2340
Langmuir 2001, 17, 2340-2345
Aggregate Structures in a Dilute Aqueous Dispersion of a Fluorinated/Hydrogenated Surfactant System. A Cryo-Transmission Electron Microscopy Study Simona Rossi,*,† Go¨ran Karlsson,‡ Sandra Ristori,† Giacomo Martini,† and Katarina Edwards‡ Dipartimento di Chimica, Universita` di Firenze, Via G. Capponi 9, 50121 Firenze, Italy, and Department of Physical Chemistry, Uppsala University, P.O. Box 532, S-751 21 Uppsala, Sweden Received October 16, 2000. In Final Form: February 1, 2001 The structural features of vesicles, micelles, and other aggregates spontaneously formed from a fluorinated surfactant (the ammonium salt of perfluoropolyether, PFPE) and a hydrogenated surfactant (ndodecylbetaine) in dilute water solution were characterized by means of cryo-transmission electron microscopy (cryo-TEM) at different betaine mole fractions. The size distribution of the aggregates was found to depend critically on surfactant composition. In a narrow range of betaine molar fractions (xbet ) 0.76-0.79), two different populations of unilamellar and spheroidal vesicles with mean radii of 70-120 and 20-30 nm, respectively, coexisted with globular and, in some cases, threadlike micelles. In the same region, vesicles with openings in the bilayer and disk-shape fragments began to appear. Further increase in betaine molar fraction resulted in an increase of the number of globular micelles and discoid aggregates and, finally, to the complete PFPE solubilization into mixed micelles. The large vesicles disappeared at xbet ) 0.81, whereas the small vesicles (mostly with open bilayers), occurred up to xbet ) 0.86. At xbet < 0.75 no micelles were detected, and the sample consisted of uni- and multilamellar vesicles with high polydispersity. Increasing the total surfactant concentration gave rise to a significant increase of the vesicle size without modification of the size distribution of the aggregates themselves.
Introduction Fluorinated surfactants, where fluorine atoms replace hydrogen atoms, show the typical amphiphilic behavior of the corresponding hydrogenated surfactants. However, with respect to their hydrogenated counterparts, fluorinated surfactants show higher hydrophobicity, as is well documented by the more pronounced lowering of the water surface tension and by the lower critical micelle concentration, cmc.1-5 Moreover they show thermal and chemical inertness, which are peculiar to C-F bond-containing compounds. The tendency of fluorosurfactants to form large and stable aggregates of anisotropic shape in water solution is well documented.1 Typically, large aggregates are considered as the precursors of lyotropic liquid crystal phases, which usually form at concentrations lower than those of the corresponding hydrogenated surfactants. Another important characteristic of fluorocarbon surfactant is their tendency to form stiff bilayer aggregates and structures with curvature lower than that of hydrocarbon surfactants.4,6 Fluorinated amphiphiles show high biocompatibility and have been investigated as carriers of oxygen and drugs and of contrast agents in magnetic resonance imaging.7-12 * To whom correspondence may be addressed. Tel.: +39-0552756315. Fax: +39-055-244102. E-mail:
[email protected]. † Universita ` di Firenze. ‡ Uppsala University. (1) Kissa, E. Fluorinated Surfactants; Surface Science Series, No. 50; Marcel Dekker: New York, 1994, and references therein. (2) Fletcher, P. D. I. In Specialist Surfactants; Robb, I. D., Ed.; Blackie Academic and Professional: London, 1997; p 104. (3) Monduzzi, M. Curr. Opin. Colloid Interface Sci. 1998, 3, 467. (4) Hoffmann, H.; Wu¨rtz, J. J. Mol. Liquids 1997, 72, 191. (5) Ravey, J. C.; Ste´be´, M. Colloids Surf., A 1994, 84, 11. (6) Wang, K.; Karlsson, G.; Almgren, M. J. Phys. Chem. B 1999, 103 (43), 9237.
The miscibility of hydrogenated and fluorinated compounds is nonideal and unfavored.1 The van der Waals interactions among fluorinated chains are much weaker than those in hydrogenated chains and this has been invoked as a possible explanation for fluorocarbon/ hydrocarbon mutual phobicity. Thus, the mixing of surfactants with hydrocarbon and fluorocarbon tails largely depends on the interactions between their hydrophilic headgroups. Almgren et al.13 have found separate populations of hydrocarbon-rich and fluorocarbon-rich surfactant micelles, and many papers have been published that strongly support a similar kind of segregation.14-19 Recently, the phase diagram of a hydrogenated surfactant (n-dodecylbetaine) and a perfluorinated surfactant (the ammonium salt of perfluoropolyether, PFPE) has been described in dilute aqueous solution by Ristori et al.20 The authors suggest that, at low total surfactant concentration, a monodisperse population of vesicles (7) Riess, J. G.; Frezard, F.; Greiner, J.; Ktafft, M. P.; Santaella, C.; Vierling, P.; Zarif, L. Handb. Nonmed. Appl. Liposomes 1996, 3, 97. (8) Riess, J. G.; Krafft, M. P. Chem. Phys. Lipids 1995, 75, 1. (9) Riess, J. G. New J. Chem. 1995, 19, 891. (10) Kunitake, T.; Okahata, Y.; Yusunami, S. J. Am. Chem. Soc. 1982, 104, 5547. (11) Gross, U.; Kolditz, L.; Papke, G.; Rudiger, S. J. Fluorine Chem. 1991, 53, 163. (12) Trabelsi, H.; Szonyi, S.; Gaysinski, M.; Cambon, A.; Watske, H. J. Langmuir 1993, 9, 1201. (13) Almgren, M.; Wang, K. Langmuir 1997, 13, 4535. (14) Mukerjee, P.; Yang, A. Y. S. J. Phys. Chem. 1976, 80, 1388. (15) Shinoda, K.; Nomura, T. J. Phys. Chem. 1980, 84, 736. (16) Funasaki, N.; Hada, S. J. Phys. Chem. 1980, 84, 736. (17) Asakawa, T.; Johten, K.; Miyagishi, S.; Nishida, M. Langmuir 1985, 1, 347. (18) Burkitt, S. J.; Ottewill, R. H.; Hayter, J. B.; Ingram, B. T. Colloid Polym Sci. 1987, 2656, 628. (19) Asakawa, T.; Hisamatsu, H.; Miyagashi, S. Langmuir 1995, 11, 478. (20) Ristori, S.; Appel, J.; Porte, G. Langmuir 1996, 12, 686.
10.1021/la001444b CCC: $20.00 © 2001 American Chemical Society Published on Web 03/24/2001
Aggregate Structures of Fluorinated Surfactants
occurs together with some polydisperse aggregates and mixed micelles. The dynamic and structural behavior of the self-organized assemblies were investigated by means of static light scattering (LS),20 quasi-elastic light scattering (QELS),20 and magnetic resonance spectroscopies (ESR and NMR).21-23 The relative amounts of monodisperse and unilamellar vesicles with respect to the other aggregates were found to depend on the betaine/PFPE molar ratio and, to a lesser extent, on the total concentration of surfactants. In particular, in a very narrow range of betaine/PFPE molar ratios (xbet from 0.7 to 0.8) and at total surfactant concentration in the range from 0.25 to 1.27% w/w, spontaneous vesicles of well-defined sizes occurred with rh ranging from 70 to 130 nm. The monodispersity was found to have its optimum value at xbet ≈ 0.75. The authors also found that vesicles invariantly coexisted with smaller aggregates, which were supposed to be micelles. Furthermore, spectroscopy data suggested the existence of different betaine- and PFPE-rich domains in the vesicle bilayer.21-23 The betaine/PFPE structures (mixed aggregates) have also been investigated as possible carriers for porphyrinderivative molecules bearing long hydrocarbon and fluorocarbon chains, which mimick the prostetic group of heme proteins.22,23 The aim of the present study was to provide a direct morphological overview of the aggregation behavior in the PFPE/betaine system. LS and QELS techniques give valuable information on particle mean diameter without readily detect subtle changes in morphology that may take place in the different types of aggregate formed by surfactants, especially when several distributions of aggregates with different shape and size coexist. The cryotransmission electron microscopy (cryo-TEM) method used in this study is not quantitative but offers unique possibilities to visualize the microstructure of the aggregates formed by amphiphilic molecules in aqueous solution.24,25 Cryo-TEM allowed us to distinguish different kinds of aggregates at different compositions of the mixture. Uni- and multilamellar vesicles, globular and threadlike micelles, and openings in vesicles were detected. Experimental Section Materials. Perfluoropolyether
(n ) 3, MW ) 681, density ) 1.8 kg/dm3, monodispersity g95% as determined by chromatographic analysis) and its sodium salt were prepared and characterized as reported in refs 20, 21, and 26. Its cmc in pure water is 2 × 10-4 mol/dm3.21 The aggregation properties and the spectroscopic features of the assembled fluorinated systems are reviewed in refs 27 and 28. The hydrocarbon surfactant n-dodecylbetaine
(21) Ristori, S.; Maggiulli, C.; Appel, J.; Marchionni, G.; Martini, G. J. Phys. Chem. B 1997, 101, 4155. (22) Ristori, S.; Rossi, S.; Ricciardi, G.; Martini, G. J. Phys. Chem. B 1997, 101, 850. (23) Rossi, S.; Ristori, S.; Pozzi, G.; Martini, G. Inorg. Chim. Acta 1998, 272, 274. (24) Talmon, Y. Ber. Bunsen-Ges. Phys. Chem. 1996, 100, 364. (25) Almgren, M.; Edwards, K.; Gustafsson, J. Curr. Opin. Colloid Interface Sci. 1996, 1, 270. (26) Caporiccio, G.; Burzio, F.; Carniselli, G.; Bianciardi, V. J. Colloid Interface Sci. 1984, 98, 202. (27) Martini, G.; Ristori, S.; Visca, M. Physical Chemistry of Ionomers; Schlick, S., Ed.; CRC Press: Boca Raton, FL, 1996; p 219. (28) Ristori, S.; Martini, G.; Schlick, S. Adv. Colloid Interface Sci. 1995, 57, 65.
Langmuir, Vol. 17, No. 8, 2001 2341
was synthesized and purified as a zwitterionic surfactant (MW ) 271, density ) 0.93 kg/dm3)29,30 and used as prepared. Its cmc is 2 × 10-3 mol/dm3.29-31 The polar head area (0.5 nm2/molecule) is almost the same for both surfactants.1,31 Solutions of the two surfactants were prepared in twicedistilled (Milli-Q) water containing 0.1 mol/dm3 NH4Cl. Pure PFPE solutions were prepared by bath sonication whereas betaine solutions were made by simply dissolving the surfactant in brine. The use of brine is required to strongly reduce the interactions among aggregates. Mixed vesicles were prepared by simply mixing the two surfactant solutions. No sonication was required to obtain the structures described in this paper. Methods. The technique used for cryo-TEM examination has been described in detail elsewhere,32-34 and it can be shortly summarized as it follows. Thin sample films (10-500 nm) of the mixed systems were prepared at controlled temperature (298 K) and relative humidity (98-99%) within a custom-built environmental chamber. The films were thereafter vitrified by quick freezing in liquid ethane and transferred to a Zeiss EM 902A transmission electron microscope for examination. Samples were protected against atmospheric conditions, and the temperature was kept below 108 K during both transfer and examination. A zero-energy loss bright-field mode was used, and the accelerating voltage was 80 kV. The electron exposures were between 5 and 15 e-/Å2, depending on the magnification. The extent of underfocus varies approximately between 1 and 3 µm. To ensure reproducibility, micrographs presented in the article were chosen from a large number of images. It should be noted that in viewing the images the twodimensional projection of a closed vesicle appears as a circular object with enhanced contrast around the rim. This is due to the fact that the projected thickness of the bilayer shell is maximum at the edges. On the other hand, the projection of a flat bilayer disk appears even in contrast right up to the edge. Larger particles tend to cluster at the edges of the film where the thickness is greater.
Results Figure 1 reports the cryo-TEM image of a PFPE 0.5% w/w in 0.1 mol/dm3 NH4Cl water solution. Several multilamellar vesicles were identified. In particular, the micrograph shows numerous small vesicles together with large vesicles (radii ranging from 10 to 120 nm). The smallest vesicles were not well resolved and most of them appeared as unstructured dots, slightly larger than typical globular micelles. At higher total surfactant concentration (e.g., 1.27% w/w) the size and the number of the multilamellar vesicles appeared practically unmodified, whereas the number of small vesicles increased (data not shown). A small number of open multilamellar vesicles could be distinguished. The low contrast shown by the aggregates in pure PFPE solutions appears to be a general feature of fluorocarbon systems. Cryo-TEM micrographs of aggregates formed by cationic fluorosurfactants, showing the same diffuse aspect, have been reported by Wang et al.6 (29) Gauthier-Fournier, F. Ph.D. Thesis, University Montpellier II, France, 1986. (30) Marignan, J.; Gauthier-Fournier, F.; Appel, J.; Akoum, Y.; Lang, L. J. Phys. Chem. 1988, 92, 440. (31) Chevalier, Y.; Storet, Y.; Pourchet, S.; Le Perchet, P. Langmuir 1991, 7, 848. (32) Almgren, M.; Edwards, K.; Karlsson, G. Colloids Surf., A 2000, 174, 3. (33) Bellare, J. R.; Davis, H. T.; Scriven, L. E.; Talmon, Y. J. Electron Microsc. Tech. 1988, 10, 87. (34) Dubochet, J.; Adrian, M.; Chang, J.; Homo, J.; Lepault, J.; Alaisdar, W. M.; Schultz, P. Q. Rev. Biophys. 1988, 21, 129.
2342
Langmuir, Vol. 17, No. 8, 2001
Rossi et al.
Figure 3. Cryo-TEM micrographs of the betaine/PFPE system (xbet ) 0.5, total surfactant concentration 0.5% w/w). Note again the presence of tube-shaped aggregated small vesicles inside the unilamellar vesicles. Bar ) 100 nm. Figure 1. Cryo-TEM micrographs of pure PFPE solutions 0.5% w/w in 0.1 M NH4Cl brine. The sample presented spherical multilamellar vesicles of different sizes. Some of them appeared open (A, B). Note the diffuse aspect of the surfactant aggregates. Bar ) 100 nm.
Figure 4. Cryo-TEM micrograph of the betaine/PFPE system (xbet ) 0.75, total surfactant concentration 0.5% w/w). Spherical unilamellar vesicles were revealed together with a few open structures (A), in coexistence with disk-shaped aggregates observed face-on (B) and edge-on (C). The inset micrograph shows some globular micelles. Bar ) 100 nm.
Figure 2. Cryo-TEM micrographs of the betaine/PFPE system (xbet ) 0.4, total surfactant concentration 0.5% w/w). Unilamellar vesicles with closed bilayer aggregates inside and high size polydispersity. Bar ) 100 nm.
Upon addition of small amounts of betaine (up to xbet ) 0.2) the diffuse aspect of the micrographs disappeared and multilamellar structures were no longer observed. A further increase of xbet to 0.4 gave rise to unilamellar vesicles with high polydispersity (Figures 2) and to a broad distribution of size with radii ranging from 10 to 300 nm. Unilamellar vesicles and closed bilayer aggregates were also present inside the large spherical vesicles. Large unilamellar vesicles enclosing tube-shaped aggregates appeared together with small vesicles in samples with xbet ) 0.5 (Figure 3). The vesicles with mean diameter of 10-20 nm were resolved well enough to show the typical contour of the bilayer. Interesting changes in the aggregate structure were observed as xbet increased to 0.75. Figure 4 revealed the formation of new structures as well as a dramatic change in the vesicle population. The small vesicles with diameters below 20 nm almost disappeared, and spherical, unila-
mellar vesicles with radii ranging from 25 to 150 nm dominate the sample. At this composition unilamellar vesicles with an open bilayer structure appeared clearly for the first time. The inset micrograph in Figure 4 also revealed globular micelles in coexistence with closed unilamellar vesicles. Occasionally we also observed threadlike micelles, similar to the one shown in Figure 5b for another system composition (see below). With an increase of xbet to 0.77 the particle shape polydispersity decreased significantly and two different vesicle populations began to appear. The sample at xbet ) 0.78 showed two coexisting populations of unilamellar vesicles with different size (Figure 5a): large vesicles with radii of 80-100 nm, which completely dominated the picture, and small vesicles with radii of 20-25 nm. Long threadlike micelles were also observed at this composition (Figure 5b) as well as in samples with xbet up to 0.81. When betaine concentration further increased, the number of small vesicles also increased whereas the number of large vesicles remained almost constant. Both populations appeared to increase in size. Figure 5c shows a micrograph of a xbet ) 0.79 solution. Unilamellar vesicles with radii of 100-120 nm appeared close to the edge of the polymer film. They were well separated from one another. The same micrograph shows two large vesicles with openings or pores through the membrane. From this surfactant composition upward, open vesicles appeared
Aggregate Structures of Fluorinated Surfactants
Langmuir, Vol. 17, No. 8, 2001 2343
Figure 6. Cryo-TEM micrograph of the betaine/PFPE system (xbet ) 0.77, total surfactant concentration ) 1.27% w/w). Large and unilamellar vesicles with radii of 120-130 nm coexisted with small ones (radii of 30-50 nm) and globular micelles. Arrow denotes an ice crystal deposited on the sample surface after vitrification (A). The inset micrograph is an enlargement of the figure and shows the small vesicle with same globular micelles and disklike fragments. Bar ) 100 nm.
Figure 7. Cryo-TEM micrograph of the betaine/PFPE system (xbet ) 0.81; total surfactant concentration ) 0.5% w/w). Large and small intact vesicles appeared to be spherical. Arrows denote flat bilayer fragments viewed either edge-on (A) or faceon (B) and an open vesicle (C). Bar ) 100 nm.
Figure 5. Cryo-TEM micrographs of the betaine/PFPE system at: (a) xbet ) 0.78; spherical and unilamellar vesicles with welldefined radius coexisted with (b) small vesicle and threadlike micelles indicated by the arrow. (c) xbet ) 0.79; arrows denote two unilamellar vesicles with an opening in the bilayer. Total surfactant concentration: 0.5% w/w. A few large vesicles, with diameters up to 300 nm, were occasionally found (not shown) in all three samples. See text for more information. Bar ) 100 nm.
also in the population of small size (results not shown). In the range xbet ) 0.77-0.79, two vesicle populations with well-defined radii were the dominant structures, even
though large vesicles with a radius up to 150 nm could be detected occasionally. The occurrence of vesicles with two different size distributions in a narrow range of compositions in the betaine/PFPE mixed system was verified by increasing the total surfactant concentration. At a total surfactant concentration of 1.27% w/w and xbet ) 0.77, two different vesicle populations were again observed (Figure 6), with radii of 120-130 and 25-50 nm, respectively. In addition to the unilamellar and spheroidal vesicles, a large number of globular micelles were also found (see the inset in the micrograph). Figure 7 shows the aggregates formed at a total surfactant concentration of 0.5% w/w and xbet ) 0.81: a large fraction of small vesicles (60 nm) coexisted with a few large vesicles (140-150 nm). Small edge-on (A arrow in Figure 7) and face-on (B arrow in Figure 7) bilayer disks were detected, and open vesicles were observed in both populations. Disks were already observed at xbet ) 0.75, and their number progressively increased with xbet. At xbet > 0.81 the large vesicles (mean radius of 140150 nm) completely disappeared (Figure 8a, xbet ) 0.82) and a large number of small bilayer fragments and disklike structures coexisted with small vesicles and globular micelles. At xbet ) 0.86 most vesicles had openings, and large disk-shaped bilayer fragments were observed together with globular micelles (Figure 8b). Finally, at xbet ) 0.92 only globular micelles were detected.
2344
Langmuir, Vol. 17, No. 8, 2001
Figure 8. Cryo-TEM micrographs of the betaine/PFPE system (total surfactant concentration ) 0.5% w/w). In (a) xbet ) 0.82, spherical small vesicles with mean diameter of 30-50 nm. Note the large number of fragments, small bilayer disks, and globular micelles in the inset micrograph. No large vesicles were found. In (b) xbet ) 0.86, unilamellar vesicles, all of them spherical, coexisted with discoidal aggregates (A) Some of the vesicles appeared open (B, C). The arrow (D) denotes an ice crystal deposited on the sample surface after vitrification. Note also the abundance of small fragments and globular micelles. Bar ) 100 nm.
Cryo-TEM analysis of pure betaine solutions at concentrations above the cmc (0.5% w/w, 1.85 × 10-2 mol/ dm3) confirmed the existence of small globular micelles. Discussion The cryo-TEM data reported above revealed a complex morphology of the system. Interestingly, it was found that, in a narrow region of the phase diagram, inside the region where two distinct populations of aggregates were detected by light scattering,20 actually two types of vesicles coexisted, one of them having extremely small size. CryoTEM images clearly provided evidence of many other structural features that were previously unknown. The interaction between betaine molecules and PFPE aggregates was characterized into three distinct, composition-dependent regions. With xbet < 0.75 heterogeneous vesicle populations were produced. The absence of micelles and open vesicles showed a highly efficient incorporation of betaine into PFPE bilayer assemblies. Higher xbet values decreased the shape polydispersity by favoring the formation of two populations of vesicles, with extremely small and much larger size, respectively. The region of composi-
Rossi et al.
tions where this happened stretched from xbet ) 0.76 to xbet ) 0.79. Interestingly, the two vesicle populations occurred together with globular and, occasionally, threadlike micelles. At last, when xbet was higher than 0.86, the vesicles disappeared and only micelles could be observed. Light scattering data in pure betaine solution, above the critical micellar concentration (cmc ) 2 × 10-3 mol/ dm3) indicate the presence of globular micelles with hydrodynamic radius rh ∼ 2 nm.30 The addition of PFPE in small amounts gives rise to a steady increase of the micelle size, up to rh ) 7-9 nm. The present cryo-TEM images of betaine solutions at the same composition also showed clearly the presence of globular micelles. Moreover, with PFPE addition up to xbet ) 0.92 the micrographs confirmed that the micelles had grown toward an elongated structure. On the other hand, on the PFPE-rich side of the phase diagram, where undefined polydisperse aggregates have been suggested to exist on the basis of scattering and magnetic resonance data,20-23 cryo-TEM showed multilamellar vesicles with either spherical or elongated shapes. In addition, cryo-TEM pictures of the pure PFPE solutions revealed the presence of open vesicles. Open structures were also detected at xbet > 0.75. It is important to stress that the formation of pores in the bilayer structure probably occurred via different mechanisms in the presence and in the absence of betaine. The geometrical shape of the PFPE molecule is more compatible with aggregates of low curvature, so that flat bilayers are highly favored. It is known that PFPE water solutions give multilamellar structures.27 After ultrasonic irradiation of pure PFPE solution, multilamellar vesicles are formed that with time open up, fuse, and gradually evolve toward a lamellar phase. Therefore open vesicles, built from perfluorinated surfactant only, represent intermediate structures on the way to reach the preferred lamellar arrangement. The mechanism of pore formation in the presence of betaine is, on the other hand, based on the insertion of a molecules that prefer aggregates of high curvature. This mechanism will be discussed in more detail later on. Addition of small amounts of betaine (xbet e 0.2) did not change the general appearance of the micrographs with respect to that of pure PFPE. However, the incorporation of low betaine amounts gave rise to a lamellarity decrease. At xbet ) 0.2 the vesicles were uni- or bilamellar. A further increase in betaine content produced heterogeneous vesicle populations composed of coexisting very large and small units. Increasing xbet up to 0.75 led to a narrower size distribution, and the vesicles were all spheroidal and unilamellar. As quoted above, the fact that fluorinated and hydrogenated surfactants possess only limited mutual affinity is well documented,1 and it is especially true for micelle formation, where maximum-type cmc curves are often observed and many examples of two different kinds of aggregates, one fluorocarbon-rich and the other hydrocarbon-rich, have been reported.13,35-37 Of particular interest for the present investigation is the observation reported by Knoblich et al.38 that the threadlike and globular micelles are simultaneously formed in a mixed (35) Akune, T.; Abe, M.; Murata, Y.; Maki, T.; Moroi, Y.; Furuya, H.; Tanaka, M. J. Colloid Interface Sci. 1996, 181, 36. (36) Asakawa, T.; Hisamatsu, T.; Miyagishi, S. Langmuir 1996, 12, 1204. (37) De Lisi, R.; Inglese, A.; Milito, S.; Pellerito, A. Langmuir 1997, 13, 192. (38) Knoblich, A.; Matsumoto, M.; Murata, K.; Fujiyoshi, Y. Langmuir 1995, 11, 2361.
Aggregate Structures of Fluorinated Surfactants
fluorinated-hydrogenated system. Comparatively, less work has been done on the investigation of mixed vesicles.39 The present investigation clearly provided evidence of the segregation of fluorinated and hydrogenated components in the PFPE/betaine system. As reported by Knobich et al.,38 we also observed that two different micellar populations occurred together: one of them composed of very long threadlike structures and the other of globular aggregates. On the basis of the preferred curvature of the respective component, it is reasonable to assume that the globular aggregates predominantly consist of betaine, whereas the threadlike micelles contain a large fraction of PFPE. The fact that, in a limited concentration range, micelles coexisted with vesicles is further proof of the segregative behavior in this system. Again, it is probable that the structures with the lowest curvatures, i.e., the vesicles, are considerably more rich in PFPE. The most unexpected finding in the present investigation was the coexistence of two distinct vesicle populations in the composition range xbet ) 0.77 to xbet ) 0.79. The appearance of two vesicle populations suggests, however, that the betaine molecules, as in the case of the micelles, are unevenly distributed between the two vesicle populations. Magnetic resonance experiments carried out by Ristori at al.21 suggest that in mixed vesicles of PFPE and betaine the betaine molecules preferentially accumulate in the highly curved outer monolayer. One could speculate that apart from this intravesicular segregation there is also an intervesicular segregation resulting in a higher betaine molar ratio in the small vesicles. The assumption that the small and more highly curved vesicles can better accommodate the betaine molecules than large vesicles of low curvatures is supported by the fact that the latter disappear at high xbet values. The important question of whether the two coexisting vesicle populations represent true equilibrium structures cannot be resolved on the basis of the present data. It is, in this context, interesting to note that the coexistence of two vesiscles populations of very different size has been predicted in thermodynamic terms by the Poisson-Boltzmann cell model,40 though it has only been experimentally observed in a few systems.41,42 In particular, it has been observed by cryo-TEM in dilute salt solutions of a cationic fluorosurfactant.6 In the present system, in the composition region where twovesicle populations coexisted, a significant growth of the vesicles could be observed with increasing betaine concentration for reasons that remain unexplored at this moment. At xbet ) 0.77 the radii of the two aggregates were 70 and 15 nm, respectively, whereas at xbet > 0.79 these values grew to 120-130 and 30 nm. Cryo-TEM micrographs at 1.27% w/w total surfactant concentration and xbet ) 0.77 agreed again with the presence of two vesicle populations, together with globular micelles. However the vesicle sizes were larger. At total surfactant concentrations of 0.5 and 1.27% w/w disk formation was observed to begin from xbet ) 0.75, where some vesicles showed stabilized openings in the bilayer structure. The transition from vesicles to bilayer disks (39) Gross, U.; Kolditz, L.; Papke, G.; Rudiger, S. J. Fluorine Chem. 1996, 71, 1853. (40) Oberdisse, J. Eur. Phys. J. B 1998, 3, 463. (41) Beguin, S.; Gabrielle-Madelmont, C.; Paternostre, M.; Ollivon, M.; Lesieur, S. Prog. Colloid Polym. Sci. 1995, 98, 206. (42) Marques, E. F.; Regev, O.; Khan, A.; Miguel, M. G.; Lindman, B. J. Phys. Chem. 1999, 103, 8353.
Langmuir, Vol. 17, No. 8, 2001 2345
upon addition of a solubilizing amphiphile is neither unique nor peculiar to the system investigated in the present study. Open structures and disklike bilayer fragments have been documented during solubilization of vesicles by a number of conventional surfactants.43-48 The formation of openings in the vesicle bilayer in the presence of micelle-forming surfactants has been explained in terms of the surfactants ability to shield the open edges and thus minimize the edge energy.45,49-51 At xbet > 0.81 most of the large vesicles had disappeared. The small vesicle population was still present at this composition, but it was now dominated by open vesicles. At xbet > 0.86 the small vesicles were completely solubilized into mixed micelles that became spherical and decreased in size with increasing betaine concentration. Conclusion The cryo-TEM investigations carried out in this study revealed new important information on the aggregate structure in the betaine/PFPE mixed systems in dilute aqueous solution. The micrographs showed that the aggregate morphology dramatically changed with surfactant composition. In particular, in the region of the betaine molar fraction 0.76-0.79 the dominant structures were unilamellar and spheroidal vesicles with well-defined radius. At total surfactant concentration 0.5% w/w, two different populations were detected, with mean radii of 80-100 and 20-30 nm, respectively, together with globular and occasionally threadlike micelles. The formation of two different size vesicle populations and two types of micelle aggregates was interpreted as a consequence of a segregative distribution of the betaine molecules between the aggregates. The threadlike micelles were detected only in the xbet ) 0.75-0.80 range, whereas the globular micelles started to appear at xbet ) 0.75 and their number increased with betaine content. No large vesicles were detected at xbet g 0.82. The transition from mixed vesicles to globular micelles occurred via intermediate structures, such as open vesicles and disks. The maximum fraction of betaine that may be incorporated without alteration of the vesicle bilayer was 0.75. Acknowledgment. Financial support from the Swedish Foundation for Strategical Research, the Swedish Research Council for Engineering Sciences, the Consorzio per lo Sviluppo di Sistemi a Grandi Interfasi (CSGI), and the University of Florence is gratefully acknowledged. Thanks are due to Ausimont SpA, Bollate, Milano, Italy, for PFPE supply. Simona Rossi is grateful to the “C. M. Lerici” Foundation for financial support to stay at the Department of Physical Chemistry, Uppsala University. LA001444B (43) Lasic, D. D.; Martin, F. In Stealth Liposomes; CRC Press: Boca Raton, FL, 1995. (44) Edwards, K.; Almgren, M.; Bellare, J.; Brown, W. Langmuir 1989, 5, 473. (45) Edwards, K.; Gustafsson, J.; Almgren, M.; Karlsson, G. J. Colloid Interface Sci. 1993, 161, 299. (46) Silvander, M.; Karlsson, G.; Edwards, K. J. Colloid Interface Sci. 1996, 179, 104. (47) Vinson, P. K.; Talmon, Y.; Walter, A. Biophys. J. 1989, 56, 669. (48) Walter, A.; Vinson, P. K.; Kaplun, A.; Talmon, Y. Biophys. J. 1991, 60, 1315. (49) Lasic. D. D. Biochim. Biophys. Acta 1982, 692, 501. (50) Fromherz, P. Chem. Phys. Lett. 1983, 94, 259. (51) Edwards, K.; Almgren, M. Langmuir 1992, 8, 824.
