Ceramides are the major lipid constituent of lamellar sheets present in the intercellular spaces of the stratum corneum. These lamellar sheets are thought to provide the barrier property of the epidermis. It is generally accepted that the intercellular lipid domain is composed of approximately equimolar concentrations of free fatty acids, cholesterol, and ceramides. Ceramides are a structurally heterogeneous and complex group of sphingolipids containing derivatives of sphingosine bases in amide linkage with a variety of fatty acids. Differences in chain length, type and extent of hydroxylation, saturation etc. are responsible for the heterogeneity of the epidermal sphingolipids. It is well known that ceramides play an essential role in structuring and maintaining the water permeability barrier function of the skin. In conjunction with the other stratum corneum lipids, they form ordered structures. An essential factor is the physical state of the lipid chains in the nonpolar regions of the bilayers. The stratum corneum intercellular lipid lamellae, the aliphatic chains in the ceramides and the fatty acids are mostly straight long-chain saturated compounds with a high melting point and a small polar head group. This means that at physiological temperatures, the lipid chains are mostly in a solid crystalline or gel state, which exhibits low lateral diffusional properties and is less permeable than the state of liquid crystalline membranes, which are present at higher temperatures. The link between skin disorders and changes in barrier lipid composition, especially in ceramides, is difficult to prove because of the many variables involved. However, most skin disorders that have a diminished barrier function present a decrease in total ceramide content with some differences in the ceramide pattern. Formulations containing lipids identical to those in skin and, in particular, some ceramide supplementation could improve disturbed skin conditions. Incomplete lipid mixtures yield abnormal lamellar body contents, and disorder intercellular lamellae, whereas complete lipid mixtures result in normal lamellar bodies and intercellular bilayers. The utilization of physiological lipids according to these parameters have potential as new forms of topical therapy for dermatoses. An alternative strategy to improving barrier function by topical application of the various mature lipid species is to enhance the natural lipid-synthetic capability of the epidermis through the topical delivery of lipid precursors.

The vesicle to micelle transition which results in the interaction of the Triton X-100 surfactant with phosphatidylcholine vesicles was studied by means of dynamic light scattering (at different reading angles) and by freeze-fracture electron microscopy techniques. Vesicle solubilization was produced by the direct formation of mixed micelles without the formation of complex intermediate aggregates. Thus, vesicle to micelle transformation was mainly governed by the progressive formation of mixed micelles within the bilayer. A subsequent separation of these micelles from the liposome surface (vesicle perforation by the formation of surfactant-stabilized holes on the vesicle surface) led to a complete solubilization of liposomes.z 1998 Federation of European Biochemical Societies.

The eosinophil cationic protein (ECP) is an antipathogen protein involved in the host defense system. ECP displays bactericidal and membrane lytic capacities [Carreras et al. (2003) Biochemistry 42, 6636-6644]. We have now characterized in detail the protein-membrane interaction process. All observed fluorescent parameters of the wild type and single-tryptophan-containing mutants, as well as the results of decomposition analysis of protein fluorescence, suggest that W10 and W35 belong to two distinct spectral classes I and III, respectively. Tryptophan residues were classified and assigned to distinct structural classes using statistical approaches based on the analysis of tryptophan microenvironment structural properties. W10 belongs to class I and is buried in a relative nonpolar, nonflexible protein environment, while W35 (class III) is fully exposed to free water molecules. Tryptophan solvent exposure and the depth of the protein insertion in the lipid bilayer were monitored by the degree of protein fluorescence quenching by KI and brominated phospholipids, respectively. Results indicate that W35 partially inserts into the lipid bilayer, whereas W10 does not. Further analysis by electron microscopy and dynamic light scattering indicates that ECP can destabilize and trigger lipid vesicle aggregation at a nanomolar concentration range, corresponding to about 1:1000 protein/lipid ratio. No significant leakage of the vesicle aqueous content takes place below that protein concentration threshold. The data are consistent with a membrane destabilization "carpet-like" mechanism.

The structural transition stages induced by the interaction of the non-ionic surfactant Triton X-100 on phosphatidylcholine unilamellar vesicles were studied by means of static and dynamic light-scattering, transmission-electron-microscopy (t.e.m.) and permeability changes. A linear correlation was observed between the effective surfactant/lipid molar ratios (Re) (‘three-stage’ model proposed for the vesicle solubilization) and the surfactant concentration throughout the process. However, this correlation was not noted for the partition coefficients of the surfactant between the bilayer and the aqueous medium (K). Thus a sharp initial K increase was observed until a maximum value was achieved for permeability alterations of 50% (initial step of bilayer saturation). Further surfactant additions resulted in a fall in the K values until 100% of bilayer permeability. Additional amounts of surfactant led to an increase in K until bilayer solubilization. Hence, a preferential incorporation of surfactant molecules into liposomes governs the initial interaction steps, leading to the initial stage of bilayer saturation with a free surfactant concentration that was lower than its critical micelle concentration (c.m.c.). Additional amounts of surfactant increased the free surfactant until the c.m.c. was reached, after which solubilization started to occur. Thus the initial step of bilayer saturation was achieved for a smaller surfactant concentration than that for the Resat, although this concentration was the minimum needed for solubilization to start. Large unilamellar vesicles began to form as the surfactant exceeded 15 mol% (50% bilayer permeability), the maximum vesicle growth being attained for 22 mol% (400 nm). Thereafter, static light-scattering started to decrease gradually, this fall being more pronounced after 40 mol%. The t.e.m. picture for 40 mol% (Resat.) showed unilamellar vesicles, although with traces of smaller structures. From 50 mol% the size distribution curves began to show a bimodal distribution. The t.e.m. pictures for 50-64 mol% revealed tubular structures, together with open bilayer fragments. Thereafter, increasing amounts of surfactant (65-69 mol%) led to planar multilayered structures which gradually tended to form concentric and helicoidal conformations. The scattered intensity decreased to a low constant value at more than 71-72 mol%. However, the surfactant concentration for the Re(sol) (72.6 mol %) still presented traces of aggregated structures, albeit with mono-modal size-distribution curves (particle size of 50 nm). This vesicle size corresponded to the liposome solubilization via mixed-micelle formation.

The transitional stages resulting in the interaction of sodium dodecyl sulfate (SDS)lphosphatidylcholine liposomes were studied by means of transmission electron microscopy (TEM), light scattering, and permeability changes. A linear correlation was observed between the surfactanfflipid molar ratio (Re) and the surfactant concentration throughout the process. However, the bilayerlaqueous phase partition coefficient (K) showed a maximum for 30% bilayer permeability (beginning of bilayer saturation). Hence, a preferential incorporation of surfactant molecules into liposomes governs the initial interaction steps, leading to the beginning of bilayer saturation with a free surfactant concentration that was lower than its critical micelle concentration (cmc). Additional surfactant amounts increased the free surfactant until the cmc is reached, after which solubilization started to occur. Large unilamellar vesicles began to form as the surfactant exceeded 10 mol % (20% permeability), the maximum vesicle growth being attained between 30 and 100% of permeability. A sharp decrease in static light scattering occurred after the Re for saturation (Resat), where TEM observations still showed vesicles although with traces of smaller structures. From 60 mol %the size curves began to show a bimodal distribution. TEM pictures for 60-68 mol % showed a gradual vesicle disintegration with formation of tubular structures. SDS concentrations greater than 74 mol % led to the solubilization of bilayers.

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The effect of dipalmitoyl phosphatidylcholine (DPPC)/dihexanoyl phosphatidylcholine (DHPC) bicelles on the microstructure of pig stratum corneum (SC) in vitro was evaluated. The physicochemical characterization of these nanoaggregates revealed small disks with diameters around 15 nm and a thickness of 5.4 nm. Upon dilution, the bicelles grow and transform into vesicles. Cryogenic scanning electron microscopy (cryo-SEM) images of the SC pieces treated with this system showed vesicles of about 200 nm and lamellar-like structures in the intercellular lipid areas. These vesicles probably resulted from the growth and molecular rearrangement of the DPPC/DHPC bicelles after penetrating the SC. The presence of lamellar-like structures is ascribed to the interaction of the lipids from bicelles with the SC lipids. The bicellar system used is suitable to penetrate the skin SC and to reinforce the intercellular lipid areas, constituting a promising tool for skin applications.

Attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy was applied to study the effects of the bicelles formed by dimyristoyl-glycero-phosphocholine (DMPC) and dihexanoyl-glycero-phosphocholine (DHPC) in porcine stratum corneum (SC) in vitro. A comparison of skin samples treated and untreated with bicelles at different temperatures was carried out. The analysis of variations after treatment in the position of the symmetric CH2 stretching, CH2 scissoring, and CH2 rocking vibrations reported important information about the effect of bicelles on the skin. Bicellar systems caused a phase transition from the gel or solid state to the liquid crystalline state in the lipid conformation of SC, reflecting the major order-disorder transition from hexagonally packed to disordered chains. Grazing incidence small and wide X-ray scattering (GISAXS and GIWAXS) techniques confirmed this effect of bicelles on the SC. These results are probably related to with the permeabilizing effect previously described for the DMPC/DHPC bicelles.