Coexistence of two domains in intercellular lipid matrix of stratum corneum

The outermost layer of the skin, the stratum corneum (SC), is composed of corneocytes and an intercellular lipid matrix. The matrix acts as both the main barrier and also as the pathway of water, drugs, etc. across the SC. In the mammalian SC, the longitudinal arrangement of the lipid molecules, consisting of long and short lamellar structures with repeat distances of about 13 nm and 6 nm, respectively, has been observed by small-angle X-ray diffraction. In the lateral arrangement of the lipid molecules, hexagonal and orthorhombic hydrocarbon-chain packing has been observed by wide-angle X-ray diffraction. From the systematic study of the temperature dependence of simultaneous small- and wide-angle X-ray diffraction patterns, we demonstrate that the intercellular lipid matrix forms two domains, which consist at room temperature of a long lamellar structure with hexagonal hydrocarbon-chain packing and a short lamellar structure with orthorhombic hydrocarbon-chain packing.     

Novel method to observe subtle structural modulation of stratum corneum on applying chemical agents

In the development of functional chemicals such as percutaneous penetration enhancers and cosmetics, the structural evidence at the molecular level in stratum corneum (SC) is highly desirable. We developed a method to observe a minute structural change of intercellular lipid matrix and corneocytes on applying the chemicals to the SC using synchrotron X-ray diffraction technique. The performance of the present method was demonstrated by applying typical chemicals, chloroform/methanol mixture, hydrophilic ethanol and hydrophobic d-limonene. From the small- and wide-angle X-ray diffraction we obtained the following results: on applying chloroform/methanol mixture the intercellular lipids were extracted markedly, on applying ethanol the intercellular lipid structure was slightly disrupted, ethanol molecules were taken into the corneocytes and in addition the pools of ethanol seem to be formed in the hydrophilic region of the intercellular lipid matrix in the SC, and on applying d-limonene the repeat distance of the long lamellar structure increased by incorporating d-limonene molecules, the intercellular lipid structure was slightly disrupted, and the pools of d-limonene were formed in the hydrophobic region of the intercellular lipid matrix in the SC.     

A possible regulation mechanism of water content in human stratum corneum via intercellular lipid matrix

We studied the water regulation mechanism in human stratum corneum which is composed of corneocytes and intercellular lipid matrix by the ex vivo small- and medium-angle X-ray diffraction. Under the normal condition water molecules are stored mainly in the corneocytes. When the water content increased, from the small-angle X-ray diffraction of the human stratum corneum we obtained the swelling behavior of the short lamellar lipid structure as a result of incorporating a very small amount of water into water layers between neighboring the lipid bilayers, and its diffraction peak width became narrow and turned to wide at the water content of 20-30wt%. In addition as evidence for uptake of water in the corneocytes, we observed the structural modification of soft keratins in the corneocytes from the medium-angle X-ray diffraction. Based upon these results we propose that the water content in the human stratum corneum is regulated to be at 20-30wt% so as to stabilize the short lamellar structure in the intercellular lipid matrix.     

Molecular mechanisms of action of different concentrations of ethanol in water on ordered structures of intercellular lipids and soft keratin in the stratum corneum

Ethanol (EtOH) is one of the bases in topically applied medicines that promote the skin permeation of drugs. Although the effects of EtOH have been attributed to structural modifications in the stratum corneum, the underlying mechanisms, especially the influence of different concentrations of EtOH, have not been examined extensively. Structural modifications in the stratum corneum of hairless mouse due to the application of EtOH/water mixture were herein investigated at the molecular level using synchrotron X-ray diffraction. The results revealed that all EtOH concentrations examined greatly modified the short lamellar structures containing the aqueous layer in intercellular lipids and the structure of keratin fibrils in corneocytes, which can take up hydrophilic compounds. However, the long lamellar and the hydrocarbon-chain packing structures were unaffected by EtOH. Changes to the short lamellar structures were not proportional to the concentration of EtOH. However, the keratin fibril structures changed gradually with increasing EtOH concentration. The X-ray diffraction experiments enabled the effects of different EtOH concentrations on the morphology of the stratum corneum to be assessed by using a number of experimental samples to avoid variations due to individual differences. The results indicated that alterations to the short lamellar structures appeared to be related to the skin permeability of drugs with the application of EtOH/water mixture, and monotonous structural changes in the keratin fibrils with an increase in EtOH concentration may contribute to this permeation as supplement. These results will be useful for the development of new drug formulations containing EtOH.     

X-ray diffraction analysis of internal wool lipids

Polarised optical microscopy (POM) and X-ray diffraction techniques were applied to intercellular lipids extracted from wool to study their structural arrangement in order to determine their role in the diffusion properties of wool fibre. Intercellular wool lipids (IWL) arranged as concentrated liposomes were shown to be a good intercellular lipid model, allowing their study by X-ray diffraction techniques. The results confirm that intercellular lipids of wool fibre are organised in a lamellar structure of 5.0–8.0 nm width, termed β-layer, which had been assumed to be lipids arranged as a bilayer. Structurally, internal wool lipids are distributed at least in two domains at low temperatures: an ordered phase made up of ceramides and free fatty acids (FFA) alone, arranged in crystal orthorhombic states separately, and a liquid crystal state when mixed together. At 40 °C there is a reversible phase transition produced by the melt of the crystal orthorhombic states, whereas the liquid crystal state remains until 65 °C.     

Swelling of intercellular lipid lamellar structure with short repeat distance in hairless mouse stratum corneum as studied by X-ray diffraction

Lamellar structures of intercellular lipids in stratum corneum of hairless mouse were studied at various water contents by small-angle X-ray diffraction. At room temperature there are at least two lamellar structures, long and short lamellar structures, with repeat distances of 13.6 and around 6 nm, respectively. The long lamellar spacing is almost constant over the water content from 0% w/w to 80% w/w that is consistent with the previously reported results. For the short lamellar structure we found that with increasing the water content the lamellar spacing becomes larger, that is, from 12 to 50% w/w the short lamellar spacing increases from 5.8 to 6.6 nm. In addition to the previously reported result that at the water content of about 20% w/w the X-ray diffraction peak for the long lamellar structure becomes sharp, we found that this is also the case for the short lamellar structure. Below the water content of about 12% w/w the X-ray diffraction peak for the short lamellar structure dies out and conversely above the water content of about 50% w/w it becomes weak and finally merges into the second-order diffraction peak for the long lamellar structure. Considering the matching of the long lamellar spacing that is unchanged with the water content and twice the short lamellar spacing that changes as a function of the water content, it is likely that the swelling of the short lamellar structure plays an important role in the regulation of water stored in stratum corneum.     

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.     

Is an orthorhombic lateral packing and a proper lamellar organization important for the skin barrier function?

The lipid organization in the stratum corneum (SC), plays an important role in the barrier function of the skin. SC lipids form two lamellar phases with a predominantly orthorhombic packing. In previous publications a lipid model was presented, referred to as the stratum corneum substitute (SCS), that closely mimics the SC lipid organization and barrier function. Therefore, the SCS serves as a unique tool to relate lipid organization with barrier function. In the present study we examined the effect of the orthorhombic to hexagonal phase transition on the barrier function of human SC and SCS. In addition, the SCS was modified by changing the free fatty acid composition, resulting in a hexagonal packing and perturbed lamellar organization. By measuring the permeability to benzoic acid as function of temperature, Arrhenius plots were constructed from which activation energies were calculated. The results suggest that the change from orthorhombic to hexagonal packing in human SC and SCS, does not have an effect on the permeability. However, the modified SCS revealed an increased permeability to benzoic acid, which we related to its perturbed lamellar organization. Thus, a proper lamellar organization is more crucial for a competent barrier function than the presence of an orthorhombic lateral packing.     

Phytosphingosine ceramide mainly localizes in the central layer of the unique lamellar phase of skin lipid model systems

Understanding the lipid arrangement within the skin’s outermost layer, the stratum corneum (SC), is important for advancing knowledge on the skin barrier function. The SC lipid matrix consists of ceramides (CERs), cholesterol, and free fatty acids, which form unique crystalline lamellar phases, referred to as the long periodicity phase (LPP) and short periodicity phases. As the SC lipid composition is complex, lipid model systems that mimic the properties of native SC are used to study the SC lipid organization and molecular arrangement. In previous studies, such lipid models were used to determine the molecular organization in the trilayer structure of the LPP unit cell. The aim of this study was to examine the location of CER N-(tetracosanoyl)-phytosphingosine (CER NP) in the unit cell of this lamellar phase and compare its position with CER N-(tetracosanoyl)-sphingosine (CER NS). We selected CER NP as it is the most prevalent CER subclass in the human SC, and its location in the LPP is not known. Our neutron diffraction results demonstrate that the acyl chain of CER NP was positioned in the central part of the trilayer structure, with a fraction also present in the outer layers, the same location as determined for the acyl chain of CER NS. In addition, our Fourier transformed infrared spectroscopy results are in agreement with this molecular arrangement, suggesting a linear arrangement for the CER NS and CER NP. These findings provide more detailed insight into the lipid organization in the SC lipid matrix.     

Structure of lamellar lipid domains and corneocyte envelopes of murine stratum corneum. An X-ray diffraction study

The lipid of the outermost layer of the skin is confined largely to the extracellular spaces surrounding the corneocytes of the stratum corneum where it forms a multilamellar adhesive matrix to act as the major permeability barrier of the skin. Knowledge of the molecular architecture of these intercellular domains is important for understanding various skin pathologies and their treatment, percutaneous drug delivery, and the cosmetic maintenance of the skin. We have surveyed by X-ray diffraction the structure of the intercellular domains and the extracted lipids of murine stratum corneum (SC) at 25, 45, and 70 degrees C which are temperatures in the vicinity of known thermal phase transitions [Rehfeld, S. J., & Elias, P. M. (1982) J. Invest. Dermatol. 79, 1-3]. The intercellular domains produce lamellar diffraction patterns with a Bragg spacing of 131 +/- 2 A. Lipid extracted from the SC and dispersed in excess water does not produce a simple lamellar diffraction pattern at any temperature studied, however. This and other facts suggest that another component, probably a protein, must be present to control the architecture of the intercellular lipid domains. We have also obtained diffraction patterns attributable to the protein envelopes of the corneocytes. The patterns suggest a beta-pleated sheet organizational scheme. No diffraction patterns were observed that could be attributed to keratin.     

Micron-scale assessment of molecular lipid organization in human stratum corneum using microprobe X-ray diffraction

Lipid and protein components of the stratum corneum (SC) are organized in complex supramolecular arrangements. Exploring spatial relations between various possible substructures is important for understanding the barrier function of this uppermost layer of epidermis. Here, we report the first study where micro-focus X-ray scattering was used for assessing fine structural variations of the human skin barrier with micrometer resolution. We found that the scattering profiles were unchanged when scanning in the direction parallel to the SC surface. Furthermore, small-angle scattering profiles did not change as a function of depth in the SC, confirming that the lipid lamellar spacings remained the same throughout the SC. However, the wide-angle scattering data showed that the orthorhombic phase was more abundant in the middle layers of the SC, whereas the hexagonal phase dominated in the surface layers both at the external and the lowest part of the SC; i.e., the lipids were most tightly packed in the middle region of the SC. Taken together, our results demonstrate that microprobe X-ray diffraction provides abundant information about spatial variations of the SC lipid structure and thus may be a promising tool for assessing the effects of topical formulations on the barrier function of skin.     

The influence of two azones and sebaceous lipids on the lateral organization of lipids isolated from human stratum corneum

The main problem with topical application of compounds to administer drugs to and regulate drug levels in a human body, is the barrier formed by the intercellular lipid matrix of the stratum corneum (SC). In a search for possibilities to overcome this barrier function, a good understanding of the organization and phase behavior of these lipids is required. SC lipid model studies especially provide a wealth of information with respect to the lipid organization and the importance of certain subclasses of lipids for the structure. Previously, we have shown that electron diffraction (ED) provides detailed information on the lateral lipid packing in both intact SC (G.S.K. Pilgram et al., J. Invest. Dermatol. 113 (1999) 403) and SC lipid models (G.S.K. Pilgram et al., J. Lipid Res. 39 (1998) 1669). In the present study, we used ED to examine the influence of two azones and sebaceous lipids on the lateral phase behavior of lipids isolated from human SC. We established that human SC lipids are arranged in an orthorhombic packing pattern. Upon mixing with the two enhancers the orthorhombic packing pattern was still observed; however, an additional fluid phase became more apparent. In mixtures with sebaceous lipids, the presence of the hexagonal lattice increased. These findings provide a basis for the mechanism by which these enhancers and sebaceous lipids interact with human SC lipids.     

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.     

A subtransition in a phospholipid with a net charge, dipalmitoylphosphatidylglycerol

Suspensions of dipalmitoylphosphatidylglycerol (DPPG) have been analyzed by differential scanning calorimetry, equilibrium and differential scanning dilatometry, and X-ray diffraction techniques. After the DPPG suspensions are stored several days at 2 degrees C, a new phase transition is observed at a lower temperature than either the main transition or the pretransition. This subtransition has an enthalpy of about 6 kcal/mol and occurs at about 20 degrees C, the exact temperature depending on the buffer used. The lipid partial specific volume increases by 0.035 mL/g upon warming through the subtransition. X-ray diffraction patterns from suspensions in the subgel phase contain orders of a lamellar repeat and several additional sharp and broad wide-angle reflections between 8 and 2 A. As the water content in the specimen is reduced, the lamellar repeat period decreases, whereas the spacings and intensities of these additional wide-angle reflections are unchanged. These data indicate that on incubation at 2 degrees C the lipid molecules crystallize in the plane of each bilayer. X-ray experiments also show that this subgel phase converts to the normal L beta' gel phase above the subtransition.     

New insight into phase behavior and permeability of skin lipid models based on sphingosine and phytosphingosine ceramides

The intercellular lipid matrix of the stratum corneum (SC), which consist mainly of ceramides (CERs), free fatty acids and cholesterol, is fundamental to the skin barrier function. These lipids assemble into two lamellar phases, known as the long and short periodicity phases (LPP and SPP respectively). The LPP is unique in the SC and is considered important for the skin barrier function. Alterations in CER composition, as well as impaired skin barrier function, are commonly observed in diseased skin, yet the understanding of this relationship remains insufficient. In this study, we have investigated the influence of non-hydroxy and α-hydroxy sphingosine-based CERs and their phytosphingosine counterparts on the permeability and lipid organization of model membranes, which were adjusted in composition to enhance formation of the LPP. The permeability was compared by diffusion studies using ethyl-p-aminobenzoate as a model drug, and the lipid organization was characterized by X-ray diffraction and infrared spectroscopy. Both the sphingosine- and phytosphingosine-based CER models formed the LPP, while the latter exhibited a longer LPP repeat distance. The ethyl-p-aminobenzoate flux across the sphingosine-based CER models was higher when compared to the phytosphingosine counterparts, contrary to the fact that the α-hydroxy phytosphingosine-based CER model had the lowest chain packing density. The unanticipated low permeability of the α-hydroxy phytosphingosine-based model is probably associated with a stronger headgroup hydrogen bonding network. Our findings indicate that the increased level of sphingosine-based CERs at the expense of phytosphingosine-based CERs, as observed in the diseased skin, may contribute to the barrier function impairment.     

Low-flux electron diffraction study on body site dependence of stratum corneum structures in human skin

For analysis of the structure of human skin stratum corneum (SC), we introduced low-flux electron diffraction (ED) and developed a new statistical analysis method for obtained ED intensity profiles. By use of this method we compared the differences in the intercellular lipid organization on the SC corneocytes collected at human forehead, cheek, and forearm by the grid-stripping method. As a result, we found a significant regional difference in the distribution of lipid hydrocarbon chain packing domains in the SC; the ring-type ED pattern with orthorhombic symmetry was more often observed in the forearm SC than in the forehead and cheek SCs. We also found that the dependence of the background electron diffraction intensity on the modulus of the scattering vector differed significantly among them. The present method for the analysis of a large number of ED patterns of noninvasively obtained SC samples could be a powerful tool to scrutinize the structural difference between the SCs under various experimental conditions.     

Characterization of a quasicrystalline phase in codispersions of phosphatidylethanolamine and glucocerebroside

Synchrotron x-ray diffraction, differential scanning calorimetry, and electron spin resonance spectroscopy have been employed to characterize a quasicrystalline phase formed in aqueous dispersions of binary mixtures of glucocerebroside and palmitoyloleoylphosphatidylethanolamine. Small- and wide-angle x-ray scattering intensity patterns were recorded during temperature scans between 20 degrees and 90 degrees C from mixtures of composition 2, 5, 10, 20, 30, and 40 mol glucocerebroside per 100 mol phospholipid. The quasicrystalline phase was characterized by a broad lamellar d-spacing of 6.06 nm at 40 degrees C and a broad wide-angle x-ray scattering band centered at approximately 0.438 nm, close to the gel phase centered at approximately 0.425 nm and distinct from a broad peak centered at 0.457 nm observed for a liquid-crystal phase at 80 degrees C. The quasicrystalline phase coexisted with gel and fluid phase of the pure phospholipid. An analysis of the small-angle x-ray scattering intensity profiles indicated a stoichiometry of one glucosphingolipid per two phospholipid molecules in the complex. Structural transitions monitored in cooling scans by synchrotron x-ray diffraction indicated that a cubic phase transforms initially into a lamellar gel. Thermal studies showed that the gel phase subsequently relaxes into the quasicrystalline phase in an exothermic transition. Electron spin resonance spectroscopy using spin labels located at positions 7, 12, and 16 carbons of phospholipid hydrocarbon chains indicated that order and motional constraints at the 7 and 12 positions were indistinguishable between gel and quasicrystalline phases but there was a significant decrease in order and increase in rate of motion at the 16 position on transformation to the quasicrystalline phase. The results are interpreted as an arrangement of polar groups of the complex in a crystalline array and a quasicrystalline packing of the hydrocarbon chains predicated by packing problems in the bilayer core requiring disordering of the highly asymmetric chains. The possible involvement of quasicrystalline phases in formation of membrane rafts is considered.     

Mechanism and kinetics of the subtransition in hydrated L-dipalmitoylphosphatidylcholine

The mechanism of the subtransitions (Lc to L beta') in L-dipalmitoylphosphatidylcholine bilayers in excess water has been investigated by time-resolved X-ray diffraction using synchrotron radiation. The temperature dependence of the diffraction patterns closely correlate with the asymmetric excess specific heat variation recorded by differential scanning calorimetry. During the subtransition two prominent wide-angle reflections, characteristic of the low-temperature crystalline phase, Lc, gradually change such that a sharp peak at a spacing of 0.430 nm decreases in intensity and ultimately disappears while a broader peak initially located at 0.375 nm progressively shifts to an eventual spacing of 0.410 nm. This behaviour is interpreted as a lateral deformation of the acyl chain packing subcell as the chains begin to rotate until a state is reached where the chains pack on a regular hexagonal array characteristic of the L beta phase. An increase in lamellar repeat distance from 6.0 to 6.4 nm takes place simultaneously with the acyl chain rearrangement at relatively low (5 K/min) as well as high (6 K/s) heating rates. As judged from the shape of the wide-angle peak, transformation to L beta' phase occurs some minutes after transition to the L beta phase. The X-ray data characterise the subtransition as a continuous (second order) phase transition in which a presumably orthorhombic subcell is transformed into a hexagonal subcell in a gradual process. In temperature jump experiments at 6 K/s between 0 degree C and 80 degrees C the relaxation time of the subtransition was found to be about 5 s while the relaxation time of the main gel to liquid-crystalline transition was about 2 s.     

Interaction among molecules in mixtures of ceramide/stearic acid,ceramide/cholesterol and ceramide/stearic acid/cholesterol

To elucidate the interaction among the molecules which constitute intercellular lipids of stratum corneum, the phase diagrams in the binary mixtures of N-octadecanoyl-phytosphingosine (CER)/stearic acid (SA) and CER/cholesterol (CHOL) were studied by differential scanning calorimetry and by small- and wide-angle X-ray diffraction. These phase diagrams are mostly expressed by a eutectic type one. However, from their detailed analyses, it was revealed that in the phase diagram of CER/SA a new solid structure is formed just above the eutectic temperature. The lamellar spacing of the new structure is nearly equal to the length given by the sum of the two molecules of CER and/or SA, that is, in the lipid bilayer the hydrocarbon chains of CER and SA lie almost perpendicular to the lipid bilayer surface and the two kinds of molecules distribute homogeneously. On the other hand, in the binary mixture of CER/CHOL, CHOL molecules are apt to be isolated from the mixture. In a ternary mixture composed of equimolar lipids of CER, CHOL and SA, it was found that a pseudo-hexagonal structure takes place even in the solid state. This fact indicates that the three components are miscible and the hydrocarbon chains lie perpendicular to the lipid bilayer surface. We can draw the conclusion that the multi-component mixtures containing ceramide are apt to form the lamellar structure where even in the solid state the hydrocarbon chains lie perpendicular to the lipid bilayer surface and the components with hydrocarbon chains distribute homogeneously.     

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.