Use of high-pressure freeze fixation and freeze fracture electron microscopy to study the influence of the phospholipid molar ratio in the morphology and alignment of bicelles.

The high-pressure freeze fixation and freeze fracture electron microscopy techniques were combined with the (31)P nuclear magnetic resonance to study the morphological transitions of two different dimyristoyl-phosphatidilcholine/dihexanoyl-phosphocholine aggregates by the effect of temperature. Through these techniques, the relationship between magnetic alignment and the morphology of alignable and non-alignable aggregates was evaluated. The micrographs related to the non-alignable dimyristoyl-phosphatidilcholine/dihexanoyl-phosphocholine sample presented rounded objects at a temperature below the dimyristoyl-phosphatidilcholine phase transition (T(m)) and, above this temperature an increase of viscosity was followed by the appearance of large elongated aggregates. The micrographs related to the alignable dimyristoyl-phosphatidilcholine/dihexanoyl-phosphocholine sample presented discoidal objects below T(m). Above T(m), when the best alignment was achieved, the images showed large areas of lamellar stacked bilayers and the presence of some multilamellar vesicles. Our results reveal that the composition of the aggregates is a key factor determining the morphological transitions of the bicellar systems. Understanding of the rules governing these transitions is crucial to modulate characteristics of these systems and to adequate them for different applications.

Liposome formation with wool lipid extracts rich in ceramides.

Internal wool lipids (IWLs) are rich in cholesterol, free fatty acids, cholesteryl sulfate, and, mainly, ceramides. The repairing effect of these lipids structured as liposomes was demonstrated by reinforcing the skin-barrier integrity and increasing the water-holding capacity when applied onto the skin. This work was focused on the formation of liposomes with IWLs rich in ceramides, obtained at pilot plant level with organic solvent extraction by using methanol and acetone. The lipid composition of the two extracts was quantitatively analyzed. IWL extracts containing different amounts of sterol sulfate were used to form liposomes at physiologic p(H). Vesicle size distribution, polydispersity index, and zeta potential of all liposomes were determined to characterize them and to study their stability. The results obtained showed that IWL extract composition, which was different depending on the extraction methodologies used, greatly influences the characteristics of the liposomes formed. Vesicular size and polydispersity index liposomes were smaller when the extract composition contained a higher proportion of either free fatty acids or sterol sulfate. Moreover, liposome stability was improved when some amount of sterol sulfate was added to the composition of methanol and acetone extracts. This natural mixture with keratinaceous origin could have a special interest for cosmetic or dermopharmaceutical companies.

New arrangement of proteins and lipids in the stratum corneum cornified envelope.

A new arrangement of proteins and lipids of stratum corneum (SC) cornified envelope (CE) is proposed. The chemical analysis of CE revealed the presence of free fatty acids (FFA), ceramides (Cer), and important percentages of glutamic acid/glutamine (Glx) and serine (Ser) residues. The molecular structure of these components suggests the existence of covalent links not only between Cer and Glx but also between FFA and Ser. The protein distribution of extracellular surface of CE, i.e., the proteins that could be involved in the bonds with lipids, was studied using post- and pre-embedding immunolabeling electron microscopy. Some loricrin (protein rich in Ser) was detected in the outermost part of the CE protein layer. The external arrangement of some domains of this protein may give rise to form linkages with FFA, yielding further insight into the CE arrangement in which Cer-Glx bonds and FFA-Ser bonds would be involved. Although the importance of fatty acids in the cohesion and barrier function of SC has been widely demonstrated, their role could be associated not only to the presence of these lipids in the intercellular lamellae but also in the CE, in the same way that Cer.

Morphological effects of ceramide on DMPC/DHPC bicelles.

Freeze fracture electron microscopy and dynamic light scattering were applied to characterize the morphological changes of DMPC/DHPC bicellar systems induced by the addition of ceramides. The results demonstrate a tendency of the DMPC/DHPC aggregates to evolve forming elongated or tubular structures with the increase of the temperature. At 20 degrees C, low concentrations of ceramide promote the appearance of elongated structures with twisted zones. Higher concentrations of this lipid lead to the formation of liposomes along the elongated structures. The increase of the temperature to 40 degrees C induces the growth of the structures containing low concentrations of ceramide forming branched aggregates. In samples with high amounts of ceramide, the increase of temperature causes phase separation and the formation of a mixed system composed by liposomes and multilamellar tubules. The morphological effects induced by ceramides in this new membrane model give new insights for the role played by this lipid in biological membranes.

Effect of bicellar systems on skin properties.

Bicelles are discoidal aggregates formed by a flat dimyristoyl-glycero-phosphocholine (DMPC) bilayer, stabilized by a rim of dihexanoyl-glycero-phosphocholine (DHPC) in water. Given the structure, composition and the dimensions of these aggregates around 10-50 nm diameter, their use for topical applications is a promising strategy. This work evaluates the effect of DMPC/DHPC bicelles with molar ratio (2/1) on intact skin. Biophysical properties of the skin, such as transepidermal water loss (TEWL), elasticity, skin capacitance and irritation were measured in healthy skin in vivo. To study the effect of the bicellar systems on the microstructure of the stratum corneum (SC) in vitro, pieces of native tissue were treated with the aforementioned bicellar system and evaluated by freeze substitution applied to transmission electron microscopy (FSTEM). Our results show that bicelles increase the TEWL, the skin elastic parameters and, decrease skin hydration without promoting local signs of irritation and without affecting the SC lipid microstructure. Thus, a permeabilizing effect of bicelles on the skin takes place possibly due to the changes in the phase behaviour of the SC lipids by effect of phospholipids from bicelles.

Changes in phosphatidylcholine liposomes caused by a mixture of Triton X-100 and sodium dodecyl sulfate.

The mechanisms governing the interaction of equimolecular mixtures of Triton X-100 (Tx-100) and sodium dodecyl sulfate (SDS) with phosphatidylcholine liposomes were investigated. Permeability alterations were determined as a change in 5(6)-carboxyfluorescein released from the interior of vesicles and bilayer solubilization as a decrease in the static light-scattered by liposome suspensions. At subsolubilizing level, a maximum bilayer/water partitioning of surfactant mixture was reached at 30% CF release, which correlated with the increased presence of SDS in the bilayers. However, transition stages between 70% CF release and 100% light-scattering corresponded to the increased presence of Tx-100 in these structures. These findings may be correlated with the reduced deleterious effects caused by this mixture in different tissues versus pure SDS, given that the presence of Tx-100 may modulate the level of SDS partitioning in the human stratum corneum. At subsolubilizing level, the mixture showed higher affinity with bilayers than those reported for single components, whereas at solubilizing level this affinity was slightly lower and higher than those reported for Tx-100 and SDS respectively. A direct relationship was established in the initial interaction steps between the growth of vesicles, the leakage of entrapped CF and the effective molar ratio of surfactant to phospholipid in bilayers (Re). This dependence was also detected during solubilization, where the decrease in the vesicle size and in the scattered light of the system depended on the Re parameter and hence on the bilayer composition. The fact that the free surfactant concentration at subsolubilizing and solubilizing levels showed respectively lower and similar values than the critical micelle concentration (c.m.c.) of the surfactant mixture indicates that permeability alterations and solubilization were determined respectively by the action of surfactant monomer and by the formation of mixed micelles. This finding supports the generally admitted assumption, for single surfactants, that the concentration of free surfactant must reach the c.m.c. for solubilization to occur and highlights the influence of the negative synergism of this surfactant mixture on the free surfactant concentration needed to saturate or solubilize liposomes.

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.

Liposomes as alternative vehicles for sun filter formulations.

The aim of our study was to determine the influence of several types of liposomes with a different lipid composition on the percutaneous absorption of one conventional sun filter with a lipophilic character (ethyl hexyl methoxycinnamate) using both in vitro and in vivo methodologies. Three different liposomes were prepared with unsaturated and saturated phosphatidylcholine (PC, HPC), and with a wool lipid mixture (IWL) with a composition similar to that of the stratum corneum lipids. Results showed that the liquid crystalline state associated with PC liposomes plays a key role in enhancing skin penetration. when liposomes with a composition and structural organization similar to that of the stratum corneum lipids (HPC and IWL) are used, the skin penetration is retarded, suggesting a certain reinforcement of the stratum corneum barrier. These two types of liposomes could be regarded as alternatives to conventional oil/water emulsions in the formulations of lipidic sun filters. Finally, an acceptable correlation was obtained using both in vitro and in vivo methodologies to evaluate the corresponding skin absorption profile.

Bicelles: New Lipid Nanosystems for Dermatological Applications.

Bicelles have emerged as promising membrane models, and due to their attractive combination of lipid composition and physicochemical characteristics, they have become new nanostructures for biomedical research. Depending on the composition, temperature and other experimental factors, these nanosystems exhibit high structural and morphological versatility. Additionally, bicelles are able to modulate the biophysical parameters and barrier function of skin. Given these properties, these nanostructures appear to be smart nanosystems with great potential in biomedicine and dermopharmacy.

Direct formation of mixed micelles in the solubilization of phospholipid liposomes by Triton X-100.

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.

Biopolymer excreted by pseudoalteromonas antarctica NF(3), as a coating and protective agent of liposomes against dodecyl maltoside

The ability of an exopolymer of glycoproteic character (GP) excreted by a new gram-negative species Pseudoalteromonas antarctica NF(3), to coat phosphatidylcholine (PC) liposomes and to protect these bilayers against the action of the nonionic surfactant dodecyl maltoside was investigated. Transmission electron microscopy (TEM) micrographs of freeze fractured liposome/GP aggregates reveal that the addition of the glycoprotein to liposomes led to the formation of a film (polymer adsorbed onto the bilayers) that tightly coated PC bilayers. The complete coating was already achieved at a PC : GP weight ratio of about 9:1. Image analysis profiles of digitalized TEM micrographs (PC : GP weight ratio 8:2) show that this film was formed by a multilayer structure. The periods of the average distance of the pattern ordering in layer structures (9-10 layers) were of about 2-3 nm and the thickness of the complete film was of about 25 nm. Higher amounts of glycoprotein resulted in a growth of this film, which exhibited at the highest proportion of this compound (50% in weight) a multifilm structure. An increasing resistance of liposomes to be affected by dodecyl maltoside both at subsolubilizing and solubilizing levels occurred as the proportion of the glycoprotein in the system rose, although this protective effect was more effective at low proportions of this compound (PC : GP weight ratios from 9:1 to 8:2). Thus, although a direct dependence was found between the growth of the enveloping structure and the resistance of the coated liposomes to be affected by the surfactant, the more effective protection occurred when this structure was a thin film formed by the assembly of various layers of GP of about 2-3 nm. Copyright 1999 John Wiley & Sons, Inc.

Transmission electron microscopy and light scattering studies on the interaction of a nonionic/anionic surfactant mixture with phosphatidylcholine liposomes.

The interaction of an equimolecular mixture of nonylphenol polyethoxylated [NP(EO)10] and sodium dodecyl sulfate (SDS) surfactants with phosphatidylcholine (PC) liposomes was studied by means of transmission electron microscopy (TEM) and changes in the mean particle size (quasielastic light scattering; QELS) and in the static light scattering (SLS) of the system during liposome solubilization. A good correlation was found between the TEM diameter of particles and the mean hydrodynamic diameter (HD) determined by QELS. The aggregates resulting in this interaction were dependent on the surfactant concentration in the system. Thus, an initial vesicle growth occurred when the surfactant concentration was 15.98 mol%, together with the formation of a very small percentage of smaller particles. Additional surfactant amounts (28.32 mol%) led first to the formation of largest vesicles (HD 418 nm) and second to a fall in the vesicle diameter and in the SLS of the system. Thus, for 38.27 mol%, the TEM picture still showed the presence of vesicles, albeit with signs of disintegration. When additional amounts of surfactant were added to the system, the size curve started to show a bimodal distribution. Thus, for 51.81 mol% surfactant concentration, a sharp curve appeared at 51 nm, corresponding to the formation of small particles and TEM pictures clearly showed vesicle disintegration with formation of tubular structures. It is noteworthy that additional surfactant amounts (from 52 to 60 mol%) led to the formation of unclosed multilayered structures together with smaller aggregates. The gradual increase in the proportion of these smaller aggregates (mixed micelles) led to the complete solubilization of liposomes.

Subsolubilizing alterations caused by alkyl glucosides in phosphatidylcholine liposomes.

The subsolubilizing alterations caused by a series of alkyl glucosides (alkyl chain lengths ranging from C8 to C12) in unilamellar phosphatidylcholine (PC) liposomes were investigated. The surfactant to phospholipid molar ratios (RE) and the normalized bilayer/aqueous phase partition coefficients (K) were determined by monitoring the increase of the fluorescence intensity of liposome suspensions due to the 5(6)-carboxyfluorescein (CF) released from the interior of vesicles to the bulk aqueous phase. Given that the free surfactant concentrations was always lower than the critical micelle concentration (CMC) of the surfactant tested we may assume that the surfactant-liposome interactions were mainly ruled by the action of surfactant monomers. In general terms, the decrease in the surfactant alkyl chain length (or the rise in the surfactant CMC) resulted in an increase in the ability of these surfactants to alter the permeability of liposomes and, inversely, in an abrupt decrease in their affinity with these bilayers structures. The overall balance of these opposite tendencies shows that at the two interaction levels studied (50 and 100% of CF release) the nonyl and the octyl glucoside showed, respectively, the highest ability to alter the release of the CF trapped in bilayers (lowest RE values), whereas the dodecyl glucoside showed the highest degree of partitioning into liposomes or affinity with these bilayer structures (highest K values).

Influence of cholesterol on liposome fluidity by EPR. Relationship with percutaneous absorption.

The influence of liposome composition on bilayer fluidity and its effect on the percutaneous absorption into the skin were investigated. Liposomes formed with saturated or unsaturated phospholipids (H-PC or PC) with varying amounts of cholesterol were prepared and their penetration behaviour into the stratum corneum was followed up by means of the stripping method. The order and dynamics of the hydrophobic domain of the vesicles were studied using electron paramagnetic resonance (EPR) methodology. Phospholipid composition and the amount of cholesterol exert a considerable influence on the penetration behaviour of the probe encapsulated in the liposomes. This behaviour is closely related to the fluidity characteristics of these liposomes studied by EPR. Therefore, a penetration mechanism of the vesicles into the skin, based on the incorporation of lipids into the skin lipids and on fluidity behaviour, is suggested.

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.

Solubilizing effects caused by alkyl pyridinium surfactants in phosphatidylcholine liposomes.

The solubilization of neutral and electrically charged liposomes by a series of alkyl pyridinium surfactants (alkyl chain lengths C10-C14) was investigated. Solubilization was detected as a decrease in static light-scattering of liposome suspensions. Two parameters were regarded as corresponding to the effective surfactant/lipid molar ratios at which the surfactant saturated the liposomes Re(sat) and led to a complete solubilization of these structures Re(sol). From these parameters the corresponding surfactant partition coefficients were determined. The Re and K parameters fell as the surfactant alkyl chain length decreased or both the critical micellar concentration (CMC) and the hydrophilic/lipophilic balance (HLB number) increased, regardless of the bilayers electrical charge. Thus, although decyl-pyridinium bromide (DePB) showed the highest ability for saturation and solubilization of bilayers, its concentration was always higher than that needed for dodecyl-pyridinium bromide (DoPB) and tetradecyl-pyridinium bromide (TePB), the last one being the most active. These results emphasize the influence of the hydrophilic/lipophilic balance of these surfactants on liposome solubilization and the minor influence of the electrostatic factors in this process.

Solubilization of phosphatidylcholine liposomes by the amphoteric surfactant dodecyl betaine.

The interaction of the amphoteric surfactant N-dodecyl-N,N-dimethylbetaine (C12-Bet) with phosphatidylcholine (PC) liposomes was investigated. Permeability alterations were detected as a change in 5(6)-carboxyfluorescein (CF) released from the interior of vesicles and bilayer solubilization as a decrease in the static light-scattering (SLS) of the system. At sublytic level a initial maximum in the bilayer/water partitioning (K) followed by an abrupt decrease of this parameter occurred as the surfactant to lipid molar ratio (Re) rose. At lytic level a direct dependence was established between both parameters. The fact that the free surfactant concentration at sublytic and lytic levels showed values lower than and similar to its critical micelle concentration indicates that permeability alterations and solubilization were determined, respectively, by the action of surfactant monomer and by the formation of mixed micelles. A direct correlation occurred in the initial interaction steps (up to 50% CF release) between the growth of vesicles their fluidity and Re. A similar direct dependence was established during solubilization (up to 30% SLS) between the fall in both the surfactant-lipid aggregate size, the SLS of the system and Re. This surfactant showed higher capacity to solubilize PC liposomes than that reported by the commonly used non-ionic surfactants octyl glucoside and Triton X-100 and by the anionic one sodium dodecyl sulfate.

Influence of the temperature in the adsorption of sodium dodecyl sulfate on phosphatidylcholine liposomes.

The influence of the temperature on the adsorption of monomeric and micellar solutions of the anionic surfactant sodium dodecyl sulfate (SDS) on phosphatidylcholine (PC) liposomes was investigated using the fluorescent probe 2-(p-toluidinyl)-naphthalene-6-sodium sulfonate (TNS). The number of adsorbed molecules was quantified by measuring changes in the electrostatic potential (Psi(o)) of the liposomes/probe during an incubation with SDS at varying temperatures. At low surfactant concentrations (from 0.05 to 0.25 mM), the increase in temperature reduced the number of surfactant molecules incorporated per vesicle regardless of the incubation time, whereas at high surfactant concentrations (from 0.50 to 1.0 mM) the incubation time has an opposite effect on this process. Thus, after 10s, the surfactant adsorption decreased with temperature, yet it increased progressively with time. The adsorption was linear with temperature below critical micellar concentration (CMC) of SDS and this linear tendency did not change above CMC. This suggests an adsorption of SDS monomers regardless of the surfactant concentration.

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.

Use of a fluorescence spectroscopy technique to study the adsorption of sodium dodecylsulfonate on liposomes.

The fluorescent probe 2-(p-toluidinyl)-naphthalene-6-sodium sulfonate was used to study the surface adsorption of sublytic concentrations of the anionic surfactant sodium dodecylsulfonate (C(12)-SO(3)) on phosphatidylcholine bilayers. The number of adsorbed molecules was quantified by determination of the electrostatic potential (psi(0)) of the bilayers. The abrupt decrease in the fluorescence intensity already detected 10 s after the surfactant addition and the slight fluorescence variations with time indicated that the surfactant adsorption was very fast and almost complete. For a given number of monomers adsorbed, a linear dependence between the lipid and C(12)-SO(3) concentrations was obtained, indicating a similar adsorption mechanism regardless of the surfactant concentration. Hence, a monomeric adsorption is assumed even in systems with a C(12)-SO(3) concentration above its critical micellar concentration (CMC). In addition, this linear correlation allowed us to determine the surfactant/lipid molar ratios (Re) (inversely related to the C(12)-SO(3) ability to be adsorbed on liposomes) and the bilayer/aqueous phase coefficients (K). The fact that the lowest values for Re were always reached after 10 s of incubation corroborates the rapid kinetic of the process. The decrease in the C(12)-SO(3) partitioning (K) when the number of surfactant molecules exceeded 15000 was possibly due to the electrostatic repulsion between the free and the adsorbed monomers, which could hinder the incorporation of new monomers on the charged surface of liposomes.