; Bras, W.; Downing, D. T. Lipid organization in pig stratum corneum. J. Lipid Res. 1995, 36, 685−695. (9) Mojumdar, E. H.; Gooris, G. S.; Barlow, D. J.; Lawrence, M. J.; Deme, B.; Bouwstra, J. A. Skin Lipids: Localization of Ceramide and Fatty Acid in the Unit Cell of the Long Periodicity Phase. Biophys. J. 2015, 108, 2670−2679. (10) Kessner, D.; Brezesinski, G.; Funari, S. S.; Dobner, B.; Neubert, R. H. H. Impact of the long chain ω-acylceramides on the stratum corneum lipid nanostructure. Part 1: Thermotropic phase behaviour of CER [EOS] and CER [EOP] studied using X-ray powder diffraction and FT-Raman spectroscopy. Chem. Phys. Lipids 2010, 163, 42−50. (11) Madison, K. C.; Swartzendruber, D. C.; Wertz, P. W.; Downing, D. T. Presence of intact intercellular lipid lamellae in the upper layers of the stratum corneum. J. Invest. Dermatol. 1987, 88, 714−718. (12) 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, 3725−3732. (13) Iwai, I.; Han, H.; den Hollander, L.; Svensson, S.; Ö fverstedt, L.G.; Anwar, J.; Brewer, J.; Bloksgaard, M.; Laloeuf, A.; Nosek, D.; Masich, S.; Bagatolli, L. A.; Skoglund, U.; Norlén, L. The human skin barrier is organized as stacked bilayers of fully extended ceramides with cholesterol molecules associated with the ceramide sphingoid moiety. J. Invest. Dermatol. 2012, 132, 2215−2225. (14) Masukawa, Y.; Narita, H.; Sato, H.; Naoe, A.; Kondo, N.; Sugai, Y.; Oba, T.; Homma, R.; Ishikawa, J.; Takagi, Y.; Kitahara, T. Comprehensive quantification of ceramide species in human stratum corneum. J. Lipid Res. 2009, 50, 1708−1719. (15) Ishikawa, J.; Narita, H.; Kondo, N.; Hotta, M.; Takagi, Y.; Masukawa, Y.; Kitahara, T.; Takema, Y.; Koyano, S.; Yamazaki, S.; Hatamochi, A. Changes in the Ceramide Profile of Atopic Dermatitis Patients. J. Invest. Dermatol. 2010, 130, 2511−2514. (16) Janssens, M.; van Smeden, J.; Gooris, G. S.; Bras, W.; Portale, G.; Caspers, P. J.; Vreeken, R. J.; Kezic, S.; Lavrijsen, A. P. M.; Bouwstra, J. A. Lamellar Lipid Organization and Ceramide Composition in the Stratum Corneum of Patients with Atopic Eczema. J. Invest. Dermatol. 2011, 131, 2136−2138. (17) t’Kindt, R.; Jorge, L.; Dumont, E.; Couturon, P.; David, F.; Sandra, P.; Sandra, K. Profiling and Characterizing Skin Ceramides Using Reversed-Phase Liquid Chromatography−Quadrupole Time-ofFlight Mass Spectrometry. Anal. Chem. 2012, 84, 403−411. (18) Elias, P. M. Epidermal lipids, membranes, and keratinization. Int. J. Dermatol. 1981, 20, 1−19. (19) Rabionet, M.; Gorgas, K.; Sandhoff, R. Ceramide synthesis in the epidermis. Biochim. Biophys. Acta, Mol. Cell Biol. Lipids 2014, 1841, 422−434. (20) Kovácǐ k, A.; Roh, J.; Vávrová, K. The Chemistry and Biology of 6-Hydroxyceramide, the Youngest Member of the Human Sphingolipid Family. ChemBioChem 2014, 15, 1555−1562. (21) Uchida, Y.; Holleran, W. M. Omega-O-acylceramide, a lipid essential for mammalian survival. J. Dermatol. Sci. 2008, 51, 77−87. (22) Rabionet, M.; Bayerle, A.; Marsching, C.; Jennemann, R.; Gröne, H.-J.; Yildiz, Y.; Wachten, D.; Shaw, W.; Shayman, J. A.; Sandhoff, R. 1-O-acylceramides are natural components of human and mouse epidermis. J. Lipid Res. 2013, 54, 3312−3321. J


Article

Langmuir

angle synchrotron X-ray diffraction study. Bioelectrochemistry 2002, 58, 87−95. (60) Bouwstra, J. A.; Gooris, G. S.; Dubbelaar, F. E. R.; Ponec, M. Phase behavior of stratum corneum lipid mixtures based on human ceramides: The role of natural and synthetic ceramide 1. J. Invest. Dermatol. 2002, 118, 606−617. (61) Danso, M. O.; van Drongelen, V.; Mulder, A.; van Esch, J.; Scott, H.; van Smeden, J.; El Ghalbzouri, A.; Bouwstra, J. A. TNF-α and Th2 Cytokines Induce Atopic Dermatitis-Like Features on Epidermal Differentiation Proteins and Stratum Corneum Lipids in Human Skin Equivalents. J. Invest. Dermatol. 2014, 134, 1941−1950. (62) Damien, F.; Boncheva, M. The extent of orthorhombic lipid phases in the stratum corneum determines the barrier efficiency of human skin in vivo. J. Invest. Dermatol. 2010, 130, 611−614. (63) Bouwstra, J. A.; Gooris, G. S.; Dubbelaar, F. E.; Ponec, M. Phase behavior of lipid mixtures based on human ceramides: Coexistence of crystalline and liquid phases. J. Lipid Res. 2001, 42, 1759−1770. (64) Kiselev, M. A.; Ermakova, E. V.; Gruzinov, A. Y.; Zabelin, A. V. Formation of the long-periodicity phase in model membranes of the outermost layer of skin (stratum corneum). Crystallogr. Rep. 2014, 59, 112−116. (65) Schröter, A.; Kessner, D.; Kiselev, M. A.; Hauß, T.; Dante, S.; Neubert, R. H. H. Basic nanostructure of stratum corneum lipid matrices based on ceramides [EOS] and [AP]: A neutron diffraction study. Biophys. J. 2009, 97, 1104−1114. (66) Imokawa, G.; Abe, A.; Jin, K.; Higaki, Y.; Kawashima, M.; Hidano, A. Decreased level of ceramides in stratum corneum of atopic dermatitis: An etiologic factor in atopic dry skin? J. Invest. Dermatol. 1991, 96, 523−526. (67) Bleck, O.; Abeck, D.; Ring, J.; Hoppe, U.; Vietzke, J.-P.; Wolber, R.; Brandt, O.; Schreiner, V. Two ceramide subfractions detectable in Cer (AS) position by HPTLC in skin surface lipids of non-lesional skin of atopic eczema. J. Invest. Dermatol. 1999, 113, 894−900. (68) Lavrijsen, A. P. M.; Bouwstra, J. A.; Gooris, G. S.; Weerheim, A.; Boddé, H. E.; Ponec, M. Reduced skin barrier function parallels abnormal stratum corneum lipid organization in patients with lamellar ichthyosis. J. Invest. Dermatol. 1995, 105, 619−624. (69) Školová, B.; Jandovská, K.; Pullmannová, P.; Tesař, O.; Roh, J.; Hrabálek, A.; Vávrová, 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, 5527−5535.

the permeability and microstructure of model stratum corneum lipid membranes. Biochim. Biophys. Acta, Biomembr. 2014, 1838, 2115− 2126. (41) Lampe, M. A.; Burlingame, A. L.; Whitney, J.; Williams, M. L.; Brown, B. E.; Roitman, E.; Elias, P. M. Human stratum corneum lipids: Characterization and regional variations. J. Lipid Res. 1983, 24, 120− 130. (42) 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, 4168−4175. (43) Wallmeyer, L.; Lehnen, D.; Eger, N.; Sochorová, M.; Opálka, L.; Kovácǐ k, A.; Vávrová, K.; Hedtrich, S. Stimulation of PPARα normalizes the skin lipid ratio and improves the skin barrier of normal and filaggrin deficient reconstructed skin. J. Dermatol. Sci. 2015, 80, 102−110. (44) Novotný, J.; Janůsǒ vá, B.; Novotný, M.; Hrabálek, A.; Vávrová, K. Short-chain ceramides decrease skin barrier properties. Skin Pharmacol. Physiol. 2009, 22, 22−30. (45) Bouwstra, J. A.; Gooris, G. S.; Salomons-de Vries, M. A.; Van der Spek, J. A.; Bras, W. Structure of human stratum corneum as a function of temperature and hydration: A wide-angle X-ray diffraction study. Int. J. Pharm. 1992, 84, 205−216. (46) Bouwstra, J. A.; Gooris, G. S.; Cheng, K.; Weerheim, A.; Bras, W.; Ponec, M. Phase behavior of isolated skin lipids. J. Lipid Res. 1996, 37, 999−1011. (47) 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, 452−456. (48) White, E. A.; Orazem, M. E.; Bunge, A. L. A critical analysis of single-frequency LCR databridge impedance measurements of human skin. Toxicol. In Vitro 2011, 25, 774−784. (49) Mantsch, H. H.; McElhaney, R. N. Phospholipid phase transitions in model and biological membranes as studied by infrared spectroscopy. Chem. Phys. Lipids 1991, 57, 213−226. (50) Craven, B. M. Crystal-structure of cholesterol monohydrate. Nature 1976, 260, 727−729. (51) Bouwstra, J. A.; Ponec, M. The skin barrier in healthy and diseased state. Biochim. Biophys. Acta, Biomembr. 2006, 1758, 2080− 2095. (52) Groen, D.; Poole, D. S.; Gooris, G. S.; Bouwstra, J. A. Investigating the barrier function of skin lipid models with varying compositions. Eur. J. Pharm. Biopharm. 2011, 79, 334−342. (53) Groen, D.; Poole, D. S.; Gooris, G. S.; Bouwstra, J. A. Is an orthorhombic lateral packing and a proper lamellar organization important for the skin barrier function? Biochim. Biophys. Acta, Biomembr. 2011, 1808, 1529−1537. (54) Školová, B.; Janůsǒ vá, B.; Vávrová, K. Ceramides with a pentadecasphingosine chain and short acyls have strong permeabilization effects on skin and model lipid membranes. Biochim. Biophys. Acta, Biomembr. 2016, 1858, 220−232. (55) Školová, B.; Hudská, K.; Pullmannová, P.; Kovácǐ k, 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, 10460−10470. (56) Mojumdar, E. H.; Gooris, G. S.; Bouwstra, J. A. Phase behavior of skin lipid mixtures: The effect of cholesterol on lipid organization. Soft Matter 2015, 11, 4326−4336. (57) Schreiner, V.; Pfeiffer, S.; Lanzendörfer, G.; Wenck, H.; Diembeck, W.; Gooris, G. S.; Proksch, E.; Bouwstra, J. Barrier characteristics of different human skin types investigated with X-ray diffraction, lipid analysis, and electron microscopy imaging. J. Invest. Dermatol. 2000, 114, 654−660. (58) Pilgram, G. S. K.; Vissers, D. C. J.; van der Meulen, H.; Pavel, S.; Lavrijsen, S. P. M.; Bouwstra, J. A.; Koerten, H. K. Aberrant lipid organization in stratum corneum of patients with atopic dermatitis and lamellar ichthyosis. J. Invest. Dermatol. 2001, 117, 710−717. (59) Uhrı ́kova,́ D.; Rapp, G.; Balgavý, P. Condensed lamellar phase in ternary DNA−DLPC-cationic gemini surfactant system: A smallK


Omega-O-Acylceramides in Skin Lipid Membranes: Effects of

Recommend Documents