Vesicular nanostructures composed of oleic acid and phosphatidylcholine: Effect of pH and molar ratio.

Phospholipids and fatty acids are the main building blocks of biological membranes. Oleic acid is a monounsaturated omega-9 fatty acid commonly found in many natural sources. Its characteristic kinked structure grants this molecule with a great number of biological properties. To better understand the role that this kind of fatty acids play into phospholipid membranes, nanostructured systems formed with hydrogenated soy phosphatidylcholine and oleic acid were studied in this work by means of cryo-electron microscopy, dynamic light scattering and differential scanning calorimetry. Differences concerning size, morphology and phase behavior were found when those systems were prepared at different conditions of pH and molar ratio between both compounds. Broadly, it was seen how alkaline mediums and high proportions of oleic acid reduced the size of the structures and increased the fluidity of the membranes. The ease of preparation of these lipid systems, and the response to pH suggests a future use of these systems as model membranes or delivery systems.

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.

Bicellar systems as vehicle for the treatment of impaired skin.

This study assesses the potential usefulness of bicellar systems to retard the penetration of drugs into damaged skin. The active compound used in this study was diclofenac diethylamine (DDEA). Initially, physicochemical characterisation of the DDEA bicellar systems was performed at different temperatures by small-angle X-ray scattering (SAXS), wide-angle X-ray scattering (WAXS) and differential scanning calorimetry (DSC) techniques. Subsequently, in vitro percutaneous absorption of bicellar systems into in vitro damaged skin was studied. SAXS results indicated a slight decrease in the width of their bilayers with increasing temperature, with no apparent stacking in those systems. WAXS patterns were compatible with an orthorhombic lateral packing of the nanoaggregates. The thermogram obtained by DSC indicated a decrease in gel-to-liquid crystalline transition temperature (Tm) when the drug was included into bicellar systems. A retardation effect for DDEA was detected by in vitro percutaneous absorption studies when DDEA was vehiculised in the bicellar systems with respect to an aqueous solution of the drug. It seems that the use of bicellar systems as a vehicle for topical application of DDEA on skin with an impaired barrier function may inhibit the penetration of DDEA to the systemic level. Such systems may consequently repair stratum corneum barrier function to some extent. The use of these systems could be considered a new alternative strategy to treat topically pathological skin with different drugs.

Bicellar systems as new delivery strategy for topical application of flufenamic acid.

In this work, bicellar systems, bilayered disc-shaped nanoaggregates formed in water by phospholipids, are proposed as a novel strategy for delivery of the anti-inflammatory flufenamic acid (FFA) to the skin. A comparative percutaneous penetration study of this drug in bicellar systems and other vehicles was conducted. The effects induced on the skin by the application of FFA in the different vehicles were analyzed by attenuated total reflectance-fourier transform infrared (ATR-FTIR). Additionally, using the microscopic technique freeze-substitution transmission electron microscopy (FSTEM) and X-ray scattering technique using synchrotron radiation (SAXS-SR), we studied the possible microstructural and organizational changes that were induced in the stratum corneum (SC) lipids and the collagen of the skin by the application of FFA bicellar systems. Bicellar systems exhibited a retarder effect on the percutaneous absorption of FFA with respect to the other vehicles without promoting disruption in the SC barrier function of the skin. Given that skin disruption is one of the main effects caused by inflammation, prevention of disruption and repair of the skin microstructure should be prioritized in anti-inflammatory formulations.

Bicellar systems as a new colloidal delivery strategy for skin.

The presented work evaluates the use of bicellar systems as new delivery vectors for controlled release of compounds through the skin. Two different active principles were introduced into the bicellar systems: diclofenac diethylamine (DDEA) and flufenamic acid (Ffa). Bicellar systems are discoidal aggregates formed by long and short alkyl chain phospholipids. Characterization of the bicellar systems by dynamic light scattering (DLS) and cryogenic transmission electron microscopy (Cryo-TEM) showed that particle size decreased when DDEA was encapsulated and increased when Ffa was included in the bicellar systems. Percutaneous absorption studies demonstrated a lower penetration of DDEA and Ffa through the skin when the drugs were included in the bicellar systems than when the drugs were applied in an aqueous solution (DDEA) and in an ethanolic solution (Ffa); the reduction in penetration was more pronounced with Ffa. These bicellar systems may have retardant effects on percutaneous absorption, which result in a promising strategy for future drug or cosmetic delivery applications.

Bicellar systems as modifiers of skin lipid structure.

The characterization of different bicellar aggregates and the effects of these systems on the stratum corneum (SC) microstructure have been studied. Dynamic light scattering (DLS) and freeze fracture electron microscopy (FFEM) techniques showed that both of the systems studied, dimyristoyl-phosphatidylcholine/dihexanoyl-phosphocholine (DMPC/DHPC) and dipalmitoyl-phosphocholine (DPPC)/DHPC, were formed by small discoidal aggregates at room temperature (20°C). Treating skin with DMPC/DHPC bicelles does not affect the SC lipid microstructure, whereas bicellar systems formed by DPPC and DHPC can promote the formation of new structures in the SC lipid domains. This indicates the passage of lipids from bicelles through the SC layers and also a possible interaction of these lipids with the SC lipids. Given the absence of surfactant in the bicellar composition and the small size of these structures, the use of these smart nano-systems offers great advantages over other lipid systems for dermatological purposes. Bicelles could be promising applications as drug carriers through the skin. This contribution, based on the new biological use of bicelles, may be useful to scientists engaged in colloid science and offers a new tool for different applications in skin and cosmetic research.

Lipid nanostructures: self-assembly and effect on skin properties.

This work evaluates the relation between the composition and the self-assembly of some lipid aggregates with their effects on the skin. To this end, liposomes, bicelles and micelles formed by dipalmitoylphosphatidylcholine (DPPC), dimyristoylphosphatidylcholine (DMPC) and dihexanoylphosphatidylcholine (DHPC) were characterized by electron microscopy and dynamic light scattering techniques, and applied on the skin. The results revealed that nanostructures with similar assembly but different composition caused different effects on the skin parameters. In general, samples containing DMPC affected the barrier function to a greater extent than systems containing DPPC. Additionally, our results showed that samples with the same lipid composition but different assembly exerted different effects on the skin. Liposomes decreased or did not modify the transepidermal water loss (TEWL), while bicelles and micelles increased this parameter. Hydration of the skin diminished especially after the application of micellar and bicellar samples. In vitro experiments showed structures like vesicles inside cutaneous SC (stratum corneum) incubated with DPPC/DHPC bicelles. These structures were not detected in SC samples incubated with DMPC/DHPC bicelles probably due to the different thermotropic behavior of DMPC and DPPC at physiological temperatures. Results reported in this work should be considered in terms of design of more efficient and specific skin delivery systems.

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.

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.

Flare up to betalactams.

BACKGROUND: The flare up phenomenon has most frequently been described with nickel. Not many cases of flare up to drugs have reported in the literature, however we have reported it with different medications. METHODS AND RESULTS: A 31-year-old woman developed an adverse reaction with an antibiotic during her childhood. Prick test with penicillin (100,000 IU/ml), penicilloyl polylysine (PPL), minor determinant mixture (MDM), amoxicillin (200 mg/ml), ampicillin (200 mg/ml) and cephalotin (200 mg/ml), and intradermal test to the same substances diluted in saline were all negative immediately. We performed an oral challenge test with 500 mg of amoxicillin. Twelve hours later, the intradermal test to PPL and MDM became positive (PPL 10 x 10 mm, MDM 8 x 7 mm). All patch tests were positive after 72 hours with erythema, vesicles and infiltration and the patient also had exanthema with pruritus on her entire body. CONCLUSIONS: We present one patient with delayed allergic reaction caused by amoxicillin and penicillin, that we all know as Flare up. We suggest that this phenomenon of Flare up occurs by a Type IV mechanism mediated by T-cells without participation of IgE antibodies. The betalactam hypersensitivity mechanism which has usually been described is an IgE mediated reaction, but there are other not very well known mechanisms that are responsible for the delayed reactions.

Influence of chemical and freezing fixation methods in the freeze-fracture of stratum corneum.

A comparison between two fixation techniques for freeze-fracture was established. Stratum corneum (SC) samples from pig epidermis were fixed using high-pressure freezing (HPF) and using plunging in propane freezing; the latter after chemical fixation. Then, frozen samples were freeze-fractured, coated with platinum-carbon, and visualized using a high-resolution low-temperature scanning electron microscope and a transmission electron microscope. Our results indicate that the plane of freeze-fracture was different depending on the fixation and freezing methodology used. In the samples frozen by HPF without chemical fixation, the fracture plane laid mainly between the lipid lamellae. However, when chemical fixation and plunging in propane freezing was used, the fracture plane did not show preference to a specific way. Plunging in propane freezing of chemically fixed samples, on the other hand, provides a more homogeneous fracture behaviour. Thus, depending on the methodology used, we can favour a visualization of either lipid or protein domains of the SC. These results could be very useful in future ultrastructural studies in order to facilitate the microscopic visualization and interpretation of the complex images such as those of SC and even of other samples in which different domains coexist.

Effect of the electrostatic charge on the mechanism inducing liposome solubilization: a kinetic study by synchrotron radiation SAXS.

The anionic surfactant sodium dodecyl sulfate (SDS) was used to induce the initial steps of the solubilization of liposomes. The structural transformations as well as the kinetics associated with this initial period were studied by means of time-resolved small-angle X-ray scattering (SAXS) using a synchrotron radiation source. Neutral and electrically charged (anionic and cationic) liposomes were used to investigate the effect of the electrostatic charges on the kinetics of these initial steps. The mechanism that induces the solubilization process consisted of adsorption of surfactant on the bilayers and desorption of mixed micelles from the liposomes surface to the aqueous medium. In all cases the time needed for desorption of the first mixed micelles was shorter than that for complete adsorption of the surfactant on the liposomes surface. The present work demonstrates that adsorption of the SDS molecules on negatively charged liposomes was slower and release of mixed micelles from the surface of these liposomes was faster than for neutral liposomes. In contrast, in the case of positively charged liposomes, the adsorption and release processes were, respectively, faster and slower than those for neutral vesicles.

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.

Assembly properties and applications of a new exopolymeric compound excreted by Pseudoalteromonas antarctica NF3.

The self assembly properties and applications of an exopolymeric compound (EC) of a glycoprotein character excreted by a new gram-negative species, Pseudoalteromonas antarctica NF3, have been reviewed. This compound exhibited surface-active properties in water, with a concentration of 0.20 mg ml(-1) being the key value associated with its physicochemical properties. Unsonicated EC aqueous dispersions showed the coexistence of concentric multilamellar and small unilamellar aggregates by transmission electron microscopy (TEM). Sonication of these dispersions revealed that each lamellae of the initial multilamellar structures were made up of various subunits coiled coils. As for the ability of this exopolymeric biomaterial to coat phosphatidylcholine (PC) liposomes and to protect these vesicles against different surfactants, freeze-fracture TEM micrographs of liposome/EC aggregates revealed that the addition of the EC to liposomes led to the formation of a film (polymer adsorbed onto the bilayers) that coated very well the PC bilayers. The complete coating was already achieved at a PC:EC weight ratio of about 9:1. An increasing resistance of PC liposomes to surfactants (in particular sodium dodecyl sulfate) occurred as the proportion of EC in the system rose, although this effect was more effective at low EC proportions (PC:EC weight ratios from 9:1 to 8:2). 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 surfactants, the best protection occurred when this structure was a thin film of about 20-25 nm formed by nine to ten layers of about 2-3 nm.

Partitioning of SDS in liposomes coated by the exopolymer excreted by Pseudoalteromonas antarctica NF3 as a measure of vesicle protection against this surfactant.

The capacity of glycoprotein (GP) excreted by Pseudoalteromonas antarctica NF3, to protect phosphatidylcholine (PC) liposomes against the action of the anionic surfactant sodium dodecyl sulfate (SDS) was studied in detail. To this end, changes in the surfactant partitioning between the lipid bilayer and the aqueous phase (partition coefficients, K) and in the effective surfactant to PC molar ratios (Re) were determined as a function of the amount of GP assembled with liposomes. The permeability of liposomes was determined by monitoring the changes in the fluorescence intensity of liposomes due to the release of the fluorescent dye 5(6)-carboxyfluorescein (CF) from the interior of vesicles to the bulk aqueous phase. Increasing GP amounts in the system resulted in the same interaction step as a rise in Re and a fall in the surfactant partitioning between the lipid bilayer and water. Hence, the higher the proportion of GP, the lower the surfactant ability to alter the permeability of liposomes and the lower its affinity with these bilayer structures. In addition, increasing GP proportions resulted in the same interaction step as a progressive increase of the free surfactant concentration (S(W)). The fact that the S(W) was always lower than the surfactant critical micelle concentration indicates that the interaction of SDS with coated liposomes was mainly ruled by the action of surfactant monomers in all cases.

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.

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 (TNS) was used to study the surface adsorption of sublytic concentrations of the anionic surfactant sodium dodecylsulfonate (C(12)-SO(3)) on phosphatidylcholine (PC) bilayers. The number of adsorbed molecules was quantified by determination of the electrostatic potential (psi(o)) of the bilayers. The abrupt decrease in the fluorescence intensity detected even 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 C12-SO3 concentrations was obtained, indicating similar adsorption mechanism regardless of the surfactant concentration. Hence, a monomeric adsorption is assumed even in systems with a C12-SO3 concentration above its CMC. In addition, this linear correlation allowed us to determine the surfactant/lipid molar ratios (Re) (inversely related to the C12-SO3 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 kinetics of the process. The decrease in the C12-SO3 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.

Liposomes as protective agents of stratum corneum against octyl glucoside: a study based on high-resolution, low-temperature scanning electron microscopy.

The ability of phosphatidylcholine (PC) liposomes to protect pig stratum corneum (SC) against the action of the nonionic surfactant octyl glucoside (OG) was investigated "in vitro" using double-layer coating for high-resolution, low-temperature scanning electron microscopy. This technique has been useful in preventing drying artifacts in the study of biological materials. The treatment of SC with OG led to a perturbation mainly in the corneocytes. However, the incubation of the tissue with liposomes prior to the OG treatment resulted in a progressive decrease in these perturbations and, consequently, in the progressive protection of the SC against the action of the surfactant.

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.

Protective effect caused by the exopolymer excreted by Pseudoalteromonas antarctica NF(3) on liposomes against the action of octyl glucoside.

The capacity of the glycoprotein (GP) excreted by Pseudoalteromonas antarctica NF(3), to protect phosphatidylcholine (PC) liposomes against the action of octyl glucoside (OG) was studied in detail. Increasing amounts of GP assembled with liposomes resulted for the same interaction step in a linear increase in the effective surfactant to PC molar ratios (Re) and in a linear fall in the surfactant partitioning between bilayer and the aqueous phase (partition coefficients K). Thus, the higher the proportion of GP assembled with liposomes the lower the surfactant ability to alter the permeability of vesicles and the lower its affinity with these bilayer structures. In addition, increasing GP proportions resulted in a progressive increase of the free surfactant concentration (S(W)) needed to produce the same alterations in liposomes. The fact that S(W) was always lower than the surfactant critical micelle concentration indicates that the interaction was mainly ruled by the action of surfactant monomers, regardless of the amount of assembled GP.