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Solubilization of phosphatidylcholine liposomes by the amphoteric surfactant dodecyl betaine
Chemistry and Physics of Lipids 94 (1998) 71 – 79
Solubilization of phosphatidylcholine liposomes by the amphoteric surfactant dodecyl betaine A. de la Maza *, O. Lopez, L. Coderch, J.L. Parra Departamento de Tensioacti6os, Consejo Superior de In6estigaciones Cientı´ficas (C.S.I.C.), Centro de In6estigacio´n y Desarrollo (C.I.D.), Calle Jordi Girona 18 -26, 08034 Barcelona, Spain Received 19 January 1998; received in revised form 28 April 1998; accepted 30 April 1998
Abstract The interaction of the amphoteric surfactant N-dodecyl-N,N-dimethylbetaine (C12-Bet) with phosphatidylcholine (PC) liposomes was investigated. Permeability alterations were detected as a change in 5(6)-carboxyfluorescein (CF) released from the interior of vesicles and bilayer solubilization as a decrease in the static light-scattering (SLS) of the system. At sublytic level a initial maximum in the bilayer/water partitioning (K) followed by an abrupt decrease of this parameter occurred as the surfactant to lipid molar ratio (Re) rose. At lytic level a direct dependence was established between both parameters. The fact that the free surfactant concentration at sublytic and lytic levels showed values lower than and similar to its critical micelle concentration indicates that permeability alterations and solubilization were determined, respectively, by the action of surfactant monomer and by the formation of mixed micelles. A direct correlation occurred in the initial interaction steps (up to 50% CF release) between the growth of vesicles their fluidity and Re. A similar direct dependence was established during solubilization (up to 30% SLS) between the fall in both the surfactant-lipid aggregate size, the SLS of the system and Re. This surfactant showed higher capacity to solubilize PC liposomes than that reported by the commonly used non-ionic surfactants octyl glucoside and Triton X-100 and by the anionic one sodium dodecyl sulfate. © 1998 Elsevier Science Ireland Ltd. All rights reserved.
Abbre6iations: PC, phosphatidylcholine; C12-Bet, N-dodecyl-N,N-dimethylbetaine; PIPES, piperazine-1,4 bis(2-ethanesulphonic acid); CF, 5(6)-carboxyfluorescein; SLS, static light-scattering; DLS, dynamic light-scattering; PI, polydispersity index; Re, effective surfactant/lipid molar ratio; ReSAT, effective surfactant/lipid molar ratio for liposome saturation; ReSOL, effective surfactant/lipid molar ratio for liposome solubilization; K, bilayer/aqueous phase surfactant partition coefficient; SW, surfactant concentration in the aqueous medium; SB, surfactant concentration in the bilayers; r2, regression coefficient; TLC-FID, thin-layer chromatography/flame ionization detection system; CMC, critical micellar concentration. * Corresponding author. Tel.: +34 3 4006161; fax: + 34 3 2045904. 0009-3084/98/$19.00 © 1998 Elsevier Science Ireland Ltd. All rights reserved. PII S0009-3084(98)00045-0
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Keywords: Phosphatidylcholine liposomes; N-dodecyl-N,N-dimethylbetaine; Permeability alterations and bilayer solubilization; Carboxyfluorescein release; Static light-scattering changes; Effective surfactant to lipid molar ratios; Surfactant partition coefficients
1. Introduction Liposomes are aqueous lipid dispersions organized as bilayers which are widely used as simplified membrane models (Lasic, 1993; Sternberg, 1995). A number of studies have been devoted to the understanding of the principles governing the interaction of surfactants with these structures (Kragh-Hansen et al., 1993; Ruiz et al., 1994; Polozava et al., 1995; Inoue, 1996; Silvander et al., 1996; Wenk et al., 1997). This interaction leads to the breakdown of lamellar structures and the formation of lipid-surfactant mixed micelles. A significant contribution in this area has been made by Lichtenberg (Lichtenberg et al., 1983) who postulated that the effective surfactant to lipid ratio (Re) producing liposome saturation and solubilization depends on the surfactant critical micelle concentration (CMC) and on the bilayer/aqueous medium surfactant distribution coefficients (K). Surface-active betaines are internally compensated quaternary ammonium compounds which differ from quaternary ammonium salts in that they do not have a mobile anion. These compounds are among the fastest-growing group of amphoteric surfactants due to their very low irritation levels and their excellent capacity to promote mildness in cosmetic formulations. These compounds have also been found to be effective in reducing the potential irritation of alkyl sulfates, alkyl ether sulfates and soaps (Cooper and Berner, 1985). However, the biological toxicity of these surfactants increases sharply with increasing chain length of their lipophile and shows an inverse correlation with the CMC of these surfactants (Ernst and Miller 1982). We previously studied the interaction of a series of alkylbetaines with PC unilamellar liposomes and with those formed by lipids modeling the stratum corneum lipid composition (de la Maza et al., 1991; de la Maza and Parra, 1993; de la Maza
et al., 1997). In the present work we seek to extend these investigations by characterizing the overall interaction of the N-dodecyl-N,N-dimethylbetaine (C12-Bet) surfactant with phosphatidylcholine (PC) liposomes. Knowledge of the Re and K parameters throughout the interaction process could be useful in improving our understanding of the complex phenomenon involved in the lamellar to micelle transitions during the solubilization of PC liposomes by this so used amphoteric surfactant and in establishing a criterion for the evaluation of its activity in biological membranes.
2. Materials and methods The amphoteric surfactant C12-Bet was specially prepared by (Albright and Wilson, Warley, West Midlands, UK) with an active matter in aqueous solution of 30% and an amino free contents of 0.20%. Piperazine-1,4 bis(2-ethanesulphonic acid) (PIPES) was obtained from (Merck, Darmstadt, Germany). PIPES buffer was prepared as 10 mM PIPES containing 110 mM Na2SO4 and adjusted to pH 7.20 with NaOH. The starting material 5(6)-carboxyfluorescein (CF), was obtained from (Eastman Kodak, Rochester, NY) and further purified by a column chromatographic method (Weinstein et al., 1986). Polycarbonate membranes and membrane holders were purchased from Nucleopore (Pleasanton, CA). PC was purified from egg lecithin (Merck) according to the method of Singleton (Singleton et al., 1965) and was shown to be pure by thin-layer chromatography (TLC). Reagent grade organic solvents was supplied by Sigma (St. Louis, MO).
2.1. Preparation of liposomes and solubilizing parameters PC liposomes (PC conc. 0.5–5.0 mM) of a defined size (about 200 nm) were prepared in
A. de la Maza et al. / Chemistry and Physics of Lipids 94 (1998) 71–79
PIPES buffer by extrusion of large unilamellar vesicles previously obtained by reverse phase evaporation (de la Maza and Parra, 1993). Liposomes containing CF (110 mM) were freed of unencapsulated fluorescent dye by passage through Sephadex G-50 medium resin (Pharmacia, Uppsala, Sweden) by column chromatography. The size distribution and polydispersity index (PI) of liposomes and surfactant-PC aggregates was determined with a photon correlator spectrometer (Malvern Autosizer 4700c PS/MV, Malvern, UK) using a Ar laser source (wavelength of 488 nm) (de la Maza and Parra, 1994a,b). The liposome PC concentration was determined by TLC-FID (Ackman et al., 1990; de la Maza and Parra, 1995a). The partition coefficients (K) of C12-Bet between the lipid bilayers and aqueous media was determined using the equation: K= Re/SW[1+ Re]
(1)
where Re is the effective molar ratio of surfactant to phospholipid in the bilayers and surfactant concentration (SW) in the aqueous medium (mM) (de la Maza and Parra, 1995a), in accordance with Lichtenberg et al. (1983) and Almog et al. (1990). The determination of Re and SW was carried out on the basis of the linear dependence existing between the surfactant concentrations required to achieve these parameters and the lipid concentration (mM) (L), which is described by the equation: ST = SW +Re. [L]
(2)
where Re and SW are in each curve, respectively, the slope and the ordinate at the origin (zero PC concentration). The permeability changes caused by C12-Bet in CF-containing PC liposomes were determined by monitoring the increase in the fluorescence intensity of liposomes due to the CF released from the interior of vesicles to the bulk aqueous phase (de la Maza et al., 1991). The solubilizing perturbation produced by C12Bet in liposomes was determined by monitoring the SLS changes of the surfactant-liposome sys-
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tem 24 h after the surfactant addition (de la Maza and Parra, 1993). This process was characterized by two parameters termed ReSAT and ReSOL, according to the nomenclature adopted by Lichtenberg et al. (1983), corresponding to the Re ratios at which the surfactant saturated liposomes and led to the complete solubilization of these structures.
3. Results and discussion
3.1. Stability of liposome suspensions The mean vesicle size of liposomes after preparation varied little (around 200 nm) and the PI remained always lower than 0.1 indicating that liposomes showed a homogeneous size distribution. The mean size of liposomes 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, liposomes appeared to be reasonably stable in the absence of surfactant under the experimental conditions used in solubilization studies.
3.2. Interaction of C12 -Bet with liposomes We previously studied the validity of the equilibrium partition model proposed by Lichtenberg et al. (1983) and Almog et al. (1990) based on the Eq. (1) for the surfactant investigated. According to these authors this equation may be expressed by: L/SB = (1/K)(1/SW)− 1. Hence, this validity requires a linear dependence between L/SB and 1/SW; this line should have a slope of 1/K, intersect with the L/SB axis at − 1 and intersect with the 1/SW at K (Almog et al., 1990). To test the validity of the model for C12-Bet unilamellar PC liposomes were mixed with varying subsolubilizing concentrations of this surfactant (ST). The resultant surfactant-containing vesicles were then spun at 140000g at 20°C for 4 h to remove the vesicles. No PC was detected in the supernatants. The surfactant concentration in the supernatants (SW) was determined by HPLC (Kondoh and Takano, 1986) and its concentration in the lipid
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A. de la Maza et al. / Chemistry and Physics of Lipids 94 (1998) 71–79
bilayers was calculated (SB =ST −SW). The results of the experiments in which SB and SW were measured (carried out in the same range of PC and surfactant concentrations used to determine K) were plotted in terms of the dependence of L/SB on 1/SW. A straight line was obtained (r2 = 0.98), which was dependent on L and intersected with the L/SB axis always at −0.97 90.10. Both the linearity of this dependence and the proximity of the intercept to − 1 support the validity of this model to determine K for this surfactant. To determine the time needed to obtain a constant level of CF release trapped into liposomes, a kinetic study of the interaction of C12-Bet with these bilayer structures was carried out (PC concentration ranging from 0.5 to 5.0 mM). Liposomes were treated with C12-Bet at sublytic concentrations (affecting only the permeability of bilayers) and subsequent changes in permeability were studied as a function of time. The CF release was always a biphasic process in which about 40 min was needed to achieve CF release plateaux and approximately 75% of CF release took place during the initial 10 min. This biphasic behavior suggests that the release of the CF trapped into the vesicles was produced through holes created in the membrane, in agreement with the concept of transient channels suggested by Edwards and Almgren in the surfactant-mediated increase in PC membrane permeability due to surfactants (Edwards and Almgren, 1990, 1992). The incorporation of C12-Bet monomers to membranes may directly induce the formation of hydrophilic pores in these structures or merely stabilize transient holes (Lasic, 1993). The long time course required to reach a constant CF release could also be related to permeation or flip-flop of surfactant across the vesicle membrane. A similar biphasic behavior has been reported for the interaction of various non-ionic surfactants with PC liposomes (de la Maza and Parra 1994a,b). Bearing in mind these findings, changes in permeability were studied 40 min after addition of surfactant to the liposomes at 25°C. The CF release in the absence of C12-Bet in this period of time was negligible. To determine the Re and K parameters at sublytic level a systematic study of CF release changes caused by the addition of C12-Bet to
liposomes was carried out (PC concentration ranging from 0.5 to 5.0 mM). The C12-Bet concentrations resulting in different CF release percentages were obtained and plotted versus PC concentration. An acceptable linear relationship was established in each case. The straight lines obtained correspond to the Eq. (2) from which the Re and K parameters were determined. The results for 100% CF release, including the SW value and the straight line regression coefficient (r2) are given in Table 1. The fact that the SW value for 100% CF release (0.72 mM) was lower than the C12-Bet CMC (1.25 mM, de la Maza and Parra, 1993) confirms for this surfactant the generally admitted assumption that permeability alterations were determined by the action of surfactant monomers (Lichtenberg et al., 1983). To determine the Re and K parameters at lytic level a systematic study of the SLS changes of the surfactant/PC systems caused by the addition of C12-Bet to liposomes was carried out (PC concentration ranging from 0.5 to 5.0 mM). An initial increase in the SLS intensity of the system was always observed due to the surfactant incorporation into bilayers. Additional surfactant amounts led to a fall in this intensity until a low constant value for liposome solubilization. The C12-Bet concentrations for different SLS percentages were obtained and plotted versus PC concentration. An acceptable linear relationship was also established in each case. The corresponding Re and K parameters were determined from these straight lines (Eq. (2)). The values for 0% SLS, including the SW value and r2 of this straight line are given in Table 1. The fact that the SW value for 0% SLS was comparable to the surfactant CMC (1.25 mM) supports that the free surfactant concentration must reach its CMC for solubilization to occur (Lichtenberg et al., 1983).
3.2.1. Relationship between the Re parameter, SW and K Fig. 1 shows the variation of SW with Re throughout the C12-Bet/liposomes interaction (vesicles or mixed micelles). A marked increase in SW occurred as Re rising up to the Re for 100% CF release (SW value 0.72 mM). The extrapolation of this curve (shaded area) led approximately
M, monomodal.
100% CF release 0% SLS
0.72 1.32
SW (mM)
0.55 1.43
Re mole/mole
0.995 0.993
r2
0.49 0.44
K (mM−1)
M M
Type
— 11
— 100
308 —
100 —
%
nm
nm
%
2nd peak
1st peak
Curve distribution (particle number)
308 11
Average (nm)
0.179 0.143
Polydispersity index
Table 1 Surfactant to Re, K, SW, nm and PIs of surfactant-lipid aggregates (vesicles or micelles) measured by DLS corresponding to 100% CF release and 0% SLS in the interaction of C12-Bet with PC liposomes. The regression coefficients (r2) of the straight lines obtained are also included
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A. de la Maza et al. / Chemistry and Physics of Lipids 94 (1998) 71–79
Fig. 1. Variation in the free SW versus the effective surfactant to Re during the overall interaction between C12-Bet and PC liposomes.
to the initial SW value for solubilization (100% SLS corresponding to the ReSAT) (SW =1.27 mM), which corresponded approximately to the surfactant CMC (1.25 mM). The increase in Re resulted in a slight increase in SW up to ReSOL, which corresponded to the complete solubilization of liposomes via formation of lipid-surfactant mixed micelles (Re=1.43, for 0% SLS).
Fig. 2. Variation in K versus the effective surfactant to Re during the overall interaction between C12-Bet and PC liposomes.
Fig. 2 shows the variation in K versus Re during the overall interaction of C12-Bet with liposomes. An initial increase in K was observed as Re rose, reaching a maximum (K= 1.31) for Re= 0.31 (20% CF release). Increasing Re values resulted in a abrupt fall in K values up to Re = 0.55 (100% CF release). This fall was more pronounced in the Re interval 0.31–0.48). Thus, the increase in Re resulted in two opposite effects on the bilayer/water partitioning of C12-Bet. 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 Re= 0.48 (abrupt fall in K). Increasing Re values, (Re between 0.50–0.55, 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. These findings are in agreement with those reported by Schubert et al. for the interaction sodium cholate with PC liposomes (Schubert et al., 1986). The Re value of 0.31 (20% CF release) may be correlated with the saturation of the outer vesicle leaflet by C12-Bet. The extrapolation of the curve (shaded area) led approximately to the initial K value for liposome solubilization. Further increase in Re resulted again in a rise in K up to ReSOL, which corresponded to the solubilization of these bilayer structures via mixed micelles formation.
3.2.2. Dependence of the surfactant-lipid aggregate size, CF release and SLS on Re A systematic DLS study of surfactant-lipid aggregates was carried out throughout the process to elucidate the dependencies of the size of these aggregates (vesicles or micelles) and the changes in both the CF release and the SLS of the system on Re. The DLS values obtained for 100% CF release and 0% SLS (PC concentration 5.0 mM) are also given in Table 1. Micellar C12-Bet solutions before they were mixed with liposomes showed a QELS peak at 8 nm in the experimental conditions used (Ar laser source with a wavelength of 488 nm), in accordance with Swarbrick and Daruwala, who studied the weight-average
A. de la Maza et al. / Chemistry and Physics of Lipids 94 (1998) 71–79
aggregation number of this surfactant in water (Swarbrick and Daruwala, 1970). The fact that this peak was not detected when this surfactant interacted with PC liposomes is attributable to the preferential formation of lipid-surfactant mixed micelles, in agreement with our previous results for the interaction of various surfactants with PC liposomes (de la Maza and Parra, 1994a,b). The maximum growth of vesicles (formation of mixed vesicles) was reached for 100% CF release (308 nm, see Table 1) and the growth of vesicles occurred in a few s with little change over a period of h. As for SLS variations, the 100% (ReSAT) resulted in a slight decrease in the vesicle size albeit with a monomodal distribution. When the SLS decreased, a sharp distribution curve appeared at 9 nm, which corresponded to a lipidsurfactant mixed micelles. The assumption that these small particles corresponded to mixed micelles is based on the fact that small unilamellar vesicles have diameters in the range of 15 – 25 nm (New, 1990) and consequently are always higher than these small particles. The curve for these small particles rose up to 0% SLS, exhibiting at this point again a monomodal distribution (11 nm) for only mixed micelles (see Table 1). Thus, in the interval 90– 10% SLS vesicle or vesicle fragments coexisted with mixed micelles, in agreement with Almog et al. (1990) when studied the interaction of PC liposomes with octyl glucoside. Fig. 3 shows the variation in both the percentage of CF release and vesicle size of liposomes versus Re at sublytic level. The increase in Re led initially to a progressive increase in both parameters. Re values exceeding 0.48 (60% CF release) resulted in a lower growth of vesicles, which coexisted with an abrupt increase in the release of the CF trapped into liposomes. Given that approximately 80% of the permeability changes occurred in the initial interaction steps and that the vesicle growth took place in a few s after surfactant addition we may assume that for Re values lower than 0.48 the growth of vesicles was related to the leakage of entrapped CF. The coexistence in the range of Re values from 0.50 to 0.55 of slight changes in the vesicle size and the abrupt increase in the fluidity of bilayers could be correlated with the increase in the rate of flip-flop of the surfactant molecules into liposomes.
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Fig. 3. Variation in the percentage of CF release and vesicle size of PC liposomes treated with C12-Bet versus Re at sublytic level. ( ) CF release, ( ) vesicle size.
Fig. 4 shows the variation in the percentage of SLS and the surfactant-lipid aggregate size (average mean) determined by DLS versus Re at lytic
Fig. 4. Variation in the percentage of static light scattering and surfactant-lipid aggregate size (measured by DLS) of PC liposomes treated with C12-Bet versus Re at lytic level. ( ) Static light scattering, ( ) Surfactant-lipid aggregate size.
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A. de la Maza et al. / Chemistry and Physics of Lipids 94 (1998) 71–79
level. The increase in Re produced a linear decrease in SLS these aggregates together with a fall in their average size up to a SLS intensity of the system of about 30%. Thus, in the range of Re values between 0.63 – 1.24 a direct correlation between the percentage of SLS and the surfactantlipid aggregate size was established. This direct dependence, together with the abrupt increase in the distribution curve for lipid-surfactant mixed micelles in the last interaction steps (see Table 1) emphasize the suitability of C12-Bet in the solubilization of these bilayer structures. Comparison of the Re values obtained for C12Bet with those reported for the interaction of the non-ionic surfactant octyl glucoside with PC liposomes (de la Maza and Parra, 1994b) reveals that the C12-Bet exhibited both at sublytic and lytic levels higher ability to interact with PC liposomes (lower Re values). It is still more surprising to verify that C12-Bet shows a clearly higher capacity than Triton X-100 to solubilize PC liposomes in the same experimental conditions (lower Re values at lytic level) (de la Maza and Parra, 1994a) Comparison of the present Re values with those reported for the interaction of the anionic surfactant sodium dodecyl sulfate (SDS) with PC liposomes in the same experimental conditions (de la Maza and Parra, 1995b) indicates that C12-Bet had higher capacity to solubilize PC liposomes, in spite of its very low irritation level and its capacity to promote mildness in cosmetic formulations. This finding is specially surprising given that the SDS produces toxicological effects in vivo tests (Singer and Pittz, 1985) and that C12-Bet has been found to be effective in reducing the potential irritation of SDS (Cooper and Berner, 1985). These findings open up new possibilities in the application of this amphoteric surfactant as promising solubilizing agent of biological membranes.
Acknowledgements This work was supported by funds from DGICYT (Direccio´n General de Investigacio´n Cientı´fica y Te´cnica) (Prog. No. PB94-0043), Spain. We are grateful to G. von Knorring for expert technical assistance.
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A. de la Maza et al. / Chemistry and Physics of Lipids 94 (1998) 71–79 Lasic, D.D., 1993. Liposomes as drug delivery system. In: Lasic, D.D. (Ed.), Liposomes: From Physics to Applications. Elsevier, Amsterdam, pp. 298–301. Lichtenberg, D., Robson, R.T., Dennis, E.A., 1983. Solubilization of phospholipids by detergents. Structural and kinetic aspects. Biochim. Biophys. Acta 737, 285–304. New, R.R.C., 1990. Liposomes: A Practical Approach. IRL Press, Oxford, p. 23. Polozava, A.I., Dubachev, G.E., Simonova, T.N., Barsukov, L.I., 1995. Temperature induced micellar-lamellar transformation in binary mixtures of saturated phosphatidylcholines with sodium cholate. FEBS Lett. 358, 17–22. Ruiz, M.B., Prado, A., Gon˜i, F.M., Alonso, A., 1994. An assessment of the biochemical applications of the non-ionic surfactant Hecameg. Biochim. Biophys. Acta 1193, 301– 306. Schubert, R., Beyer, K., Wolburg, H., Schmidt, K.H., 1986. Structural changes in membranes of large unilamellar vesicles after adding of sodium cholate. Biochemistry 25, 5263 – 5269. Silvander, M., Karlsson, G., Edwards, K., 1996. Vesicle solubilization by alkyl sulfate surfactants: A Cryo-TEM study of the vesicle to micelle transition. J. Colloid Interface Sci. 179, 104 – 113. Singer, E.J., Pittz, E.P., 1985. Interaction of surfactants with epidermal tissues. Biochemical and toxicological aspects.
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Solubilization of phosphatidylcholine liposomes by the amphoteric surfactant dodecyl betaine A. de la Maza *, O. Lopez, L. Coderch, J.L. Parra Departamento de Tensioacti6os, Consejo Superior de In6estigaciones Cientı´ficas (C.S.I.C.), Centro de In6estigacio´n y Desarrollo (C.I.D.), Calle Jordi Girona 18 -26, 08034 Barcelona, Spain Received 19 January 1998; received in revised form 28 April 1998; accepted 30 April 1998
Abstract The interaction of the amphoteric surfactant N-dodecyl-N,N-dimethylbetaine (C12-Bet) with phosphatidylcholine (PC) liposomes was investigated. Permeability alterations were detected as a change in 5(6)-carboxyfluorescein (CF) released from the interior of vesicles and bilayer solubilization as a decrease in the static light-scattering (SLS) of the system. At sublytic level a initial maximum in the bilayer/water partitioning (K) followed by an abrupt decrease of this parameter occurred as the surfactant to lipid molar ratio (Re) rose. At lytic level a direct dependence was established between both parameters. The fact that the free surfactant concentration at sublytic and lytic levels showed values lower than and similar to its critical micelle concentration indicates that permeability alterations and solubilization were determined, respectively, by the action of surfactant monomer and by the formation of mixed micelles. A direct correlation occurred in the initial interaction steps (up to 50% CF release) between the growth of vesicles their fluidity and Re. A similar direct dependence was established during solubilization (up to 30% SLS) between the fall in both the surfactant-lipid aggregate size, the SLS of the system and Re. This surfactant showed higher capacity to solubilize PC liposomes than that reported by the commonly used non-ionic surfactants octyl glucoside and Triton X-100 and by the anionic one sodium dodecyl sulfate. © 1998 Elsevier Science Ireland Ltd. All rights reserved.
Abbre6iations: PC, phosphatidylcholine; C12-Bet, N-dodecyl-N,N-dimethylbetaine; PIPES, piperazine-1,4 bis(2-ethanesulphonic acid); CF, 5(6)-carboxyfluorescein; SLS, static light-scattering; DLS, dynamic light-scattering; PI, polydispersity index; Re, effective surfactant/lipid molar ratio; ReSAT, effective surfactant/lipid molar ratio for liposome saturation; ReSOL, effective surfactant/lipid molar ratio for liposome solubilization; K, bilayer/aqueous phase surfactant partition coefficient; SW, surfactant concentration in the aqueous medium; SB, surfactant concentration in the bilayers; r2, regression coefficient; TLC-FID, thin-layer chromatography/flame ionization detection system; CMC, critical micellar concentration. * Corresponding author. Tel.: +34 3 4006161; fax: + 34 3 2045904. 0009-3084/98/$19.00 © 1998 Elsevier Science Ireland Ltd. All rights reserved. PII S0009-3084(98)00045-0
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Keywords: Phosphatidylcholine liposomes; N-dodecyl-N,N-dimethylbetaine; Permeability alterations and bilayer solubilization; Carboxyfluorescein release; Static light-scattering changes; Effective surfactant to lipid molar ratios; Surfactant partition coefficients
1. Introduction Liposomes are aqueous lipid dispersions organized as bilayers which are widely used as simplified membrane models (Lasic, 1993; Sternberg, 1995). A number of studies have been devoted to the understanding of the principles governing the interaction of surfactants with these structures (Kragh-Hansen et al., 1993; Ruiz et al., 1994; Polozava et al., 1995; Inoue, 1996; Silvander et al., 1996; Wenk et al., 1997). This interaction leads to the breakdown of lamellar structures and the formation of lipid-surfactant mixed micelles. A significant contribution in this area has been made by Lichtenberg (Lichtenberg et al., 1983) who postulated that the effective surfactant to lipid ratio (Re) producing liposome saturation and solubilization depends on the surfactant critical micelle concentration (CMC) and on the bilayer/aqueous medium surfactant distribution coefficients (K). Surface-active betaines are internally compensated quaternary ammonium compounds which differ from quaternary ammonium salts in that they do not have a mobile anion. These compounds are among the fastest-growing group of amphoteric surfactants due to their very low irritation levels and their excellent capacity to promote mildness in cosmetic formulations. These compounds have also been found to be effective in reducing the potential irritation of alkyl sulfates, alkyl ether sulfates and soaps (Cooper and Berner, 1985). However, the biological toxicity of these surfactants increases sharply with increasing chain length of their lipophile and shows an inverse correlation with the CMC of these surfactants (Ernst and Miller 1982). We previously studied the interaction of a series of alkylbetaines with PC unilamellar liposomes and with those formed by lipids modeling the stratum corneum lipid composition (de la Maza et al., 1991; de la Maza and Parra, 1993; de la Maza
et al., 1997). In the present work we seek to extend these investigations by characterizing the overall interaction of the N-dodecyl-N,N-dimethylbetaine (C12-Bet) surfactant with phosphatidylcholine (PC) liposomes. Knowledge of the Re and K parameters throughout the interaction process could be useful in improving our understanding of the complex phenomenon involved in the lamellar to micelle transitions during the solubilization of PC liposomes by this so used amphoteric surfactant and in establishing a criterion for the evaluation of its activity in biological membranes.
2. Materials and methods The amphoteric surfactant C12-Bet was specially prepared by (Albright and Wilson, Warley, West Midlands, UK) with an active matter in aqueous solution of 30% and an amino free contents of 0.20%. Piperazine-1,4 bis(2-ethanesulphonic acid) (PIPES) was obtained from (Merck, Darmstadt, Germany). PIPES buffer was prepared as 10 mM PIPES containing 110 mM Na2SO4 and adjusted to pH 7.20 with NaOH. The starting material 5(6)-carboxyfluorescein (CF), was obtained from (Eastman Kodak, Rochester, NY) and further purified by a column chromatographic method (Weinstein et al., 1986). Polycarbonate membranes and membrane holders were purchased from Nucleopore (Pleasanton, CA). PC was purified from egg lecithin (Merck) according to the method of Singleton (Singleton et al., 1965) and was shown to be pure by thin-layer chromatography (TLC). Reagent grade organic solvents was supplied by Sigma (St. Louis, MO).
2.1. Preparation of liposomes and solubilizing parameters PC liposomes (PC conc. 0.5–5.0 mM) of a defined size (about 200 nm) were prepared in
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PIPES buffer by extrusion of large unilamellar vesicles previously obtained by reverse phase evaporation (de la Maza and Parra, 1993). Liposomes containing CF (110 mM) were freed of unencapsulated fluorescent dye by passage through Sephadex G-50 medium resin (Pharmacia, Uppsala, Sweden) by column chromatography. The size distribution and polydispersity index (PI) of liposomes and surfactant-PC aggregates was determined with a photon correlator spectrometer (Malvern Autosizer 4700c PS/MV, Malvern, UK) using a Ar laser source (wavelength of 488 nm) (de la Maza and Parra, 1994a,b). The liposome PC concentration was determined by TLC-FID (Ackman et al., 1990; de la Maza and Parra, 1995a). The partition coefficients (K) of C12-Bet between the lipid bilayers and aqueous media was determined using the equation: K= Re/SW[1+ Re]
(1)
where Re is the effective molar ratio of surfactant to phospholipid in the bilayers and surfactant concentration (SW) in the aqueous medium (mM) (de la Maza and Parra, 1995a), in accordance with Lichtenberg et al. (1983) and Almog et al. (1990). The determination of Re and SW was carried out on the basis of the linear dependence existing between the surfactant concentrations required to achieve these parameters and the lipid concentration (mM) (L), which is described by the equation: ST = SW +Re. [L]
(2)
where Re and SW are in each curve, respectively, the slope and the ordinate at the origin (zero PC concentration). The permeability changes caused by C12-Bet in CF-containing PC liposomes were determined by monitoring the increase in the fluorescence intensity of liposomes due to the CF released from the interior of vesicles to the bulk aqueous phase (de la Maza et al., 1991). The solubilizing perturbation produced by C12Bet in liposomes was determined by monitoring the SLS changes of the surfactant-liposome sys-
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tem 24 h after the surfactant addition (de la Maza and Parra, 1993). This process was characterized by two parameters termed ReSAT and ReSOL, according to the nomenclature adopted by Lichtenberg et al. (1983), corresponding to the Re ratios at which the surfactant saturated liposomes and led to the complete solubilization of these structures.
3. Results and discussion
3.1. Stability of liposome suspensions The mean vesicle size of liposomes after preparation varied little (around 200 nm) and the PI remained always lower than 0.1 indicating that liposomes showed a homogeneous size distribution. The mean size of liposomes 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, liposomes appeared to be reasonably stable in the absence of surfactant under the experimental conditions used in solubilization studies.
3.2. Interaction of C12 -Bet with liposomes We previously studied the validity of the equilibrium partition model proposed by Lichtenberg et al. (1983) and Almog et al. (1990) based on the Eq. (1) for the surfactant investigated. According to these authors this equation may be expressed by: L/SB = (1/K)(1/SW)− 1. Hence, this validity requires a linear dependence between L/SB and 1/SW; this line should have a slope of 1/K, intersect with the L/SB axis at − 1 and intersect with the 1/SW at K (Almog et al., 1990). To test the validity of the model for C12-Bet unilamellar PC liposomes were mixed with varying subsolubilizing concentrations of this surfactant (ST). The resultant surfactant-containing vesicles were then spun at 140000g at 20°C for 4 h to remove the vesicles. No PC was detected in the supernatants. The surfactant concentration in the supernatants (SW) was determined by HPLC (Kondoh and Takano, 1986) and its concentration in the lipid
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bilayers was calculated (SB =ST −SW). The results of the experiments in which SB and SW were measured (carried out in the same range of PC and surfactant concentrations used to determine K) were plotted in terms of the dependence of L/SB on 1/SW. A straight line was obtained (r2 = 0.98), which was dependent on L and intersected with the L/SB axis always at −0.97 90.10. Both the linearity of this dependence and the proximity of the intercept to − 1 support the validity of this model to determine K for this surfactant. To determine the time needed to obtain a constant level of CF release trapped into liposomes, a kinetic study of the interaction of C12-Bet with these bilayer structures was carried out (PC concentration ranging from 0.5 to 5.0 mM). Liposomes were treated with C12-Bet at sublytic concentrations (affecting only the permeability of bilayers) and subsequent changes in permeability were studied as a function of time. The CF release was always a biphasic process in which about 40 min was needed to achieve CF release plateaux and approximately 75% of CF release took place during the initial 10 min. This biphasic behavior suggests that the release of the CF trapped into the vesicles was produced through holes created in the membrane, in agreement with the concept of transient channels suggested by Edwards and Almgren in the surfactant-mediated increase in PC membrane permeability due to surfactants (Edwards and Almgren, 1990, 1992). The incorporation of C12-Bet monomers to membranes may directly induce the formation of hydrophilic pores in these structures or merely stabilize transient holes (Lasic, 1993). The long time course required to reach a constant CF release could also be related to permeation or flip-flop of surfactant across the vesicle membrane. A similar biphasic behavior has been reported for the interaction of various non-ionic surfactants with PC liposomes (de la Maza and Parra 1994a,b). Bearing in mind these findings, changes in permeability were studied 40 min after addition of surfactant to the liposomes at 25°C. The CF release in the absence of C12-Bet in this period of time was negligible. To determine the Re and K parameters at sublytic level a systematic study of CF release changes caused by the addition of C12-Bet to
liposomes was carried out (PC concentration ranging from 0.5 to 5.0 mM). The C12-Bet concentrations resulting in different CF release percentages were obtained and plotted versus PC concentration. An acceptable linear relationship was established in each case. The straight lines obtained correspond to the Eq. (2) from which the Re and K parameters were determined. The results for 100% CF release, including the SW value and the straight line regression coefficient (r2) are given in Table 1. The fact that the SW value for 100% CF release (0.72 mM) was lower than the C12-Bet CMC (1.25 mM, de la Maza and Parra, 1993) confirms for this surfactant the generally admitted assumption that permeability alterations were determined by the action of surfactant monomers (Lichtenberg et al., 1983). To determine the Re and K parameters at lytic level a systematic study of the SLS changes of the surfactant/PC systems caused by the addition of C12-Bet to liposomes was carried out (PC concentration ranging from 0.5 to 5.0 mM). An initial increase in the SLS intensity of the system was always observed due to the surfactant incorporation into bilayers. Additional surfactant amounts led to a fall in this intensity until a low constant value for liposome solubilization. The C12-Bet concentrations for different SLS percentages were obtained and plotted versus PC concentration. An acceptable linear relationship was also established in each case. The corresponding Re and K parameters were determined from these straight lines (Eq. (2)). The values for 0% SLS, including the SW value and r2 of this straight line are given in Table 1. The fact that the SW value for 0% SLS was comparable to the surfactant CMC (1.25 mM) supports that the free surfactant concentration must reach its CMC for solubilization to occur (Lichtenberg et al., 1983).
3.2.1. Relationship between the Re parameter, SW and K Fig. 1 shows the variation of SW with Re throughout the C12-Bet/liposomes interaction (vesicles or mixed micelles). A marked increase in SW occurred as Re rising up to the Re for 100% CF release (SW value 0.72 mM). The extrapolation of this curve (shaded area) led approximately
M, monomodal.
100% CF release 0% SLS
0.72 1.32
SW (mM)
0.55 1.43
Re mole/mole
0.995 0.993
r2
0.49 0.44
K (mM−1)
M M
Type
— 11
— 100
308 —
100 —
%
nm
nm
%
2nd peak
1st peak
Curve distribution (particle number)
308 11
Average (nm)
0.179 0.143
Polydispersity index
Table 1 Surfactant to Re, K, SW, nm and PIs of surfactant-lipid aggregates (vesicles or micelles) measured by DLS corresponding to 100% CF release and 0% SLS in the interaction of C12-Bet with PC liposomes. The regression coefficients (r2) of the straight lines obtained are also included
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Fig. 1. Variation in the free SW versus the effective surfactant to Re during the overall interaction between C12-Bet and PC liposomes.
to the initial SW value for solubilization (100% SLS corresponding to the ReSAT) (SW =1.27 mM), which corresponded approximately to the surfactant CMC (1.25 mM). The increase in Re resulted in a slight increase in SW up to ReSOL, which corresponded to the complete solubilization of liposomes via formation of lipid-surfactant mixed micelles (Re=1.43, for 0% SLS).
Fig. 2. Variation in K versus the effective surfactant to Re during the overall interaction between C12-Bet and PC liposomes.
Fig. 2 shows the variation in K versus Re during the overall interaction of C12-Bet with liposomes. An initial increase in K was observed as Re rose, reaching a maximum (K= 1.31) for Re= 0.31 (20% CF release). Increasing Re values resulted in a abrupt fall in K values up to Re = 0.55 (100% CF release). This fall was more pronounced in the Re interval 0.31–0.48). Thus, the increase in Re resulted in two opposite effects on the bilayer/water partitioning of C12-Bet. 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 Re= 0.48 (abrupt fall in K). Increasing Re values, (Re between 0.50–0.55, 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. These findings are in agreement with those reported by Schubert et al. for the interaction sodium cholate with PC liposomes (Schubert et al., 1986). The Re value of 0.31 (20% CF release) may be correlated with the saturation of the outer vesicle leaflet by C12-Bet. The extrapolation of the curve (shaded area) led approximately to the initial K value for liposome solubilization. Further increase in Re resulted again in a rise in K up to ReSOL, which corresponded to the solubilization of these bilayer structures via mixed micelles formation.
3.2.2. Dependence of the surfactant-lipid aggregate size, CF release and SLS on Re A systematic DLS study of surfactant-lipid aggregates was carried out throughout the process to elucidate the dependencies of the size of these aggregates (vesicles or micelles) and the changes in both the CF release and the SLS of the system on Re. The DLS values obtained for 100% CF release and 0% SLS (PC concentration 5.0 mM) are also given in Table 1. Micellar C12-Bet solutions before they were mixed with liposomes showed a QELS peak at 8 nm in the experimental conditions used (Ar laser source with a wavelength of 488 nm), in accordance with Swarbrick and Daruwala, who studied the weight-average
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aggregation number of this surfactant in water (Swarbrick and Daruwala, 1970). The fact that this peak was not detected when this surfactant interacted with PC liposomes is attributable to the preferential formation of lipid-surfactant mixed micelles, in agreement with our previous results for the interaction of various surfactants with PC liposomes (de la Maza and Parra, 1994a,b). The maximum growth of vesicles (formation of mixed vesicles) was reached for 100% CF release (308 nm, see Table 1) and the growth of vesicles occurred in a few s with little change over a period of h. As for SLS variations, the 100% (ReSAT) resulted in a slight decrease in the vesicle size albeit with a monomodal distribution. When the SLS decreased, a sharp distribution curve appeared at 9 nm, which corresponded to a lipidsurfactant mixed micelles. The assumption that these small particles corresponded to mixed micelles is based on the fact that small unilamellar vesicles have diameters in the range of 15 – 25 nm (New, 1990) and consequently are always higher than these small particles. The curve for these small particles rose up to 0% SLS, exhibiting at this point again a monomodal distribution (11 nm) for only mixed micelles (see Table 1). Thus, in the interval 90– 10% SLS vesicle or vesicle fragments coexisted with mixed micelles, in agreement with Almog et al. (1990) when studied the interaction of PC liposomes with octyl glucoside. Fig. 3 shows the variation in both the percentage of CF release and vesicle size of liposomes versus Re at sublytic level. The increase in Re led initially to a progressive increase in both parameters. Re values exceeding 0.48 (60% CF release) resulted in a lower growth of vesicles, which coexisted with an abrupt increase in the release of the CF trapped into liposomes. Given that approximately 80% of the permeability changes occurred in the initial interaction steps and that the vesicle growth took place in a few s after surfactant addition we may assume that for Re values lower than 0.48 the growth of vesicles was related to the leakage of entrapped CF. The coexistence in the range of Re values from 0.50 to 0.55 of slight changes in the vesicle size and the abrupt increase in the fluidity of bilayers could be correlated with the increase in the rate of flip-flop of the surfactant molecules into liposomes.
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Fig. 3. Variation in the percentage of CF release and vesicle size of PC liposomes treated with C12-Bet versus Re at sublytic level. ( ) CF release, ( ) vesicle size.
Fig. 4 shows the variation in the percentage of SLS and the surfactant-lipid aggregate size (average mean) determined by DLS versus Re at lytic
Fig. 4. Variation in the percentage of static light scattering and surfactant-lipid aggregate size (measured by DLS) of PC liposomes treated with C12-Bet versus Re at lytic level. ( ) Static light scattering, ( ) Surfactant-lipid aggregate size.
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level. The increase in Re produced a linear decrease in SLS these aggregates together with a fall in their average size up to a SLS intensity of the system of about 30%. Thus, in the range of Re values between 0.63 – 1.24 a direct correlation between the percentage of SLS and the surfactantlipid aggregate size was established. This direct dependence, together with the abrupt increase in the distribution curve for lipid-surfactant mixed micelles in the last interaction steps (see Table 1) emphasize the suitability of C12-Bet in the solubilization of these bilayer structures. Comparison of the Re values obtained for C12Bet with those reported for the interaction of the non-ionic surfactant octyl glucoside with PC liposomes (de la Maza and Parra, 1994b) reveals that the C12-Bet exhibited both at sublytic and lytic levels higher ability to interact with PC liposomes (lower Re values). It is still more surprising to verify that C12-Bet shows a clearly higher capacity than Triton X-100 to solubilize PC liposomes in the same experimental conditions (lower Re values at lytic level) (de la Maza and Parra, 1994a) Comparison of the present Re values with those reported for the interaction of the anionic surfactant sodium dodecyl sulfate (SDS) with PC liposomes in the same experimental conditions (de la Maza and Parra, 1995b) indicates that C12-Bet had higher capacity to solubilize PC liposomes, in spite of its very low irritation level and its capacity to promote mildness in cosmetic formulations. This finding is specially surprising given that the SDS produces toxicological effects in vivo tests (Singer and Pittz, 1985) and that C12-Bet has been found to be effective in reducing the potential irritation of SDS (Cooper and Berner, 1985). These findings open up new possibilities in the application of this amphoteric surfactant as promising solubilizing agent of biological membranes.
Acknowledgements This work was supported by funds from DGICYT (Direccio´n General de Investigacio´n Cientı´fica y Te´cnica) (Prog. No. PB94-0043), Spain. We are grateful to G. von Knorring for expert technical assistance.
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