6218
Langmuir 1996, 12, 6218-6223
Subsolubilizing Effects of Alkyl Sulfates on Liposomes Modeling the Stratum Corneum Lipid Composition A. de la Maza* and J. L. Parra Consejo Superior de Investigaciones Cientı´ficas (C.S.I.C.), Centre de Investigacio´ n y Desarrollo (C.I.D.), Departamento de Tensioactivos, Calle Jorge Girona 18-26, 08034 Barcelona, Spain Received March 8, 1996. In Final Form: October 1, 1996X The subsolubilizing interactions of three alkyl sulfates (chain lengths C10, C12, and C14), with liposomes modeling the stratum corneum lipid composition, were investigated. Liposomes were formed from a lipid mixture containing by weight 40% ceramides, 25% cholesterol, 25% palmitic acid and 10% cholesteryl sulfate. The surfactant/lipid molar ratios (Re) and the bilayer/aqueous phase partition coefficients (K) were determined by monitoring the increase in the fluorescence intensity of liposomes due to the 5(6)carboxyfluorescein (CF) released from the interior of vesicles. The free surfactant concentration was always lower than the critical micelle concentration. At the two interaction levels studied (50% and 100% CF release) the tetradecyl sulfate showed the highest ability to release the CF trapped in the liposomes (lowest Re values) and the highest degree of partitioning into the bilayers (highest K values). Whereas stratum corneum liposomes were more resistant to the action of alkyl sulfates than were phosphatidylcholine (PC) liposomes, the degree of partitioning of these surfactants into stratum corneum bilayers was always greater than that reported for PC ones.
Introduction The interaction of alkyl sulfates with skin induces structural changes in the epidermal surfaces and in the stratum corneum transcutaneous permeability barrier1,2 the sodium dodecyl sulfate being the most irritating. As a consequence, this surfactant has frequently been used as a model substance to study the irritant dermatitis.3-5 A number of studies have been devoted to the interaction of alkyl sulfates with phospholipid bilayers as membrane models.6-8 This interaction leads to the formation of lipid/ surfactant mixed micelles. A significant contribution in this area has been made by Lichtenberg,9 who postulated that the critical effective surfactant/lipid molar ratio (Re) producing saturation and solubilization of bilayer structures depends on the surfactant critical micelle concentration (cmc) and on the bilayer/aqueous medium distribution coefficients (K) rather than on the nature of the surfactants. The stratum corneum (SC), the outermost layer of mammalian epidermis, consists of flat cells (corneocytes) that are separated by an intercellular matrix mainly composed of lipids. These lipids are organized into bilayers that have been postulated to account for the permeability properties of SC and possibly to ensure the cohesiveness X Abstract published in Advance ACS Abstracts, December 1, 1996.
(1) Wilheim, K. P.; Surber, C.; Maibach, H. I. J. Invest. Dermatol. 1991, 97, 927-932. (2) Wilheim, K. P.; Surber, C.; Maibach, H. I. Arch. Dermatol. Res. 1989, 281, 293-295. (3) Moon, K. C.; Maibach H. I. Exogenous Dermatoses: Environmental Dermatitits; Menne´ T., Maibach H. I., Eds.; CRC Press: Boca Raton, FL, 1991; pp 217-226. (4) Wilheim, K. P.; Surber, C.; Maibach, H. I. J Invest. Dermatol. 1991, 96, 963-967. (5) Scha¨fer-Korting, M. Clinical Trial Protocols for Anti-Inflammatory and Other Liposome Dermatics. In Liposome Dermatics (Griesbach Conference); Braun-Falco, O., Korting, H. C., Maibach, H., Eds.; Springer-Verlag: Berlin, 1992; pp 299-307. (6) Inuoe, T.; Yamahata, T.; Shimozawa, R. J. Colloid Interface Sci. 1992, 149, 345-358. (7) Urbaneja, M. A.; Alonso, A.; Gonza´lez-Man˜as, J. M.; Gon˜i, F. M.; Partearroyo, M. A.; Tribout, M.; Paredes, S. Biochem. J. 1990, 270, 305-308. (8) Ruiz, J.; Gon j i, F. M.; Alonso, A. Biochim. Biophys. Acta 1989, 937, 127-134. (9) Lichtenberg, D. Biochim. Biophys. Acta 1985, 821, 470-478.
S0743-7463(96)00219-3 CCC: $12.00
between corneocytes.10-12 The composition of lipids and proteins forming the SC has been extensively analyzed.13-16 SC has been shown to be virtually devoid of phospholipids. In order to find out whether SC lipids could form bilayers, Wertz et al.,17 Wertz18 and Abraham et al.19 prepared liposomes from lipid mixtures approximating the composition of SC lipids at physiological pH. Downing et al. investigated the interaction of these bilayer structures with the anionic surfactant sodium dodecyl sulfate (SDS) to study the deleterious effect of this surfactant on human skin.20 Furthermore, Blume et al. reported the permeability of the skin to lipid vesicles.21 We have previously studied the subsolubilizing and solubilizing interactions of alkyl sulfates, in particular sodium dodecyl sulfate, with PC unilamellar liposomes.22-24 Likewise, we investigated the formation and characterization of liposomes formed with different mixtures of four commercially available synthetic lipids approximating the composition of stratum corneum.25 In the present work (10) Friberg, S. E.; Goldsmith, L. B.; Kayali, I.; Suhaimi, H. Interfacial Phenomena in Biological Systems; Bender, M., Ed.; Surfactant Science Series, Volume 39; Marcel Dekker, Inc.: New York, 1991; Chapter 1. (11) Downing, D. T. J. Lipid Res. 1992, 33, 301-313. (12) Bouwstra, J. A.; Gooris, G. S.; Bras, W.; Downing, D. T. J. Lipid Res. 1995, 36, 685-695. (13) Abraham, W.; Wertz, P. W.; Downing, D. T. J. Lipid Res. 1985, 26, 761-766. (14) Ranasingle, A. W.; Wertz, P. W.; Downing, D. T.; Mackeine, J. C. J. Invest. Dermatol. 1986, 86, 187-190. (15) Imokawa, G.; Abe, A.; Jin, K.; Higaki, Y.; Kamashima, M.; Hidano, A. J. Invest. Dermatol. 1991, 96, 523-526. (16) Wertz, P. W.; Downing, D. T., Transdermal Drug Delivery. Developmental Issues and Research Iniciatives; Hadgraft, J., Guy, R. H., Eds.; Marcel Dekker: New York, 1989; pp 1-22. (17) Wertz, P. W.; Abraham, W.; Landman, L.; Downing, D. T. J. Invest. Dermatol. 1986, 87, 582-584. (18) Wertz, P. W. Liposome Dermatics, Chemical Aspects of the Skin Lipid Approach. In Liposome Dermatics (Griesbach Conference); BraunFalco, O., Korting, H. C., Maibach, H., Eds.; Springer-Verlag: Berlin, 1992; pp 38-43. (19) Abraham, W.; Wertz, P. W.; Landman, L.; Downing, D. T., J. Invest. Dermatol. 1987, 88, 212-214. (20) Downing, D. T.; Abraham, W.; Wegner, B. K.; Willman, K. W.; Marshall, J. M. Arch. Dermatol. Res. 1993, 285, 151-157. (21) Blume, A.; Jansen, M.; Ghyczy, M.; Gareiss, J. Int. J. Pharm. 1993, 99, 219-228. (22) de la Maza, A.; Parra, J. L. Langmuir 1992, 8, 2422-2426. (23) de la Maza, A.; Parra, J. L. Langmuir 1993, 9, 870-873. (24) de la Maza, A.; Parra, J. L. Langmuir 1995, 11, 2435-2441. (25) de la Maza, A.; Manich, A. M.; Coderch, L.; Bosch, P.; Parra, J. L. Colloids Surf. A 1995, 101, 9-19.
© 1996 American Chemical Society
Alkyl Sulfates on Stratum Corneum Liposomes
we seek to extend these investigations by characterizing the surfactant to lipid molar ratios and the partition coefficients of a series of alkyl sulfate surfactants between these bilayers and the aqueous medium. Materials and Methods The anionic surfactants sodium decyl sulfate, sodium dodecyl sulfate, and sodium tetradecyl sulfate (C10-SO4, C12-SO4 and C14-SO4) were obtained from Merck and were purified by column chromatography.26 Triton X-100 was purchased from Rohm and Haas (Lyon France). Piperazine-1,4-bis(2-ethanesulfonic acid) (PIPES) was obtained from Merck (Darmstadt, Germany). 5(6)Carboxyfluorescein (CF) was obtained from Eastman Kodak (Rochester, NY) and was purified by column chromatography.27 Polycarbonate membranes and membrane holders were purchased from Nucleopore (Pleasanton, CA). Reagent grade organic solvents, ceramides type III (Cer) and cholesterol (Chol) were supplied by Sigma Chemical Co. (St. Louis, MO), and palmitic acid (PA) (reagent grade) was purchased from Merck. Cholesteryl sulfate (Chol-sulf) was prepared by reaction of cholesterol with excess chlorosulfonic acid in pyridine and purified chromatographically. The molecular weight of ceremide type III used in the lipid mixture was determined by low-resolution fast atom bombardment mass spectrometry using a Fisons VG Auto Spec Q (Manchester U.K.) with a cesium gun operating at 20 KV. The lipids of the highest purity grade available were stored in chloroform/methanol 2:1 under nitrogen at -20 °C until use. Liposome Preparation. We previously reported the formation and characterization of liposome suspensions formed by a weight mixture of lipids modeling the composition of the SC (40% Cer, 25% Chol, 25% PA, and 10% Chol-sulf),25 which was prepared following the method described by Wertz et al.17 Individual lipids were dissolved in chloroform/methanol 2:1, and appropriate volumes were combined. The solvent was removed with a stream of nitrogen and then under high vacuum at room temperature. The lipid mixture was suspended in 20 mM PIPES/ NaOH buffer pH 7.20, containing 110 mM Na2SO4 and supplemented with 100 mM CF to provide a final lipid concentration ranging between 0.5 and 5.0 mM. The lipids were left to hydrate for 30 min under nitrogen with occasional shaking. The suspension was then sonicated in a bath sonicator (514 ECT Selecta) at 60 °C for about 15 min until the suspension became clear. Vesicles of about 200 nm size were obtained by extrusion through 800-200 nm polycarbonate membranes at 60 °C using a thermobarrel extruder equipped with a thermoregulated cell compartment (Lipex, Biomembranes Inc. Vancouver, Canada). The preparations were then kept at the same temperature for 30 min and incubated at 25 °C under nitrogen atmosphere. Vesicles were freed of unencapsulated fluorescent dye on a Sephadex G-50 medium gel bed (Pharmacia Biotech, Uppsala, Sweden). Chemical Analysis and Phase Transition Temperature of Bilayer Lipid Mixture. The liposome lipid composition was determined using thin-layer chromatography coupled to an automated flame ionization detection system (Iatroscan MK-5, Iatron Laboratories Inc., Tokyo, Japan).28 Liposome suspensions were directly spotted onto silica-gel-coated chromarods (type S-III) in 0.5, 1.0, and 1.5 µL aliquots using a SES 3202/IS-02 semiautomatic sample spotter with a precision 2 µL syringe. The rods were developed for a distance of 10 cm using the solvent mixture n-hexane/ethyl ether/formic acid (50/20/0.3) to separate the nonpolar lipids PA and Chol from the rest of the compounds. A partial scan of 80% of the rods was performed to quantify and eliminate them. Redevelopment of the rods with chloroform/ methanol/ammonia (58/10/2.5) of 7 cm leads to a good separation of the polar lipids (Cer and Chol-sulf) from the buffer, which remains at the spotting position. A total scan was performed to (26) Rosen, M. J. J. Colloid Interface Sci. 1981, 79, 587-588. (27) Weinstein, J. N.; Ralston, E.; Leserman, L. D.; Klausner, R. D.; Dragsten, P.; Henkart, P.; Blumenthal, R. Self-Quenching of Carboxyfluorescein Fluorescence: Uses in Studying Liposome Stability and Liposome Cell Interaction, In Liposome Technology; Gregoriadis G., Ed.; CRC Press: Boca Raton, FL. Vol III, 1986 Chapter 13. (28) Ackman, R. G.; Mc Leod, C. A.; Banerjee, A. K, J. Planar Chromatogr.sMod. TLC., 1990, 3, 450-490.
Langmuir, Vol. 12, No. 26, 1996 6219 quantify Cer and Chol-sulf. The same procedure was applied to different standard solutions of these lipids dissolved in chloroform/methanol 2:1 to obtain the calibration curves for the quantification of each compound. In order to find out whether all the components of the lipid mixture formed liposomes, vesicular dispersions were analyzed for these lipids.28 The dispersions were then spun at 140 000 g at 25 °C for 4 h to remove the vesicles.29 No lipids were detected in any of the supernatants. 1H NMR was carried out at 25-90 °C at intervals of 5 °C to determine the phase transition temperature of the lipid mixture forming liposomes on a Varian Unity at 300 MHz (Palo Alto, California). The line widths of the CH2 band at 1.3 ppm were measured, and 1024 scans were accumulated each time. The different line widths were plotted versus the temperature, and the inflexion point of the curve was taken as the phase transition temperature, which was 55-56 °C. Vesicle Size Distribution. The vesicle size distribution and the polydispersity index (PI) of liposome suspensions after preparation was determined with dynamic light-scattering measurements using a photon correlator spectrometer (Malvern Autosizer 4700c PS/MV). The studies were made by particle number measurement. The sample was adjusted to the appropriate concentration with PIPES buffer, and the measurements were taken at 25 °C at a reading angle of 90°. The vesicle size distribution varied very little at lipid concentrations from 0.5 to 5.0 mM around 200 nm (PI lower than 0.1). The size of vesicles after the addition of equal volumes of PIPES buffer and equilibration for 60 min showed in all cases values similar to those obtained after preparation, with a slight increase in the PI (between 0.10 and 0.12). Hence, the liposome preparations appeared to be reasonably stable in the absence of surfactant under the experimental conditions used. Parameters Involved in the Interaction of Surfactants with SC Liposomes. In the analysis of the equilibrium partition model proposed by Schurtenberger et al.30 for bile salt/lecithin systems, Lichtenberg9 and Almog et al.29 have shown that for a mixture of lipids (at a lipid concentration L (mM)) and surfactant (at a concentration ST (mM)) in dilute aqueous media the distribution of surfactant between lipid bilayers and aqueous media obeys a partition coefficient K given (in mM-1) by
K ) SB/[(L + SB)SW]
(1)
where SB is the concentration of surfactant in the bilayers (mM) and SW is the surfactant concentration in the aqueous medium (mM). For L . SB, the definition of K 30 applies:
K ) SB/(LSW) ) Re/SW
(2)
where Re is the effective molar ratio of surfactant to lipid in the bilayers (Re ) SB/L). Under any other conditions, eq 2 has to be employed to define K. This yields
K ) (Re/SW) [1 + Re]
(3)
This approach is consistent with the experimental data offered by Lichtenberg9 and Almog et al.29 for different surfactant lipid mixtures over wide ranges of Re values. Given that the range of lipid concentrations used in our mixture is similar to that used by Almog et al., the K parameter has been determined using this equation. These parameters can be determined on the basis of the linear dependence between the surfactant concentrations required to achieve 50% and 100% of CF release and the SC lipid concentration (SCL), which can be described by the equations
ST,50%CF ) SW,50%CF + Re50%CF[SCL]
(4)
ST,100%CF ) SW,100%CF + Re100%CF[SCL]
(5)
(29) Almog, S.; Litman, B. J.; Wimley, W.; Cohen, J.; Wachtel, E. J.; Barenholz, Y.; Ben-Shaul, A.; Lichtenberg, D. Biochemistry 1990, 29, 4582-4592. (30) Schurtenberger, P.; Mazer, N.; Ka¨nzig, W. J. Phys. Chem. 1985, 89, 1042-1049.
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Table 1. Surfactant-to-Lipid Molar Ratios (Re), Partition Coefficients (K), and Surfactant Concentrations in Aqueous Medium (SW) Resulting in Subsolubilizing Interaction (50% and 100% CF Release) of Alkyl Sulfate Surfactants with SC Liposomesa
C10-SO4 C12-SO4 C14-SO4
cmc (mM)
SW,50%CF (mM)
SW,100%FC (mM)
Re50%CF mol/mol
Re100%CF mol/mol
K50%CF (mM-1)
K100%CF (mM-1)
r2 (50% CF)
r2 (100% CF)
2.4 0.50 0.17
1.10 0.083 0.036
1.70 0.289 0.098
1.735 0.350 0.193
4.9 1.0 0.55
0.57 3.12 4.49
0.48 1.73 3.62
0.996 0.994 0.998
0.995 0.996 0.992
a The critical micelle concentrations of each surfactant tested are also included together with the regression coefficients of the straight lines obtained.
where ST,50%CF and ST,100%CF are the total surfactant concentrations. The surfactant/lipid molar ratios Re50%CF and Re100%CF and the aqueous concentrations of surfactant SW,50%CF and SW,100%CF are, in each curve, the slope and the ordinate at the origin (zero lipid concentration), respectively. Surfactant Critical Micelle Concentration (cmc). The surface tensions of buffered solutions containing increasing concentrations of surfactants were measured by the ring method31 using a Kru¨ss tensiometer. The critical micelle concentration (cmc) of each alkyl sulfate was determined from the abrupt change in the slope of the surface tension values versus surfactant concentration. The fact that the surface tension curves did not show a minimum in the vicinity of the surfactant cmc’s indicated the high purity level of these compounds.27 The cmc values obtained are given in Table 1. Monitoring the Release of CF from Liposomes. Liposomes entrapping concentrated CF hardly fluoresce, but the fluorescence strongly increases when CF is released to the bulk aqueous phase.32 Therefore, the release of the CF trapped into SC vesicles due to the action of the alkyl sulfates was determined quantitatively by monitoring the increase in the fluorescence intensity.22 The measurements were made at 25 °C with a spectrofluorophotometer (Shimadzu RF-540, Kyoto Japan) equipped with a thermoregulated cell. On excitation at 495 nm, a fluorescence maximum emission of CF was obtained at 515.4 nm. The presence of surfactants did not affect the direct quenching of the aforementioned spectrofluorophotometric CF signal. Liposomes were adjusted to the appropriate lipid concentration (from 1.0 to 10.0 mM). A volume of the appropriate surfactant solution (2.0 mL) was added to these liposomes, and the resulting systems was left to equilibrate for 60 min. This interval was chosen as the minimum period of time needed to achieve a constant level of CF release for the lipid concentration range investigated. The experimental determination of this interval is indicated in the Results and Discussion section. The percentage of CF released was calculated by means of the equation
%CF release ) (IT - I0)/(I∞ - I0)100
(6)
where I0 is the initial fluorescence intensity of CF-loaded liposome suspensions in the absence of surfactant and IT is the final fluorescence intensity measured after 60 min. I∞ corresponds to the fluorescence intensity remaining after the complete destruction of liposomes by the addition of 60 µL of 10% v/v aqueous solution of Triton X-100. The assays were carried out in triplicate, and the results given are the averages.
Results and Discussion Subsolubilizing Interaction of Surfactants with SC Liposomes. Wertz et al.17 demonstrated that SC lipids form liposomes when hydrated at 80 °C. The Cer used in this work is composed primarily of simple sphingosines linked to largely monounsaturated fatty acids. It therefore has a much lower bulk melting temperature than SC ceramides, which contain only saturated fatty acids including hydroxyacids. In preliminary experiments we determined the suitable sonication temperature of the lipid mixture investigated by preparing liposomes at temperatures approximating its (31) Lunkenheimer, K.; Wantke, D. Colloid Polym. Sci. 1981, 259, 354-366. (32) Parker, C. A. Photoluminiscence of Solutions; Elsevier: New York, 1968; p 303.
Figure 1. Time curves of the release of CF trapped into SC liposomes caused by the addition of a constant concentration (1.0 mM) of C10-SO4 (b), C12-SO4 (0), and C14-SO4 (9). The lipid concentration was (A) 1.0 and (B) 5.0 mM.
phase transition temperature (55-56 °C). It was found that temperatures exceeding this temperature by more than 10 °C caused alterations in Cer and Chol-sulf. As a consequence, the lipid mixture was sonicated at 60 °C. In surfactant/lipid systems, complete equilibrium may take several hours.7,9 However, in subsolubilizing interactions a substantial part of the surfactant effect takes place within approximately 30 min.8 The time needed to obtain a constant level of CF release of SC liposomes was investigated. Liposomes were treated with a subsolubilizing surfactant concentration of 1.0 mM, and the subsequent CF release was studied as a function of time. The results obtained for 1.0 and 5.0 mM lipid concentrations are indicated in parts A and B of Figure 1, respectively. About 60 min was needed to achieve a CF release plateau at the two interaction levels investigated. The incorporation of surfactant monomers in membranes may induce the formation of hydrophilic pores in these structures or merely stabilize transient holes, in agreement with the concept of transient channels suggested by Edwards et al. in the surfactant-mediated increase in phospholipid membrane permeability for nonionic and
Alkyl Sulfates on Stratum Corneum Liposomes
Langmuir, Vol. 12, No. 26, 1996 6221
Figure 2. Percentage changes in CF release of SC liposomes (lipid concentration ranging from 0.5 to 5.0 mM) induced by the presence of increasing concentrations of C12-SO4. Lipid concentrations were 0.5 (O), 1.0 (0), 2.0 (4), 3.0 (9), 4.0 (3), and 5.0 (b) mM.
alkyl sulfate surfactants.33-35 Howewer, the time needed to obtain a constant level of CF release for SC liposomes was slightly longer (60 min) than that for PC ones (40 min).22 From these findings, the aforementioned CF release changes were studied 60 min after addition of surfactants to the liposome suspensions at 25 °C. The spontaneous release of the fluorescent agent encapsulated into SC liposomes in the absence of surfactant in this period of time was negligible. To determine the Re and SW parameters, the CF release from SC liposomes caused by the addition of the alkyl sulfates was studied for SC lipid concentrations from 0.5 to 5.0 mM. The curves obtained for C12-SO4 are given in Figure 2. The surfactant concentrations resulting in 50% and 100% CF release for each surfactant tested were graphically obtained and plotted versus lipid concentration. A linear relationship was established in each case. These results are plotted in part A (50% CF release) and part B (100% CF release) of Figure 3. The error bars given in the figure are SD and represent the error of three replicates. The straight lines obtained corresponded to eqs 4 and 5 from which Re and SW were determined. These parameters including the regression coefficients (r2) of the straight lines are also given in Table 1. As the SW values were increased, the CF release percentage rose. These findings are in agreement with those reported for subsolubilizing interactions of these surfactants with PC unilamellar liposomes.22 As Re was increased, the CF release percentage also rose, regardless of the alkyl chain length of the surfactant tested. Given that the surfactant capacity to release the encapsulated dye is inversely related to the Re, the maximum activity at 50% and 100% CF release corresponded to the C14-SO4 and the minimum activity to the C10-SO4. Thus, the higher the surfactant hydrophobic moiety the higher its ability to alter the permeability of the bilayer structures. Comparison of the Re values with those reported by our research group for the interaction of the same alkyl sulfate surfactants with PC unilamellar liposomes22 reveals that the ability of these surfactants to alter the release of the dye trapped into SC bilayers (permeability alterations for 50% of CF release) appears to be less (higher Re values) (33) Edwards, K.; Almgen, M. Progr. Colloid Polym. Sci. 1990, 82, 190-197. (34) Edwards, K.; Almgen, M. Langmuir 1992, 8, 824-832. (35) Silvander, M.; Karlsson, G.; Edwards, K. J. Colloid Interface Sci. 1996, 179, 104-113.
Figure 3. Surfactant concentrations resulting in (A) 50% CF release and (B) 100% CF release versus lipid concentration of liposome suspensions C10-SO4 (b), C12-SO4 (0), and C14-SO4 (9).
than that reported for PC bilayers in all cases, although they show similar tendencies with respect to the influence of the surfactant alkyl chain length. As a consequence, SC bilayer structures exhibit more resistance to the surfactant perturbations than PC ones at the sublytic interaction level investigated. This different behavior could be explained by the more hydrophilic nature of PC, which could facilitate the permeation of water and some other molecules (as surfactants) in PC liposomes either through the hydrophilic holes created by the surfactants on the PC polar heads or via formation of short-lived complexes of surfactants/PC polar heads and subsequent transfer the bilayers via flip-flop.36 The surfactant partition coefficients between SC bilayers and the aqueous medium at both 50% and 100% CF release show that the C14-SO4 molecules had the highest degree of partitioning into bilayers (maximum K values), whereas the C10-SO4 showed the lowest (minimum K values) (Table 1). The fact that these surfactants showed at 100% CF release lower K values than those for 50% could be explained assuming that at low Re possibly only the outer vesicle leaflet was available for interaction with surfactant molecules, the binding of additional molecules to bilayers being hampered at slightly higher Re values. These findings are in agreement with those reported by Schubert et al. for sodium cholate/PC liposomes37 and with our previous investigations involving the overall interaction of C12-SO4 with PC liposomes.24 (36) Lasic, D. D. Liposomes: from Physics to Applications; Elsevier Science Publishers B. V.: Amsterdam, 1993; Chapter 2. (37) Schubert, R.; Beyer, K.; Wolburg, H.; Schmidt, K. H. Biochemistry 1986, 25, 5263-5269.
6222 Langmuir, Vol. 12, No. 26, 1996
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(A)
number of surfactant molecules was needed to produce alterations in SC bilayers, these molecules showed increased affinity with these structures. However, a similar influence of the alkyl chain length of each surfactant tested in the Re and K parameters could be observed for both bilayered structures, in spite of their different lipid compositions and physicochemical characteristics. The lipids used in this work are not exactly the same as those existing in the stratum corneum. Nevertheless, the comparison of the Re and K subsolubilizing parameters obtained in the present work on SC liposomes with those reported for the same surfactants with PC liposomes may be useful, given the high level of toxicity of surfactants in biological membranes.
(B)
Conclusions
Figure 4. Variation in Re (A) and K (B) of C10-SO4, C12-SO4 and C14-SO4, versus surfactant cmc’s. (O) 50% CF release; (b) 100% CF release.
In a parallel way, comparison of the present K values with those reported for the interaction of the same surfactants with PC liposomes shows that the degree of partitioning of these surfactants into SC bilayers (or bilayer affinity) was always greater (higher K values) than that for PC ones. However, the influence of the hydrophobic moiety of the surfactant in this affinity was also similar in both cases in spite of the different compositions and properties of these two bilayer structures.22 The increased degree of partitioning of these surfactants into the SC bilayers may be explained bearing in mind the physicochemical meaning of the partition coefficient concept, which is the balance between the surfactant concentration in bilayers SB (mM) per mole of lipids and that in the aqueous phase. The fact that the Re and SW values for SC liposomes showed values respectively higher than and similar to those reported for PC ones in all cases may explain these differences. From a structural viewpoint the different chain lengths, degrees of saturation, and nature of polar heads of the lipids building these structures appear to be responsible for this different behavior. The Re and K parameters for each surfactant at the two sublytic levels investigated were plotted as a function of the cmc values in parts A and B of Figure 4. A progressive increase in Re occurred as the surfactant cmc increased (or the surfactant alkyl chain length decreased), this rise being more pronounced at high cmc values. However, the increase of the surfactant cmc resulted in a fall in the K parameter which was more pronounced at low cmc values (Figure 4B). In general terms, different trends in the interaction of these surfactants with SC and PC liposomes were observed at subsolubilizing levels when comparing the corresponding Re and K parameters. Thus, although a greater
The C14-SO4 surfactant showed at the two interaction levels investigated (50% and 100% CF release) the highest ability to alter the release of the CF encapsulated in SC bilayers (lowest Re values) and the highest degree of partitioning into these structures (highest K values), whereas the C10-SO4 showed the lowest tendencies. Thus, the larger the surfactant hydrophobic moiety, the higher the ability to alter the permeability of the bilayer structures. These findings are in agreement with those reported for the interaction of these surfactants with PC unilamellar liposomes in spite of the different composition and characteristics of these two types of liposomes.22 Given that the free surfactant concentrations were always lower than the corresponding cmc values, we may assume, in agreement with the aforementioned results reported for PC liposomes, that the surfactant/SC liposome interactions were mainly ruled by the action of surfactant monomers. Thus, SC liposomes appeared to be more resistant to the action of surfactant monomers (higher Re values), whereas the affinity of each alkyl sulfate to SC structures was greater than that to PC ones (higher K values for SC liposomes). The differences could be attributable to the different gel/liquid crystal phase transition temperature of the lipids, which depends on chain length, degree of saturation, and nature of the polar heads. These differences affect both the assembly properties and mobility of these lipids and, consequently, their ability to interact with surfactants. Abbreviations SC PIPES CF Re Re50%CF Re100%CF K K50%CF K100%CF SW SW,50%CF SW,100%CF SB
stratum corneum piperazine-1,4-bis(2-ethanesulfonic acid) 5(6)-carboxyfluorescein effective surfactant/lipid molar ratio effective surfactant/lipid molar ratio for 50% of CF release effective surfactant/lipid molar ratio for 100% of CF release bilayer/aqueous phase surfactant partition coefficient bilayer/aqueous phase surfactant partition coefficient for 50% of CF release bilayer/aqueous phase surfactant partition coefficient for 100% of CF release surfactant concentration in the aqueous medium surfactant concentration in the aqueous medium for 50% of CF release surfactant concentration in the aqueous medium for 100% of CF release surfactant concentration in the bilayers
Alkyl Sulfates on Stratum Corneum Liposomes SCL PC Cer Chol PA Chol-sulf C10-SO4 C12-SO4 C14-SO4 PI
stratum corneum lipids phosphatidylcholine ceramides type III cholesterol palmitic acid cholesteryl sulfate decyl sulfate dodecyl sulfate tetradecyl sulfate polydispersity index
Langmuir, Vol. 12, No. 26, 1996 6223 cmc r2
critical micellar concentration regression coefficient
Acknowledgment. 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. (Direccio´n General de Investigacio´n Cientı´fica y Te´cnica) (Prog. no. PB940043), Spain. LA960219K
