Stratum Corneum Lipid Matrix Research Articles

TNF-α and Th2 Cytokines Induce Atopic Dermatitis–Like Features on Epidermal Differentiation Proteins and Stratum Corneum Lipids in Human Skin Equivalents

Atopic dermatitis (AD) is a chronic inflammatory skin disease in which the skin barrier function is disrupted. In this inflammatory AD environment, cytokines are upregulated, but the cytokine effect on the AD skin barrier is not fully understood. We aimed to investigate the influence of Th2 (IL-4, IL-13, IL-31) and pro-inflammatory (tumor necrosis factor alpha (TNF-α)) cytokines on epidermal morphogenesis, proliferation, differentiation, and stratum corneum lipid properties. For this purpose, we used the Leiden epidermal model (LEM) in which the medium was supplemented with these cytokines. Our results show that IL-4, IL-13, IL-31, and TNF-α induce spongiosis, augment TSLP secretion by keratinocytes, and alter early and terminal differentiation-protein expression in LEMs. TNF-α alone or in combination with Th2 cytokines decreases the level of long chain free fatty acids (FFAs) and ester linked ω-hydroxy (EO) ceramides, consequently affecting the lipid organization. IL-31 increases long chain FFAs in LEMs but decreases relative abundance of EO ceramides. These findings clearly show that supplementation with TNF-α and Th2 cytokines influence epidermal morphogenesis and barrier function. As a result, these LEMs show similar characteristics as found in AD skin and can be used as an excellent tool for screening formulations and drugs for the treatment of AD.

Formation of the long-periodicity phase in model membranes of the outermost layer of skin (Stratum corneum)

Model SC membranes (lipid membranes fabricated from the main components of the lipid matrix of the outermost layer of mammalian skin (stratum corneum (SC)) are used to study the general regularities of the nanostructurization of SC lipid matrix. A model membrane from the outermost layer of skin SC with a composition ceramide 1/ceramide 6/cholesterol/palmitic acid/cholesterol sulfate and a component weight ratio of 30/30/20/15/5 has been investigated by X-ray diffraction. It is shown that the membrane structure at pH = 7.2 consists of two short-periodicity phases with repeat distances d = 47 and 35.7 A and a long-periodicity phase with d = 127 A. An increase in pH to 9.0 leads to destruction of the long-periodicity phase, while the short-periodicity phase is retained with d = 48.3 A.

In vitro model systems for studying the impact of organic chemicals on the skin barrier lipids

This paper describes two synthetic lipid models designed to replace human stratum corneum (SC) in studies of the impact of volatile organic chemicals on the molecular organization of the skin barrier lipids. The models built upon previously developed self-assembled lipid membranes which have composition and 3D organization similar to those of the lipid matrix in SC. In one model the target chemicals were incorporated in the lipids before their self-assembly, and in the other one they were applied on top of a preformed lipid membrane. The chemicals could be incorporated within the model membranes in quantities close to those reached within human SC upon heavy surface loading. The dose-dependent effects of the chemicals on the lateral molecular organization in the models were qualitatively identical to those observed by infrared spectroscopy in human SC. The models facilitated the interpretation of X-ray diffraction profiles used to determine the nature of the interactions between the chemicals and the lipid lamellae and the position of the exogenous molecules within the unit cell of the lipid phases. These model systems are suitable for in vitro studies in the areas of skin biophysics, dermatology, transdermal drug delivery, and risk assessment.

Impact of intravenous lipid emulsion infusion on the stratum corneum barrier function in patients receiving parenteral nutrition.

The importance of the lipid matrix of stratum corneum (SC) in epidermal barrier function is well documented. Intravenous lipid emulsions (ILE) provide essential fatty acids (EFAs), main components of the SC lipid matrix. The objective of this study was to investigate the influence of ILE upon SC barrier function. The skin barrier was assessed by measuring transepidermal water loss (TEWL). Patients receiving lipid-containing parenteral nutrition (LCPN) were compared to patients receiving lipid-free PN (LFPN). In addition, a before/after LCPN introduction study was set up to limit the influence of inter-individual variability. Twenty-six patients receiving LCPN and seven patients receiving LFPN were included. Medianagewas not significantly different between the two groups. The TEWL of theLCPNgroup(9.05g/m2/h) was significantly lower than the TEWL of the LFPN group(12.1g/m2/h; Wilcoxon test: p=0.016). The relative variation of TEWL before and after ILE treatment of 5studied patients was 21.29±10.28 %. ILE improve epidermal barrier function when compared to lipid-free parenteral treatments. Results of the before/after study confirm this conclusion and the usefulness of ILE intake for preventing excessive TEWL. SC barrier function improvement could be a choice criterion between the different ILE generations, in particular in burn patients and premature neonates.

Study of the Influence of the Penetration Enhancer Isopropyl Myristate on the Nanostructure of Stratum Corneum Lipid Model Membranes Using Neutron Diffraction and Deuterium Labelling

In order to elucidate the mode of action of the lipophilic penetration enhancer isopropyl myristate (IPM) on a molecular scale, we investigated oriented quaternary stratum corneum (SC) lipid model membranes based on ceramide AP, cholesterol, palmitic acid and cholesterol sulfate containing 10 wt% IPM by means of neutron diffraction. Our results indicate that IPM affects the lamellar lipid assembly in terms of bilayer perturbation and disordering. Phase segregation occurred, indicating that IPM is not likely to mix properly with the other SC lipids due to its branched structure. We used selective deuterium labelling to localize the penetration enhancer, and could successfully prove the presence of IPM in the two coexisting lamellar phases. We conclude that IPM’s mode of action as penetration promoter is presumably based on incorporation into the SC lipid matrix, extraction of certain SC lipids into a separate phase and perturbation of the multilamellar lipid assembly.

Study of the miscibility of cholesteryl oleate in a matrix of ceramide, cholesterol and fatty acid

Cholesteryl esters (CE) are not generally abundant but are ubiquitous in living organisms and have markedly different properties from cholesterol because of their acyl chain. The miscibility/immiscibility of CE with biological lipid structures is a key property for their functions. In this work we study the solubility of cholesteryl oleate (ChO) in a model of the stratum corneum lipid matrix composed of ceramide C16, cholesterol and palmitic acid in excess water. Experiments were done in conditions of fully ionized (pH = 9.0) and fully neutralized fatty acid (pH = 4.0), and differential scanning calorimetry of the ternary mixtures with added ChO at pH = 9.0 clearly displayed a main transition with the same maximum temperature, peak shape, and enthalpy, suggesting that ChO was excluded from the remaining lipids. This technique is not conclusive at pH = 4.0 because the transitions of the lipid matrix and ChO overlap. The insolubility of ChO at both pH values is supported by X-ray diffraction. Adding the ceramide:cholesterol:fatty acid lipid mixture to ChO did not change the X-ray pattern of the mixture nor that of the ChO. To supplement the above physical techniques, we applied 13C MAS NMR spectroscopy with C-13 carbonyl-labeled ChO. A single 13C carbonyl peak from the ChO at 171.5 ppm was observed, indicating exposure to only one environment. The chemical shift was identical to pure ChO below and above the temperature of isotropic liquid formation. Taken together, our results lead to the conclusion that the solubility of ChO is negligible in the ceramide:cholesterol:fatty acid lipid mixture.

The thermotropism and prototropism of ternary mixtures of ceramide C16, cholesterol and palmitic acid. An exploratory study

Mixtures of ceramides with other lipids in the presence of water are key components of the structure of the lipid matrix of the stratum corneum and are involved in lateral phase separation processes occurring in lipid membranes. Besides their structural role, ceramides are functional for cell signaling and trafficking. We elected, as our object of study, a mixture of N-hexadecanoylceroyl- d- erythro-sphyngosine, C16-Cer, with cholesterol, Ch, in a molar proportion 54:46 in excess water to which palmitic acid, PA, is added in varying amounts. The chosen C16-Cer:Ch proportion replicates the relative abundance of ceramides and cholesterol found in the stratum corneum lipid matrix. For each lipidic composition, we identify the phases in equilibrium and study the thermotropism of the system, using differential scanning calorimetry and temperature-dependent small and wide-angle X-ray powder diffraction. Since the molecular aggregation of the system and its mesoscopic properties are affected by the degree of protonation of the PA, we explore mixtures with several PA contents at two extreme pH values, 9.0 and 4.0. A specific C16-Cer:Ch:PA composition forms at pH 9.0 a lamellar crystalline aggregate, to which we attribute the stoichiometry C16-Cer 5Ch 4PA 2, that melts at 88–90 °C to give a H II phase. For pH values at which there is partial or total protonation of PA another L C C16-Cer:Ch (2:3) stoichiometric aggregate is observed, identical to that previously reported for C16-Cer:Ch mixtures ( Souza et al., 2009, J. Phys. Chem. B, 113, 1367–1375), coexisting with a lamellar fluid phase. For pH 4.0 and 7.0, the existing lamellar liquid crystalline converts into a isotropic fluid phase at high temperatures. It is also found that the miscibility of PA in the C16-Cer:Ch mixture at pH 4.0 does not exceed ca. 18 mol%, but for pH 9.0 no free PA is detected at least until 60 mol%.

Correlation between the properties of the lipid matrix and the degrees of integrity and cohesion in healthy human Stratum corneum

The correlation between the degrees of integrity and cohesion in healthy human Stratum corneum (SC) and the properties of the SC lipid matrix could be examined non-invasively in vivo using ATR-FTIR spectroscopy and measurements of pH, conductance, and transepidermal water loss (TEWL) taken in the course of tape-stripping. The change of TEWL following the removal of a SC layer with a predefined thickness served as a measure for the SC integrity, and the amount of protein removed by predefined number of tapes - as a measure for the SC cohesion. The extent of lipids organized in orthorhombic lattices and the pH in the inner SC emerged as the main factors that determine the degree of integrity. The amounts and molecular organization of the SC lipids did not correlate with the degree of cohesion, while the pH and the hydration of SC correlated well with the degree of cohesion in the superficial but not in the inner SC layers. This study evidenced the variability of SC integrity and cohesion existing in healthy human skin, demonstrated the importance of the lipid molecular organization for the SC integrity, and illustrated the limitations in the determination the degree of corneodesmolysis in SC based only on the protein content of tape-strips.

Synthesis and Characterization of Long-Chain Tartaric Acid Diamides as Novel Ceramide-Like Compounds

Ceramides play a crucial role in the barrier function of the skin as well as in transmembrane signaling. In this study long aliphatic chain tartaric acid diamides able to replace ceramides in an in vitro model of the stratum corneum lipid matrix due to their similar physico-chemical properties were synthesized from diacetoxysuccinic anhydride in four steps. Their pro-apoptotic effect on fibroblast cells was also investigated.

Investigation of stratum corneum lipid model membranes with free fatty acid composition by neutron diffraction

The outermost epidermal layer, the stratum corneum (SC), is the main skin barrier. Studies of SC model systems enable characterization of the influence of individual lipids on the organization of the SC lipid matrix, which is the main pathway of water through the skin. This work presents a neutron diffraction study of the SC model membranes based on short-chain ceramide 6 with nearly realistic composition of free fatty acids (FFA) at physiological temperature of the SC. The influence of FFA and the effect of cholesterol-cholesterol sulfate substitution on the structure and hydration of the SC model membranes are described. The structure of the SC membrane with FFA is close to the structure of the earlier studied SC membrane based on short-chain palmitic acid (PA) and does not vary significantly under changes of the ratio of the main membrane components. FFA accelerates membrane swelling at the same low level of hydration of both PA- and FFA-containing membranes. The substitution of cholesterol sulfate by cholesterol in the membrane composition decreases membrane swelling and leads to phase separation in the model system.

Impact of the long chain ω-acylceramides on the stratum corneum lipid nanostructure. Part 1: Thermotropic phase behaviour of CER[EOS] and CER[EOP] studied using X-ray powder diffraction and FT-Raman spectroscopy

The stratum corneum (SC), the outermost layer of the mammalian skin, is the main skin barrier. Ceramides (CERs) as the major constituent of the SC lipid matrix are of particular interest. At the moment, 11 classes of CERs are identified, but the effect of each single ceramide species is still not known. Therefore in this article, the thermotropic behaviour of the long chain omega-acylceramides CER[EOS] and CER[EOP] was studied using X-ray powder diffraction and FT-Raman spectroscopy. It was found that the omega-acylceramides CER[EOS] and CER[EOP] do not show a pronounced polymorphism which is observed for shorter chain ceramides as a significant feature. The phase behaviour of both ceramides is strongly influenced by the extremely long acyl-chain residue. The latter has a much stronger influence compared with the structure of the polar head group, which is discussed as extremely important for the appearance of a rich polymorphism. Despite the strong influence of the long chain, the additional OH-group of the phyto-sphingosine type CER[EOP] influences the lamellar repeat distance and the chain packing. The less polar sphingosine type CER[EOS] is stronger influenced by the long acyl-chain residue. Hydration is necessary for the formation of an extended hydrogen-bonding network between the polar head groups leading to the appearance of a long-periodicity phase (LPP). In contrast, the more polar CER[EOP] forms the LPP with densely packed alkyl chains already in the dry state.

Transition processes in stratum corneum model lipid membranes with a mixture of free fatty acids

The hydration of model membranes based on ceramide 6 with a mixture of free fatty acids the most commonly encountered the native lipid matrix of Stratum corneum, the outermost layer of the mammalian skin has been studied by neutron diffraction. Membrane hydration with water vapour at a temperature of 25 degrees C is characterized by a small increase in the repeat distance delta d0 = 1.0 A, which is comparable with membrane swelling in the presence of excess water. The kinetics changes in the repeat distance, connected with an increase of the water layer between bilayers during hydration, and water exchange during the processes of hydration and H-D isotopic substitution consists of a fast initial and a subsequent slow stage and is well described by exponential with two characteristic times lying in the range from a few tens of minutes to several hundreds of minutes. During hydration at a temperature of 57 degrees C, the repeat distance increases by delta d0 = 1.6 A, after which the membrane irreversibly separates into two phases. One of the phases is formed mainly by long-chain free fatty acids and is characterized by a large decrease in the repeat distance delta d(ph) = 8.3 A on dehydration. The investigation of the structure of model membranes in the temperature range 20-72 degrees C indicated that the system with 20% (w) of cholesterol in the range of 63-67 degrees C undergoes a structural phase transition caused by the melting of hydrocarbon chains of lipids. In the system with a smaller content of cholesterol, no phase transition was observed up to a temperature of 72 degrees C.

Phase Behavior of Aqueous Dispersions of Mixtures of N-Palmitoyl Ceramide and Cholesterol: A Lipid System with Ceramide−Cholesterol Crystalline Lamellar Phases

Ceramides are particularly abundant in the stratum corneum lipid matrix, where they determine its unusual mesostructure, are involved in the lateral segregation of lipid domains in biological cell membranes, and are also known to act as signaling agents in cells. The importance attributed to ceramides in several biological processes has heightened in recent years, demanding a better understanding of their interaction with other membrane components, namely, cholesterol. Structural data concerning pure ceramides in water are relatively scarce, and this is even more the case for mixtures of ceramides with other lipids commonly associated with them in biological systems. We have derived the thermotropic binary phase diagram of mixtures of N-palmitoyl- D-erythro-sphingosine, C16:0-ceramide, and cholesterol in excess water, using differential scanning calorimetry and small- and wide-angle X-ray diffraction. These mixtures are self-organized in lamellar mesostructures that, between other particularities, show two ceramide to cholesterol crystalline phases with molar proportions that approach 2:3 and 1:3. The 2:3 phase crystallizes in a tetragonal arrangement with a lamellar repeat distance of 3.50 nm, which indicates an unusual lipid stacking, probably unilamellar. The uncommon mesostructures formed by ceramides with cholesterol should be considered in the rationalization of their singular structural role in biological systems.

Use of “Bricks and Mortar” Model To Predict Transdermal Permeation: Model Development and Initial Validation

Presented in this paper is a general purpose computer model for predicting transdermal permeation of solutes in vivo. The bricks and mortar model is employed to represent the stratum corneum (SC), the main barrier to transdermal permeation. Transdermal permeation and absorption is modeled as a dynamic process of mass transfer in the heterogeneous stratum corneum including both the tortuous lipid pathway and the transcellular corneocytes pathway. The partition and diffusion properties of solutes in SC lipid matrix and corneocytes are calculated from the fundamental physical chemical properties of octanol-water partition coefficient, molecular size, and diffusion coefficients in water and lipid, using equations established elsewhere. To test the model, the in vivo tape striping data of 4-cyanophenol is simulated. Using the calculated partition and diffusion properties of 4-cyanophenol in SC lipids and corneocytes, the predicted dynamic profiles of 4-cyanophenol in the SC agreed very well with the experimental data. Results show that for a moderately hydrophobic solute like 4-cyanophenol, the transcellular pathway is also an important route of percutaneous absorption with about 2/3 of the absorbed 4-cyanophenol partitioned into the corneocytes. © 2008 American Chemical Society.

Arrangement of ceramide [EOS] in a stratum corneum lipid model matrix: new aspects revealed by neutron diffraction studies

The lipid matrix in stratum corneum (SC) plays a key role in the barrier function of the mammalian skin. The major lipids are ceramides (CER), cholesterol (CHOL) and free fatty acids (FFA). Especially the unique-structured omega-acylceramide CER[EOS] is regarded to be essential for skin barrier properties by inducing the formation of a long-periodicity phase of 130 angstroms (LPP). In the present study, the arrangement of CER[EOS], either mixed with CER[AP] and CHOL or with CER[AP], CHOL and palmitic acid (PA), inside a SC lipid model membrane has been studied for the first time by neutron diffraction. For a mixed CER[EOS]/CER[AP]/CHOL membrane in a partly dehydrated state, the internal membrane nanostructure, i.e. the neutron scattering length density profile in the direction normal to the surface, was obtained by Fourier synthesis from the experimental diffraction patterns. The membrane repeat distance is equal to that of the formerly used SC lipid model system composed of CER[AP]/CHOL/PA/ChS. By comparing both the neutron scattering length density profiles, a possible arrangement of synthetic long-chain CER[EOS] molecules inside a SC lipid model matrix is suggested. The analysis of the internal membrane nanostructure implies that one CER[EOS] molecule penetrates from one membrane layer into an adjacent layer. A 130 angstroms periodicity phase could not be observed under experimental conditions, either in CER/CHOL mixtures or in CER/CHOL/FFA mixture. CER[EOS] can be arranged inside a phase with a repeat unit of 45.2 angstroms which is predominately formed by short-chain CER[AP] with distinct polarity.

Localisation of partially deuterated cholesterol in quaternary SC lipid model membranes: a neutron diffraction study

This letter presents our first results in using the benefit of selective deuteration in neutron diffraction studies on stratum corneum (SC) lipid model systems. The SC represents the outermost layer of the mammalian skin and exhibits the main skin barrier. It is essential for studying drug penetration through the SC to know the internal structure and hydration behaviour on the molecular level. The SC intercellular matrix is mainly formed by ceramides (CER), cholesterol (CHOL) and long-chain free fatty acids (FFA). Among them, CHOL is the most abundant individual lipid, but a detailed knowledge about its localisation in the SC lipid matrix is still lacking. The structure of the quaternary SC lipid model membranes composed of either CER[AP]/CHOL-D6/palmitic acid (PA)/cholesterol sulphate (ChS) or CER[AP]/CHOL-D7/PA/ChS is characterized by neutron diffraction. Neutron diffraction patterns from the oriented samples are collected at the V1 diffractometer of the Hahn-Meitner-Institute, Berlin, measured at 32 degrees C, 60% humidity and at different D2O contents. The neutron scattering length density profile in the direction normal to the surface is restored by Fourier synthesis from the experimental diffraction patterns. The analysis of scattering length density profile is a suitable tool for investigating the internal structure of the SC lipid model membranes. The major finding is the experimental proof of the CHOL localisation in SC model membrane by deuterium labelling at prominent positions in the CHOL molecules.

Synergetic effects of isopropyl alcohol (IPA) and isopropyl myristate (IPM) on the permeation of betamethasone-17-valerate from semisolid Pharmacopoeia bases

Betamethasone-17-valerate (BM-17-V) is a corticosteroid commonly administered in semisolid preparations for cutaneous use. In this study, an ointment and a cream from the German Pharmacopoeia, i.e. wool fat alcohol ointment (WS) and basis cream DAC were loaded separately with BM-17-V. Two conventional penetration enhancers, namely isopropyl alcohol (IPA) and isopropyl myristate (IPM) as sole additives, as well as a mixture of both, IPA-IPM 1:1 (w/w) were also incorporated into the different semisolid formulations and in vitro permeation experiments of BM- 17-V were carried out in Franz diffusion cells using excised human stratum corneum (SC). The effect of the additives on the thermal behavior of the SC lipid matrix was evaluated by differential scanning calorimetry (DSC). The saturation concentrations of BM-17-V within the formulations were estimated based on microscopical characterization. BM-17-V is transformed into its hydrolization product, betamethasone (BM), during the permeation experiments by the effect of the SC using the BM-21-V pathway. Furthermore the microstructure of the lipids and protein complexes located at the membrane of the corneocytes was altered by the use of the enhancers. The combination of IPA and IPM resulted in a synergetic enhancement of the BM flux. The BM permeation was augmented by approximately 12 times when a combination of IPA-IPM 1:1 (w/w) was used in wool fat alcohol ointment.

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.

FTIR spectroscopic studies of lipid dynamics in phytosphingosine ceramide models of the stratum corneum lipid matrix

IR spectroscopic studies are reported for N-stearyl- d- erythro-phytosphingosine (Cer NP) and N-stearyl-2-hydroxy- d- erythro-phytosphingosine (Cer AP) in a hydrated model of the skin lipid barrier comprised of equimolar mixtures of each ceramide with cholesterol and d 35-stearic acid. Examination of the methylene stretching, rocking and bending modes reveal some rotational freedom and hexagonal packing in both the ceramide and stearic acid chains. Analysis of the acid carbonyl stretch and the ceramide Amide I modes show both shift to higher frequencies, indicating weaker hydrogen bonding, in the mixed systems compared to the pure materials. For both systems, the fatty acid chain disordering temperatures are significantly increased from those of the pure acids. The observed behaviors of these phytosphingosine ceramide systems are fundamentally different from the previously reported analogous sphingosine ceramide systems. The implications of these observations for lipid organization in the stratum corneum are briefly discussed.

PH‐induced alterations in stratum corneum properties

Skin-cleansing compositions based on alkyl carboxylates (soaps) have a higher irritation potential than those based on syndet surfactants such as alkyl isethionates or alkyl ether sulphates. Contributing factors include inherent differences in the irritation potential of soaps and syndet surfactants, pH-induced changes in surfactant solution chemistry, and the direct effects of pH on the physical properties of the stratum corneum (SC). Past work has not directly addressed the effect of solution pH on the SC itself and its potential role in cleanser-induced skin irritation. In the current work, alterations to SC properties induced by buffered pH solutions and two strongly ionizable surfactants, sodium dodecyl sulphate and sodium lauryl ether sulphate, at different pH values are measured. By utilizing optical coherence tomography (OCT) and infrared (IR) spectroscopy we have directly measured physical changes in SC proteins and lipids. Our results indicate that SC swelling, which reflects alterations to SC structural proteins, is increased significantly at pH 10, compared to pH 4 and 6.5. The transition temperature (T(m)) of SC lipids is found to increase at pH 10, compared to pH 4 and 6.5, suggesting a more rigid SC lipid matrix. Surfactants cause a further increase in swelling and lipid rigidity. Some aspects of what these results mean for SC physical properties as well as their implications to potential mechanisms of surfactant-induced skin irritation are discussed.