Lipid organization in human and porcine stratum corneum differs widely, while lipid mixtures with porcine ceramides model human stratum corneum lipid organization very closely

The conformational disordering and lateral packing of lipids in porcine and human isolated stratum corneum (SC) was compared using Fourier transform infrared spectroscopy (FTIR). It was shown that SC of both species differ markedly, porcine SC lipids being arranged predominantly in a hexagonal lattice while lipids in human SC are predominantly packed in the denser orthorhombic lattice. However, the lipid organization of equimolar ceramide:cholesterol:free fatty acid (CER:CHOL:FFA) mixtures prepared with isolated porcine CER or human CER is very similar, only the transition temperatures differed being slightly lower in mixtures with porcine CER. Therefore, the difference in lateral packing between human and porcine stratum corneum is not due to the difference in CER composition. Furthermore, it is possible to use more readily available porcine CER in model lipid mixtures to mimic lipid organization in human SC. As the equimolar porcine CER:CHOL:FFA mixtures closely mimic the lipid organization in human SC, both human SC and this mixture were selected to examine the effect of glycerol on the lipid phase behaviour. It was found that high concentrations of glycerol change the lamellar organization slightly, while domains with an orthorhombic lateral packing are still observed.     

Lipid organization in human and porcine stratum corneum differs widely, while lipid mixtures with porcine ceramides model human stratum corneum lipid organization very closely

The conformational disordering and lateral packing of lipids in porcine and human isolated stratum corneum (SC) was compared using Fourier transform infrared spectroscopy (FTIR). It was shown that SC of both species differ markedly, porcine SC lipids being arranged predominantly in a hexagonal lattice while lipids in human SC are predominantly packed in the denser orthorhombic lattice. However, the lipid organization of equimolar ceramide:cholesterol:free fatty acid (CER:CHOL:FFA) mixtures prepared with isolated porcine CER or human CER is very similar, only the transition temperatures differed being slightly lower in mixtures with porcine CER. Therefore, the difference in lateral packing between human and porcine stratum corneum is not due to the difference in CER composition. Furthermore, it is possible to use more readily available porcine CER in model lipid mixtures to mimic lipid organization in human SC. As the equimolar porcine CER:CHOL:FFA mixtures closely mimic the lipid organization in human SC, both human SC and this mixture were selected to examine the effect of glycerol on the lipid phase behaviour. It was found that high concentrations of glycerol change the lamellar organization slightly, while domains with an orthorhombic lateral packing are still observed.     

FTIR studies show lipophilic moisturizers to interact with stratum corneum lipids, rendering the more densely packed

Lipophilic moisturizers are widely used to treat dry skin. However, their interaction with the lipids in the upper layer of the skin, the stratum corneum (SC), is largely unknown. In the present study this interaction of three moisturizers, isostearyl isostearate (ISIS), isopropyl isostearate (IPIS) and glycerol monoisostearate (GMIS), has been elucidated using lipid mixtures containing isolated ceramides (CER), cholesterol (CHOL) and free fatty acids (FFA), mimicking the lipid composition and organization in SC. The conformational ordering and the lateral packing of the lipid mixtures were examined by Fourier transformed infrared spectroscopy. Equimolar CER:CHOL:FFA mixtures show an orthorhombic to hexagonal phase transition between 22 and 30 degrees C and an ordered-disordered phase transition between 46 and 64 degrees C. Addition of 20% m/m ISIS or IPIS increased the thermotropic stability of the orthorhombic lateral packing, while GMIS had no influence. Furthermore, small amounts of all three moisturizers are incorporated into the CER:CHOL:FFA lattice, while the majority of the moisturizer exists in separate domains. Especially the thermotropic stabilization of the orthorhombic lateral packing, which might reduce water loss from the skin, is considered to contribute to the moisturizing effect of IPIS and ISIS in stratum corneum.     

FTIR studies show lipophilic moisturizers to interact with stratum corneum lipids, rendering the more densely packed

Lipophilic moisturizers are widely used to treat dry skin. However, their interaction with the lipids in the upper layer of the skin, the stratum corneum (SC), is largely unknown. In the present study this interaction of three moisturizers, isostearyl isostearate (ISIS), isopropyl isostearate (IPIS) and glycerol monoisostearate (GMIS), has been elucidated using lipid mixtures containing isolated ceramides (CER), cholesterol (CHOL) and free fatty acids (FFA), mimicking the lipid composition and organization in SC. The conformational ordering and the lateral packing of the lipid mixtures were examined by Fourier transformed infrared spectroscopy. Equimolar CER:CHOL:FFA mixtures show an orthorhombic to hexagonal phase transition between 22 and 30 °C and an ordered-disordered phase transition between 46 and 64 °C. Addition of 20% m/m ISIS or IPIS increased the thermotropic stability of the orthorhombic lateral packing, while GMIS had no influence. Furthermore, small amounts of all three moisturizers are incorporated into the CER:CHOL:FFA lattice, while the majority of the moisturizer exists in separate domains. Especially the thermotropic stabilization of the orthorhombic lateral packing, which might reduce water loss from the skin, is considered to contribute to the moisturizing effect of IPIS and ISIS in stratum corneum.     

Infrared spectroscopy studies of mixtures prepared with synthetic ceramides varying in head group architecture: Coexistence of liquid and crystalline phases

The barrier function of the skin is provided by the stratum corneum (SC), the outermost layer of the skin. Ceramides (CERs), cholesterol (CHOL) and free fatty acids (FFAs) are present in SC and form highly ordered crystalline lipid lamellae. These lamellae are crucial for a proper skin barrier function. In the present study, Fourier transform infrared spectroscopy was used to examine the lipid organization of mixtures prepared from synthetic CERs with CHOL and FFAs. The conformational ordering and lateral packing of these mixtures showed great similarities to the lipid organization in SC and lipid mixtures prepared with native CERs. Therefore, mixtures with synthetic CERs serve as an excellent tool for studying the effect of molecular architecture of CER subclasses on the lipid phase behavior. In SC the number of OH-groups in the head groups of CER subclasses varies. Furthermore, acylCERs with a linoleic acid chemically bound to a long acyl chain are also identified. The present study revealed that CER head group architecture affects the lateral packing and conformational ordering of the CER:CHOL:FFA mixtures. Furthermore, while the majority of the lipids form a crystalline packing, the linoleate moiety of the acylCERs participates in a “pseudo fluid” phase.     

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.     

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'.     

Human stratum corneum lipid organization as observed by atomic force microscopy on Langmuir-Blodgett films

The barrier function of skin ultimately depends on the physical state and structural organisation of the stratum corneum extracellular lipid matrix. Ceramides, cholesterol and a broad distribution of saturated long-chain free fatty acids dominate the stratum corneum lipid composition. Additionally, smaller amounts of cholesterol sulfate and cholesteryl oleate may be present. A key feature determining skin barrier capacity is thought to be whether or not different lipid domains coexist laterally in the stratum corneum extracellular lipid matrix. In this study, the overall tendency for lipid domain formation in different mixtures of extracted human stratum corneum ceramides, cholesterol, free fatty acids, cholesterol sulfate and cholesteryl oleate were studied using atomic force microscopy (AFM) on Langmuir-Blodgett (LB) films on mica. It is shown that the saturated long-chain free fatty acid distribution of human stratum corneum prevents hydrocarbon chain segregation. Further, LB-films of human stratum corneum ceramides express a pattern of connected elongated domains with a granular domain interface. The dominating effect of both cholesterol and cholesterol sulfate is that of increased ceramide domain dispersion. This effect is counteracted by the presence of free fatty acids, which preferentially mix with ceramides and not with cholesterol. Cholesteryl oleate does not mix with other skin lipid components, supporting the hypothesis of an extra-endogenous origin. In the system composed of endogenous human ceramides and cholesterol plus 15 wt% stratum corneum distributed free fatty acids, i.e., the system mimicking most closely the lipid composition of the stratum corneum extracellular space, LB-films on mica express lateral domain formation.     

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.     

Effect of the ω-acylceramides on the lipid organization of stratum corneum model membranes evaluated by X-ray diffraction and FTIR studies (Part I)

The lipid organization in the outermost layer of the skin, the stratum corneum, is important for the skin barrier function. The stratum corneum lipids are composed of ceramides (CER), free fatty acids (FFA) and cholesterol (CHOL). In the present study Fourier transform infrared (FTIR) and small-angle X-ray scattering (SAXS) techniques were utilized to evaluate the effect of three C18 fatty acid esterified ω-acylceramides (CER EOS) on the lipid organization of stratum corneum model membranes. FTIR spectra (scissoring and rocking bands) showed as a function of temperature significant line-shape changes for both components assigned to the orthorhombic phase. Second-derivative analyzes revealed a significant decrease in the interchain coupling strength (Δν values) for the samples formed by CER EOS with the linoleate (CER EOS-L) and oleate (CER EOS-O) moiety around 28.5 °C. However, only a gradual decrease in the Δν values was noticed for the mixture formed with CER EOS with the stearate moiety (CER EOS-S) over the whole temperature range. In the absence of CER EOS the decrease started already at 25.5 °C, demonstrating that CER EOS stabilized the orthorhombic lattice. This stabilization was most pronounced for the CER EOS-S. Spectral fittings allowed to evaluate the orientation changes of the skeletal plane within the orthorhombic unit cell (θ values) for a given temperature range. From the best-fit parameters (peak area values), a decrease in the orthorhombic phase contribution to the scissoring band was also monitored as a function of the temperature. SAXS studies showed the coexistence of two lamellar phases with a periodicity of ∼5.5 nm (short periodicity phase, SPP) and ∼12 nm (LPP) in the presence of the CER EOS-L and CER EOS-O. However, no diffraction peaks associated to the LPP were detected for CER EOS-S. While CER EOS-S most efficiently stabilized the orthorhombic phase, CER EOS-L and CER EOS-O promoted the presence of the LPP. Therefore, the presence of all three CER EOS as observed in human stratum corneum may contribute to a proper skin barrier function.     

The phase behaviour of skin lipid mixtures based on synthetic ceramides

The lipid lamellae present in the outermost layer of the skin, the stratum corneum (SC), form the main barrier for diffusion of molecules across the skin. The main lipid classes in SC are cholesterol (CHOL), free fatty acids (FFA) and at least nine classes of ceramides (CER), referred to as CER1 to CER9. In the present study the phase behaviour of four synthetic CER, either single or mixed with CHOL or CHOL and FFA, has been studied using small and wide angle X-ray diffraction. The lipid mixtures showed complex phase behaviour with coexistence of several phases. The results further revealed that the presence of synthetic CER1 as well as a proper composition of the other CER in the mixture were crucial for the formation of a phase with a long periodicity, characteristic for SC lipid phase behaviour. Only a mixture containing synthetic CER1 and CER3, CHOL and FFA showed similar phase behaviour to that of SC.     

The role of ceramide composition in the lipid organisation of the skin barrier.

The lipid lamellae in the stratum corneum (SC) play a key role in the barrier function of the skin. The major lipids are ceramides (CER), cholesterol (CHOL) and free fatty acids (FFA). In pig SC at least six subclasses of ceramides (referred to as CER 1, 2-6) are present. Recently it was shown that in mixtures of isolated pig SC ceramides (referred to as CER(1-6)) and CHOL two lamellar phases are formed, which mimic SC lipid organisation very closely [J.A. Bouwstra et al., 1996, J. Lipid Res. 37, 999-1011] [1]. Since the CER composition in SC originating from different sources/donors often varies, information on the effect of variations in CER composition on the SC lipid organisation is important. The results of the present study with mixtures of CHOL including two different CER mixtures that lack CER 6 (CER(1-5) mixtures) revealed that at an equimolar molar ratio their lipid organisation was similar to that of the equimolar CHOL:CER(1-6) and CHOL:CER(1,2) mixtures, described previously. These observations suggest that at an equimolar CHOL:CER ratio the lipid organisation is remarkably insensitive toward a change in the CER composition. Similar observations have been made with equimolar CHOL:CER:FFA mixtures. The situation is different when the CHOL:CER molar ratio varies. While in the CHOL:CER(1-6) mixture the lamellar organisation hardly changed with varying molar ratio from 0.4 to 2, the lamellar organisation in the CHOL:CER(1-5) mixtures appeared to be more sensitive to a change in the relative CHOL content, especially concerning the changes in the periodicities of the lamellar phases. In summary, these findings clearly indicate that at an equimolar CHOL:CER molar ratio the lamellar organisation is least sensitive to a variation in CER composition, while at a reduced CHOL:CER molar ratio the CER composition plays a more prominent role in the lamellar phases. This observation may have an implication for the in vivo situation when both the CER composition and the CHOL:CER molar ratio change simultaneously.     

Effect of cholesterol on miscibility and phase behavior in binary mixtures with synthetic ceramide 2 and octadecanoic acid. Infrared studies

The three main lipid components of the stratum corneum, namely ceramides, free fatty acids and cholesterol, play a fundamental role in the maintenance of the skin barrier. The current investigation is aimed toward understanding the miscibility and intermolecular interactions of these lipids. Toward this end, Fourier transform infrared spectroscopic studies of the three possible equimolar binary mixtures of cholesterol, a synthetic non-hydroxylated fatty acid N-acyl sphingosine with a C18 chain length (N-stearoylsphingosine, approximating human ceramide 2), and stearic acid were undertaken. The thermotropic responses of the methylene stretching and scissoring vibrations were used to evaluate chain conformation and packing respectively. Selective perdeuteration, of either the stearic acid or the ceramide acid chains, permitted separate and simultaneous evaluation of the conformational order and packing properties of the sphingosine chain, the amide linked fatty acid chains and/or the stearic acid chain. Whereas cholesterol mixed well with ceramide at physiological temperatures, the stearic acid was miscible with the cholesterol only at relatively high temperatures where the fatty acid is disordered. A complex interaction between stearic acid and ceramide was detected. A separate fatty acid-rich phase persisted until at least 50 degrees C, whereas at higher temperatures the components appear to be quite miscible. However, a preferential association of the fatty acid with the ceramide base chain is indicated. None of the binary systems studied exhibit miscibility and interactions resembling those in the ternary mixtures of these substances, which is widely used to model stratum corneum. The role of cholesterol in controlling the miscibility characteristics in the ternary system is evident.     

Cholesterol sulfate and Ca(2+) modulate the mixing properties of lipids in stratum corneum model mixtures

The influence of cholesterol sulfate (CS) and calcium on the phase behavior of lipid mixtures mimicking the stratum corneum (SC) lipids was examined using vibrational spectroscopy. Raman microspectrocopy showed that equimolar mixtures of ceramide, palmitic acid, and cholesterol underwent a phase transition in which, at low temperatures, lipids formed mainly a mosaic of microcrystalline phase-separated domains, and above 45 degrees C, a more fluid and disordered phase in which the three lipid species were more miscible. In the presence of Ca(2+), there was the formation of fatty acid-Ca(2+) complexes that led to domains stable on heating. Consequently, these lipid mixtures remained heterogeneous, and the fatty acid molecules were not extensively involved in the formation of the fluid lipid phase, which included mainly ceramide and cholesterol. However, the presence of CS displaced the association site of Ca(2+) ions and inhibited the formation of domains formed by the fatty acid molecules complexed with Ca(2+) ions. This work reveals that CS and Ca(2+) modulate the lipid mixing properties and the lipid order in SC lipid models. The balance in the equilibria involving Ca(2+), CS, and fatty acids is proposed to have an impact on the organization and the function of the epidermis.     

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.     

Structure of the skin barrier and its modulation by vesicular formulations

The natural function of the skin is to protect the body from unwanted influences from the environment. The main barrier of the skin is located in the outermost layer of the skin, the stratum corneum. Since the lipids regions in the stratum corneum form the only continuous structure, substances applied onto the skin always have to pass these regions. For this reason the organization in the lipid domains is considered to be very important for the skin barrier function. Due to the exceptional stratum corneum lipid composition, with long chain ceramides, free fatty acids and cholesterol as main lipid classes, the lipid phase behavior is different from that of other biological membranes. In stratum corneum crystalline phases are predominantly present, but most probably a subpopulation of lipids forms a liquid phase. Both the crystalline nature and the presence of a 13 nm lamellar phase are considered to be crucial for the skin barrier function. Since it is impossible to selectively extract individual lipid classes from the stratum corneum, the lipid organization has been studied in vitro using isolated lipid mixtures. These studies revealed that mixtures prepared with isolated stratum corneum lipids mimic to a high extent stratum corneum lipid phase behavior. This indicates that proteins do not play an important role in the stratum corneum lipid phase behavior. Furthermore, it was noticed that mixtures prepared only with ceramides and cholesterol already form the 13 nm lamellar phase. In the presence of free fatty acids the lattice density of the structure increases. In stratum corneum the ceramide fraction consists of various ceramide subclasses and the formation of the 13 nm lamellar phase is also affected by the ceramide composition. Particularly the presence of ceramide 1 is crucial. Based on these findings a molecular model has recently been proposed for the organization of the 13 nm lamellar phase, referred to as "the sandwich model", in which crystalline and liquid domains coexist. The major problem for topical drug delivery is the low diffusion rate of drugs across the stratum corneum. Therefore, several methods have been assessed to increase the permeation rate of drugs temporarily and locally. One of the approaches is the application of drugs in formulations containing vesicles. In order to unravel the mechanisms involved in increasing the drug transport across the skin, information on the effect of vesicles on drug permeation rate, the permeation pathway and perturbations of the skin ultrastructure is of importance. In the second part of this paper the possible interactions between vesicles and skin are described, focusing on differences between the effects of gel-state vesicles, liquid-state vesicles and elastic vesicles.     

Phase behaviour of skin barrier model membranes at pH 7.4.

The main function of the skin is to protect the body against exogenous substances. The skin barrier is located in the outermost layer of the skin, the stratum corneum (SC). This layer consists of keratin enriched cells embedded in lipid lamellae that form the main barrier for diffusion of substances through the skin. The main lipid classes in this barrier are ceramides, cholesterol and free fatty acids. Cholesterol sulfate and calcium are also present in SC. Furthermore it has been suggested that a pH gradient exists. In a previous paper the effect of cholesterol sulfate and calcium on the lipid phase behaviour of mixtures prepared from cholesterol, ceramides and free fatty acids at pH 5 was reported (approximate pH at the skin surface). In the present study the phase behaviour of mixtures prepared from cholesterol, ceramides and free fatty acids prepared at pH 7.4 (the pH of viable cells) has been examined between 25 and 95 degrees C. Our studies reveal that a reversed hexagonal phase has been formed at elevated temperatures. Addition of calcium inhibits the formation of the reversed hexagonal phase, while cholesterol sulfate promotes the presence of the reversed hexagonal phase at increased temperatures. From our results we can conclude that the lipid mixtures prepared at pH 5 resemble more closely the lipid phase behaviour in intact SC than the lipid mixtures prepared at pH 7.4.     

The effect of the chain length distribution of free fatty acids on the mixing properties of stratum corneum model membranes

The stratum corneum (SC) plays a fundamental role in the barrier function of the skin. The SC consists of corneocytes embedded in a lipid matrix. The main lipid classes in the lipid matrix are ceramides (CERs), cholesterol (CHOL) and free fatty acids (FFAs). The aim of this study was to examine the effect of the chain length of FFAs on the thermotropic phase behavior and mixing properties of SC lipids. Fourier transform infrared spectroscopy and Raman imaging spectroscopy were used to study the mixing properties using either protonated or deuterated FFAs. We selected SC model lipid mixtures containing only a single CER, CHOL and either a single FFA or a mixture of FFAs mimicking the FFA SC composition. The single CER consists of a sphingoid base with 18 carbon atoms and an acyl chain with a chain length of 24 carbon atoms. When using lignoceric acid (24 carbon atoms) or a mixture of FFAs, the CER and FFAs participated in mixed crystals, but hydration of the mixtures induced a slight phase separation between CER and FFA. The mixed crystalline structures did not phase separate during storage even up to a time period of 3 months. When using palmitic acid (16 carbon atoms), a slight phase separation was observed between FFA and CER. This phase separation was clearly enhanced during hydration and storage. In conclusion, the thermotropic phase behavior and the mixing properties of the SC lipid mixtures were shown to strongly depend on the chain length and chain length distribution of FFAs, while hydration enhanced the phase separation.     

Modelling the stratum corneum lipid organisation with synthetic lipid mixtures: the importance of synthetic ceramide composition

Cholesterol (CHOL), free fatty acids (FFA) and nine classes of ceramides (CER1-CER9) form the main constituents of the intercellular lipid lamellae in stratum corneum (SC), which regulate the skin barrier function. Both the presence of a unique 13-nm lamellar phase, of which the formation depends on the presence of CER1, and its dense lateral packing are characteristic for the SC lipid organisation. The present study focuses on the lipid organisation in mixtures prepared with CHOL, FFA and a limited number of synthetic CER, namely CER1, CER3 and bovine brain CER type IV (SigmaCERIV). The main objective is to determine the optimal molar ratio of CER3 to SigmaCERIV for the formation of the 13-nm lamellar phase. CER3 contains a uniform acyl chain length, whereas SigmaCERIV contains fatty acids with varying chain lengths. Using small angle X-ray diffraction (SAXD), it is demonstrated that the CER3 to SigmaCERIV ratio affects the formation of the 13-nm lamellar phase and that the optimal ratio depends on the presence of FFA. Furthermore, the formation of the 13-nm lamellar phase is not very sensitive to variations in the total CER level, which is similar to the in vivo situation.     

Direct observation of domains in model stratum corneum lipid mixtures by Raman microspectroscopy

Several studies on intact and model stratum corneum (SC), the top layer of the epidermis, have suggested the presence of crystalline domains. In the present work, we used micro-Raman mapping to detect lipid domains in model lipid mixtures formed by an equimolar mixture of ceramides, cholesterol, and palmitic acid, the three main lipid species of SC. We were able to determine the spatial distribution of the three compounds individually based on the systematic analysis of band areas. As a control, we studied freeze-dried lipid mixtures, and the Raman microspectroscopy reported faithfully the homogeneous distribution of the three compounds. Spectral mapping was then performed on hydrated equimolar mixtures carefully annealed. In this case, clear phase separations were observed. Domains enriched in cholesterol, ceramides, or palmitic acid with a size of a few tens of square microns were detected. These findings constitute the first direct evidence of the formation of heterogeneous domains in the SC lipid models in a bulk phase. Raman microspectroscopy is an innovative approach to characterize the conditions leading to the formation of domains and provides new insights into the understanding of the skin barrier.