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Mechanisms of Solubilization of Mixed Liposomes: Preferential Dissolution of Liposome Components Namita Deo,† P. Somasundaran,*,† and Yasuhiro Itagaki‡ NSF IUCR Center for Advanced Studies in Novel Surfactants, Langmuir Center for Colloid and Interfaces, and Department of Chemistry, Columbia University, New York, New York 10027
The mechanism of vesicle-to-micelle transformation due to the interactions with sodium dodecylsulfate (SDS) has been studied by monitoring changes in the optical density, surface tension, SDS monomer concentrations, pyrene fluorescence, and mass spectrometry. Two inflection points appeared on the optical density as well as effective hydrodynamic diameter curves. Based on the results of the surface tension and SDS monomer measurements, the first inflection point is attributed to the saturation of bilayers by SDS monomers and the onset of liposome solubilization processes. The second inflection point corresponds to the onset of complete disruption of bilayers and the critical micelle concentration of the mixed systems. The fluorescence results show the core of the mixed micelles to be more hydrophobic than that of the SDS micelles, suggesting that liposome solubilization is a micellization process. From the individual phospholipid liposome solubilization studies, it was found that phosphatidic acid (PA) molecules are more susceptible toward SDS. Interestingly, it has been detected from the mass spectra that the disruption of liposome bilayers is a preferential dissolution process. It is proposed that, at low SDS concentration, PA molecules preferentially exit first from the mixed liposome bilayers (1:1 PA/phosphatidylcholine), causing liposome solubilization. In contrast, at high SDS concentrations, the breakdown of liposome takes place instantaneously. Introduction The vesicle-to-micelle transformation induced by the addition of surfactants and surfactant mixtures is currently attracting much interest.1-5 This is mainly due to its relevance to the solubilization of cell membranes. The interaction of many surfactants with liposomes eventually leads to disintegration of the liposomes structure, resulting in solubilization of its components. It is known that this involves an initial growth of vesicles, followed by the formation of complex lipid-surfactant aggregates.6 The exact mechanism by which liposomes transform to micelles is, however, still unknown. In this paper, we describe the basic mechanisms behind the liposome destabilization processes due to the presence of an anionic surfactant, sodium dodecylsulfate (SDS), which is a well-known solubilizing agent of membrane lipids and proteins. In an earlier paper,7 we proposed a model for solubilization of a 1:1 phosphatidic acid (PA)/phosphatidylcholine (PC) mixed liposome by SDS. In this paper, we have further verified the proposed model experimentally using different analytical techniques including mass spectrometry (MS). It was found that at low SDS concentrations liposome solubilization is a preferential dissolution process, while at high SDS concentrations preferential dissolution can be instantaneous. Experimental Section SDS (99% pure) was obtained from Fluka and used as received. Surface tension data give an indirect * To whom correspondence should be addressed. † NSF IUCR Center for Advanced Studies in Novel Surfactants, Langmuir Center for Colloid and Interfaces. ‡ Department of Chemistry.
measure of the purity of the SDS sample. PA and PC from egg lecithin were purchased from Sigma. A phosphate buffer of pH 7.0 with an ionic strength of 0.1 M was used to maintain the pH. Liposome Preparation. Unilamellar liposomes of uniform size were prepared following the method of Fendler.8 A lipidic film was formed by removing the organic solvent from the individual lipid (1 mM), or lipid mixtures (lipid composition 1:1 PC/PA molar ratio), by rotary evaporation and then dispersed in the phosphate buffer and sonicated at 40 °C for 1 h. The resulting liposomes were separated through a polycarbonate filter of 1.5-m pore size. Absorbance Measurements. Absorbance measurements were made at 25 °C with a Shimadzu UV-240 spectrophotometer (l ) 350 nm; cell length ) 5 cm). The absorbance of each vesicle suspension (1 mM) was monitored before and upon interaction with SDS (112 mM). The absorbance measurements were taken after surfactant addition and subsequent vigorous agitation for different intervals of time. Light Scattering. The diameter and polydispersity index of the liposome particles before and upon interaction with SDS of each sample were measured by a Brookhaven BIC model photon correlation spectrometer. All the experiments were performed at an acute angle of 90°. SDS Concentration in Bilayers. The SDS concentration in lipid bilayers was determined after interaction of individual vesicles with SDS for 1 h. The above mixtures were then filtered through an Amicon filter (molecular weight cutoff of 3000) in order to separate SDS monomers from vesicles and micelles in the bulk. It has been tested that only SDS monomers can enter through the above filter and not liposomes or micelles. The monomer concentration of SDS in each filtrate was
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determined by a two-phase titration method.9 The SDS concentration in the bilayer was calculated by subtracting the monomer concentration in the filtrate from the initial concentration. Surface Tension Measurements. The surface tension of SDS solutions in a phosphate buffer was measured before and after interaction with liposomes (1-12.0 mM) by a drop-volume method10 at 25 °C. The critical micelle concentration (cmc) of the surfactants in the absence and the presence of liposomes was determined from a plot of surface tension versus concentration. Sample Preparation for MS Study. Liposome of 1 mM was mixed with 4 and 10 mM SDS for 1 h and then filtered through the Amicon filter (molecular weight cutoff of 30 000) in order to separate mixed micelles from vesicles and mixed vesicles. The above time was fixed based on the kinetics of the liposome dissolution study. Two-Phase Separation Method. Because the SDS molecules do interfere with mass spectroscopic measurements of phospholipid molecules, the SDS molecules were separated from the mixed micelles by a two-phase separation method. A total of 10 mL of chloroform was mixed in a separating funnel with 1 mL of a mixed micelles solution and 10 mL of water. After 1 h of phase separation, the organic phase was removed, and the procedure was repeated until all of SDS was separated from the phospholipids. The organic phase was evaporated overnight. The dried paste of phospholipids was hydrated with a phosphate buffer, and mass spectra were taken. MS. Mass spectra were obtained with a JMS-HX110A/ HX110A tandon mass spectrometer (JEOL, Tokyo, Japan). Xenon was used as the particle source for fast atom bombardment (FAB). A FAB gun (Ion Tech) was operated at 2 mA with an acceleration voltage of 10 keV. Argon at a pressure of 0.5-1 mTorr (1 Torr ) 133.3 Pa) was used, and the collision energy offset was 15-20 eV. These conditions ensured multiple collisions between the target gas and the ion exiting the first quadruple mass spectrometer. Approximately 1-2 µL of glycerol and 3-nitrobenzyl alcohol (1:1, v/v) was used as the matrix for FAB. A total of 1 µL of aliquot was added to the matrix, which was already in place on the probe tip, and allowed to evaporate for 1 min at room temperature before analysis. Fluorescence Spectroscopy. Fluorescence spectra were recorded on a Photon Technology LS-100 spectrophotometer. The excitation wavelength was 335 nm. For micropolarity measurements, fluorescence intensities at 373 and 383 nm were recorded. The sample cells were of 10-mm path length, and a suitable correction was applied to avoid interference from the lamp. Pyrene of a desired concentration (10-6 M) in chloroform was added to the PA/PC mixtures in chloroform, and the solution was evaporated overnight to remove all chloroform from the mixtures. The dried paste was dispersed in a phosphate buffer saline at pH 7.0 and sonicated for 1 h at 40 °C. For comparison purposes, pyrene (10-6 M) solutions in the buffer were also prepared as controls following the same procedure. The fluorescence spectra of pyrene in liposome were measured before and upon interaction with SDS, and I3/I1 was calculated. Results and Discussion Figure 1 gives the optical density and effective hydrodynamic diameter of 1 mM liposome upon interac-
Figure 1. Change in the optical density and effective hydrodynamic diameter of 1 mM liposome after interaction with SDS for 1 h.
Figure 2. Change in the surface tension and monomer concentration of SDS after 1 h of interaction with 1 mM liposome.
tion with SDS for 1 h. Increases in the optical density and hydrodynamic diameter were observed at low SDS concentrations. These increases are attributed to the incorporation of SDS monomers on liposome vesicles, which is in agreement with the literature.1-4,11 The points where the hydrodynamic diameter and optical density reached a maximum correspond to the saturation of bilayers of the first inflection point. Upon further increase in the SDS concentration above 1 mM, continuous decreases in the optical density and hydrodynamic diameter were observed, indicating disruption of the liposome. After 1 h of interaction at 10 mM SDS, the optical density approached almost zero and the hydrodynamic diameter reached 20-25 nm and remained almost constant upon further increase in the SDS concentration, indicating complete disruption of the bilayers corresponding to the second inflection point. Light scattering measurements also suggested that the vesicles and the mixed micelles are coexisting at intermediate SDS concentrations. However, as the SDS concentration increased, the liposome concentration gradually decreased and an increase in the mixed micelle concentration was observed, which further supports the stepwise solubilization behavior of liposomes. The results for the surface tension and monomer concentration of SDS upon 1 h of interaction with 1 mM liposome are shown in Figure 2. The surface tension of SDS decreased gradually with an increase in the SDS concentration and reached equilibrium at 10 mM surfactant concentration. In contrast, the SDS monomer concentration increased with an increase in the total SDS concentration and reached equilibrium at the same
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Figure 3. Fluorescence spectra of pyrene in different environments.
surfactant concentration (10 mM SDS). However, the surface tension and monomer concentration of SDS alone in buffer remained constant after 1.5 mM SDS, which corresponds to the cmc of SDS in the buffer solution. This suggests that SDS is being consumed by the liposomes by the bilayer disintegration processes. The onset of a plateau corresponds to complete disruption of bilayers and the cmc of the mixed systems. This observation is in agreement with the optical density and hydrodynamic diameter results. From the above studies, it is clear that liposome solubilization is a micellization process. However, it is not possible to differentiate between mixed micelles and SDS micelles using the above results. To monitor the difference between the two types of micelles, fluorescence tests were performed. Fluorescence spectra of pyrene in different environments are illustrated in Figure 3. The ratio of the third peak (λ ) 383 nm) to the first peak (λ ) 372 nm) of pyrene fluorescence spectra is a well-established parameter, which gives information about the polarity experienced by the pyrene probe.12 A low value of I3/I1 reflects a more polar environment than a high value does. The observed value typically ranges from 0.6 for water as the solvent to 1.4 for hydrocarbon as the solvent. In the absence of the liposome, the I1 peak is much higher than the I3 peak, indicating that the pyrene molecule was surrounded by an aqueous environment. However, in the presence of 1 mM liposome, the I3 peak became more intense, suggesting a transfer of the pyrene molecules to the hydrophobic core of the lipid bilayers. The intensity of the fluorescence spectrum was enhanced when 1 mM liposome interacted with 10 mM SDS, possibly because the pyrene molecules are concentrated in a smaller area of mixed micelles upon the breakdown of huge liposome
bilayers. I3/I1 was calculated from the above spectra and is plotted in Figure 4. It can be seen from Figure 4 that, in the absence of the liposome, I3/I1 increases gradually upon interaction with SDS and remains constant above 2 mM surfactant, suggesting a transfer of the pyrene molecules into the hydrophobic core of the SDS micelles.13 However, in the presence of 1 mM liposome, I3/I1 remains essentially constant upon interaction with SDS. It has been known from the light scattering study that the diameter of mixed micelles is almost double (8 nm) that of micelles (4 nm). In addition, phospholipid molecules are also more hydrophobic than SDS. Thus, the bulky pyrene molecules can accommodate themselves easily inside the hydrophobic core of the mixed micelles because of its bigger size. I3/I1 remains constant even after interaction with SDS because the pyrene molecules are essentially transferred from the hydrophobic core of the lipid bilayars to the hydrophobic core of the mixed micelles13 upon breakdown of the liposome bilayers. However, I3/ I1 of pyrene in mixed micelles is much higher than that in SDS micelles, suggesting that the size of mixed micelles was much larger than that of the SDS micelles because of the presence of the long-chain phospholipid molecules, which gives further evidence about the size of the mixed micelles. All of the above results suggest that liposome solubilization is a micellization process. However, the mechanism of the destabilization process of liposome was not clear. Our previous study7 has shown that solubilization of PA liposomes is much faster than that of PC liposomes and the rate of solubilization of the mixed liposome (1:1 PA/PC) was intermediate between that of PC and PA liposomes. On the basis of these results, we proposed a mechanism for the mixed liposome destabi-
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Figure 4. I3/I1 of pyrene in buffer and in liposome before and after interaction with SDS for 1 h.
Figure 5. FAB-MS spectrum of the filtrate separated from a liposome/SDS mixture (1 mM liposome/4 mM SDS, after 1 h of interaction).
lization process. Because of the different responses toward SDS, one of the phospholipid components preferentially exits first, causing destabilization and enhanced solubilization of liposomes. To test the proposed model, a MS study was performed, and the results obtained are illustrated in Figures 5 and 6. At 4 mM SDS, after 1 h of interaction, only PA (molecular weight of 697.5) was found to be present in the supernatant, suggesting the preferential dissolution of the liposome (Figure 5). A small amount of the PA/SDS complex (molecular weight of 860; Figure 5) was observed at 4 mM SDS, indicating the incomplete separation of surfactant from the mixed micelles. However, at 10 mM SDS, upon 1 h of interaction, both PA and PC (molecular weight of 760.6) are present in the supernatant, suggesting the complete disruption of bilayers. At 10 mM SDS, the PA peak was found to be smaller than that of PC because the ionization of PC molecules is faster than that of PA molecules. On the basis of the MS results, a model is proposed for liposome solubilization processes and illustrated in
Figure 7. At low SDS concentrations, the surfactant molecules form a monolayer on the surface of the liposome, as represented in Figure 7A, in agreement with the optical density and light scattering results corresponding to the first inflection point. During the SDS/liposome interaction process, the hydrophobic chains of SDS gradually penetrate into the hydrophobic bilayers and preferentially displace some of the PA molecules out of the liposome bilayers because of its compatibility with the SDS molecules, simultaneously causing destabilization of the liposomes. The PA molecule is more compatible with SDS because of its simpler molecular structure.7 In contrast with the above, at higher SDS concentrations, no preferential dissolution was observed because of the high SDS/phospholipids molar ratio. Under these conditions, many of the SDS molecules possibly penetrate simultaneously into bilayers, causing instantaneous breakdown of liposome vesicles (Figure 7B). All of these results suggest that mechanisms of liposome destabilization depend on the surfactant con-
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Figure 6. FAB-MS spectrum of the filtrate separated from a liposome/SDS mixture (1 mM liposome/10 mM SDS, after 1 h of interaction).
ever, at high surfactant concentrations, well above the saturation point of the liposome, liposome solubilization is an instantaneous process due to the presence of numerous amounts of SDS molecules. Conclusions (1) The SDS monomer was found to play a major role in the liposome solubilization processes. (2) The cmc of SDS was shifted to a higher value because of the presence of the liposome. (3) The core of the mixed micelles is more hydrophobic than that of the SDS micelles because of the presence of hydrophobic phospholipid molecules. (4) At low SDS concentrations, solubilization of the liposome was found to be a stepwise process and PA was found to exit first. (5) At high SDS concentrations, solubilization of the liposome was an instantaneous process. Acknowledgment Authors acknowledge the support of the National Science Foundation (Grant 9804618 Industrial/University cooperation research center for adsorption studies in novel surfactants) and Unilever Research Laboratory. Literature Cited
Figure 7. (A) Model of the preferential dissolution process of PA at low SDS concentrations. (B) Model of the coexistence of vesicles and mixed micelles at high SDS concentrations.
centration. At low surfactant concentrations, the liposome components, which are more compatible with surfactant molecules, exit first, destabilize the whole system, and enhance the solubilization processes. How-
(1) Paternostre, M.; Meyer, O.; Grbielle-Madelmont, C.; Lesieur, S.; Ghanam, M.; Oliivon, M. Biophys. J. 1995, 69, 2476. (2) Polozova, A. I.; Dubachev, G. E.; Simonova, T. N.; Barsukov, L. I. FEBS Lett. 1995, 358, 17. (3) Silvander, M.; Karlsson, G.; Edwards, K. J. Colloid Interface Sci. 1996, 179, 104-113. (4) Partearroyo, M. A.; Alonso, A.; Goni, F. M.; Trbout, M.; Paredes, S. J. Colloid Interface Sci. 1996, 178, 156. (5) Surfactants in Cismetics; Rieger, M. M., Eds.; Marcel Dekker: New York, 1985; p 133. (6) de la Maza, A.; Juez Para, J. L. J. Am. Oil Chem. Soc. 1993, 70 (7), 699. (7) Deo, N.; Somasundaran, P. Colloids Surf. A 2001, 186, 33. (8) Fendler, J. H. Membrane Mimetic Chemistry; John Wiley and Sons: New York, 1982; p 113.
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(9) Li, Z.; Rosen, M. J. Anal. Chem. 1981, 53, 1516. (10) Dennis, E. A. Arch. Biochem. Biophys. 1974, 165, 764. (11) Lo´pez, O.; de la Maza, A.; Coderch, L.; Lo´pez-Iglesias, C.; Wehrli, E.; Parra, J. L. FEBS Lett. 1998, 426, 314. (12) Kalyanasundaram, K.; Thomas, J. K. J. Chem. Soc., Faraday Trans. 1977, 99, 1312.
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Received for review March 22, 2004 Revised manuscript received October 18, 2004 Accepted October 19, 2004 IE040082Q
