J. L. Parra

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.

EEMCO Guidance for the in vivo Assessment of Skin Surface pH

The pH of the skin follows a sharp gradient across the stratum corneum (SC), which is suspected to play an important role in controlling the enzymatic activities involved in cellular metabolism and renewal. This gradient is maintained by several systems, such as sweat and sebum secretion and degradation as well as cellular metabolism. At the surface of the skin, what is measured is in fact an apparent skin pH due to extracted material from the SC diffusing into water applied at the surface. pH values recorded at the surface of a semi hydrophobic milieu such as the SC should be interpreted with great caution because it is obvious that hydrogen ions are not in a pure solution at the surface of the skin. For a correct measurement of skin surface pH, it is recommended to follow all practical operating conditions. Care must be taken in identifying the skin site, healthy controls (age, gender, skin type), the time of day of the measurement and the environmental conditions. Also, subjects should receive precise instructions before the test, mainly in terms of hygiene procedure or use of topical products. The interpretation of data should not overlook the fact that even small differences in pH may reflect significant modifications at the molecular level. Although it is usually agreed that the pH of the skin surface may influence the cutaneous microflora, much remains to be learnt about the role of the acid mantle of the skin with regard to defensins and other protective mechanisms.

Efficacy of stratum corneum lipid supplementation on human skin

Recent studies suggest that supplementing intercellular lipids of the stratum corneum in ageing populations or in people with dry skin can stimulate the functioning of the skin. This work lends support to the reinforcement capacity of two different stratum corneum lipid mixtures (synthetic stratum corneum lipid mixtures, SSCL, and internal wool lipid extracts, IWL) formulated as liposomes on healthy skin of two differently aged groups of individuals. Protection of healthy skin against detergent‐induced dermatitis was evaluated. Transepidermal water loss and capacitance were used to evaluate the effect of these formulations in in vivo long‐term studies. Increase in water‐holding capacity is obtained only when the formulations applied are structured as liposomes. This is slightly more pronounced for aged skin. Subsequent SLS exposure reflected the protection of healthy human skin against detergent‐induced dermatitis. Slightly better results were obtained with IWL containing a mixture of natural ceramides than with SSCL with only one ceramide present in the formulation. All these results support the beneficial effects of skin lipid supplementation given their resemblance to the lipids in the stratum corneum both in composition and in the structuring of the formulation.

Chromatographic characterization of internal polar lipids from wool

Wool internal polar lipids were isolated and separated into different fractions based on polarity. Qualitative and quantitative analyses of the different fractions were performed by thin-layer chromatography and thin-layer chromatography coupled to flame-ionization detection, respectively. Cholesterol esters, free fatty acids, sterols, ceramides, glycosylceramides, and cholesterol sulfate were the main components, with ceramides being in the highest proportion. The fatty acid composition of ceramides and glycosylceramides was determined by gas chromatography/mass spectrometry. As for other keratinized tissues, long-chain fatty acids predominated in comparison to either free fatty acids or phospholipid-linked fatty acids; in both cases, stearic and lignoceric acids were the most abundant fatty acids, and a low amount of 18-methyleicosanoic acid was found. This work opens new avenues in the study of lipid rearrangement in more complex and realistic vesicle structures than conventional liposomes.

Solubilizing effects caused by the nonionic surfactant dodecylmaltoside in phosphatidylcholine liposomes

The interaction of the nonionic surfactant dodecylmaltoside (DM) with phosphatidylcholine liposomes was investigated. Permeability alterations were detected 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. This surfactant showed higher capacity to saturate and solubilize PC liposomes and greater affinity with these structures than those reported for the octyl glucoside. At subsolubilizing level an initial maximum in the bilayer/water partitioning (K) followed by an abrupt decrease of this parameter occurred as the effective molar ratio of surfactant to phospholipid in bilayers (Re) rose. However, at solubilizing level a direct dependence was established between both parameters. A direct correlation took place in the initial interaction steps (Re up to 0.28) between the growth of vesicles, their fluidity, and Re. A similar direct dependence was established during solubilization (Re range from 0.9 to 1.7) between the decrease in both the surfactant-PC aggregate size, the light scattering of the system, and Re (composition of aggregates). The fact that the free DM concentration at subsolubilizing and solubilizing 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.

The formation of liposomes in vitro by mixtures of lipids modeling the composition of the stratum corneum

Abstract The influence of different synthetic lipid mixtures approximating the composition of stratum corneum on the formation and physicochemical properties of liposomes was investigated with the aim of characterizing these structures. To this end, a lipid mixture containing ceramide (40%), cholesterol (25%), palmitic acid (25%) and cholesteryl sulfate (10%) was chosen as a central composition of the optimizing Box and Behnken experimental model for three variables. From these compositions, the optimizing lipid percentage range was varied for each lipid ±15%, except for the cholesteryl sulfate in which case the variation was extended to ±100%. Liposomes were prepared by sonication at 63 °C in a buffered medium (100 mM NaCl, 5 mM TRIS) at pH 7.5 and supplemented in some cases with 10 mM of 5(6)-carboxyfluorescein. The vesicle size distribution of liposome suspensions (nm) was determined by photon correlation spectrometry, whereas the internal volume (ml per mmol lipid) was determined by spectrofluorometry. Transmission electron microscopy (TEM) reveals the presence of unilamellar liposomes in all the lipid mixtures investigated. With regard to the physicochemical properties of the systems, increasing concentrations of ceramide, palmitic acid and cholesterol (or low cholesteryl sulfate concentrations) result in a decrease in both the size of the vesicles and the internal volume of these structures. This direct correlation confirms the formation of similar bilayer structures in all cases.

Percutaneous penetration of liposomes using the tape stripping technique

The reservoir capacity of the stratum corneum was studied by topical application of sodium fluorescein encapsulated in vesicles in order to elucidate the mechanism involved in the (trans)dermal transport of drugs when vesicles are applied to the skin. The penetration profile of sodium fluorescein in the different strips was found to be logarithmic with both the constant and the slope of the regression curves, accounting for the superficial non-penetration content and for the penetration rate inside the stratum corneum, respectively. The results show a small but significantly enhanced penetration of these vesicle structures for the release of hydrophilic substances. Moreover, similar values obtained when the same liposomes with a varying encapsulation content were applied could lend support to the penetration mechanism where the vesicle enhancement is mainly due to stratum corneum structural modification.

Permeability investigations of phospholipid liposomes by adding cholesterol

Abstract The sublytic changes caused by sodium dodecyl sulfate (SDS) in egg phosphatidylcholine (PC) liposomes with rising concentrations of cholesterol (CH) (PC:CH mole ratios from 9:1 to 7:3) were investigated. The effective molar ratios Re of membrane-bound surfactant to lipid and the bilayer/aqueous phase partition coefficients (K) were determined. A linear increase and decrease in Re and K, respectively, take place as the CH proportion increase. Given that the surfactant capacity to interact with liposomes is inversely related to the Re value, the rise of CH reduces linearly both the SDS activity against vesicles (increase in Re) and its affinity with these structures (decrease in K). These linear variations may be due to the linear increase of hydrophobicity in the interfacial region of bilayers and to their reduced translational fluidity by changes in the bilayer packing and ordering. The increase in Re always results in two opposite effects on K. At low Re, K firstly increases because only the outer vesicle leaflet is available for interaction with SDS. The following abrupt fall in K is due to the fact that the binding of additional SDS molecules to bilayer is hampered. The K peaks obtained are correlated in all cases with the saturation of the outer vesicle leaflet by SDS. Increasing Re values, lead to an increased rate of flip–flop of the surfactant molecules (or permeabilization of the bilayers to SDS), thus also making the inner monolayer available for interaction with added SDS. The free SDS concentrations are always lower than its critical micelle concentration indicating that the SDS–liposome interaction is mainly ruled by the action of surfactant monomers regardless of the CH concentration in bilayers.

Physicochemical characteristics of liposomes formed with internal wool lipids

The bilayer-forming capability of internal wool lipids and their physicochemical properties were studied in an attempt to enhance our understanding of the lipid structure, present in wool and other keratinized tissues. Internal wool lipids were extracted and analyzed, and the mixture obtained [sterol esters (10%), free fatty acids (24%), sterols (11%), ceramides (46%), and cholesteryl sulfate (9%)] was shown to form stable liposomes. A phase-transition temperature of 60°C was obtained from nuclear magnetic resonance spectra for this lipid mixture. The spontaneous permeability of these vesicles was lower than that of phosphatidylcholine liposomes but slightly higher than that of the vesicles formed with lipids extracted from other keratinized tissues with higher amounts of cholesterol. The transmission electron micrographs showed large vesicular aggregates of approximately 300 nm, which seem to be made up of smaller structures of approximately 20 nm in size. This particular structure could account for the large diameters and small internal volumes found by dynamic light-scattering and spectrofluorometric measurements.

Bicellar systems for in vitro percutaneous absorption of diclofenac

This work evaluates the effect of different bicellar systems on the percutaneous absorption of diclofenac diethylamine (DDEA) using two different approaches. In the first case, the drug was included in bicellar systems, which were applied on the skin and, in the second case, the skin was treated by applying bicellar systems without drug before to the application of a DDEA aqueous solution. The characterization of bicellar systems showed that the particle size decreased when DDEA was encapsulated. Percutaneous absorption studies demonstrated a lower penetration of DDEA when the drug was included in bicellar systems than when the drug was applied in an aqueous solution. This effect was possibly due to a certain rigidity of the bicellar systems caused by the incorporation of DDEA. The absorption of DDEA on skin pretreated with bicelles increased compared to the absorption of DDEA on intact skin. Bicelles without DDEA could cause certain disorganization of the SC barrier function, thereby facilitating the percutaneous penetration of DDEA subsequently applied. Thus, depending on their physicochemical parameters and on the application conditions, these systems have potential enhancement or retardant effects on percutaneous absorption that result in an interesting strategy, which may be used in future drug delivery applications.