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Interaction of Equimolecular Mixtures of Nonionic/Anionic Surfactants with Liposomes Alfonso de la Maza* and Jose Luis Parra Departamento de Tensioactivos, Centro de Investigacio´ n y Desarrollo (C.I.D.), Consejo Superior de Investigaciones Cientı´ficas (C.S.I.C.), C/.Jorge Girona, 18-26, 08034 Barcelona, Spain Received October 16, 1995. In Final Form: April 2, 1996X The mechanisms governing the interaction of equimolecular mixtures of nonylphenol polyethoxylated with 10 mol of ethylene oxide (NP(EO)10) and sodium dodecyl sulfate (SDS) with phosphatidylcholine (PC) liposomes were investigated. Permeability alterations were detected as a change in 5(6)-carboxyfluorescein (CF) released from the interior of vesicles and bilayer solubilization was detected as a decrease in the static light scattered by liposome suspensions. At the subsolubilizing level, a maximum bilayer/water partitioning of surfactant mixture was reached at 30-50% CF release, which correlated with the increased presence of SDS in the bilayers. However, transition stages between 60% CF release and 100% light scattering corresponded to the increased presence of NP(EO)10 in these structures. The surfactant mixture showed throughout the interaction a much higher affinity with bilayers and a higher ability to saturate and solubilize these structures than that reported for the anionic component. A direct dependence was established in the initial interaction steps (effective molar ratio of surfactant to phospholipid in bilayers (Re) lower than 0.190) between the growth of vesicle fluidity and the maximum bilayer/water partitioning of the surfactant mixture (K). These parameters also depended on the surfactant saturation of the outer vesicle leaflet. A linear dependence was also established during solubilization between the decrease in both the surfactant-PC aggregate size and the scattered light of the system and the composition of these aggregates (Re). The fact that the free surfactant concentration at subsolubilizing and solubilizing levels was respectively lower than and similar to the critical micelle concentration (cmc) of the surfactant mixture indicates that permeability alterations and solubilization were determined respectively by the action of the surfactant monomer and by the formation of mixed micelles. This finding supports the generally admitted assumption, for single surfactants, that the concentration of free surfactant must reach the cmc for solubilization to occur and highlights the influence of the negative synergism of this surfactant mixture on the free surfactant concentration needed to saturate or solubilize liposomes.
Introduction A number of investigations have been devoted to the understanding of the principles governing the interaction of the anionic surfactant sodium dodecyl sulfate (SDS) with simplified membrane models such as phospholipid or stratum corneum lipid bilayers.1-4 This interaction in excess water leads to the breakdown of lamellar structures and to the formation of lipid-surfactant mixed micelle systems. A significant contribution to these investigations has been made by Lichtenberg,5 who postulated that the critical effective surfactant/lipid ratio (Re) producing saturation and solubilization depends on the surfactant critical micellar concentration (cmc) and on the bilayer/ aqueous medium distribution coefficients (K). Mixtures of nonylphenol polyethoxylated with 10 mol of ethylene oxide (NP(EO)10) nonionic surfactant and SDS in aqueous solution exhibit negative deviation from ideal solution behaviour.6-10 These mixtures show changes in the surface properties which improve the wetting ability * To whom correspondence should be addressed. Telephone: (34-3) 400.61.61. Telex: 97977 IDEB E. Fax: (34-3) 204.59.04. X Abstract published in Advance ACS Abstracts, June 15, 1996. (1) 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. (2) Inuoe, T.; Yamahata, T.; Shimozawa, R. J. Colloid Interface Sci. 1992, 149, 345-358. (3) Downing, D. T.; Abraham, W.; Wegner, B. K.; Willman, K. W.; Marshall, J. L. Arch. Dermatol. Res. 1993, 285, 151-157. (4) Ruiz, J.; Gon˜i, F. M.; Alonso, A. Biochim. Biophys. Acta 1988, 937, 127-134. (5) Lichtenberg, D. Biochim. Biophys. Acta 1985, 821, 470-478. (6) Rathman, J. F.; Scamehorn, J. F. Langmuir 1988, 4, 474-481. (7) Rathman, J. F.; Scamehorn, J. F. Langmuir 1987, 3, 372-377. (8) Scamehorn, J. F.; Schechter, R. S.; Wade, W. H. J. Dispersion Sci. Technol. 1982, 3, 261-278. (9) Zourab, Sh. M.; Sabet, V. M.; Abo-El Dahad, H. J. Disper. Sci. Technol. 1991, 12, 25-36.
S0743-7463(95)00878-X CCC: $12.00
of water on hydrophobic surfaces11,12 and the salinity tolerance with regard to that of the anionic component.13 The presence of NP(EO)10 in anionic/cationic surfactant mixtures also exerts a control in the precipitation of the SDS.14 The change in the physico chemical properties of mixed micelles and their additional stability arise from the charge separation of the ionic head groups, which affects the mean aggregation numbers of these micelles.8,15 The interaction of SDS with skin induces structural changes in the epidermal surfaces16-18 and in the stratum corneum transcutaneous permeability barrier.19,20 However, the “in vitro” tests involving the equilibrium monomer-micelle aggregates in the diffusion of NP(EO)10/ SDS mixtures through biological films reveal a reduction of their irritancy power with respect to that of pure anionic surfactant.21 (10) Nguyen, C. M.; Rathman, J. F.; Scamehorn, J. F. J. Colloid Interface Sci. 1986, 112, 438-446. (11) Carrio´n, F. J. J. Am. Oil. Chem. Soc. 1991, 68, 272-277. (12) Carrio´n, F. J. Text. Res. J. 1994, 64, 49-55. (13) Steliner, K. L.; Scamehorn, J. F. J. Am. Oil. Chem. Soc. 1986, 63, 566-574. (14) Shiau, B. J.; Harwell, J. H.; Scamehorn, J. F. J. Colloid Interface Sci. 1994, 167, 332-345. (15) Hollang P. M. In Phenomena in Mixed Surfactant Systems; Scamehorn, J. F., Ed.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986; Vol. 311. (16) Moon, K. C.; Maibach, H. I. In Exogenous Dermatoses: Environmental Dermatitis; Menne´, T., Maibach, H. I., Eds.; CRC Press: Boca Raton, FL, 1991; pp 217-226. (17) Wilhelm, K. P.; Surber, C.; Maibach, H. I. J. Invest. Dermatol. 1991, 96, 963-967. (18) Braun-Falco O.; Korting, H. C.; Maibach, H. I. In Liposome Dermatitis (Griesbach Conference); Braun-Falco, O., Korting, H. C., Maibach, H. I., Eds.; Springer-Verlag: Berlin, 1992; p 301. (19) Wilhelm, K. P.; Surber, C.; Maibach, H. I. J. Invest. Dermatol. 1991, 97, 927-932. (20) Wilhelm, K. P.; Surber, C.; Maibach, H. I. Arch. Dermatol. Res. 1989, 281, 293-295.
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In recent papers, we studied the phase transitions involved in the individual interaction of SDS with liposomes.22,23 In the present work we seek to extend these investigations by characterizing the overall interaction of the NP(EO)10/SDS equimolecular mixture with phosphatidylcholine liposomes. Knowledge of the effective surfactant/phospholipid molar ratios and the partition coefficients of these surfactants between lipid bilayers and the aqueous phase could be useful in improving our understanding of the synergism existing between these two surfactants and in establishing a criterion for the evaluation of the activity of this mixture in biological membranes. Materials and Methods Phosphatidylcholine (PC) was purified from egg lecithin (Merck, Darmstadt, Germany) according to the method of Singleton24 and was shown to be pure by thin-layer chromatography (TLC). The nonionic surfactant nonylphenol polyethoxylated with 10 units of ethylene oxide [NP(EO)10] was supplied by Tenneco Espan˜a S.A. as a 100% active matter product. Triton X-100 was purchased from Rohm and Haas (Lyon, France). The anionic surfactant sodium dodecyl sulfate (SDS) was purchased from Merck and further purified by a column chromatographic method.25 Piperazine-1,4-bis(2-ethanesulphonic acid) (PIPES buffer) obtained from Merck was prepared as 20 mM PIPES adjusted to pH 7.20 with NaOH, containing 110 mM Na2SO4. The starting material 5(6)-carboxyfluorescein (CF) was obtained from Eastman Kodak (Rochester, NY) and further purified by a column chromatographic method.26 Unilamellar liposomes of a defined size (about 200 nm) and PC concentration, ranging from 0.5 to 5.0 mM, were prepared by extrusion of large unilamellar vesicles previously obtained by reverse-phase evaporation.27,28 The PC concentration was determined by TLC-FID.29 To determine the distribution of surfactants (single surfactants or mixture) between the lipid phase and the aqueous media, equilibrated surfactant/PC mixed vesicular dispersions (containing subsolubilizing surfactant concentrations) were analyzed for PC.29 The dispersions were then spun at 140000g at 25 °C for 4 h to remove the vesicles.30 The supernatants of all the mixed dispersions were tested again for PC and surfactants. Surfactant analyses were carried out by spectrophotometric methods.31 All the samples were assayed in quadruplicate. No PC was detected in any of the supernatants. The size distribution and the polydispersity index (PI) of liposomes and surfactant-PC aggregates were determined with a photon correlator spectrometer (Malvern Autosizer 4700c PS/ MV).23 The critical micelle concentration values (cmc) for the surfactant mixture and the single components in PIPES buffer were determined from the abrupt change in the slope of the surface tension values versus surfactant concentration.32 (21) Garcı´a, M. T.; Ribosa, I.; Sanchez Leal, J.; Comelles, F. J. Am. Oil Chem. Soc. 1992, 69, 25-29. (22) De la Maza, A.; Parra, J. L. Langmuir 1993, 9, 870-873. (23) De la Maza, A.; Parra, J. L. Langmuir 1995, 11, 2435-2441. (24) Singleton, W. S.; Gray, M. S.; Brown, M. L.; White, J. L. J. Am. Oil Chem. Soc. 1965, 42, 53-57. (25) Rosen, M. J.; Hua, X. Y. J. Colloid Interface Sci. 1982, 86, 164172. (26) Weinstein, J. N.; Ralston, E.; Leserman, L. D.; Klausner, R. D.; Dragsten, P.; Henkart, P.; Blumenthal, R. In Liposome Technology, Gregoriadis, G., Ed.; CRC Press: Boca Raton, FL, 1986; Vol. III, Chapter 13. (27) De la Maza, A.; Parra, J. L. Biochem. J. 1994, 303, 907-914. (28) Lasic, D. D. Liposomes: From Physics to Applications; Elsevier/ North-Holland: Amsterdam, 1994; pp 63-107. (29) Ackman, R. G.; Mc Leod, C. A.; Banerjee, A. K. J. Planar Chromatogr. 1990, 3, 450-490. (30) Almog, S.; Litman, B. J.; Wimley, W.; Cohen, J.; Wachtel, E. J.; Barenholz, Y.; Ben-Shaul, A.; Lichtenberg, D. Biochemistry 1990, 29, 4582-4592. (31) Standard Methods for the Examination of Water and Wastewater; American Public Health Association: Washington, DC, 1985; Methods 512 B and 512 C, pp 581-588. (32) Lunkenheimer, K.; Wantke, D. Colloid and Polym. Sci. 1981, 259, 354-366.
de la Maza and Parra In the analysis of the equilibrium partition model proposed by Schurtenberger33 for bile salt/lecithin systems, Lichtenberg5 and Almog et al.30 have shown that, for a mixing of lipids in dilute aqueous media, the distribution of surfactant between the lipid phase and aqueous media obeys a partition coefficient K, given (in mM-1) by
K ) Re/[SW(1 + Re)]
(1)
where Re is the effective molar ratio of surfactant to phospholipid in bilayers: (Re ) SB/PL) and SW is the surfactant concentration in the aqueous medium (mM). Given that the range of phospholipid concentrations used in our investigation is similar to that used by Almog to test his equilibrium partition model, the K parameter has been determined using this equation. The determination of the Re, SW, and K parameters can be carried out on the basis of the linear dependence existing between the surfactant concentrations required to achieve these parameters and the phospholipid concentration in liposomes,23 which can be described by the equation
ST ) SW + RePL
(2)
where Re and the aqueous concentration of surfactant SW are in each curve respectively the slope and the ordinate at the origin (zero phospholipid concentration). The permeability alterations caused by the surfactant mixture were determined by monitoring the increase in the fluorescence intensity of the liposome suspensions due to the CF released from the interior of vesicles to the bulk aqueous phase. The fluorescence intensity measurements were taken 40 min after the addition of surfactant mixture to liposomes at 25 °C.26 The solubilizing perturbation produced by the surfactant mixture in PC liposomes was monitored by measuring the variations in static light scattering of these systems during solubilization.22 The overall solubilization can be mainly characterized by two parameters termed Re(sat) and Re(sol), according to the nomenclature adopted by Lichtenberg5 corresponding to the Re ratios at which light scattering starts to decrease with respect to the original value and shows no further decrease. These parameters corresponded to the surfactant/lipid molar ratios at which the surfactant (a) saturated liposomes and (b) led to a complete solubilization of these structures. Light-scattering measurements were made spectrofluorophotometrically 24 h after the addition of surfactant mixture to liposomes at 25 °C.22
Results and Discussion (A) Mean Vesicle Size and Stability of Liposome Suspensions. The mean vesicle size of liposome suspensions after preparation (phospholipid concentration ranging from 0.5 to 5.0 mM) varied little (around 200 nm). The polydispersity index (PI), defined as a measure of the width of the particle size distribution obtained from the “cumulants analysis”, remained in all cases lower than 0.1, indicating that the liposome suspensions showed a homogeneous size distribution in all cases. The size of the vesicles after the addition of equal volumes of PIPES buffer and equilibration for 24 h showed in all cases values similar to those obtained after preparation, with a slight increase in the PI (between 0.12 and 0.14). Hence, the liposome preparations appeared to be reasonably stable in the absence of surfactants under the experimental conditions used in solubilization studies. (B) Critical Micelle Concentration of Surfactant Mixture. Previously we determined the cmc’s of the surfactant mixture (mole ratio 1:1) and the single components in PIPES buffer. To this end, we studied the variation of the surface tensions of surfactant solutions as a function of the surfactant concentration. The values obtained were 0.050, 0.035, and 0.50 mM, respectively, for the surfactant mixture, NP(EO)10, and SDS. (33) Schurtenberger, P.; Mazer, N.; Ka¨nzig, W. J. Phys. Chem. 1985, 89, 1042-1049.
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Assuming that the thermodynamics of the micellation process for this surfactant mixture obeys the ideal solution theory, when monomer and micelles are in equilibrium in the system, the cmc value would fall on the line predicted by the relationship34
1/C12 ) X/C1 + (1 - X)/C2
(3)
where C12 is the cmc for the mixed micelle system of surfactant 1 (NP(EO)10) and the surfactant 2 (SDS), C1 is the cmc of surfactant 1, C2 is the cmc of surfactant 2, and X is the mole fraction of surfactant 1 in the mixture. From the values experimentally obtained the theoretical cmc for the mole fraction XNP(EO)10/SDS ) 0.5 was 0.065 mM. This value was higher than that experimentally obtained for the surfactant mixture (0.050 mM), and consequently, its mixed micelle formation showed a negative deviation with respect to the ideal behavior, in agreement with the results reported for this mixture.6-10 (C) Interaction of the NP(EO)10/SDS Mixture with Liposomes. To determine the time needed to obtain a constant level of CF release of liposomes in the range of PC concentration investigated (0.5 and 5.0 mM), a kinetic study of the interaction of the surfactant mixture with liposomes was carried out. Liposome suspensions were treated with this mixture at subsolubilizing concentration, and subsequent changes in permeability were studied as a function of time. Aproximately 40 min was necessary to achieve a constant level of CF release in all cases despite the fact that approximately 80% of the CF release took place during the initial 10 min. Thus, changes in permeability were studied 40 min after addition of surfactant to the liposomes at 25 °C. The CF release of liposome suspensions in the absence of surfactant in this period of time was negligible. To determine the K values at the subsolubilizing level, a systematic study of permeability changes caused by the addition of the surfactant mixture to liposomes was carried out for different lipid concentrations. Changes in CF release were determined 40 min after surfactant addition at 25 °C. The surfactant concentrations resulting in different percentages of CF release were graphically obtained and plotted versus the phospholipid concentration. An acceptable linear relationship was established in each case. The straight lines obtained correspond to the aforementioned eq 2, from which the Re and K parameters were determined. The results obtained including the free surfactant concentration (SW) and the regression coefficient of each straight line (r2) are given in Table 1. Different trends in the evolution of the Re and K parameters were observed as the percentage of CF increased. Thus, whereas Re progressively increased, the K values showed a maximum for 40% CF release. Furthermore, SW increased as the percentage of CF rose. Bearing in mind the cmc experimentally obtained for the surfactant mixture was 0.050 mM, SW showed always lower values than its cmc, thereby confirming for this mixture the generally admitted assumption for single surfactants that permeability alterations were determined by the action of surfactant monomers.5 The solubilizing interaction of these surfactant mixtures with liposomes was studied through the changes in the static light scattered by these systems 24 h after the addition of surfactant.1,35 An initial increase in the scattered intensity of the system was always observed (34) Cox, M. F.; Borys, N. F.; Matson, T. P. J. Am. Oil Chem. Soc. 1985, 62, 1139-1144. (35) Partearroyo, M. A.; Urbaneja, M. A.; Gon˜i, F. M. FEBS Lett. 1992, 302, 138-140.
Table 1. Surfactant to Phospholipid Molar Ratios (Re), Partition Coefficients (K) and Surfactant Concentrations in the Aqueous Medium (SW) Resulting in the Overall Interaction of the NP(EO)10/SDS Mixture with PC Liposomes (The regression coefficients of the straight lines obtained are also included) CF release %
SW (mM)
Re (mol/mol)
r2
K (mM-1)
10 20 30 40 50 60 70 80 90 100
0.003 0.006 0.008 0.009 0.011 0.014 0.018 0.021 0.024 0.026
0.040 0.088 0.128 0.150 0.188 0.230 0.276 0.315 0.354 0.390
0.996 0.991 0.998 0.993 0.996 0.992 0.995 0.998 0.995 0.993
12.82 13.49 14.19 14.56 14.46 13.37 12.05 11.41 10.92 10.80
light scattering %
SW (mM)
Re (mol/mol)
r2
K (mM-1)
100 90 80 70 60 50 40 30 20 10 0
0.050 0.050 0.051 0.051 0.052 0.052 0.053 0.053 0.053 0.053 0.053
0.61 0.74 0.85 0.97 1.08 1.19 1.30 1.42 1.53 1.64 1.75
0.995 0.997 0.992 0.993 0.999 0.994 0.992 0.996 0.998 0.992 0.995
7.57 8.50 9.01 9.66 9.99 10.45 10.66 11.07 11.41 11.72 12.01
due to the surfactant incorporation into bilayers. Additional surfactant amounts resulted in a fall in this intensity until a low constant value for bilayer solubilization. The surfactant concentrations for different lightscattering percentages were obtained by graphical methods for each PC concentration. Plotting these surfactant concentrations versus lipid concentration, curves were obtained, in which an acceptable linear relationship was also established in each case. The corresponding Re and K parameters were determined from these straight lines (eq 2) and are also given together with their regression coefficients (r2) in Table 1. It should be noted that both the Re and K parameters progressively increased as the percentage of static light scattering decreased. From these findings, we may also assume that an increasing degree of partitioning of surfactant molecules into the liposomes governs their association with the PC building these structures to form mixed micelles. Comparison of surfactant mixture partition coefficients with those reported for SDS,23 shows that the mixture has a much higher affinity with bilayers (approximately 5-7 times more) than that shown by the anionic components. Similar comparison for the Re parameters shows that the ability of this mixture to saturate and solubilize PC liposomes is also clearly higher than that reported for SDS.22,23 These findings open new avenues in the biological application of this mixture with respect to the use of the SDS enhancing its efficacy in the interaction with phospholipid bilayers both at subsolubilizing and solubilizing levels. The use of this surfactant mixture may also be considered as a new alternative procedure in the denaturation and potential solubilization of biological membranes. There appears to be some discrepancy between the increased ability of this mixture to saturate or solubilize liposomes and the reduction of its irritancy power versus the anionic component.21 The complex biochemical mechanisms involved in the skin irritancy process, in which the stratum corneum lipids (SCL) play an essential role in controlling the permeability to water penetration,
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together with the fact that the SCL are virtually devoid of phospholipids,36 may in part account for this discrepancy. The finding that the free surfactant concentration of the surfactant mixture was always comparable to its cmc supports, for this mixture, the generally admitted assumption (for single surfactants) that the concentration of free surfactant must reach the cmc for solubilization to occur5 and underlines the influence of the negative synergism of the NP(EO)10/SDS mixed micelles on the aqueous surfactant concentration needed to saturate or solubilize PC liposomes. (i) Relationship between the Re Parameter, K, and SW. Figure 1A shows the variation in K versus Re during the overall surfactant/liposome interaction. A marked initial increase in K was observed as Re rose, reaching a maximum (K ) 14.56) for Re ) 0.150 (corresponding to 40% CF release). Increasing Re values resulted in a fall in K values up to 100% CF release, this decrease being more pronounced in the interval 50-70% CF release. Thus, the increase in Re resulted in two opposite effects on the bilayer/water partitioning of the surfactant mixture. At low Re, K first increased possibly because only the outer
vesicle leaflet was available for interaction with surfactant molecules, the binding of additional surfactant to the bilayer being hampered up to approximately 70% CF release (abrupt fall in K). The 40% CF release may be correlated with the saturation of the outer vesicle leaflet by the surfactant. Increasing Re (Re between 0.276 and 0.390 corresponding to a low decrease in K) led to an increased rate of flip-flop of the surfactant molecules (or permeabilization of the bilayers to surfactant), thus also making the inner monolayer available for interaction with added surfactant mixture. These findings are in agreement with the results reported by Schubert et al. for the interaction of sodium cholate/PC liposomes.37 The extrapolation of the curve (discontinuous line) led approximately to the initial K value for solubilization (100% light scattering, for Re(sat) ) 0.61). The subsequent increase in Re resulted again in a rise in K up to Re(sol) ) 1.75, which corresponded to the solubilization of liposomes. The most striking result is the observation that although the evolution in K was similar to that observed for SDS/PC liposomes, the maximum bilayer/ water partitioning of the surfactant mixture was reached in a more wide Re range (Re values between 0.130 and 0.190) and for K values approximately 5-6 times higher than those reported for SDS.23 Figure 1B shows the variation in SW versus Re throughout the surfactant/liposome interaction. A progressive increase in SW was observed as Re rose up to 100% CF release. The extrapolation of the curve (discontinuous line) led approximately to the initial SW value for solubilization (100% light scattering, for Re(sat)), which corresponded to the cmc of the surfactant mixture. As discussed above, these findings confirm that permeability alterations were determined by the action of surfactant monomer and support the generally admitted assumption for single surfactants that the concentration of free surfactant must reach the cmc for solubilization to occur.5 The increase in Re resulted again in a slight rise in SW up to Re(sol), which corresponded to the complete solubilization of liposomes via mixed formation. (ii) Influence of Each Surfactant Mixture Component in the Subsolubilizing Process. Systematic analysis of NP(EO)10 and SDS concentrations in the aqueous medium (SW(NP(EO)10) and SW(SDS)) was carried out for different transition stages at the subsolubilizing level in order to determine the influence of each component in this interaction. Equilibrated surfactant/PC mixed vesicular dispersions for different percentages of CF release and 100% light scattering (Re(sat)) were analyzed for PC.29 The dispersions were then spun at 140.000G for 4 h to remove the vesicles.30 The supernatants of all the mixed dispersions were tested again for PC and single surfactants. A similar pattern was observed for various PC concentrations (0.5-5.0 mM): up to the surfactant concentration for bilayer saturation (Re(sat)) no PC became solubilized. These findings are in agreement with the results reported by Almog et al. for octyl glucoside/PC systems30 and confirm that the total surfactant concentration must reach one corresponding to Re(sat) for PC solubilization to occur.5 Figure 2 shows the variation in free surfactant concentration for NP(EO)10 and SDS, versus surfactant concentration at the subsolubilizing level (PC concentration 5.0 mM). The percentage of CF released is also indicated. It is noteworthy that in the range of CF release between 10% and 50% the free surfactant concentrations for NP(EO)10 and SDS showed respectively higher and
(36) Long, S. A.; Wertz, P. W.; Strauss, J. S.; Downing, D. T. Arch. Dermatol. Res. 1985, 277, 284-292.
(37) Schubert, R.; Beyer, K.; Wolburg, H.; Schmidt, K. H. Biochemistry 1986, 25, 5263-5269.
Figure 1. (A, top) Variation in the partition coefficients (K) versus the effective surfactant to phospholipid molar ratio (Re) during the overall interaction between the NP(EO)10/SDS mixture and PC liposomes. (B, bottom) Variation in the free surfactant concentration versus the effective surfactant to phospholipid molar ratio (Re) during the overall interaction between the NP(EO)10/SDS mixture and PC liposomes.
Liposome Solubilization by NP(EO)10/SDS Mixtures
Langmuir, Vol. 12, No. 14, 1996 3397 Table 2. Mean Size Distributions (nm) and Polydispersity Indexes of Surfactant-PC Aggregates (Vesicles or Micelles) Resulting in the Overall Interaction of the NP(EO)10/SDS Mixture with PC Liposomes curve distribution (particle number)
CF release %
typea
0 10 20 30 40 50 60 70 80 90 100
M M M M M M M M M M M
1st peak nm %
2nd peak nm % 200 249 301 342 368 380 397 400 408 414 413
23.0 23.1 23.2 23.3 23.0 23.3 23.2 22.8 23.1 23.3 23.2
curve distribution (particle number)
Figure 2. Variation in the free surfactant concentration for the NP(EO)10/SDS mixture (9) and the single surfactants NP(EO)10 (O) and SDS (b) versus the total surfactant concentration at the subsolubilizing level (PC concentration 5.0 mM).
lower values than those theoretically predicted for the mixture, whereas their concentrations between 60% CF release and SW(sat) showed inversely lower and higher values than those predicted. The theoretical SW values for each component were calculated from those corresponding to the surfactant mixture (eq 2, given in Table 1), which are also indicated in Figure 2 (curve (9)). On the basis of these findings we may assume that, between 10 and 50% CF release, NP(EO)10 and SDS exhibited respectively lower and higher affinities with bilayers than those theoretically predicted, whereas in the subsequent subsolubilizing steps their affinities with bilayers showed the opposite tendency. These results highlight the major role played by SDS in the initial sublytic steps as well as the dominant influence of NP(EO)10 in the subsequent steps up to bilayer saturation. The surfactant composition during liposome solubilization was not determined given that these analyses were hindered by the presence of PC in the aqueous medium. Comparison of Figures 1A and 2 shows that the maximum bilayer/water partitioning of surfactant mixture (CF release ranging from 30 to 50%) may be correlated with the increased presence of SDS in the bilayers (outer vesicle leaflet), in agreement with the results reported by Schubert et al. for sodium cholate,37 whereas the decrease in K between 60 and 100% CF release would be correlated with the major presence of NP(EO)10 in bilayers. These findings could explain, in part, the reduced deleterious effect caused by the NP(EO)10/SDS mixture in different tissues compared with that of pure SDS,21 in spite of the increased activity of this mixture (from PC liposomes) with respect to that of SDS. As reported by Downing et al.,3 the deleterious effect caused by SDS on human skin is due to the high degree of partitioning of this surfactant into stratum corneum lipid liposomes, which affects the properties of the lipid lamellae that constitute the epidermal permeability barrier. Assuming a certain parallelism in the behavior of the stratum corneum lipid liposomes and PC liposomes, the presence of NP(EO)10 in the mixture could control the level of SDS partitioning in the human stratum corneum. (iii) Dependence of the Surfactant-PC Aggregate Size, CF Release, and Static Light Scattering on Re. A sys-
light 1st peak scattering % typea nm % 100 90 80 70 60 50 40 30 20 10 0 a
M B B B B B B B B M M
51 51 51 51 51 51 51 51 51 51
2.0 4.0 5.4 5.9 6.5 7.6 9.5 13.6 23.3 23.2
2nd peak nm % 361 358 345 318 293 260 228 181 143
23.3 21.4 19.4 18.1 17.6 16.9 16.0 14.0 10.0
average mean (nm)
polydispersity index
200 249 301 342 368 380 397 400 408 418 417
0.107 0.123 0.135 0.141 0.146 0.157 0.162 0.167 0.173 0.181 0.188
average mean polydispersity (nm) index 361 332 295 257 232 202 171 128 90 51 51
0.202 0.218 0.248 0.242 0.237 0.226 0.214 0.210 0.203 0.169 0.154
M, monomodal; B, bimodal.
tematic investigation based on dynamic light-scattering measurements of surfactant-PC aggregates was carried out throughout the process to elucidate the dependencies between the size of these aggregates (vesicles or micelles), the changes in the percentages of both CF release and the static light scattering of the system, and Re. The values obtained for 5.0 mM PC are given in Table 2. A progressive growth of vesicles was detected as the percentage of CF release rose; the maximum increase was attained for 90100%. The growth of vesicles occurred in a few seconds with not much change over a time scale of hours. As for static light-scattering variations, the 100% corresponding to Re(sat) produced a slight fall in the vesicle size albeit with a monomodal distribution. When the light scattered by the system decreased, a sharp distribution curve appeared approximately at 51 nm, which corresponded to a new particle size distribution (surfactant-PC mixed micelles). The curve for these small particles rose until 10% scattered light, exhibiting at this point again a monomodal distribution, which corresponded to the surfactant/PC mixed micelles (particles of 51 nm). Figure 3A shows the variation in both the percentage of CF release and the vesicle size of the liposomes versus Re at the subsolubilizing level. The initial increase in Re (Re up to 0.190) led to a marked increase in both the percentage of CF release (linear increase) and the size of the vesicles. However, Re values exceeding 0.200 resulted in a lower growth of vesicles, which achieved a maximum for 90-100% CF (Re ranging between 0.35-0.40). Considering that more than 80% of the permeability alterations occurred in the initial interaction step (10 min) and that the growth of vesicles took place in a few seconds after the addition of surfactant to liposomes, we may assume that, for Re values lower than 0.190, the growth
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Langmuir, Vol. 12, No. 14, 1996
de la Maza and Parra
Figure 3B shows the variation in the percentage of static light scattering and the surfactant-PC aggregate size (average mean) versus Re at the solubilizing level. The increase in Re produced a similar linear decrease in both parameters. Thus, during solubilization a direct dependence between these parameters and the composition of the surfactant-PC aggregates (Re) was established.
Figure 3. (A) Variation in the percentage of CF release (0) and the vesicle size (O) of the liposomes versus Re at subsolubilizing level. (B) Variation in the percentage of static light scattering (0) and the surfactant-PC aggregate size (O) of the surfactant-PC aggregates versus Re at the solubilizing level.
of vesicles was directly correlated with the leakage of entrapped CF, both parameters being dependent on the bilayer composition (Re). Given that the saturation of the outer vesicle leaflet was achieved in the same Re range (K maximum for Re values ranging from 0.130 to 0.190 in Figure 1A), we may assume that the increasing presence of surfactant molecules in the outer vesicle leaflet (particularly SDS) was responsible for the changes in both the fluidity and the growth of the vesicles. However, the subsequent increase in Re (increase in the rate of flip-flop of the surfactant molecules) does not produce significant changes in the vesicle fluidification trend (subsequent linear increase in the percentage of CF release). The low growth of vesicles in this phase transition could also be attributed to the aforementioned increase in the permeabilization of the bilayers to surfactant.
Conclusions The experimental results provide some new insights concerning the aggregative states of equimolecular mixtures of NP(EO)10/SDS with phosphatidylcholine liposomes. This mixture showed during the overall process clearly increased affinity with bilayers and higher ability to saturate or solubilize PC liposomes than that reported for the anionic component.23 At the subsolubilizing level, a maximum bilayer/water partitioning of the surfactant mixture (K) appeared at Re ranging from 0.130 to 0.190 (30-50% CF release). This maximum may be correlated with the increased presence of SDS in the outer vesicle leaflet, whereas the decrease in K between 60 and 100% CF release may be related to the greater presence of NP(EO)10 in bilayers. These findings could explain, in part, the reduced deleterious effect caused by the surfactant mixture in different tissues compared with that of pure SDS, in spite of its increased activity (from PC liposomes) with respect to that of SDS. When Re was lower than 0.190, the growth of vesicles was directly correlated with the leakage of entrapped CF, both parameters being dependent on the bilayer composition (Re). In this interacion step, changes in the fluidity and size of vesicles were due to the increasing presence of surfactant molecules in the outer vesicle leaflet (particularly SDS). A linear dependence was also established at the solubilizing level between the decrease in both the surfactant-PC aggregate size and the static light scattering of the system and the composition of these aggregates (Re). The fact that the free surfactant concentration at subsolubilizing and solubilizing levels was respectively lower than and similar to the surfactant mixture cmc indicates that permeability alterations and solubilization were determined respectively by the action of the surfactant monomer and by the formation of mixed micelles. This finding supports the generally admitted assumption, for single surfactants, that SW must reach the cmc for solubilization to occur and highlights the influence of the negative synergism of this mixture on the SW needed to saturate or solubilize liposomes. The application of this mixture in biological domains opens up new avenues with respect to the use of SDS and may be considered as a new alternative procedure in solubilization and reconstitution of biological membranes. Acknowledgment. We are grateful to Mr. G. von Knorring for expert technical assistance. This work was supported by funds from DGICYT (Direccio´n General de Investigacio´n Cientı´fica y Te´cnica) (Prog. no. PB94-0043), Spain. LA950878H
