Influence of the Level of Cholesteryl Sulfate Present in Stratum Corneum Lipid Liposomes on Their Stability Against Triton X-100

Journal of Colloid and Interface Science 215, 183–189 (1999) Article ID jcis.1999.6265, available online at http://www.idealibrary.com on

Influence of the Level of Cholesteryl Sulfate Present in Stratum Corneum Lipid Liposomes on Their Stability Against Triton X-100 O. Lopez, M. Co´cera, J. L. Parra, and A. de la Maza 1 Departamento de Tensioactivos, Centro de Investigacio´n y Desarrollo (C.I.D.), Consejo Superior de Investigaciones Cientı´ficas (C.S.I.C.), Calle Jorge Girona 18-26, 08034 Barcelona, Spain Received December 15, 1998; accepted April 2, 1999

portions than that existing in skin lipids (7). However, a physico-chemical study of the role played by this lipid on the SC lipid properties using a simplified membrane model such as SC lipid liposomes is still lacking. Wertz and Downing prepared liposomes from lipid mixtures modeling the SC composition and studied their interaction with sodium dodecyl sulfate to determine its effect on human skin (8 –10). Stratum corneum lipid liposomes have been also used as membrane models to study the adsorption of enhancer agents and to compare these results with those obtained in skin studies (11–14). The interaction of Triton X-100 (T X-100) with liposomes leads to the breakdown of lamellar structures and to the formation of lipid–surfactant mixed micelles (15–18). A significant contribution in this area has been made by Lichtenberg (19), who postulated that the surfactant/lipid molar ratio (Re) producing liposome solubilization depends on the surfactant critical micelle concentration and on the bilayer/aqueous medium partition coefficients (K). We studied the formation of liposomes using a mixture of four lipids modeling the SC composition and the sublytic interaction of octylphenols with these liposomes (20, 21). Here, we seek to extend these studies by characterizing the influence of the cholesteryl sulfate (Chol-sulf) on the resistance of SC lipid liposomes against T X-100. To this end, we determined the Re and K parameters of this interaction at sublytic level and studied the liposome solubilization (using a dynamic light-scattering technique) varying the proportion of Chol-sulf in bilayers. This information may shed light on the possible correlation between the level of Chol-sulf in bilayers and the abnormalities in the skin barrier function and in the SC cohesion.

The interaction of Triton X-100 (T X-100) with stratum corneum (SC) lipid liposomes varying the proportion of cholesteryl sulfate (Chol-sulf) was investigated. The surfactant/lipid molar ratios and the bilayer/aqueous phase partition coefficients were determined at sublytic level by monitoring the changes in the fluorescence intensity of liposomes due to the 5(6) carboxyfluorescein released from the interior of vesicles. The fact that the free surfactant concentration was always lower than the surfactant CMC indicates that permeability changes were mainly ruled by the action of surfactant monomers in all cases. The lowest surfactant ability to alter the permeability of SC liposomes and highest surfactant affinity with these bilayer structures was reached when the proportion of Chol-sulf in bilayers was 10%. Futhermore, the highest resistance of SC liposomes to be solubilized by T X-100 (via mixed micelle formation) also occurred at this Chol-sulf proportion, which corresponds to that existing in the intercellular SC lipids. These surfactant effects may be related to the reported dependencies between the level of Chol-sulf in the intercellular lipids and the abnormalities in the skin properties as the barrier function. © 1999 Academic Press Key Words: stratum corneum lipid liposomes; Triton X-100; stratum corneum lipid liposomes/Triton X-100 interactions; influence of cholesteryl sulfate on the liposome stability; carboxyfluorescein release; dynamic light-scattering; surfactant/lipid molar ratios; surfactant partition coefficients.

INTRODUCTION

The stratum corneum (SC) forms a continuous sheath of alternating squamae (protein-enriched corneocytes) embedded in an intercellular matrix enriched in unpolar lipids displayed as lamellar sheets. The proportion of cholesterol and cholesteryl sulfate (Chol-sulf) in these lipids is claimed to play an important role in the stability properties of the SC (cohesion and desquamation) and in the regulation of the skin barrier function (1–5). Thus, patients with recessive X-linked ichthyosis show elevated proportions of Chol-sulf due to steroid sulfatase deficiency (6), whereas tissues with extremely tenacious intercellular cohesion also present higher Chol-sulf pro1

MATERIALS AND METHODS

To whom correspondence should be addressed.

Triton X-100, octylphenol polyethoxylated with 10 units of ethylene oxide and active matter of 100% was purchased from Rohm and Hass (Lyon France). The starting material 5(6)carboxyfluorescein (CF) was obtained from Eastman Kodak (Rochester, NY) and further purified by column chromatogra-

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0021-9797/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.

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TABLE 1 Liposome Lipid Composition Corresponding to the Six Experiments in which the Percentage of Chol-sulf Varied from 1 to 25% and the Relative Proportions of the Other Lipids Remained Constant Liposome lipid composition % Exp no.

Cer

Chol

PA

Chol-sulf

1 2 3 4 5 6

44.0 42.2 40.0 37.8 35.6 33.4

27.5 26.4 25.0 23.6 22.2 20.8

27.5 26.4 25.0 23.6 22.2 20.8

1.0 5.0 10.0 15.0 20.0 25.0

phy (22). Piperazine-1,4-bis(2-ethanesulphonic acid) (PIPES) was obtained from Merck (Darmstadt, Germany). PIPES buffer was prepared as 20 mM PIPES containing 110 mM Na 2SO 4, adjusted to pH 7.20 with NaOH. This buffer was supplemented with 110 mM CF when studied permeability variations. Reagent grade organic solvents, ceramides type III (Cer), cholesterol (Chol) and palmitic acid (PA) were supplied by Sigma Chemical Co. (St Louis, MO). Cholesteryl sulfate (Chol-sulf) was prepared by reaction of cholesterol with excess chlorosulphonic acid in pyridine and purified chromatographically. The molecular weight of ceramide type III (Sigma) was determined by low resolution fast atom bombardment mass spectrometry using a Fisons VG Auto Spec Q (Manchester, UK) with a caesium gun operating at 20 Kv. From this analysis a molecular weight of 671 g/mol was obtained for the majority compound used. This value was similar to the molecular weight of ceramide III (667 g) calculated from the structure of this compound reported by Wertz (9), despite the fact that the ceramide type III used was a mixture of ceramides of different chain lengths (mainly containing stearic and nervonic acids, purity approx 99%). As a consequence, we used the molecular weight obtained to calculate the molarity of the lipid mixtures investigated. The lipids of the highest purity grade available were stored in chloroform/methanol 2:1 under nitrogen at 220°C until use. Liposome Preparation and Characterization Liposomes formed by mixtures of SC lipids varying the percentage of Chol-sulf from 1 to 25%, the relative proportions of the other lipids remaining constant, were prepared following the method described by Wertz (8, 23). The lipid compositions investigated are given in Table 1 (final lipid conc ranging from 0.5 to 5.0 mM). Experiment 3 corresponded to the composition of the intercellular lipids. The lipid composition and concentration of liposomes after preparation were determined by thin-layer chromatography coupled to an automated flame ionization detection system (Iatroscan MK-5, Iatron Lab. Inc., Tokyo, Japan) (20, 24).

In order to find out whether all the lipid mixture components formed liposomes, vesicular dispersions were analyzed for these lipids (24). The dispersions were then spun at 140,000 g at 25°C for 4 h to remove the vesicles (25). The supernatants were tested again for these components. No lipids were detected in any of the supernatants. The phase transition temperatures of the lipid mixtures forming liposomes were determined by proton magnetic resonance ( 1H NMR), showing values ranging from 55 to 59°C (20). Parameters Involved in the Interaction of T X-100 with SC Lipid Liposomes In the analysis of the equilibrium partition model proposed by Schurtenberger (26) for bile salt/lecithin systems, Lichtenberg et al., and Almog et al. (19, 25) have shown that for a mixing of lipids (at a conc. L (mM)) and surfactant (at a conc. S T (mM)), in dilute aqueous media, the distribution of surfactant between lipid bilayers and aqueous media obeys a partition coefficient K, given (in mM 21) by K 5 S B/@~L 1 S B! z S W#,

[1]

where S B is the surfactant concentration in the bilayers (mM) and S W is that in the aqueous medium (mM). For L @ S B, the definition of K, as given by Schurtenberger, applies K 5 S B/~L z S W! 5 Re/S W,

[2]

where Re is the effective surfactant to lipid molar ratio in the bilayers (Re 5 S B/L). Under any other conditions, Eq. [2] has to be employed to define K; this yields K 5 Re/S W@1 1 Re#.

[3]

This approach is consistent with the experimental data offered by Lichtenberg et al. and Almog et al. (19, 25) for different surfactant lipid mixtures over wide ranges of Re values. The validity of this model for the systems investigated has been studied in the Results and Discussion section. The Re, S W, and K parameters were determined on the basis of the linear dependence between the surfactant concentrations required to achieve 50 and 100% CF release and the lipid concentration (SCL), which is described by S T 5 S W 1 Re z @SCL#,

[4]

where S T (S T,50%CF, S T,100%CF) are the total surfactant concentrations. The surfactant to lipid molar ratios Re (Re 50%CF, Re 100%CF) and the surfactant aqueous concentration S W (S W,50%CF, S W,100%CF) are in each curve the slope and the ordinate at the origin (zero lipid concentration), respectively. The K 50%CF and K 100%CF parameters (bilayer/aqueous phase partition

STRATUM CORNEUM LIPID LIPOSOMES/TRITON X-100

coefficient for 50 and 100% CF release) were determined from Eq. [3]. Permeability Alterations and Solubilization of SC Lipid Liposomes The permeability alterations of SC lipid liposomes due to the action of T X-100 were quantitatively determined by monitoring the release of the CF trapped into these vesicles (21). Changes in the fluorescence intensity were measured at 25°C using the spectrofluorophotometer (Shimadzu RF-540, Kyoto, Japan). Liposomes of different composition were adjusted to the appropriate lipid concentration (from 1.0 to 10.0 mM). Equal volumes of T X-100 solutions (2.0 ml) were added to these liposomes and the resulting systems were left to equilibrate for periods of time ranging from 40 to 75 min, depending on the Chol-sulf amounts in bilayers. These intervals were chosen as the minimum periods of time needed to achieve a constant level of CF release for each lipid composition (see Results and Discussion section). As for the solubilization studies, the hydrodynamic diameter (HD) of pure T X-100 micelles, SC lipid liposomes and the aggregates resulting in the interaction of T X-100 with these bilayer structures (SC conc. 1.0 mM) was determined using a dynamic light scattering technique (DLS). To this end, a photon correlator spectrometer (Malvern Autosizer 4700c PS/MV) equipped with an Ar laser source (wavelength 488 nm) was used. This laser source is suitable to elucidate the size of small and large particles present in the same system. Equal volumes of T X-100 solutions (2.0 ml) were added to the liposomes and the resulting systems were left to equilibrate for 24 h. The DLS measurement of each mixture was made with a scattering angle of 90° at 25°C (controlled temperature). The optimum signal was obtained in each case by varying the laser intensity and the aperture of the photomultiplier selector. In order to acquire the full range of decay time necessary to determine the signal from both the large and the small particles, a low sample time value (2 ms) and a dilatation of 3 with parallel subcorrelators was used. The analysis of the data was performed using CONTIN software provided by Malvern Instruments, England. The goodness of CONTIN results was tested by fitting a single or a biexponential to the correlation function. If a biexponential had to be fitted, first a single exponential was fitted to a long time range and the second exponential was then fitted to the residual. Both methods agreed fairly well. The results are given as diameter and the percentages correspond to the intensity values. The permeability and DLS assays were both carried out in triplicate and the results given are the averages. RESULTS AND DISCUSSION

We previously reported the critical micelle concentrations (CMC) of T X-100 in the working medium, which was 0.15 mM

185

(18). The characterization of the geometric properties of liposomes used in the present study was previously reported (20). This study demonstrated that these liposomes were formed by unilamellar vesicles in all cases. Furthermore, the vesicle size distribution of liposomes after preparation varied very little, showing in all cases a similar value of about 100 nm (PI lower than 0.1), thereby indicating that the size distribution was very homogeneous. The size of the vesicles after addition of equal volumes of PIPES buffer and equilibration for 2 h always showed values similar to those obtained after preparation with a slight PI increase (between 0.11 and 0.13). Hence, the SC lipid liposomes investigated were reasonably stable in the absence of T X-100 under the experimental conditions used. Influence of Chol-Sulf in the Interaction of T X-100 with SC Lipid Liposomes A systematic study based on the CF release of liposomes varying the level of Chol-sulf and due to the action of T X-100 was performed to shed light on the possible dependencies between the level of Chol-sulf in skin lipids and the function barrier abnormalities. To this end, the percentage of Chol-sulf varied from 1 to 25% (lower and higher values than that in SC lipids, which was 10%), the relative proportion of the other lipids remained constant (Table 1). To determine the time needed to obtain a constant level of CF release, SC liposomes at various lipid compositions were treated with T X-100 at the sublytic level and the subsequent CF release changes were studied as a function of time. The CF release was in all cases a biphasic process, in which increasing amounts of Chol-sulf in bilayers resulted in increased periods of time to achieve CF release plateaux. The maximum period (75 min) corresponded to the liposomes containing 10% Cholsulf, which was to that existing in the intercellular lipids. This period remained unaffected by the presence of increasing Cholsulf amounts in bilayers. Thus, periods from 40 to 75 min were needed to achieve this plateau from Experiment 1 to the Experiment 6 (Table 1). To determine the partition coefficients of T X-100 between bilayers and the aqueous phase, we first studied the validity of the equilibrium partition model proposed by Lichtenberg et al. and Almog et al. (19, 25), based on Eq. [1] for the systems investigated. This equation may be expressed by L/S B 5 (1/K)(1/S W) 2 1. Hence, this validity requires a linear dependence between L/S B and 1/S W; this line should have a slope of 1/K, intersect with the L/S B axis at 21 and intersect with the 1/S W at K. To test the validity of this model for the liposomes investigated, liposomes were mixed with varying sublytic T X-100 concentrations (S T). The resultant surfactant-containing vesicles were then spun at 140000 g at 25°C for 4 h to remove the vesicles. No lipids were detected in the supernatants (24). The T X-100 concentration in the supernatants (S W) was determined by HPLC (28) and its concentration in the lipid bilayers was calculated (S B 5 S T 2 S W). The results of the experiments in

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LOPEZ ET AL.

FIG. 1. Percentage changes in CF release of SC lipid liposomes (lipid composition for the Experiment 3, Table 1), the lipid concentration ranging from 0.5 to 5.0 mM, induced by the presence of increasing concentrations of T X-100. Lipid concentrations: 0.5 mM (E), 1.0 mM (h), 2.0 mM (‚), 3.0 mM (■), 4.0 mM (ƒ), 5.0 mM (F).

which S B and S W were measured (at the same range of lipid and surfactant concentrations used to determine K) were plotted in terms of the dependence of L/S B on 1/S W. Straight lines were obtained for each lipid mixture tested (r 2 5 0.992, 0.992, 0.993, 0.991, 0.991, and 0.992 for Experiments 1, 2, 3, 4, 5, and 6, respectively, Table 1). These straight lines were dependent on L and intersected with the L/S B axis always at 20.96 6 0.12. Both the linearity of these dependences and the proximity of the intercept to 21 support the validity of this model to determine K for these surfactant/liposome systems. To determine the Re and S W parameters the variation of CF release due to the action of T X-100 was studied (lipid conc. ranging from 0.5 to 5.0 mM). To this end, the CF release changes were measured after that a constant level of CF release was reached in each case. The spontaneous release of the fluorescent agent encapsulated into liposomes in the absence of T X-100 in these periods of time was negligible in all cases. The curves obtained for Experiment 3 (10% Chol-sulf) are given in

the Fig. 1. The T X-100 concentrations resulting in 50 and 100% of CF release for each system were plotted versus the lipid concentration. A linear relationship was established in each case. The straight lines obtained corresponded to the Eq. [4] from which Re and S W were determined. The Re and S W values for the six experiments investigated, including the regression coefficients (r 2) of the straight lines, are given in Table 2. The increase in the percentage of Chol-sulf in liposomes resulted in a rise in the Re values up to a maximum was reached for the Chol-sulf proportion 10%. Given that the surfactant capacity to release the encapsulated dye is inversely related to the Re values, the lowest T X-100 activity at the two levels investigated corresponded to the lipid mixture containing 10% Chol-sulf. As aforementioned this proportion corresponded to that existing in the intercellular SC lipids. The partitioning of T X-100 between bilayers and water increased as the percent of Chol-sulf in liposomes rose (increase in the K values) up to a maximum was reached also for the Chol-sulf proportion 10%. As for the free surfactant concentrations, the increasing presence of Chol-sulf in bilayers resulted in a decrease in both S W,50%CF and S W,100%CF, being this fall specially pronounced at low Chol-sulf proportions. However, these values were always smaller than that of the surfactant CMC (0.15 mM) (18). This finding 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 (29). The variations of Re and K versus the percentage of Cholsulf in liposomes at the two interaction levels investigated are plotted in Figs. 2 and 3, respectively. The Re values increased with the Chol-sulf concentration in bilayers up to a maximum was achieved for 10% Chol-sulf (Fig. 2). Both the K 50%CF and K 100%CF values sharply increased as the proportion of Chol-sulf in bilayers rose up to a maximum was achieved for 10% Chol-sulf in both cases. Higher Chol-sulf amounts led to a fall in the K values, being this tendency more pronounced for K 50%CF (Fig. 3). Hence, despite the reduced partitioning of surfactant mole-

TABLE 2 Surfactant to Lipid Molar Ratios (Re), Partition Coefficients (K) and Surfactant Concentrations in the Aqueous Medium (S W) Resulting in the Interaction of T X-100 with SC Lipid Liposomes at the Two Interaction Levels Investigated (50 and 100% CF Release), Varying the Liposome Lipid Composition (Table 1)

Exp. no.

S W,50%CF (mM)

S W,100%CF (mM)

Re 50%CF mole/mole

Re 100%CF mole/mole

K 50%CF (mM 21)

K 100%CF (mM 21)

r2 (50%CF)

r2 (100%CF)

1 2 3 4 5 6

0.072 0.046 0.037 0.033 0.033 0.033

0.113 0.094 0.086 0.084 0.083 0.083

0.035 0.10 0.12 0.10 0.09 0.06

0.34 0.42 0.44 0.42 0.39 0.34

0.47 1.97 2.89 2.75 2.50 1.71

2.24 3.14 3.55 3.52 3.38 3.05

0.994 0.998 0.995 0.992 0.998 0.994

0.996 0.994 0.993 0.997 0.996 0.993

187

STRATUM CORNEUM LIPID LIPOSOMES/TRITON X-100

cules in liposomes containing low proportions of Chol-sulf (low affinity with these bilayer structures) their ability to alter these bilayer structures was higher than that for bilayers approximating the SC lipid composition (percentage of Chol-sulf 10%). This finding emphasizes the low resistance of these bilayers against the action of T X-100 at sublytic level. Inversely, the increased surfactant partitioning (K) and Re values at the Chol-sulf level corresponding to that of the SC lipids reveals that although a increased number of surfactant molecules were incorporated into bilayers these molecules were less able to alter the permeability of these bilayes structures. These increased Re values are also in line with the increased period of time required by these liposomes to achieve a constant level of CF release. These findings may be related to the reported dependencies between the alterations in the level of Chol-sulf in the intercellular lipids and the abnormalities in the SC barrier function (1, 2, 6). In fact, insufficient or excessive Chol-sulf content would alter the liquid-crystalline “melting point” of these lipids, thereby producing nonphysiologic phase transitions. These alterations in the intercellular lipids would affect the skin barrier function. Dynamic Light Scattering Experiments A series of dynamic light scattering (DLS) studies were performed to shed light on the correlation between the level of Chol-sulf in skin lipids and their resistance to be solubilized by T X-100. To this end, we studied the size distribution of the aggregates formed during the solubilization of SC liposomes (lipid conc. 1.0 mM) varying the level of Chol-sulf (Table 1). The DLS curves (at a scattering angle of 90°) of micellar T X-100 solutions (T X-100 conc. ranging from 1 to 5 mM) always showed a monomodal distribution with a peak at 10 nm. The DLS data for SC lipid vesicles and for some surfactant/lipid

FIG. 3. Partition coefficients (K 50%CF and K 100%CF) for T X-100 versus the percentage of Chol-sulf in SC lipid liposomes: K 50%CF (E) and K 100%CF (F).

systems (Experiments 1 and 3) are given in Table 3. SC liposomes showed a monomodal distribution with a hydrodynamic diameter (HD) of 105 nm in both cases. The addition of low T X-100 amounts to liposomes did not produce noticeable changes in the size of surfactant–lipid mixed vesicles formed. When the T X-100 concentration exceeded 0.5 and 0.9 mM (Experiments 1 and 3, respectively) a new peak in the size distribution curve appeared (about 12 nm) corresponding to the formation of surfactant–lipid mixed micelles. Increasing surfactant amounts led to a progressive rise in the proportion of mixed micelles and to a fall in that for mixed vesicles (large

TABLE 3 Dynamic Light Scattering Data (Scattering Angle of 90°) Corresponding to the Interaction of T X-100 with SC Lipid Liposomes (1.0 mM) for Experiments 1 and 3 Experiment No. 1 Large particles

FIG. 2. Effective surfactant to lipid molar ratios (Re 50%CF and Re 100%CF) for T X-100 versus the percentage of Chol-sulf in SC lipid liposomes: Re 50%CF (E) and Re 100%CF (F).

Experiment No. 3

Mixed micelles

Large particles

Mixed micelles

T X-100 [mM]

nm

%

nm

%

nm

%

nm

%

0 0.5 0.9 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0

105 104 100 78 57 47 35 22 — — — —

100 94 77 75 56 37 20 2 — — — —

— 12 12 13 13 14 14 15 15 16 16 15

— 6 23 25 44 63 80 98 100 100 100 100

105 105 104 100 82 73 59 45 35 22 — —

100 100 99 98 83 68 53 36 20 5 — —

— — 12 12 13 13 14 14 15 15 16 16

— — 1 2 17 32 47 64 80 95 100 100

Note. The T X-100 concentration varied from 0.5 to 5.0 mM.

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to the SC tissue, in which changes in the proportion of this lipid are associated to alterations in its stability properties and to abnormalities in its barrier function. Hence, a potential pharmaceutical application of liposomes formed by skin lipids could be envisaged in order to enhance the stability properties of the stratum corneum. APPENDIX: NOMENCLATURE

FIG. 4. Variation in the percentage of large particles during the solubilization of SC lipid liposomes (1.0 mM) by T X-100. The proportion of Chol-sulf in bilayers varied from 1 to 25% (Table 1). Experiment simbols: No. 1 (F), No. 2 (E), No. 3 (■), No. 4 (h), No. 5 (Œ), and No. 6 (‚).

particles). It is noteworthy that in this interval the HD of mixed micelles slightly increased, whereas that for large particles progressively decreased, up to the complete solubilization of liposomes. Hence, in this interval large particles and mixed micelles coexisted in different proportions (bimodal size distribution curves). The T X-100 concentrations of 3.1 and 4.2 mM (Experiments 1 and 3, respectively) showed again a monomodal size distribution curve for mixed micelles (about 15 nm). Figure 4 shows the variation in the percentage of large particles versus the T X-100 concentration during the process of liposome solubilization for all the lipid compositions studied. It is noteworthy that Experiment 3 (10% Chol-sulf) required the highest surfactant concentration to solubilize liposomes, whereas Experiment 1 (1% Chol-sulf) needed the lowest. Thus, the liposomes modeling the SC lipid composition exhibited the highest resistence to be solubilized by T X-100. Hence, no correlation is found between these findings and the reported dependencies of high Chol-sulf proportions in the intercellular SC lipids and its increased cohesion (1, 2). This divergence may be explained, taking into account the complexity of the SC, in which the tissue cohesion is also associated to the structural proteins and to the corneocyte envelopes (1–5). Comparison of the data of Table 2 and the results plotted in Fig. 4 shows that the Chol-sulf concentration for the highest stability against T X-100 at sublytic level (10% Chol-sulf) also corresponded to that for the highest resistance of liposomes to be solubilized by this surfactant via mixed micelles formation. In conclusion, this membrane model has shown to be useful in establishing a correlation between the proportion of Cholsulf in liposomes and the stability properties of these bilayer structures against T X-100. The fact that the Chol-sulf proportion that exibited the highest stability corresponded to that of the intercellular lipids allows us to extrapolate the present findings

SC PC CF Cer Chol PA Chol-sulf PIPES T X-100 Re Re 50%CF Re 100%CF K K 50%CF K 100%CF S W,50%CF S W,100%CF PI CMC r2 DLS HD

stratum corneum phosphatidylcholine 5(6)-carboxyfluorescein ceramides type III cholesterol palmitic acid cholesteryl sulfate piperazine-1,4 bis(2-ethanesulphonic acid) Triton X-100 effective surfactant/lipid molar ratio effective surfactant/lipid molar ratio for 50% CF release effective surfactant/lipid molar ratio for 100% CF release bilayer/aqueous phase surfactant partition coefficient bilayer/aqueous phase surfactant partition coefficient for 50% CF release bilayer/aqueous phase surfactant partition coefficient for 100% CF release surfactant concentration in the aqueous medium for 50% CF release surfactant concentration in the aqueous medium for 100% CF release polydispersity index critical micelle concentration regression coefficient dynamic light scattering hydrodynamic diameter ACKNOWLEDGMENTS

We are grateful to Mr. G. von Knorring for expert technical assistance. This work was supported by funds from D.G.I.C.Y.T. (Prog. No. PB94-0043), Spain.

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