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Langmuir 1999, 15, 2230-2233
Electrokinetic Study of the Sublytic Interaction of Alkyl Sulfates with Phosphatidylcholine Liposomes M. Co´cera,† O. Lo´pez,† A. de la Maza,*,† J. L. Parra,† and J. Estelrich‡ Departamento de Tensioactivos, Centro de Investigacio´ n y Desarrollo (CID), Consejo Superior de Investigaciones Cientı´ficas (CSIC), C/Jordi Girona, 18-26, 08034 Barcelona, Spain, and Departamento de Fisicoquı´mica, Facultat de Farma` cia, Universitat de Barcelona, Avenida Joan XXIII s/n, 08028 Barcelona, Spain Received October 6, 1998. In Final Form: December 9, 1999
Introduction The sublytic action of surfactants on the phospholipid bilayers leads to the incorporation of surfactant molecules into these structures because of the partition equilibrium between bilayers and the aqueous phase.1-5 This incorporation involves complex perturbations in the physical properties of vesicle membranes, which depend on the type and the amount of surfactant partitioned. Thus, a vesicle growth not explained by only the surfactant addition was reported by Edwards et al.6,7 in the sublytic interaction of lecithin vesicles with nonionic surfactants attributable to the vesicle fusion and closure of surfactantstabilized bilayer disks. The interpretation of this phenomenon using theoretical models of mixed aggregates is still not very well understood because simple models predict a decrease in the vesicle size because of the added surfactant.8 In earlier papers we studied the structural changes resulting in the solubilization of phosphatidylcholine (PC) liposomes with alkyl sulfates.9-12 Here, we seek to extend these investigations by studying the electrokinetic behavior involved in the sublytic interactions of these surfactants (chain lengths C10, C12, and C14) with unilamellar PC vesicles. To this end, a combination of zeta potential (ζ), fluorescence spectroscopy [measurement of surface potential (ψ0) on the liposome surface], and dynamic light-scattering (DLS) techniques has been used. The comparative use of these three techniques may shed light on the not very well understood mechanism of incorporation of surfactant molecules into vesicles up to the saturation of these structures. * To whom correspondence should be addressed. Telephone: (34-93) 400-61-61. Fax: (34-93) 204-59-04. † CID-CSIC. ‡ Universitat de Barcelona. (1) Liu, Y.; Regen, S. L. J. Am. Chem. Soc. 1993, 115, 708-713. (2) Paternostre, M.; Meyer, O.; Gabielle-Madelmont, C.; Lesieur, S.; Ghanam, M.; Ollivon, M. Biophys. J. 1995, 69, 2476-2488. (3) Inoue, T. Interaction of Surfactant with Phospholipid Vesicles. In Vesicles; Rosoff, M., Ed.; Surfactant Science Series 62; Marcel Dekker Inc.: New York, 1996; Chapter 5. (4) Wenk, M. R.; Alt, T.; Seelig, A.; Seelig, J. Biophys. J. 1997, 72, 1719-1731. (5) Wenk, M. R.; Seelig, J. Biophys. J. 1997, 73, 2565-2574. (6) Edwards, K.; Almgren M. Prog. Colloid Polym. Sci. 1990, 82, 190-197. (7) Edwards, K.; Almgren, M. Langmuir 1992, 8, 824-832. (8) Petrow, A. G.; Bivas, M. Prog. Surf. Sci. 1984, 16, 389-412. (9) de la Maza, A.; Parra, J. L.; Sanchez Leal, J. Langmuir 1992, 8, 2422-2426. (10) de la Maza, A.; Parra, J. L. Langmuir 1993, 9, 870-873. (11) de la Maza, A.; Parra, J. L. Langmuir 1995, 11, 2435-2441. (12) de la Maza, A.; Parra, J. L. Langmuir 1996, 12, 3393-3398.
Materials and Methods PC was purified from egg lecithin (Merck, Darmstadt, Germany) by the method of Singleton13 and was shown to be pure by thin-layer chromatography. Sodium decyl sulfate (C10-SO4), dodecyl sulfate (C12-SO4), and tetradecyl sulfate (C14-SO4) were supplied by Lancaster Synthesis Ltd. (Strasbourg, France) and further purified by column chromatography.14 Tris(hydroxymethyl)aminomethane (TRIS buffer) obtained from Merck was prepared as 5.0 mM TRIS buffer adjusted to pH 7.4 with HCl and containing 100 mM NaCl. The fluorescent agent 2-(ptoluidinyl)naphthalene-6-sodium sulfonate (TNS) purchased from Sigma was prepared as 100 µM TNS in a TRIS buffer and stored at 4 °C. Polycarbonate membranes were purchased from Nucleopore (Pleasanton, CA). The experiments were performed at 25 °C, except for C14-SO4, which were carried out at 40 °C, given its low solubility at 25 °C. The surface tension of the surfactant solutions in TRIS buffer was determined by the ring method15 using a Kru¨ss tensiometer (Processor Tensiometer K-12), which determines directly the real surface tension values at equilibrium. The surfactant critical micelle concentrations (cmc’s) were determined from the abrupt change in the slope of the surface tension values versus surfactant concentration. Preparation of PC Liposomes and Interaction with Surfactants. Unilamellar liposomes of a defined size (200 nm) were prepared from large unilamellar vesicles obtained by means of a reverse-phase evaporation technique9 followed by a 10-fold passage through 800-200 nm polycarbonate membranes.16 Equal volumes of appropriate surfactant solutions were added to liposome suspensions (1.0 mM PC), and the resulting mixtures were left to equilibrate for 24 h. After that, ζ and fluorescence measurements were performed. Particle Size and Microelectrophoretic Mobility Measurements. The hydrodynamic diameter (HD) of the initial particles and those resulting in the interaction of surfactants with liposomes was determined by means of a DLS technique using a photon correlation spectrometer (Malvern Autosizer 4700c PS/MV, U.K.) equipped with an Ar laser source (wavelength 488 nm).17 Electrophoretic measurements were performed in a Zetasizer 4 (Malvern, U.K.) based on the laser-Doppler microelectrophoresis technique. Samples containing variable surfactant concentrations in TRIS buffer (with or without 0.5 mM PC) were injected into the glass capillary tube. The ζ of particles was calculated from their electrophoretic mobility by means of the Henry correction of Smoluchowski’s equation:18
µ ) (2ζ/3η)f(κa)
(1)
where µ is the particle electrophoretic mobility, is the dielectric constant (78), ζ is the zeta potential, η is the aqueous solution viscosity (10-2 Pa‚s), and f(κa) is the Henry coefficient, the value of which 1.11, 1.13, 1.16, and 1.5 for C10-SO4, C12-SO4, C14SO4, and liposomes, respectively.18 Experiments were performed in triplicate, and the results given are the average of four measurements at the stationary level. Fluorescence Measurements. The main pathway in the excited state of TNS is a twisted intramolecular charge transfer, which is quite fast in the highly polar aqueous media. Hence, the quantum yield of fluorescence of this probe is extremely low in (13) Singleton, W. S.; Gray, M. S.; Brown, M. L.; White, J. L. J. Am. Oil Chem. Soc. 1965, 42, 53-57. (14) Rosen, M. J.; Hua, X. Y. Colloid Interface Sci. 1982, 86, 164168. (15) Lunkenheimer, K.; Wantke, D. Colloid Polym. Sci. 1981, 259, 354-366. (16) Dorovska-Taran, V.; Wich, R.; Walde, P. Anal. Biochem. 1996, 240, 37-47. (17) Lo´pez, O.; de la Maza, A.; Coderch, L.; Lopez-Iglesias, C.; Wehrli, E.; Parra, J. L. FEBS Lett. 1998, 426, 314-318. (18) Hunter, J. R. Zeta potential in Colloid Science; Academic Press: London, 1981; p 69.
10.1021/la981394z CCC: $18.00 © 1999 American Chemical Society Published on Web 02/19/1999
Notes
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water.19 The addition of TNS to liposomes led to the adsorption of these molecules on the bilayers surface. This adsorption produces a marked increase in their fluorescence, which is quenched by the presence of negative charges as those of alkyl sulfates. This variation allows one to calculate the surface potential (ψ0) of the charged vesicles. This probe has not been used to date as an alternative to other markers to characterize the incorporation of surfactants to bilayers.20 Fluorescence measurements were performed on a spectrofluorophotometer (Shimadzu RF-540, Japan) with excitation wavelength (λexc) at 321 nm and emission (λem) at 446 nm. The probe was added to PC liposomes (0.5 mM PC) and liposome/surfactant systems at various surfactant concentrations, just 30 min before the reading. We first checked the fluorescence lifetime of the probe and the optimal ratio lipid/probe. Fluorescence intensity barely varied within 6 h after the mixing of liposomes and liposome/surfactant systems with the probe, and a molar ratio PC/TNS of 100 was chosen as the most appropriate. Around this ratio, the net TNS fluorescence was proportional to the number of probe molecules adsorbed to the membrane, and its fluorescence in aqueous solution was negligible with respect to that of the TNS bound to lipid vesicles. The surface potential (ψ0) was calculated on the basis of the ratio of fluorescence of pure liposomes and those containing surfactant molecules at the same lipid concentration using the relation
f(-)/f(0) ) exp{Fψ0/RT}
(2)
where f(0) and f(-) are the fluorescence intensity in the absence and in the presence of quencher, F is the Faraday constant, R is the gas constant, and T is the absolute temperature. This equation is useful when a small fraction of binding sites are occupied by TNS at the liposome surface and when the aggregates provide very similar surface environments for TNS to give equal lifetimes, as occurred in our experimental conditions. From the surface potential values (ψ0) (expressed in volts), the surface charge density (σ0) expressed in µC‚cm-2 for symmetrical electrolytes may be calculated by means of21
σ0 ) 11.74xc sinh(zeψ0/2kT)
(3)
where c is the electrolyte concentration in mol‚L-1, z the valence of ions, e the elementary charge, k the Boltzmann constant, and T the absolute temperature. The number of charged molecules (n) can be obtained from
n)
σ0 1.60219 × 10-19
Sext × 10-22
(4)
where Sext is the external surface of a vesicle expressed in Å2.
Results and Discussion Particle Size and Electrokinetic Behavior of Liposome/Surfactant Systems. The cmc values of the surfactants tested in TRIS buffer were 10.0, 0.75, and 0.15 mM for C10-SO4, C12-SO4, and C14-SO4, respectively. After 24 h of mixing of PC liposomes (PC concentrated to 0.5 mM) with surfactants, the ζ values were obtained from electrophoretic mobility measurements. These variations and those for pure surfactants vs the surfactant concentration are plotted in Figure 1. The addition of surfactants to liposomes always led to a ζ increase (in absolute values), up to a maximum value. This increase indicates the incorporation of surfactant molecules in the bilayers with the corresponding rise in their surface density charge (σ0). This behavior is in line with that reported by Needman,22 who claimed that when lipid (19) Chang, L.; Cheung, H. C. Chem. Phys. Lett. 1990, 173, 343-346. (20) Sujatha, J.; Mishra, A. K. Photochem. Photobiol. A: Chem. 1997, 104, 173-178. (21) Hunter, J. R. Zeta potential in Colloid Science; Academic Press: London, 1981; p 28.
Figure 1. Variation of ζ potentials of C10-SO4 solutions and liposome/C10-SO4 systems, C12-SO4 solutions and liposome/ C12-SO4 systems, and C14-SO4 solutions and liposome/C14SO4 systems as a function of the surfactant concentrations, with the PC concentration of liposomes remaining constant (0.5 mM): (]) surfactant solutions, ([) liposome/surfactant systems. The arrows represent the effective surfactant concentrations for the maximum ζ potentials, which correspond to the cmc’s of the liposome/surfactant systems (cmcSYSTEM).
bilayers are in contact with surfactants, the mass and composition of bilayers change in seconds, affecting their σ0. Increasing surfactant amounts led to a slight ζ decrease in all cases. To elucidate if the maximum ζ values (see arrows) corresponded to the initial steps of vesicle to micelle transformation, a series of DLS measurements were performed in each system, and the results are given in Table 1. Liposomes after preparation showed a monodisperse and monomodal curve with a diameter of 190 nm (expressed as z average). The unilamellarity of these vesicles was demonstrated on the basis of the extrusion process used16 and taking into account our previous structural study based on freeze-fracture electron microscopy.17 After 24 h of mixing of liposomes with surfactants at concentrations up to those for the maximum ζ values, only one peak for mixed vesicles (175-185 nm) was detected. However, slightly higher concentrations exhibited two peaks in all cases. The large particles corresponded to mixed vesicles and the small ones to the mixed micelles (HD of pure micelles 3.2, 4.0, and 4.8 nm for C10-SO4, C12-SO4, and C14-SO4, respectively). No growth was observed in the mixed vesicles formed. Increasing surfactant amounts led to a rise and a fall in the percent of the small and large particles, respectively. These findings are in agreement with those recently reported for kinetic studies of PC liposome solubilization by C12-SO4.23 Hence, the maximum ζ values were as(22) Needman, D.; Stoicheva, N.; Zhelev, D. V. Biophys. J. 1997, 73, 2615-2629.
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Notes
Table 1. Mean Size (Expressed as z Average) of Liposomes and Liposome/Surfactant Systems at Different Surfactant Concentrationsa surfactant conc. [mM] 0 0.5 1.0 1.25 1.50 2.0 2.5 5.0 10.0 10.25 11.50 12.50 13.50
liposome/ C10-SO4 first peak [nm]
5.5 (12%) 5.5 (14%) 5.7 (40%) 4.5
second peak [nm] 190 184 183 177 178 178 176 178 178 178 176 176
liposome/ C12-SO4 first peak [nm]
6.0 (15%) 6.0 (16%) 6.2 (40%) 5.3 5.3 5.2 5.2 5.2 5.2
second peak [nm] 190 184 182 176 175 176
liposome/ C14-SO4 first peak [nm]
6.5 (15%) 6.4 (17%) 6.6 (35%) 6.6 (49%) 6.0 5.5 5.5 5.5 5.4 5.4
second peak [nm] 190 182 188 176 176
a Value points out the average and the standard deviation (n ) 3). In all the cases, the PC concentration remained constant (0.5 mM).
sociated to the effective surfactant concentrations for bilayer saturation in all cases (charges of -52.5 mV for 10 mM C10-SO4, -50.6 mV for 1.25 mM C12-SO4, and -50.0 mV for 1.0 mM C14-SO4). These surfactant concentrations were defined as the cmc of each liposome/ surfactant system (cmcsystem), in accordance with our investigations involving the interaction of liposomes with surfactants.24 Given that the surfactant electrical charge was always the same, the fact that different surfactant amounts were needed to achieve the ζ values for bilayer saturation indicates that the surfactant incorporation in the bilayers was dependent on its hydrophobic characteristics. When the affinity of surfactants with liposomes is defined as the inverse of the effective surfactant concentration needed to saturate these structures, C10SO4 showed the lowest affinity, whereas C14-SO4 exhibited the highest. In the absence of liposomes low surfactant amounts did not produce changes in the electrokinetic properties of the aqueous solutions. However, at concentrations preceding their cmc’s, a clear rise in ζ (in absolute values) occurred in all cases. The maximum ζ values corresponded exactly to the surfactant cmc’s determined by surface tension measurements. Increasing surfactant amounts led to almost constant ζ values. Hence, a premicellar association of surfactant molecules (concentrated range from 8 to 10 mM for C10-SO4, from 0.50 to 0.75 mM for C12-SO4, and from 0.08 to 0.15 mM for C14-SO4) not detected by the surface tension method used to determine the surfactant cmc’s may be envisaged. This fact is based on the particular characteristics of each technique. Thus, whereas surface tension measures changes in the interface water-air, zeta potential measures the electric potential between the aggregates and the ionic atmosphere, i.e., changes in the interface aggregate-water. Variation of Surface Potential Determined from Fluorescence Intensity Changes. To study “in situ” the variation in the charge surface density of liposomes due to the incorporation of alkyl sulfates, the anionic probe TNS was used. Thus, fluorescence changes of PC bilayers containing increasing surfactant amounts and due to the (23) Lo´pez, O.; Co´cera, M.; Pons, R.; Azemar, N.; de la Maza, A. Langmuir 1998, 14, 4671-4674. (24) de la Maza, A.; Parra, J. L. J. Am. Oil Chem. Soc. 1993, 70, 699-706.
Figure 2. Variation of the fluorescence intensity percentages of liposome/C10-SO4, liposome/C12-SO4, and liposome/C14-SO4 systems versus surfactant concentration due to the adsorption of a constant amount of TNS on these charged vesicles. The PC concentration of liposomes remained constant (0.5 mM).
adsorption of a constant amount of TNS were studied vs surfactant concentration and are plotted in Figure 2. Fluorescence intensity of 100% corresponded to that of liposomes lacking surfactant at a liposome/probe molar ratio of 100. An abrupt decrease in the fluorescence intensity always occurred already at very low surfactant concentrations (markedly lower than their cmc’s), although the surfactant amounts needed to produce this variation sharply decreased as the surfactant alkyl chain length rose. These findings are in line with the ζ determinations, in which the affinity of these surfactants with liposomes was directly dependent on their alkyl chain length. It is noteworthy that no variations in the fluorescence intensity were detected in the effective surfactant concentrations for bilayer saturation, in contrast with the ζ measurements. This behavior may be explained by taking into account that the transition vesicle-micelle did not produce changes in the quenching of the TNS with the negative charge of surfactants despite the structural changes that occurred in the system. The theoretical ψ0 values calculated from the fluorescence intensity values applyied in eq 2 allow one to determine the charge surface density of liposomes (eq 3) and, consequently, the number of molecules of surfactant inserted in the membrane (eq 4). Assuming that liposomes were unilamellar and formed by spherical vesicles of 190 nm of HD, the surface area on the lipid molecules was 70 Å2, and the thickness of the bilayers was 4 nm,25 a total vesicle surface of 22.2 × 106 Å2 was obtained with 320 000 molecules of lipid in this bilayer. (25) Lasic, D. D. In Liposomes: from Physics to Applications; Elsevier Science Publishers: Amsterdam, The Netherlands, 1993; p 554.
Notes
The theoretical ψ0 values for the effective surfactant concentration for bilayers saturation (maximum ζ values) were similar in all cases (about -60 mV). Hence, a similar number of surfactant molecules (about 41 000 molecules adsorbed in each vesicle) was needed to saturate liposomes, regardless of the surfactant hydrophobic tail. This amount represents approximately 13% of the total number of lipid molecules present in the vesicle. As a consequence, for the alkyl sulfates investigated the surfactant amount needed for bilayer saturation was strongly dependent on the physicochemical characteristics of liposomes. We are aware of the fact that the surfactant concentrations used to calculate the number of surfactant molecules adsorbed into liposomes was determined from the theoretical ψ0 values for bilayers saturation (fluorescence measurements for the maximum ζ). Hence, this number of inserted
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surfactant molecules was related to the effective surfactant charge (concentration of added surfactant) and not to the structural charge. Nevertheless, the present findings lead to the conclusion that although the hydrophobic surfactants, characteristics play an important role in its affinity with liposomes, the surfactant amount needed for bilayer saturation was strongly dependent on the physicochemical characteristics of liposomes. Acknowledgment. We are grateful to Mr. G. von Knorring for expert technical assistance. This work was supported by funds from DGICYT, Spain (Prog. No. PB94-0043). LA981394Z
