Spontaneous Vesicles Formed in Aqueous Mixtures of Two Cationic

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Spontaneous Vesicles Formed in Aqueous Mixtures of Two Cationic Amphiphiles Maria Isabel Viseu,*,† Katarina Edwards,‡ Claúdia S. Campos,† and Sı´lvia M. B. Costa† Centro de Quı´mica Estrutural, Complexo I, Instituto Superior Te´ cnico, Av. Rovisco Pais, 1049-001 Lisboa, Portugal, and Department of Physical Chemistry, Uppsala University, Box 532, S-751 21 Uppsala, Sweden Received June 25, 1999. In Final Form: October 28, 1999 The spontaneous formation of vesicles was detected in aqueous mixtures of two cationic amphiphiles: the double-tailed didodecyldimethylammonium bromide, DDAB, and the single-tailed dodecyltrimethylammonium chloride, DTAC. These aggregates appear in a high-dilution region of the system, intermediate between those where monomers and micelles prevail, and are most easily formed at a considerable excess of the single-chained surfactant (DTAC/DDAB molar ratios ≈ 2-20). Vesicles were characterized at 25 °C by cryogenic transmission electron microscopy (cryo-TEM) and dynamic light-scattering (DLS) measurements: they present a well-defined contour, are mostly spherical and unilamellar, but show a large size polydispersity, with the more frequent population distributions of diameters from ≈40-50 to ≈500-600 nm. Apart from intact vesicles (and in most cases coexisting with them), vesicles with ruptured membranes, small bilayer disks (probably discoidal micelles, rarely found), and globular micelles were also visualized by cryo-TEM. Ruptured vesicles and disks were assigned to intermediate structures between intact vesicles and globular micelles. We propose that the main factor which drives the appearance of vesicles in this bicationic system is the difference in the spontaneous curvature (or packing parameter) of the two long-chained surfactant ions.

Introduction Spontaneous vesicles prepared from aqueous mixtures of commercial, widely available surfactants of simple structures have attracted a great interest in the last few years for several reasons:1 they are quite stable when compared with “conventional”, sonicated vesicles, and their size, charge, or permeability can be readily adjusted by varying the relative amounts and/or chain lengths of the two surfactants. Most spontaneous vesicles reported in the literature are formed in aqueous mixtures of oppositely charged amphiphiles, either with both single-tailed surfactants,1-4 or with double-tailed and single-tailed surfactants,5 or even with two double-tailed surfactants.6 The thermodynamic stability of spontaneous vesicles formed in amphiphile mixtures was explained by Safran et al.,7,8 considering the free energy of elastic curvature of the two monolayers: the vesicle bilayer will be the more stable phase in solution (as compared with the micellar or lamellar phases) only for those special compositions of the inner and outer monolayers which give equal (in * Corresponding author. Telephone: 00-351-21-8419389. Fax: 00-351-21-8464455. E-mail: [email protected]. † Instituto Superior Te ´ cnico. ‡ Uppsala University. (1) Kaler, E. W.; Murthy, A. K.; Rodriguez, B. E.; Zasadzinski, J. A. N. Science 1989, 245, 1371. (2) Kaler, E. W.; Herrington, K. L.; Murthy, A. K.; Zasadzinski, J. A. N. J. Phys. Chem. 1992, 96, 6698. (3) Herrington, K. L.; Kaler, E. W.; Miller, D. D.; Zasadzinski, J. A. N.; Chiruvolu, S. J. Phys. Chem. 1993, 97, 13792. (4) Yatcilla, M. T.; Herrington, K. L.; Brasher, L. L.; Kaler, E. W.; Chiruvolu, S.; Zasadzinski, J. A. J. Phys. Chem. 1996, 100, 5874. (5) Marques, E.; Khan, A.; Miguel, M. G.; Lindman, B. J. Phys. Chem. 1993, 97, 4729. (6) Caria, A.; Khan, A. Langmuir 1996, 12, 6282. (7) Safran, S. A.; Pincus, P.; Andelman, D. Science 1990, 248, 354. (8) Safran, S. A.; Pincus, P. A.; Andelman, D.; MacKintosh, F. C. Phys. Rev. A 1991, 43, 1071.

magnitude) but opposite spontaneous curvatures. This implies an asymmetric distribution of surfactants in the two vesicle monolayers; that is, these aggregates only form at different monolayer compositions. The above stability criterium seems therefore to exclude the formation of vesicles from a pure surfactant in water or, in mixtures, vesicles with equal monolayer compositions, because opposite (nonzero) curvatures cannot be reached spontaneously in such cases. On the other hand, no experimental evidence was found in the literature for the spontaneous formation in water of “bicationic” or “bianionic” vesicles (i.e., vesicles formed from two equally charged long-tailed ions). However, the thermodynamic stability of these vesicles does not seem to be excluded by the theory of Safran et al.7,8 or by other theoretical treatments concerning the same subject.9-16 In our laboratory, vesicles were obtained easily in the bicationic amphiphile system DDAB-DTAC in water, where DDAB stands for the double-tailed (“lipid” type) didodecyldimethylammonium bromide and DTAC for the single-tailed (“detergent” type) dodecyltrimethylammonium chloride. These aggregates were formed by gently dissolving DDAB in an aqueous DTAC solution and apparently remained unaltered for several months. Therefore, they are believed to be spontaneous. They were caracterized at 25 °C by means of the complementary cryogenic transmission electron microscopy (cryo-TEM) and dynamic light-scattering (DLS) techniques. A pre(9) Kumaran, V. J. Chem. Phys. 1993, 99, 5490. (10) Porte, G.; Ligoure, C. J. Chem. Phys. 1995, 102, 4290. (11) Yuet, P. K.; Blankschtein, D. Langmuir 1995, 11, 1925. (12) Bergstro¨m, M.; Eriksson, J. C. Langmuir 1996, 12, 624. (13) Bergstro¨m, M. Langmuir 1996, 12, 2454. (14) Yuet, P. K.; Blankschtein, D. Langmuir 1996, 12, 3802. (15) Yuet, P. K.; Blankschtein, D. Langmuir 1996, 12, 3819. (16) Bergstro¨m, M.; Eriksson, J. C. Langmuir 1998, 14, 288.

10.1021/la990831m CCC: $19.00 © 2000 American Chemical Society Published on Web 01/08/2000

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liminary phase diagram of the system DDAB-DTACWater was obtained in a composition range where vesicles appear. Bicationic amphiphile mixtures present quite distinct phase behavior from that of their catanionic couterparts: two stricking differences are the absence of a precipitate in the former case (which forms around equimolar surfactant ratios in catanionic mixtures) and the “asymmetric” sequence of phases observed from one pure amphiphile to the other, at constant total surfactant concentrations. Reporting on the experimental evidence for the spontaneous formation of bicationic vesicles and describing their properties seems therefore important to stimulate theoretical or other experimental developments on these systems. Experimental Section Materials. Didodecyldimethylammonium bromide (DDAB) was obtained from Fluka, with purity higher than 98%. Dodecyltrimethylammonium chloride (DTAC) was an ion-pair chromatographic reagent, purchased from Tokyo Kasei, Japan. The amphiphiles were used as obtained, without further purification. The water used as solvent was distilled twice and purified with the Millipore Milli-Q system. Preparation of Samples. Solutions of the amphiphile mixtures were prepared by dissolving DDAB in an aqueous DTAC solution, or by the simultaneous dissolution in water of both solid surfactants. In any case, DDAB dissolved more easily in the presence of DTAC than in pure water, and therefore no external energy input was applied except for a gentle manual mixing or slight heating, when necessary. Solutions containing vesicles could be obtained in two different ways: directly, as described above, or by dilution, to the final sample volume, of stock solutions prepared in the region of mixed micelles. All concentrations are given in a molar basis. Most samples were prepared with DDAB mole fractions [xDDAB ) nDDAB/(nDDAB + nDTAC)] from 0.05 to 0.60 and with total surfactant concentrations (C0 ) CDDAB + CDTAC) from 1 to 30 or to 50 mM, covering a composition range where monomers, vesicles, and micelles appear. Pure DDAB solutions, up to 10 mM, and pure DTAC solutions, up to 40 mM, were also made. In the case of pure DDAB, homogeneous “solutions” (or dispersions) could only be obtained after some heating and/or vigorous mixing; these dispersions are metastable, and a phase separation occurs in a few days. Solutions were allowed to equilibrate a few days at room temperature (≈20-25 °C) before they were further analyzed, and at least 1 h at 25 °C just before measurements. These periods of time were considered sufficient to obtain the equilibrium structures because kinetic light-scattering experiments showed that most DDAB-DTAC vesicles appear within a few minutes after dilution of an initial mixed micellar solution.17 Characterization Techniques. The first procedure used to identify the presence of vesicles (and/or lamellae) was a visual inspection of the solutions’ turbidity: samples containing vesicles (or vesicles + lamellae) were more or less turbid, while those containing monomers or micelles were transparent. Two complementary techniques were then used to characterize the vesicles: (1) transmission electron microscopy at cryogenic temperatures (cryo-TEM), to view the vesicles’ morphology and size; (2) quasi-elastic (dynamic) light-scattering (DLS), to obtain their dimensional statistics. Mixed micelles, globular or disklike, could also be detected by cryo-TEM. 1. Cryogenic Transmission Electron Microscopy (cryo-TEM). The technique used for the cryo-TEM examination has been described in detail elsewhere,18,19 but in short consisted of the following. A small drop of sample solution was deposited on a copper grid covered by a holey polymer film. Excess liquid was (17) Viseu, M. I. Unpublished results, 1998. (18) Dubochet, J.; Adrian, M.; Chang, J. J.; Homo, J. C.; Lepault, J.; McDowall, A. W.; Schultz, P. Q. Rev. Biophys. 1988, 21, 129. (19) Bellare, J. R.; Davis, H. T.; Scriven, L. E.; Talmon, Y. J. Electron Microsc. Technol. 1988, 10, 87.

Viseu et al. blotted away with filter paper. By this process (which was performed under controlled temperature (25 °C) and humidity conditions within a custom-built environmental chamber), thin, ≈20-500 nm, sample films spanning the holes in the polymer film were formed. The films were thereafter vitrified by quick freezing in liquid ethane and transferred to a Zeiss EM 902 transmission electron microscope for examination. To prevent sample perturbation and the formation of ice crystals, the specimens were kept cool, below 108 K, during both the transfer and viewing procedures. All observations were made in zero-loss bright-field mode and at an accelerating voltage of 80 kV. To ensure reproducibility, the selection of micrographs presented in the article was chosen from a large number of negatives. 2. Dynamic Light Scattering (DLS). Before the dynamic lightscattering experiments, samples were filtered through Millipore Millex HV13 filters (with pore sizes of ≈450 nm) to remove dust particles in suspension. DLS measurements were performed at 25.0 ( 0.2 °C with an apparatus of standard design, equipped with a 35-mW He-Ne laser, model 127 from Spectra Physics (providing light of 632.8 nm), a variable angle spectrometer from Brookhaven Instruments Corp. (BIC), USA, and an autocorrelator with 136 channels (model BI-2030AT), also from BIC. Scattered light from the sample was analyzed at an angle of 90° from incident light. Two main methods of correlation analysis were used: a 4th-order cumulants expansion (4th CUMFIT) method,20 to calculate mean diameters (〈D〉) and polydispersity indices (σ), and a multiple pass, nonnegatively constrained least-squares (NNLS) method,21,22 to obtain diameter probability distribution functions. The correlation analysis affords directly the aggregates diffusion coefficients, from which diameters were calculated using the Stokes-Einstein relationship.

Results and Discussion General Behavior of DTAC and DDAB Amphiphiles. Even though DTAC and DDAB have alkyl chains of equal length (C12) and similar headgroups (tri- and dimethylammonium, respectively), their pure aqueous solutions present quite different behaviors. The single-chained “detergent-type” DTAC is easily soluble in water, forming micelles above a critical micelle concentration (CMC) of ≈20-22 mM at 25 °C.23,24 The double-chained “lipid-type” DDAB is only sparingly soluble in water, making difficult the evaluation of its critical aggregation concentration (CAC) in true equilibrium conditions. This may be the reason there is some discrepancy in the values obtained for the CAC of this surfactant, at 25 °C: while some references indicate a CAC ≈ 0.05 mM,25,26 other works (including our own conductivity and equilibrium surface tension measurements) indicate higher values, ≈0.14-0.18 mM.24,27 Also, the nature of the spontaneous aggregates formed by DDAB in water beyond the CAC does not seem to be fully established yet. Slightly above the CAC and up to ≈3 wt % DDAB, uni- and bilamellar vesicles have been observed using the cryo-TEM technique.25,26,28 However, these are probably metastable because energy is required (20) Koppel, D. E. J. Chem. Phys. 1972, 57, 4814. (21) Lawson, C. L.; Hanson, R. J. Solving Least Squares Problems; Society for Industrial and Applied Mathematics: Philadelphia, 1995; Chapter 23. (22) Richards, R. W., Ed. Scattering Methods in Polymer Science; Ellis Horwood Limited: London, 1995; Chapter 2. (23) Mukerjee, P.; Mysels, K. J. Critical Micelle Concentrations of Aqueous Surfactant Systems; National Standards Reference Data Series; U.S. National Bureau of Standards: Washington, D.C., 1971. (24) Viseu, M. I.; Vela´zquez, M. M.; Campos, C. S.; Garcıá-Mateos, I.; Costa, S. M. B. Submitted for publication. (25) Marques, E. J. F. Catanionic Surfactant Mixtures. Ph.D. Thesis, Coimbra, 1998. (26) Caria, A.; Regev, O.; Khan, A. J. Colloid Interface Sci. 1998, 200, 19. (27) Treiner, C.; Makayssi, A. Langmuir 1992, 8, 794. (28) Regev, O.; Khan, A. Prog. Colloid Polym. Sci. 1994, 97, 298.

Spontaneous Vesicles in Bicationic Amphiphile Mixtures

in the sample preparation (generally described as a prolonged shaking of the samples). Beyond a concentration of ≈3 wt %, DDAB “swells” in water, forming hydrated lamellar liquid crystals.29-33 These may transform into closed bilayer structures (vesicles) if an adequate energy input (like sonication) is applied to the system.34 However, sonicated DDAB vesicles are metastable and tend to revert to planar lamellae. At room temperature, two lamellar phases have been characterized for DDAB in water:29,31,33 LR1 (“swollen” lamellae) at lower concentrations, ≈3-30 wt % DDAB, and LR2 (“collapsed” lamellae) at higher concentrations, ≈83-91 wt % DDAB, with a two-phase region in between. At temperatures above ≈75 °C, only one lamellar phase appears.31,33 Except for the coexistence of very large, multilayered vesicles with the flat bilayers of LR1, in the case of DDAB,33 none of these surfactants alone seems to form vesicles spontaneously. However, vesicles were easily obtained from DDAB-DTAC aqueous mixtures without any energy input except for a gentle mixing. The first sign of vesicles formation in this system was the quick dissolution of DDAB in water, in the presence of DTAC, and the slight turbidity of the final solutions. These vesicles where then characterized by cryo-TEM and DLS techniques. Cryo-TEM Results. Cryo-TEM is a technique wellsuited to observing small- and medium-sized vesicles with diameters up to about 500 nm.35 Because of the limited thickness of the liquid film spanning the holes in the polymer film, larger vesicles may not be accurately visualized by the technique. Furthermore, very large vesicles can be disrupted because of shear imposed during the blotting step in the sample preparation procedure, in particular those built from bilayers of low cohesive strength. Several solutions of the present system were analyzed by cryo-TEM, most of them in a region where vesicles appear but a few also in the monomer or micellar regions. Table 1 summarizes the aggregates found in each sample, as a function of the DDAB molar ratio (xDDAB) and total surfactant concentration (C0). It is worthwhile to compare the sequence of aggregates found in samples 7-11 of Table 1, at constant surfactants ratio (xDDAB ) 0.20), and in order of increasing C0 (from 1.25 to 50 mM). No aggregates were seen in the very diluted sample 7 (0.25 mM DDAB; 1.00 mM DTAC), which probably contains mainly monomers; however, because the DDAB concentration is slightly higher than its CAC, small aggregates (whose sizes are quite below the resolution limit of the cryo-TEM technique), such as mixed dimers, may also exist in this sample. Vesicles are the only structures observed in samples 8 and 9 (see Figure 2b,c below, for sample 9). In sample 10 a few vesicles are still present, coexisting with globular micelles (Figure 1a), but in the more concentrated sample 11 only globular micelles were found (Figure 1b). Spheroidal micelles in mixed samples (including sample 4, not shown herein) are seen as dots, in the resolution (29) Fontell, K.; Ceglie, A.; Lindman, B.; Ninham, B. Acta Chem. Scand. A 1986, 40, 247. (30) Dubois, M.; Zemb, Th. Langmuir 1991, 7, 1352. (31) Zemb, Th.; Gazeau, D.; Dubois, M.; Gulik-Krzywicki, Th. Europhys. Lett. 1993, 21, 759. (32) Evans, D. F.; Wennerstro¨m, H. The Colloidal Domain; VCH Publishers: New York, 1994; Chapter 6. (33) Caboi, F.; Monduzzi, M. Langmuir 1996, 12, 3548. (34) Kunitake, T.; Okahata, Y. J. Am. Chem. Soc. 1977, 99, 3860. (35) Frederik, P. M.; Stuart, M. C. A.; Bomans, P. H. H.; Lasic, D. D. Cryo-Electron Microscopy of Liposomes. In Handbook of Nonmedical Applications of Liposomes; Lasic, D. D., Barenholz, Y., Eds.; CRC Press: Boca Raton, FL, 1996; Chapter 15.

Langmuir, Vol. 16, No. 5, 2000 2107 Table 1. Aggregates Observed by Cryo-TEM in the System DDAB-DTAC-Water, after Quenching the Samples from 25 °C, as a Function of the DDAB Molar Ratio xDDAB and Total Surfactant Concentration C0 C0 aggregates sample xDDAB (mM) detected 1 2 3 4 5 6 7 8 9 10

0.00 0.05 0.09 0.10 0.11 0.15 0.20 0.20 0.20 0.20

40.00 10.50 11.00 44.45 9.00 11.75 1.25 10.00 12.50 25.00

11 12 13 14

0.20 0.26 0.27 0.31

50.00 13.50 11.00 14.50

15

0.35

15.50 vesicles

16

1.00

2.50 vesicles

vesicles vesicles micelles vesicles vesicles vesicles vesicles micelles +vesicles micelles vesicles vesicles vesicles

comments

figure

a +open membranes +open membranes seen as dots +open membranes b

2a

+open membranes micelles as dots

2b,c 1a

seen as dots +open membranes +open membranes +open membranes +bilayer fragments +open membranes +bilayer fragments quite large +open membranes

1b 2d

2e,f 3

a Pure DTAC micelles must be present in this sample because CDTAC ≈ 2 × CMCDTAC. This CMC was detected by conductivity and equilibrium surface tension measurements.24 b Monomers should be the main species present in this sample. Small aggregates (such as dimers) may also be present because CDDAB > CACDDAB.

limit of the cryo-TEM technique (≈50 Å).36 On the other hand, pure DTAC micelles should be present in sample 1, which contains pure DTAC in a concentration near twice its CMC (CMC ≈ 22 mM, as obtained by conductivity and equilibrium surface tension measurements).24 However, pure DTAC micelles were not visualized by cryo-TEM, probably being smaller than ≈40-50 Å in diameter. Consequently, we infer that micelles formed in the mixed systems are bigger than pure DTAC micelles, and because the amount of DDAB in these solutions is well above its CAC, it is likely that both amphiphiles participate in the micelles structure. Br- counterions, which associate more strongly with the surfactants headgroups than Cl-, may also contribute to lowering the micelles curvature (and thus increasing their aggregation number) as compared to a hypothetical DDAC-DTAC system. Samples 6, 9, 12, and 15 of Table 1, with increasing CDDAB (1.75-5.5 mM) at constant CDTAC (10 mM), provide an interesting sequence where vesicles are the main structures observed (see Figure 2a-f). Pure DDAB vesicles, obtained from the turbid top layer of sample 16 which only contains this surfactant (see Table 1), are shown in Figure 3 for comparison. Apart from vesicles, some other structures appear in a few micrographs, such as small bilayer fragments (Figure 2f) which were found only rarely. Most vesicles observed herein (and in other micrographs, not shown) present a well-defined contour and, when not constrained, have a spherical shape. This is the case of the smaller vesicles seen in the micrographs and of vesicles inside other vesicles. Sphericity is lost for vesicles with diameters comparable to or larger than the film thickness, which are squeezed to the area near the edge of the hole, in the polymer-covered grid supporting the film (see, e.g., the bigger vesicles in Figures 2a,c and 3). Vesicles are mainly unilamellar, but a few multilamellar aggregates were found, as seen in Figures 1a, 2a,c-f, and (36) Almgren, M.; Edwards, K.; Gustafsson, J. Curr. Opin. Colloid Interface Sci. 1996, 1, 270.

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Figure 1. Cryo-TEM micrographs for samples with xDDAB ) 0.20, showing (a) globular micelles and a few vesicles, for C0 ) 25 mM, and (b) globular micelles, for C0 ) 50 mM. Arrows point out some globular micelles. Scale bar ) 100 nm.

3. In the case of the mixed DDAB-DTAC vesicles, multilamellar structures correspond mostly to large vesicles enclosing smaller ones (up to seven vesicles, as seen in Figure 2e), while the pure DDAB bilamellar vesicles show nearly concentric bilayer shells (Figure 3). Most vesicles have intact membranes. But open membranes were also found in several samples, such as those with CDTAC ) 10 mM (see the curved arrows in Figure 2). Ruptured membranes may indicate the onset of a phase or a structural transition. In several lecithin-surfactant systems, open vesicles have been found as an intermediate structure during the vesicles f micelles transition.37-39 These systems resemble the present DDAB-DTAC one in that they contain both double- and single-chained amphiphiles. In the DDAB-DTAC system, however, ruptured membranes were found for C0 values considerably lower than those at which micelles were detected by DLS (see line 1 in the phase diagram of Figure 6 below). But because the showing up of open membranes seems to depend mostly on the DTAC (rather than DDAB) sample content, it is possible that these structures also indicate herein the onset of the vesicles f micelles transition. If this is the case, the transition is not abrupt; that is, vesicles and micelles coexist in an extensive composition region (where DLS only detects vesicles). We are currently investigating this possibility by static light-scattering measurements. The apparent data discrepancy may also be due to some artifacts in the film preparation, before cryogenation (e.g., the preferential migration of the more surface-active DDAB to the newly formed thin film surface), which lower the DDAB/DTAC ratio of the structures visualized by cryoTEM. Small bilayer fragments were observed in a few micrographs of samples 14 (not shown) and 15 (Figure 2f). Those viewed from an edge-on position with respect to the electron beam (marked E in Figure 2f) show a clear rim contrast (they are seen as small lines); others, viewed from a face-on position (marked F in the same Figure), (37) Edwards, K.; Almgren, M.; Bellare, J.; Brown, W. Langmuir 1989, 5, 473. (38) Edwards, K.; Gustafsson, J.; Almgren, M.; Karlsson, G. J. Colloid Interface Sci. 1993, 161, 299. (39) Silvander, M.; Karlsson, G.; Edwards, K. J. Colloid Interface Sci. 1996, 179, 104.

display a rounded, low-contrast contour; and still others, seen from intermediate positions, show eliptical shapes. These fragments are clearly distinguishable from vesicles, which always exhibit a high-contrast rounded contour. They have a disklike shape, showing diameters of ≈2030 nm (smaller than those of the vesicles coexisting with them) and thicknesses of ≈4-5 nm (near the resolution limit of the cryo-TEM technique). If they are flat, as it seems, they can be called disklike or discoidal micelles and may be an intermediate structure in the vesicles f spherical micelles transition. However, similarly to ruptured vesicles, bilayer disks were found for C0 values much lower than those at which micelles were detected by DLS. This discrepancy remains to be clarified. Both structures, open vesicles and disklike micelles, can have their curved edges stabilized by the single-chained DTAC, as discussed later for disks. We should note that open vesicles (but not discoidal micelles) were observed also in the pure DDAB sample 16. In Figure 3, the unilamellar vesicle at the bottom of the picture and the inner membrane of the bilamellar vesicle, just above and to the right of it, are both ruptured. In the pure DDAB system, the exposed hydrophobic edges of ruptured vesicles cannot be stabilized by a singlechained surfactant, as happens in the mixed DDABDTAC system (see the discussion below, for disks). But even for the pure DDAB system, this result is not unexpected: because the picture was taken from the upper phase of a sample that contains both swollen lamellae and vesicles, probably not in thermodynamic equilibrium, the open vesicles may represent short-lived transition structures. Alternatively, open membranes could have been formed because of rupture of some of the extremely large DDAB vesicles, during the cryo-TEM sample preparation. A prominent feature of the present DDAB-DTAC vesicles is their high size polydispersity, with diameters from ≈30-40 nm up to ≈500-600 nm. As a general trend, vesicles tend to be larger as the DDAB/DTAC ratio increases, but exceptions are common, such as the big vesicles in Figure 2a, compared to the small vesicles of Figure 2b. Also, small and large vesicles were found for the same sample, either in different micrographs (e.g., Figure 2b,c, sample 9), or in the same micrograph (e.g., Figure 2e, sample 15).


Vesicles show a greater tendency to aggregate as the DDAB concentration increases: very large vesicles and clusters of vesicles (which could not be entirely depicted by cryo-TEM because their size was comparable to the film thickness) were found in samples containing the highest DDAB concentrations (samples 14 and 15) and, in greater quantity, in the pure DDAB sample 16. Figure 3, for example, shows very large pure DDAB vesicles. Here, we should recall that pure DDAB homogeneous

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“solutions” (or dispersions) were only obtained after some vigorous mixing, and so the vesicles observed may not be the true equilibrium structures. Also, micrograph 3 was obtained from the turbid top layer in the solution, which began to separate from a clear bottom layer ≈1-2 days after sample preparation. It therefore seems that pure DDAB vesicles, whether spontaneous or metastable, belong to a biphasic L1 + LR1 region, in which LR1 designates a “swollen” lamellar phase and L1 an isotropic solution

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Figure 2. Cryo-TEM micrographs for samples with increasing xDDAB, at constant CDTAC (10 mM), showing (a) medium-size and large vesicles and a multilamellar vesicle, for xDDAB ) 0.15, (b) mainly small vesicles, for xDDAB ) 0.20, (c) small and large vesicles, also for xDDAB ) 0.20, (d) small vesicles, a multilamellar vesicle, and a peanut-shaped vesicle, for xDDAB ) 0.26, (e) small vesicles and large, multilamellar, vesicles, for xDDAB ) 0.35, and (f) small vesicles, a multilamellar vesicle, and disklike micelles, also for xDDAB ) 0.35. Curved arrows in (a)-(e) identify some open vesicles; arrows in (f) point out some disklike micelles, viewed edge-on (E) or face-on (F) relative to the electron beam. Scale bar ) 100 nm.

phase containing vesicles. (If vesicles are metastable, they will revert later to LR1, and L1 will be simply water.) DDAB-DTAC samples were more easily prepared than

pure DDAB ones, and a phase separation was not observed in the former system, except for a few samples with high xDDAB ratios, several months after preparation. However,


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Figure 4. Vesicles mean diameters 〈D〉 obtained with a 4thorder CUMFIT correlation analysis of DLS data as a function of total surfactant concentration C0, at several DDAB molar ratios (indicated in the inserted legend).

Figure 3. Cryo-TEM micrograph for the turbid top layer removed from a 2.5 mM pure DDAB sample, showing mainly bilamellar, large and very large vesicles (note the smaller magnification scale, scale bar ) 200 nm).

the large polydispersity and the existence of very large aggregates in these samples strongly suggest that we may not be dealing with a single-phase region. Therefore, in the mixed samples (especially those with the higher xDDAB), vesicles may also belong to a biphasic region, L1 + LR1. If this is the case, it is likely that the lamellae of LR1 contain only the double-tailed DDAB and the vesicles are composed of both surfactants, as discussed later. DLS Results. DLS measurements were performed in a wide composition range, to obtain a general view of the aggregates surrounding the vesicles region. In fact, DLS easily distinguishes samples with monomers and/or micelles, which produce almost no scattering (low signal/ noise ratio in the data aquisition), from those containing vesicles and/or lamellae, which originate considerable scattering (high signal/noise ratio). A second purpose of DLS was to quantify the size and polydispersity of the vesicles because a sampling of the whole solution cannot be made from a few individual structures imaged by cryoTEM. In agreement with the above cryo-TEM analysis, vesicles were found for a total surfactant concentration range C0 intermediate between those where monomers (at low C0) or micelles (at high C0) appear. The extension of this concentration regime increases with the DDAB/DTAC molar ratio. Vesicles mean diameters 〈D〉 evaluated with a 4th-order CUMFIT analysis of the scattering curves are shown in Figure 4, as a function of C0 and xDDAB. We should stress that polydispersity indices are high, and therefore the meaning of mean diameters is not straightforward. As a general trend, mean diameters, which vary from ≈100 to 200 nm, are nearly independent of C0 at constant xDDAB, but tend to increase with the DDAB content of the samples,

Figure 5. Typical probability distribution profile of vesicle diameters D obtained with the NNLS correlation analysis of DLS data for a sample with xDDAB ) 0.20 and C0 ) 10 mM.

for xDDAB ≈ 0.20-0.60. For xDDAB ≈ 0.05-0.15, mean diameters are slightly higher than expected from this trend (not shown). These results show the same trend as the cryo-TEM data. Because polydispersity indices are high, a 4th-order CUMFIT correlation analysis, besides affording only mean diameters, may be quite incorrect.22 A more meaningful representation of size polydispersity can be obtained by directly evaluating the statistical distribution of diameters, for example, with the NNLS option.21,22 Diameters from ≈40-50 to ≈300-400 nm were generally found by the NNLS analysis, with the most significant populations in the range of D ≈ 100-220 nm, but less frequent populations of very small (D ≈ 20-30 nm) or very large aggregates (D ≈ 400-600 nm) were also encountered. A typical population distribution of diameters is presented in Figure 5. The NNLS results also agree with the cryo-TEM analysis, as far as the comparison is possible. The accuracy of DLS results is limited mainly by the following: (i) high polydispersity indices, which make the correlation analysis difficult; (ii) vesicles inside other vesicles (as those of Figure 2e), which do not contribute to the scattered light; (iii) prefiltration of solutions, which may disrupt large vesicles or lamellae. Consequently, the 〈D〉 values and the diameter frequency distributions obtained should be regarded only in a semiquantitative way. DDAB-DTAC-Water Phase Diagram. On the basis of the ensemble of DLS and cryo-TEM results, Figure 6 shows a preliminary phase diagram of the system DDAB-

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Figure 6. DDAB-DTAC-water phase diagram at 25 °C, in a region of high dilution and from surfactants equimolarity to pure DTAC in water (representation in a molar basis). Legend: Open symbols, DLS results; closed symbols, cryo-TEM results; triangles (at the top of the diagram, visible in the zoom, on the right), monomers; circles, vesicles and/or lammellae; squares, micelles. Line 1: approximate separation of vesicles from micelles, obtained from DLS.

DTAC-water at 25 °C, around the region where vesicles and micelles were found. A convenient representation of the system composition, in this highly diluted region, is in terms of the surfactants molar concentrations, instead of weight percentages. Because there is no need to indicate explicitly the water concentration, the base of the triangle shows directly the DDAB molar fraction relative to total surfactant, xDDAB, from zero to equimolarity. Lines at constant total surfactant concentration, C0, are horizontal. Solutions containing only monomers are located at the top of the triangle (lower C0) and those with micelles are displayed at the bottom right (higher C0 and higher xDTAC). Solutions containing vesicles (and/or lamellae) are located in the intermediate region, in a range of C0 which increases in extension with xDDAB. If a single-phase of DDAB-DTAC vesicles exists (the isotropic solution phase, L1), it should be located on the DTAC-rich side of the diagram. However, as referred to above and suggested by the existence of very large and polydisperse aggregates, vesicles may also belong to the biphasic region L1 + LR (which would be located in the left part of the diagram), with an increasing proportion of LR1 as the DDAB/DTAC ratio increases. However, because the cryo-TEM or DLS techniques do not provide effective means to distinguish between vesicle phases and conventional lamellar phases, it was not possible to obtain herein the limit between these mono- and biphasic regions. The transitions monomers f vesicles and vesicles f micelles depend on both C0 and xDDAB, but could not be completely defined from the present cryo-TEM and DLS results. A region of coexistence of vesicles and micelles belonging to the isotropic L1 phase is anticipated, as referred to above. Experiments are in progress to better define these structural transitions. Bilayer Structure of the Vesicles. As stated above, the thermodynamic stability of vesicles formed in amphiphile mixtures implies an asymmetric surfactant distribution in the two monolayers; that is, these aggregates only form spontaneously at different monolayer compositions.7,8 This also implies nonideal mixing of the two components within the membrane; that is, it requires the presence of complexing surfactant interactions (or a nonzero interaction parameter, β).7,8 These interactions can arise from either the surfactants polar heads or chains.7

Because of the high synergism between oppositely charged surfactants in catanionic systems,40 the two components should be present in both the inner and outer vesicle layers. Therefore, the membrane asymmetry must result from a different excess of one component in each layer (in the limit, the inner layer may be neutral). On the other hand, in the case of bicationic (or bianionic) vesicles, strong interactions between the two likely charged components are not expected. In fact, equilibrium surface tension measurements performed in DDABDTAC mixtures showed only a moderate synergism between both amphiphiles (most likely arising from chain interactions), which operates in the concentration regimes where vesicles and/or micelles are formed.24 Consequently, there is no evidence that the two cationic surfactants are present in both of the vesicle layers. An asymmetric bilayer with adequate inner (cin) and outer (cout) curvatures (cin ≈ -cout) may be reached spontaneously when DDAB is the main (even exclusive) component of the inner layer, but it is mixed with DTAC in the outer layer. In fact, DTAC and DDAB have quite different geometrical packing parameters, PP, defined by the ratio vc/ahlc (where ah stands for the headgroup surface area and vc and lc are, respectively, the chain volume and length):41 PP ≈ 1/3 for DTAC (assumed similar to DTAB)42 and PP ≈ 0.6-0.8 for DDAB.42,43 Because both surfactants have the same charge, similar headgroups, and equally sized chains, their mixtures should present an average, or effective, PP approximately weighted by their molar fractions in the two vesicle monolayers. Consequently, if DTAC is mixed with DDAB in the outer layer, PP < 1 and a positive spontaneous curvature is reached therein. (We use the convention that micelles in water have a positive spontaneous curvature). The inner layer can reach a negative curvature if DDAB is its exclusive component (PP > 1 for partially compressed chains). It is interesting to note that vesicles have been anticipated to form spontaneously in the similar bicationic system DDABDTAB-water, based only on an effective PP evaluation.43 Therefore, the main factor that seems to be the driving force for the formation of bicationic vesicles (as compared (40) Zhu, B. Y.; Rosen, M. J. J. Colloid Interface Sci. 1984, 99, 435. (41) Israelachvili, J. N.; Mitchell, D. J.; Ninham, B. W. J. Chem. Soc., Faraday Trans. 2 1976, 72, 1525. (42) Warr, G. G.; Sen, R.; Evans, D. F.; Trend, J. E. J. Phys. Chem. 1988, 92, 774. (43) Evans, D. F.; Ninham, B. W. J. Phys. Chem. 1986, 90, 226.


to the pure surfactants solutions) is a general one which also operates in the formation of other spontaneous vesicles: the possibility of changing the effective packing parameter of the two vesicle monolayers in such a way that equal (in magnitude) but opposite spontaneous curvatures are reached. But in bicationic (or bianionic) vesicles, where there seems to be no special synergism between the likely charged headgroups, the two components must have quite different PPs, as happens with single- and double-tailed amphiphiles. The above condition (different PPs) is not a requisite for catanionic systems because the effective PP of the mixture is not the molar weighted average of the individual surfactants PPs, and therefore vesicles may be formed from surfactants of very similar PPs.1-4 In these systems, electrostatic attraction between oppositely charged headgroups seems to be the main factor for changing the effective monolayers PPs. On the other hand, in catanionic systems containing double- and single-chained amphiphiles, such as DDAB-SDS (where SDS stands for sodium dodecyl sulfate),5 both effects must operate. It is worthwhile to compare the DDAB-DTAC system with lecithin-surfactant systems, in which the micelle f vesicle transition has been widely investigated in view of its application in the reconstitution of membrane proteins.37-39,44-46 Even though with longer chains and zwitterionic phospholipid headgroups, these systems resemble the present one in that they also contain a doubletailed, lamellar-forming amphiphile (lecithin) and a singletailed, micellar-forming surfactant (which may be ionic or nonionic). In lecithin-surfactant systems, vesicles may be obtained spontaneously by dilution of a mixed micellar solution; an example is provided by the lecithin-CTAC system (CTAC standing for hexadecyltrimethylammonium chloride), where vesicles could be obtained with only a gentle shaking, instead of sonication.38 The thermodynamic stability of liposome systems containing a diacyl polar lipid (e.g., phosphatidylcholine) and a single-tailed surfactant has been reviewed recently,47 and it seems that the question whether the vesicles formed “spontaneously” in these systems represent “true equilibrium” or “kinetically trapped” structures is still unresolved. But if the same principles are operating in both types of systems, lecithin-surfactant and DDAB-DTAC, as it seems, vesicles appear in the former type of system mainly because of the different spontaneous curvatures (or PPs) of the lipid and surfactant molecules. A small charge effect (interaction of the ionic surfactant with the oppositely charged part of the lipid headgroup) may also be operating in this case. The different nature of Br- and Cl- counterions is another factor to consider in the mechanism of vesicle formation, in the present DDAB-DTAC system. This effect is difficult to analyze because the similar (and simpler) DDAB-DTAB system was not studied herein. Because DTAC should have a slightly lower PP than DTAB (due to the weaker binding of Cl- to the aggregate, as compared to Br-), we expect that it would be more prone than DTAB to change the curvature of DDAB and form vesicles. But because Br-, rather than Cl-, will preferentially bind to the aggregates, DDAB-DTAC mixtures will probably behave similarly to DDAB-DTAB ones until Br- is exausted as the binding counterion (i.e., Br- will behave as the “limiting” bound counterion). (44) Edwards, K.; Almgren, M. J. Colloid Interface Sci. 1991, 147, 1. (45) Edwards, K.; Almgren, M. Langmuir 1992, 8, 824. (46) De la Maza, A.; Parra, J. L. Langmuir 1995, 11, 2435. (47) Lasic, D. D. J. Liposome Res. 1999, 9, 43.

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Because no special synergism is expected between the likely charged headgroups of the present DDAB-DTAC system, it can be a useful model to study hydrophobic interactions, and therefore to separate the effects of headgroup and chain synergism which operate in different extents in the widely investigated lecithin-surfactant and catanionic amphiphile mixtures. Discoidal Micelles. In the present DDAB-DTACwater system, small bilayer fragments were found in 2 (out of 16) samples analyzed by cryo-TEM. From the different orientations in which these structures were viewed, we concluded that they have a disk shape, showing diameters of ≈20-30 nm and thicknesses of ≈4-5 nm. Their small thicknesses also indicate that they have no water inside, and so they are probably discoidal micelles, an intermediate structure between flat bilayers and spherical micelles. These aggregates have been poorly documented in the literature. Ever since the advent of cryo-TEM, bilayer fragments (also named discoidal micelles or flat nanodisks) have been characterized in different types of amphiphile mixtures, such as lecithin-surfactant systems,37-39 phospholipid mixtures of unequal chain lengths,48 phospholipid-cholesterol systems sterically stabilized by poly(ethylene glycol)-phospholipids,49,50 catanionic mixtures (DDAB-SDS),51 and cationic surfactant in the presence of salt.52 Very recently, they were also detected in other catanionic surfactant mixtures by small-angle neutron scattering53 and freeze-fracture microscopy,54 and in a lecithin-bile salt system by time-resolved static and dynamic light and small-angle neutron scattering.55 In some lecithin-surfactant systems, small bilayer disks were interpreted as intermediates between vesicles and micelles, during the vesicles f cylindrical micelles transition.37-39 This may also be the case of the present DDAB-DTAC system, except that globular, rather than cylindrical, micelles are formed. It is interesting to note that most (may be all) of the amphiphile systems where bilayer fragments were found are also able to form vesicles spontaneously. Indeed, in most of these systems, including the present DDABDTAC one, disks have been observed to coexist with vesicles. However, the concentration domain where disks appear seems to always be much more restricted than that of vesicles. The formation of a large number of small bilayer disks in pure lipid systems is very unfavorable energetically because of the exposure of an enormous hydrophobic area around the fragment edges. However, they can be stabilized in amphiphile mixtures containing a single-tailed surfactant, which has an adequate curvature to protect the hydrophobic tails around the bilayer edges. This could be the role of the surfactant in the lecithin-surfactant systems,37-39 of SDS in the anionic-rich DDAB-SDS system,51 and of DTAC in the present DDAB-DTAC system. In disklike micelles the two component surfactants (48) Vinson, P. K.; Bellare, J. R.; Davis, H. T.; Miller, W. G.; Scriven, L. E. J. Colloid Interface Sci. 1991, 142, 74. (49) Edwards, K.; Johnsson, M.; Karlsson, G.; Silvander, M. Biophys. J. 1997, 73, 258. (50) Beugin, S.; Edwards, K.; Karlsson, G.; Ollivon, M.; Lesieur, S. Biophys. J. 1998, 74, 3198. (51) Marques, E. F.; Regev, O.; Khan, A.; Miguel, M. G.; Lindman, B. J. Phys. Chem. B 1998, 102, 6746. (52) Swanson-Vethamuthu, M.; Feitosa, E.; Brown, W. Langmuir 1998, 14, 1590. (53) Bergstro¨m, M.; Pedersen, J. S. Langmuir 1998, 14, 3754. (54) Zemb, Th.; Dubois, M.; Deme´, B.; Gulik-Krzywicki, Th. Science 1999, 283, 816. (55) Egelhaaf, S. U.; Schurtenberger, P. Phys. Rev. Lett. 1999, 82, 2804.

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are even more segregated than they are in vesicles because the central (flat) part would be constituted almost exclusively by the double-tailed surfactant (or by the equimolar catanionic surfactant, in catanionic systems). Consequently, disks have been anticipated to form only at rather low temperatures (to suppress the effect of the entropy of mixing) or for lipid-surfactant mixtures with unusual curvature properties.56 The identification of a significant fraction of ruptured vesicles and discoidal micelles in vesicle preparations can have important pratical consequences, for example, in systems where vesicles are potential vehicles for drug delivery.49,50 In fact, these open structures are unable to retain water-soluble drugs, and therefore the evaluation of critical concentrations where these aggregates appear (from vesicles) is of most importance.49,50 Conclusion

Viseu et al.

of the vesicles. DLS confirms this high polydispersity (diameters from ≈40-50 up to ≈400-500 nm) and gives semiquantitative values for mean diameters (≈100-200 nm) which are mostly invariant with the total surfactant concentration, at a constant surfactant ratio, but tend to increase slightly with the DDAB molar ratio. Pure DDAB vesicles were found to be rather large as compared to the mixed ones and are probably metastable. It is proposed that the main factor which drives the appearance of vesicles in the system DDAB-DTAC-water is the difference in the spontaneous curvature (or packing parameter) of the two surfactants. Bilayer fragments with diameters of ≈20-30 nm and thicknesses of ≈4-5 nm were found in a few mixed samples, coexisting with vesicles. They were interpreted as discoidal micelles, an intermediate structure between vesicles and spherical micelles.

This paper presents evidence for the formation of vesicles in the system DDAB-DTAC-water, containing two cationic amphiphiles, the double-chained, lamellarforming DDAB and the single-chained, micellar-forming DTAC. The vesicles were obtained by gently dissolving DDAB in an aqueous solution of DTAC and therefore are believed to be spontaneous. Mostly spherical and unilamellar DDAB-DTAC vesicles were found by cryo-TEM, presenting a well-defined and intact contour: exceptions include a few multilamellar aggregates (vesicles inside other vesicles) and some open vesicles. A high polydispersity in size is a main feature

Acknowledgment. The authors are most grateful to Mr. G. Karlsson, at the Department of Physical Chemistry, Uppsala University, for running the electron microscope, to Prof. J. Martinho, from Centro de Quı´mica-Fı´sica Molecular, Instituto Superior Tećnico, Lisbon, for providing the dynamic light-scattering apparatus, and to Dr. E. Marques, from the University of Coimbra, for helpful discussions. This work received financial support from the Junta Nacional de Investigaç aõ Cientı´fica, JNICT (under Project PRAXIS XXI 2/2.1/QUI/443/94), from The Swedish Research Council for Engineering Sciences, and from The Swedish Foundation for Strategical Research. One of the authors (C. Campos) acknowledges a grant from Project PRAXIS XXI.

(56) Kozlov, M. M.; Lichtenberg, D.; Andelman, A. J. Phys. Chem. B 1997, 101, 6600.

LA990831M

Spontaneous Vesicles Formed in Aqueous Mixtures of Two Cationic

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