Trapping of Intermediate Structures of the Morphological Transition of

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Trapping of Intermediate Structures of the Morphological Transition of Vesicles to Inverted Hexagonally Packed Rods in Dilute Solutions of PS-b-PEO Kui Yu, Carl Bartels, and Adi Eisenberg* Department of Chemistry, McGill University, 801 Sherbrooke Street West, Montreal, Quebec, Canada H3A 2K6 Received December 7, 1998. In Final Form: June 1, 1999 The morphological transition from vesicles to inverted hexagonally packed (hollow) rods was studied for asymmetric diblock copolymers of polystyrene-b-poly(ethylene oxide) (PS-b-PEO) in dilute solutions. The self-assembled aggregates were prepared by the addition of water to the copolymer solutions in tetrahydrofuran (THF) to induce the aggregation of the PS blocks, and the morphological transition was induced by an increase in the water content. Many intermediates, such as vesicles with hollow regions in the wall running parallel to the surfaces, as well as various quasihexagonal structures, were trapped. The mechanism of the transition involves a thickening of the vesicle walls accompanied by the formation of the hollow rods in the walls and a decrease in the size of the original water core. Evidence is presented that the transition proceeds in three steps. In the first step, hollow regions form in the walls of the vesicles. The second step involves further thickening of the walls and some alignment of the rods in a hexagonal pattern leading to the formation of quasihexagonal structures. Finally, the fully developed structures of inverted hexagonally packed rods form. This mechanism, especially in the initial step, is different from those found either in polystyrene-b-poly(acrylic acid) (PS-b-PAA) diblocks or in small molecule amphiphile systems, both of which involve fusion of vesicles. A comparison of the mechanisms is presented. Because of the ease of trapping of intermediates, the present study of the morphological transition may improve our understanding of the later stages of the process of the biomembrane fusion and the lipid lamellar (LR) to inverted hexagonal (HII) phase transition.

1. Introduction The morphologies of phase-separated block copolymers in the bulk have attracted considerable interest for several decades.1,2 Depending on the copolymer composition and the value of the interaction parameter, three classical microstructures have been identified in the earliest experimental studies of AB diblocks: spheres of block A, hexagonally packed cylinders (H) of block A, lamellae (L), hexagonally packed cylinders (H) of block B, and spheres of block B. Recently, additional microphases, such as the bicontinuous gyroid (G), were found in the composition range between the H and L phases. In addition to the work on block copolymers in the bulk, the self-assembly of amphiphilic block copolymers in dilute solution into spherical aggregates (or micelles) has also been studied for many years.3 Usually, the preparation involves the dissolution of an asymmetric copolymer in a solvent which is good for the long block; under some * To whom correspondence should be addressed. (1) (a) Noshay, A.; McGrath, J. E. Block Copolymers: Overview and Critical Survey; Academic Press: New York, 1977. (b) Cowie, J. M. G. In Developments in Block Copolymers; Goodman, I., Ed.; Applied Science Publishers: London, 1982; Vol. 1, p 1. (c) Brown, R. A.; Masters, A. J.; Price, C.; Yuan, X. F. In Comprehensive Polymer Science: Polymer Properties; Allen, S. G., Bevington, J. C., Booth, C., Price, C., Eds.; Pergamon: Oxford, U.K., 1989; Vol. 2, p 155. (2) (a) Kinning, D. J.; Winey, K. I.; Thomas, E. L. Macromolecules 1988, 21, 3502. (b) Hashimoto, T.; Tanaka, T.; Hasegawa, H. Macromolecules 1990, 23, 4378. (c) Tanaka, T.; Hasegawa, H.; Hashimoto, T. Macromolecules 1991, 24, 240. (d) Bates, F. S.; Fredrickson, G. H. Annu. Rev. Chem. 1994, 41, 525. (e) Matsen, M. W.; Bates, F. S. Macromolecules 1996, 29, 1091. (3) (a) Price, C. In Developments in Block Copolymers; Goodman, I., Ed.; Applied Science Publishers: London, 1982; Vol. 1, p 39. (b) Selb, J.; Gallot, Y. In Developments in Block Copolymers; Goodman, I., Ed.; Applied Science: London, 1985; Vol. 2, p 27. (c) Tuzar, Z.; Kratochvil, P. In Surface and Colloid Science; Matijevic, E., Ed.; Plenum Press: New York, 1993; Vol. 15, p 1.

conditions, an equilibrium may exist between micelles and single chains. The resulting structures are often called star micelles, with relatively large coronae consisting of the long soluble block and relatively small cores consisting of the short insoluble block. During the process of micellization, the solvent quality does not change, and the Flory-Huggins χ parameter remains constant. It has been suggested that there are three major contributions to the free energy of micellization, namely the interactions in the core, in the corona, and at the core-solvent interface.3 To date, most experimental and theoretical work has been devoted to this type of micelle. Aggregates of another kind, namely crew-cuts,4,5 in which the dimensions of the cores are large relative to those of the coronae, have recently received experimental attention, and a wide range of morphologies has been observed.5-15 Crew-cut aggregates are frequently prepared by the dissolution of the block copolymer, such as (4) (a) de Gennes, P. G. In Solid State Physics; Liebert, L., Ed.; Academic Press: New York, 1978; supplement 14, p 1. (b) Halperin, A.; Tirrel, M.; Lodge, T. P. Adv. Polym. Sci. 1992, 100, 31. (5) Gao, Z.; Varshney, S. K.; Wong, S.; Eisenberg, A. Macromolecules 1994, 27, 7923. (6) Zhang, L.; Eisenberg, A. Science 1995, 268, 1728. (7) Zhang, L.; Eisenberg, A. J. Am. Chem. Soc. 1996, 118, 3168. (8) (a) Zhang, L.; Yu, K.; Eisenberg, A. Science 1996, 272, 1777. (b) Zhang, L.; Eisenberg, A. Macromolecules 1996, 29, 8805. (9) Zhang, L.; Bartels, C.; Yu, Y.; Shen, H.; Eisenberg, A. Phys. Rev. Lett. 1997, 79, 5034. (10) Zhang, L.; Eisenberg, A. Polym. Adv. Technol. 1998, 9, 677. (11) (a) Yu, Y.; Eisenberg, A. J. Am. Chem. Soc. 1997, 119, 8383. (b) Yu, Y.; Zhang, L.; Eisenberg, A. Macromolecules 1998, 31, 1144. (12) Yu, K.; Eisenberg, A. Macromolecules 1996, 29, 6359. (13) Yu, K.; Zhang, L.; Eisenberg, A. Langmuir 1996, 12, 5980. (14) Yu, K.; Eisenberg, A. Macromolecules 1998, 31, 3509. (15) (a) Ding, J.; Liu, G. Macromolecules 1997, 30, 655. (b) Ding, J.; Liu, G.; Yang, M. Polymer 1997, 38, 5497. (c) Iyama, K.; Nose, T. Polymer 1998, 39, 651. (d) Massey, J.; Power, N.; Manners, I.; Winnik, M. A. J. Am. Chem. Soc. 1998, 120, 9533.

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polystyrene-b-poly(vinylpyridinium methyl iodide),5 polystyrene-b-poly(acrylic acid) (PS-b-PAA),6-11 or polystyreneb-poly(ethylene oxide) (PS-b-PEO),12-14 in a common solvent which is good for both blocks, such as N,Ndimethylformamide (DMF), followed by the addition of water to induce the aggregation of the longer PS block. The method of direct dissolution of copolymers in mixtures of DMF and water is also used.13,14 The study on the morphologies of self-assembled aggregates of PS-b-PAA and PS-b-PEO in dilute solution shows that a number of factors and the interplay between them affect the observed morphologies. These factors include the copolymer composition, its concentration in the common solvent, the nature of the common solvent and the precipitant, the temperature, as well as the presence of additives such as electrolytes. For example, as the corona-forming PEO or PAA blocks get shorter, the morphologies of the self-assembled aggregates change from spheres, to rods and bicontinuous rods, to bilayers, and to large compound micelles (LCMs). The LCMs consist of assemblies of inverted micelles with PEO or PAA cores and PS coronae; each LCM has a hydrophilic surface. The observed bilayer aggregates include lamellae, vesicles, tubules, and large compound vesicles (LCVs or compartmented spheroids). Possible mechanisms of the formation of large vesicles from lamellae, as well as tubules and LCVs from vesicles, have been described recently.14 A number of nonequilibrium morphologies, such as spheres with protruding rods (“pincushions”) and lamellae with protruding rods (“pancakes with fingers”) have also been observed.13 Many of the aggregates, such as spheres, vesicles, tubules, and LCVs, may have potential applications, for instance as drug delivery vehicles.6,12,16 During the water addition stage in the preparation of the aggregates, the Flory-Huggins parameter, χ, between the major-component hydrophobic block (e.g. PS) and the solvent increases gradually, and micellization is induced at some point. Because a decrease of the solvent quality is involved during the formation of the crew-cuts, this system is more complicated than that involved in the formation of star micelles. However, a dynamic equilibrium between micelles and single chains exists in the initial stages of the aggregation at low water contents. Although the interplay between thermodynamics and kinetics is complicated, some of the final morphologies of the crew-cuts (which are finally isolated in water), such as spheres and rods, are manifestations of those encountered under thermodynamic equilibrium conditions. Under these conditions, it is believed that the formation of various morphologies of crew-cuts is mainly controlled by the balance of the same three interactions as for star micelles. Very recently, a new morphology consisting of hexagonally packed hollow hoops (HHH) made from PS-b-PAA in dilute solution was described.9 The HHHs are lined with PAA chains and are packed in a hexagonal array in the solid copolymer matrix. They bear some similarity to copolymer microdomains of hexagonally packed cylinders (H) in the bulk; however, the hollow rods in the HHH case form hoops because the end-capping energy of the rods in the size range studied is greater than the curvature energy. The preparation involves the addition of NaCl to induce (16) (a) Bader, H.; Ringsdorf, H.; Schmidt, B. Angew. Makromol. Chem. 1984, 123, 457. (b) Pratten, M. K.; Lloyd, J. B.; Horpel, G.; Ringsdorf, H. Makromol. Chem. 1985, 186, 725. (c) Yokoyama, M.; Miyauchi, M.; Yamada, N.; Okano, T.; Sakurai, Y.; Kataoka, K.; Inoue, S. J. Controlled Release 1990, 11, 269. (d) Kabanov, A. V.; Batrakova, E. V.; Melik-Nubarov, N. S.; Fedoseev, N. A.; Dorodnich, T. Tu.; Alakhov, V. Y.; Chekhonin, V. P.; Nazarova, I. R.; Kabanov, V. A. J. Controlled Release 1992, 22, 141. (e) Allen, C.; Yu, Y.; Maysinger, D.; Eisenberg, A. Bioconjugate Chem. 1998, 9, 564.

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the aggregation of preformed small PS-b-PAA vesicles in a DMF-water mixture. As a result of the adhesion of individual small vesicles, large compound vesicles (LCVs) form initially and subsequently rearrange to the HHH form. The potential application of particles with the HHH morphology might be as vehicles for the timed release of drugs. The continued discovery of novel morphologies, such as tubules and HHHs, in the self-assembled crew-cuts in dilute solution demonstrates that the study of this field is in the early stages and that our understanding of the system is far from complete. In addition to aggregates of multiple morphologies from macromolecules, various structures resulting from the selfassembly of small molecule amphiphiles have been known for a long time.17 Usually the morphologies of the associated or aggregated structures include spherical and cylindrical micelles, planar and spherical bilayers, and inverted micelles. These various morphologies can be predicted from the value of a dimensionless packing parameter, v/a0lc, in which v and lc are the volume and the maximum effective length of the hydrocarbon chain, respectively, while a0 is the optimal area per headgroup at the apolar-polar interface. For example, when v/a0lc is between 1/2 and 1, bilayers can be formed.18,19 When v/a0lc is larger than 1, an inverted phase, such as the inverted hexagonally packed cylinders, called HII, can be formed. The morphological transition of lamellae (LR) to inverted hexagonally packed cylinders (HII) can be induced when the solution conditions, such as pH and temperature, are changed. When large unilamellar vesicles are subjected to the conditions under which the lamellar-to-inverted phase transition occurs, intervesicular mixing and fusion are observed. The initial intermediates of the LR/HII transition are believed to be the same as those of biomembrane fusion.18,19 The fact that small molecule bilayers share many properties of the cell membrane, such as the aggregation of vesicles and the LR/HII transition, is one of the reasons for the intense interest in bilayers. Recently, PS-b-PEO vesicles with hollow regions in the wall running parallel to the surfaces were described in a preliminary report.20 We suggested that they were intermediates in the morphological transition from vesicles to inverted hexagonally packed rods or hoops (HHRs or HHHs). This morphology had not been observed before. Therefore, from a fundamental point of view, a detailed study of the formation of the vesicles with hollow regions in the wall and of the vesicle-to-inverted aggregate transition is of interest. In the present publication, we give detailed evidence that the transition proceeds in three steps. In the first step, a wall thickening and the development of hollow regions in the walls are involved. The formation of the hollow regions may be related to presence of wavy surfaces. The second step involves the formation of quasihexagonal domains. Finally, HHHs form. The present vesicle-to-inverted rod transition mech(17) (a) Silver, B. L. The Physical Chemistry of Membranes, an Introduction to the Structure and Dynamics of Biological Membranes; The Solomon Press: New York, 1985. (b) Cevc, G.; Marsh, D. Phospholipid Bilayers, Physical Principles and Models; John Wiley & Sons: New York, 1985. (c) Mitchell, D. J.; Ninham, B. W. J. Chem. Soc., Faraday Trans. 1981, 77, 609. (d) Ringsdorf, H.; Schlarb, B.; Venzmer, J. Angew. Chem., Int. Ed. Engl. 1988, 27, 113. (e) Israelachvili, J. N. Intermolecular and Surface Forces, 2nd ed.; Academic Press: London, San Diego, 1992. (f) Seddon, J. M. Ber. Bunsen-Ges. Phys. Chem. 1996, 3, 380. (18) (a) Chu, C. J.; Szoka, F. C., Jr. J. Liposome Res. 1994, 4 (1), 361. (b) Lai, M. Z. Characterization of Cholesterylhemisuccinate- and Tocopherol Acid Succinate-Phospholipid Membranes: Phase Behavior of pH-sensitive Liposomes. Ph.D. Thesis, University of California, San Francisico, CA, 1984. (19) Siegel, D. P.; Epand, R. M. Biophys. J. 1997, 73, 3089. (20) Yu, K.; Bartels, C.; Eisenberg, A. Macromolecules 1998, in press.

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anism (especially the early stages) does not depend on vesicle fusion and is, therefore, different from that found in either the PS-b-PAA diblocks or the small molecule amphiphile systems. A comparison of the mechanisms is presented. 2. Experimental Section The copolymer samples used in the present study are polystyrene-b-poly(ethylene oxide) (PS-b-PEO) diblock copolymers. The diblocks were synthesized by sequential anionic polymerization of styrene monomer followed by ethylene oxide monomer. Tetrahydrofuran (THF) was the solvent and cumylpotassium the initiator. A detailed description can be found elsewhere.14,21 Each of the diblock copolymers gave one narrow size exclusion chromatography (SEC) peak. The polydispersity of PS block was less than 1.1 for all the samples used in the study. The copolymers were recovered by precipitation into a large excess of methanol, filtered out, washed, and dried under vacuum at room temperature to a constant weight. The composition of the copolymers was determined by nuclear magnetic resonance (1H NMR). The notation used to describe the copolymers is PS(a)-b-PEO(c), where a and c are the number of polystyrene and poly(ethylene oxide) repeat units, respectively. The PS-b-PEO aggregates were prepared by the addition of water to copolymer solutions in THF. THF is a common solvent for both PS and PEO blocks, but water is only good for the PEO block and is a precipitant for the PS block. First, the diblocks were stirred in THF at 1 wt % for more than 4 h to obtain molecularly dispersed and homogeneous solutions. Subsequently, deionized water was added dropwise to the copolymer solutions with stirring to induce the micellization of the PS blocks. As the water content in the solution increased, the solvent quality for the PS block decreased. Usually, when the water content reached ca. 17-20 wt %, turbidity with a light bluish tinge appeared, indicating the aggregation or micellization of the PS block. After the water content reached ca. 90 wt %, the solutions were placed in dialysis tubes and dialyzed against distilled water for ca. 3 days to get rid of THF and to obtain aqueous solutions of copolymer aggregates. This method will be referred as the continuous water addition method (CWA method). An alternative way involved stirring the solutions for some time (e.g. overnight) once a desired water content (e.g. 35 wt %) had been reached. Then more water was added dropwise till the water content reached ca. 90 wt %. Dialysis was performed, as before, to obtain aqueous solutions of the aggregates. This alternative method will be referred as the annealing method. For electron microscopy, the aqueous solutions, after dialysis, were diluted about 10-fold. A drop of the diluted solution was placed onto a copper EM grid, which had been precoated with a thin film of Formvar (polyvinyl formaldehyde plastic, J. B. EM Services Inc.) and then with carbon. A description of the preparation of the Formvar- and carbon-coated copper grids for the transmission electron microscopy (TEM) measurements can be found elsewhere.5-14 After a few minutes, excess solution on the sample grid was blotted away with a strip of filter paper. After the sample grid was dried, it was shadowed with a palladium/platinum alloy. The TEM images were obtained using a Phillips EM400 or 400T transmission electron microscope operating at an acceleration voltage of 80 kV.

3. Results and Discussion In general, the words “micelle” and “associate” are usually used when a system is under equilibrium conditions, while the word “aggregate” is adopted under nonequilibrium conditions. However, “aggregate” will be used in the present work to describe vesicles, vesicles with hollow regions in the walls, and inverted aggregates, as this study does not address the problem of equilibrium and most of the morphologies described here are trapped. The term “nonclassical vesicle” will be used to describe a (21) (a) Ziegler, K.; Dislich, H. Chem. Ber. 1957, 90, 1107. (b) O’Malley, J. J.; Marchessault, R. H. Macromol. Synth. 1972, 4, 35. (c) Hruska, Z.; Hurtrez, G.; Walter, S.; Riess, G. Polymer 1992, 33, 2447.

Figure 1. Classical vesicles.

vesicle with hollow regions in the walls. The results and discussion section consists of three parts. The TEM study of the vesicle-to-inverted rod transition is presented in the first part. A proposed mechanism and confirmation of the mechanism are also contained in this part. A discussion of the morphological transition from vesicles to inverted hexagonally packed rods or hoops (HHRs or HHHs) is given in the second part. In the third part, this morphological transition is compared to those in other systems. 3.1. TEM Study. 3.1.1. Observation of the Intermediates. Aggregates prepared from PS(100)-b-PEO(30) (FEO, the mole fraction of EO repeat units, is 0.23), both by the CWA and the annealing methods, are characterized by the coexistence of several morphologies, such as classical and nonclassical vesicles, as well as HHHs or HHRs. Nonclassical vesicles are observed more frequently by the annealing method than by the CWA method. In addition, the morphologies are not sensitive to the details of the annealing conditions, i.e., the annealing period (overnight to 1 week) or the water content (from the onset of micellization to 70 wt %) during annealing. This indicates that the complete morphological transition from vesicles to HHRs for this sample under the present experimental conditions is much slower than the time scale of the experiment. If not mentioned otherwise, annealing was performed overnight at a 35 wt % water content. Typical examples of the aggregates prepared by the annealing method are shown in Figures 1-3. Figure 1 shows several vesicles, most of which are classical. For these aggregates, the relatively low optical density in the central parts compared with that on their periphery clearly indicates the vesicular nature, which means that the structures are hollow. In addition, from the shapes of their shadowed regions, it is seen that they are three-dimensional. The shadowed regions cannot be seen because, for this picture, the exposure time for printing was such as to optimize the visibility of the aggregates and not that of their shadowed regions. The same situation will be encountered in some of the other pictures, although the aggregates in the present study were all shadowed before the TEM studies. Indentations are seen in the aggregates in Figure 1. For vesicles, partial indentation or collapse may occur due to the existence of a pressure differential between the inside and the outside during the TEM sample preparation or due to osmotic effects during the early stages of self-assembly. The vesicle sizes are polydisperse, with the outside radii (Rout) ranging from 35 to 500 nm; the majority is in the range of 150300 nm. The wall thicknesses are of the order of 23 ( 3 nm. Besides classical vesicles, nonclassical vesicles are also observed in this sample. Some examples are shown in Figure 2. The identification of the structures of the nonclassical vesicles has been given in our short com-

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Figure 2. Examples of nonclassical vesicles.

Figure 3. (A) Quasihexagonal or hexagonal domains. (B) Domain of hexagonally packed hoops appearing as bright spots. (C) Domain of striation. (D) Inverted hexagonally packed hoops (HHHs).

munication.20 In general, the walls of the nonclassical vesicles are thicker than those of the classical ones and contain hollow regions running parallel to the surfaces in the walls. Although the nonclassical vesicles appear to be very different from each other, they have in common both the vesicular nature and the presence of hollow regions. A hexagonally packed pattern of rods develops when there is more than one set of hollow rods in the wall. In addition, the degree of regularity in the alignment of the substructures in the walls can be high, which suggests that an ordering process resembling crystallization may be operative during their formation. Figure 2A shows a typical nonclassical vesicle with only 1 set of hollow rods, which appear to be circular and running parallel to the “equator” of the sphere. The average wall thickness is ca. 45 nm, as measured directly from the TEM micrograph. In the regions of the alternating darkness and brightness, the dark regions are thought to be “PS supports”, the width of which are ca. 15-20 nm. The diameters of the bright regions, which are identified as hollow rods, are ca. 5-10 nm. The average wall thickness of the structure shown in Figure 2B is ca. 90 nm. Four sets of hollow rods are seen in the wall. The relative location of the hollow rods implies hexagonal packing similar to the H (rod) phase of block copolymers in the bulk,1,2 the PS-b-PAA HHH morphology,9 and the inverted hexagonal phase (HII) of small molecule amphiphiles.16-19 Both the outer and inner

surfaces are rippled; the crests are located above the hollow regions in the first set counting from the surface, and the valleys above the PS “supports” between the hollow regions of the first set or above the hollow regions in the second set. The waviness may indicate the incipient formation of the next layer of the hollow rods, although the details of the formation are not clear. For the hollow rods in one set (parallel to the surfaces), the center to center distance of two neighbors is 65 nm, but that of two nearest neighbors in different sets is ca. 35 nm. Usually, the average diameter of the hollow rods is ca. 10 nm. For this structure at the periphery, the bright regions appear as circular spots and short bent rods. This different appearance is caused by changes in the viewing angle relative to the curvature of the rod. Moreover, in the central part, the bright lines appear to be crossing or intersecting because the hollow rod patterns at both the front and back of the vesicle are seen simultaneously. The nonclassical vesicle shown in Figure 2C is of interest because there are linear alignments of small “subvesicles” or bubbles in the wall. As measured directly from this TEM micrograph, the average inside diameter of the “subvesicles” is ca. 15 nm and the “wall thickness” is ca. 20 nm, while the outside diameter (Dout ) 2Rout) of the “mother” vesicle is ca. 350 nm. Such vesicles with bubbles are seen only occasionally; the aggregation of the bubbles forming strings is shown in the insert. This phenomenon will be discussed in section 3.3.1. Besides the classical and nonclassical vesicles, inverted quasihexagonal and hexagonal aggregates are also observed, as shown in Figure 3A-C. The word “quasihexagonal” has been used in a small molecule amphiphile system to describe the domains of striations and hexagonally arranged spots.13 In that study, it was pointed out that the striation spacing and the diameter of the hexagonally packed circular spots are usually larger than those of the HII phase. Thus, the quasihexagonal structure is not an equilibrium HII phase, although it resembles an HII-like morphology and is probably an HII precursor. The optical density of the images of the inverted quasihexagonal and hexagonal aggregates, as shown in Figure 3A, is usually high. In addition, the density in the central part of an aggregate is often too high to reveal the internal structure. However, the fine structure at the periphery of the aggregate is usually visible and resembles the quasihexagonal morphology. Dark regions represent the block copolymer, while the bright regions represent hollow rods. In general, the diameters of the inverted rods are approximately equal. Figure 3B is a high magnification image of the regions of bright “spots”. It is seen that every bright spot in the interior of the structure has six neighboring ones and that the distances to the six neighbors are approximately equal,


Figure 4. Nonclassical vesicles and quasihexagonal structures viewed from two directions.

with the six neighboring spots forming an equilateral hexagon. The structure thus appears to be very similar to that of the HHH morphology.9 As measured directly from Figure 3B, the average diameter of the inverted rods is ca. 15 nm, and the average center to center distance of two nearest neighbor rods is ca. 45 nm; both of these numbers are different from those of the structure shown in Figure 2B. An example of an aggregate with a striation domain is shown in Figure 3C. The striation spacing, i.e., the diameter of the hollow rods, is ca. 10 nm. For this image, this striation region only appears at the right side of the structure and seems to consist of nearly concentric circles. The different appearance at the right and left sides suggests that this particle contains two parts, in which the hollow rods are running in different directions. The lower optical density in the center part than that of its surroundings indicates the remaining vesicular nature. We suggest that the hexagonally packed spots and the striations are similar structures despite their different appearance; the different appearance is due to difference in the viewing angles relative to the orientation of the hollow rods in different sections of the aggregate. This recognition is important because it helps us to identify these structures. Aggregates made from PS(215)-b-PEO(37) (FEO ) 0.15) by the CWA method are shown in Figure 3D. The hollow rods appear as rings arranged in a hexagonal array. The external shape of the aggregates appears to be conical, at least in part. Here inverted quasihexagonally and/or hexagonally packed rods seem to be the dominant morphology. Thus, under these preparation conditions, the formation of HHHs appears to be faster for PS(215)b-PEO(37) than for PS(100)-b-PEO(30). It is necessary to keep in mind that the appearance of the same aggregate with substructures can be quite different when viewed from different directions. In addition, the aggregates are randomly distributed on the EM grid. Thus, they are viewed from different directions relative to the orientation of the hollow rods within the particles. For the aggregates with high optical density, such as the quasihexagonal structures labeled A-D in Figure 4, an image with relatively good resolution of the internal structure can usually be achieved when viewed

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from an optimal angle, such as +45° in the example of Figure 4. The figure shows that the appearance of the hollow regions (hoops) is more clear when viewed at the angle of +45° than at -22.5°. 3.1.2. Proposed Mechanism for the Vesicle-to-HHH Transition. On the basis of the above observations, it appears that the vesicles with hollow regions in the walls (shown in Figure 2) and the quasihexagonal domains (shown in parts A-C in Figure 3) are trapped intermediates in the morphological transition from vesicles (shown in Figure 1) to inverted hexagonally packed hoops or rods (HHHs or HHRs) (shown in Figure 3D or ref 7). It is also suggested that the vesicles with hollow regions in the walls form in the early stage and the quasihexagonal structures in the later stage of the transition. Therefore, it seems reasonable that a three-step mechanism is operative in the morphological transition from vesicles to inverted hexagonally packed rods. The first step involves the formation of vesicles with hollow regions in the walls from classical vesicles. This process is accompanied by a thickening of the vesicle walls and the appearance of the hollow regions or rods running parallel to the surfaces of the walls, as well as a decrease of the overall sizes of the vesicles. This step will be compared in section 3.3.1 with that seen in the lipid systems. As more hollow rods form and the vesicle walls become thicker, the hollow rods arrange themselves into a hexagonal array. Therefore, the second step is the transition of the nonclassical vesicles to inverted quasihexagonal structures. The last step consists of the formation of inverted hexagonally packed rods (or hoops), with the total disappearance of the original vesicular hollow cores. Physical parameters, such as the diameter of the hollow rods and the center to center distance between them, characterizing the regions with a hexagonal packing of rods or hoops, change during the transition. 3.1.3. Confirmation of the Proposed Mechanism. To confirm the proposed mechanism of the transition, it is helpful to study the morphogenic effect of copolymer composition. Previous results have shown and the present study confirms that the morphologies of the self-assembled aggregates can change from spheres, to rods, to lamellae and vesicles, and to LCMs as the EO component decreases.12-14 Therefore, by studying block copolymers within a certain (narrow) range of compositions, we should be able to trap intermediates in different stages of a transition, such as the present vesicle-to-inverted rod transition, provided the transition in those samples is slow. Thus, trapping the intermediates for the samples with finely tuned compositions can provide a deeper insight into the process of the vesicle-to-HHH transition and is therefore of interest. For a particular PS block length, the range of optimum PEO block lengths which allows trapping of the intermediates under the experimental conditions is probably narrow. To target this range, blends are utilized rather than samples prepared by further syntheses. To prepare diblocks with the same styrene block length but different ethylene oxide block lengths, two copolymers with the same PS but a different PEO block length, specifically, PS(125)-b-PEO(94) (FEO ) 0.43) and PS(125)-b-PEO(30) (FEO ) 0.19) or PS(215)-b-PEO(100) (FEO ) 0.32) and PS(215)-b-PEO(37) (FEO ) 0.15), were mixed in different proportions. In the unmixed state, by the CWA method, PS(125)-b-PEO(94) gives rods and PS(125)-b-PEO(30) gives HHHs, while PS(215)-b-PEO(100) gives rods as well as bilayers and PS(215)-b-PEO(37) gives HHHs. When the samples are treated in the exactly same way, the morphologies of the copolymer mixtures depend on the

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Table 1. Frequency of Observation of Vesicles with Hollow Regions (VHR) and Quasihexagonal Structures (QHS) from the Mixtures by the CWA and Annealing Methods frequency of observn of various structures prepared by

copolymer mixture (wt ratio) PS(125)-b-PEO(94): PS(125)-b-PEO(30)

annealing methodd

equiv composn (FEO)

VHR

QHS

VHR

QHS

2:3 1:4

PS(125)-b-PEO(51) (0.29) PS(125)-b-PEO(43) (0.26) PS(215)-b-PEO(62) (0.22) PS(215)-b-PEO(50) (0.18)

rarely freqa freqb less freqc

rarely rarely rarely freqc

freqa freqa,b freqb less freqc

rarely freq freq freq

1:2 1:4

d

CWA methodd

PS(215)-b-PEO(100): PS(215)-b-PEO(37)

a The thickening appears in parts of vesicles. b The thickening appears over the whole vesicles. c QHS becomes the dominant morphology. Freq ) frequently.

proportion of the two original copolymers. The composition (and FEO) of a binary mixture is determined by that of the original diblocks and their proportions. In general, with increasing relative amounts of the copolymer with the shorter PEO chains, i.e., PS(125)-b-PEO(30) or PS(215)b-PEO(37) in the mixtures, the concentration of rods decreases and the bilayers, such as vesicles and lamellae, become the dominant morphologies. In addition, it was observed that when a particular morphology, such as vesicles, is prepared, the greater the length of the PS chains, the smaller the FEO needed to obtain that morphology. These two trends in the morphogenic effect of the composition are the same as those found for unmixed diblocks. Thus, although blending may have some effect on the phase behavior as a result of the extra degree of freedom,22 we assume that in the present case blending makes little difference, because each mixture is composed of two copolymers with the exactly the same PS block and different PEO block lengths. We now describe the trapping of intermediates (in the early and later stages of the vesicle-to-HHH transition) in the binary mixtures. The effect of FEO on the morphological transition was studied on the two series of mixtures mentioned above. In general, the aggregates prepared are characterized by the coexistence of several morphologies. As shown in Table 1, the frequency of observation of the intermediates depends on the samples used. As the EO component (FEO) decreases in the mixtures, the frequency of observation of the various structures increases in the order of vesicles, to nonclassical vesicles (VHR), to quasihexagonal structures (QHS), and to HHHs. This means that the intermediates trapped are closer to the HHH morphology as the EO contents decrease. For example, by the method of continuous water addition, most of the aggregates prepared from the mixture with FEO ) 0.29 were classical bilayers, while vesicles with hollow regions in the wall were observed only occasionally. For the mixture with FEO ) 0.26, nonclassical vesicles were observed frequently (together with classical ones). While many pictures were taken during this aspect of the study, only a few are shown in the present publication to illustrate most clearly the details of the transition. Examples of the trapped intermediates (from the mixture with FEO ) 0.26) are shown in Figures 5-7. Figure 5A shows an example of a segment of a nonclassical bilayer in a flat form, which is seen only rarely. It is probably a fragment of a very large broken nonclassical vesicle. The dark lines are the PS “supports”, and the regions with lower optical density (than that of the dark lines) are hollow regions. Careful observation of the hollow regions shows that the optical density is not homogeneous, in that the optical density is relatively low for the hollow regions (22) Zhao, J.; Majumdar, B.; Schulz, M. F.; Bates, F. S.; Almdal, K.; Mortensen, K.; Hajduk, D. A.; Gruner, S. M. Macromolecules 1996, 29, 9, 1204.

Figure 5. Nonclassical vesicles: (A) double wall in a flat form from a large broken nonclassical vesicle; (B) nonclassical vesicle with only parts of the wall thickened.

near the PS “supports” on the side which the metal vapor comes from. This suggests that the surface of the layer is not flat but slightly wavy, with the height of the parts above the hollow regions slightly higher than that above the PS “supports”. The relative location of the “higher” regions and the hollow regions is somewhat similar to that of the aggregate with the rippled surface shown in Figure 2B. Additional observation of the edge of the image confirms that the structure consists of two overlapping bilayers with the PS “supports” bridging them. The average height of the structure over the hollow regions is ca. 80 nm, while that over the PS “supports” is ca. 70 nm, as measured from the length of the shadow at the edge of the structure. The width of the PS “supports” is quite uniform and is ca. 20 nm. A few discontinuous PS “supports” or ends are observed. For the nonclassical vesicles prepared from the mixture with FEO ) 0.26 by the method of continuous water addition, it is a general phenomenon that thickening occurs in only parts of the vesicles. An example is shown Figure 5B. This aggregate consists of a classical bilayer part on the left side and a nonclassical part on the right side. The different optical density in the border regions (of the two parts) indicates the thickening; in addition, the degree of thickening can be judged by comparing the wall thicknesses of the two parts. It is necessary to point out that the location of the nonclassical part within a vesicle can be different from this one; for instance, the thicker part can occupy just the middle of a vesicle. For most of the vesicular aggregates with both the classical and nonclassical parts, the average wall thickness of the nonclassical parts is ca. 75 nm, which is almost the same as the thickness of the flat structure shown in Figure 4A. In addition, the average width of the PS “supports” is also ca. 20 nm. This confirms that the structure shown in Figure 5A is a piece of the nonclassical part of a broken vesicle. At the same time, this recognition helps us to identify the structure shown in Figure 5B, in that the crossed or


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Figure 6. Nonclassical vesicle viewed from three different angles with the direction of tilting designed to increase the visibility from the open part.

Figure 7. Nonclassical vesicle viewed in directions tilted around its long axis.

intersecting appearance is due to the visibility of both the front and the back sides (i.e. hollow regions and the PS “supports”) of the vesicle. Furthermore, it is noticed that, in general, the average wall thickness of the original classical bilayers is almost the same as the width of the PS “supports”. Two sets of TEM tilt images of two nonclassical vesicles, viewed from different angles, are shown in Figures 6 and 7. The direction of tilting in Figure 6 was designed to increase the view from the open side, while in Figure 7 the vesicle was tilted around its long axis (perpendicular to the plane of the open side). When viewed from the open side, the features of only one nonclassical layer are observed. The different appearance of the edge of the structure in Figure 7 from different viewing directions is worthy of notice. Figures 6 and 7 also illustrate the fact that the appearance of the same nonclassical vesicle can be quite different when viewed from different directions. For the mixture with FEO ) 0.26, quasihexagonal structures were observed only rarely if the samples were prepared by the CWA method but frequently if prepared by the annealing method. The observed quasihexagonal structures are very similar to those shown in Figures 3AC. However, it is necessary to point out that the frequent coalescence of several aggregates is deduced from the irregular external shapes of the aggregates. In addition, the coexistence of the nonclassical vesicle layer (with relatively low optical density) and the quasihexagonal structure in some aggregates was noticed. This suggests that the nonclassical vesicle layer may be the precursor of the inverted quasihexagonal aggregate. For the mixture with FEO ) 0.22 (but longer PS), the phenomena encountered in trapping nonclassical vesicles and quasihexagonal structures are similar to those for PS(125)-b-PEO(43) (FEO ) 0.26). For the nonclassical vesicles prepared by the CWA method, the wall thickening usually occurred over the whole vesicle rather than just a part. Examples of such nonclassical structures are shown in Figure 8. As measured, the width of the PS “supports” is 27 nm, the same as the wall thickness of the classical

Figure 8. Nonclassical vesicles made from the mixture with FEO ) 0.22 by the continuous water addition method showing different stages of the transition.

Figure 9. Quasihexagonal structures made from a mixture with FEO ) 0.18 by the continuous water addition method.

vesicles observed from this sample; the average wall thickness is ca. 90 nm. These values are larger than those for PS(125)-b-PEO(43) aggregates shown in Figures 5-7. In addition, the trapped intermediates shown in Figure 8 are at different stages of the transition, as judged from the wall thickening. The growth of quasihexagonal domains from nonclassical vesicles shown toward the left side of Figure 8 is of interest. Quasihexagonal structures made from the mixture with FEO ) 0.18 and shown in Figure 9 were observed frequently when prepared by the CWA method. Here the quasihexagonal structures are quite well developed. Aggregates very similar to those shown in Figures 3A-C were also observed. Not all the aggregates were fully transformed to HHRs or HHHs by either of the two methods of water addition. The presence of domains with striations and those with hexagonally packed hollow spots on the same aggregate is clearly seen in Figure 9. This phenomenon also suggests that the two domains are similar despite their different appearance, differing only in the angle which these different segments present to the viewer. In the regions of hexagonally packed spots in Figure 9, the

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average diameter of the bright spots is ca. 25 nm, and center to center distance of two nearest neighboring spots is ca. 60 nm. In general, by the CWA method, nonclassical vesicles, which are the intermediates in the initial stage of the transition, are often trapped from mixtures with FEO of 0.22 and 0.26. The wall thickening is usually seen over the entire vesicles with FEO of 0.22, and the quasihexagonal structures are seen in some of nonclassical vesicles. But the wall thickening only occurs in parts of the vesicles with FEO of 0.26. Quasihexagonal structures, which are the intermediates in the later stage of the transition, are often trapped from the mixture with FEO of 0.18. The annealing method may be a good alternative method depending on the sample used. For example, nonclassical vesicles can be often trapped for the mixture with FEO ) 0.29, and quasihexagonal structures for the mixtures with FEO ) 0.22 and 0.26. The morphogenic effect of the composition supports the three-step mechanism of the morphological transition from vesicles to inverted hexagonally packed rods. The three steps involve the transition from classical vesicles to nonclassical vesicles, to inverted quasihexagonal structures, and then to inverted hexagonally packed rods. The formation of inverted quasihexagonal structures from nonclassical vesicles is strongly evidenced by Figure 8. During these steps, the particle size decreases if there is no coalescence; also, the volume of the water core decreases and eventually disappears. In general, the rate of the first two steps might be faster than the last one, since not all aggregates were transformed to HHRs for the mixture with FEO of 0.18. 3.2. Discussion of the Vesicle-to-HHH Transition. 3.2.1. Trapping Intermediates. Intermediates encountered during morphological transitions can be trapped if the kinetics are slow. For amphiphilic diblocks with PS as the hydrophobic block, THF appears to be useful as a solvent for trapping of intermediates, because the range of water contents over which morphological activity is seen in THF is wider than that in DMF.23 A recent study dealt with the solvent content in the PS-rich phase of a phase-separated ternary system (consisting of PS, common solvent, and water).11 It showed that the solvent content in the PS-rich phase decreases as the water content increases for all the solvents studied. This decrease is much faster in DMF than that in THF. Thus the effect of water content on the solvent content in the PS-rich phase is much weaker in THF than in DMF, which means that a higher degree of control for trapping can be exercised for copolymer solutions in THF than in DMF. 3.2.2. Formation of Inverted Aggregates. The formation of the present inverted hexagonally packed rods or hoops bears some resemblance to that of the H phase of block copolymers in the bulk. In parallel with the H phase behavior, the minority-component PEO blocks form the hexagonally packed cylinders. However, the resulting hollow rods, such as those shown in Figure 3D, form rings without ends. Ring versus rod formation is a function of the relative magnitude of the end-capping energy compared with the curvature energy for these inverted rods.9 Rings form when the end-capping energy is relatively large, which is not unreasonable for particles of the size seen here. The effect of particle size on block copolymer morphologies in the bulk was described briefly by Thomas et al.24 When symmetric diblocks which exhibit lamellae (23) Yu, K.; Eisenberg, A. A Manuscript in preparation. (24) Thomas, E. L.; Reffner, J. R.; Bellare, J. Colloq. Phys. 1990, 51 (23), C7, 363.

Yu et al.

in the bulk were confined to bulk microdroplets, concentric spherical shells or a chaotic bicontinuous structure were obtained, while for diblocks which exhibit an ordered bicontinuous structure, a distorted network was observed.24 The morphogenic effect of the balance of the rim energy versus the curvature energy is also reflected in the formation of vesicles versus flat lamellae.25 The external shape of the aggregates shown in Figure 3D is conical (truncated). This implies a strong interrelation between the external shape and the internal structure. This interrelationship was also seen in the PS-b-PAA HHHs.9 During the formation of the inverted rods, a process similar to crystallization leads to both the highly regular internal structure and the change in the external shape. 3.2.3. Deformation of the PS Chains in the PS “Supports”. The degree of deformation (stretching) of PS chains (Sc) is usually defined as the ratio of the radius (or half-dimension) of the PS regions to the end-to-end distance of the PS chain in the unperturbed state (R0). R0 can be calculated by26

R0 ) 0.067M1/2

(1)

where M (g/mol) is the molecular weight of the PS block. In the present study, “the radius (or half-dimension) of the PS regions” is the half-thickness of the vesicle walls and the half-width of the PS “support”. As mentioned before, the width of the PS “supports” is ca. 20 and 27 nm, for the aggregates made from PS(125)-b-PEO(43) and PS(215)-b-PEO(62), respectively; in addition, the wall thickness of the classical vesicle is almost the same as the width of the PS “supports”. Therefore, for the nonclassical vesicles of the two copolymers, the degree of deformation of the PS chains is almost the same, ca. 1.35. Accordingly, we conclude that the degree of deformation of the PS chains appears to be constant both during the morphological transition from classical to nonclassical vesicles and between the different polymers in the present study. 3.2.4. Coexistence of Multiple Morphologies. The coexistence of the morphologies has to be addressed because the presence of simultaneous multiple morphologies is a characteristic of the self-assembly of PS(100)b-PEO(30) and the binary mixtures under the preparation condition of self-assembly. For these samples, it is highly likely that the morphologies, i.e., classical vesicles and inverted hexagonal aggregates, coexist in some areas of the phase diagram, due to the relatively small differences in their free energy under the preparation conditions. During the morphological transition, various intermediates are trapped. Another possible reason for the coexistence may be related to the fact that, in the process of the water addition, boundaries between regions of stability of the various morphologies may be crossed as the water concentration changes. This boundary crossing may be due to the fact that many factors, such as the concentration of the copolymer single chains and the THF content in the PS cores, change during the water addition. 3.2.5. Observations for Blended Mixtures. It was noticed that during the addition of water to a single component PS(125)-b-PEO(30) or PS(215)-b-PEO(37) solution in THF, some precipitate formed after micellization. However, when there is blending of PS(125)-b-PEO(94) (25) (a) Lasic, D. D. Biochim. Biophys. Acta 1982, 692, 501. (b) Hargreave, W. R.; Deamer, D. W. Biochemistry 1987, 18, 3759. (c) Lasic, D. D. J. Theor. Biol. 1987, 124, 35. (d) Thompson, T. E. Hepatology 1990, 12, 51S. (26) Brandrup, J.; Immergut, E. H. Polymer Handbook, 3rd ed.; WileyInterscience: New York, 1989; pp VII 38, VII 526.


with PS(125)-b-PEO(30) or PS(215)-b-PEO(100) with PS(215)-b-PEO(37), even in small proportions, for example 20/80 w/w %, no precipitate was observed. Therefore, the addition of the copolymer with longer PEO chains seems to have stabilized the aggregates. The phenomenon is similar to the well-known stabilization of lipid bilayers by detergents.16 3.3. Comparison of the Morphological Transition to Those in Other Systems. 3.3.1. Comparison with Small Molecule Amphiphile System. For small molecule amphiphiles, extensive studies have been performed on the transition of lamellae (LR) to inverted hexagonally packed cylinders (HII).17-19 By contrast, the present study of the transition of PS-b-PEO vesicles to HHHs or HHRs in dilute solution is only in its initial stages. However, because of the similarity in the amphiphilic character of the two systems and the resulting behavior, it is instructive to compare the two. For small molecule amphiphiles, a decrease in the size of the headgroup favors the formation of inverted aggregates.18,19 As mentioned in the Introduction, HII phases are formed when the value of v/a0lc is larger than 1. The morphological transition from vesicles to the HII phase can be induced by any factor which increases the value of v/a0lc (from the range of 0.5-1 to >1). Three interactions are involved which determine the morphology.17 Two are repulsive, involving headgroups and hydrocarbon chains, and one is attractive, arising from the interfacial tension. This attraction is assumed to act at the apolar-polar interface. Evidently, the attraction tends to decrease the area per headgroup at the interface, and the two repulsions tend to increase the area per headgroup in contact with water. Therefore, a decrease of the headgroup size, which may cause the hydrocarbon chain repulsion to dominate the repulsive interactions and also lead to an increase of the value of v/a0lc, favors the formation of inverted aggregates. For a particular lipid, the area per headgroup at the lipid-water interface of the LR phase is bigger than that in the HII phase; also, the ratio of these two areas is of importance for the kinetics of the formation of the HII phase. For example, if the ratio is less than 1.2, the rate of formation of HII would be slow. When the ratio is as large as 1.8, the LR/HII transition can be as fast as 1 s or even less.17 For the present system, the effect of the size of the hydrophilic part (the PEO block) on the morphological transition from vesicles to inverted aggregates shows the same trend as that of the headgroup size in small molecule systems. As the length of the corona-forming block (PEO) decreases, the morphology changes from vesicles to inverted aggregates. A shorter PEO block length means a smaller area per corona chain, which implies a bigger value of v/a0lc. Again, in parallel with small molecule systems, three sources of interactions are involved here, i.e., in the hydrophobic core, in the hydrophilic corona, and at the interface.10 A shorter PEO block length leads to a smaller inter-PEO repulsion, which favors the formation of inverted aggregates. For a particular copolymer, during the transition from classical to nonclassical vesicles, the degree of the PS chain stretching is almost the same, as seen in the TEM study. Thus the strength of the PS interaction should be about the same. If the relationship between the transition rate and the degree of reduction of the area per corona chain is similar to that for small molecule amphiphiles, it can be argued that the slow rate of the transition could be indicative of a small decrease in the area per PEO chain on the PS core surface. Although the morphogenic effect of the relative size of the hydrophilic segments is similar for the two systems,

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the mechanisms of the transitions are different. It is wellknown that the formation of the lipid HII phases begins with vesicle fusion,18,19 which is different from that seen in the present system. A recent study pointed out that the LR/HII transition in phosphatidylethanolamine proceeds in three steps.19 First, small connections are formed (by lipid rearrangement) between apposed liposome bilayers. This step, therefore, is fusion-dependent; i.e., vesicle fusion is involved. In the second step, quasihexagonal domains, which consist of two regions of hexagonal packing and equally spaced striations, are formed. These two regions are always associated; therefore, they are assumed to represent two different views of the same or related structures. TEM micrographs of the quasihexagonal domains given in ref 16 are very similar to those shown in Figure 3B,C as well as in Figure 9; in addition, the coexistence of the lipid vesicles with the quasihexagonal structures is noticed. Finally, in the third step, the quasihexagonal structures are transformed into the HII phase. For the present system, the transition also proceeds in three steps but all within one vesicle. [The quantity of copolymer in a typical vesicle of 400 nm diameter and 25 nm wall thickness is ca. 107 nm3. This is sufficient to form an HHH structure with an outside diameter of 200 nm and a height of 300 nm, which is typical of the sizes observed for a complete HHH aggregate of PS-b-PAA.] Thus, the process of the present transition is not fusiondependent. As mentioned before, in the first step, namely the formation of the initial intermediates (nonclassical vesicles), a thickening of the vesicle walls and the formation of the hollow regions in the walls running parallel to the surfaces are involved. Therefore, the present transition starts differently from that found in the lipid system, and the resulting intermediates (after the first step) are obviously also different. However, the intermediates formed in the second step in the polymers are similar to those formed in the second step of the transition in the lipid system, the quasihexagonal structures;19 thus, we also name them quasihexagonal structures. In our final step, inverted rods with a hexagonal array form; however, the inverted rods usually appear as hoops. In our study, detailed appearance of aggregates with substructures depends strongly on the viewing direction. Thus, the domains appearing as hexagonally packed spots and as striations are the same structures. Similar to what is seen in the lipid system, the coexistence of aggregates is also observed in the present system. The reason the transition mechanisms of the two systems are different is of interest. To understand why the transition mechanism of the lipid system depends on vesicle fusion, it would be helpful to review briefly the experimental procedure followed in the lipid system, which begins with large unilamellar vesicles (LUVs).19 When these LUVs are prepared, they do not aggregate, due to the strong electrostatic repulsion between the charged headgroups. However, a pH decrease induces the LUV aggregation, because the headgroups are not charged any more and the steric repulsion between the headgroups is not strong enough to keep the LUVs apart. Afterward, lipid rearrangement occurs, and quasihexagonal domains are observed, infrequently at relatively low temperatures but frequently at relatively high ones. For the present system, the underlying reason the transition mechanism is not fusion-dependent may be due to the nature of the PEO chains. Due to the relatively large size of the PEO chains compared to that of the headgroups, the strength of the steric repulsion among the PEO chains is relatively high compared to that among the uncharged headgroups (after the pH decrease).

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Therefore, it appears that the strength of the inter PEO chain repulsion is high enough to prevent fusion. Our vesicles may aggregate; however, the degree of aggregation will be relatively low compared to that in the lipid system, and no rearrangement of the copolymer chains occurs between the two approaching or contacting bilayers. Here, we focus only on the aggregation in solutions and not in the process of the TEM grid preparation. We now turn our attention to a proposed method of the formation of the inverted (hollow) rods in another small molecule amphiphile system of succinate phospholipids.18 A transient structure termed an inverted micellar intermediate (IMI) was proposed as the initial intermediate for two apposed bilayers.18 The IMIs are reverse micelles (with hydrophilic cores) forming in the hydrophobic parts of bilayers. The IMIs tend to aggregate and form strings which later elongate to inverted cylinders. For the present system, some of the TEM images (as shown in Figure 2C) may suggest that similar inverted micellar intermediates (IMIs) are formed first. Afterward, they also aggregate to form strings, which may later transform into rods. But in our work, the formation of the “IMIs” appears to happen inside the wall of one vesicle. Although we do not know how these hollow regions (IMIs) originate initially inside the vesicle walls, the formation may be related to the wavy surfaces, as shown in Figures 2B and 5A. The sizes of the initial inverted regions change during the transition. Finally, it is necessary to point out that, generally, fusion processes and rates of morphological transitions of small molecule surfactants are fast. Consequently, it is not easy to trap the intermediate structures. However, the morphological transitions of macromolecules are much slower because of the lower mobility of the high molecular weight molecules. Therefore, it becomes much easier to trap intermediate structures. This aspect facilitates the study of the morphological transitions. As mentioned before, the intermediates, i.e., the quasihexagonal structures of the later stages of the transitions in the two systems, are similar. Therefore, the present study may be particularly instructive and may even shed some light on the process of biomembrane fusion and the lipid lamellae (LR) to inverted hexagonal (HII) phase transition. Thus, a better understanding of the present system may provide a deeper insight into the general process of amphiphile selfassembly. 3.3.2. Comparison with PS-b-PAA. Obviously, both the PS-b-PAA and PS-b-PEO copolymers are amphiphilic; in addition, their hydrophobic parts are PS. The formation of the PS(410)-b-PAA(13) HHHs involves fusion (i.e. aggregation and rearrangement) of preformed vesicles,9 as mentioned in the Introduction. Therefore, the transition is also fusion-dependent and resembles that found in the lipid system. After vesicle aggregation and fusion, it is observed that ordering first appears in the outer regions and then moves toward the interior of the aggregates in the process of the formation of the PS-b-PAA HHHs. After 12-24 h (under the specific conditions of the experiment), HHHs appears in most particles, which assume an external external shape cylinders or cones.9 For the present system, no fusion of preformed vesicles is involved in the early stages of the transition; therefore, the present mechanism differs from that found in the PSb-PAA system. Wall thickening and the concurrent formation of the hollow regions in the wall are involved in the early stage of the transition. Further thickening accompanies the formation of more sets of hollow rods arranged in a hexagonal array; finally, the original water core disappears. Therefore, it appears that the ordering

Yu et al.

moves from outer regions toward the interior, as has also been found in the PS-b-PAA system. This aspect is seen from some of our micrographs. Moreover, conical and cylindrical external shapes are also observed for the present HHHs. Therefore, the PS-b-PAA and PS-b-PEO systems show very different starting mechanisms but considerable similarity in the latter stage of the transition. The underlying reason for the different transition mechanisms seems to be related to the nature of the hydrophilic parts (PAA and PEO) and the common solvents in which the copolymers are first dissolved. The PS-b-PAA HHHs are prepared from DMF-water mixtures, while the PS-b-PEO HHHs are made from THF-water mixtures. It is known that the polarity of DMF (dielectric constant ) 38.2) is greater than that of THF ( ) 7.5);26 furthermore, PAA is ionizable while PEO is not. Consequently, the PAA interchain repulsion in DMF/water solutions is, qualitatively, much larger than the PEO interchain repulsion in THF-water solutions. The strength of the corona repulsion is also (semiquantitatively) reflected by the area per corona chain (Ac) on the PS core surface. A small Ac means a weak intercorona repulsion.7,10 For the PS(410)-b-PAA(16) vesicles made by the addition of water to a DMF solution,7,10 Ac is ca. 6.6 nm2, as calculated from a modified version of the equations given in ref 7,

Ac ) 2VsNPS/(Rout - Rin)

(2)

where Vs is the volume of polystyrene repeat unit (0.167 nm3), NPS is the degree of polymerization of the PS block, and Rout and Rin are the outer and inside radii, respectively. In eq 2, the equivalent lamellar thickness is taken to be Rout - Rin. For the present PS(100)-b-PEO(30) vesicles (with a wall thickness of 23 nm), Ac is ca. 1.5 nm2, as calculated from eq 2. In addition to Ac, the strength of the corona repulsion is also semiquantitatively reflected by the value of σNcorona6/5, where Ncorona is the degree of polymerization of the corona chain and σ is a dimensionless parameter, the value of which is taken as

σ ) a2/Ac

(3)

where a is the size of a repeat unit of the corona chain.7,10 The utility of the term σNcorona6/5 is due to the fact that, in the study of the conformation of flexible polymers (with the degree of polymerization N) immersed in good solvents with one-end covalently grafted onto solid surfaces, the surface density is described by a dimensionless parameter σN6/5.27 When σN6/5 is larger than 1, the density is considered to be high. Therefore, a large value of σNcorona6/5 means a high density of corona chains on the core surface and a small intercorona repulsion. For the PS(410)-b-PAA(16) vesicles made by the addition of water to a DMF solution, the value of σNPAA6/5 is ca. 0.26.7,10 In general, the values of σNPAA6/5 for PS-b-PAA aggregates prepared in a DMF ( ) 38.2) solution fall in the range of 0.2-0.5, and when made in a THF ( ) 7.5) or dioxane ( ) 2.2) solution, they fall in the range of 1-2.10 Thus it is instructive to use the value of σNcorona6/5 to judge the strength of the intercorona repulsion. For the present PS(100)-b-PEO(30) vesicles (with the wall thickness of 23 nm), the value of σNPEO6/5 is ca. 2.6 when Ac is estimated by eq 2. (27) (a) de Gennes, P. G. Macromolecules 1980, 13, 1069. (b) Hadziioannou, G.; Patel, S.; Granick, S.; Tirrell, M. J. Am. Chem. Soc. 1986, 108, 2869.


Therefore, by comparison of the value of Ac and σNcorona6/5 of the two systems, it appears that the density of ionizable PAA chains on the surface of the PS cores is low, yet the corona-corona repulsion is strong since it comes mainly from electrostatic interactions, which are long range. By contrast, the density of nonionizable PEO chains on the surface of the PS cores is high; the weak, short-range steric repulsion dominates the corona-corona repulsion. In the PS-b-PAA system, added electrolyte (NaCl) screens the electrostatic repulsion among the PAA chains;7-10 therefore, the inter-PAA repulsion decreases. Consequently, individual PS-b-PAA vesicles aggregate. Due to the small sizes of the PAA chains and the low density of the PAA chains on the surface of the PS cores, the purely steric repulsion between the PAA chains is weak, and vesicle fusion takes place. The HHH structure forms in the process of establishing a new balance of forces after the perturbation by NaCl. Thus, the added salt induces the vesicle aggregation and fusion and drives the morphology from bilayers to the next stage, namely the inverted hexagonally packed hoops. The later effect is similar to that of a temperature increase in the lipid system. For the present system, an increase in the water concentration accomplishes the same goal as the addition of NaCl in the PS-b-PAA system; namely, it drives the morphology from vesicles to inverted rods in a hexagonal array. In general, the increase of water content causes a decrease of miscibility between the PS block and the solvent; therefore, the morphogenic effect of the increase of water content is equivalent to that of increasing the PS block length or decreasing the PEO block length, both of which favor the transition. Since the vesicle-to-inverted rod transition or the formation of HHHs begins with vesicles, it is helpful to describe the relative size and polydispersity of the vesicles of the two systems. Usually, the PS-b-PAA vesicles prepared from DMF solutions (only by the addition of water, without additives) are small (with Rout smaller than 100 nm) and have a narrow size distribution.6-10 On the other hand, the PS-b-PEO vesicles (formed from THF solutions) are large and highly polydisperse.12-14 The reason for the different vesicle properties of the two systems is also related to the different strength of the inter-PAA and inter-PEO repulsion. As mentioned before, the balance of the three sources of interactions, i.e., in the hydrophobic core, in the hydrophilic corona, and at the interface, determines the morphologies. In parallel with small molecule amphiphiles,17 the three forces do not act in the same plane, and bilayers possess a spontaneous tendency to curve. Because the repulsion arising from the corona favors bending from the start and the repulsion arising from the core acts to oppose bending, large corona repulsion favors small vesicles with a narrow size distribution and small corona repulsion leads to large vesicles with a broad size distribution. Therefore, the weak steric

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inter-PEO repulsion may be responsible, in part, for the fact that the PS-b-PEO vesicles made from THF-water system are usually large and highly polydisperse, and the strong electrostatic inter-PAA repulsion may be responsible, in part, for the fact that the PS-b-PAA vesicles made from DMF-water system are usually small and less polydisperse. 4. Conclusions The morphological transition from vesicles to inverted hexagonally packed rods or hoops (HHRs or HHHs) was studied for diblock copolymers of polystyrene-b-poly(ethylene oxide) (PS-b-PEO) in dilute solutions. The selfassembly as well as the morphological change were induced by the addition of water to the copolymer solutions in tetrahydrofuran (THF). Many intermediates, such as vesicles with hollow regions in the walls and quasihexagonal structures, were trapped. The transition is proposed to proceed in three steps. In the first step, vesicles with hollow regions in the walls form. This step involves a thickening of vesicle walls accompanied by the formation of the hollow rods in the walls running parallel to the surfaces. The method of the thickening and formation of the inverted regions may be related to the observed wavy surfaces and “subvesicles”. The degree of deformation of the PS block in nonclassical vesicles is almost the same as that in classical vesicles. The second step involves the formation of quasihexagonal structures. Finally, inverted hexagonally packed rods form. The present mechanism is not fusion-dependent. A comparison of the present morphological transition with those seen in the PS-b-PAA diblock and in the small molecule amphiphiles is presented. The present mechanism of the vesicle-to-inverted rod transition is different from those found either in the PS-b-PAA diblock or in the small molecule amphiphiles, both of which operate by vesicle fusion. The reason for the difference in the mechanisms may be due to the strength of the corona or headgroup repulsion of the precursor vesicles and not to the size of the precursor vesicles. The present morphological transition was induced by an increase in water content. The formation of hollow rods or hoops arranged in a hexagonal array in polymer matrix has now been observed in two diblock copolymer systems and may thus be general for amphiphilic diblocks in dilute solution. The present study of the morphological transition may help us to understand further the process of biomembrane fusion and the lipid lamella (LR) to inverted hexagonal (HII) phase transition. Acknowledgment. We thank the Natural Sciences and Engineering Research Council of Canada (NSERC) for financial support of this research. We are also indebted to Dr. J. K. Cox and Mr. N. Cameron for useful discussions. LA981688K

Trapping of Intermediate Structures of the Morphological Transition of

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