Ceramide-induced alterations in the lateral organization of membrane proteins can be involved in several biological contexts, ranging from apoptosis to viral infections. In order to investigate such alterations in a simple model, we used a combined approach of atomic force microscopy, scanning fluorescence correlation spectroscopy and confocal fluorescence imaging to study the partitioning of different membrane components in sphingomyelin/dioleoyl-phosphatidylcholine/cholesterol/ceramide supported bilayers. Such model membranes exhibit coexistence of liquid-disordered, liquid-ordered (raft-like) and ceramide-rich lipid phases. Our results show that components with poor affinity toward the liquid-ordered phase, such as several fluorescent lipid analogues or the synaptic protein Synaptobrevin 2, are excluded from ceramide-rich domains. Conversely, we show for the first time that the raft-associated protein placental alkaline phosphatase (GPI-PLAP) and the ganglioside GM1 are enriched in such domains, while exhibiting a strong decrease in lateral diffusion. Analogue modulation of the local concentration and dynamics of membrane proteins/receptors by ceramide can be of crucial importance for the biological functions of cell membranes.

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In mixed monolayers with ganglioside GM1, ceramide induces a non-ideal increase of the monolayer collapse pressure, a reduction of the mean molecular area and a decrease of the surface potential per molecule at all surface pressures. The critical packing parameter and van der Waals interaction energy calculated from monolayer data predict the transduction of changes from the molecular to the supramolecular level, such as formation of bilayers and possible subsequent facilitation of non-bilayer structures as the ceramide concentration increases, along with a greater thermal stability of the lipid structures. In agreement with the expectations from monolayer data, calorimetry, dynamic light scattering and electron microscopy data reveal the actual presence of phases with high phase-transition temperatures; at about 5 mol% ceramide in the mixture, the aggregates change their topology from micelles to multilamellar vesicles of increasing size and finally to long, thin tubules as the amount of ceramide in the system increases.

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The adequate formulation of topical vehicles to treat skin diseases is particularly complex. A desirable formulation should enhance the accumulation of the active drugs in the target tissue (the skin), while avoiding the penetration enhancement to be so large that the drugs reach the systemic circulation in toxic amounts. We have evaluated the transcutaneous penetration of three drugs chosen for their widely variable physicochemical properties: Amphotericin B, Imiquimod and Indole. We incorporated the drugs in fluid or ultra-flexible liposomes. Ultra-flexible liposomes produced enhancement of drug penetration into/through human skin in all cases in comparison with fluid liposomes without detergent, regardless of drug molecular weight. At the same time, our results indicate that liposomes can impede the transcutaneous penetration of molecules, in particular small ones.

An important part of natural ceramides contain asymmetric hydrocarbon chains. We have used calorimetry, atomic force microscopy, and electron paramagnetic resonance to study the effect of ceramide chain asymmetry in mixtures of C8Cer with DMPC as a model system of hydrocarbon chain disparity. A phase diagram is provided along with information on the thickness of the membrane and the mobility of the chains at different temperatures both below and above the phase transition temperature of the mixtures. The results indicate a partial interdigitation of C8Cer chains in the gel phase, producing a correlation between the organization of both hemilayers. Our data suggest that the effects of ceramides on biomembranes may be bimodal and similar to those of cholesterol.

In mixtures with dipalmitoylphosphatidylcholine, ceramide induces broadening of the calorimetric main phase transition that could be deconvoluted into at least three components: the first represents isothermal melting of a phosphatidylcholine-enriched phase; the second and third represent phases with increasing proportions of ceramide melting at progressively higher temperatures. The partial phase diagram (up to 40 mole % ceramide) indicates complete or partial gel-phase immiscibility, and complete gel-and liquid-phase miscibility depending on the ceramide content. Cluster distribution function analysis of each individual transition reveals decreased cooperativity and domain size with increased amounts of ceramide. Compared to individual lipids, mixed monolayers with dipalmitoylphosphatidylcholine show unchanged mean molecular areas or slight expansions at 24 ؇ C with dipole potentials exhibiting hyperpolarization; by contrast, already at 27 ؇ C the mean molecular areas become condensed and dipole potentials show little changes or are slightly depolarized. This suggests that favorable ceramide-phosphatidylcholine dipolar matching in the liquid state can be one of the local determinants for close molecular interactions while unfavorable matching may explain lateral domain segregation of ceramideenriched gel phases. The changes are detected at relatively low proportions of Cer (1-12 mole %) which are comparable to variations of Cer levels in membranes of cultured cells undergoing functional responses mediated by the sphingomyelin signaling pathway. -Carrer, D. C., and B. Maggio. Phase behavior and molecular interactions in mixtures of ceramide with dipalmitoylphosphatidylcholine. J.

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