Thermodynamic Size Control of Block Copolymer Vesicles in Solution

in Solution. Laibin Luo and Adi Eisenberg*. Department of Chemistry, McGill University, 801 Sherbrooke Street West,. Montreal, Quebec, H3A 2K6, Canada...
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Thermodynamic Size Control of Block Copolymer Vesicles in Solution Laibin Luo and Adi Eisenberg* Department of Chemistry, McGill University, 801 Sherbrooke Street West, Montreal, Quebec, H3A 2K6, Canada Received March 23, 2001. In Final Form: August 14, 2001 Poly(styrene)-b-poly(acrylic acid) (PS-b-PAA) diblock copolymer vesicles are thermodynamically stable in solutions of dioxane/THF/H2O or DMF/THF/H2O. The vesicle sizes can be changed reversibly by changing the solvent composition, especially the water content. The size change with water content is primarily driven by the interfacial energy contribution to the free energy, such that with increasing water content, as the interfacial energy increases, the system minimizes the total interfacial area by increasing vesicle sizes. For low vesicle sizes, the interfacial area is relatively strongly size-dependent, which also induces a narrow size distribution. By contrast, for large vesicle sizes, the interfacial area is very weakly sizedependent, and a wide size distribution is observed. In a previous study, it had been shown that the segregation of hydrophilic PAA blocks is responsible for the thermodynamic stabilization of vesicles, with the long PAA chains preferentially segregated to the outside, and the short chains to the inside. In the present study, fluorescence quenching experiments have shown that this segregation is size dependent. The larger the vesicles, the lower the degree of segregation becomes. Furthermore, the extent of segregation is reversible with changing vesicles size. In addition, the mechanism of reequilibration of vesicles is explored, and is shown to involve fusion and fission. During these processes, the PAA block chains can diffuse through a PS wall so that a corona chain segregation equilibrium can be reestablished.

1. Introduction The self-assembly of block polymers in solution into aggregates of a range of morphologies has attracted considerable interest for several years.1-10 Although it is possible to prepare such aggregates from a wide range of block copolymers, amphiphilic block copolymers in selective solvents are perhaps the most versatile in that connection. If the soluble block in such aggregates, for example micelles, is very long, they are referred to as starlike,1 whereas if the corona block is short relative to the size of core, one speaks of crew-cut aggregates.3 In * To whom correspondence should be addressed. E-mail: adi.eisenberg@ mcgill.ca. (1) (a)Tuzar, Z.; Kratochvil, P. Adv. Colloid Interface Sci. 1976, 6, 201. (b)Tuzar, Z.; Kratochvil, P. In Surface and Colloid Science; Matijievic, E., Ed.; Plenum Press: New York, 1993; Vol. 15, p 1. (c) Price, C. In Developments in Block Copolymers; Goodman, I., Ed.; Applied Science Publishers: London, 1982; Vol. 1, p 39. (d) Selb, J.; Gallot, Y. In Developments in Block Copolymers; Goodman, I., Ed.; Applied Science Publishers: London, 1985; Vol. 2, p 27. (e) Riess, G.; Hurtrez, G.; Bahadur, P. Encyclopedia of Polymer Science and Engineering, 2nd ed.; Wiley: New York, 1985; Vol. 2, p 324. (2) Hilfiker, R.; Wu, D. Q.; Chu, B. J. Colloid Interface Sci. 1990, 135, 573. (3) (a) Halperin, A.; Tirrell, M.; Lodge, T. P. Adv. Polym. Sci. 1992, 100, 31. (b) Gao, Z.; Varshney, S. K.; Wong, S.; Eisenberg, A. Macromolecules 1994, 27, 7923. (c) Honda, C.; Sakaki, K.; Nose, T. Polymer 1994, 35, 5309. (4) (a) Zhang, L.; Eisenberg, A. Science 1995, 268, 1728. (b) J. Am. Chem. Soc. 1996, 118, 3168. (c) Zhang, L.; Bartels, C.; Yu, Y.; Shen, H.; Eisenberg, A. Phys. Rev. Lett. 1997, 79, 5034. (5) (a) Zhang, L.; Yu, K.; Eisenberg, A. Science 1996, 272, 1777. (b) Zhang, L.; Eisenberg, A. Macromolecules 1996, 29, 8805. (6) (a) Guo, A.; Liu, G.; Tao, J. Macromolecules 1996, 29, 2487. (b) Ding, J.; Liu, G. Macromolecules 1997, 30, 655. (c) Ding, J.; Liu, G. J. Phys. Chem. B 1998, 102, 6107. (d) Henselwood, F.; Liu, G. Macromolecules 1998, 31, 4213. (7) (a) Spatz, J. P.; Mo¨ssmer, S.; Mo¨ller, M. Angew. Chem., Int. Ed. Engl. 1996, 35, 1510. (b) Spatz, J. P.; Sheiko, S.; Mo¨ller, M. Macromolecules 1996, 29, 3220. (8) Massey, J.; Power, K. N.; Manners, I.; Winnik, M. A. J. Am. Chem. Soc. 1998, 120, 9533. (9) (a) Jenekhe, S. A.; Chen, X. L. Science 1998, 279, 1903. (c) Jenekhe, S. A.; Chen, X. L. Science 1999, 283, 372. (10) Won, Y.; Davis, H. T.; Bates, F. S. Science 1999, 283, 960.

1995, Zhang and Eisenberg reported that, in solution, vesicles are a part of morphological continuum of crewcut aggregates, which includes spheres, rods, vesicles, inverse micellar aggregates,4a and, in some cases, also bicontinuous and hollow-rod structures.4c The morphologies of crew-cut aggregates, including vesicles, are governed by a balance of contributions to the free energy involving the interfacial energy between the core and the outside solution, the stretching of the core forming blocks, and the repulsive interactions among corona chains.11 Thus, morphologies can be controlled by many factors, such as relative block length, ion content, solvent composition, etc., all of which influence one or more of the three free energy contributions.4,5,11-13 It has been suggested that under some circumstances vesicles are equilibrium structures because of the reversibility of sizes in response to changes in solvent composition.14a In recent work from this group, the mechanism of thermodynamic stabilization of diblock copolymer vesicles has been elucidated. Utilizing fluorescently labeled block copolymers,15 it was shown that the curvature in block copolymer vesicles is stabilized by preferential segregation of the short hydrophilic blocks to the inside of the vesicles, and the long chains to the outside. The repulsion among the longer corona chains is clearly greater than that among the shorter chains. Therefore, segregation of the hydrophilic blocks, which allows the formation of an asymmetric lamella, stabilizes the curvature of the vesicles. Polymeric vesicles and other hollow spheres have many potential applications in areas such as microreactors, (11) (a) Zhang, L.; Eisenberg, A. Polym. Adv. Technol. 1998, 9, 677. (b) Zhang, L.; Eisenberg, A. Macromolecules 1999, 32, 2239. (12) (a) Yu, K.; Eisenberg, A. Macromolecules 1996, 29, 6359. (b)Yu, K.; Bartels, C.; Eisenberg, A. Macromolecules 1998, 31, 9399. (13) (a) Yu, Y.; Eisenberg, A. J. Am. Chem. Soc. 1997, 119, 8383. (b)Yu, Y.; Zhang, L.; Eisenberg, A. Macromolecules 1998, 31, 1144. (14) (a) Shen, H.; Eisenberg, A. J. Phys. Chem. B 1999, 103, 9473. (b) Chen, L.; Shen, H.; Eisenberg, A. J. Phys. Chem. B 1999, 103, 9488. (c) Shen, H.; Eisenberg, A. Macromolecules 2000, 33, 2561. (15) Luo, L.; Eisenberg, A. J. Am. Chem. Soc. 2001, 123, 1012.

10.1021/la0104370 CCC: $20.00 © 2001 American Chemical Society Published on Web 10/04/2001

Size Control of Block Copolymer Vesicles

microcapsules, and drug delivery systems, and many groups have studied such structures in the past few years.2,6b,c,15-17 On the basis of synchrotron SAXS measurements on solutions of poly(styrene-isoprene) block copolymers in aniline, Hilfiker et al. suggested the presence of vesicle-like aggregates when the PS block was sufficiently short.2 Ding and Liu reported that polyisoprene-b-poly(2-cinnamonylethyl methacrylate) can selfassemble into vesicles in a hexane/THF solvent mixture as revealed by TEM.6b,6c More recently, Kabanov et al. reported the spontaneous formation of vesicles from complexes of ionic block copolymers and surfactants.16 Discher et al. have studied vesicles made from poly(ethylene oxide)-b-polyethylene amphiphilic diblock copolymers.17 In a publication of Ilhan et al., it was reported that giant vesicles could be formed through self-assembly of complementary random copolymers, in which the complementarity was achieved using a diaminopyridinethymine three-point hydrogen-bonding interaction.18 In addition, utilizing very different approaches, a number of polymers were used to prepare hollow nanospheres.6a,d,9,19,20 Vesicles can also be prepared from small amphiphilic molecules.21-29 It is widely accepted that catanionic vesicles, which are formed in aqueous mixtures of oppositely charged surfactants, are equilibrium structures. The partitioning of surfactant molecules between inner and outer monolayers is thought to be responsible for stabilizing the vesicles,23-27 although some theoretical models exist which do not invoke asymmetry.30 Although a number of studies on polymeric vesicles have been published, some fundamental questions regarding their equilibrium nature still remain. In this paper, we report on an investigation concerning the thermodynamic size control of poly(styrene)-b-poly(acrylic acid) (PS-b-PAA) diblock copolymer vesicles in solution. The reversibility of vesicle sizes with solvent composition, the relationship between the degree of segregation of hydrophilic block lengths and the vesicle size, as well as the reversibility (16) Kabanov, A. V.; Bronich, T. K.; Kabanov, V. A.; Yu, K.; Eisenberg, A. J. Am. Chem. Soc. 1998, 120, 9941. (17) Discher, B. M.; Won, Y.; Ege, D. S.; Lee, J. C.-M.; Bates, F. S.; Discher, D. E.; Hammer, D. A. Science 1999, 284, 1143. (18) Ilhan, F.; Galow, T. H.; Gray, M.; Clavier, G.; Rotello, V. M. J. Am. Chem. Soc. 2000, 122, 5895. (19) (a) Thurmond, K. B., II; Kowalewski, T.; Wooley, K. L. J. Am. Chem. Soc. 1997, 119, 6656. (b) Huang, H.; Remsen, E. E.; Kowalewski, T.; Wooley, K. L. J. Am. Chem. Soc. 1999, 121, 3805. (20) Donath, E.; Sukhorukov, G. B.; Caruso, F.; Davis, S. A.; Mo¨hwald, H. Angew. Chem., Int. Ed. Engl. 1998, 37, 2202. (21) Lasic, D. D. Liposomes: From Physics to Applications; Elsevier: Amsterdam, 1993. (22) Laughlin, R. G. The Aqueous Phase Behavior of Surfactants; Academic Press: San Diego, 1994. (23) (a) Kaler, E. W.; Murthy, A. K.; Rodriguez, B. E.; Zasadzinski, J. A. N. Science 1989, 245, 1371. (b) Kaler, E. W.; Herrington, K. L.; Murthy, A. K.; Zasadzinski, J. A. J. Phys. Chem. 1992, 96, 6698. (b) Herrington, K. L.; Kaler, E. W.; Miller, D. D.; Zasadzinski, J. A.; Chiruvolu, S. J. Phys. Chem. 1993, 97, 13 792. (c) Yatcilla, M. T.; Herrington, K. L.; Brasher, L. L.; Kaler, E. W.; Chiruvolu, S.; Zasadzinski, J. A. J. Phys. Chem. 1996, 100, 5874. (24) (a) Watzke, H. J. Prog. Colloid Polym. Sci. 1993, 93, 15. (b) Ambu¨hl, M.; Bangerter, E.; Luisi, P. L.; Skrabal, P.; Watzke, H. J. Prog. Colloid Polym. Sci. 1993, 93, 183. (c) Regev, O.; Khan, A. J. Colloid Interface Sci. 1996, 182, 95. (25) (a) Marques, E. F.; Regev, O.; Khan, A.; Miguel, M. G.; Lindman, B. J. Phys. Chem. B 1998, 102, 6746. (b) Marques, E. F.; Regev, O.; Khan, A.; Miguel, M. G.; Lindman, B. J. Phys. Chem. B 1999, 103, 8353. (c) Marques, E. F. Langmuir 2000, 16, 4798. (26) Helfrich, W. Z. Naturforsch 1978, A33, 305. (27) Safran, S. A.; Pincus, P.; Andelman, D. Science 1990, 248, 354. (28) Jung, H. T.; Coldren, B.; Zasadzinski, J. A.; Iampietro, D. J.; Kaler, E. W. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 1353. (29) (a) Joannic, R.; Auvray, L.; Lasic, D. D. Phys. Rev. Lett. 1997, 78, 3402. (b) Szleifer, I.; Gerasimov, O. V.; Thompson, D. H. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 1032. (30) (a) Bergstro¨m, M. Langmuir 1996, 12, 2454. (b) Yuet, P. K.; Blankschtein, D. Langmuir 1996, 12, 3802.

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of block length segregation with vesicle size are demonstrated. In addition, the mechanism of reequilibration of vesicles in response to changing solution conditions is explored, and is shown to involve fusion and fission. These studies prove conclusively that under the conditions of self-assembly of diblock copolymers of polystyrene and poly(acrylic acid) in mixed solvents, the vesicles are true equilibrium structures, which can then be frozen by any one of several methods for studies by various techniques. This approach is analogous to what is done in the study of bulk morphologies. 2. Experimental Section 2.1. Polymer Synthesis. Synthesis of PS-b-PAA Diblock Copolymers. Polystyrene-b-poly(tert-butylacrylate) (PS-b-PBuA) diblock copolymers were synthesized by anionic polymerization in tetrahydrofuran (THF) at -78 °C using sec-butyllithium as the initiator for styrene polymerization.31 After the formation of the polystyrene block, an aliquot of the reaction mixture was withdrawn for characterization of the polystyrene block. Then, freshly distilled tert-butylacrylate was allowed to drop into the reaction mixture. The reaction was quenched by addition of an excess of degassed methanol. The degree of polymerization of the PS and the polydispersity of both the PS and the diblock copolymers were determined by gel permeation chromatography (GPC). NMR was used to determine the degree of polymerization of the PBuA block relative to that of the PS block. The PBuA block was hydrolyzed to its acid form, poly(acrylic acid) (PAA), in toluene using p-toluenesulfonic acid as the catalyst, as described in the literature.31 The polymers are denoted as PSxb-PAAy, where x and y stand for the degrees of polymerization of the PS and the PAA blocks, respectively. Synthesis of Pyrene Labeled Diblock Copolymers PSPy-b-PAA. 1-Phenyl-1-(1′-pyrenyl)ethylene was synthesized as reported elsewhere.32 The synthesis of the PS-Py-b-PBuA follows that of PS-b-PAA: following the styrene polymerization, approximately a 2-fold excess of 1-phenyl-1-(1′-pyrenyl)ethylene, dissolved in THF, was added to the system. After an aliquot of the polymerization mixture had been removed for analysis, the t-BuA was allowed to drop into the flask. A series of diblock copolymers with the same polystyrene block length but different PbuA lengths were obtained by withdrawing aliquots of the mixture after successive t-BuA additions. As before, the degree of polymerization of the PBuA block was determined by NMR relative to that of the PS block. The procedure for the determination of polydispersities, as well as for the hydrolysis of the PbuA, were same as those given above. 2.2. Micellization. Preparation of Unlabeled Vesicles. Dioxane, THF and DMF are well-known common solvents for both PS and PAA blocks. PS300-b-PAA44 was used for the preparation of vesicles in the range of 90-200 nm. The polymer was initially dissolved in a THF/dioxane mixture (44.4/55.6 w/w) at a concentration of 10 wt %; PS310-b-PAA28 was utilized for vesicle sizes in the rage of 100-1000 nm. The polymer was initially dissolved in various DMF/THF mixtures, with THF contents of 20.4 wt % to 30.6 wt %, 40.8 wt %, 51.0 wt % and 61.2 wt % at a polymer concentration of 2 wt %. To prepare the vesicles, deionized water, as a precipitant, was added at a rate of 0.1 wt %/30s with stirring to the copolymer solutions. In the system of PS300-b-PAA44/dioxane/THF, the final water content was varied between 20 wt % and 67 wt % to change the vesicle sizes. In the system of PS310-b-PAA28/DMF/THF, the final water content was kept constant at 50 wt % and vesicle sizes were varied by changing the THF/DMF ratios. After the formation of the vesicles, all solutions were dropped into excess water to quench the polymer aggregates, which were then dialyzed against water for 2 days. Preparation of Pyrene Labeled Vesicles. To prepare the labeled vesicles, 5 wt % of the pyrene labeled diblock copolymers, PS295-Py-b-PAA12 or PS295-Py-b-PAA74, were added to either PS300-b-PAA44 or PS310-b-PAA28 diblock copolymers. The polymer mixtures were then handled as described above. (31) Zhong, X. F.; Varshney, S. K.; Eisenberg, A. Macromolecules 1992, 25, 7160. (32) Quirk, R. P.; Schock, L. E. Macromolecules 1991, 24, 1237.

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2.3. Reversibility Studies. Reversibility of Vesicle Sizes. The PS300-b-PAA44 diblock copolymers were dissolved in dioxane/ THF (44.4/55.6 w/w) at a polymer concentration of 10 wt %. Deionized water was added to the polymer solution at a rate of 0.1 wt %/30s with stirring. Aliquots of the solution were withdrawn at water contents of 20.0 wt %, 24.5 wt %, 28.6 wt %, 39.4 wt %, 50.0 wt % and 66.7 wt %. It had been found in preliminary studies with the present copolymer that these water contents correspond to approximate sizes of 90, 100, 120, 150, and 200 nm. After the water content reached 66.7 wt %, the water content was reduced progressively by adding the THF/ dioxane (44.4/55.6 w/w) mixture to the colloid solution. Aliquots of the solution were again withdrawn at water contents of 50.0 wt %, 39.4 wt %, 28.6 wt %, 24.5 wt %, and 20.0 wt %. The resulting solutions could not be freeze-dried because of the low melting point of THF. Therefore, all solutions were dropped into an excess of water to quench the aggregates, and then dialyzed against water to remove dioxane and THF. Reversibility of Segregation of Hydrophilic Blocks by Vesicle Size. Pyrene labeled vesicles containing 5 wt % of the pyrene labeled diblock copolymer PS295-Py-b-PAA12 in PS300b-PAA44 were used for these studies. The polymer mixtures were handled as was done in the studies of reversibility of vesicle size and of fluorescence (see below). 2.4. Transmission Electron Microscopy. Transmission electron microscopy (TEM) was carried out on a Philips EM410 microcope operating at an acceleration voltage of 80 kv. Copper EM grids were precoated with a thin film of Formvar and then coated with carbon. 0.01 mL of the diluted colloid solutions were deposited on the resulting grids. After drying in air overnight, the samples were used for TEM studies. 2.5. Fluorescence. Steady-state fluorescence spectra were recorded on a fluorescence spectrophotometer, FluoroMax-2, with excitation at λ ) 343 nm. The pyrene labeled vesicles for the studies of segregation of hydrophilic block lengths by vesicle size and the studies of reversibility of hydrophilic block length segregation with vesicle size were prepared as described above. After dialysis, aqueous vesicle solutions were diluted to 0.05 wt %. Fluorescence quenching experiments were performed by adding aqueous solutions of TlNO3 (10-100 mM) to the diluted vesicle aqueous solutions to bring the Tl+ concentration into the range of 0 mM to 1mM. The total dilution resulting from the addition of the Tl+ solution to the vesicle solution was always less than 2.5% v/v.

3. Results and Discussion 3.1. Changes in the Distribution of Hydrophilic Block Lengths Resulting from the Addition of the Fluorescently Labeled Polymers. To illustrate the effect of the addition of the fluorescently labeled polymer on the molecular weight distribution of the hydrophilic block, the distributions were calculated for two mixtures containing 5% of the short and of the long labeled hydrophilic blocks. For this calculation, it was assumed that both the labeled and unlabeled hydrophilic blocks have a Gaussian distributions of 1.10. The cumulative distribution of unlabeled polymer is given as the solid line in Figure 1, whereas those of the mixtures containing the short and long labeled hydrophilic blocks are given as the dotted and dashed lines, respectively. Although the assumption of a Gaussian distribution is arbitrary, the plots show that the distribution is perturbed only insignificantly by the addition of the labeled polymer. 3.2. Reversibility of PS300-b-PAA45 Vesicle Sizes in Dioxane/THF Solutions. It was reported that morphologies of PS-b-PAA diblock copolymers aggregates are related to the nature of initial common solvent in which the aggregates are prepared. In DMF/THF solvent mixtures, vesicles with varied sizes can be prepared by changing the DMF/THF ratios.13 In the exploration of the phase diagram of the PS310-b-PAA52/dioxane/water system, it was found that the vesicle size is related to the water content in the solvent.14a Thus, both the nature of the

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Figure 1. Hypothetical Gaussian distributions of hydrophilic block lengths for labeled and unlabeled samples.

common solvent or the mixture and solvent/nonsolvent ratio affect vesicle sizes. In the present work, we explore in detail the reversibility of PS300-b-PAA44 vesicle sizes in response to changing water contents in THF/dioxane (44.4/ 55.6 w/w) solutions. Initially, water was added to a 10.0 wt % PS300-b-PAA44 diblock copolymer solution in the THF/ dioxane mixture to increase the water content progressively to 20.0 wt %, 24.5 wt %, 28.6 wt %, 39.4 wt %, 50.0 wt % and finally 66.7 wt %. Subsequently, the THF/dioxane (44.4/55.6 w/w) solvent mixture was added to the solution with the highest water content to decrease the water content progressively to the same percentages as utilized above, i.e., 50 wt %, 39.4 wt %, 28.6 wt %, 24.5 wt % and finally 20.0 wt %. At each step, an aliquot of solution of the aggregate was quenched by excess water and dialyzed against water to remove any residual organic solvent; finally, TEM was used to monitor the morphologies of the aggregates and to determine the sizes of the vesicles. As shown in Figure 2, PS300-b-PAA44 diblock copolymers selfassemble into a coexisting mixture of rods and vesicles at a water content 20.0 wt %. When the water content is increased to 24.5 wt %, vesicles with a diameter 91 ( 3 nm and wall thickness 35 ( 3 nm are observed. The sizes and distributions are based on an analysis of 50 vesicles in each case. When the water content is increased progressively to 28.6 wt %, 39.4 wt %, 50.0 wt %, and 66.7 wt %, the vesicle sizes are increased to 100 ( 4, 119 ( 4, 151 ( 8, and 201 ( 12 nm, respectively. The wall thicknesses of vesicles are 35 ( 3, 35 ( 3, 36 ( 3, and 36 ( 3 nm; obviously, no significant changes in the vesicle wall thicknesses are seen. The THF/dioxane mixture is then added to decrease the water content progressively to 50.0 wt %, 39.4 wt %, 28.6 wt %, and 24.5 wt %; in the process, the vesicle sizes decreased to 151 ( 8, 119 ( 4, 99 ( 4, and 91 ( 3 nm, respectively. The wall thicknesses of vesicles remained at 36 ( 3, 35 ( 3, 36 ( 3, 35 ( 3 nm, respectively. When the water content was decreased further to 20.0 wt %, a mixture of rods and vesicles reappeared in the solution. These results show that vesicle sizes are dependent on the water content in solution, and can be changed reversibly by changing the water content, which confirms that vesicles are equilibrium structures under thermodynamic control. Vesicle wall thicknesses do not appear to change in that range of water contents. The reason for the increase in vesicle sizes in response to increasing water contents is probably related to the

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Figure 2. Reversibility of vesicle sizes in response to increasing or decreasing water contents for PS300-b-PAA44 vesicles in a THF/Dioxane (44.4/55.6) solvent mixture.

Figure 3. Observed polydispersity and calculated interfacial area of vesicles versus vesicle radius. (The surface area calculation is based on 1 cm3 of PS for a wall thickness of 35 nm.)

increase in the interfacial energy in the system, which would drive the system to decrease the total interfacial area. The interfacial area of the vesicles as a function of their sizes is shown in Figure 3 for 1 cm3 of PS chains for a PS wall thickness of 35 nm. The details of the calculation are given in the Supporting Information. It is seen clearly that the total interfacial area decreases as the vesicle diameter increases, as expected, in response to increasing water contents. Other factors, such as a change of the corona repulsion, probably also contribute to the final vesicle size, but it is likely that the reduction of the interfacial area is the major component. A possible consequence of the suggestion that the changes in vesicle sizes are driven by the changes in the interfacial area is possibility that the polydispersities are also related to the vesicle sizes. The polydispersities are defined here as the standard deviation divided by average size, and are also plotted in Figure 3 (the vesicles sizes and standard deviations are given in the Supporting Information, Tables, S1 to S5). As can be seen in that figure, at small vesicle sizes, the surface areas drop rapidly with vesicle radii, which implies that the driving forces are relatively large. By contrast, for large vesicle sizes, the surface areas change only slightly with vesicle sizes, which implies a relatively weaker driving force. It should be noted that the vesicle sizes in Figure 3 are plotted logarithmically. For small vesicle sizes, where the steep

Figure 4. Size distribution of the PS310-b-PAA28 diblock copolymer vesicles determined by TEM. (A) vesicles prepared in DMF/TMF (79.6/20.4 w/w). The diameter is 90 ( 3 nm; (B) vesicles prepared in DMF/TMF (38.8/61.2 w/w). The diameter is 1010 ( 340 nm.

slope suggests a large driving force, fluctuations are relatively less important and polydispersities are small. By contrast, for large vesicles, where the surface areas are only very weakly dependent on sizes, the polydispersities are very large. Two examples of the change of polydispersities with vesicle sizes are shown in Figure 4 for samples showing the smallest and largest vesicles. In both cases, the distributions were divided into 10 equally wide sections, and the numbers in each section are plotted. For sample A, the diameter ( standard deviation is 90 ( 3 nm, while, for sample B it is 1010 ( 340 nm. In the first case, the standard deviation represents 3.3% of the diameter, while in the second it is 34%. The interrelation between vesicle sizes, polydispersities, and slope of interfacial area vs vesicle size is clear. 3.3. Segregation of Hydrophilic Blocks Lengths by Size of Vesicle. As was mentioned above, the vesicle curvature is stabilized by the preferential segregation of hydrophilic blocks by length.15 Because the curvature energy is related to the vesicle size, the degree of segregation of the hydrophilic blocks by length in diblock copolymer vesicles may also be related to the vesicle size. The proof of the variation in the degree of segregation of hydrophilic blocks by vesicle size was again based on the

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Figure 5. Steady-state quenching by Tl+ for PS300-b-PAA44 diblock copolymer vesicles. Sizes are indicated on the right.

fluorescence quenching method. 5 wt % of pyrene labled polymers, PS295-Py-b-PAA12 or PS295-Py-b-PAA74 were mixed with PS300-b-PAA44 to prepare vesicles with different sizes in THF/dioxane solutions at water contents of 24.5 wt %, 28.6 wt %, 39.4 wt %, 50.0 wt %, and 66.7 wt %, respectively, as discussed above. The sizes of vesicles containing the labeled PS295-Py-b-PAA12 are 91 ( 3, 99 ( 4, 120 ( 5, 151 ( 6, and 201 ( 13 nm, respectively, with wall thicknesses of 35 ( 3, 36 ( 3, 36 ( 3, 36 ( 3, and 36 ( 3 nm. The sizes of the vesicles containing PS295-Pyb-PAA74 are 91 ( 3, 99 ( 4, 119 ( 4, 151 ( 5, and 201 ( 13 nm, respectively, with wall thicknesses of 35 ( 3, 36 ( 3, 36 ( 3, 35 ( 3, and 36 ( 3 nm. No significant changes in either the sizes or the wall thicknesses of the vesicles are observed upon incorporation of 5 wt % of pyrene labled polymers. As before, fluorescence quenching by Tl+ ions was used to study the location of pyrene molecules on the vesicles.15 If the pyrene molecules are located on the outside interface of the vesicles, they can be quenched by Tl+ ions in solutions. By contrast, if the pyrene molecules are located on the inside interface of vesicles, they cannot be quenched, because the Tl+ ions are confined to the outside aqueous phase of the vesicles. Figure 5 shows the steady-state fluorescence quenching of pyrene in PS300-b-PAA44/PS295Py-b-PAA74 vesicle solutions, i.e., those in which most of the labeled chains are on the outside. As the Tl+ concentrations in the vesicle solutions are increased, the fluorescence intensities decrease because the pyrene fluorescence is quenched by Tl+. Furthermore, the amount of fluorescence that can be quenched decreases, i.e., the degree of preferential segregation decreases with increasing vesicle size at the same Tl+ concentration. By contrast, in PS300-b-PAA44/PS295-Py-b-PAA12 vesicle solutions, i.e., in those in which the most of the fluorescent chains are on the inside, the amount of fluorescence that can be quenched increases with increasing vesicle size (graph given in the Supporting Information, Figure S1), again showing that the degree of preferential segregation decreases. In the case where there are two fluorescence sites, one accessible to quenchers and the other not accessible, the Stern-Volmer equation can be utilized.33-35 The equation is

I0 1 1 ) + I0 - I φK[Tl +] φ

(1)

Figure 6. Size-dependent segregation of hydrophilic blocks in PS-b-PAA diblock copolymer vesicles as seen from the percentage of quenchable chromophore for different vesicle sizes and hydrophobic block lengths in the labeled chains.

where K is the Sterm-Volmer constant for the accessible chromophore, and φ is the fraction of accessible chromopores. The equation can be changed to eq 2 for convenience in plotting the fluorescence quenching data in terms of I0/I. The modified equation is

I0 φK[Tl +] )1+ I 1 + (1 - φ)K[Tl +]

(2)

The results are given in Figure 5 as the smooth lines, with values of φ and K given in Table S1 of the Supporting Information for the mixture PS300-b-PAA44/PS295-Py-bPAA74. For the mixture PS300-b-PAA44/PS295-Py-b-PAA12, the results are given in Figure S1 of the Supporting Information as the smooth lines, with values of φ and K given in Table S2 of the Supporting Information. The calculated fractions of pyrene molecules which are accessible in both PS300-b-PAA44/PS295-Py-b-PAA74 and PS300-b-PAA44/PS295-Py-b-PAA12 vesicles are plotted versus vesicle size in Figure 6. The accessibility of Tl+ to pyrene molecules for PAA44/PS295-Py-b-PAA74 vesicles decreases, whereas in PS300-b-PAA44/PS295-Py-b-PAA12, vesicles it increases with increasing vesicle size. These results show that the degree of segregation of the long PAA chains to the outside of the vesicles, and of the short PAA chains to the inside of the vesicles decreases with increasing vesicle size. Clearly, the larger the vesicle size, the smaller the degree of segregation. Because the sizes of PS300-b-PAA44 vesicles vary only between 90 and 200 nm, whereas in those prepared from PS310-b-PAA28 the sizes vary between 100 and 1000 nm (as shown in Figure 4 and in the Supporting Information, Table S3), the latter family was selected for further study. Plots similar to that given Figure 5 but for both mixtures of PS310-b-PAA28/PS295-Py-b-PAA74 and of PS310-b-PAA28/ PS295-Py-b-PAA12, as well as the tabulated data are given in the Supporting Information, Figure S2 and Tables S4 and S5. As shown in Figure 6, the vesicle size dependent distribution of pyrene molecules on the outside and inside (33) Lakowicz, J. R. Principle of Fluorescence Spectroscopy; Plenum Press: New York, 1983. (34) Cao, T.; Yin, W.; Armstrong, J. L.; Webber, S. E. Langmuir 1994, 10, 1841. (35) Kawamoto, T.; Morishima, Y. Langmuir 1998, 14, 6669.

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Langmuir, Vol. 17, No. 22, 2001 6809

Figure 7. Reversibility of segregation in PS300-b-PAA44 diblock copolymer vesicles revealed by Tl+ steady-state quenching experiments. Water contents and the chronological run numbers are given on the right.

of the vesicles of PS310-b-PAA28 is similar to that of PS300b-PAA44. These results give the further proof of the vesicle size dependent segregation of hydrophilic block lengths. 3.4. Reversibility of Segregation. So far, we have proven the reversibility of vesicle sizes in response to increasing or decreasing water contents in THF/dioxane (44.4/55.6) solvent mixtures, as well as the size dependence of the degree of segregation of the hydrophilic block lengths. To tie together these two aspects of the thermodynamic stabilization of PS-b-PAA diblock copolymer vesicles, we have investigated the reversibility of segregation in response to changes in vesicle size as induced by increasing or decreasing water contents. Again, the fluorescence quenching method was utilized. PS300-bPAA44/PS295-Py-b-PAA12 vesicles were chosen for the study. Samples were prepared by the slow addition of water to a 10 wt % PS300-b-PAA44/PS295-Py-b-PAA12 solution in THF/dioxane (44.4/55.6, w/w). Aliquots of the vesicle solutions at water contents 24.5 wt %, 28.6 wt %, 39.4 wt %, 50.0 wt % and 66.7 wt % were withdrawn. When the water content reached 66.7 wt %, a THF/dioxane (44.4/55.6 w/w) mixture was added to the solution to decrease the water content progressively. To demonstrate the reversibility of segregation, samples were withdrawn at water contents of 66.7 wt %, 50.0 wt %, 39.4 wt %, 28.6 wt %, and 24.5 wt %. It should be recalled that in all cases the sizes and fluorescence intensities were measured after quenching into pure water. The results of the Tl+ steadystate quenching experiments are shown in Figure 7. The

Stern-Volmer equation was again used to fit the experimental data and to calculate the accessibilities of the pyrene molecules in vesicles. The accessibilities, together with vesicles sizes as revealed by TEM, the water contents at which they were determined, as well as the polymer contents in each solution are summarized in Table 1. It is clear that changing of vesicle sizes induced by either decreasing or increasing water content results in a reversible change of the amount of fluorescence that can be quenched. The accessibilities of pyrene molecules by Tl+ are dependent on the vesicle size, but, more importantly, the accessibilities are independent of whether the sizes were achieved by decreasing larger vesicles or increasing smaller vesicles. Therefore, it can be concluded that the segregation of hydrophilic blocks by lengths in vesicles is reversible. 3.5. Mechanism of Size Equilibration. Because the PS-b-PAA diblock copolymer vesicles are under thermodynamic control in solutions, it is of interest to inquire about the mechanism of size equilibration. Before starting the discussion of the mechanisms, it is important to review what is known about the fraction of single chains vs aggregates in these solutions. Equations are available which allow us to calculate the single chain contents in dioxane/water mixtures. The equation for PS310-b-PAA52 is14a

C - cmc ) 1 - exp[-2.303([H2O] - cwc)/b] C where C is the total polymer concentration, cmc is the critical micellization concentration, cwc is the critical water content for micellization, [H2O] represents the water content, and b is the slope obtained from plot of cwc vs polymer concentration. Because the hydrophilic block lengths are similar although slightly longer, it is reasonable to assume that the equation applies to the present system also as an upper limit because PS solubility increases with increasing PAA block length. Utilizing the equation, one can extrapolate the single chain fractions (relative to total polymer content) at water contents of 24.5%, 28.6%, 39.4%, 50.0%, and 66.7% to be 10-14, 10-17, 10-24, 10-32, and 10-43, respectively. These should be considered as upper limits. Therefore, the single chain involvement in the equilibration of vesicles is probably negligible in the present system. Thus, the fusion and fission of vesicles is probably responsible for reequilibration following a change in the solvent conditions. The transition states are clearly nonequilibrium structures; however, those structures can be quenched by excess water or by dropping the temperature and subsequent freezedrying, and can then be studied by TEM. In this study, the morphologies were preserved by quenching into excess water.

Table 1. Reversibility of Chromophore Segregation in Response to Change in Vesicle Size Induced by Increasing or Decreasing Water Contents for PS300-b-PAA44 with 5% PS295-Py-b-PAA12 in 44.4/55.6 THF/Dioxane Mixture experiment# (direction)

1

water content/ % vesicle size/nm φ (accessibility)/% K/mM-1 polymer concentration/%

24.5 91 ( 3 4.82 ( 0.06 9.0 ( 0.5 7.55

2

f

experiment# (direction)

9

water content/% vesicle size/nm φ (accessibility)/% K/mM-1 polymer concentration/%

24.5 91 ( 2 4.95 ( 0.04 9.3 ( 0.2 1.22

3

f

28.6 100 ( 4 9.73 ( 0.09 9.4 ( 0.3 7.14 r

8 28.6 99 ( 5 9.87 ( 0.06 9.3 ( 0.2 1.43

39.4 120 ( 5 13.8 ( 0.2 9.3 ( 0.4 6.06 r

4

f

7 39.4 120 ( 4 13.9 ( 0.2 9.2 ( 0.4 1.97

50.0 151 ( 6 19.8 ( 0.2 9.1 ( 0.3 5.00 r

5

f

66.7 201 ( 3 25.9 ( 0.3 9.1 ( 0.4 3.33 6

50.0 150 ( 6 20.2 ( 0.2 9.1 ( 0.3 2.50

r

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Langmuir, Vol. 17, No. 22, 2001

Luo and Eisenberg

Figure 8. Possible mechanisms of (a) fusion of vesicles and (b) fission of a vesicle.

Several intermediate structures isolated during the fusion and fission of vesicles are shown in Figure 8. These pictures were obtained by examining a large number of micrographs and then arranged in what appears to be a reasonable sequence. Only very few such intermediate structures were seen, and of those few the best were selected for presentation. The mechanisms are, thus, highly speculative. We believe that they are, nonetheless, useful in that they suggest a realistic mechanism. The first step of the fusion process appears to involve the contact and adhesion of two vesicles. This is followed by coalescence and formation of a center wall. The latter is then destabilized, which eventually results in an asymmetric detachment, retraction of the thickened part of the center wall into the outer wall, and finally smoothing of the outer wall to give uniform vesicles. On the other hand, the mechanism of fission of vesicles involves in the elongation of the vesicles, followed by formation of an internal waist, narrowing the external waist, at which point a connection between the two internal compartments can still be seen, and finally, complete separation. It seems reasonable that fusion and fission of vesicles proceed simultaneously in equilibrated vesicle solutions. This suggestion is made on the basis of the speed with which vesicle sizes adjust in response to changes in solvent composition. Previously, it was shown that rod to vesicle transition occurs with relaxation time of 300 to 5000 seconds.14b More recently, the vesicle to rod transition was shown to occur much more rapidly, i.e., with a relaxation time of 4 to 13 s,36 whereas the sphere to rod and reverse transition have time scale of 10 to 2000 seconds.37 If vesicles are truly equilibrium structures, then one should be able to monitor the speed of vesicle enlargement or diminution in a similar way. Such a study is now underway.38 Preliminary results suggest the enlargement of vesicles in response to water addition occurs with a relaxation time of approximately 10-50 s, whereas the reverse process has a relaxation time of less than 5 s. Such rapid processes in response to minor perturbation in water content suggest that vesicle size changes occur in both directions all the time, and that perturbations allow us to measure the speed of those processes. This parallels a wide range of other situations at equilibrium in which processes move in both directions. The speeds become measurable in relaxation experiments in response to minor perturbations in the environment. A simple parallel example would involve liquid-vapor equilibria in a one-component system such as water. Water molecules (36) Burke, S.; Eisenberg, A. Polymer, in press. (37) Burke, S.; Eisenberg, A. Langmuir, in press. (38) Choucair A. et al., to be published.

move both ways between liquid and vapor all the time. A rapid small perturbation, e.g. of the temperature, would allow us to measure the relaxation time of the process. In that specific case, it would be quite rapid. The speed with which an individual water molecule crosses the boundary between one phase and another would, however, be much more rapid than that. In parallel, the fission and fusion of any vesicles or pair would be expected to be much faster that the relaxation time of the whole process. The applicability of the mechanism of vesicle size changes described above to the reversibility of block length segregation in response to size changes needs to be considered. At first glance, one could conclude that no reversibility should be observed if the mechanism is valid because, at first glance, in the process of fusion and fission, the inner surface remains inside, the outer remains outside. However, we do know that the segregation is size dependent and that corona chains can switch from inside to outside, and vice versa, in response to a size change. This must mean that the corona chains can diffuse through the PS wall with the same time scale as that of the experiment. 3.6. Comparison of the Present Thermodynamic Stabilization Mechanism with that Encountered in Vesicles from Small Molecule Surfactants. Several studies have been performed to explore thermodynamic stabilization in small molecule surfactant vesicles.21-29 Catanionic vesicles, which are prepared from an aqueous mixture of cationic and anionic surfactants, are under thermodynamic control.23-28 Since the initial discovery of spontaneous formation of vesicles from aqueous mixtures of simple, commercially available, single tailed cationic and anionic surfactants,23a extensive studies have shown that when the mixing ratios of the two surfactants are not equimolar, the systems are thermodynamically stable, and partitioning of surfactant molecules between the inner and outer monolayer is responsible for the stabilization of bilayer curvature.23-25 Theoretical models accounting for the stability of catanionic vesicles have also been proposed. When the bending constant of the vesicles is of the order of kBT, thermal bilayer fluctuations induce a repulsive potential between bilayers.26 The net repulsive interaction between the bilayers can overcome the van der Waals attraction between them, and the vesicles are stabilized. In the case that the bending constant is larger than kBT, the mixing of two oppositely charged surfactants may form surfactant bilayers with a different surfactants composition in each monolayer. The difference of the composition may result in equal but opposite spontaneous curvatures of the interior and exterior monolayers of the bilayer. If such bilayers are sufficiently rigid, the

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Langmuir, Vol. 17, No. 22, 2001 6811

curvature energy can overcome the attractive interaction between the bialyers, leading to thermodynamically stable vesicles.27 Very recently, it was reported that cetyltrimethylammonium bromide/sodium octyl sulfonate catanionic vesicles have a broad size distribution. The bending constant of those vesicles was calculated from the size distribution to be 0.7kBT. Undulation repulsions thus have been suggested as being responsible for the stabilization of those vesicles. By contrast, in the cetyltrimethylammonium bromide/sodium perfluorooctanoate system, the vesicles have a narrow size distribution, and the bending constant calculated from the size distribution is 6kBT. It was suggested that in this case, the spontaneous curvature is responsible for the stabilization of vesicles and the particular vesicle sizes. Other radii are disfavored energetically.28 Small molecule surfactant vesicles can also be stabilized by grafting polymer to the outer surface.29 Both experimental evidence and theoretical models have been reported.29 The grafting of polymer chains to the outer surface induces a form of asymmetry, which favors the formation of vesicles. For a relatively small graft density, the polymer does not influence the membrane rigidity, but it locks the curvature of the vesicles.

segregated to the outside, and the short chains to the inside. In this study, we have proven that this segregation is related to the vesicle size. The larger is the vesicle, the lower the degree of segregation. The vesicle sizes are reversible in response to change in the solvent composition, and are determined mainly by the interfacial energy between the core and the outside solution, since the interfacial area of vesicles is related to the vesicle sizes. For small vesicle sizes, the interfacial area is relatively strongly size-dependent, which induces narrow size distribution. By contrast, for large vesicle sizes, the interfacial area is very weakly size-dependent, which induces wide size distribution. Furthermore, the size dependent segregation is also reversible. Fusion and fission of vesicles appear to be the mechanisms involved in the reequilibration. Finally, during the fusion and fission of the vesicles, the PAA block chains can diffuse through PS wall so that a corona chain segregation equilibrium can be reestablished with changing vesicle sizes.

4. Conclusions

Supporting Information Available: Calculation of total surface area of vesicles, summaries of results of quenching experiments on vesicles and vesicle sizes (Tables S1-S5), Steadystate quenching by Tl+ for PS300-b-PAA44 diblock copolymer vesicles (Figures S1 and S2). This material is available free of charge via the Internet at http://pubs.acs.org.

TEM and fluorescence quenching studies have demonstrated that PS-b-PAA diblock copolymer vesicles are under thermodynamic control in solutions under a wide range of conditions. As reported previously, the curvature of the vesicles is stabilized by the segregation of hydrophilic PAA blocks, with the long PAA chains preferentially

Acknowledgment. We thank the Natural Science and Engineering Research Council of Canada (NSERC) for continuing support of this research.

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