Stratum Corneum Lipid Composition Research Articles

Stratum corneum lipid profile and maturation pattern of corneocytes in the outermost layer of fresh scars: the presence of immature corneocytes plays a much more important role in the barrier dysfunction than do changes in intercellular lipids.

The functional characteristics of the stratum corneum (SC) of fresh scars as well as keloids and hypertrophic scars are characterized by elevated transepidermal water loss (TEWL) and increased SC hydration. To study the composition of the intercellular lipids and maturation properties of the cornified envelope (CE) of the SC, as these are the most important components for the SC barrier function in fresh scars. SC lipids were extracted from the donor site for split-thickness skin grafting soon after re-epithelialization using a cup method, and were analysed with high-performance thin-layer chromatography. CEs, which were prepared from superficial layers of the SC, were double stained with Nile red and anti-involucrin. We found a significant decrease in the proportion of ceramide (CER) in the SC lipids of fresh scars. We also observed changes in the SC CER profile that consisted of an increase in CER 4 and CER 7 and a decrease in CER 3, without any significant change in the proportion of CER 1. These changes were insufficient to explain the remarkably high TEWL recorded in the early stage of fresh scars. In contrast, with double staining of CE with Nile red and anti-involucrin, we detected the presence of numerous immature and less hydrophobic corneocytes in the outermost layer of the SC of fresh scars. Scanning electron microscopy of such corneocytes revealed numerous fine wrinkles on their enlarged surface area. Most of all, we found a closely similar, time-dependent, exponential decrease in the ratio of immature corneocytes with a poorly hydrophobic CE and in the recorded TEWL values in fresh scars. There was a highly significant positive correlation between the proportion of immature corneocytes in the outermost layer of the SC and TEWL values. These results suggest that the SC barrier dysfunction of the fresh scars is attributable to the presence of immature corneocytes with a less hydrophobic CE, rather than to the changes in SC lipid composition.

Lipid Composition of Outer Stratum corneum in Hereditary Palmoplantar Keratodermas

Background: The analysis of lipid composition of the outer stratum corneum is a promising approach to study the pathophysiology of inherited disorders of keratinization. Objective: The purpose of the study was the search for biochemical alterations of stratum corneum lipids in hereditary palmoplantar keratoderma (PPK). Methods: Using high-performance thin-layer chromatography, we performed an analysis of all major stratum corneum lipid classes in scales of 29 patients with 8 different types of hereditary PPK. Results: In comparison to the controls, slight differences in the lipid pattern were found in all keratodermas. Reduced amounts of total ceramides and increased levels of free fatty acids were noted in nearly all types. Conclusions: The study indicates that the abnormal composition of stratum corneum lipids in PPK is probably not caused by genetic defects of the epidermal lipid metabolism, but it appears to represent an epiphenomenon of a disturbed cornification.

Dodecyl maltoside as a solubilizing agent of stratum corneum lipid liposomes

The lytic interactions of the nonionic surfactant dodecyl maltoside (DM) with liposomes formed by a mixture of lipids modeling the stratum corneum (SC) lipid composition were investigated. To this end, the surfactant to lipid molar ratios (Re) and the normalized bilayer/aqueous phase partition coefficients (K) were determined by monitoring the changes in the static light-scattering (SLS) of the system during solubilization. The fact that the free surfactant concentration was always similar to its critical micelle concentration indicates that the liposome solubilization was mainly ruled by formation of mixed micelles. In addition, the linear dependence established between the level of SLS and Re indicates a progressive incorporation of DM in the liposomes as well as the progressive formation of mixed micelles. DM showed in all cases lower bilayer activity (higher Re values) and greater affinity with vesicles (higher K values) than those reported for its interaction with phosphatidylcholine (PC) liposomes. Thus, whereas the SC lipid liposomes were more resistant to the action of this surfactant, its degree of partitioning into SC bilayers was higher throughout the solubilization process than that exhibited in PC vesicles. Comparison of the present Re values with those reported for the lytic interaction of dodecyl glucoside (DG) with SC liposomes reveals that in the case of DM the bilayer activity was more than three times higher than that for DG in spite of the identical alkyl chain length.

Influence of the hydrophobic tail of alkyl glucosides on their ability to solubilize stratum corneum lipid liposomes

The lytic interactions of a series of alkyl glucosides (alkyl chain lengths ranging from C8 to C12) with liposomes formed by a mixture of lipids modeling the stratum corneum (SC) lipid composition were investigated. The surfactant-to-lipid molar ratios (Re) and the normalized bilayer/aqueous phase partition coefficients (K) were determined by monitoring the changes in the static light-scattering (SLS) of the system during solubilization. The fact that the free surfactant concentrations were always similar to their critical micelle concentrations indicates that the liposome solubilization was mainly ruled by the formation of mixed micelles. At the two interaction levels studied (100 and 0% SLS) the nonyl glucoside showed the highest ability to saturate and to solubilize liposomes (lowest Re values), whereas the dodecyl glucoside showed the highest degree of partitioning into liposomes or affinity with these structures (highest K values). Comparison of the data for octyl glucoside with that reported for the interaction of this surfactant with phosphatidylcholine (PC) liposomes shows that whereas the SC lipid liposomes were more resistant to the action of this surfactant (higher Re values), its degree of partitioning into SC bilayers was both in the saturation and solubilization of liposomes similar to that exhibited in PC vesicles (similar K values).

Determination of stratum corneum lipid profile by tape stripping in combination with high-performance thin-layer chromatography.

Intercellular lipids in the stratum corneum (SC) are responsible for the barrier function of mammalian skin. The main components of the SC lipids are ceramides, cholesterol, and free fatty acids, as established by thin-layer chromatographic analysis of lipids extracted from the human and mammalian SC. Up to now, for lipid analysis the extracts of the entire SC has been used and information on whether the lipid composition changes with the depth in the SC is scarce. Tape stripping is a technique which removes corneocyte layers step by step with an adhesive film. The use of this technique for lipid analysis was hampered by the contamination of lipid extracts with compounds co-extracted from the tape with organic solvents used for the extraction of SC lipids. The aim of the present study was to establish a suitable analytical method for the determination of the local SC lipid composition. For this purpose, the SC samples were collected by sequential stripping with Leukoplex tape in five healthy volunteers. The lipids were extracted with ethyl acetate:methanol mixture (20:80) and separated by means of HPTLC. The results of this study revealed that the free fatty acid level is highest and the cholesterol and ceramide levels lowest in the uppermost SC layers (about 4 strippings). The levels remained unchanged in the underlying SC layers. In these layers, the ceramide level was about 60 wt% and the free fatty acid and cholesterol levels were about 20 wt% each. Ceramides could be separated into seven different fractions and the relative amounts of individual ceramide fractions did not significantly change with the SC depth. Cholesterol sulfate levels were about 5% of total cholesterol and did not change with the SC depth, except for the for the first strip where the level was about 1%. The method developed makes it possible to study the differences in the SC lipid profile in healthy and diseased human skin with relation to the SC lipid organization and to the skin barrier function in vivo.

Sublytic alterations caused by alkyl glucosides in stratum corneum lipid liposomes

The sublytic interactions of a series of alkyl glucosides (alkyl chain lengths ranging from C 8 to C 12) with liposomes formed by a mixture of lipids modeling the stratum corneum (SC) lipid composition were investigated. The surfactant to lipid molar ratios ( R e) and the normalized bilayer/aqueous phase partition coefficients ( K) were determined by monitoring the increase in the fluorescence intensity of liposomes due to the 5(6)-carboxyfluorescein (CF) released from the interior of vesicles to the bulk aqueous phase. Given that the free surfactant concentrations were always lower than their critical micelle concentrations (CMC) we may assume that the surfactant–liposome interactions were mainly ruled by the action of surfactant monomers. At the two interaction levels studied (50 and 100% CF release) the nonyl and the octyl glucoside showed respectively the highest ability to alter the liposome permeability (lowest R e values), whereas the dodecyl glucoside showed the highest degree of partitioning into liposomes or affinity with these structures (highest K values). Different trends in the interaction of these surfactants with SC and PC liposomes were detected when comparing the present R e and K values with those reported for PC vesicles. Thus, whereas SC lipid liposomes were more resistant to the action of alkyl glucosides (higher R e values), the degree of partitioning of these surfactants into SC bilayers was always greater than that exhibited for PC vesicles (higher K values).

Alterations in stratum corneum lipid liposomes due to the action of Triton X-100: Influence of the level of ceramides on this process

The role played by the ceramides (Cer) in the interaction of Triton X-100 (T X-100) with liposomes modeling the stratum corneum (SC) lipid composition was studied. The surfactant/lipid molar ratios ( Re) and the bilayer/aqueous phase partition coefficients ( K) were determined at sublytic level by monitoring the changes in the fluorescence intensity of liposomes due to the 5(6)-carboxyfluorescein (CF) released from the interior of vesicles. Higher and lower Cer proportions than those existing in the SC (40%) led respectively to a fall and to a rise in the surfactant ability to alter these liposomes. However, the surfactant partitioning between bilayers and water (bilayer affinity with vesicles) exhibited a maximum for 40% Cer. Thus, at low Cer proportions the ability of T X-100 molecules to alter these bilayers was maximum despite their reduced partitioning into bilayers, in line with the reported interaction of the anionic surfactant model sodium dodecyl sulfate with these vesicles. These findings underline the fragility of these bilayers as an effective barrier and could explain in part the reported dependencies of low level of Cer in skin lipids and function barrier abnormalities. The fact that the free surfactant concentration needed to achieve the two interaction levels investigated was lower than the surfactant CMC indicates that permeability alterations were mainly ruled by the action of surfactant monomers, regardless of Cer proportion in bilayers.

Barrier Characteristics of Different Human Skin Types Investigated with X-Ray Diffraction, Lipid Analysis, and Electron Microscopy Imaging

The stratum corneum requires ceramides, cholesterol, and fatty acids to provide the cutaneous permeability barrier. The lipids are organized in intercellular membranes exhibiting short- and long-periodicity lamellar phases. In recent years, the phase behavior of barrier lipid mixtures has been studied in vitro. The relationship of human stratum corneum lipid composition to membrane organization in vivo, however, has not been clearly established. Furthermore, the special function of the different ceramide species in the stratum corneum is largely unknown. We examined lipid organization and composition of stratum corneum sheets from different subtypes of healthy human skin (normal, dry, and aged skin). Lipid organization was investigated using X-ray diffraction and demonstrated that the 4.4 nm peak attributed to the long periodicity phase was frequently missing for skin with a low Cer(EOS)/Cer(total) ratio, indicating an important part for Cer(EOS), which contains omega-hydroxy fatty acid (O) ester-linked to linoleic acid (E) and amide-linked to sphingosine (S). A deficiency in the 4. 4 nm peak was predominantly observed in young dry skin. In one case of aged skin, however, and less often in young normal skin this peak was also missing. Furthermore, the ceramide composition of samples without the 4.4 nm peak showed a deficiency of Cer(EOH), which contains 6-hydroxy-4-sphingenine (H), and an increase in Cer(NS) and Cer(AS), which contain nonhydroxy (N) or alpha-hydroxy fatty acids (A). In addition, a 3.4 nm peak attributed to crystalline cholesterol occurred in most cases of aged and dry skin, but was not observed in young normal skin. Our results do not indicate a definite pattern of correlation between lipid organization and types of human skin. They demonstrate, however, that Cer(EOS) and Cer(EOH) are key elements for the molecular organization of the long periodicity lamellar phase in the human stratum corneum.

Influence of the level of ceramides on the permeability of stratum corneum lipid liposomes caused by a C 14-alkyl betaine/sodium dodecyl sulfate mixture

The role played by the ceramides in the interaction of a mixture of tetradecylbetaine (C 14-Bet)/sodium dodecyl sulfate (mole fraction of the zwitterionic surfactant, 0.6) with liposomes modeling the stratum corneum (SC) lipid composition was studied. The surfactant/lipid molar ratios (Re) and the bilayer/aqueous phase partition coefficients ( K) were determined by monitoring the changes in the fluorescence intensity of liposomes due to the 5(6)-carboxyfluorescein released from the interior of vesicles. The presence in liposomes of higher and lower ceramide proportions than that existing in the SC lipids led to a fall and to a rise in the sublytic activity of the surfactant mixture on these structures, respectively. However, the surfactant mixture partitioning into liposomes (or affinity with these bilayer structures) increased as the proportion of ceramides type III (Cer) increased to achieve the highest value for a Cer proportion similar to that in the SC lipids (about 40%). Thus, at low Cer proportions, the ability of the surfactant mixture to alter the permeability of these bilayer structures was higher than that for liposomes approximating the SC lipid composition, despite their reduced partitioning into liposomes. These findings are in agreement with the recently reported dependencies of the level of ceramides in skin lipids and function barrier abnormalities, and could explain, in part, these dependencies. The fact that the free surfactant concentration needed to achieve the interaction levels investigated was lower than the surfactant mixture critical micellar concentration indicates that permeability alterations were mainly ruled by the action of surfactant monomers, regardless of the liposome lipid composition.

Influence of ceramides in the solubilization of stratum corneum lipid liposomes by C 12-betaine/sodium dodecyl sulfate mixtures

The solubilization of liposomes modeling the stratum corneum (SC) lipid composition and those obtained varying the proportion of ceramides by means of dodecyl betaine (C 12-Bet)/sodium dodecyl sulfate (SDS) mixtures was studied. The surfactant/lipid molar ratios (Re) and the bilayer/aqueous phase partition coefficients ( K) were determined by monitoring the changes in the static light scattering of the system during solubilization. The fact that the free surfactant concentration was always similar to its critical micelle concentration (CMC) indicates that the liposome solubilization was mainly ruled by the formation of mixed micelles. The mole fraction of the zwitterionic component ( X zwitter) of 0.4 showed the lowest ability to saturate or solubilize liposomes, although exhibiting the highest degree of partitioning into liposomes. This X zwitter corresponded to the highest derivation of the CMCs of these mixtures (negative synergism) and to the highest reduction in the skin irritation with respect to the anionic component. Higher and lower proportion of ceramides in the mixture led to a fall and to a rise in both the activity and the partitioning of a specific surfactant mixture ( X zwitter=0.4). This finding could be related to the recently reported dependences of the level of ceramides in skin and function barrier abnormalities. Comparison of the present Re and K values with those reported for phosphatidylcholine (PC) liposomes shows that, although SC liposomes were more resistant to the action of surfactant mixtures, the surfactant partitioning into SC bilayers was similar to that reported for PC ones in all cases.

Normal Ultrastructure, but Altered Stratum Corneum Lipid and Protein Composition in a Mouse Model for Epidermolytic Hyperkeratosis

Recently, we established keratin 10-deficient mice, serving as a model for the hyperkeratotic skin disorder epidermolytic hyperkeratosis. The considerable ichthyosis in these mice suggested alterations in terminal differentiation and in the formation of a functional epidermal barrier. Here, we report on the ultrastructural organization and composition of the stratum corneum lipids and on the expression of two major cornified envelope proteins. Electron microscopy of ruthenium tetroxide postfixed skin samples demonstrated a normal extrusion and morphology of lamellar bodies as well as the formation of bona fide lamellar layers in neonatal keratin 10-deficient mice. When we studied the composition of the major stratum corneum lipids, however, we found significant changes. Most importantly, the analysis of ceramide subpopulations revealed that the total amount of ceramide 2 was elevated in keratin 10-deficient mice, whereas ceramides 1, 3, 4, and 5 were decreased among total stratum corneum lipids. The amount of the ceramide precursors sphingomyelin and glucosylceramide was reduced in the stratum corneum without accompanying changes in the mRNA coding for acid sphingomyelinase. Notably, we found an increased mRNA and protein content for involucrin in neonatal keratin 10-deficient mice, whereas the expression of loricrin was not changed. Our data demonstrate that, although the formation of lipid layers in the stratum corneum appeared to be normal, its lipid composition is significantly altered in keratin 10-deficient mice.

Influence of the level of ceramides on the permeability of stratum corneum lipid liposomes caused by a C 12-betaine/sodium dodecyl sulfate mixture

The sublytic interactions of a mixture of N-dodecyl- N, N-dimethylbetaine dodecyl betaine (C 12-Bet)/sodium dodecyl sulfate (SDS) (mole fraction of the zwitterionic surfactant=0.6) with stratum corneum (SC) lipid liposomes varying the proportion of ceramides type III (Cer) were investigated. The surfactant/lipid molar ratios (Re) and the bilayer/aqueous phase partition coefficients ( K) were determined by monitoring the changes in the fluorescence intensity of liposomes due to the 5(6) carboxyfluorescein (CF) released from the interior of vesicles. The fact that the free surfactant mixture concentration was always lower than its critical micelle concentration indicates that permeability changes were ruled by the action of surfactant monomers in all cases. Higher and lower Cer proportions than that of the SC lipids led to a fall and to a rise in the activity of the surfactant mixture on these bilayer structures. However, the surfactant partitioning into liposomes (or affinity with these bilayer structures) increased as the proportion of Cer increased up to the highest value was achieved for a Cer proportion similar to that in the SC lipids (about 40–45%). Thus, at low Cer proportions the ability of the surfactant mixture to alter the permeability of these bilayer structures was higher than that for liposomes approximating the SC lipid composition despite their reduced partitioning into liposomes. These findings are in agreement with the recently reported dependencies of the level of ceramides in skin lipids and function barrier abnormalities and could explain in part these dependencies.

Alkyl sulfate surfactants as solubilizing agents of liposomes modeling the composition of the stratum corneum lipids

The interaction of a series of alkyl sulfates (alkyl chain lengths C-10 (C 10–SO 4), C-12 (C 12–SO 4) and C-14 (C 14–SO 4)) with liposomes modeling the stratum corneum (SC) lipid composition (40% ceramides, 25% cholesterol, 25% palmitic acid and 10% cholesteryl sulfate) was investigated. The surfactant/lipid molar ratios (Re) and the bilayer/aqueous phase partition coefficients ( K) were determined by monitoring the changes in the static light scattering of the system during solubilization. The fact that the free concentration for each surfactant tested was always similar to its critical micelle concentration (in the experimental working medium) indicates that the liposome solubilization was mainly ruled by the formation of mixed micelles. The K parameter progressively rose as the surfactant CMC decreased or its alkyl chain length increased, whereas the Re showed a minimum for C 12–SO 4. Thus, although the maximum ability to saturate or solubilize SC liposomes corresponded to the C 12–SO 4, the C 14–SO 4 showed the highest degree of partitioning into liposomes or affinity with these bilayer structures. The overall balance of these two tendencies shows that the C 14–SO 4 and the C 12–SO 4 had, respectively the highest power to saturate and solubilize SC liposomes in terms of total surfactant amount needed to produce these effects. Different trends in the interaction of these surfactants with SC liposomes were observed when comparing the Re and K parameters with those reported for PC ones. Whereas SC liposomes were more resistant to the surfactant action, the affinity of these surfactants with these bilayer structures was higher in all cases.

Inter- and Intra-Individual Differences in Human Stratum Corneum Lipid Content Related to Physical Parameters of Skin Barrier Function In Vivo

For a full understanding of the properties of the human skin barrier, physical macroscopic parameters of barrier function must be correlated to the structural organization of the barrier on a molecular level. This study was undertaken to relate differences in the relative composition of the three main lipid classes of human stratum corneum, i.e., free fatty acids, cholesterol, and ceramides, to differences in transepidermal water loss, stratum corneum electrical impedance, and corneometer value. A new high performance liquid chromatography/light scattering detection-based analysis method recently developed was used for collection of quantitative lipid data in conjunction with gas chromatography/mass spectrometry/flame ionization detection measurements on the free fatty acid fraction. After subtraction of contaminating lipid fractions we have estimated the molar ratio of the human skin barrier lipid composition to be, respectively, 15% cholesterol esters, 16% saturated long chain free fatty acids, 32% cholesterol, and 37% ceramides. The inter-individual difference in the relative amount of free fatty acids, cholesterol, and ceramides, respectively, can be >100% in the individual case. It was found that the relative amount of ceramides to cholesterol is larger in the wrist area, paralleled by a higher transepidermal water loss and corneometer value as well as different skin electrical impedance values as compared with the upper forearm area. We conclude that the site-dependent differences in the stratum corneum lipid composition are small compared with the large inter-individual variation. Interestingly, in the individual case, no correlation was registered between relative ceramide content and barrier properties.

Interactions of oxyethylenated nonylphenols with liposomes mimicking the stratum corneum lipid composition

The interactions of oxyethylenated nonylphenols (ethylene oxide units (EO) average between 5 and 30) with liposomes modeling the stratum corneum lipid composition were investigated. Liposomes were formed by a lipid mixture containing 40% ceramides, 25% cholesterol, 25% palmitic acid and 10% of cholesteryl sulfate. The surfactant/lipid molar ratios (Re) and the bilayer/aqueous phase partition coefficients (K) were determined at three interaction levels: 50% release of the 5(6) carboxyfluorescein (CF) trapped into the vesicles, saturation, and complete liposome solubilization. The fact that the free surfactant concentrations for each surfactant tested were at sublytic and lytic levels lower than and similar to its critical micelle concentration (in the experimental working medium) indicated that these interactions were mainly governed by the action of surfactant monomers and the formation of mixed micelles respectively. The nonylphenols with 15 EO units always showed the highest activity on SC bilayers (lowest Re values), whereas that with 5 EO units exhibited the highest degree of partitioning into liposomes or affinity with these bilayer structures (highest K values). Different trends in the interaction of nonylphenols with SC and phosphatidylcholine (PC) liposomes were observed when comparing the present Re and K parameters with those reported for PC ones. Thus, whereas SC liposomes were more resistant to the action of nonylphenols, the degree of partitioning of these surfactants into SC bilayers was always greater than that reported for PC ones.

Solubilization of model stratum corneum liposomes by quaternary ammonium surfactants

AbstractThe solubilizing interactions of a series of quaternary ammonium surfactants [alkyl chain lengths C‐12 (DoTAB), C‐14 (TeTAB), and C‐16 (HeTAB)] with liposomes formed by a mixture of lipids modeling the stratum corneum (SC) lipid composition (40% ceramides, 25% cholesterol, 25% palmitic acid, and 10% of cholesteryl sulfate) were investigated. Surfactant/lipid molar ratios (Re) and bilayer/aqueous phase partition coefficients (K) were determined by monitoring changes in static light scattering of the system during solubilization. Free surfactant concentration was always similar to the critical micelle concentration (CMC). A general assumption for phosphatidylcholine (PC) liposomes suggests that the free surfactant concentration must reach CMC for solubilization to occur. This assumption can be applied to SC liposomes in this study, and indicates that liposome solubilization was mainly driven by mixed micelle formation. The Re and K parameters fell as the surfactant alkyl chain length decreased or CMC increased. Thus, a higher CMC corrsponds to an increased ability of these surfactants to saturate or solubilize SC liposomes and to a lower degree of partitioning into liposomes or affinity with these bilayer structures. The overall balance of these opposing tendencies shows that TeTAB had the highest effectiveness with respect to the saturation and solubilization of SC structures in terms of total surfactant needed to produce these effects. Different trends in surfactant interaction with SC liposomes were observed when comparing Re and K parameters with those for PC liposomes. Because SC liposomes were more resistant to the surfactant action, the affinity of surfactants with these bilayer structures was higher in all cases.

Influence of the level of ceramides in the permeability of stratum corneum lipid liposomes caused by sodium dodecyl sulfate

The role played by the ceramides in the sublytic interactions of sodium dodecyl sulfate (SDS) with liposomes modeling the stratum corneum (SC) lipid composition was studied. The surfactant/lipid molar ratios ( Re) and the bilayer/aqueous phase partition coefficients ( K) were determined by monitoring the changes in the fluorescence intensity of liposomes due to the 5(6) carboxyfluorescein (CF) released from the interior of vesicles. The presence in liposomes of higher and lower ceramide proportions than that existing in the SC lipids led to a fall and to a rise in the sublytic activity of SDS on these structures. However, the SDS partitioning into liposomes (or affinity with these bilayer structures) increased as the proportion of Cer increased up to achieve almost a constant value for a Cer proportion similar to that in the SC lipids (about 40%). Thus, at low Cer proportions the ability of SDS molecules to alter these bilayer structures was higher than that for liposomes approximating the SC lipid composition despite their reduced partitioning into liposomes. These findings are in agreement with the recently reported dependencies of the level of ceramides in skin lipids and function barrier abnormalities and could explain in part these dependencies. The fact that the free surfactant concentration needed to achieve the two interaction levels investigated was lower than the surfactant critical micellar concentration (CMC) indicates that permeability alterations were mainly ruled by the action of surfactant monomers, regardless of the liposome lipid composition.

Stratum corneum lipid composition and structure in cultured skin substitutes is restored to normal after grafting onto athymic mice.

Restoration of an epidermal barrier is a definitive requirement for wound closure. Cultured skin substitutes grafted onto athymic nude mice were used as a model for a long-term study of stratum corneum barrier lipid metabolism and organization. Samples of stratum corneum collected after 12 and 21 d in vitro and 6, 11, and 24 mo postgrafting were examined for their lipid and fatty acid composition, and their lipid organization and structure using electron microscopy and small angle X-ray diffraction, respectively. All of these methods confirm the impaired barrier function of cultured skin substitutes in vitro, as judged from the deviations in lipid composition and from poor organization of the stratum corneum lipids that show no lamellar structure. At 6 mo postgrafting, the total stratum corneum lipid profiles of the epidermal grafts is close to that of the human stratum corneum with the exception of the presence of mouse specific lipids. The increase of ceramides 4-7 in cultured skin substitutes after grafting indicates restored activity of processes involved in the hydroxylation of fatty acids and sphingoid bases. Conversely, the ceramide profile still reveals some abnormalities (elevated content of ceramide 2 and slightly lower content of ceramide 3) and the content of long-chain fatty acids remains below its physiologic level at 6 mo postgrafting, but normalizes by 2 y postgrafting. The ultramicroscopic observations revealed the formation of lamellar extracellular lipid domains by 4 mo postgrafting. Despite these findings, the X-ray diffraction showed differences in the diffraction pattern at 2 y after grafting, suggesting that the organization of stratum corneum lipids in all epidermal grafts differs from that of the native skin.

Permeability changes in liposomes modeling the stratum corneum lipid composition due to C 12-alkyl betaine/sodium dodecyl sulfate mixtures

The interaction of dodecyl betaine (C 12-Bet)/sodium dodecyl sulfate (SDS) mixtures at different mole fractions of the zwitterionic component ( X zwitter) with liposomes modeling the stratum corneum (SC) lipid composition were investigated. Liposomes were formed by a lipid mixture of 40% ceramides, 25% cholesterol, 25% palmitic acid and 10% cholesteryl sulfate. The surfactant/lipid molar ratios ( R e) and the bilayer/aqueous phase partition coefficients ( K) were determined at two sublytic levels (50 and 100% CF release) by monitoring the increase in the fluorescence intensity of liposomes due to the 5(6)-carboxyfluorescein (CF) released from the interior of vesicles. The fact that the free surfactant concentrations were always lower than their critical micelle concentrations suggests that the liposomes–surfactant sublytic interactions were mainly ruled by the action of surfactant monomers. At the two interaction levels studied the surfactant mixture for X zwitter=0.6 showed the highest ability to alter the release of the CF entrapped in the interior of vesicles (lowest R e values), whereas that for X zwitter=0.4 showed the highest degree of partitioning into liposomes or affinity with these structures (highest K values). Different trends in the interaction of these mixtures with SC lipids and phosphatidylcholine (PC) liposomes were observed when comparing the present R e and K parameters with those reported for PC ones. Thus, whereas SC lipid liposomes were more resistant to the action of C 12-Bet/SDS mixtures, the partitioning of these mixtures into SC lipid bilayers was always greater than that reported for PC ones.

Solubilization of liposomes modelling the stratum corneum lipid composition by betaine-type zwitterionic surfactants

The interaction of a series of alkylbetaines (alkyl chain lengths C-10 (C 10-Bet), C-12 (C 12-Bet) and C-14 (C 14-Bet)) with liposomes modelling the stratum corneum (SC) lipid composition (40% ceramides, 25% cholesterol, 25% palmitic acid and 10% cholesteryl sulphate) was investigated. The surfactant/lipid molar ratios Re and the bilayer/aqueous phase partition coefficients K were determined by monitoring the changes in the static light scattering of the system during solubilization. The fact that the free concentration for solubilization by each surfactant tested was always similar to its critical micelle concentration (CMC) indicates that the liposome solubilization was mainly ruled by the formation of mixed micelles. The K parameter rose as the surfactant alkyl chain length increased or its CMC decreased, whereas the Re parameter showed a minimum for C 12-Bet. Thus, although the highest ability to saturate or solubilize SC liposomes corresponded to the C 12-Bet, the highest degree of partitioning into liposomes (or affinity with these bilayer structures) was reached for C 14-Bet. The overall balance of these two tendencies shows that C 12-Bet had the highest activity at the two interaction levels investigated in terms of the total surfactant amount needed to produce these effects. Different trends in the interaction of these surfactants with SC liposomes were observed when comparing the Re and K parameters with those reported for phosphatidylcholine. Thus, whereas SC liposomes were more resistant to the surfactant action (especially for C 10-Bet), the affinity of these surfactants with these bilayer structures was higher in all cases.