Temperature-Dependent Vesicle Formation of Aqueous Solutions of

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Temperature-Dependent Vesicle Formation of Aqueous Solutions of Mixed Cationic and Anionic Surfactants Koji Tsuchiya,† Hisanori Nakanishi,† Hideki Sakai,*,†,‡ and Masahiko Abe†,‡ Faculty of Science and Technology, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-8510, Japan and Institute of Colloid and Interface Science, Tokyo University of Science, 1-3 Kagurazaka, Shinjuku, Tokyo 162-8601, Japan Received July 15, 2003. In Final Form: November 12, 2003 The phase behavior of aqueous solutions of mixed cetyltrimethylammonium bromide (CTAB) and sodium octyl sulfate (SOS) was examined at different temperatures (20, 30, 40, and 50 °C). While stable vesicles were formed in a narrow composition range on the SOS-rich side at 20 °C, the range widened remarkably when the temperature was raised to 30 °C. Thus, the vesicle region extended to cover almost the entire composition range, CTAB:SOS ) 0.5:9.5-5.0:5.0, at the total surfactant concentrations of 50-70 mM on the SOS-rich side. To analyze the temperature dependence of this phase behavior of the mixed surfactant system, DSC and fluorescence polarization measurements were performed on the system. The experimental findings obtained revealed that pseudo-double-tailed CTAB/SOS complex, the major component of the bimolecular membrane formed by the surfactant mixture, undergoes a gel (Lβ)-liquid crystal (LR) phase transition at about 26 °C. This phenomenon was interpreted as showing that the bimolecular membrane has no curvature and is rigid and easy to precipitate at temperatures below the phase transition point, whereas it has a curvature and is loose enough to disperse in the solution as vesicles at temperatures above the phase transition point. Vesicles formed by the anionic/cationic surfactant complex were then stable at temperatures above the phase transition temperature of the complex.

Introduction Vesicles have a bimolecular membranous structure consisting of amphiphilic substances with an aqueous phase as its core and are expected to be used as a drug carrier in drug delivery systems,1 a biomembrane model,2 a microfield for chemical reactions,3-5 and so forth. Yet, there still remain many problems to be solved before they are practically used because the method of vesicle preparation usually needs the aid of external forces such as ultrasonication, and vesicles thus prepared are a metastable nonequilibrium system that tends to deposit on standing in aqueous solution.6 These circumstances have prompted many researchers to improve the preparation and stability of vesicles. In recent years, spontaneous vesicle formation in the absence of applied external force has been reported in aqueous solutions of various amphiphilic substances. Spontaneously formed vesicles have many advantages such as the simplicity of their preparation and their good dispersibility in water. So far, spontaneous vesicle formation has been found in various aqueous solutions including those of cationic and anionic surfactant mixtures,7-20 mixtures of didodecyldimethylammonium bromide (DDAB), a double-tailed cationic * To whom all correspondence should be addressed. Phone: +814-7124-1501 (ext. 3621); fax: +81-4-7122-1442; e-mail: hisakai@ rs.noda.tus.ac.jp. † Faculty of Science and Technology. ‡ Institute of Colloid and Interface Science. (1) Ostro, M. J.; Cullis, P. R. Am. J. Hosp. Pharm. 1989, 46, 1576. (2) Singer, S. J.; Nicolson, G. L. Science 1972, 175, 720. (3) Moss, R. A.; Bizzigotti, G. O. J. Am. Chem. Soc. 1981, 103, 6512. (4) Mann, S.; Hanningtom, J. P.; Williams, R. J. P. Nature 1986, 324, 565. (5) Yaacob, I. I.; Nunes, A. C.; Shah, D. O. J. Colloid Interface Soc. 1994, 168, 289. (6) Deasy, P. B. Microencapsulation and related drug processes; Marcel Dekker Inc: New York, 1984. (7) Kaler, E. W.; Murthy, A. K.; Rodriguez, B. E.; Zasadzinski, J. A. N. Science 1989, 245, 1371. (8) Herrington, K. L.; Kaler, E. W.; Miller, D. D.; Zasadzinski, J. A. N. J. Phys. Chem. 1993, 97, 13792.

surfactant, and didodecyldimethylammonium acetate or hydroxide (DDAA or DDAOH),21-25 mixtures of fatty acids and lysolecithin,26,27 lecithin-lysolecithin mixture,28 oleic acid-linoleic acid mixture at pH 8-9,29,30 and ganglioside GM3.31 Many investigations have been conducted mainly on aqueous solutions of mixed cationic and anionic (9) Yatcilla, M. T.; Herrington, K. L.; Brasher, L. L.; Kaler, E. W.; Zasadzinski, J. A. N. J. Phys. Chem. 1996, 100, 5874. (10) Kaler, E. W.; Herrington, K. L.; Murthy, A. K.; Zasadzinski, J. A. N. J. Phys. Chem. 1992, 96, 6698. (11) Brasher, L. L.; Herrington, K. L.; Kaler, E. W. Langmuir 1995, 11, 4267. (12) Chiruvolu, S.; Israelachvile, J. N.; Naranjo, E.; Xu, Z.; Zasadzinski, J. A.; Kaler, E. W.; Herrington, K. L. Langmuir 1995, 11, 4256. (13) Backlund, S.; Friman, R.; Karlsson, S. Colloids Surf., A 1997, 123, 125. (14) Marques, E. F.; Regev, O.; Khan, A.; Miguel, M. G.; Lindman, B. J. Phys. Chem. B 1998, 102, 6746. (15) Marques, E. F.; Regev, O.; Khan, A.; Miguel, M. G.; Lindman, B. J. Phys. Chem. B 1999, 103, 8353. (16) Marques, E. F.; Khan, A.; Miguel, M. G.; Lindman, B. J. Phys. Chem. 1993, 97, 4729. (17) Kondo, Y.; Uchiyama, H.; Yoshida, N.; Nishiyama, K.; Abe, M. Langmuir 1995, 11, 2380. (18) Sakai, H.; Imamura, H.; Kakizawa, Y.; Abe, M.; Kondo, Y.; Yoshino, N.; Harwell, J. H. Denki Kagaku 1997, 65, 669. (19) Sakai, H.; Matsumura, A.; Yokoyama, S.; Saji, T.; Abe, M. J. Phys. Chem. B 1999, 103, 10737. (20) Tsuchiya, K.; Sakai, H.; Kwon, K.; Takei, T.; Abe, M. J. Oleo Sci. 2002, 51, 133. (21) Talmoon, Y.; Evans, D. F.; Ninham, B. W. Science 1983, 221, 1047. (22) Ninham, B. W.; Evans, D. F.; Wei, G. J. J. Phys. Chem. 1983, 87, 5020. (23) Brady, J. E.; Evans, D. F.; Kachar, R.; Ninham, B. W. J. Am. Chem. Soc. 1984, 106, 4279. (24) Brady, J. E.; Evans, D. F.; Warr, G. G.; Grieser, F.; Ninham, B. W. J. Phys. Chem. 1986, 90, 1853. (25) Miller, D. D.; Magid, L. J.; Evans, D. F. J. Phys. Chem. 1990, 94, 5921. (26) Jain, M. K.; van Echteld, C. J. A.; Ramirez, F.; de Gier, J.; de Hass, G. H.; van Deenen, L. L. M. Nature 1980, 284, 486. (27) Jain, M. K.; de Hass, G. H.; van Deenen, L. L. M. Biochim. Biophys. Acta 1981, 642, 203. (28) Hauser, H. Chem. Phys. Lipids 1987, 43, 283. (29) Gebicki, J. M.; Hicks, M. Nature 1973, 243, 232. (30) Gebicki, J. M.; Hicks, M. Chem. Phys. Lipids 1976, 16, 142.

10.1021/la0302908 CCC: $27.50 © 2004 American Chemical Society Published on Web 02/11/2004

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surfactants since Kaler et al. reported spontaneous vesicle formation in the system.7 In mixed systems of cationic and anionic surfactants, however, the region in which stable vesicles are formed is rather narrow because the oppositely charged surfactants generally form an insoluble salt (catanionic surfactant) that precipitates around the equimolar mixing ratio as a result of the charge neutralization between their hydrophilic groups. This is a large obstacle to putting cationic/anionic mixed surfactants to practice even though they have excellent solution properties. The development is then demanded of such mixed surfactant systems that allow formation of stable vesicles in a wide composition range. Meanwhile, it is well known that the bimolecular membrane fluidity of vesicles (liposomes) consisting of phospholipids changes significantly and the vesicles undergo a gel-liquid crystal phase transition with variations in temperature. The hydrophobic groups of phospholipids have a trans-type conformation in the gel phase and the molecules form close-packed rigid bimolecular membranes, whereas the groups take a gauche-type conformation in the liquid crystal phase and the molecules form loose bimolecular membranes. However, few studies have been made on the temperature dependence of spontaneous vesicle formation in aqueous solutions of mixed cationic and anionic surfactants. Then, it will be worthwhile to examine the effect of temperature on the phase behavior of the system because this would provide us with a clue to the formation mechanism of stable vesicles in mixed cationic/anionic surfactant systems. We report in this article the experimental results on the effect of temperature upon the phase behavior, especially spontaneous vesicle formation, of aqueous mixtures of cetyltrimethylammonium bromide (CTAB) and sodium octyl sulfate (SOS), a most frequently studied system.

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Figure 1. Ternary phase diagram for CTAB/SOS/H2O at 20 °C. (M, spherical micelles; R, rodlike micelles; V, vesicles; L, lamellar liquid crystals; and P, precipitate.) steel pan was used as the sample vessel. The reference was distilled water with the same weight as that of the sample. Fluorescence Polarization Measurement. Examination of the microviscosity of bimolecular membranes was made through fluorescence polarization measurements using 1,6diphenyl-1,3,5-hexatriene (DPH) as a fluorescence probe. DPH was dissolved in tetrahydrofuran and the resultant solution was added to each of the aqueous CTAB/SOS solutions to make the molar ratio of DPH to surfactant 1:500. The DPH containing solutions were then incubated at 37 °C for 1 h to solubilize the probe and the fluorescence intensity was measured with a fluorescence spectrophotometer (RF-5000, Shimadzu Co.). The excitation and emission wavelengths were 350 and 450 nm, respectively. Fluorescence polarization (P) was calculated using the following equation.

P ) (Ip - Iv)/(Ip + Iv)

Materials and Methods Materials and Preparation. Cetyltrimethylammonium bromide (CTAB, Sigma-Aldrich Chemical Co.) was used as the cationic surfactant after being purified by recrystallization from 1:1 mixture of ethanol and acetone. Sodium octyl sulfate (SOS, Sigma-Aldrich Chemical Co.) was used as supplied as the anionic surfactant. The water used was distilled water for injection (Otsuka Pharmaceutical Co.). Solutions of mixed surfactants were prepared by mixing stock solutions of CTAB and SOS at various molar ratios at different total concentrations under stirring with a vortex mixer for 5 s at room temperature. No strong external forces such as ultrasonic irradiation were applied in the preparation. The mixed solutions thus prepared were allowed to stand at different temperatures (20, 30, 40, and 50 °C). Freeze-Fracture Transmission Electron Microscopy (FF-TEM). TEM observations of the sample were conducted using the freeze-replica method. The sample was rapidly frozen in liquid propane with a cryo-preparation system (LEICA EM CPC, LEICA microsystems), and the frozen sample was transferred into a freeze-replica preparing apparatus (FR-7000A, Hitachi Science Systems) and fractured with a glass knife at -120 °C. A replica film was prepared by evaporating platinumcarbon at 45° and then carbon at 90° on the fractured sample. The replica film prepared was washed several times with acetone and distilled water after being taken out of the freeze-replica preparing apparatus and transferred onto a 300-mesh copper grid. The replica thus prepared was observed with a transmission electron microscope (JEM-1200EX, JEOL). Differential Scanning Calorimetry (DSC). DSC measurements were performed with a differential scanning calorimeter (DSC 8230, Rigaku Co.) at a heating rate of 1 K/min. A stainless (31) Cantu, L.; Corti, M.; Musolino, M.; Salina, P. Europhys. Lett. 1990, 13, 561.

where Ip and Iv are the fluorescence intensities of the emitted light polarized parallel and vertical to the exciting light, respectively.

Results and Discussion Phase Behavior of Aqueous Mixture of CTAB and SOS at 20 °C. The phase behavior of aqueous solutions of CTAB/SOS mixtures was examined in detail first at 20 °C. Figure 1 is a pseudoternary phase diagram8.9 for a dilute solution of CTAB/SOS left standing for at least a week at 20 °C after preparation. The phase behavior was examined by visual, differential interference optical microscopic, and FF-TEM observations. Solutions with CTAB-rich compositions in the phase diagram are taken up first. White crystalline precipitates were observed in the solution containing CTAB alone and the precipitates disappeared with a small addition of SOS to give a transparent solution. Since the Krafft point of CTAB is 25.4 °C as revealed by the results of DSC measurement described later, the white precipitates are likely to be a hydrated CTAB solid. With further addition of SOS, the viscosity of the solution increased, a fact suggesting the formation of rodlike micelles (R).9 When the ratio of SOS to CTAB was increased beyond those in the R region, cloudlike aggregates (cloud wisps)9-11 were observed in the highly viscous solution, which separated into two layers with further increase in the ratio up to 1:1. The upper layer contained white cloudlike aggregates while the lower layer was a highly viscous turbid solution. FF-TEM observations of the aggregates in the upper layer showed the formation of a lamellar liquid crystal, the layer

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Figure 2. Freeze-fracture TEM micrographs of an aqueous mixture of CTAB and SOS. (Total surfactant concentration: 120mM, CTAB: SOS ) 5.5:4.5, R + L region.)

Figure 3. Freeze-fracture TEM micrographs of an aqueous mixture of CTAB and SOS. (Total surfactant concentration: 50mM, CTAB: SOS ) 3.0:7.0, V region.)

thickness of which was about 8 nm (Figure 2, left). The presence of multilamellar vesicles was also verified (Figure 2, right). These regions were designated as R + L region on the basis of the results described above and the pseudoternary phase diagram already reported for CTAB/SOS/ H2O system at 25 °C.9 The V region (vesicle lobe) was not seen on the CTAB-rich side of our phase diagram. However, the vesicle lobe is expected to exist in the more dilute CTAB-rich solutions (at concentrations below 20 mM) as in the phase diagram for CTAB/SOS/H2O at 25 °C reported by Kaler et al.9 On the other hand, the region denoted by M on the SOS-rich side of the diagram was for transparent mixed micellar solutions. Highly viscous rodlike micelles (R) were observed in a narrow area located at total surfactant concentrations and CTAB ratios higher than those for the M region. Crystalline precipitates (P) were found in a wide range of CTAB/SOS ratio from equimolar to SOS-rich compositions. Solutions in the region denoted by V were bluish and turbid and kept their dispersion stability for more than a month. This region was identified as a vesicle region (vesicle lobe) because the presence of monodisperse vesiclelike particles of about 100 nm size was recognized by FFTEM (Figure 3). Despite this finding, the region in which stable vesicles are formed at 20 °C was narrow, in accordance with the results obtained at 25 °C.9 In addition, solutions with compositions nearer to the equimolar one than those in the V region separated into two layers, the upper one containing white cloudlike aggregates and the lower one being a bluish and turbid solution. Figure 4 shows typical FF-TEM pictures of the aggregates seen in the upper layer, indicating that they are lamellar aggregates piling up irregularly in contrast to those observed in the R + L region on the CTAB-rich side.

Figure 4. Freeze-fracture TEM micrographs of an aqueous mixture of CTAB and SOS. (Total surfactant concentration: 120mM, CTAB: SOS ) 4.0:6.0, V + L region.)

Various types of molecular aggregates were formed in aqueous solutions of CTAB/SOS mixture (20 °C) as mentioned above and reported in the earlier papers.8-10 However, spontaneous formation of stable vesicles (V) was observed only in solutions with compositions in a narrow range on the SOS-rich side. Temperature Dependence of Phase Behavior of Aqueous CTAB/SOS Mixture. The phase behavior of aqueous solutions of mixed CTAB and SOS was examined at 30, 40, and 50 °C. Figure 5 shows the pseudoternary phase diagrams of CTAB/SOS system at the three temperatures. Comparison of this figure and Figure 1 indicates that the temperature rise from 20 to 30 °C causes significant changes in the phase behavior of the system, notably a remarkable expansion of the V region where stable vesicles are formed. Actually, stable vesicles were formed at almost all compositions (CTAB:SOS ) 5.0:5.0-

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Figure 6. DSC heating curves of aqueous mixtures of CTAB and SOS at a concentration of 50 mM.

Figure 5. Ternary phase diagrams for CTAB/SOS/H2O at (a) 30 °C, (b) 40 °C, and (c) 50 °C. (M, spherical micelles; R, rodlike micelles; V, vesicles; L, lamellar liquid crystals; and P, precipitate.)

0.5:9.5) on the SOS-rich side when the total surfactant concentration ranged from 40 to 60 mM. As far as we know, no paper has reported the formation of stable vesicles in aqueous solutions of mixed cationic and anionic surfactants with such a wide range of compositions. The area of the V region widened to a small extent at 40 °C, whereas it shrank slightly at 50 °C. This may imply that the thermal motion of molecular aggregates becomes more brisk at higher temperatures to increase the frequency of collisions between the aggregates, thereby causing their coagulation to occur more easily. While precipitate formation was observed in a considerably wide CTAB/SOS ratio range from equimolar to SOS-rich compositions at 20 °C, it was limited to an extremely narrow range around the equimolar composition and a turbid solution was obtained at the other compositions at 30-50 °C. In particular, no precipitate formation was observed in the total surfactant concentration range of 50-90 mM even at the equimolar composition and the solution was turbid (V + L region). This turbid solution at the equimolar composition was verified to contain vesicles by differential interference optical microscopic and FF-TEM observations. While precipitates were formed in solution of CTAB alone at 20 °C, a temperature lower than its Krafft point of 25.4 °C, the solution turned a transparent and isotropic micellar one at temperatures higher than 30 °C. The area of the M region observed at SOS-rich compositions expanded with increasing temperature while that of the V + L region shrank. This would be brought about by an increase in the solubility of surfactant molecules in water produced by the temperature rise to make the formation of mixed micelles easier than that of bimolecular membranes. The above findings demonstrate that the phase behavior of aqueous solution of CTAB/SOS mixture changes with temperature, the change being remarkable especially when the composition lies on the SOS-rich side, and the area of the V region greatly expands if the temperature rises from 20 to 30 °C.

Differential Scanning Calorimetry (DSC). The phase behavior of aqueous solution of CTAB/SOS mixture was shown to significantly change with temperature in the foregoing section. The temperature dependence of the phase behavior was then examined using differential scanning calorimetry (DSC). Figure 6 shows the results of DSC measurements in the heating process for aqueous CTAB/SOS mixture at the total surfactant concentration of 50 mM. The temperature at which endothermic peak starts was taken as the phase transition point. An endothermic peak was observed at 25.4 °C for CTAB solution, corresponding to its Krafft point32,33 (the temperature of phase transition from hydrated solid to spherical micelle). This endothermic peak disappeared with addition of SOS to the solution and an endothermic peak (∆) appeared at around 11 °C. When the mole fraction of SOS, XSOS, changed from 0.05 to 0.30, the endothermic peak shifted to the lower temperature side. This composition range corresponds to that for highly viscous solutions containing rodlike micelles at temperatures above the phase transition point (Figures 1 and 5). Precipitate formation was observed when the phase state of the solution was examined at 0 °C, a temperature below the phase transition point. Hence, the endothermic peak can be considered to be due to the phase transition from precipitate to rodlike micelle. An endothermic peak appeared at about 3 °C (O) for solutions at XSOS ) 0.300.40. Precipitate formation was observed in solutions at these compositions at 0 °C. Lamellar liquid crystals (cloud wisps) dispersed in water at temperatures above the phase transition point (Figures 1 and 5). These findings indicate that the endothermic peaks are ascribable to the phase transition from precipitate to lamellar liquid crystal. An endothermic peak (*) appeared at about 26 °C at the equimolar composition (XSOS ) 0.50). This peak is due presumably to the phase transition from precipitated salt (CTA+OS-) to dispersed vesicle (V). This temperature corresponded to the temperature at which the phase state significantly changed as shown in Figures 1 and 5. When the SOS mole fraction, XSOS, changed from 0.50 to 0.80, the phase transition temperature gradually shifted to the lower temperature side (down to 24 °C). This corresponds to the loosening of membrane packing caused by the change in the composition in the bimolecular membrane with an increase in the SOS ratio. Judging from the (32) Kaneshima, S.; Yamanaka, M J. Colloid Interface Sci. 1989, 131, 493. (33) Kaneshima, S.; Yamanaka, M J. Colloid Interface Sci. 1990, 140, 474.

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pseudoternary phase diagrams shown in Figures 1 and 5, the endothermic peaks observed at XSOS ) 0.50-0.65 and 0.70-0.80 are due to the phase transitions from precipitate (P) to vesicle (V) and from vesicle + lamellar liquid crystal (V + L) to vesicle (V), respectively. This would mean that crystalline precipitate observed at temperatures below the phase transition point (at XSOS ) 0.50-0.65) consists of lamellae because the phase transition temperature at this SOS mole fraction is almost equal to that at XSOS ) 0.70-0.80. Pearson et al. reported that phosphatidylcholine (PC) molecules, which form liposome in water, are arranged in a lamellar structure in the crystal.34 Further, bimolecular membranes undergo a gel-liquid crystal phase transition with variations of temperature. Surprisingly, no DSC peak was observed in the temperature range used when XSOS was higher (0.85-0.90). In addition, no precipitate was formed even at 20 °C and instead stable vesicles (V) were formed (Figure 1). Vesicles formed at these compositions were dispersed stably even at 0 °C. This means that the system exhibits no clear phase transition at these compositions because the bimolecular membrane is enough loose to have a spontaneous curvature as mentioned later. The results of DSC measurements described above strongly suggest that the expansion of the V region caused by the temperature rise from 20 to 30 °C arises from the phase transition at 24-26 °C from precipitates formed at the equimolar composition to vesicles. Fluorescence Polarization Measurement. The microviscosity of the interior of bimolecular vesicle membranes formed in aqueous mixed CTAB/SOS solutions was examined through measurements of the temperature dependence of fluorescence polarization using DPH as fluorescence probe. DPH is located in the hydrophobic region of the bimolecular membrane and thus allows to evaluate the microviscosity in its vicinity.35-37 The microviscosity of bimolecular membranes is determined by fluorescence polarization according to the Perrin-Weber equation.38 When the degree of fluorescence polarization of DPH is high or low, the microviscosity of the interior of bimolecular vesicle membrane is also high or low. Figure 7 shows the effect of temperature on the degree of fluorescence polarization of DPH in 50 mM aqueous mixed CTAB/SOS solutions (SOS-rich side). The results on the solutions with SOS mole fractions ranging from 0.6 to 0.9 are considered first. Vesicles are formed in these solutions at temperatures between 20 and 50 °C as indicated in the pseudoternary phase diagrams in Figures 1 and 5 (V or V + L region). The microviscosity of the bimolecular vesicle membrane in these solutions was lower than that (ca. 0.1) of the liquid crystal phase of DPPC (L-R-dipalmitoylphosphatidylcholine), a typical phospholipid,39 which suggests a loose structure of the vesicle membrane. The temperature rise caused the degree of fluorescence polarization to decrease, thus making the membrane looser. Let us see next the effect of temperature on the degree of fluorescence polarization of DPH in 50 mM aqueous mixed CTAB/SOS solution at XSOS ) 0.5. The degree of fluorescence polarization was almost zero at 20 and 25 (34) Pearson, R. H.; Pascher, I. Nature 1979, 281, 499. (35) Kinoshita, K.; Kataoka, R.; Kimura, Y.; Gotoh, O.; Ikegami, A. Biochemistry 1981, 20, 4270. (36) Kinoshita, K.; Ikegami, A. Biophys. Soc. 1982, 37, 461. (37) Zolese, G.; Gratton, E.; Curatola, G. Chem. Phys. Lipids 1990, 55, 29. (38) Yagi, K.; Sekine, T. Application of Fluorescence Spectrometry to Biochemistry Research; Gakkai Shuppan Press: 1976; p 34. (39) Fukuzawa, K.; Chiba, H.; Suzuki, A. J. Nutr. Sci. Vitaminal 1980, 26, 427.

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Figure 7. Relationship between fluorescence polarization and temperature for CTAB/SOS aqueous mixtures at a concentration of 50 mM. Fluorescence probe: 1,6-diphenyl-1,3,5hexatriene (DPH).

°C, both being below the temperature corresponding to the endothermic peak (*) in Figure 6. This would indicate that no DPH molecule is incorporated into the molecular aggregates from the bulk phase because of the formation of precipitates at the equimolar composition. The degree of fluorescence polarization increased at temperatures above that corresponding to the endothermic peak (*) and became almost identical with the degree in the V region (XSOS ) 0.6-0.9), a fact implying that the bimolecular membranes disperse in the solution in the form of liquid crystal above that temperature. The results mentioned above suggest that vesicles formed in aqueous mixed CTAB/SOS solutions consist of loosely packed bimolecular membranes whose state is comparable to or looser than that of the liquid crystal of nonequilibrium vesicles formed by ordinary double-tailed surfactants. On the other hand, CTAB/SOS complex is in the form of a crystal or a hydrated solid below the phase transition temperature (ca. 26 °C) at compositions around the equimolar one, while it takes the form of a liquid crystal above the temperature, thereby contributing to vesicle formation. Effect of Temperature on Vesicle Formation in Aqueous CTAB/SOS Mixture. A great increase was shown previously in the region of vesicle formation (vesicle lobe) in aqueous mixed CTAB/SOS solution produced by a temperature rise from 20 to 30 °C. The major cause of this event is discussed in this section in terms of the relationship between spontaneous vesicle formation and temperature. Many papers have dealt with vesicle formation in aqueous solutions of mixed cationic and anionic surfactants from both theoretical and experimental viewpoints.7,10,11,39,40 Kaler et al. described the formation of a pseudo-double-tailed complex (catanionic surfactant) with a geometrical structure (critical packing parameter, CPP) favorable to bimolecular membrane formation because of the electrostatic attraction between the hydrophilic groups of both types of surfactants as a major cause of vesicle formation.7,11 The nonideal mixing of pseudo-double-tailed complexes and excess surfactant molecules in the inner and outer monolayers of the bimolecular membrane allows the formation of spontaneous curvatures,10,40,41 that is, excess surfactant molecules that are easy to have a curvature orient themselves preferentially in the outer (40) Safran, S. A.; Pincus, P.; Andelman, D. Science 1990, 248, 354. (41) Safran, S. A.; Mackintosh, F. C.; Pincus, P. A.; Andelman, D. A. Prog. Colloid Polym. Sci. 1991, 84, 3.

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monolayer. This is one of the reasons for spontaneous formation of vesicles. The results of DSC and fluorescence polarization measurements on aqueous mixed CTAB/SOS solution revealed that an endothermic peak appears at about the equimolar composition because of the phase transition from precipitate (or V + L) to vesicle, and the bimolecular membrane of vesicles formed at temperatures above this phase transition point is in a liquid crystalline state. This would suggest that the endothermic peak observed at about the equimolar composition is ascribable to the phase transition from gel state to liquid crystal state of the bimolecular membrane. The main component of vesicles formed around the equimolar composition is CTAB/SOS complex and very few excess SOS molecules are present. The bimolecular membrane formed at temperatures below this phase transition point is predicted to be in a state corresponding to the gel phase (Lβ) of nonequilibrium vesicles formed by double-tailed surfactants39 and CTAB/ SOS complex, the major component of the membrane, forms a rigid layer. Since several papers have reported an important contribution of the bending energy of bimolecular membrane to the criterion for determining whether the membrane forms vesicle or planar lamella from a theoretical viewpoint,10,40,41 rigid membranes are unlikely to disperse in water as vesicles because of their high bending energy. In addition, the hydrophilicity of CTAB/ SOS complex is extremely low since the electric charges on the hydrophilic groups are screened because of the electrostatic attraction between them. In view of these considerations, the dispersibility of bimolecular membranes (gel phase, Lβ) formed at temperatures below the phase transition point would be poor, thereby leading easily to precipitate formation. On the other hand, the bimolecular membrane would be in a liquid crystal state (LR) at temperatures above the phase transition point. The packing of molecules would then be loose enough for the membrane to acquire a curvature with ease and consequently it disperses forming vesicles. Hargreaves et al. examined the temperature dependence of spontaneous vesicle formation using aqueous mixtures of partially ionized fatty acids with C8 to C18 hydrocarbon chains.42 These mixtures formed crystals at temperatures below a certain dissolution temperature (Tm) and permitted spontaneous vesicle formation at temperatures above Tm. Similarly, stable vesicles are expected to be spontaneously formed in aqueous solutions of mixed cationic and anionic surfactants above the dissolution temperature (gel-liquid crystal phase transition temperature) of the mixed surfactants (catanionic surfactant) that constitute the bimolecular membrane. Liposomes composed of phospholipids are also dispersed more stably when they are in the liquid crystal state than in the gel state. Although bimolecular membranes consisting of phospholipids form liposomes in the gel state, this formation is achieved by the aid of external forces including ultrasonic wave and the liposomes formed are dispersed in water only temporarily. These nonequilibrium vesicles in both gel and liquid crystal states eventually separate from the aqueous (42) Hargreaves, W. R.; Deamer, D. W. Biochemistry 1978, 17, 3759.

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medium as precipitates with the passage of time, just as bimolecular membranes consisting of cationic/anionic surfactant mixtures in the gel state. This would suggest that bimolecular membranes in the liquid crystal state and spontaneous curvature formation through nonideal mixing in the membrane are necessary conditions for spontaneous vesicle formation to occur in cationic/anionic surfactant mixtures. Hence, the great increase in the vesicle region produced by the temperature rise from 20 to 30 °C should be caused by the fact that bimolecular membranes formed at about the equimolar composition aggregate and deposit as precipitates at temperatures below the phase transition (gel-liquid crystal) point while they disperse in water as vesicles at temperatures above the phase transition point. The endothermic peak (*) observed at about the equimolar composition in the DSC measurement gradually diminished with increasing molar ratio of SOS and shifted to the lower temperature side. This would mean that excess SOS molecules are incorporated into the outer monolayer of the bimolecular membrane consisting of CTAB/SOS complex and mix nonideally with the complex to make the membrane acquire a curvature spontaneously with increasing molar ratio of SOS10,40,41 and the membrane components gain a two-dimensional mobility, thus approaching a liquid crystal-like state. Moreover, the increase in the molar ratio of SOS would bring the membrane to a state where the fluidity is high to allow it to acquire a curvature spontaneously even at 20 °C to form stable vesicles. The present work demonstrated that the temperature of phase transition from gel to liquid crystal of cationic/ anionic surfactant complex greatly affects the formation of stable vesicles in aqueous solutions of mixed cationic and anionic surfactants. This finding would suggest the possibility of obtaining stable vesicles in a wide composition range at room temperature if the combination of cationic and anionic surfactants with a low phase transition (gel-liquid crystal) temperature is used. Conclusions Examinations of the phase behavior of aqueous solutions of mixed CTAB and SOS at 20, 30, 40, and 50 °C revealed that the phase behavior is significantly affected by temperature at compositions on the SOS-rich side. In particular, a slight temperature rise to 30 °C from room temperature caused a shrink in the precipitation region at about the equimolar composition and an appreciable expansion in the vesicle region. This was interpreted as showing that the temperature rise causes the bimolecular membrane to undergo a phase transition from gel (Lβ) to liquid crystal (LR). The bimolecular membrane was rigid and unstable enough to precipitate at temperatures below the phase transition point, whereas it was loose enough to acquire a curvature and disperse as vesicles at temperatures above the phase transition point. The low phase transition temperature of CTAB/SOS mixture was thus considered to be the major cause of the formation of stable vesicles in the aqueous medium. LA0302908