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Langmuir 1997, 13, 5076-5081
Electrostatic Control of Spontaneous Vesicle Aggregation Scott A. Walker† and Joseph A. Zasadzinski* Department of Chemical Engineering, University of California, Santa Barbara, Santa Barbara, California 93106-5080 Received January 29, 1997. In Final Form: July 11, 1997X Aqueous mixtures of cationic cetyltrimethylammonium p-toluenesulfonate (CTAT) and anionic sodium dodecylbenzenesulfonate (SDBS) spontaneously form equilibrium “catanionic” vesicles whose charge depends on the relative amounts of each surfactant. Aggregation of these vesicles was induced by incorporating a small concentration of biotin-lipid in the bilayer, followed by addition of streptavidin as a cross-linking receptor. Freeze-fracture transmission electron microscopy shows that almost no aggregation occurred in solutions with no added electrolyte, in which the Debye length was much larger than the dimensions of the streptavidin biotin linkage, about 2.5 nm. Much higher levels of aggregation (multivesicle aggregates) were observed for solutions with 0.1 M NaCl, in which the Debye length was smaller than the linkage dimensions. At an electrolyte concentration of 0.025 M, in which the Debye length is comparable to the linkage dimensions, significant aggregation, which depended on vesicle concentration, occurred. The short-range nature of the specific recognition interaction makes controlling aggregation via electrostatics possible. These results also suggest that electrostatic interactions are at least somewhat responsible for the stability of the spontaneous vesicles against aggregation. As these catanionic vesicles do not flatten upon aggregation, they can maintain an applied osmotic stress across their bilayer much like phospholipid vesicles.
Introduction T/bond1)
Biotin binds with a very high affinity (∼30 kB in an aqueous solution to one of four streptavidin binding sites; the pairs of binding sites lie on opposite faces of streptavidin, allowing for the cross-linking of biotinlabeled surfaces.2,3 What renders this system so versatile is that biotin can be conjugated to the headgroup of a phospholipid while (1) the biotin maintains its ability to bind to streptavidin and (2) the phospholipid maintains its ability to be incorporated into bilayers.4,5 Hence, vesicles labeled with biotin lipids can be subsequently aggregated by adding streptavidin.6 Uncharged, biotin labeled phosphatidylcholine vesicles aggregate quickly and at specific contact points upon addition of streptavidin to form multimicron, multivesicle clusters that sediment under gravity. This aggregation is complete within minutes and irreversible due to the strong binding between biotin and streptavidin. The vesicles are undamaged by this aggregation process and retain their contents.6 However, mainly due to steric effects, lipid-conjugated biotin (15-16 kT/bond) has a lower binding energy with streptavidin7 than does free biotin and vesicle aggregation can be reversed. Added soluble-free biotin6 competes for the binding sites on the streptavidin and causes the vesicles to redisperse. While rapid, complete aggregation, or total redispersion is easily achieved, for certain ap* To whom correspondence should be sent. E-mail: gorilla@ engineering.ucsb.edu. Telephone: 805-893-4769. Fax: 805-89347831. † Current address: Imation Corporation, Advanced Technology Center, 3M Center Building 201-3S-01, St. Paul, Minnesota 551441000. X Abstract published in Advance ACS Abstracts, September 1, 1997. (1) Green, N. M. Meth. Enzymol. 1990, 184, 51. (2) Green, N. M. Adv. Protein Chem. 1975, 29, 85. (3) Blankenburg, R.; Meller, P.; Ringsdorf, H.; Salesse, C. Biochemistry 1989, 28, 8214. (4) Bayer, E. A.; Rivnay, B.; Skutelsky, E. Biochim. Biophys. Acta 1979, 550, 464. (5) Plant, A. L.; Brizgys, M. V.; Locasio-Brown, L.; Durst, R. A. Anal. Biochem. 1989, 176, 420. (6) Chiruvolu, S.; Walker, S.; Israelachvili, J. N.; Schmitt, F.-J.; Leckband, D.; Zasadzinski, J. A. Science 1994, 264, 1753. (7) Powers, D. D.; Willard, B. L.; Carbonell, R. G.; Kilpatrick, P. K. Biotechnol. Prog. 1992, 8, 436.
S0743-7463(97)00094-2 CCC: $14.00
plications, it would be useful to be able to control the vesicle aggregation to make clusters of a given size. One way to do this is to slow the aggregation kinetics; it might be possible to grow aggregates irreversibly at a slower rate but still get compact, highly stable aggregates. One way to slow the aggregation rate is to use charged vesicles. Charged bilayers provide an added barrier to vesicle “contact”. The specific recognition reaction is retarded by electrostatic “double-layer” repulsions that reduce the number of collisions between vesicles.8,9 The double-layer repulsion creates an energy barrier to aggregation, somewhat analogous to the existence of an activation energy barrier for chemical reactions. Classical theory shows that the rate of colloidal aggregation is governed by the maximum of the total interaction energy between the aggregating colloids, that is, the height of the energy barrier along the line of approach.8 When electrolyte is added to a system of charged catanionic vesicles, the maximum of the interaction potential can be decreased10 and hence the rate of specific aggregation can be increased; aggregation increases with decreasing electrostatic repulsion. The range of the electrostatic interaction is also decreased with increasing electrolyte concentration as given by the Debye length (see eqn 2).9,10 The short-range nature of the specific recognition interaction11 means that biotin-streptavidin binding can only occur when the two come into close contact. In this study, we have used equilibrium, charged vesicles formed from mixtures of anionic and cationic single-tailed surfactants. Equilibrium unilamellar vesicles (ULVs) form spontaneously in dilute aqueous mixtures of these mixed surfactants12-14 and other mixed surfactants in(8) Hunter, R. J. Foundations of Colloid Science; Cambridge Press: Oxford, U.K., 1986; Vol. 1. (9) Israelachvili, J. N. Intermolecular and Surface Forces; Academic Press: London, 1992. (10) Chiruvolu, S.; Israelachvili, J. N.; Naranjo, E.; Xu, Z.; Zasadzinski, J. A.; Kaler, E. W.; Herrington, K. L. Langmuir 1995, 11, 4256. (11) Leckband, D. E.; Schmitt, F.-J.; Israelachvili, J. N.; Knoll, W. Biochemistry 1994, 33, 4611. (12) Kaler, E. W.; Herrington, K. L.; Zasadzinski, J. A. Structure and Dynamics of Strongly Interacting Colloids and Supermolecular Aggregates in Solution; Chen, S. H., Huang, J. S., Tartaglia, P., Eds.; Kluwer Academic Publishers: Dordrecht, The Netherlands, 1992.
© 1997 American Chemical Society
Control of Spontaneous Vesicle Aggregation
cluding double-tailed gangliosides,15 single-chain amino acid surfactants,16 and surfactant-alcohol cosurfactant mixtures.17,18 Equilibrium vesicles are formed from mixtures of cetyltrimethylammonium p-toluenesulfonate (CTAT), a single-tailed, cationic surfactant, and sodium dodecylbenzenesulfonate (SDBS), a single-tailed, anionic surfactant, hence, the shortened term “catanionic” vesicle. The vesicles form by adding dry surfactants to water or mixing micellar solutions of the two surfactants.12-14 CTAT-rich vesicles (overall positive charge) have a diameter, D, ranging from 10 to 250 nm with a mean size of D ≈ 70 nm.14 SDBS-rich vesicles (overall negative charge) are somewhat smaller, with fewer larger (>200 nm) vesicles, and have a mean size of D ≈ 60 nm.14 Single-equilibrium phases of spontaneous vesicles have only been observed in mixtures of surfactants or surfactant-cosurfactant mixtures. Pure CTAT forms rod micelles, while pure SDBS forms spherical micelles in dilute aqueous solution. On mixing, however, the critical aggregation concentration (cac) of CTAT/SDBS (cac ≈ 0.000 17 wt %) measured by changes in surface tension with concentration12 is about 100 times lower than the critical micelle concentration (cmc) of CTAT (cmc ≈ 0.01 wt %) and 1000 times lower than that of SDBS (cmc ≈ 0.1 wt %). Because of the surprising drop in the cac in the mixed system, it is believed14,16 that the oppositely charged surfactants form a neutral dimer complex that resembles a zwitterionic double-tailed surfactant that is able to form bilayers. Any excess of positive or negative surfactant in the vesicle gives it an overall charge; these unpaired surfactants also tend to help fluidize the bilayer.19 Conductivity measurements, however, show that, in addition to fluidizing the bilayers, a portion of the excess cationic (CTAT-rich) or anionic (SDBS-rich) surfactant forms a micellar phase in equilibrium with the vesicle phase.12,19 Vesicles do not form in equimolar mixtures of CTAT and SDBS; at this 1:1 ratio, a CTA+:DBS- complex precipitates from solution.14 The pseudo double-tailed surfactants that are formed in this system effectively have a smaller headgroup and larger hydrophobic tailgroup than the individual surfactants.14 The unpaired surfactants and surfactant complexes assemble in the bilayer to create a mixture of surfactant species, one set with a larger headgroup area (unpaired surfactant) and another with a smaller headgroup area (complex). One explanation as to why vesicles form in mixed surfactant systems is that both monolayers of the vesicle need not have equal concentrations of both surfactant species.20 Small vesicles are favored if the outer monolayer has an excess of the unpaired, larger headgroup surfactants and the inner monolayer has an excess of the smaller headgroup surfactants. This nonideal mixing leads to different spontaneous curvatures of the inner and outer monolayers. Safran and co-workers proposed a Helfrich-type model21-23 for the bilayer curvature free (13) Kaler, E. W.; Herrington, K. L.; Murthy, A. K.; Zasadzinski, J. A. J. Phys. Chem. 1992, 96, 6698. (14) Kaler, E. W.; Murthy, A. K.; Rodriguez, B. E.; Zasadzinski, J. A. Science 1989, 245, 1371. (15) Cantu, L.; Corti, M.; Del Favero, E.; Raudino, A. J. Phys. II (France) 1994, 4, 1585. (16) Ambu¨hl, M.; Bangerter, F.; Luisi, P. L.; Skrabal, P.; Watzke, H. J. Langmuir 1993, 9, 36. (17) Chiruvolu, S.; Warriner, H. E.; Naranjo, E.; Idziak, S. H. J.; Ra¨dler, J. O.; Plano, R. J.; Zasadzinski, J. A.; Safinya, C. R. Science 1994, 266, 1222. (18) Herve, P.; Roux, D.; Bellocq, A. M.; Nallet, F.; Gulik-Krzywicki, T. J. Phys. II (France) 1993, 3, 1255. (19) Herrington, K. Ph.D. Thesis, University of Delaware, Newark, DE, 1994. (20) Safran, S. A.; Pincus, P.; Andelman, D. Science 1990, 248, 354. (21) Helfrich, W. Z. Naturforsch. C 1973, 28C, 693. (22) Helfrich, W. Z. Naturforsch. A 1978, 33A, 305.
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energy, E, per unit area, A, for bilayers with monolayers of differing spontaneous curvatures:20,24
E 1 ) K[(c + co)2 + (c - ci)2] A 2
(1)
where K is the bilayer bending modulus, c the vesicle curvature (the reciprocal of the vesicle radius, c ) 1/R), co the outer monolayer spontaneous curvature, and ci the inner monolayer spontaneous curvature. For singlecomponent bilayers, ci necessarily equals co and the minimum energy occurs when c ) 0; the spontaneous curvatures of the opposing monolayers are in competition, and the net result is a “flat” or zero spontaneous curvature bilayer. Nonideal mixing of surfactants in two or more component bilayers can lead to monolayers with equal and opposite spontaneous curvatures, i.e., ci ) -co) c, in which a vesicle of radius 1/c is the minimum-energy structure.20,24 However, nonideal mixing cannot account for the observed stability against aggregation of CTAT/SDBS or other spontaneous vesicles, except in the limit of a very large bending modulus, K. Interactions between the bilayers must play an important role in the transition from unilamellar vesicles to multilamellar phases. Surface force apparatus (SFA) measurements of adsorbed bilayers of CTAT and SDBS10 show that electrostatic double-layer forces dominate all other interactions at low surfactant concentrations and that the stability of the vesicles in dilute solutions is at least partially due to the double-layer repulsion between charged bilayers.10 Added electrolyte, up to a point, does not alter the stability or unilamellarity of the vesicles.10 Too much electrolyte (g0.5 M NaCl for dilute solutions) leads to multilayer formation in SFA experiments. In CTAT-rich solutions, added NaCl also changes the distribution of the surfactants in the bilayers; the added electrolyte increases the surface charge density of the vesicles because the hydrophobic p-tolunesulfonate ion (presumably buried in the bilayer) is displaced from the vicinity of the CTA+-rich bilayers by the more hydrated chloride ion.10 This ion exchange may also effectively decrease the headgroup area of the charged surfactants by altering the charged headgroup interactions of individual surfactants within each bilayer. Decrease of the headgroup area also contributes to multilamellar vesicle (MLV) formation because it leads to a lower bilayer curvature and thus larger structures. However, the SFA cannot detect undulation repulsions, which are also hypothesized to be important in these systems, and can act as an additional stabilizing force against aggregation,18,19,25 especially for more concentrated vesicle solutions in which the electrostatic interactions are screened. For example, concentrated (≈4 wt %) solutions of CTAT/ SDBS consist of close-packed equilibrium vesicles, as shown by cryo-transmission electron microscopy,10 and show no signs of aggregation. Hence, examining the streptavidin-mediated aggregation of biotin-lipid-labeled catanionic vesicles as a function of added salt can provide a great deal of information about the stability and interactions of catanionic vesicles as well as the specific recognition process. We find that little or no aggregation occurs when the Debye length is significantly larger than the dimensions of the streptavidinbiotin linkage, which we calculate to be about 2.5 nm. (23) Helfrich, W.; Servuss, R. M. Nuovo Cimento Soc. Ital. Fis., D 1984, 3D, 137. (24) Safran, S. A.; Pincus, P.; Andelman, D.; MacKintosh, F. C. Phys. Rev. A 1991, 43, 1071. (25) Brasher, L. L.; Herrington, K. L.; Kaler, E. W. Langmuir 1995, 11, 4267.
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Table 1. Vesicle Concentrations, Weight Ratios, Average Diameters, and Number of Biotin Molecules Protruding from an Average Vesicle for the Standard Solutions Useda molarity (M), mean vesicle accessible mole ratio diameter (nm) biotins 1.0 wt % 7/3 CTAT/SDBS 1.0 wt % 3/7 CTAT/SDBS 0.3 wt % 7/3 CTAT/SDBS 0.3 wt % 3/7 CTAT/SDBS
0.024, 1.8/1 0.027, 1/3 0.0075, 1.8/1 0.0083, 1/3
120 70 115 70
190 60 170 60
a
The ratios represent amount of CTAT vs amount of SDBS. The 1.0 wt %, 7/3 wt ratio sample vesicle diameter was estimated from diameters obtained from the other 7/3 and 3/7 systems.
Rapid aggregation is observed when the Debye length is less than the dimensions of this linkage, leading to large, sedimenting flocs, similar to the uncharged phospholipid vesicles.6 Finally, when the Debye length is on the order of the linkage length, small aggregates are formed over the course of several days. This control of aggregate growth rates in charged vesicle solution leads to a method of controlling aggregate sizes. The catanionic vesicles are undamaged by the aggregation process, at least at low salt concentrations, showing that these vesicles are longlived and can maintain their integrity against the stresses imposed by aggregation, similar to phospholipid vesicles. In this system, we can proceed from molecular solution to quite complex, highly organized structures through sequential self-assembly. The control of aggregation with Debye length also shows that long-range electrostatic interactions are important to the stability against aggregation of catanionic vesicles, in agreement with the results of Chiruvolu et al.10 Experimental Section CTAT (Sigma, St. Louis, MO), SDBS (Tokyo Kasei, Japan), and N-(((6-biotinoyl)amino)hexanoyl)-1,2-dihexadecanoyl-snglycero-3-phosphoethanolamine, triethylammonium salt (BiotinX-DHPE from Molecular Probes, Eugene, OR) were mixed in various aqueous electrolyte solutions to form equilibrium vesicles. The addition of the Biotin-X-DHPE lipid at the low concentration used (0.20 mol % of total surfactant for each solution) did not disrupt formation of catanionic vesicles. Vesicles were prepared at concentrations of 0.3 and 1.0 total wt % surfactant (including Biotin-X-DHPE), at 7/3 and 3/7 CTAT/SDBS weight ratios, and in 0, 0.025, and 0.10 M NaCl aqueous solutions. All the solutions have added Biotin-X-DHPE at 0.2 mol % total surfactant. The concentrations and weight ratios were chosen because (1) they were within the equilibrium vesicle lobe on the ternary phase diagram and (2) they corresponded to a monodisperse vesicle population.10,12-14 The number of biotin ligands protruding from the vesicles was estimated by assuming an average headgroup area of 0.48 nm2 19 for the surfactants in the bilayer. CTAT-rich vesicles are larger and thus have a greater number of protruding ligands per vesicle. Table 1 displays the molar concentrations of the surfactants by weight percents and weight ratios with their corresponding molar concentrations, compositions, average vesicle diameters,12-14 and the approximate number of biotins protruding from an average vesicle surface. The number of ligands on the vesicle surface, the number density of the vesicles in solution, and the average size of the vesicles were all independent (to a good approximation) of the total ionic strength of the solution; hence, the only relevant parameter influencing the aggregation kinetics was the electrostatic interaction energy as measured by the Debye length.8 Because these vesicles are charged, they exhibit a characteristic electrostatic double-layer repulsive force which hinders vesiclevesicle aggregation.8 The magnitude and range of the electrostatic interaction is best characterized by the Debye length, κ-1; the Debye length is generally taken as the range over which electrostatic interactions are significant.8 Added electrolyte screens the electrostatic repulsion between the charged surfaces and reduces κ-1. There is also a contribution to the electrolyte concentration from the ionization of the surfactants themselves.
However, only partial ionization occurs (≈30-80%) as shown by surface force measurements of Chiruvolu et al.;10 we took a value of 30% ionization of the surfactants for the results in Table 1. For 1:1 monovalent electrolyte at a total concentration of CM (mol) at 25 °C, the Debye length (nm) is9
κ-1 )
0.304
xCM
nm
(2)
The relevant length scale to compare to κ-1 is the distance from the vesicle surface to where the biotin molecule binds to the streptavidin. In Biotin-X-DHPE, biotin is connected to DHPE by a 6-carbon spacer group.4 Assuming about 1.3-1.5 Å per carbon-carbon bond, the spacer is ≈1 nm in length; the biotin molecule is ≈1 nm in length.26 Thus, the biotin protrudes (biotin + spacer) about 2 nm from the vesicle surface. The biotinstreptavidin interaction has been shown by surface force measurements to be very short-ranged, e0.5 nm.11 This distance plus the biotin protrusion distance, ≈2.5 nm, is the distance from the vesicle surface that the biotin-streptavidin interaction can “lock-in”. Table 2 shows the respective κ-1 of each of the solutions, including values assuming the surfactant had partially ionized (30%); i.e., the salt plus surfactant reflects fully ionized NaCl and partially ionized (30%) surfactant. The salt concentrations chosen were such that the Debye length was significantly larger (no added salt) than the estimated distance from the vesicle surface of the biotin-streptavidin bond; on the order of the bond dimensions (0.025 M salt); or less than the bond dimensions (0.1 M salt). The best method of determining the extent of aggregation, the size of the aggregates formed, and the extent of deformation of the aggregating vesicles is freeze-fracture electron microscopy.27,28 Freeze-fracture TEM can provide bilayer resolution while simultaneously providing information about the size distribution and the extent of aggregation of the vesicle solution. Freeze-fracture was performed by standard techniques as described elsewhere27,28 using a custom-built environmental chamber for rapid freezing, a Balzers 400 freeze-fracture apparatus for sample preparation, and a JEM 100 CX transmission electron microscope for imaging. The important features to look for in the micrographs are the nature of the contact zones between adhering vesicles as well as the extent of aggregation. Biotin-streptavidin coupling leads to localized contacts without significant deformation of the vesicles.6 Coupling due to nonspecific forces such as van der Waals, hydrophobic, etc., leads to a flattened contact zone between the vesicles and a significant deformation of the vesicles away from spheroidal.6,29
Results and Discussion The predominant structures in each vesicle solution prior to streptavidin addition were ULVs as expected.12-14 MLVs were more common in the salt solutions and at higher surfactant concentrations. Figure 1 is a freezefracture electron micrograph of a salt-free 7/3 CTAT/SDBS vesicle solution at 1 wt % total surfactant (with BiotinX-DHPE at 0.2 mol % of total), showing that the unilamellar nature of the vesicles is not altered by addition of the “impurity” Biotin-X-DHPE at low concentrations. These vesicles are qualitatively similar to catanionic vesicles reported originally.12-14 The vesicles are not aggregated prior to streptavidin addition. Streptavidin (Molecular Probes) was added (in the same concentration of salt solution) at an overall 8/1 biotin/ streptavidin mole ratio to the vesicle solutions (resulting in slight dilution) to induce aggregation. The biotin/ streptavidin ratio corresponded roughly to a 1:1 match (26) DeTitta, G. T.; Edmonds, J. W.; Stallings, W.; Donohue, J. J. Am. Chem. Soc. 1976, 98, 1920. (27) Chiruvolu, S.; Naranjo, E.; Zasadzinski, J. A. In Structure and Flow in Surfactant Solutions; Herb, C. A., Prud’homme, R. K., Eds.; American Chemical Society: Washington, DC, 1994; Chapter 5. (28) Zasadzinski, J. A.; Bailey, S. M. J. Electron Microsc. Tech. 1989, 13, 309. (29) Bailey, S. M.; Chiruvolu, S.; Israelachvili, J. N.; Zasadzinski, J. A. N. Langmuir 1990, 6, 1326.
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Table 2. Debye Lengths of Solutions Studied, including Values Assuming the Partial Ionization (30%) of Surfactant in Solutiona Debye length (nm) partially ionized surfactant 0.025 M NaCl 0.025 M NaCl + surfactant 0.01 M NaCl 0.01 M NaCl + surfactant 1.0 wt % surfactant 0.3 wt % surfactant a
4 6
1.9 1.9
1.7 1.8
1.0 1.0
1 1
Salt + surfactant reflects fully ionized NaCl and partially ionized (30%) surfactant.
Figure 1. Freeze-fracture electron micrograph of a salt-free 7/3 CTAT/SDBS vesicle solution at 0.3 wt % total surfactant with added Biotin-X-DHPE (0.2 mol % of total). Vesicles are freely floating and unilamellar, indicating Biotin-X-DHPE did not alter their equilibrium structure.
between streptavidin binding sites and biotins on the outer vesicle monolayer. In each of the systems, aggregation was not nearly as fast nor as extensive as in the phospholipid vesicle systems,6 where aggregation occurred within 30 min for similar ligand-receptor ratios. In the salt-free solutions, very little aggregation of the charged vesicles was noticeable even after 1 week of incubation with streptavidin; the bluish spontaneous vesicle solution did not change significantly after addition of the receptor. Freeze-fracture transmission electron microscopy (FFTEM) confirmed that very little aggregation (free vesicles plus a few dimers and trimers) had occurred. In fact, in many cases it was difficult to tell whether the adhesion of two vesicles was due to specific interactions; some adhesions appeared to be nonspecific in nature as evidenced by a flat bilayer (Figure 2). Figure 2 shows the state of the salt-free 7/3 CTAT/SDBS vesicle solution at 0.3 wt % total surfactant after about 2 h; the degree of aggregation did not change over the course of a week. The other salt-free vesicle solutions (7/3, 1.0%; 3/7, 0.3%; 3/7, 1.0%) showed similar levels of aggregation. Increasing the surfactant concentration added more vesicles to solution but aggregation was the same; dimers and trimers were the only aggregates present. The low aggregation levels in the 0 M NaCl solutions can be explained by noting that charged vesicles experience a long-range electrostatic double-layer repulsive force and rarely approach close enough to aggregate. Assuming the surfactants were at most 30% ionized, the Debye length, κ-1, was about 4 nm for 1.0 wt % solutions and about 6 nm for 0.3 wt % solutions (cf. Table 2). Apparently, screening by surfactant ionization is not enough to induce vesicle aggregation. To increase aggregation, vesicles were prepared in a 0.1 M NaCl solution. Added electrolyte screens the electrostatic double layer repulsions, decreasing the Debye length. Too much electrolyte (g0.5 M NaCl) destroys the unilamellarity of the vesicles.10 Including the partially ionized surfactants, the Debye length decreased to κ-1 ≈ 1 nm for the 0.1 M NaCl solutions (see Table 2). This
Figure 2. Freeze-fracture electron micrograph of a salt-free 7/3 CTAT/SDBS vesicle solution at 0.3 wt % total surfactant 2 h after addition of streptavidin at 8/1 biotin/streptavidin mole ratio. Only a small fraction of the vesicles (center) are aggregated. The larger arrow points to a flattened contact, most likely due to nonspecific aggregation. The smaller arrow points to a localized contact, most likely due to ligand-receptor aggregation. Most vesicles remain freely floating. The degree of aggregation did not change over the course of a week.
Debye length is much less than the distance the ligand protrudes from the bilayer; hence, binding should therefore proceed more rapidly as observed. The CTAT-rich solution (Figure 3A, 0.3 wt % total surfactant) had high degrees of aggregation (20+ vesicle clusters) 1 week after streptavidin addition. Vesicles appeared to aggregate at specific contact points, similar to phospholipid vesicles.6 The SDBS-rich solutions (Figure 3B, 0.3 wt % total surfactant)had very little aggregation (a few dimers and trimers) after 1 week, comparable to the salt-free samples. In the CTAT-rich solutions, there were higher degrees of aggregation in the 0.1 M salt samples after even 2 h than in salt-free samples after 1 week. One interesting feature of the 0.1 M salt solutions was that the unilamellarity of the vesicles seemed disrupted as more MLVs were seen (cf. Figure 3A). This indicated that the electrolyte may have effectively screened surfactant headgroup repulsions enough to allow MLVs to form.10 Comparable results are seen in the 1.0 wt % solutions. Aggregation is low after a few hours for CTAT-rich vesicles but is substantial after 1 week. Even at the higher surfactant concentration, SDBS-rich vesicles have not aggregated significantly after 1 week (Figure 3C). Our goal was to produce a system in which we can control the extent of specific aggregation of equilibrium unilamellar vesicles. Aggregation in the salt-free samples was minimal, though the vesicles were unilamellar. Aggregation in the 0.1 M NaCl samples was much more controllable (low aggregation at a few hours and high aggregation at 1 week), yet MLVs were forming. Transition of ULVs to MLVs would be disastrous for applications in which vesicle stability was important. To satisfy both unilamellarity and controllable aggregation, we prepared the solutions at an intermediate salt concentration, 0.025 M NaCl. This concentration represented a Debye length about midway between the solutions: κ-1 ≈ 1.7 nm for 1.0 wt % solutions
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Figure 4. Freeze-fracture electron micrographs of vesicles in 0.025 M NaCl solutions 1 week after streptavidin addition. (A) 7/3 CTAT/SDBS vesicle solution at 0.3 wt % total surfactant contains vesicles that exist as multimers (pictured) or remain freely floating. Only a few large aggregates such as those prevalent in the 0.1 M solutions (Figure 3A) were present. Note the vesicles have aggregated at specific points and not in flattened (nonspecific) regions (small arrows). (B) 7/3 CTAT/ SDBS vesicle solution at 1.0 wt % total surfactant contains several large MLV/ULV aggregates. The flattening between vesicles may indicate nonspecific aggregation is occurring, leading to fusion of the vesicles into multilamellar structures.
Figure 3. Freeze-fracture electron micrographs of vesicles in 0.1 M NaCl solutions 1 week after streptavidin addition. (A) 7/3 CTAT/SDBS vesicle solution at 0.3 wt % total surfactant contains vesicles that are highly aggregated. Some vesicles appear to be adhering to each other at specific points (small arrow), similar to the phospholipid vesicles in ref 6.29 (B) 3/7 CTAT/SDBS vesicle solution at 0.3 wt % total surfactant contains vesicles that exist mainly as dimers (pictured), trimers, and freely floating vesicles. Note the nonspecific, flattened contact region (large arrow).6,29 Little specific aggregation was observed because the excess SDBS likely denatures the streptavidin.30-33 (C) 3/7 CTAT/SDBS vesicle solution at 1.0 wt % total surfactant also contains vesicles that exist mainly as dimers, trimers (pictured), and free ULVs. Aggregation is slightly higher than that in 0 M solutions. Note that the vesicles are adhering at flat interfaces (large arrow), indicating nonspecific interactions are likely causing the aggregation rather than specific interactions due to biotin-streptavidin. The added salt reduces the electrostatic repulsion sufficiently that the vesicles can adhere due to van der Waals or other nonspecific attractive forces.6,29
and κ-1 ≈ 1.8 nm for 0.3 wt % solutions, assuming partial (30%) surfactant ionization (see Table 2). The Debye lengths for these solutions were comparable to the distance the biotin protrudes from the bilayer and within the lockin distance, meaning the vesicles can approach within aggregation range. Therefore, one would expect aggregation levels between those of the 0 and 0.1 M samples. After a few hours, aggregation proceeded such that there were a few dimers, trimers, and multimers (aggregates of 5-15 vesicles and e1 µm in size), comparable to the 0.10 M NaCl solutions. There was not much difference between the 0.3 and 1.0 wt % solutions or the CTAT- and SDBS-rich solutions. After 1 week, though, there was a difference in aggregation between the solutions. Several multimers of ULVs (nearly the state of aggregation desired) were present in the CTAT-rich 0.3 wt % sample (Figure 4A). The vesicles in the multimers had aggregated at specific contact points, consistent with ligand-receptor coupling.6,29 Free ULVs were also seen. Note that each of the vesicles in the aggregate is unilamellar in nature and the aggregate has a finite size, even after 1 week of incubation. The comparable SDBS-rich solution had little aggregation (not shown); free ULVs, dimers, and trimers were most common. Even more interesting was that the 1.0 wt % 7/3 solutions with 0.025 M added salt were
Control of Spontaneous Vesicle Aggregation
comparable to the 0.1 M NaCl samples (Figure 4B); large aggregates of flattened MLVs and ULVs were common. FF-TEM of the SDBS-rich solutions again showed significantly lower levels of aggregation (dimers/trimers and freely floating ULVs) after 1 week. Screening the electrostatic repulsion was a critical factor in determining the degree of aggregation in the catanionic vesicle systems. A Debye length greater than the lock-in distance from the bilayer (≈2.5 nm) led to low aggregation levels; a Debye length much less than this distance removed the influence of the electrostatic repulsion and vesicles aggregated readily in the CTAT-rich solutions. In the 0 M salt solutions aggregation is low. In the 0.025 M solutions, aggregation proceeds slowly but to a noticeable extent. In the 0.1 M solutions, aggregation nears “completion”. It appears that an “optimal” Debye length of κ-1 ≈ 1.8 nm leads to increased aggregation levels (multimers) while maintaining the unilamellar structure of the vesicles. This Debye length is comparable to the biotin-streptavidin interaction distance. The lack of specific aggregation in all the SDBS-rich vesicle solutions at all added salt concentrations suggests that the anionic SDBS denatures the streptavidin. This is consistent with other anionic surfactants that are known to denature proteins. For example, sodium dodecyl sulfate (SDS) is used often as a denaturing or unfolding agent for proteins.30-33 In the SDBS-rich vesicle solutions, micellar SDBS coexists with the vesicles12 and is likely reponsible for denaturing the streptavidin. This denaturing is likely due to (1) the surfactant aliphatic chains imbedding in the hydrophobic regions of proteins and (2) the strong electrostatic charges interfering with the hydrogen bonding interactions that help the protein maintain its tertiary structure.30-33 However, the protein does not seem to be affected in the CTAT-rich vesicles because aggregation consistent with specific binding was observed. In the CTAT-rich solutions, all the SDBS is tied up in CTA+DBS- complexes within the vesicle bilayers and unable to interact with streptavidin; very little free or micellar SDBS is available to interact with the streptavidin. Conclusions Specific aggregation of catanionic vesicles can be controlled by adding electrolyte to the solutions. If too much electrolyte is added, the unilamellarity of the vesicles is destroyed. If no electrolyte is added, aggregation is minimal. A near-optimal aggregation state (aggregates of ULVs of 1 µm in size) is achieved with added electrolyte that screens the electrostatic double layer repulsion just enough such that the solution Debye length is about the same as the distance the ligand protrudes from the bilayer (and less than the lock-in distance). Aggregation in this system is much slower than that in phospholipid vesicle systems; it takes days instead of minutes for similar aggregation states to be achieved. The catanionic vesicles have one advantage over the phospholipid vesicles previously used in the aggregation (30) Ibel, K.; May, R. P.; Sandberg, M.; Mascher, E.; Greijer, E.; Lundahl, P. Biophys. Chem. 1994, 53, 77. (31) Ibel, K.; May, R. P.; Kirschner, K.; Szadkowski, H.; Mascher, E.; Lundahl, P. Eur. J. Biochem. 1990, 190, 311. (32) Jones, M. N.; Chapman, D. Micelles, Monolayers and Biomembranes; Wiley-Liss: New York, 1995. (33) Parker, W.; Song, P. S. Biophys. J. 1992, 61, 1435.
Langmuir, Vol. 13, No. 19, 1997 5081
process: catanionic vesicles are equilibrium structures that form by simply mixing the two surfactants in solution. These vesicles can then be controllably aggregated by simply adding receptors to the solution (with added electrolyte). There are no chemical or physical techniques required to form ULVs from MLV dispersions. This work effectively demonstrates how we can go from molecular solution (mixed surfactants) to higher-order structures (aggregated vesicles) by simple self-assembly. We can also modify the short-range, specific aggregation process by controlling electrostatic interactions. One inherent difficulty with the vesicles in this system is that they are not biologically compatible; they are charged soaps that are typically toxic to biological cells. Thus, these particular catanionic vesicles cannot be used in biological applications, and other nonbiological applications must be found. However, this work did demonstrate that specific aggregation of charged vesicles can be controlled by adding electrolyte to the solution. Therefore, charged biologically compatible vesicles could be used and their aggregation controlled. The ideal system would be a biological surfactant mixture that forms equilibrium vesicles spontaneously when dispersed in solution; this way, the vesicles would be biocompatible and their aggregation could be controlled. The alternative is to mechanically create phospholipid vesicles with a small amount of charged lipid to control the aggregation process. The short-range nature of the specific recognition interaction makes controlling aggregation via electrostatics possible. Although the purpose of this study was to create higher order unilamellar vesicle aggregates by simple selfassembly processes, another interesting result indicated that the spontaneous vesicles resembled phospholipid vesicles, as they appeared to be stable against the small osmotic stress imposed by the biotin-streptavidin binding. Catanionic vesicles had been thought to be “floppy” and very susceptible to changes in the osmotic conditions of the medium. Our results indicate that if this were the case, upon aggregation, the vesicles would not maintain their spherical shape and would instead continually flatten out as fluid was pumped out of the vesicle due to deformation caused by several binding points. The vesicles, though, appear to maintain their osmotic stress and do not flatten out, which is similar to the behavior of aggregated lipid vesicles. These results also suggest that electrostatic interactions are important to the stability of spontaneous catanionic vesicles against aggregation. This system demonstrates that higher order organization can proceed from molecular solution to quite complex, highly organized structures through sequential steps of selfassembly. Acknowledgment. We thank Eric Kaler, Kathleen Herrington, Laura Brasher, Michael Kennedy, and Shivkumar Chiruvolu for ongoing discussions on spontaneous vesicles and their properties. Financial support for this project was provided by the National Institutes of Health (GM47334), the National Science Foundation (CTS9305868 and CTS-9319447), and the MSERC program of the NSF under Grant DMR-96-32716. LA970094Z
