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Ceramides are essential constituents of the skin barrier that allow humans to live on dry land. Reduced levels of ceramides have been associated with ...
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Ceramides in the Skin Lipid Membranes: Length Matters Barbora Školová,† Barbora Janůsǒ vá,† Jarmila Zbytovská,†,‡ Gert Gooris,§ Joke Bouwstra,§ Petr Slepička,‡ Pavel Berka,† Jaroslav Roh,† Karel Palát,† Alexandr Hrabálek,† and Kateřina Vávrová*,† †

Charles University in Prague, Faculty of Pharmacy, Heyrovského 1203, 50005 Hradec Králové, Czech Republic Institute of Chemical Technology Prague, Technická 5, 16628 Prague, Czech Republic § Skin Research Group, Leiden/Amsterdam Center for Drug Research, Einsteinweg 55, 2333 CC Leiden, The Netherlands ‡

S Supporting Information *

ABSTRACT: Ceramides are essential constituents of the skin barrier that allow humans to live on dry land. Reduced levels of ceramides have been associated with skin diseases, e.g., atopic dermatitis. However, the structural requirements and mechanisms of action of ceramides are not fully understood. Here, we report the effects of ceramide acyl chain length on the permeabilities and biophysics of lipid membranes composed of ceramides (or free sphingosine), fatty acids, cholesterol, and cholesterol sulfate. Short-chain ceramides increased the permeability of the lipid membranes compared to a long-chain ceramide with maxima at 4−6 carbons in the acyl. By a combination of differential scanning calorimetry, Fourier transform infrared spectroscopy, X-ray diffraction, Langmuir monolayers, and atomic force microscopy, we found that the reason for this effect in short ceramides was a lower proportion of tight orthorhombic packing and phase separation of continuous short ceramide-enriched domains with shorter lamellar periodicity compared to native long ceramides. Thus, long acyl chains in ceramides are essential for the formation of tightly packed impermeable lipid lamellae. Moreover, the model skin lipid membranes are a valuable tool to study the relationships between the lipid structure and composition, lipid organization, and the membrane permeability.

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INTRODUCTION Ceramides (Cer) are essential constituents of the skin barrier that protect the body against water loss and the entrance of toxic compounds, allergens, irritants, and microbes, thereby permitting life on dry land.1 These sphingolipids are composed of a sphingoid base (sphingosine (Sph), phytosphingosine, dihydrosphingosine or 6-hydroxysphingosine) N-acylated with a long-chain fatty acid (mostly lignoceric, C24). Cer, together with free fatty acids (FFA), cholesterol (Chol), and cholesterol sulfate (CholS), form multiple membranes filling the intercellular spaces of the uppermost skin layer, the stratum corneum (SC).2−10 Reduced or altered Cer levels have been recognized as a prominent feature of skin diseases, such as atopic dermatitis, psoriasis, or ichtyoses.5,11−13 Topically applied Cer or their synthetic analogues showed great potential in correcting these lipid barrier abnormalities.14−17 However, the structural requirements and mechanisms of action of exogenous Cer and their analogues are seldom defined. This lack of explanation is because it is impossible to precisely control and vary the amount of Cer that penetrate into the SC because of the complexity of skin. Hence, a suitable screening tool for Cer analogues is needed to study the structure−activity relationships in skin barrier Cer. Our general aim is to study the impact of Cer structure on permeability and biophysical characteristics of model membrane systems mimicking several aspects of the SC lipid barrier. In this study, we focus on the Cer acyl chain length because © 2013 American Chemical Society

short-chain Cer analogues have been widely used as more soluble Cer mimics;15 however, several studies have shown that the behavior of Cer is chain-length-dependent.18−23 We have recently shown that the long acyl chain in non-hydroxy acyl sphingosine-type (NS) Cer is essential for a competent skin barrier and that shortening the chain increased the skin permeability with maxima at Cer with 4−6C chain length.24,25 In this work, we use the same series of Cer analogues to determine if we can reproduce their effects on the skin barrier that we observed previously24 using a simple SC lipid model composed of sphingolipid (free Sph or Cer with acyl chain of 2, 4, 6, 8, 12, 18:1 (oleoyl), or 24C; Figure 1A), FFA, Chol, and CholS. We also study several biophysical parameters to explain the structure−permeability relationships using differential scanning calorimetry (DSC), attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR), smallangle X-ray diffraction (SAXD), Langmuir monolayers at air/ water interface, and atomic force microscopy (AFM). This knowledge could help to lay the design criteria for a more rational development of Cer analogues for potential use in therapy of skin diseases. In addition, understanding the biophysical mechanisms important for the permeability of the skin barrier could lead to better targeting of treatment or identification of parameters for skin disease diagnostics.21 Received: September 27, 2013 Revised: November 22, 2013 Published: November 27, 2013 15624

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was stirred at 32 °C; the precise volume was measured for each cell and included in the calculation. After a 1 h equilibration, the electrical impedance was measured;24 see the Supporting Information. Then, 100 μL of the donor sampleeither 5% theophylline (TH) or 2% indomethacin (IND) suspensions in 60% propylene glycol (see the Supporting Information for comments)was applied to the membrane. This setup ensured sink conditions for the selected drugs. Samples of the acceptor phase (300 μL) were withdrawn every 2 h over 8 h and replaced with the same volume of PBS. During this period, a steady state situation was reached. The samples were analyzed by HPLC;24,25 see the Supporting Information. Differential Scanning Calorimetry (DSC). The basic thermotropic behavior of the model membranes prepared in the same way as those for permeability studies without the supporting filters was determined by DSC. DSC thermograms were recorded from 20 to 120 °C using a DSC 200PC calorimeter (NETSCH, Selb, Germany) at a scan rate of 1 °C/min. An empty aluminum crucible was used as a standard. The transition temperatures were determined from the peak onset using the NETSCH Proteus Thermal Analysis software. Infrared (IR) Spectroscopy. To study the lipid chain conformation and packing and the behavior of the polar headgroup regions at skin temperature and during the phase transitions, attenuated total reflectance Fourier-transform IR (ATR-FTIR) spectroscopy was used. Infrared spectra of the model SC lipid membranes (same multilayers on a support filter as those used for permeability experiments, with the lipid film facing the ATR crystal) were collected on a Nicolet IMPACT 400 spectrometer (Thermo Scientific, Waltham, MA, USA) equipped with a single-reflection MIRacle ATR ZnSe crystal (PIKE technologies, Madison, WI, USA). A clamping mechanism with constant pressure was used. The spectra were generated by coaddition of 256 scans collected at 2 cm−1 resolution. The temperature dependence of the IR spectra was studied over the range 28−100 °C with 2 °C steps using a temperature control module (PIKE technologies, Madison, WI, USA). After each temperature increment, the sample was allowed to stabilize for 6 min before the spectrum was measured. The spectra were analyzed with the Bruker OPUS software. The exact peak positions were determined from the second derivative spectra and by peak fitting. Small-Angle X-ray Diffraction (SAXD). SAXD was used to determine the repeat distance of the lipid lamellae. For this, the same lipid multilayers on a filter support as for the permeability studies were used. The scattering intensity (I) was measured as a function of the scattering vector q (in reciprocal nm) defined as q = (4π sin θ)/λ, in which θ is the scattering angle and λ is the wavelength. From the positions of a series of peaks (qn) attributed to the same phase, the periodicity, or d-spacing, was calculated using the equation qn = 2nπ/d, n is the order number of the diffraction peak, and d is the repeat distance. All measurements were performed at the European Synchrotron Radiation Facility (ESRF, Grenoble) using station BM26B. The X-ray wavelength and the sample-to-detector distance were 0.113 and 0.419 nm, respectively. The lipid membrane was mounted parallel to the primary beam in a sample holder with mica windows. Static diffraction patterns were collected for 5 min. Langmuir Monolayers. Next, we studied the lipid monolayers at the air−water interface to compare their areas per molecule and compressibilities. Surface pressure−area isotherms were measured using a small Langmuir−Blodgett trough (KSV-NIMA, Espoo, Finland). Lipids (1 mg/mL, 10 μL) were mixed in chloroform/ methanol 3:1 and spread at the air−water interface, and the solvents were allowed to evaporate for 20 min. The lipid film was compressed at 20 mm/min at 23 °C, and the surface pressure recorded using a platinum plate at least in triplicate. The molecular areas per lipid were calculated at 1.5 mN/m (onset) and 20 mN/m. Maximum surface compression moduli were calculated according to Cs−1 = −A(∂π/∂A), where A is the area per lipid and π is the surface pressure. Atomic Force Microscopy (AFM). To visualize domains in the model membranes, we used AFM. The lipid monolayer at the air− water interface was compressed to 20 mN/m surface pressure and then transferred on freshly cleaved mica (12 × 15 mm2) by raising the mica support vertically through the air−water interface at 2 mm/min.

Figure 1. Structures of the studied sphingolipids with varying acyl chain length (A) and their effects on the electrical impedance (B), steady-state flux of TH (C), and IND (D) of the model SC lipid membranes containing Cer (or Sph)/FFA/Chol/CholS. Impedance and flux values of the membranes without Cer (No Cer) are included for comparison. Data are presented as the means ± SEM, n ≥ 6, * statistically significant against Cer24-containing membrane (p < 0.05), + statistically significant against No Cer membrane (p < 0.05).

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EXPERIMENTAL SECTION

Chemicals. Cer and Sph (synthetic, over 99% stereochemically pure, i.e., (2S,3R,4E)) were purchased from Avanti Polar Lipids (Alabaster, AL, USA) or synthesized as described previously.25 Deuterated lignoceric acid (DFFA) was obtained from C/D/N isotopes (Pointe-Claire, Canada). All other chemicals and solvents were from Sigma-Aldrich (Schnelldorf, Germany). Water was deionized, distilled, and filtered through a Millipore Q purification system. Preparation of Model SC Lipid Membranes. The model SC lipid membranes (multilayers) were prepared as equimolar mixtures of the Cer (or Sph), Chol, and lignoceric acid (most abundant FFA in SC) with addition of 5 wt % CholS. The lipids were dissolved in hexane/96% ethanol 2:1 (v/v) at 4.5 mg/mL (note: use of 96% (not absolute) ethanol is necessary to dissolve CholS). These lipid solutions (3 × 100 μL/cm2) were slowly sprayed on Nuclepore polycarbonate filters (Whatman, Kent, U.K.) under a stream of nitrogen using Linomat IV (Camag, Muttenz, Switzerland) equipped with additional y-axis movement. The lipid layer thickness was approximately 11 μm (measured by a digital micrometer gauge). For the initial comparisons, filters with pore diameters of 15 and 50 nm were used, and the filters with 15 nm pores were selected for all the other experiments (see the Supporting Information). These lipid films were heated to 90 °C, which is well over the main phase transition, equilibrated for 10 min, and slowly (∼3 h) cooled to room temperature. Then, they were incubated at 32 °C for 24 h at 30% air humidity. The effect of incubation for 0−14 days on the membrane permeability was also investigated. The membranes for ATR-FTIR and SAXD were prepared in the same way. The lipid membranes for DSC were prepared without the supporting filters. Permeation Experiments. The permeability of the model SC lipid membranes was evaluated using Franz diffusion cells with an available diffusion area of 0.5 cm2 and an acceptor volume of approximately 6 mL. The membranes were mounted into the diffusion cells with the lipid film facing the donor compartment. The acceptor compartment of the cell 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 15625

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properties, displayed IND flux of 0.32 ± 0.08 μg/cm2/h, while those with Cer4 and Cer6 were up to 6.5 times more permeable for this relatively large lipophilic substance. The other sphingolipids including free Sph did not produce any significant changes in IND permeability. Interestingly, the permeability of the membrane without Cer was 0.70 ± 0.15 μg/ cm2/h, which is significantly lower than that of Cer4 membrane. Hence, incorporation of the native long-chain Cer24 into the FFA/Chol/CholS membrane significantly increased its barrier properties toward the movement of ions (as reflected by the increased impedance) and small compounds with limited lipophilicity (as probed by TH flux). The flux of a large lipophilic substance IND was also lower but not significantly. The 4−6C chain Cer increased the membrane permeability compared to Cer24 for all three markers; however, their effect is mixed when compared to the No Cer membrane: these short Cer still reinforce the barrier for ions (and possibly other small hydrophilic compounds) but disturb the barrier for larger lipophilic compounds. When Cer24 was replaced by its hydrolysis product Sph, the membrane permeability did not increase. In fact, the impedance suggested slightly lower permeability for ions, which may be explained by the partial positive charge of Sph. Nevertheless, the low permeability of the Sph membrane remained also in the case of TH, which is largely neutral at pH 7.4. Permeability of the Model SC Lipid Membranes Correlates with Permeability of Skin. Figure S2 (Supporting Information) shows the correlations between the skin permeability after the application of the studied Cer (i.e., how Cer with various acyl chain lengths influence skin permeability when applied topically; data from our previous work24) and permeability of the model membranes containing these Cer. The correlations were statistically significant for all three markers at p < 0.05. Given the small number of compounds and the fact that in the case of a topical application Cer were only added to the existent SC lipid mixture, these data proved that such a simple four-component SC lipid model can yield physiologically relevant data; i.e., Cer that increased skin permeability after their topical application also yielded more permeable model membranes. Short-Chain Cer Broaden and Downshift Phase Transitions of the Model SC Membranes. To identify the molecular mechanisms underlying the observed differences in permeability, in particular the bell-shaped relationship between Cer chain length and the membrane/skin permeability, we studied the SC membranes using a variety of biophysical techniques. First, DSC was used to detect the phase transitions that may be indicative of cohesive forces and lipid mixing in the membrane and serve as a base for temperaturedependent ATR-FTIR studies. The DSC thermograms are given in Figure 2 and Figure S3 (Supporting Information). Mixtures containing Cer24 and Cer2 displayed narrow endothermic transitions with Tm at 71 and 77 °C, respectively. The transitions in the other membranes, i.e., containing Cer418:1 or free Sph, were broader and contained more components, suggesting the presence of compositionally distinct domains or several structures as a function of temperature. The main effect of Cer with 4−8C acyl was not only to broaden but also to downshift the transition. In these membranes, additional transitions at lower temperatures were also present. The broadest transition with the lowest onset was found in the Cer6- and Cer4-containing samples. This suggests

The surface morphology of the samples was examined by the AFM technique using a VEECO CP II device (Bruker Corp., Karlsruhe, Germany) in contact mode with CONT20A-CP Si probe. The domain areas, height differences, and surface roughness were determined using Veeco DI SPMLab NT 6.0.2 software. For additional processing and HPTLC verification of the lipid transfer, see the Supporting Information. Data Analysis. One way analysis of variance with Dunnett’s posttest method or Kruskal−Wallis one way analysis of variance on ranks with Dunn’s posttest where appropriate was used for the statistical analysis. Data are presented as the means ± SEM, and the number of replicates is given in the pertinent figure. Pearson product− moment correlation analysis was used to determine the dependences between the skin and membrane permeabilities.

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RESULTS

Short-Chain Cer Increase Permeability of the Model SC Membranes. The SC lipid membrane model was based on the “SC substitute”,26−28 i.e., SC lipid membrane reconstituted on a filter support. For the initial validation, see Figure S1 in the Supporting Information. To characterize the permeability of the model SC membranes, we used three complementary markers: (a) electrical impedance, which reflects the movement of ions through the membrane, (b) flux of TH, a small molecule (molar mass of 180 g/mol) with balanced lipophilicity (log P ∼ 0) that is likely to cross the membrane via free-volume diffusion, and (c) flux of IND, a large lipophilic molecule (358 g/mol and log P 4.3) that will prefer lateral diffusion along lipid bilayers.29 The support filters without any lipids did not significantly contribute to the barrier properties of the studied model SC membranes (Figure S1, Supporting Information). The electrical impedance values of the membranes containing the studied sphingolipids (i.e., Cer2, 4, 6, 8, 12, 18:1, 24, or free Sph, for chemical structures, see Figure 1A) are shown in Figure 1B. The impedance of the control membrane without Cer (i.e., FFA/Chol/CholS, labeled No Cer) was 2.1 ± 0.5 kΩ × cm2. The addition of the natural long-chain Cer24 resulted in an impedance of 334 ± 43 kΩ × cm2, which means a significantly decreased permeability for ions. Substitution of Cer24 by an equimolar amount of Cer2, Cer4, or Cer6 decreased the membrane impedance, with the lowest value (57 ± 17 kΩ × cm2) in the Cer6-containing membrane. Cer18:1, i.e., N-oleoylsphingosine, produced no significant changes compared to Cer24, while Cer8, Cer12, and Sph rather slightly increased the membrane electrical impedance. Nevertheless, all of the membranes containing the studied sphingolipids were significantly more resistant to the permeation of ions than the control sample without Cer. The second marker of the membrane permeability was a flux of TH (Figure 1C). Without Cer, the TH flux through the membrane was 6.6 ± 1.4 μg/cm2/h. Upon the addition of Cer24, the permeability for TH significantly decreased to 0.36 ± 0.04 μg/cm2/h. The replacement of Cer24 by the shorter, but still relatively long, Cer18:1, Cer12, or Cer8, or the shortest Cer2 or free Sph did not lead to any significant changes in TH flux. In contrast, Cer4 and Cer6 were not able to maintain the membrane barrier properties toward TH permeation. The TH flux through the Cer6-containing membrane reached 3.6 ± 0.4 μg/cm2/h. See Figure S1 in the Supporting Information for permeation profiles. A similar, roughly bell-shaped relationship between the Cer chain length and membrane permeability was also found for the third marker, the flux of IND (Figure 1D). The Cer24/FFA/ Chol/CholS membrane, mimicking the SC lipid barrier 15626

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the lipid chains and phase transitions can be deduced. All the membranes contained well-ordered lipid chains with high proportions of all-trans conformers at skin temperature, as indicated by the wavenumber below 2850 cm−1 (from 2846.5 to 2847.9 cm−1 in the sphingolipid containing membranes and 2848.6 cm−1 in the No Cer sample).37,38 Although the wavenumbers and bandwidths of both CH2 stretching vibrations varied slightly, no trend that could explain the differences in the membrane permeabilities was found. Thus, in these membranes with various Cer acyl chain lengths, the presence of ordered chains does not ensure low membrane permeability. Upon heating, the proportion of gauche conformers increased as the lipids in the membranes underwent order−disorder transition characterized by an increase in the νsCH2 frequency up to 2852 cm−1. In the Cer24-containing membrane, a sharp transition suggesting highly cooperative melting of the lipid chains was observed at 59 °C, which is in good agreement with previous studies. The spectra of all other membranes revealed transitions that took place over a wider temperature range. This transition was composed of two temperature regions, except in the Cer2 membrane. The transition temperatures calculated from the first derivative of the plots are given in Figure 3A and are represented by the dotted lines in panels B−D of this figure as a guide for the eye. In accordance with the DSC experiments, the most permeable membranes, i.e., those with Cer4−8,

Figure 2. DSC thermograms of the model SC lipid membranes composed of Cer (or Sph)/FFA/Chol/CholS showing that shortchain Cer broaden and downshift the main phase transition.

weaker cohesive forces in the most permeable membranes and decreased lipid miscibility. Lipid Chain Conformational Order Is Not Indicative of Model SC Membrane Permeability. FTIR spectroscopy is a powerful, accurate, reproducible, and non-perturbative technique with high resolution, which enables detection of even small changes in the lipid membranes including those mimicking the skin barrier30−36 either at skin temperature or during the phase transitions. We first examined the model SC lipid membranes containing unlabeled FFA. Figure 3A shows the temperature dependence of the methylene symmetric stretching (νsCH2) vibration wavenumber from which the conformational order of

Figure 3. ATR-FTIR spectroscopy of the model SC lipid membranes: the temperature dependence of the methylene symmetric stretching (A and B), scissoring (C and D), and rocking (C) wavenumbers in the model SC lipid membranes containing unlabeled FFA (A and C) and perdeuterated DFFA (B and D). 15627

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showed the onset of the main phase transitions at the lowest temperatures, which may indicate the weakest intermolecular interactions. To examine the origin of the chain order and phase transitions, the unlabeled FFA in the membranes was substituted by perdeuterated DFFA. This deuteration enabled us to follow simultaneously the behavior of both Cer and DFFA chains because CD2 vibrations occur at different wavenumbers than CH2 bands. Figure 3B shows the CH2 symmetric stretching, which originates mostly from the Cer chains, and the CD2 symmetric stretching of the DFFA chains as a function of temperature. Only the Cer24 and Cer12 membranes displayed associated Cer and DFFA phase transitions; the other membranes were composed of separated domains rich in either Cer (or Sph) or DFFA. In the case of the most permeable membranes, the DFFA behavior was almost identical to the No Cer membrane composed of DFFA/Chol/CholS (Figure S4, Supporting Information), suggesting that Cer having acyl chains with 4−8 carbons barely mix with FFA. Such phase separation may explain the high permeability of these membranes, especially for the hydrophilic permeability markers, as these are able to diffuse readily along the domain boundaries. The Most Permeable Membranes with Cer4−6 Contain Fewer Orthorhombically Packed Lipids. Another important parameter of the membrane function is the chain packing. In human skin, orthorhombic chain packing was found.39 It has been regarded as an indicator of good barrier function,40 although the change from orthorhombic to hexagonal packing in human SC and the model lipid membranes did not have an effect on the permeability.28 Infrared scissoring and rocking modes provide a convenient way to detect this type of lipid chain packing because polymethylene chains that are packed in an orthorhombic subcell are vibrationally coupled and appear as a doublet of these particular modes.37,38 Figure 3C shows that orthorhombic chain packing was detected in all the studied membranes at the skin temperature (a scissoring doublet at ∼1463 and 1471 cm−1 and a rocking doublet at ∼718 and 729 cm−1). Both doublets collapsed during the chain disordering phase transition except for Cer24, where the orthorhombic packing disappeared during the pretransition at approximately 37 °C. To obtain more detailed information on the chain packing, we determined the extent of orthorhombic phases by fitting the scissoring peak and comparing the areas of the individual components (doublet at 1461 and 1471 cm−1 characteristic of an orthorhombic phase, peak at 1468 cm−1 typical of a hexagonal packing, and peak at 1465−1466 cm−1 of disordered chains, which increased upon heating). While the membrane with Cer24 contained 90% of the orthorhombic phase, the most permeable membranes contained only 52−55% of these tightly packed lipids and up to 37% of disordered phase (Figure 4A). Interestingly, the membrane having free Sph instead of Cer contained 68% of the orthorhombic phase. Short-Chain Cer and FFA Form Separate Domains; Only FFA-Rich Domains Show Prevailing Orthorhombic Packing. The possible reason for the result that even the short Cer-containing membranes still display 52−55% of the very tight orthorhombic chain packing may be the fact that this orthorhombic packing is found only in certain lipid domains. This hypothesis was examined by using membranes with deuterated DFFA (Figure 3D). In the Cer24 membrane, the scissoring doublet disappeared upon exchange of FFA by DFFA. This disappearance is because the short-range vibra-

Figure 4. The relative extent of the orthorhombic, hexagonal, and disordered phases and the relative intensities of the νs CH2 ratio to νs CD2 of the Cer (or Sph)/DFFA/Chol/CholS model SC lipid membranes at 32 °C. On the left side, the data are presented as the means; SEM were omitted for clarity. On the right side, data are presented as the means ± SE, n ≥ 3, * statistically significant against Cer24-containing membrane (p < 0.05).

tional coupling occurs only between the same isotopes and not between mixed protonated and deuterated chains.41 Thus, the native long-chain Cer24 was well mixed with DFFA within this membrane most likely due to their matching chains. In contrast, the behavior of CH2 and CD2 scissoring modes confirmed the phase separation in the short-chain Cer membranes and those with free Sph. The orthorhombic chain packing in these membranes originated mainly from the DFFA chains (a doublet at ∼1085 and 1092 cm−1), whereas in Cerrich domains only hexagonal packing with the scissoring band at ∼1466 cm−1 was found. The magnitude of the CD2 splitting suggests the presence of relatively large domains of DFFA.41 This phase separation may explain the presence of conformationally ordered, orthorhombically packed chains in the short Cer-containing membranes despite them being much more permeable: the well-organized FFA-rich domains may form isolated “islands” that contribute to the overall permeability only a little. Polar Head Region Is Similar in All SC Membranes. To find a possible explanation of the different behavior of the individual short Cer, we also examined the polar head region of the membranes, which includes the FFA carboxyl and Cer amide modes (Figure S5, Supporting Information). However, these vibrations were very similar. In all of the membranes, the Cer amide modes displayed relatively broad distributions of Hbond strengths, which collapsed during the main phase transition. The carboxyl groups were, except for the Cer24 membrane, involved in strong H-bonding, as suggested by their relatively low wavenumbersapproximately 1700 cm−1. This may be connected to the observed FFA phase separation in the shorter chain Cer membranes. In the Sph membrane, a doublet at 1700 and 1723 cm−1 was found, indicating different Hbonding strengths of the COOH group. Relative CH2 Intensity at 32 °C Is Weaker in the Most Permeable Membranes with Cer4−6. By closer examination of the spectra at 32 °C, we found that the intensity of the CH2 symmetric stretching (and also of the other methylene modes) was much lower in Cer4 and Cer6 membranes than in those with the longer chain Cer or Cer2, while the intensities of CD2 modes were similar in all of the membranes. The relative CH 2 /CD2 intensities are shown in Figure 4B. When considering Cer4 to Cer24 membranes, their CH2/CD2 intensities reflected the decreased chain length. On the other hand, with further acyl shortening in Cer2 or its shortening in Sph, the relative methylene intensities increased. This result suggests that Cer2 behaves as a single chain amphiphile similar to free Sph; i.e., its small acyl chain is accommodated within the 15628

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polar head region of the membrane. This observation, together with the orthorhombic phase content, may be the reason for the bell-shaped relationship between the Cer acyl chain length and permeability. The Most Permeable Membranes with Cer4−6 Contain Short Cer-Enriched Phases with the Shortest Lamellar Periodicities. To learn more about the internal structure of the membrane models, the lipid phase arrangement, the role of Chol, and the repeat distances of the lamellar phases,42,43 we used SAXD on the multilamellar lipid models on the filter supports. The diffraction profile of the FFA/Chol/ CholS membrane (labeled as “No Cer”) revealed lamellar phases with repeat distances of 5.2 and 3.4 nm (Figure 5). The

Figure 6. The pressure−area isotherms of selected lipid monolayers at the air−water interface (A), mean molecular areas per lipid at the onset of condensation and at 20 mN/m (B), and monolayer compression modulus (1/Cs, panel C). Data are presented as the means ± SEM, n ≥ 4, * statistically significant against Cer24containing membrane (p < 0.05), + statistically significant against No Cer membrane (p < 0.05).

Figure 5. SAXD profiles of selected stratum corneum lipid membranes containing Cer2, Cer4, and Cer24 (A) and the lamellar repeat distances of the Cer (or Sph)/FFA/Chol/CholS model SC lipid membranes (B). Data are presented as the means, n = 2.

Information. The FFA/Chol/CholS sample, i.e., No Cer, formed a relatively dense monolayer that lifted off at 31.7 ± 0.4 Å2 (area per lipid at 1.5 mN/m) and reached 27.1 ± 0.1 Å2 at 20 mN/m at laboratory temperature (23 °C; data at 32 °C are given in the Supporting Information). The isotherm shape revealed a liquid condensed−solid transition at 22 mN/m and 26.6 Å2, similar to that found in pure FFA, confirming their limited mixing. The maximum compression modulus was 534 ± 27 mN/m, suggesting a relatively rigid condensed structure. Addition of the long-chain Cer24 to FFA/Chol/CholS mixture produced a highly condensed monolayer with no apparent phase transitions. The areas at the onset of condensation and at 20 mN/m were 36.3 ± 1.7 and 32.5 ± 1.5 Å2, respectively, and the maximum compression modulus of the membrane was 508 ± 35 mN/m. This result is in good agreement with previous studies on Cer/FFA/Chol monolayers that reported areas of 38.7 and 41 Å2 at 20 mN/m.45,46 In contrast, the monolayers containing the short-chain Cer4−6 did not condense spontaneously at the air−water interface; they lift off at ∼40−44 Å2 and form a liquid expanded phase (Figure 6). This is similar to previous results with shorter Cer.47 Their maximum compression moduli were less than 200 mN/m, i.e., 3 times lower than that of a native long Cercontaining monolayer. On the other hand, further shortening of the Cer acyl to 2C led to a steep isotherm with a molecular area of 25.4 ± 0.04 Å2, which is comparable to the monolayer without Cer. These monolayers also displayed a transition similar to the No Cer sample. In the monolayer containing Sph, an area per molecule of 21.1 ± 0.4 Å2 was found with no apparent phase transition. These results indicate a decrease in the ability of the Cer4and Cer6-containing lipid mixtures to form condensed monolayers. Despite the fact that Cer may only be able to adopt the hairpin conformation in these monolayer studies, i.e., with both chains pointing in the same direction, this result may

latter corresponds to separated Chol. Upon addition of Cer24, the profile did not change markedly; the repeat distance of the longer periodicity phase increased to 5.4 nm, revealing one lamellar phase, which suggests that this long-chain Cer mixes with C24-FFA in one lamellar phase. Here, it should be noted that these lipid models form only the so-called “short periodicity phase” because they do not contain the ωlinoleyloxyacylsphingosines, i.e., EO-type Cer. The fact that Chol does not fully mix with long-chain Cer24 is in agreement with previous studies on model systems and isolated SC.44 Shortening of the Cer acyl chain or its removal in Sph resulted in the formation of at least one additional lamellar phase with a periodicity that decreases almost linearly with chain length to C4. In all of these membranes, phase separated Chol was observed. With further shortening (or the removal of the acyl chain and only having Sph), the repeat distances of the Cer-rich phase began to rise again, giving a bell-shaped profile with the shortest repeat distance of the Cer-rich phase in the most permeable membranes. In the Sph, Cer2, and Cer4 membranes, an additional phase with a periodicity at approximately 4 nm was visible. The Cer2 membrane was the only sample that did not show any phase-separated Chol, which may explain the difference in behavior compared to the Cer4− 6-containing membranes (Figure 5 and Figure S6, Supporting Information). The Most Permeable Membranes with Cer4−6 Form Liquid Expanded Monolayers at Low Surface Pressures. To compare the areas per lipid in the membranes, the pressure−area isotherms of the lipid monolayers at the air− water interface were studied (Figure 6 and Figures S7 and S8, Supporting Information). The parameters of the isotherms of the individual lipid components are given in the Supporting 15629

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chain Cer-containing membranes, the higher domains occupy less total area, are larger (1−10 μm), and, most importantly, are discontinuous.

explain the comparatively decreased ability to form tightly packed monolayers in the ATR-FTIR studies (which does not force the hairpin conformation). The Area of the FFA-Rich Phase Is the Smallest and Discontinuous in the Cer4 and Cer6 Membranes. Monolayers at an air−water interface were then transferred to a mica support at 20 mN/m and studied using AFM to visualize the different lipid domains. This technique provides a highresolution, three-dimensional surface profile of the monolayers (Figure 7A). Here, the lighter areas are higher (thicker

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DISCUSSION Cer are essential components of mammalian skin. Although their (patho)biochemical and biophysical effects have been widely explored, it is surprising how little we know about the basic structure−permeability relationships of Cer-containing membranes. Together with our limited information on lipid composition of diseased skin, this lack of knowledge is most likely the main obstacle for the rational design of skin barrier repair agents or a broader use of transdermal drug delivery. One of the reasons is the complexity and variability of skin that makes the experiments time-consuming and hard to interpret. Previous studies in atopic eczema patients,8,12 in vivo mice models of atopic skin,48,49 and in vitro skin tissue24,25 showed that the long acyl chains in Cer are essential for the proper organization and permeability of the skin lipid barrier. To study the direct effect of Cer chain length on the lipid membrane organization and permeability, we prepared a simplified model from the major skin barrier lipids, i.e., an equimolar mixture of Cer, FFA, and Chol, plus 5% (w/w) of CholS. The major advantage of this model is its simplicity: it enables an easy substitution of the native lipids by their analogues, the evaluation of the changes in the permeability of the membrane, and the determination of the biophysical parameters that can help to relate the changes in chemical structure to the changes in permeability. However, we also realize that the simplicity of this model is also its disadvantage because it mimics several but not all aspects of the skin barrier. First, the model lacks corneocytes; thus, it does not have the tortuous pathway. Second, the amount of lipids and, consequently, the thickness of the lipid layer is approximately 10 times higher than that in SC to partially compensate for the lack of tortuosity (for a detailed comparison with SC and discussion, see ref 26). Finally, the lamellar phases are different from those detected in vivo. First, we compared the permeabilities of the model SC lipid membranes with and without the native long-chain Cer24. This experiment confirmed that the long-chain Cer are indispensable for the barrier toward penetration of small hydrophilic to moderately lipophilic compounds. In contrast, penetration of larger lipophilic compounds is relatively well blocked by the other lipids. This observation is in agreement with the central role of Cer in preventing desiccation.1 On the other hand, the short-chain Cer do not maintain the barrier properties of the native long-chain Cer despite possessing the same polar head structure and hydrogen bonding ability. This result is most likely because only the long Cer mix well with the long FFA, while their shorter analogues phase-separate. Such phase separation of short Cer may explain the high permeability of these membranes, especially for the hydrophilic permeability markers, as these markers are able to diffuse readily along the domain boundaries. The highest permeability was consistently found in membranes with Cer containing fatty acids with 4−6 carbon chains, suggesting that their conformation has an adverse effect on tight lipid packing necessary for a good barrier (see Figure S10, Supporting Information, for the different geometry of energyminimized models). Thus, the short acyl chains in Cer4−6 are probably not long enough to pack efficiently with the other lipid chains by hydrophobic interactions and wobble between

Figure 7. The AFM images of the lipid monolayers composed of Cer (or Sph)/FFA/Chol/CholS (A), height differences between the monolayer domains with a schematic representation of their origin using 3D formulas of selected lipids (from left to right: Chol, Cer4, and FFA; panel B), and % area covered by the higher (FFA-rich) domain (C). Data are presented as the means ± SEM, n ≥ 6, * statistically significant against Cer24-containing membrane (p < 0.05), + statistically significant against No Cer membrane (p < 0.05).

monolayer), and the darker are lower. The height differences were between 0.7 and 1.2 nm (Figure 7B). This difference corresponds to the theoretical difference between FFA (max. length 3.1 nm) and Sph chain (2.3 nm) or between FFA and Chol (1.7 nm). The greatest height difference was found in the Cer6-containing membrane. Considered together with the aforementioned data, the lighter domains are rich in FFA (or FFA with Cer24 in the long-chain Cer24 membrane), and the darker areas are rich in Chol, CholS, and short Cer or Sph. This observation agrees well with the relative areas occupied by the higher domain: ∼52% in the long-chain Cer24 membrane corresponds to the areas of FFA and Cer24, while, in the Cer4−8-containing membranes, ∼20% of the higher area indicates an almost pure FFA domain (Figure 7C). Together with the FTIR and SAXD data, the lipids in the lighter domains are well ordered and tightly packed and should be less permeable. Importantly, only in the long-chain Cer24-based membrane, the lighter domain forms continuous, 0.2−2 μm thick “chains” (for details, see Figure S9, Supporting Information). In contrast, in the short15630

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applies to fluorescent or radiolabeled Cer used to study the fate of Cer because the use of the shorter commercial analogues may yield misleading information.53 On the other hand, the properties of 4−6C acyl in combination with one lipophilic chain may be explored in the field of penetration enhancers,14,54 i.e., compounds increasing transdermal drug delivery. Moreover, the model SC lipid membranes reconstituted on a filter support developed originally as a SC substitute26,27 proved to be a valuable tool to study the relationships between the Cer structure (and also of the other SC lipids), membrane permeability, and the underlying mechanisms on a molecular level.

the hydrophobic and hydrophilic region of the membranes, preventing tight lipid packing in their vicinity. When shortened further to two carbons, such acyl seems to be short enough to be accommodated well in the polar head region without perturbing the lipid packing.20 The hydrogen bonding ability of the short Cer polar head was not strong enough to overcome the unfavorable molecular shape and inadequate chain−chain interactions and, thus, maintain the tight membrane packing. Interestingly, similar bell-shaped relationships between Cer acyl chain length and their behavior were also found in phospholipid/sphingomyelin membranes by different techniques.19,20,50 These data generated by this simplified model are in good agreement with our previous work on skin tissue.24 In addition, the significance of long acyl chains in Cer has also been demonstrated in vivo in mouse models of diseased skin48,49 and also in atopic dermatitis8,12,51 and ichthyosis patients.52 In atopic eczema, the reduced chain length of Cer compared to healthy individuals correlated with the skin barrier function.12 To obtain a link on a molecular level (and a possible predictive tool) between the change in Cer structure and membrane permeability, we used a variety of techniques including DSC, ATR-FTIR, and SAXD on multilamellar lipid films, Langmuir monolayers, and AFM on monolayers. The initial DSC and FTIR studies showed differences in thermotropic behavior of the membranes, suggesting lower cohesive forces in the most permeable membranes. Nevertheless, at skin temperature, the membranes were surprisingly similara much-unexpected finding was that even the most permeable membranes showed high lipid chain conformational order and the presence of orthorhombic chain packing at physiological temperature. However, closer examination revealed that the relative amount of the orthorhombic phases was lower in the most permeable membranes, which is in good agreement with in vivo data.40 Our data suggest that these FTIR parameters must be treated cautiously: although the absence of well-ordered lipids and orthorhombic packing is a good indicator of a more permeable barrier, their presence cannot guarantee a good one. In addition, FTIR spectroscopy using deuterated lipids, SAXD, and Langmuir monolayers showed phase separation of the short Cer-enriched domains. The FFA-rich domains showed prevailing orthorhombic packing, while the short Cer-rich domains were less well ordered, formed lamellar phases with shorter repeat distances, and did not spontaneously condense. Although the data obtained on monolayers must be treated cautiously because Cer can only adopt the hairpin conformation in a monolayer, the results seem to reasonably correlate with the data obtained on multilayers. Final visualization by AFM showed that the separated, lesspermeable, FFA-rich phase is discontinuous in the short-chain Cer membranes. Thus, the permeating molecules, in particular the relatively hydrophilic ones, may cross the membrane through the less tightly packed short Cer-rich phase, while the FFA-rich phases form isolated domains that only increase the pathway tortuosity. In conclusion, this work showed that the exceptionally long Cer acyl chains are essential for the formation of tightly packed impermeable lipid lamellae. We suggest that such long chains should be included when designing Cer analogues for the prospective treatment of skin barrier disorders with decreased Cer levels because short-chain Cer increase the permeability of Cer/FFA/Chol/CholS membranes. This suggestion also

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ASSOCIATED CONTENT

* Supporting Information S

Supporting methods (electrical impedance, HPLC, HPTLC, and additional AFM measurements) and results (comparison of different support filters and incubation conditions during the preparation of lipid membranes, correlations between skin and membrane permeability, DSC, ATR-FTIR data on the polar region of the membranes, SAXD diffractograms, Langmuir isotherms of pure lipids and lipid monolayers at 23 and 32 °C, and detailed AFM images). This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Phone: +420 495 067 497. Fax: +420 495 067 166. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the Czech Science Foundation projects 207/11/0365 and 13-23891S. B.Š. and B.J. thank SVV 2013-267-001.

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ABBREVIATIONS AFM, atomic force microscopy; ATR, attenuated total reflectance; Cer, ceramide/s; Chol, cholesterol; CholS, cholesterol sulfate; DFFA, perdeuterated fatty acid; DSC, differential scanning calorimetry; FFA, free fatty acid; FTIR, Fourier transform infrared spectroscopy; IND, indomethacin; NS, nonhydroxyacyl sphingosine; SAXD, small-angle X-ray diffraction; SC, stratum corneum; Sph, sphingosine; TH, theophylline

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REFERENCES

(1) Attenborough, D. Life on Earth - A Natural History; Little, Brown & Co.: Boston, MA, 1980. (2) Bouwstra, J. A.; Ponec, M. The skin barrier in healthy and diseased state. Biochim. Biophys. Acta 2006, 1758 (12), 2080−2095. (3) Elias, P. M. Epidermal lipids, barrier function, and desquamation. J. Invest. Dermatol. 1983, 80, 44−49. (4) Feingold, K. R. Thematic review series: skin lipids. The role of epidermal lipids in cutaneous permeability barrier homeostasis. J. Lipid Res. 2007, 48 (12), 2531−2546. (5) Holleran, W. M.; Takagi, Y.; Uchida, Y. Epidermal sphingolipids: metabolism, function, and roles in skin disorders. FEBS Lett. 2006, 580 (23), 5456−5466. (6) Wartewig, S.; Neubert, R. H. Properties of ceramides and their impact on the stratum corneum structure: a review. Part 1: ceramides. Skin Pharmacol. Physiol. 2007, 20 (5), 220−229. 15631

dx.doi.org/10.1021/la4037474 | Langmuir 2013, 29, 15624−15633

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Article

(25) Novotný, J.; Janůsǒ vá, B.; Novotný, M.; Hrabálek, A.; Vávrová, K. Short-chain ceramides decrease skin barrier properties. Skin Pharmacol. Physiol. 2009, 22 (1), 22−30. (26) de Jager, M.; Groenink, W.; ABielsa 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), 951−960. (27) de Jager, M.; Groenink, W.; van der Spek, J.; Janmaat, C.; Gooris, G.; Ponec, M.; Bouwstra, J. Preparation and characterization of a stratum corneum substitute for in vitro percutaneous penetration studies. Biochim. Biophys. Acta 2006, 1758 (5), 636−644. (28) 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), 4168−4175. (29) Mitragotri, S. Modeling skin permeability to hydrophilic and hydrophobic solutes based on four permeation pathways. J. Controlled Release 2003, 86 (1), 69−92. (30) Mao, G.; VanWyck, D.; Xiao, X.; Mack Correa, M. C.; Gunn, E.; Flach, C. R.; Mendelsohn, R.; Walters, R. M. Oleic acid disorders stratum corneum lipids in Langmuir monolayers. Langmuir 2013, 29 (15), 4857−4865. (31) Rodriguez, G.; Rubio, L.; Cocera, M.; Estelrich, J.; Pons, R.; de la Maza, A.; Lopez, O. Application of bicellar systems on skin: diffusion and molecular organization effects. Langmuir 2010, 26 (13), 10578−10584. (32) Zhu, Y.; Imae, T.; Saiwaki, T.; Oka, T. Damage/recovery by additive on lipid membrane as a mimicry of human stratum corneum. Langmuir 2010, 26 (7), 4951−4957. (33) Chen, H.; Mendelsohn, R.; Rerek, M. E.; Moore, D. J. Fourier transform infrared spectroscopy and differential scanning calorimetry studies of fatty acid homogeneous ceramide 2. Biochim. Biophys. Acta 2000, 1468 (1−2), 293−303. (34) Rerek, M. E.; Chen, H.; Markovic, B.; Van Wyck, D.; Patrick 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), 9355−9362. (35) Moore, D. J.; Rerek, M. E.; Mendelsohn, R. FTIR spectroscopy studies of the conformational order and phase behavior of ceramides. J. Phys. Chem. B 1997, 101 (44), 8933−8940. (36) Moore, D. J.; Rerek, M. E.; Mendelsohn, R. Role of ceramides 2 and 5 in the structure of the stratum corneum lipid barrier. Int. J. Cosmet. Sci. 1999, 21 (5), 353−368. (37) Mendelsohn, R.; Moore, D. J. Vibrational spectroscopic studies of lipid domains in biomembranes and model systems. Chem. Phys. Lipids 1998, 96 (1−2), 141−157. (38) Snyder, R. G.; Schachtschneider, J. H. Vibrational analysis of the n-paraffinsI: Assignments of infrared bands in the spectra of C3H8 through n-C19H40. Spectrochim. Acta 1963, 19 (1), 85−116. (39) Gay, C. L.; Guy, R. H.; Golden, G. M.; Mak, V. H.; Francoeur, M. L. Characterization of low-temperature (i.e., < 65 degrees C) lipid transitions in human stratum corneum. J. Invest. Dermatol. 1994, 103 (2), 233−239. (40) Boncheva, M.; Damien, F.; Normand, V. Molecular organization of the lipid matrix in intact Stratum corneum using ATR-FTIR spectroscopy. Biochim. Biophys. Acta 2008, 1778 (5), 1344−1355. (41) Mendelsohn, R.; Liang, G. L.; Strauss, H. L.; Snyder, R. G. IR spectroscopic determination of gel state miscibility in long-chain phosphatidylcholine mixtures. Biophys. J. 1995, 69 (5), 1987−1998. (42) 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), 3725−3732. (43) 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 (12), 3649−3653. (44) 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 (2), 334−342.

(7) Wertz, P. W. Lipids and barrier function of the skin. Acta Derm.Venereol., Suppl. 2000, 208, 7−11. (8) 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 (7), 1466−1476. (9) 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 (6), 1211−1221. (10) Wertz, P. W. Current understanding of skin biology pertinent to skin penetration: skin biochemistry. Skin Pharmacol. Physiol. 2013, 26 (4−6), 217−226. (11) Choi, M. J.; Maibach, H. I. Role of ceramides in barrier function of healthy and diseased skin. Am. J. Clin. Dermatol. 2005, 6 (4), 215− 223. (12) Janssens, M.; van Smeden, J.; Gooris, G. S.; Bras, W.; Portale, G.; Caspers, P. J.; Vreeken, R. J.; Hankemeier, T.; Kezic, S.; Wolterbeek, R.; Lavrijsen, A. P.; Bouwstra, J. A. Increase in shortchain ceramides correlates with an altered lipid organization and decreased barrier function in atopic eczema patients. J. Lipid Res. 2012, 53 (12), 2755−2766. (13) Janssens, M.; van Smeden, J.; Gooris, G. S.; Bras, W.; Portale, G.; Caspers, P. J.; Vreeken, R. J.; Kezic, S.; Lavrijsen, A. P.; Bouwstra, J. A. Lamellar lipid organization and ceramide composition in the stratum corneum of patients with atopic eczema. J. Invest. Dermatol. 2011, 131 (10), 2136−2138. (14) Chamlin, S. L.; Kao, J.; Frieden, I. J.; Sheu, M. Y.; Fowler, A. J.; Fluhr, J. W.; Williams, M. L.; Elias, P. M. Ceramide-dominant barrier repair lipids alleviate childhood atopic dermatitis: changes in barrier function provide a sensitive indicator of disease activity. J. Am. Acad. Dermatol. 2002, 47 (2), 198−208. (15) Man, M. Q.; Feingold, K. R.; Elias, P. M. Exogenous lipids influence permeability barrier recovery in acetone-treated murine skin. Arch. Dermatol. 1993, 129 (6), 728−738. (16) Vávrová, K.; Hrabálek, A.; Mac-Mary, S.; Humbert, P.; Muret, P. Ceramide analogue 14S24 selectively recovers perturbed human skin barrier. Br. J. Dermatol. 2007, 157 (4), 704−712. (17) Vávrová, K.; Zbytovská, J.; Palát, K.; Holas, T.; Klimentová, J.; Hrabálek, A.; Doležal, P. Ceramide analogue 14S24 ((S)-2tetracosanoylamino-3-hydroxypropionic acid tetradecyl ester) is effective in skin barrier repair in vitro. Eur. J. Pharm. Sci. 2004, 21 (5), 581−587. (18) Grosch, S.; Schiffmann, S.; Geisslinger, G. Chain length-specific properties of ceramides. Prog. Lipid Res. 2012, 51 (1), 50−62. (19) Chiantia, S.; Kahya, N.; Schwille, P. Raft domain reorganization driven by short- and long-chain ceramide: a combined AFM and FCS study. Langmuir 2007, 23 (14), 7659−7665. (20) Nybond, S.; Bjorkqvist, Y. J.; Ramstedt, B.; Slotte, J. P. Acyl chain length affects ceramide action on sterol/sphingomyelin-rich domains. Biochim. Biophys. Acta 2005, 1718 (1−2), 61−66. (21) Khazanov, E.; Priev, A.; Shillemans, J. P.; Barenholz, Y. Physicochemical and biological characterization of ceramide-containing liposomes: paving the way to ceramide therapeutic application. Langmuir 2008, 24 (13), 6965−6980. (22) Sot, J.; Aranda, F. J.; Collado, M. I.; Goni, F. M.; Alonso, A. Different effects of long- and short-chain ceramides on the gel-fluid and lamellar-hexagonal transitions of phospholipids: a calorimetric, NMR, and x-ray diffraction study. Biophys. J. 2005, 88 (5), 3368−3380. (23) Sot, J.; Goni, F. M.; Alonso, A. Molecular associations and surface-active properties of short- and long-N-acyl chain ceramides. Biochim. Biophys. Acta 2005, 1711 (1), 12−19. (24) 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 2011, 1811 (3), 129−137. 15632

dx.doi.org/10.1021/la4037474 | Langmuir 2013, 29, 15624−15633

Langmuir

Article

(45) Eeman, M.; Francius, G.; Dufrene, Y. F.; Nott, K.; Paquot, M.; Deleu, M. Effect of cholesterol and fatty acids on the molecular interactions of fengycin with Stratum corneum mimicking lipid monolayers. Langmuir 2009, 25 (5), 3029−3039. (46) Lopez-Castellano, A.; Cortell-Ivars, C.; Lopez-Carballo, G.; Herraez-Dominguez, M. The influence of Span20 on stratum corneum lipids in langmuir monolayers: comparison with Azone. Int. J. Pharm. 2000, 203 (1−2), 245−253. (47) Dupuy, F.; Fanani, M. L.; Maggio, B. Ceramide N-acyl chain length: a determinant of bidimensional transitions, condensed domain morphology, and interfacial thickness. Langmuir 2011, 27 (7), 3783− 3791. (48) Sassa, T.; Ohno, Y.; Suzuki, S.; Nomura, T.; Nishioka, C.; Kashiwagi, T.; Hirayama, T.; Akiyama, M.; Taguchi, R.; Shimizu, H.; Itohara, S.; Kihara, A. Impaired epidermal permeability barrier in mice lacking elovl1, the gene responsible for very-long-chain fatty acid production. Mol. Cell. Biol. 2013, 33 (14), 2787−2796. (49) Park, Y. H.; Jang, W. H.; Seo, J. A.; Park, M.; Lee, T. R.; Park, Y. H.; Kim, D. K.; Lim, K. M. Decrease of ceramides with very long-chain fatty acids and downregulation of elongases in a murine atopic dermatitis model. J. Invest. Dermatol. 2012, 132 (2), 476−479. (50) Gidwani, A.; Brown, H. A.; Holowka, D.; Baird, B. Disruption of lipid order by short-chain ceramides correlates with inhibition of phospholipase D and downstream signaling by FcepsilonRI. J. Cell. Sci. 2003, 116 (Pt15), 3177−3187. (51) Tawada, C.; Kanoh, H.; Nakamura, M.; Mizutani, Y.; Fujisawa, T.; Banno, Y.; Seishima, M. Interferon-gamma decreases ceramides with long-chain fatty acids: Possible involvement in atopic dermatitis and psoriasis. J. Invest. Dermatol. 2013, DOI: 10.1038/jid.2013.364. (52) Eckl, K. M.; Tidhar, R.; Thiele, H.; Oji, V.; Hausser, I.; Brodesser, S.; Preil, M. L.; Onal-Akan, A.; Stock, F.; Muller, D.; Becker, K.; Casper, R.; Nurnberg, G.; Altmuller, J.; Nurnberg, P.; Traupe, H.; Futerman, A. H.; Hennies, H. C. Impaired epidermal ceramide synthesis causes autosomal recessive congenital ichthyosis and reveals the importance of ceramide acyl chain length. J. Invest. Dermatol. 2013, 133 (9), 2202−2211. (53) Novotný, J.; Pospěchová, K.; Hrabálek, A.; Č áp, R.; Vávrová, K. Synthesis of fluorescent C24-ceramide: evidence for acyl chain length dependent differences in penetration of exogenous NBD-ceramides into human skin. Bioorg. Med. Chem. Lett. 2009, 19 (24), 6975−6977. (54) Barry, B. W. Novel mechanisms and devices to enable successful transdermal drug delivery. Eur. J. Pharm. Sci. 2001, 14 (2), 101−114.

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