Lipid mixtures prepared with well-defined synthetic ceramides closely mimic the unique stratum corneum lipid phase behavior

Lipid lamellae present in the outermost layer of the skin, the stratum corneum, form the main barrier for the diffusion of molecules through the skin. The presence of a unique 13 nm lamellar phase and its high crystallinity are characteristic for the stratum corneum lipid phase behavior. In the present study, small-angle and wide-angle X-ray diffraction were used to examine the organization in lipid mixtures prepared with a unique set of well-defined synthetic ceramides, varying from each other in head group architecture and acyl chain length. The results show that equimolar mixtures of cholesterol, free fatty acids, and synthetic ceramides (resembling the composition of pig ceramides) closely resemble the lamellar and lateral stratum corneum lipid organization, both at room and higher temperatures. Exclusion of several ceramide classes from the mixture does not affect the lipid organization. However, complete substitution of ceramide 1 (acylceramide with a sphingosine base) with ceramide 9 (acylceramide with a phytosphingosine base) reduces the formation of the long periodicity lamellar phase. This indicates that the head group architecture of acylceramides affects the lipid organization. In conclusion, lipid mixtures prepared with well-defined synthetic ceramides offer an attractive tool with which to unravel the importance of the molecular structure of individual ceramides for proper lipid organization.     

The skin barrier in healthy and diseased state

The primary function of the skin is to protect the body for unwanted influences from the environment. The main barrier of the skin is located in the outermost layer of the skin, the stratum corneum. The stratum corneum consists of corneocytes surrounded by lipid regions. As most drugs applied onto the skin permeate along the lipid domains, the lipid organization is considered to be very important for the skin barrier function. It is for this reason that the lipid organization has been investigated quite extensively. Due to the exceptional stratum corneum lipid composition, with long chain ceramides, free fatty acids and cholesterol as main lipid classes, the lipid organization is different from that of other biological membranes. In stratum corneum, two lamellar phases are present with repeat distances of approximately 6 and 13 nm. Moreover the lipids in the lamellar phases form predominantly crystalline lateral phases, but most probably a subpopulation of lipids forms a liquid phase. Diseased skin is often characterized by a reduced barrier function and an altered lipid composition and organization. In order to understand the aberrant lipid organization in diseased skin, information on the relation between lipid composition and organization is crucial. However, due to its complexity and inter-individual variability, the use of native stratum corneum does not allow detailed systematic studies. To circumvent this problem, mixtures prepared with stratum corneum lipids can be used. In this paper first the lipid organization in stratum corneum of normal and diseased skin is described. Then the role the various lipid classes play in stratum corneum lipid organization and barrier function has been discussed. Finally, the information on the role various lipid classes play in lipid phase behavior has been used to interpret the changes in lipid organization and barrier properties of diseased skin.     

The skin barrier in healthy and diseased state

The primary function of the skin is to protect the body for unwanted influences from the environment. The main barrier of the skin is located in the outermost layer of the skin, the stratum corneum. The stratum corneum consists of corneocytes surrounded by lipid regions. As most drugs applied onto the skin permeate along the lipid domains, the lipid organization is considered to be very important for the skin barrier function. It is for this reason that the lipid organization has been investigated quite extensively. Due to the exceptional stratum corneum lipid composition, with long chain ceramides, free fatty acids and cholesterol as main lipid classes, the lipid organization is different from that of other biological membranes. In stratum corneum, two lamellar phases are present with repeat distances of approximately 6 and 13 nm. Moreover the lipids in the lamellar phases form predominantly crystalline lateral phases, but most probably a subpopulation of lipids forms a liquid phase. Diseased skin is often characterized by a reduced barrier function and an altered lipid composition and organization. In order to understand the aberrant lipid organization in diseased skin, information on the relation between lipid composition and organization is crucial. However, due to its complexity and inter-individual variability, the use of native stratum corneum does not allow detailed systematic studies. To circumvent this problem, mixtures prepared with stratum corneum lipids can be used. In this paper first the lipid organization in stratum corneum of normal and diseased skin is described. Then the role the various lipid classes play in stratum corneum lipid organization and barrier function has been discussed. Finally, the information on the role various lipid classes play in lipid phase behavior has been used to interpret the changes in lipid organization and barrier properties of diseased skin.     

The Lipid Organisation of the Skin Barrier: Liquid and Crystalline Domains Coexist in Lamellar Phases

The superficial layer of the skin, the stratum corneum, is the main barrier for diffusion of substances across the skin. The stratum corneum is composed of corneocytes embedded in lipid lamellae. In previous studies two lamellar phases have been identified with periodicities of 6.4 and 13.4 nm of which the 13.4 nm phase (long periodicity phase = LPP) is considered to be very important for the skin banier function. The main lipid classes in stratumcorneum are ceramides, free fatty acids and cholesterol. Until now 8 subclassesof ceramides are identified in human stratum corneum referred to as ceramide 1 to 8. Studies with mixtures prepared with isolated human ceramides revealed that cholesterol and ceramides are very important for the formation of the lamellar phases. After addition of free fatty acids the lipids are organised in an orthorhombic packing with a small proportion of lipids in a liquid phase. Our most recent results show that the presence of ceramide 1 and the formation of a liquid phase are crucial elements for the formation of the LPP. These observations and the broad-narrowbroad sequence of lipid layers in the LPP led us to propose a molecular model for this phase. This consists of one narrow central lipid layer with fluid domains with on both sides a broad layer with a crystalline structure. This model is referred to as `the sandwich model'.     

Disposition of ceramide in model lipid membranes determined by neutron diffraction

The lipid matrix present in the uppermost layer of the skin, the stratum corneum, plays a crucial role in the skin barrier function. The lipids are organized into two lamellar phases. To gain more insight into the molecular organization of one of these lamellar phases, we performed neutron diffraction studies. In the diffraction pattern, five diffraction orders were observed attributed to a lamellar phase with a repeat distance of 5.4 nm. Using contrast variation, the scattering length density profile could be calculated showing a typical bilayer arrangement. To obtain information on the arrangement of ceramides in the unit cell, a mixture that included a partly deuterated ceramide was also examined. The scattering length density profile of the 5.4-nm phase containing this deuterated ceramide demonstrated a symmetric arrangement of the ceramides with interdigitating acyl chains in the center of the unit cell.     

Novel lipid mixtures based on synthetic ceramides reproduce the unique stratum corneum lipid organization

Lipid lamellae present in the outermost layer of the skin protect the body from uncontrolled water loss. In human stratum corneum (SC), two crystalline lamellar phases are present, which contain mostly cholesterol, free fatty acids, and nine types of free ceramides. Previous studies have demonstrated that the SC lipid organization can be mimicked with model mixtures based on isolated SC lipids. However, those studies are hampered by low availability and high interindividual variability of the native tissue. To elucidate the role of each lipid class in the formation of a competent skin barrier, the use of synthetic lipids would offer an alternative. The small- and wide-angle X-ray diffraction results of the present study show for the first time that synthetic lipid mixtures, containing only three synthetic ceramides, reflect to a high extent the SC lipid organization. Both an appropriately chosen preparation method and lipid composition promote the formation of two characteristic lamellar phases with repeat distances similar to those found in native SC. From all synthetic lipid mixtures examined, equimolar mixtures of cholesterol, ceramides, and free fatty acids equilibrated at 80 degrees C resemble to the highest extent the lamellar and lateral SC lipid organization, both at room and increased temperatures.     

Influence of different ceramides on the structure of in vitro model lipid systems of the stratum corneum lipid matrix

Human stratum corneum (SC) consists of several layers of keratinized corneocytes embedded in a lipid matrix of ordered lamellar structure which is considered to constitute the major barrier to percutaneous penetration. Artificial mixtures of SC lipids are often used as model systems to mimic the skin barrier or to investigate the effects of substances on the phase behaviour of the models. In the present study a SC lipid model composed of cholesterol, fatty acids and ceramides was used to investigate the effect of three different commercially available ceramide types on the microstructure and the physicochemical behaviour of the lipids. Polarized light microscopy, transmission electron microscopy, small-angle X-ray diffraction, wide-angle X-ray diffraction and differential scanning calorimetry (DSC) were used for physicochemical characterization. The results revealed a lamellar structure for all models but showed differences with regard to the thermal and optical behaviour depending obviously on the composition of the ceramide mixtures. A model containing a mixture of Cer[AS] was comparable to human SC lipids.     

Phase behavior of lipid mixtures based on human ceramides: coexistence of crystalline and liquid phases.

The lipid regions in the outermost layer of the skin (stratum corneum) form the main barrier for diffusion of substances through the skin. In this layer the main lipid classes are ceramides, cholesterol (CHOL), and FFA. Previous studies revealed a coexistence of two crystalline lamellar phases with periodicities of approximately 13 nm (referred to as long periodicity phase) and 6 nm (short periodicity phase). Additional studies showed that lipid mixtures prepared with isolated pig ceramides (pigCER) mimic lipid phase behavior in stratum corneum closely. Because the molecular structure of pigCER differs in some important aspects from that of human ceramides (HCER), in the present study the phase behavior of mixtures prepared with HCER has been examined. Phase behavior studies of mixtures based on HCER revealed that in CHOL:HCER mixtures the long periodicity phase dominates. In the absence of HCER1 the short periodicity phase is dominant. Addition of FFA promotes the formation of the short periodicity phase and induces a transition from a hexagonal sublattice to an orthorhombic sublattice. Furthermore, the presence of FFA promotes the formation of a liquid phase. Finally, cholesterol sulfate, a minor but important lipid in the stratum corneum, reduces the amount of cholesterol that phase separates in crystalline domains. From these observations it can be concluded that the phase behavior of mixtures prepared from HCER differs in some important aspects from that prepared from pigCER. The most prevalent differences are the following: i) the addition of FFA promotes the formation of the short periodicity phase; and ii) liquid lateral packing is obviously present in CHOL:HCER:FFA mixtures. These changes in phase behavior might be due to a larger amount of linoleic acid moiety in HCER mixtures compared with that in pigCER mixtures.     

The permeability enhancing mechanism of DMSO in ceramide bilayers simulated by molecular dynamics

The lipids of the topmost layer of the skin, the stratum corneum, represent the primary barrier to molecules penetrating the skin. One approach to overcoming this barrier for the purpose of delivery of active molecules into or via the skin is to employ chemical permeability enhancers, such as dimethylsulfoxide (DMSO). How these molecules exert their effect at the molecular level is not understood. We have investigated the interaction of DMSO with gel-phase bilayers of ceramide 2, the predominant lipid in the stratum corneum, by means of molecular dynamics simulations. The simulations satisfactorily reproduce the phase behavior and the known structural parameters of ceramide 2 bilayers in water. The effect of DMSO on the gel-phase bilayers was investigated at various concentrations over the range 0.0-0.6 mol fraction DMSO. The DMSO molecules accumulate in the headgroup region and weaken the lateral forces between the ceramides. At high concentrations of DMSO (> or =0.4 mol fraction), the ceramide bilayers undergo a phase transition from the gel phase to the liquid crystalline phase. The liquid-crystalline phase of ceramides is expected to be markedly more permeable to solutes than the gel phase. The results are consistent with the experimental evidence that high concentrations of DMSO fluidize the stratum corneum lipids and enhance permeability.     

Cholesterol sulfate and calcium affect stratum corneum lipid organization over a wide temperature range

The main diffusion barrier for drugs penetrating through the skin is located in the intercellular lipid matrix in the upper layer of the skin, the stratum corneum (SC). The main lipid classes in the SC are ceramides (CER), free fatty acids (FFA) and cholesterol (CHOL). The lipids in SC are organized into two lamellar phases with periodicities of approximately 13 and 6 nm, respectively. Similar lipid organization has been found with equimolar CHOL:CER:FFA mixtures in SAXD studies performed at room temperature. However, one may conclude that the phase behavior of the mixtures is similar to that in SC only when the lipid organization of the lipid mixtures resembles that in SC over a wide temperature range. Therefore, in the present study, the organization of the lipid mixtures has been studied in a temperature range between 20 degrees and 95 degrees C. From these experiments it appeared that at elevated temperatures in equimolar CHOL:CER:FFA mixtures a new prominent 4.3 nm phase is formed between 35;-55 degrees C, which is absent or only weakly formed in intact human and pig SC, respectively. As it has been suggested that gradients of pH and cholesterol sulfate exist in the SC and that Ca(2+) is present only in the lowest SC layers, the effect of pH, cholesterol sulfate, and Ca(2+) on the lipid phase behavior has been investigated with lipid mixtures. Both an increase in pH from 5 (pH at the skin surface) to 7.4 (pH at the SC;-stratum granulosum interface) and the presence of cholesterol sulfate promote the formation of the 13 nm lamellar phase. Furthermore, cholesterol sulfate reduces the amount of CHOL that is present in crystalline domains, causes a shift in the formation of the 4.3 nm phase to higher temperatures, and makes this phase less prominent at higher temperatures. The finding that Ca(2+) counteracts the effects of cholesterol sulfate indicates the importance of a proper balance of minor SC components for appropriate SC lipid organization. In addition, when the findings are extrapolated to the in vivo situation, it seems that cholesterol sulfate is required to dissolve cholesterol in the lamellar phases and to stabilize SC lipid organization. Therefore, a drop in cholesterol sulfate content in the superficial layers of the SC is expected to destabilize the lipid lamellar phases, which might facilitate the desquamation process.     

Comparison of rat epidermal keratinocyte organotypic culture (ROC) with intact human skin: lipid composition and thermal phase behavior of the stratum corneum.

The present report is a part of our continuing efforts to explore the utility of the rat epidermal keratinocyte organotypic culture (ROC) as an alternative model to human skin in transdermal drug delivery and skin irritation studies of new chemical entities and formulations. The aim of the present study was to compare the stratum corneum lipid content of ROC with the corresponding material from human skin. The lipid composition was determined by thin-layer chromatography (TLC) and mass-spectrometry, and the thermal phase transitions of stratum corneum were studied by differential scanning calorimetry (DSC). All major lipid classes of the stratum corneum were present in ROC in a similar ratio as found in human stratum corneum. Compared to human skin, the level of non-hydroxyacid-sphingosine ceramide (NS) was increased in ROC, while alpha-hydroxyacid-phytosphingosine ceramide (AP) and non-hydroxyacid-phytosphingosine ceramides (NP) were absent. Also some alterations in fatty acid profiles of ROC ceramides were noted, e.g., esterified omega-hydroxyacid-sphingosine contained increased levels of oleic acid instead of linoleic acid. The fraction of lipids covalently bound to corneocyte proteins was distinctly lower in ROC compared to human skin, in agreement with the results from DSC. ROC underwent a lipid lamellar order to disorder transition (T2) at a slightly lower temperature (68 degrees C) than human skin (74 degrees C). These differences in stratum corneum lipid composition and the thermal phase transitions may explain the minor differences previously observed in drug permeation between ROC and human skin.     

Effect of ceramide acyl chain length on skin permeability and thermotropic phase behavior of model stratum corneum lipid membranes

Stratum corneum ceramides play an essential role in the barrier properties of skin. However, their structure-activity relationships are poorly understood. We investigated the effects of acyl chain length in the non-hydroxy acyl sphingosine type (NS) ceramides on the skin permeability and their thermotropic phase behavior. Neither the long- to medium-chain ceramides (8-24 C) nor free sphingosine produced any changes of the skin barrier function. In contrast, the short-chain ceramides decreased skin electrical impedance and increased skin permeability for two marker drugs, theophylline and indomethacin, with maxima in the 4-6C acyl ceramides. The thermotropic phase behavior of pure ceramides and model stratum corneum lipid membranes composed of ceramide/lignoceric acid/cholesterol/cholesterol sulfate was studied by differential scanning calorimetry and infrared spectroscopy. Differences in thermotropic phase behavior of these lipids were found: those ceramides that had the greatest impact on the skin barrier properties displayed the lowest phase transitions and formed the least dense model stratum corneum lipid membranes at 32°C. In conclusion, the long hydrophobic chains in the NS-type ceramides are essential for maintaining the skin barrier function. However, this ability is not shared by their short-chain counterparts despite their having the same polar head structure and hydrogen bonding ability.     

Comparison of rat epidermal keratinocyte organotypic culture (ROC) with intact human skin: Lipid composition and thermal phase behavior of the stratum corneum

The present report is a part of our continuing efforts to explore the utility of the rat epidermal keratinocyte organotypic culture (ROC) as an alternative model to human skin in transdermal drug delivery and skin irritation studies of new chemical entities and formulations. The aim of the present study was to compare the stratum corneum lipid content of ROC with the corresponding material from human skin. The lipid composition was determined by thin-layer chromatography (TLC) and mass-spectrometry, and the thermal phase transitions of stratum corneum were studied by differential scanning calorimetry (DSC). All major lipid classes of the stratum corneum were present in ROC in a similar ratio as found in human stratum corneum. Compared to human skin, the level of non-hydroxyacid-sphingosine ceramide (NS) was increased in ROC, while α-hydroxyacid-phytosphingosine ceramide (AP) and non-hydroxyacid-phytosphingosine ceramides (NP) were absent. Also some alterations in fatty acid profiles of ROC ceramides were noted, e.g., esterified ω-hydroxyacid-sphingosine contained increased levels of oleic acid instead of linoleic acid. The fraction of lipids covalently bound to corneocyte proteins was distinctly lower in ROC compared to human skin, in agreement with the results from DSC. ROC underwent a lipid lamellar order to disorder transition (T₂) at a slightly lower temperature (68 °C) than human skin (74 °C). These differences in stratum corneum lipid composition and the thermal phase transitions may explain the minor differences previously observed in drug permeation between ROC and human skin.     

Structural Transitions in Ceramide Cubic Phases during Formation of the Human Skin Barrier

The stratum corneum is the outermost layer of human skin and the primary barrier toward the environment. The barrier function is maintained by stacked layers of saturated long-chain ceramides, free fatty acids, and cholesterol. This structure is formed through a reorganization of glycosylceramide-based bilayers with cubic-like symmetry into ceramide-based bilayers with stacked lamellar symmetry. The process is accompanied by deglycosylation of glycosylceramides and dehydration of the skin barrier lipid structure. Using coarse-grained molecular dynamics simulation, we show the effects of deglycosylation and dehydration on bilayers of human skin glycosylceramides and ceramides, folded in three dimensions with cubic (gyroid) symmetry. Deglycosylation of glycosylceramides destabilizes the cubic lipid bilayer phase and triggers a cubic-to-lamellar phase transition. Furthermore, subsequent dehydration of the deglycosylated lamellar ceramide system closes the remaining pores between adjacent lipid layers and locally induces a ceramide chain transformation from a hairpin-like to a splayed conformation.     

Direct visualization of lipid domains in human skin stratum corneum's lipid membranes: effect of pH and temperature

The main function of skin is to serve as a physical barrier between the body and the environment. This barrier capacity is in turn a function of the physical state and structural organization of the stratum corneum extracellular lipid matrix. This lipid matrix is essentially composed of very long chain saturated ceramides, cholesterol, and free fatty acids. Three unsolved key questions are i), whether the stratum corneum extracellular lipid matrix is constituted by a single gel phase or by coexisting crystalline (solid) domains; ii), whether a separate liquid crystalline phase is present; and iii), whether pH has a direct effect on the lipid matrix phase behavior. In this work the lateral structure of membranes composed of lipids extracted from human skin stratum corneum was studied in a broad temperature range (10 degrees C-90 degrees C) using different techniques such as differential scanning calorimetry, fluorescence spectroscopy, and two-photon excitation and laser scanning confocal fluorescence microscopy. Here we show that hydrated bilayers of human skin stratum corneum lipids express a giant sponge-like morphology with dimensions corresponding to the global three-dimensional morphology of the stratum corneum extracellular space. These structures can be directly visualized using the aforementioned fluorescence microscopy techniques. At skin physiological temperatures (28 degrees C-32 degrees C), the phase state of these hydrated bilayers correspond microscopically (radial resolution limit 300 nm) to a single gel phase at pH 7, coexistence of different gel phases between pH 5 and 6, and no fluid phase at any pH. This observation suggests that the local pH in the stratum corneum may control the physical properties of the extracellular lipid matrix by regulating membrane lateral structure and stability.     

New Insights into the Stratum Corneum Lipid Organization by X-Ray Diffraction Analysis

The characteristic 13-nm lamellar phase that is formed by lipids in the outermost layer of the skin, the stratum corneum (SC), is very important for the barrier function of the skin. To gain more insight into the molecular organization of this lamellar phase, we performed small-angle x-ray diffraction (SAXD) using various lipid mixtures mimicking the lipid composition in SC. In the SAXD pattern of each mixture, at least seven diffraction orders were observed, attributed to the lamellar phase with a repeat distance ranging from 12.1 to 13.8 nm. Using the sampling method based on the variation in repeat distance, we selected phase angles for the first six diffraction orders. Using these phase angles for the lamellar phase, a high-resolution electron density distribution could be calculated. Subsequently, from SAXD patterns of isolated SC, the electron density distribution of the lamellar phase was also calculated and appeared to be very similar to that in the lipid mixtures. This demonstrates that the lipid mixtures serve as an excellent model for the lipid organization in SC, not only with respect to the repeat distance, but also in terms of the electron density distribution within the unit cell.     

Deuterium NMR investigation of polymorphism in stratum corneum lipids

The intercellular lipid lamellae of stratum corneum constitute the major barrier to percutaneous penetration. Deuterium magnetic resonance and freeze-fracture electron microscopic investigation of hydrated lipid mixtures consisting of ceramides, cholesterol, palmitic acid and cholesteryl sulfate and approximating the stratum corneum intercellular lipid composition, revealed thermally induced polymorphism. The transition temperature of bilayer to hexagonal transition decreased as the ratio of cholesterol to ceramides in these mixtures was lowered. Lipid mixtures in which the stratum corneum ceramides were replaced by synthetic dipalmitoylphosphatidylcholine did not show any polymorphism throughout the temperature range used in the present study. The ability of the ceramide-containing samples to form hexagonal structures establishes a plausible mechanism for the assembly of the stratum corneum intercellular lamellae during the final stages of epidermal differentiation. Also, the bilayer to hexagonal phase transition of these nonpolar lipid mixtures could be used to enhance the penetration of drugs through skin.     

Role of ceramide structure and its microenvironment on the conformational order of model stratum corneum lipids mixtures: an approach by FTIR spectroscopy

The aim of this study is to investigate the influence of ceramide head group architecture and free fatty acid (another main class of stratum corneum lipids) or protein (keratin), on the lamellar organization of the ceramide auto-associated in model films mimicking lipid organization within the stratum corneum. FTIR spectroscopy is a powerful technique for investigating the structure of such systems. This technique has already been used to characterize phase transitions of the SC and of related model systems. As temperature is known to modify the conformational order of lipids, we used it as a variable parameter to monitor the differences in the conformational stability of ceramides. Our study included four ceramides: ceramide 2, 3, 5 and 6 which differ by their head group architecture. Two kinds of lipid-lipid interactions were studied: non-polar and polar. We noted some structural factors which participated to the organizational behavior: insaturation of alkyl chain, alpha-hydroxyl on fatty acid moiety and sphingosine or phytosphingosine head group. There is a direct interaction of palmitic acid on alkyl chains organization and a weak interaction with polar head group in presence of keratin, both provoking a destabilization of the ceramidic orthorhombic organization.     

Integrity and barrier function of the epidermis critically depend on glucosylceramide synthesis

Ceramides are vital components of the water barrier in mammalian skin. Epidermis-specific, a major ceramide portion contains omega-hydroxy very long chain fatty acids (C30-C36). These omega-hydroxy ceramides (Cers) are found in the extracellular lamellae of the stratum corneum either as linoleic acyl esters or protein bound. Glucosylceramide is the major glycosphingolipid of the epidermis. Synthesized from ceramide and UDP-glucose, it is thought to be itself an intracellular precursor and carrier for extracellular omega-hydroxy ceramides. To investigate whether GlcCer is an obligatory intermediate in ceramide metabolism to maintain epidermal barrier function, a mouse with an epidermis-specific glucosylceramide synthase (Ugcg) deficiency has been generated. Four days after birth animals devoid of GlcCer synthesis in keratinocytes showed a pronounced desquamation of the stratum corneum and extreme transepidermal water loss leading to death. The stratum corneum appeared as a thick unstructured mass. Lamellar bodies of the stratum granulosum did not display the usual ordered inner structure and were often irregularly arranged. Although the total amount of epidermal protein-bound ceramides remained unchanged, epidermal-free omega-hydroxy ceramides increased 4-fold and omega-hydroxy sphingomyelins, almost not detectable in wild type epidermis, emerged in quantities comparable with lost GlcCer. We conclude that the transient formation of GlcCer is vital for a regular arrangement of lipids and proteins in lamellar bodies and for the maintenance of the epidermal barrier.