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.     

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.     

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.     

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.     

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.     

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.     

Different stratum corneum lipid liposomes as models to evaluate the effect of the sodium dodecyl sulfate

The stability of stratum corneum (SC) liposomes against the action of surfactants has been revised. To this end, two types of vesicles were used; vesicles formed with the lipid and protein material extracted from SC, and lipid mixtures approximating the SC composition. In this case, the proportion of ceramides (Cer) and cholesteryl sulfate (Chol-sulf) was varied and the relative proportion of the other lipids remained constant. The increasing presence of these two lipids increased the resistance of liposomes against the action of the anionic surfactant sodium dodecyl sulfate (SDS). The rise in the cell-to-cell cohesion that occurred in recessive X-linked ichthyosis due to the accumulation of Chol-sulf could be associated in part to the enhanced stability of (Chol-sulf)-enriched bilayers. It is noteworthy that the surfactant partitioning between bilayers and the aqueous phase increased and decreased, respectively, as the proportion of Cer and Chol-sulf increased. This effect may be attributed to the variations in both the electrostatic interactions lipid-surfactant (electrostatic repulsion between the sulfate groups of both Chol-sulf and SDS), and the hydrophilic lipophilic balance of the lipid mixtures, in which Cer is replaced by the major polar lipid of the mixture (Chol-sulf). The fact that the free surfactant concentration was always smaller than its critical micelle concentration indicates that the permeability alterations were mainly ruled by the action of surfactant monomers, in agreement with the results reported for sublytic interactions of this surfactant with PC liposomes.     

Interaction between corneocytes and stratum corneum lipid liposomes in vitro

Small unilamellar vesicles were made from a mixture of epidermal ceramides (45%), cholesterol (35%), free fatty acids (15%) and cholesteryl sulfate (5%). Isolated corneocytes prepared from pig epidermis were added to the liposomes and the interaction between corneocytes and liposomes was studied by (1) thin-section electron microscopy and (2) monitoring the release of aqueous contents of the vesicles by following the fluorescence intensity of carboxyfluorescein entrapped in the vesicles. The vesicles adsorbed readily onto the corneocytes and slowly transformed into lamellar sheets. Enhanced fluorescence intensity indicated a corneocyte-induced membrane fusion process that resulted in the release of aqueous contents of the vesicles. The results suggest a cohesive role for the corneocyte cell envelope, which consists of a monomolecular layer of lipids covalently bound to the outside of a cross-linked protein envelope. This may be one of the major factors in the reassembly of extruded membranous disks into lamellar sheets which occurs during the final stages of epidermal differentiation.     

Local temperature rises influence in vivo electroporation pore development: a numerical stratum corneum lipid phase transition model

Electroporation is an approach used to enhance transdermal transport of large molecules in which the skin is exposed to a series of electric pulses. Electroporation temporarily destabilizes the structure of the outer skin layer, the stratum corneum, by creating microscopic pores through which agents, ordinarily unable to pass into the skin, are able to pass through this outer barrier. Long duration electroporation pulses can cause localized temperature rises, which result in thermotropic phase transitions within the lipid bilayer matrix of the stratum corneum. This paper focuses on electroporation pore development resulting from localized Joule heating. This study presents a theoretical model of electroporation, which incorporates stratum corneum lipid melting with electrical and thermal energy equations. A transient finite volume model is developed representing electroporation of in vivo human skin, in which stratum corneum lipid phase transitions are modeled as a series of melting processes. The results confirm that applied voltage to the skin results in high current densities within the less resistive regions of the stratum corneum. The model captures highly localized Joule heating within the stratum corneum and subsequent temperature rises, which propagate radially outward. Electroporation pore development resulting from the decrease in resistance associated with lipid melting is captured by the lipid phase transition model. As the effective pore radius grows, current density and subsequent Joule heating values decrease.     

Improved low pH bicelle system for orienting macromolecules over a wide temperature range

We have prepared and characterized a novel bicelle system composed of 1,2-di-O-dodecyl-sn-glycero-3-phos- phocholine (DIODPC) and 3-(chloramidopropyl)dimethylammonio-2-hydroxyl-1-propane sulfonate (CHAPSO). At the optimal DIODPC/CHAPSO molar ratio of 4.3:1, this medium becomes magnetically oriented from pH 6.5 down to pH 1.0. Unlike previously reported bicelle preparations, these bicelles are chemically stable at low pH and are capable of inducing protein alignment, as illustrated by the large residual dipolar couplings measured for rusticyanin from Thiobacillus ferrooxidans at pH 2.1. The DIODPC/CHAPSO system is particularly useful for measuring residual dipolar couplings of macromolecules that require very acidic conditions.     

An effective iodide formulation for killing Bacillus and Geobacillus spores over a wide temperature range

AIMS: To develop a sporicidal reagent which shows potent activity against bacterial spores not only at ambient temperatures but also at low temperatures. METHODS AND RESULTS: Suspension tests on spores of Bacillus and Geobacillus were conducted with the reagent based on a previously reported agent (N. Kida, Y. Mochizuki and F. Taguchi, Microbiology and Immunology 2003; 47: 279-283). The modified reagent (tentatively designated as the KMT reagent) was composed of 50 mmol l(-1) EDTA-2Na, 50 mmol l(-1) ferric chloride hexahydrate (FeCl(3).6H(2)O), 50 mmol l(-1) potassium iodide (KI) and 50% ethanol in 0.85% NaCl solution at pH 0.3. The KMT reagent showed significant sporicidal activity against three species of Bacillus and Geobacillus spores over a wide range of temperature. The KMT reagent had many practical advantages, i.e. activity was much less affected by organic substances than was sodium hypochlorite, it did not generate any harmful gas and it was stable for a long period at ambient temperatures. The mechanism(s) of sporicidal activity of the KMT reagent was considered to be based on active iodine species penetrating the spores with enhanced permeability of the spore cortex by a synergistic effect of acid, ethanol and generated active oxygen. CONCLUSIONS: The data suggest that the KMT reagent shows potent sporicidal activity over a wide range temperatures and possesses many advantages for practical applications. SIGNIFICANCE AND IMPACT OF THE STUDY: The results indicate development of a highly applicable sporicidal reagent against Bacillus and Geobacillus spores.     

Thermotaxis of human sperm cells in extraordinarily shallow temperature gradients over a wide range

On the basis of the finding that capacitated (ready to fertilize) rabbit and human spermatozoa swim towards warmer temperatures by directing their movement along a temperature gradient, sperm thermotaxis has been proposed to be one of the processes guiding these spermatozoa to the fertilization site. Although the molecular mechanism underlying sperm thermotaxis is gradually being revealed, basic questions related to this process are still open. Here, employing human spermatozoa, we addressed the questions of how wide the temperature range of thermotaxis is, whether this range includes an optimal temperature or whether spermatozoa generally prefer swimming towards warmer temperatures, whether or not they can sense and respond to descending temperature gradients, and what the minimal temperature gradient is to which they can thermotactically respond. We found that human spermatozoa can respond thermotactically within a wide temperature range (at least 29-41°C), that within this range they preferentially accumulate in warmer temperatures rather than at a single specific, preferred temperature, that they can respond to both ascending and descending temperature gradients, and that they can sense and thermotactically respond to temperature gradients as low as <0.014°C/mm. This temperature gradient is astonishingly low because it means that as a spermatozoon swims through its entire body length (46 μm) it can sense and respond to a temperature difference of <0.0006°C. The significance of this surprisingly high temperature sensitivity is discussed.     

Molecular properties of a stratum corneum model lipid system: large unilamellar vesicles.

Stratum corneum lipids are relatively complex, and there is little detailed understanding of their chemical and physical properties at the molecular level. Large unilamellar vesicles (LUVs) with lipid compositions similar to those of stratum corneum were prepared at pH 9 with commercially available lipids. This system was used as a model system for molecular studies of stratum corneum lipids. LUVs were chosen as the model system as they are comparatively more stable and can be characterized more quantitatively in terms of lipid concentration, surface area, and volume than model systems such as lipid mixture suspensions, lipid films, and small unilamellar vesicles. Results from freeze-fracture and cryo electron microscopy studies of our LUVs showed spherical vesicles. Quasi-elastic light scattering measurements revealed a narrow size distribution, centering around 119 nm. At room temperature, the LUVs were stable for several weeks at pH 9 and for more than 15 h but less than 24 h at pH 6. Differential scanning calorimetry measurements indicated broad endothermic transitions centered near 60-65 degrees C, closely matching the transition temperature reported for stratum corneum lipid extracts. Spin probes, 5-doxylstearic acid and 12-doxylstearic acid, were used for electron paramagnetic resonance (EPR) studies of the molecular dynamics of the lipids. EPR results indicated more restricted motion near the polar headgroup region than near the center of the alkyl chain region. Motional profiles of the spin labels near the polar headgroup and within the alkyl chain region in the LUVs were obtained as a function of temperature, ranging from 25 to 90 degrees C. We also found that the partitioning between the lipid and aqueous phases for each spin probe was temperature dependent and was generally correlated with phase transitions observed by differential scanning calorimetry and with alkyl chain mobility observed by EPR. Thus, this LUV system is well suited for additional molecular studies under different experimental conditions.     

Investigation of the interaction between modified ISCOMs and stratum corneum lipid model systems

The modified ISCOMs, so-called Posintro™ nanoparticles, provide an opportunity for altering the surface charge of the particles, which influences their affinity for the negatively charged antigen sites, cell membranes and lipids in the skin. Hypothetically, this increases the passage of the ISCOMs (or their components) and their load through the stratum corneum. The subsequent increase in the uptake by the antigen-presenting cells results in enhanced transcutaneous immunization. To understand the nature of penetration of Posintro™ nanoparticles into the intercorneocyte space of the stratum corneum, the interaction between the nanoparticles and lipid model systems in form of liposomes and/or supported lipid bilayer was studied. As a lipid model we used Stratum Corneum Lipid (SCL), a mixture similar in composition to the lipids of the intercorneocyte space. By Förster Resonance Energy Transfer (FRET), Atomic Force Microscopy (AFM), Electrochemical Impedance Spectroscopy (EIS) and cryo-Transmission Electron Microscopy (cryo-TEM) it was shown that application of nanoparticles to the SCL bilayers results in lipid disturbance. Investigation of this interaction by means of Isothermal Titration Calorimetry (ITC) confirmed existence of an enthalpically unfavorable reaction. All these methods demonstrated that the strength of electrostatic repulsion between the negatively charged SCL and the nanoparticles affected their interaction, as decreasing the negative charge of the Posintro™ nanoparticles leads to enhanced disruption of lipid organization.     

Molecular dynamics simulations of stratum corneum lipid models: fatty acids and cholesterol

We report the results of an investigation on stratum corneum lipids, which present the main barrier of the skin. Molecular dynamics simulations, thermal analysis and FTIR measurements were applied. The primary objective of this work was to study the effect of cholesterol on skin structure and dynamics. Two molecular models were constructed, a free fatty acid bilayer (stearic acid, palmitic acid) and a fatty acid/cholesterol mixture at a 1:1 molar ratio. Our simulations were performed at constant pressure and temperature on a nanosecond time scale. The resulting model structures were characterized by calculating surface areas per headgroup, conformational properties, atom densities and order parameters of the fatty acids. Analysis of the simulations indicates that the free fatty acid fraction of stratum corneum lipids stays in a highly ordered crystalline state at skin temperatures. The phase behavior is strongly influenced when cholesterol is added. Cholesterol smoothes the rigid phases of the fatty acids: the order of the hydrocarbon tails (mainly of the last eight bonds) is reduced, the area per molecule becomes larger, the fraction of trans dihedrals is lower and the hydrophobic thickness is reduced. The simulation results are in good agreement with our experimental data from FTIR analysis and NIR-FT Raman spectroscopy.     

ATR-FTIR spectroscopy: a chemometric approach for studying the lipid organisation of the stratum corneum

The barrier function of skin resides in the lipid components of the stratum corneum, particularly their spatial organisation. FTIR spectroscopy has already been used as a relevant tool to study this lipid organisation: IR vibration band shifts have been attributed to the variations in lipid organisation induced by temperature. Our study included a stratum corneum model, composed of the three main lipids: palmitic acid as an example of fatty acids, cholesterol and ceramide III as an example of ceramide. Different films with various ratios of these lipids were studied. In our analytical strategy, the interest of using a chemometric analysis of global data obtained from ATR-FTIR spectra to highlight the main interactions involved in the molecular organisation of lipids has been demonstrated. Two kinds of interaction between the three main lipids have been shown: a non polar interaction between the long hydrocarbon chains and a polar interaction as the hydrogen bonding between polar functional groups. By varying the lipid ratio, we have shown first that the relative importance of each interaction was modified, second, that the induced modification of organisation can be detected by chemometric analysis of the ATR-FTIR spectra. The role of each kind of lipid in the organisation has been discussed. In conclusion, associating the ATR-FTIR with chemometric treatment is a promising tool: firstly, to understand the consequence of lipid relative compositions on the structural organisation of the stratum corneum, secondly, to show the relationship between lipid organisation and percutaneous penetration data. Indeed, this methodology will be transposed to in vivo studies with IR measurements through a probe.     

MD simulation of self-diffusion and structure in some n-alkanes over a wide temperature range at high pressures

Self-diffusion and structural properties of n-alkanes have been studied by molecular dynamics simulation in the temperature range between the melting pressure curve and 600 K at pressures up to 300 MPa. The simulated results of lower n-alkanes are in good agreement with the existing experimental data, and support the reliability of results of the simulations of self-diffusion coefficients obtained at the extreme conditions. We predict the self-diffusion coefficients for methane, ethane, propane and n-butane at the similar reduced temperatures and pressures to draw a comparison between them. Then the correlation between self-diffusion and structural properties are further investigated by calculating the coordination numbers. Moreover, we define four distances and their corresponding relative deviations to characterize the flexibility of long-chain n-alkanes. The simulated results show that the self-diffusion of n-alkane molecules is mainly affected by the close packing, and the flexibility has a strong impact on the self-diffusion of longer n-alkane molecules.