Lipid Monolayer on Cell Surface Protein Templates Functional Extracellular Lipid Assembly.

When the ancestors of men moved from aquatic habitats to the drylands, their evolutionary strategy to restrict water loss is to seal the skin surface with lipids. It is unknown how these rigid ceramide-dominated lipids with densely packed chains squeeze through narrow extracellular spaces and how they assemble into their complex multilamellar architecture. Here it is shown that the human corneocyte lipid envelope, a monolayer of ultralong covalently bound lipids on the cell surface protein, templates the functional barrier assembly by partly fluidizing and rearranging the free extracellular lipids in its vicinity during the sculpting of a functional skin lipid barrier. The lipid envelope also maintains the fluidity of the extracellular lipids during mechanical stress. This local lipid fluidization does not compromise the permeability barrier. The results provide new testable hypotheses about epidermal homeostasis and the pathophysiology underlying diseases with impaired lipid binding to corneocytes, such as congenital ichthyosis. In a broader sense, this lipoprotein-mediated fluidization of rigid (sphingo)lipid patches may also be relevant to lipid rafts and cellular signaling events and inspire new functional materials.

The intriguing molecular dynamics of Cer[EOS] in rigid skin barrier lipid layers requires improvement of the model.

Omega-O-acyl ceramides such as 32-linoleoyloxydotriacontanoyl sphingosine (Cer[EOS]) are essential components of the lipid skin barrier, which protects our body from excessive water loss and the penetration of unwanted substances. These ceramides drive the lipid assembly to epidermal-specific long periodicity phase (LPP), structurally much different than conventional lipid bilayers. Here, we synthesized Cer[EOS] with selectively deuterated segments of the ultralong N-acyl chain or deuterated or 13C-labeled linoleic acid and studied their molecular behavior in a skin lipid model. Solid-state 2H NMR data revealed surprising molecular dynamics for the ultralong N-acyl chain of Cer[EOS] with increased isotropic motion toward the isotropic ester-bound linoleate. The sphingosine moiety of Cer[EOS] is also highly mobile at skin temperature, in stark contrast to the other LPP components, N-lignoceroyl sphingosine acyl, lignoceric acid, and cholesterol, which are predominantly rigid. The dynamics of the linoleic chain is quantitatively described by distributions of correlation times and using dynamic detector analysis. These NMR results along with neutron diffraction data suggest an LPP structure with alternating fluid (sphingosine chain-rich), rigid (acyl chain-rich), isotropic (linoleate-rich), rigid (acyl-chain rich), and fluid layers (sphingosine chain-rich). Such an arrangement of the skin barrier lipids with rigid layers separated with two different dynamic "fillings" i) agrees well with ultrastructural data, ii) satisfies the need for simultaneous rigidity (to ensure low permeability) and fluidity (to ensure elasticity, accommodate enzymes, or antimicrobial peptides), and iii) offers a straightforward way to remodel the lamellar body lipids into the final lipid barrier.

Cholesterol sulfate fluidizes the sterol fraction of the stratum corneum lipid phase and increases its permeability.

Desulfation of cholesterol sulfate (CholS) to cholesterol (Chol) is an important event in epidermal homeostasis and necessary for stratum corneum (SC) barrier function. The CholS/Chol ratio decreases during SC maturation but remains high in pathological conditions, such as X-linked ichthyosis, characterized by dry and scaly skin. The aim of this study was to characterize the influence of the CholS/Chol molar ratio on the structure, dynamics, and permeability of SC lipid model mixtures. We synthesized deuterated CholS and investigated lipid models with specifically deuterated components using 2H solid-state NMR spectroscopy at temperatures from 25°C to 80°C. Although the rigid acyl chains in ceramides and fatty acids remained essentially rigid upon variation of the CholS/Chol ratio, both sterols were increasingly fluidized in lipid models containing higher CholS concentrations. We also show the X-ray repeat distance of the lipid lamellar phase (105 Å) and the orthorhombic chain packing of the ceramide's acyl chains and long free fatty acids did not change upon the variation of the CholS content. However, the Chol phase separation visible in models with high Chol concentration disappeared at the 50:50 CholS/Chol ratio. This increased fluidity resulted in higher permeabilities to model markers of these SC models. These results reveal that a high CholS/Chol ratio fluidizes the sterol fraction and increases the permeability of the SC lipid phase while maintaining the lamellar lipid arrangement with an asymmetric sterol distribution.

Effects of (R)- and (S)-α-Hydroxylation of Acyl Chains in Sphingosine, Dihydrosphingosine, and Phytosphingosine Ceramides on Phase Behavior and Permeability of Skin Lipid Models.

Ceramides (Cers) with α-hydroxylated acyl chains comprise about a third of all extractable skin Cers and are required for permeability barrier homeostasis. We have probed here the effects of Cer hydroxylation on their behavior in lipid models comprising the major SC lipids, Cer/free fatty acids (C 16-C 24)/cholesterol, and a minor component, cholesteryl sulfate. Namely, Cers with (R)-α-hydroxy lignoceroyl chains attached to sphingosine (Cer AS), dihydrosphingosine (Cer AdS), and phytosphingosine (Cer AP) were compared to their unnatural (S)-diastereomers and to Cers with non-hydroxylated lignoceroyl chains attached to sphingosine (Cer NS), dihydrosphingosine (Cer NdS), and phytosphingosine (Cer NP). By comparing several biophysical parameters (lamellar organization by X-ray diffraction, chain order, lateral packing, phase transitions, and lipid mixing by infrared spectroscopy using deuterated lipids) and the permeabilities of these models (water loss and two permeability markers), we conclude that there is no general or common consequence of Cer α-hydroxylation. Instead, we found a rich mix of effects, highly dependent on the sphingoid base chain, configuration at the α-carbon, and permeability marker used. We found that the model membranes with unnatural Cer (S)-AS have fewer orthorhombically packed lipid chains than those based on the (R)-diastereomer. In addition, physiological (R)-configuration decreases the permeability of membranes, with Cer (R)-AdS to theophylline, and increases the lipid chain order in model systems with natural Cer (R)-AP. Thus, each Cer subclass makes a distinct contribution to the structural organization and function of the skin lipid barrier.

Effects of omega-O-acylceramide structures and concentrations in healthy and diseased skin barrier lipid membrane models.

Ceramides (Cers) with ultralong (∼32-carbon) chains and ω-esterified linoleic acid, composing a subclass called omega-O-acylceramides (acylCers), are indispensable components of the skin barrier. Normal barriers typically contain acylCer concentrations of ∼10 mol%; diminished concentrations, along with altered or missing long periodicity lamellar phase (LPP), and increased permeability accompany an array of skin disorders, including atopic dermatitis, psoriasis, and ichthyoses. We developed model membranes to investigate the effects of the acylCer structure and concentration on skin lipid organization and permeability. The model membrane systems contained six to nine Cer subclasses as well as fatty acids, cholesterol, and cholesterol sulfate; acylCer content-namely, acylCers containing sphingosine (Cer EOS), dihydrosphingosine (Cer EOdS), and phytosphingosine (Cer EOP) ranged from zero to 30 mol%. Systems with normal physiologic concentrations of acylCer mixture mimicked the permeability and nanostructure of human skin lipids (with regard to LPP, chain order, and lateral packing). The models also showed that the sphingoid base in acylCer significantly affects the membrane architecture and permeability and that Cer EOP, notably, is a weaker barrier component than Cer EOS and Cer EOdS. Membranes with diminished or missing acylCers displayed some of the hallmarks of diseased skin lipid barriers (i.e., lack of LPP, less ordered lipids, less orthorhombic chain packing, and increased permeability). These results could inform the rational design of new and improved strategies for the barrier-targeted treatment of skin diseases.

The Sphingosine and Acyl Chains of Ceramide [NS] Show Very Different Structure and Dynamics That Challenge Our Understanding of the Skin Barrier.

The lipid phase of the uppermost human skin layer is thought to comprise highly rigid lipids in an orthorhombic phase state to protect the body against the environment. By synthesizing sphingosine-d28 deuterated N-lignoceroyl-d-erythro-sphingosine (ceramide [NS]), we compare the structure and dynamics of both chains of that lipid in biologically relevant mixtures using X-ray diffraction, 2 H NMR analysis, and infrared spectroscopy. Our results reveal a substantial fraction of sphingosine chains in a fluid and dynamic phase state at physiological temperature. These findings prompt revision of our current understanding of the skin lipid barrier, where an extended ceramide [NS] conformation is preferred and a possible domain structure is proposed. Mobile lipid chains may be crucial for skin elasticity and the translocation of physiologically important molecules.

Long and very long lamellar phases in model stratum corneum lipid membranes.

Membrane models of the stratum corneum (SC) lipid barrier, either healthy or affected by recessive X-linked ichthyosis, constructed from ceramide [Cer; nonhydroxyacyl sphingosine N-tetracosanoyl-d-erythro-sphingosine (CerNS24) alone or with omega-O-acylceramide N-(32-linoleyloxy)dotriacontanoyl-d-erythro-sphingosine (CerEOS)], FFAs(C16-24), cholesterol (Chol), and sodium cholesteryl sulfate (CholS) were investigated. X-ray diffraction (XRD) revealed a previously unreported polymorphism of the membranes. In the absence of CerEOS, the membranes formed a short lamellar phase (SLP; the repeat distance d = 5.3 nm), a medium lamellar phase (MLP; d = 10.6 nm), or very long lamellar phases (VLLP; d = 15.9 and 21.2 nm). An increased CholS-to-Chol ratio modulated the membrane polymorphism, although the CholS phase separated at ≥ 7 weight% (of total lipids). The presence of CerEOS led to the stable long lamellar phase (LLP) with d = 12.2 nm and prevented VLLP formation. Our XRD results agree well with recently published cryo-electron microscopy data for vitreous skin sections, while also revealing new structures. Thus, lamellar phases with long repeat distances (MLP and VLLP) may be formed in the absence of omega-O-acylceramide, whereas these ultralong Cer species likely stabilize the final SC lipid architecture of LLP by riveting the adjacent lipid layers.

Probing the role of ceramide hydroxylation in skin barrier lipid models by 2H solid-state NMR spectroscopy and X-ray powder diffraction.

In this work, we studied model stratum corneum lipid mixtures composed of the hydroxylated skin ceramides N-lignoceroyl 6-hydroxysphingosine (Cer[NH]) and α-hydroxylignoceroyl phytosphingosine (Cer[AP]). Two model skin lipid mixtures of the composition Cer[NH] or Cer[AP], N-lignoceroyl sphingosine (Cer[NS]), lignoceric acid (C24:0) and cholesterol in a 0.5:0.5:1:1 molar ratio were compared. Model membranes were investigated by differential scanning calorimetry and 2H solid-state NMR spectroscopy at temperatures from 25 °C to 80 °C. Each component of the model mixture was specifically deuterated for selective detection by 2H NMR. Thus, the exact phase composition of the mixture at varying temperatures could be quantified. Moreover, using X-ray powder diffraction we investigated the lamellar phase formation. From the solid-state NMR and DSC studies, we found that both hydroxylated Cer[NH] and Cer[AP] exhibit a similar phase behavior. At physiological skin temperature of 32 °C, the lipids form a crystalline (orthorhombic) phase. With increasing temperature, most of the lipids become fluid and form a liquid-crystalline phase, which converts to the isotropic phase at higher temperatures (65-80 °C). Interestingly, lignoceric acid in the Cer[NH]-containing mixture has a tendency to form two types of fluid phases at 65 °C. This tendency was also observed in Cer[AP]-containing membranes at 80 °C. While Cer[AP]-containing lipid models formed a short periodicity phase featuring a repeat spacing of d = 5.4 nm, in the Cer[NH]-based model skin lipid membranes, the formation of unusual long periodicity phase with a repeat spacing of d = 10.7 nm was observed.

Effects of Ceramide and Dihydroceramide Stereochemistry at C-3 on the Phase Behavior and Permeability of Skin Lipid Membranes.

Ceramides (Cer) are key components of the skin permeability barrier. Sphingosine-based CerNS and dihydrosphingosine-based CerNdS (dihydroCer) have two chiral centers; however, the importance of the correct stereochemistry in the skin barrier Cer is unknown. We investigated the role of the configuration at C-3 of CerNS and CerNdS in the organization and permeability of model skin lipid membranes. Unnatural l-threo-CerNS and l-threo-CerNdS with 24-C acyl chains were synthesized and, along with their natural d-erythro-isomers, incorporated into membranes composed of major stratum corneum lipids (Cer, free fatty acids, cholesterol, and cholesteryl sulfate). The membrane microstructure was investigated by X-ray powder diffraction and infrared spectroscopy, including deuterated free fatty acids. Inversion of the C-3 configuration in CerNS and CerNdS increased phase transition temperatures, had no significant effects on lamellar phases, but also decreased the proportion of orthorhombic packing and decreased lipid mixing in the model membranes. These changes in membrane organization resulted in membrane permeabilities that ranged from unchanged to 5-fold higher (depending on the permeability markers, namely, water loss, electrical impedance, flux of theophylline, and flux of indomethacin) compared to membranes with natural CerNS/NdS isomers. Thus, the physiological d-erythro stereochemistry of skin Cer and dihydroCer appears to be essential for their correct barrier function.

DNA-DOPE-gemini surfactants complexes at low surface charge density: from structure to transfection efficiency.

DNA condensation, structure and transfection efficiency of complexes formed by gemini surfactants alkane-α,ω-diyl-bis(dodecyldimethylammonium bromide)s (CnGS12, n = 3, 6 and 12 is the number of alkane spacer carbons), dioleoylphosphatidylethanolamine (CnGS12/DOPE = 0.3 mol/mol) and DNA at low surface charge density were investigated through different techniques. Small angle X-ray diffraction showed a condensed lamellar phase with marked dependence of DNA-DNA distance on (+/-) charge ratio. High ionic strength of hydrating medium screens the interaction DNA - CnGS12/DOPE and complexed DNA represented maximally ~ 45-60% of total DNA in the solution as derived from fluorescence and UV-VIS spectroscopy. The in vitro transfection efficiency of CnGS12/DOPE liposomes on mammalian HEK 293 cell line was spacer length-dependent. C12GS12/DOPE/DNA complexes exhibited the best transfection efficiency (~ 18% GFP-expressing cells relative to all viable cells) accompanied by ~ 89% cell viability.

Effects of 6-Hydroxyceramides on the Thermotropic Phase Behavior and Permeability of Model Skin Lipid Membranes.

Ceramides (Cer) based on 6-hydroxysphingosine are important components of the human skin barrier, the stratum corneum. Although diminished concentrations of 6-hydroxyCer have been detected in skin diseases such as atopic dermatitis, our knowledge on these unusual sphingolipids, which have only been found in the skin, is limited. In this work, we investigate the biophysical behavior of N-lignoceroyl-6-hydroxysphingosine (Cer NH) in multilamellar lipid membranes composed of Cer/free fatty acids (FFAs) (C16-C24)/cholesterol/cholesteryl sulfate. To probe the Cer structure-activity relationships, we compared Cer NH membranes with membranes containing Cer with sphingosine (Cer NS), dihydrosphingosine, and phytosphingosine (Cer NP), all with the same acyl chain length (C24). Compared with Cer NS, 6-hydroxylation of Cer not only increased membrane water loss and permeability in a lipophilic model compound but also dramatically increased the membrane opposition to electrical current, which is proportional to the flux of ions. Infrared spectroscopy revealed that Cer hydroxylation (in either Cer NH or Cer NP) increased the main transition temperature of the membrane but prevented good Cer mixing with FFAs. X-ray powder diffraction showed not only lamellar phases with shorter periodicity upon Cer hydroxylation but also the formation of an unusually long periodicity phase (d = 10.6 nm) in Cer NH-containing membranes. Thus, 6-hydroxyCer behaves differently from sphingosine- and phytosphingosine-based Cer. In particular, the ability to form a long-periodicity lamellar phase and highly limited permeability to ions indicate the manner in which 6-hydroxylated Cer contribute to the skin barrier function.

Stimuli responsive polymorphism of C12NO/DOPE/DNA complexes: Effect of pH, temperature and composition.

N,N-dimethyldodecylamine-N-oxide (C12NO) is a surfactant that may exist either in a neutral or cationic protonated form depending on the pH of aqueous solutions. Using small angle X-ray diffraction (SAXD) we observe the rich structural polymorphism of pH responsive complexes prepared due to DNA interaction with C12NO/dioleoylphosphatidylethanolamine (DOPE) vesicles and discuss it in view of utilizing the surfactant for the gene delivery vector of a pH sensitive system. In neutral solutions, the DNA uptake is low, and a lamellar Lα phase formed by C12NO/DOPE is prevailing in the complexes at 0.2≤C12NO/DOPE<0.6 mol/mol. A maximum of ~30% of the total DNA volume in the sample is bound in a condensed lamellar phase LαC at C12NO/DOPE=1 mol/mol and pH7.2. In acidic conditions, a condensed inverted hexagonal phase HIIC was observed at C12NO/DOPE=0.2 mol/mol. Commensurate lattice parameters, aHC≈dLC, were detected at 0.3≤C12NO/DOPE≤0.4 mol/mol and pH=4.9-6.4 suggesting that LαC and HIIC phases were epitaxially related. While at the same composition but pH~7, the mixture forms a cubic phase (Pn3m) when the complexes were heated to 80°C and cooled down to 20°C. Finally, a large portion of the surfactant (C12NO/DOPE>0.5) stabilizes the LαC phase in C12NO/DOPE/DNA complexes and the distance between DNA strands (dDNA) is modulated by the pH value. Both the composition and pH affect the DNA binding in the complexes reaching up to ~95% of the DNA total amount at acidic conditions.

Effects of sphingomyelin/ceramide ratio on the permeability and microstructure of model stratum corneum lipid membranes.

The conversion of sphingomyelin (SM) to a ceramide (Cer) by acid sphingomyelinase (aSMase) is an important event in skin barrier development. A deficiency in aSMase in diseases such as Niemann-Pick disease and atopic dermatitis coincides with impaired skin barrier recovery after disruption. We studied how an increased SM/Cer ratio influences the barrier function and microstructure of model stratum corneum (SC) lipid membranes. In the membranes composed of isolated human SC Cer (hCer)/cholesterol/free fatty acids/cholesteryl sulfate, partial or full replacement of hCer by SM increased water loss. Partial replacement of 25% and 50% of hCer by SM also increased the membrane permeability to theophylline and alternating electric current, while a higher SM content either did not alter or even decreased the membrane permeability. In contrast, in a simple membrane model with only one type of Cer (nonhydroxyacyl sphingosine, CerNS), an increased SM/Cer ratio provided a similar or better barrier against the permeation of various markers. X-ray powder diffraction revealed that the replacement of hCer by SM interferes with the formation of the long periodicity lamellar phase with a repeat distance of d=12.7nm. Our results suggest that SM-to-Cer processing in the human epidermis is essential for preventing excessive water loss, while the permeability barrier to exogenous compounds is less sensitive to the presence of sphingomyelin.

The role of the trans double bond in skin barrier sphingolipids: permeability and infrared spectroscopic study of model ceramide and dihydroceramide membranes.

Dihydroceramides (dCer) are members of the sphingolipid family that lack the C4 trans double bond in their sphingoid backbone. In addition to being precursors of ceramides (Cer) and phytoceramides, dCer have also been found in the extracellular lipid membranes of the epidermal barrier, the stratum corneum. However, their role in barrier homeostasis is not known. We studied how the lack of the trans double bond in dCer compared to Cer influences the permeability, lipid chain order, and packing of multilamellar membranes composed of the major skin barrier lipids: (d)Cer, fatty acids, cholesterol, and cholesteryl sulfate. The permeability of the membranes with long-chain dCer was measured using various markers and was either comparable to or only slightly greater than (by up to 35%, not significant) that of the Cer membranes. The dCer were less sensitive to acyl chain shortening than Cer (the short dCer membranes were up to 6-fold less permeable that the corresponding short Cer membranes). Infrared spectroscopy showed that long dCer mixed less with fatty acids but formed more thermally stable ordered domains than Cer. The key parameter explaining the differences in permeability in the short dCer and Cer was the proportion of the orthorhombic phase. Our results suggest that the presence of the trans double bond in Cer is not crucial for the permeability of skin lipid membranes and that dCer may be underappreciated members of the stratum corneum lipid barrier that increase its heterogeneity.

The DNA-DNA spacing in gemini surfactants-DOPE-DNA complexes.

Gemini surfactants from the homologous series of alkane-α,ω-diyl-bis(dodecyldimethylammonium bromide) (CnCS12, number of spacer carbons n=2-12) and dioleoylphosphatidylethanolamine (DOPE) were used for cationic liposome (CL) preparation. CLs condense highly polymerized DNA creating complexes. Small-angle X-ray diffraction identified them as condensed lamellar phase L(α)(C) in the studied range of molar ratios CnGS12/DOPE in the temperature range 20-60°C. The DNA-DNA distance (d(DNA)) is studied in dependence to CnGS12 spacer length and membrane surface charge density. The high membrane surface charge densities (CnGS12/DOPE=0.35 and 0.4mol/mol) lead to the linear dependence of d(DNA) vs. n correlating with the interfacial area of the CnGS12 molecule.

Polymorphism, Nanostructures, and Barrier Properties of Ceramide-Based Lipid Films.

Ceramides belong to sphingolipids, an important group of cellular and extracellular lipids. Their physiological functions range from cell signaling to participation in the formation of barriers against water evaporation. In the skin, they are essential for the permeability barrier, together with free fatty acids and cholesterol. We examined the periodical structure and permeability of lipid films composed of ceramides (Cer; namely, N-lignoceroyl 6-hydroxysphingosine, CerNH24, and N-lignoceroyl sphingosine, CerNS24), lignoceric acid (LIG; 24:0), and cholesterol (Chol). X-ray diffraction experiments showed that the CerNH24-based samples form either a short lamellar phase (SLP, d ∼ 5.4 nm) or a medium lamellar phase (MLP, d = 10.63-10.78 nm) depending on the annealing conditions. The proposed molecular arrangement of the MLP based on extended Cer molecules also agreed with the relative neutron scattering length density profiles obtained from the neutron diffraction data. The presence of MLP increased the lipid film permeability to the lipophilic model permeant (indomethacin) relative to the CerNS24-based control samples and the samples that had the same lipid composition but formed an SLP. Thus, the arrangement of lipids in various nanostructures is responsive to external conditions during sample preparation. This polymorphic behavior directly affects the barrier properties, which could also be (patho)physiologically relevant.

The microstructure of DNA-egg yolk phosphatidylcholine-gemini surfactants complexes: effect of the spacer length.

BACKGROUND: The length of spacer of gemini surfactants affects the DNA packing in DNA-neutral phospholipid-gemini surfactant complexes. METHODS: The microstructure of complexes DNA-egg yolk phosphatidylcholine (EYPC)-alkane-α,ω-diyl-bis(dodecyl\xaddimethylammonium bromides) (CnGS, spacer n=2-12, n is even) was studied using small angle X-ray diffraction. RESULTS: At EYPC:CnGS=1:1 mol/mol, the condensed lamellar phase was identified in complexes with CnGS, n=2-4, whereas longer spacer (n≥6) induced a hexagonal phase. The condensed lamellar phase Lαc was observed in the range 2\≤ EYPC:GnGS≤10 (mol/mol) in all complexes. The distance between adjacent DNA strands increases linearly with decreasing surface charge density of EYPC-CnGS vesicles. We determined the increase in dDNA 0.40±0.03 nm/1 mol of EYPC from the slope of dDNA=f (molEYPC/molCnGS) in the range of molar ratios 2≤EYPC:CnGS≤5. At lower surface charge density, EYPC:CnGS>5 mol/mol, the length of CnGS spacer (n=6-10) modulates the DNA-DNA distance. CONCLUSIONS: Both the short spacer of CnGS and the low molar ratio EYPC:CnGS result in the closest DNA-DNA packing. A high surface charge density of membrane was reported as a key parameter for transfection efficiency of Lαc phase-forming complexes.

Study of interaction of long-chain n-alcohols with fluid DOPC bilayers by a lateral pressure sensitive fluorescence probe.

The excimer 1,2-dipyrenedecanoyl-sn-glycero-3-phosphatidylcholine (dipy10PC) fluorescence probe was used to determine effects of aliphatic alcohols (CnH2n+1OH, n = 12-18 is the even number of carbons in alkyl chain) on fluid dioleoylphosphatidylcholine (DOPC) +dioleoylphosphatidylserine (DOPS) bilayers in multilamellar vesicles at molar ratio DOPC/DOPS = 24.7. The excimer to monomer fluorescence intensity ratio increases with the increase of CnH2n+1OH/DOPC molar ratio and decreases with the CnH2n+1OH alkyl chain length n at a constant CnH2n+1OH/DOPC = 0.4 molar ratio. These effects indicate changes in the bilayer lateral pressure on the level of pyrenyl moieties location.

Acidic pH Is Required for the Multilamellar Assembly of Skin Barrier Lipids In Vitro.

Lipid membrane remodeling belongs to the most fundamental processes in the body. The skin barrier lipids, which are ceramide dominant and highly rigid, must attain an unusual multilamellar nanostructure with long periodicity to restrict water loss and prevent the entry of potentially harmful environmental factors. Our data suggest that the skin acid mantle, apart from regulating enzyme activities and keeping away pathogens, may also be a prerequisite for the multilamellar assembly of the skin barrier lipids. Atomic force microscopy on monolayers composed of synthetic or human stratum corneum lipids showed multilayer formation (approximately 10-nm step height) in an acidic but not in a neutral environment. X-ray diffraction, Fourier transform infrared spectroscopy, and permeability studies showed markedly altered lipid nanostructure and increased water loss at neutral pH compared with that at acidic pH. These findings are consistent with the data on the altered organization of skin lipids and increased transepidermal water loss under conditions such as inadequate skin acidification, for example, in neonates, the elderly, and patients with atopic dermatitis.

Behavior of 1-Deoxy-, 3-Deoxy- and N-Methyl-Ceramides in Skin Barrier Lipid Models.

Ceramides (Cer) are essential components of the skin permeability barrier. To probe the role of Cer polar head groups involved in the interfacial hydrogen bonding, the N-lignoceroyl sphingosine polar head was modified by removing the hydroxyls in C-1 (1-deoxy-Cer) or C-3 positions (3-deoxy-Cer) and by N-methylation of amide group (N-Me-Cer). Multilamellar skin lipid models were prepared as equimolar mixtures of Cer, lignoceric acid and cholesterol, with 5 wt% cholesteryl sulfate. In the 1-deoxy-Cer-based models, the lipid species were separated into highly ordered domains (as found by X-ray diffraction and infrared spectroscopy) resulting in similar water loss but 4-5-fold higher permeability to model substances compared to control with natural Cer. In contrast, 3-deoxy-Cer did not change lipid chain order but promoted the formation of a well-organized structure with a 10.8 nm repeat period. Yet both lipid models comprising deoxy-Cer had similar permeabilities to all markers. N-Methylation of Cer decreased lipid chain order, led to phase separation, and improved cholesterol miscibility in the lipid membranes, resulting in 3-fold increased water loss and 10-fold increased permeability to model compounds compared to control. Thus, the C-1 and C-3 hydroxyls and amide group, which are common to all Cer subclasses, considerably affect lipid miscibility and chain order, formation of periodical nanostructures, and permeability of the skin barrier lipid models.