Fully Collapsed (Kippah) Vesicles: Preparation and Characterization

May 5, 2010 - Kyriaki Pafiti , Zhengxing Cui , Daman Adlam , Judith Hoyland , Anthony J. Freemont , and Brian R. Saunders. Biomacromolecules 2016 17 (...
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Fully Collapsed (Kippah) Vesicles: Preparation and Characterization Tony Azzam and Adi Eisenberg* Department of Chemistry and Centre for Self Assembled Chemical Structures (CSACS), McGill University, 801 Sherbrooke Street West, Montreal, Quebec H3A 2K6, Canada Received February 4, 2010. Revised Manuscript Received March 26, 2010 A study is presented of the formation of a kippah or hemispherical dome structure, a new morphology generated when a vesicle completely collapses to a hollow hemisphere. Justification for the new name is given in the Introduction. Relatively large vesicles of ca. ∼500 nm in diameter were prepared from poly(acrylic acid)-block-polystyrene (PAA-bPS) amphiphilic copolymer in the dioxane/water system. The vesicle specimens for transmission electron microscopy (TEM) were prepared using four different methods: drying under ambient conditions, freeze-drying, freeze-drying and subsequent resuspension in water, and drying under vacuum. The formation of the kippah was found to be strongly influenced by the method of preparation. When the vesicles were allowed to dry on the grid, either by drying under ambient conditions or by direct freeze-drying, “normal” vesicles (i.e., not kippah) with the classical indentation pattern were the only structures to be observed. Kippah vesicles, on the other hand, were obtained only by freeze-drying and subsequent rehydration in water or by direct drying under vacuum where no freezing is involved. The cause of the kippah vesicle formation is not yet completely understood for all methods of preparation; however, it was postulated to be strongly influenced by one or more of the following parameters: the relative flexibility of the vesicle wall, pressure gradient, and surface tension. Unlike “normal” vesicles, which exhibit, in TEM, a classical indentation pattern, kippah vesicles appear nearly round but with average wall thickness twice as large as in the “normal” vesicles. The study illustrates also the usefulness of specimen tilting in the analysis of the kippah. In addition, specimen tilting was found to allow the unambiguous determination of the orientation of the kippah on the surface (i.e., open-side-up or open-side-down).

Introduction Highly asymmetric amphiphilic block copolymers can selfassemble in selective solvents to form aggregates of a wide range of morphologies, such as spherical micelles, rods, vesicles, and others.1-7 In these aggregates, the long hydrophobic block forms either the core in micelles and rods or the walls in bilayer structures, while the short hydrophilic block forms the corona.8,9 A number of research groups have studied block copolymer aggregates of *Corresponding author: Tel (þ1)-514-3986934; Fax (þ1)-514-3983797; e-mail [email protected]. (1) van Hest, J. C. M.; Delnoye, D. A. P.; Baars, M. W. P. L.; van Genderen, M. H. P.; Meijer, E. W. Polystyrene-dendrimer amphiphilic block copolymers with a generation-dependent aggregation. Science (Washington, DC, U.S.) 1995, 268 (5217), 1592-5. (2) Zhang, L.; Eisenberg, A. Multiple morphologies of “crew-cut” aggregates of polystyrene-b-poly(acrylic acid) block copolymers. Science (Washington, DC, U.S.) 1995, 268 (5218), 1728-31. (3) Zhang, L.; Yu, K.; Eisenberg, A. Ion-induced morphological changes in “crew-cut” aggregates of amphiphilic block copolymers. Science (Washington, DC, U.S.) 1996, 272 (5269), 1777-1779. (4) Yu, K.; Eisenberg, A. Multiple Morphologies in Aqueous Solutions of Aggregates of Polystyrene-block-poly(ethylene oxide) Diblock Copolymers. Macromolecules 1996, 29 (19), 6359-6361. (5) Zhang, L.; Eisenberg, A. Multiple Morphologies and Characteristics of “Crew-Cut” Micelle-like Aggregates of Polystyrene-b-poly(acrylic acid) Diblock Copolymers in Aqueous Solutions. J. Am. Chem. Soc. 1996, 118 (13), 3168-81. (6) Hillmyer, M. A.; Bates, F. S.; Almdal, K.; Mortensen, K.; Ryan, A. J.; Fairclough, J. P. A. Complex phase behavior in solvent-free nonionic surfactants. Science (Washington, DC, U.S.) 1996, 271 (5251), 976-8. (7) Won, Y.-Y.; Davis, H. T.; Bates, F. S. Giant wormlike rubber micelles. Science (Washington, DC, U.S.) 1999, 283 (5404), 960-963. (8) Luo, L.; Eisenberg, A. Thermodynamic Stabilization Mechanism of Block Copolymer Vesicles. J. Am. Chem. Soc. 2001, 123 (5), 1012-1013. (9) Discher, D. E.; Eisenberg, A. Materials science: Soft surfaces: Polymer vesicles. Science (Washington, DC, U.S.) 2002, 297 (5583), 967-973. (10) Davis, K. P.; Lodge, T. P.; Bates, F. S. Vesicle Membrane Thickness in Aqueous Dispersions of Block Copolymer Blends. Macromolecules 2008, 41 (22), 8289-8291. (11) Du, B.; Mei, A.; Yin, K.; Zhang, Q.; Xu, J.; Fan, Z. Vesicle Formation of PLAx-PEG44 Diblock Copolymers. Macromolecules 2009, 42 (21), 8477-8484.

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various morphologies in recent years.10-13 Among these morphologies, vesicles, as also liposomes made of phospholipids, are of great interest due to potential applications as encapsulation agents, particularly in fields such as biomedicine, drug delivery, and catalysis.14-16 Block copolymer vesicles, frequently also referred to as polymersome, can be thermodynamically stable structures under a range of conditions.1,17-33 The thermodynamic stabilization (12) Du, J.; Armes, S. P. Preparation of Biocompatible Zwitterionic Block Copolymer Vesicles by Direct Dissolution in Water and Subsequent Silicification within Their Membranes. Langmuir 2009, 25 (16), 9564-9570. (13) Kim, K. T.; Cornelissen, J. J. L. M.; Nolte, R. J. M.; van Hest, J. C. M. A Polymersome Nanoreactor with Controllable Permeability Induced by StimuliResponsive Block Copolymers. Adv. Mater. (Weinheim, Ger.) 2009, 21 (27), 2787-2791. (14) Nallani, M.; de Hoog, H. P. M.; Cornelissen, J.; Palmans, A. R. A.; van Hest, J. C. M.; Nolte, R. J. M. Polymersome nanoreactors for enzymatic ringopening polymerization. Biomacromolecules 2007, 8 (12), 3723-3728. (15) Shum, H. C.; Kim, J.-W.; Weitz, D. A. Microfluidic Fabrication of Monodisperse Biocompatible and Biodegradable Polymersomes with Controlled Permeability. J. Am. Chem. Soc. 2008, 130 (29), 9543-9549. (16) Blanazs, A.; Armes, S. P.; Ryan, A. J. Self-assembled block copolymer aggregates: from micelles to vesicles and their biological applications. Macromol. Rapid Commun. 2009, 30 (4-5), 267-277. (17) Ding, J. F.; Liu, G. J. Polyisoprene-block-poly(2-cinnamoylethyl methacrylate) vesicles and their aggregates. Macromolecules 1997, 30 (3), 655657. (18) Discher, B. M.; Won, Y. Y.; Ege, D. S.; Lee, J. C. M.; Bates, F. S.; Discher, D. E.; Hammer, D. A. Polymersomes: Tough vesicles made from diblock copolymers. Science (Washington, DC, U.S.) 1999, 284 (5417), 1143-1146. (19) Discher, B. M.; Hammer, D. A.; Bates, F. S.; Discher, D. E. Polymer vesicles in various media Curr. Opin. Colloid Interface Sci. 2000, 5 (1-2), 125-131. (20) Luo, L.; Eisenberg, A. Thermodynamic Size Control of Block Copolymer Vesicles in Solution. Langmuir 2001, 17 (22), 6804-6811. (21) Luo, L.; Eisenberg, A. Thermodynamic Size Control of Block Copolymer Vesicles in Solution [ Erratum for Langmuir 2001, 17, 6804-6811 ]. Langmuir 2002, 18 (5), 1952. (22) Nardin, C.; Widmer, J.; Winterhalter, M.; Meier, W. Amphiphilic block copolymer nanocontainers as bioreactors. Eur. Phys. J. E 2001, 4 (4), 403-410. (23) Discher, B. M.; Bermudez, H.; Hammer, D. A.; Discher, D. E.; Won, Y. Y.; Bates, F. S. Cross-linked polymersome membranes: Vesicles with broadly adjustable properties. J. Phys. Chem. B 2002, 106 (11), 2848-2854.

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mechanism of poly(acrylic acid)-b-polystyrene (PAA-b-PS) diblock copolymer vesicles has been elucidated. It was shown that the curvature in this type of vesicles is stabilized by preferential segregation of short hydrophilic blocks to the inside of the vesicles and of the long blocks to the outside of the vesicle bilayer. The repulsion among the longer corona chains is greater than that among the shorter ones. Therefore, segregation of the hydrophilic blocks by length, which allows the formation of asymmetric lamellae, stabilizes the curvature of the vesicles.20,21 To explore block copolymer vesicles for many potential applications, the control of vesicular characteristics, such as size, shape, wall thickness, and inner volume, is of great importance. It is assumed that the parameters which control the morphologies of the aggregates, i.e., core chain stretching, interfacial tension, and corona repulsion, are also responsible for the thermodynamic control of the vesicular architecture.3,34-40 Solution properties such as solvent composition,35 presence of additives (e.g., salts, acids, or bases),39 water content,40 and hydrophilic/hydrophobic block length ratios41 were also shown to have a strong influence on vesicle size. For example, the addition of a base during the preparation of the vesicles containing acrylic acid in the corona decreases the size of the vesicles due to an increase in the electrostatic repulsion among the corona chains.39 Addition of (24) Kukula, H.; Schlaad, H.; Antonietti, M.; Forster, S. The formation of polymer vesicles or “peptosomes” by polybutadiene-block-poly(L-glutamate)s in dilute aqueous solution. J. Am. Chem. Soc. 2002, 124 (8), 1658-1663. (25) Vriezema, D. M.; Hoogboom, J.; Velonia, K.; Takazawa, K.; Christianen, P. C. M.; Maan, J. C.; Rowan, A. E.; Nolte, R. J. M. Vesicles and polymerized vesicles from thiophene-containing rod-coil block copolymers. Angew. Chem., Int. Ed. 2003, 42 (7), 772-776. (26) Du, J. Z.; Armes, S. P. pH-responsive vesicles based on a hydrolytically selfcross-linkable copolymer. J. Am. Chem. Soc. 2005, 127 (37), 12800-12801. (27) Chen, D. Y.; Jiang, M. Strategies for constructing polymeric micelles and hollow spheres in solution via specific intermolecular interactions. Acc. Chem. Res. 2005, 38 (6), 494-502. (28) Thibault, R. J.; Uzun, O.; Hong, R.; Rotello, V. M. Recognition-controlled assembly of nanoparticles using photochemically crosslinked recognition-induced polymersomes. Adv. Mater. 2006, 18 (16), 2179. (29) Li, Y.; Lokitz, B. S.; McCormick, C. L. Thermally responsive vesicles and their structural “locking” through polyelectrolyte complex formation. Angew. Chem., Int. Ed. 2006, 45 (35), 5792-5795. (30) Edmonds, W. F.; Hillmyer, M. A.; Lodge, T. P. Block copolymer vesicles in liquid CO2. Macromolecules 2007, 40 (14), 4917-4923. (31) Hu, Z. J.; Verheijen, W.; Hofkens, J.; Jonas, A. M.; Gohy, J. F. Formation of vesicles in block copolymer-fluorinated surfactant complexes. Langmuir 2007, 23 (1), 116-122. (32) Mai, Y. Y.; Zhou, Y. F.; Yan, D. Y. Real-time hierarchical setf-assembly of large compound vesicles from an amphiphilic hyperbranched multiarm copolymer. Small 2007, 3 (7), 1170-1173. (33) Houga, C.; Giermanska, J.; Lecommandoux, S.; Borsali, R.; Taton, D.; Gnanou, Y.; Le Meins, J. F. Micelles and Polymersomes Obtained by SelfAssembly of Dextran and Polystyrene Based Block Copolymers. Biomacromolecules 2009, 10 (1), 32-40. (34) Zhang, L.; Eisenberg, A. Morphogenic Effect of Added Ions on Crew-Cut Aggregates of Polystyrene-b-poly(acrylic acid) Block Copolymers in Solutions. Macromolecules 1996, 29 (27), 8805-8815. (35) Yu, Y.; Zhang, L.; Eisenberg, A. Morphogenic Effect of Solvent on CrewCut Aggregates of Amphiphilic Diblock Copolymers. Macromolecules 1998, 31 (4), 1144-1154. (36) Chen, L.; Shen, H.; Eisenberg, A. Kinetics and Mechanism of the Rod-toVesicle Transition of Block Copolymer Aggregates in Dilute Solution. J. Phys. Chem. B 1999, 103 (44), 9488-9497. (37) Shen, H.; Zhang, L.; Eisenberg, A. Multiple pH-Induced Morphological Changes in Aggregates of Polystyrene-block-poly(4-vinylpyridine) in DMF/H2O Mixtures. J. Am. Chem. Soc. 1999, 121 (12), 2728-2740. (38) Bronich, T. K.; Ouyang, M.; Eisenberg, A.; Kabanov, V.; Szoka, F. C., Jr.; Kabanov, A. V. Reactive stabilization of vesicles from cationic surfactants selfassembled on anionic block ionomer template. Abstracts of Papers, 220th ACS National Meeting, Washington, DC, Aug 20-24, 2000; POLY-199. (39) Choucair, A.; Lavigueur, C.; Eisenberg, A. Effect of additives and solvent composition on polystyrene-b-poly(acrylic acid) vesicle size. Abstracts of Papers, 225th ACS National Meeting, New Orleans, LA, March 23-27, 2003; COLL-328. (40) Choucair, A.; Lavigueur, C.; Eisenberg, A. Polystyrene-b-poly(acrylic acid) Vesicle Size Control Using Solution Properties and Hydrophilic Block Length. Langmuir 2004, 20 (10), 3894-3900. (41) Azzam, T.; Eisenberg, A. Control of vesicular morphologies through hydrophobic block length. Angew. Chem., Int. Ed. 2006, 45 (44), 7443-7.

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acids or salts, on the other hand, increases the vesicle size due to decreasing repulsion resulting from protonation of the PAA segments or the screening of the electrostatic repulsion among the corona chains.39 The inner volume of the vesicles is usually dictated by their size, wall thickness, and shape. As noted earlier, vesicle sizes and wall thicknesses are affected by many parameters which, under certain conditions, can be controlled. For example, a linear increase (R2 = 0.986) in the vesicle wall thickness was found in a series of vesicles made from PAA-b-PS copolymers, where the PAA block length was kept fixed and the PS block length was increased. However, when the values of the wall thicknesses of PAA-b-PS vesicles series having different PAA and PS block lengths were combined, the quality of the linear fit was poor, which suggests that the wall thickness is not a function of the PS block length alone. A direct correlation with the PS block length was shown to be reasonable only when a constant PAA block was utilized.41 To exploit polymeric vesicles fully as potential carriers for bioactive encapsulates, it is important to maintain their integrity, stability, and spherical shape. From a geometric point of view, maximum loading is expected with spherically shaped hollow particles, rather than deformed or indented ones. Most of the large polymer vesicles studied by our group showed by TEM some type of structural deformation.42-45 Under certain conditions, however, which will be described below, vesicles were obtained which appeared not to have any indentations. The only apparent anomaly was their wall thickness, which was approximately double that obtained for other vesicles prepared from the same block copolymer. Sometimes the two types of vesicles are seen in the same micrograph, as shown in Figure 1. A more extensive investigation revealed that the thickwalled vesicles were completely collapsed into the shape of a dome or “kippah”.46 In this study we explore the experimental conditions under which spherical vesicles collapse completely to yield hemispheres with zero enclosed volume, utilizing primarily the technique of specimen tilting in TEM to detect this artifact. More importantly, the method of surface tilting was successfully used in elucidating the alignments of these fully collapsed vesicles on the TEM grid. PAA-b-PS copolymer vesicles were chosen for this specific study due to their physical stability and robustness of the PS wall. In addition, the high electron density of PS polymer allows easy visualization by TEM without any need of staining agents. For the collapsed vesicles, we prefer not to use the word “bowl” because that word was applied to a very different morphology in earlier studies.47,48 In giving the name “kippah” to this unique morphology, we follow a long tradition in polymer science in naming morphologies or phenomena using common words and (42) Liu, F.; Eisenberg, A. Preparation and pH Triggered Inversion of Vesicles from Poly(acrylic Acid)-block-Polystyrene-block-Poly(4-vinyl Pyridine). J. Am. Chem. Soc. 2003, 125 (49), 15059-15064. (43) Terreau, O.; Bartels, C.; Eisenberg, A. Effect of Poly(acrylic acid) Block Length Distribution on Polystyrene-b-poly(acrylic acid) Block Copolymer Aggregates in Solution. 2. A Partial Phase Diagram. Langmuir 2004, 20 (3), 637-645. (44) Wu, J.; Eisenberg, A. Proton Diffusion across Membranes of Vesicles of Poly(styrene-b-acrylic Acid) Diblock Copolymers. J. Am. Chem. Soc. 2006, 128 (9), 2880-2884. (45) Yu, S.; Azzam, T.; Rouiller, I.; Eisenberg, A. “Breathing” Vesicles. J. Am. Chem. Soc. 2009, 131 (30), 10557-10566. (46) Kippah is the Hebrew word for both dome and the traditional skullcap. A more extensive justification for the use of the word is given in the last paragraph of the Introduction. (47) Riegel, I. C.; Eisenberg, A.; Petzhold, C. L.; Samios, D. Novel bowl-shaped morphology of crew-cut aggregates from amphiphilic block copolymers of styrene and 5-(N,N-diethylamino)isoprene. Langmuir 2002, 18 (8), 3358-3363. (48) Liu, X.; Kim, J.-S.; Wu, J.; Eisenberg, A. Bowl-Shaped Aggregates from the Self-Assembly of an Amphiphilic Random Copolymer of Poly(styrene-comethacrylic acid). Macromolecules 2005, 38 (16), 6749-6751.

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Article method D, on the other hand, involves direct drying under high vacuum (without freezing) of a dilute vesicle solution droplet placed on an EM grid. For method C, the vesicle solution was freeze-dried to obtain the vesicles in the form of a white powder. Then, this powder was resuspended in water (0.05 wt %) and added to EM grids as per method A. For additional experimental details, see the Supporting Information.

Results and Discussion

Figure 1. PAA34-b-PS324 block copolymer vesicles prepared in pure water showing the two different wall thicknesses (images A and B). Note the circular shape in the thick-walled vesicle (image A) and the typical indentation in the thin-walled one (image B) after tilting the grid by þ50° (clockwise).

expressions, which are sometimes of ethnic origin. For example, “shish kebab” (Persian-Arabic origin) has been used for nearly four decades to describe the polymer crystal morphology of a long rod periodically decorated with lamellar crystals.49 More recently, the words “knedel” (east-central European origin)50 and spaghetti (Italian origin)51 were given to describe unique forms of supramolecular structures of amphiphilic block copolymers. Other names and expressions include “crew-cut”,2,52,53 “star”,54 “flower”,55 “bottle brush”,56 “plumber’s nightmare”,57 and “Janus” (from Roman mythology),58 just to name a few frequently used examples.

Experimental Section PAA34-b-PS324, PAA47-b-PS434, and PAA47-b-PS307 block copolymers were prepared by atom-transfer radical polymerization (ATRP).41 The subscripts refer to the average degree of polymerization (DP) of each block and were estimated by NMR and size exclusion chromatography (SEC). Vesicle samples from the above block copolymers were prepared in the dioxane/water system (Supporting Information). The specimens for TEM in this study were prepared using the following four methods: (A) evaporation under ambient conditions, (B) direct freeze-drying, (C) freezedrying and subsequent resuspension in water, and (D) direct drying of the vesicle solution under vacuum. In method A, a droplet of the diluted vesicle solution was allowed to dry under ambient temperature and atmospheric pressure. Method B involves freeze-drying the sample directly on the EM grid, while (49) Lindenmeyer, P. H., Molecular theory of polymer chain folding. J. Polym. Sci., Polym. Symp. 1967, 20, 145-58. (50) Thurmond, K. B., II; Kowalewski, T.; Wooley, K. L. Water-Soluble Knedel-like Structures: The Preparation of Shell-Cross-Linked Small Particles. J. Am. Chem. Soc. 1996, 118 (30), 7239-7240. (51) Devereaux, C. A.; Baker, S. M. Surface Features in Langmuir-Blodgett Monolayers of Predominantly Hydrophobic Poly(styrene)-Poly(ethylene oxide) Diblock Copolymer. Macromolecules 2002, 35 (5), 1921-1927. (52) Vilgis, T.; Halperin, A. Aggregation of coil-crystalline block copolymers: equilibrium crystallization. Macromolecules 1991, 24 (8), 2090-5. (53) Honda, C.; Sakaki, K.; Nose, T. Micellization of an asymmetric block copolymer in mixed selective solvents. Polymer 1994, 35 (24), 5309-18. (54) Zilliox, J. G.; Rempp, P.; Parrod, J. Preparation of star-shaped macromolecules by anionic copolymerization. J. Polym. Sci., Part C 1968, No. 22 (Pt. 1), 145-56. (55) Saito, R.; Yoshida, S.; Ishizu, K. Synthesis of poly(vinyl alcohol) corepolystyrene shell-type flower microgels. J. Appl. Polym. Sci. 1997, 63 (7), 849-854. (56) Hong, S. C.; Neugebauer, D.; Inoue, Y.; Lutz, J.-F.; Matyjaszewski, K. Preparation of segmented copolymers in the presence of an immobilized/soluble hybrid ATRP catalyst system. Macromolecules 2003, 36 (1), 27-35. (57) Yu, K.; Eisenberg, A. Bilayer Morphologies of Self-Assembled Crew-Cut Aggregates of Amphiphilic PS-b-PEO Diblock Copolymers in Solution. Macromolecules 1998, 31 (11), 3509-3518. (58) Erhardt, R.; Zhang, M.; Boker, A.; Zettl, H.; Abetz, C.; Frederik, P.; Krausch, G.; Abetz, V.; Muller Axel, H. E. Amphiphilic Janus micelles with polystyrene and poly(methacrylic acid) hemispheres. J. Am. Chem. Soc. 2003, 125 (11), 3260-7.

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Liposomes are usually formed by rehydration and sonication of a phospholipid film, previously cast by the evaporation of a phospholipid solution in a hydrocarbon solvent (mainly chloroform). This method, however, while most useful for liposomes, has not been utilized for many polymer vesicles due to the extremely low solubility of their constituent polymers in water as well as their high molecular weights. In many cases, block copolymer aggregates of highly amphiphilic copolymers are prepared by first dissolving the copolymer in a common watermiscible organic solvent. The self-assembly is then induced by a slow addition of a precipitant (e.g., water) for the hydrophobic block. The rate of water addition is usually optimized to prevent precipitation of the copolymer while allowing equilibration to be reached between water addition increments. Eventually, when the vesicles are obtained, a stage easily detected by a major increase in solution turbidity, the aggregates are usually “quenched” in an excess of water in order to prevent any morphological changes. Finally, residual organic solvent is removed, usually by dialysis. In an alternative method of preparation, the block copolymer is initially dissolved in a water-miscible solvent (dioxane, THF, DMF, etc.), followed by dialysis against large amounts of water to induce self-assembly. The latter method is more practical for scaling up; however, for the present system, it is accompanied by the formation of large aggregations. Therefore, and in order to minimize such undesired aggregation, the method of stepwise addition of water was adopted for the current study. Figure 2a shows a typical TEM image of the vesicles obtained from the PAA34-b-PS324 block copolymer. The EM sample was dried under ambient conditions as described in method A of the Experimental Section. The average vesicle diameter and wall thickness was 435 ( 140 (n = 200) and 44 ( 4 (n = 100) nm, respectively. The value n in parentheses following the vesicle diameter and the wall thickness represents the sample size used in the statistics. In all cases, only vesicles of ca. >200 nm in diameter were taken into an account for the statistics. Vesicles smaller than 200 nm in diameter, which accounted for less than 1% of the total number of aggregates, were omitted from the calculations since they could not be identified unambiguously as vesicles. Figure 2b represents PAA34-b-PS324 vesicles prepared by direct freezedrying on the EM grid (method B, Experimental Section). The average vesicle diameter and wall thickness were 500 ( 90 (n = 300) and 40 ( 4 nm (n = 100), respectively. Additional TEM images of both samples showing very similar indentation patterns can be found in Figures S1 and S2 of the Supporting Information. It is evident from Figures 2a,b that there are no appreciable differences, either in vesicles size or in the wall thickness, between samples prepared by the two different methods (i.e., drying under ambient condition vs freeze-drying). Parts c and d of Figure 2 show the TEM images of vesicles prepared from another copolymer, PAA47-b-PS434, by drying under ambient conditions and direct freeze-drying, respectively. The average vesicle diameter and wall thickness of the sample which was dried under ambient conditions (Figure 2c) was 350 ( 125 nm (n = 200) and 46 ( 4 nm (n = 100), respectively. The values obtained from vesicles prepared by the direct freeze-drying method (Figure 2d) were 380 ( 120 nm DOI: 10.1021/la1004837

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Figure 2. TEM images of vesicles made from PAA34-b-PS324 block copolymer (a, b) and PAA47-b-PS434 block copolymer (c, d). Drying under ambient condition was employed for samples a and c (method A of the Experimental Section), whereas the direct freeze-drying on the EM grids was utilized for samples b and d (method B of the Experimental Section).

(n = 300) and 50 ( 3 nm (n = 150) for the diameter and wall thickness, respectively. The slight difference in the wall thicknesses between the two samples (i.e., Figures 2a,b) is within experimental error. It is worth noting in this connection that only data on near spherically shaped vesicles were included in the calculation of the vesicles size as well as the wall thickness. Vesicles having severe indentation and distortion were excluded from the statistics. The preceding pictures (Figure 2) and the statistical summaries clearly show that the both the wall thickness and type of indentation are maintained as the method of preparation changes from method A to B (Experimental Section). The reason behind using two copolymers of different block lengths, i.e., PAA34-b-PS324 and PAA47-bPS434, was to emphasize the fact that the indentation of large vesicles is not related to the specific chemical composition of the copolymer but the method of preparation. The same indentation pattern was also observed for vesicles made from other copolymers such as poly(ethylene oxide)-b-polystyrene,4,57 poly(4-vinylpyridine)-b-polystyrene,37 poly(4-vinylpyridine)-b-polystyrene-b-poly(acrylic acid),42 polybutadiene-b-poly(2-vinylpyridine),59 and poly(acrylic acid)-b-poly(n-butyl acrylate) (to be published), just to name a few examples. The reason behind vesicle indentation is not fully understood; however, it is believed to happen during the process of vesicle formation. As described earlier, the block copolymer is initially dissolved in dioxane, followed by a stepwise addition of water. During water addition to the copolymer solution, the vesicles are usually formed at ca. 20 wt % water or even lower,40 depending on the degrees of polymerization (DP) and block length ratio. After reaching 50 wt % water content, the vesicles are usually quenched in an excess of water to yield a total dioxane content of less than 5 wt %. Quenching is usually achieved by adding the freshly formed vesicle solution to a large excess of water under continuous (59) Walther, A.; Goldmann, A. S.; Yelamanchili, R. S.; Drechsler, M.; Schmalz, H.; Eisenberg, A.; Mueller, A. H. E. Multiple Morphologies, Phase Transitions, and Cross-Linking of Crew-Cut Aggregates of Polybutadiene-blockpoly(2-vinylpyridine) Diblock Copolymers. Macromolecules 2008, 41 (9), 3254-3260.

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stirring. During the addition of water to the external solution, the dioxane content inside the vesicles has to decrease as well through diffusion of the dioxane to the exterior and of the water to the interior. Since the rate of dioxane diffusion through the hydrophobic PS wall toward the exterior is considerably faster than the rate of water diffusion toward the interior, a negative pressure is generated on the inside, which leads to indentation. By the time the pressure has equalized, the Tg of the PS wall has risen sharply which, in turn, prevents the recovery and leads to the permanent setting of the indentation. Smaller vesicles, on the other hand, exhibit considerably less surface area compared to large vesicles; therefore, the negative forces generated during quenching in water are much smaller. Since the volume of the wall in small vesicles is occupying a major portion of the total vesicle volume, and given the fact that the generated negative forces are small, the process of quenching of small vesicles does not deform their spherical shape, as is clearly shown in Figure S3 of the Supporting Information. We investigated the resuspension of freeze-dried vesicles in water, in connection with another project, and found that the resuspended vesicles are different from the original vesicles in two important features. They showed no evidence of any kind of indentation in that the vast majority of the vesicles appeared round or slightly oval. In addition, and more importantly, the resuspended vesicles have a wall that was 60-80% thicker than the normal vesicle wall. Figure 3 shows typical TEM images of PAA34-b-PS324 and PAA47-b-PS434 vesicles after freeze-drying and subsequent resuspension in water (method C, Experimental Section). A quick glimpse at the vesicles PAA34-b-PS324 and PAA47-b-PS434 (Figure 3, a and d, respectively) shows that after such treatment, i.e. freeze-drying and subsequent resuspension in water, the TEM images of the vesicles show them to be devoid of the usual indentations. Freeze-drying and subsequent resuspension in water of vesicles from other batches, for both PAA34-bPS324 and PAA47-b-PS434, showed them to behave in the same way, as illustrated in Figures S4 and S5 (Supporting Information). Most importantly, a closer look at these round vesicles in Figure 3b reveals an unusually thick vesicle wall. The average wall thickness was estimated to be 70 ( 10 nm (n = 200), while the average vesicle diameter remained essentially unchanged (i.e., 465 ( 120, n = 200) compared to the vesicles made by methods A and B (i.e., under ambient conditions as well as direct freezedrying). The wall thickness of the vesicles obtained after freezedrying and resuspension in water, i.e. 70 ( 10 nm, thus is seen to exhibit a 1.8-fold increase compared to that of the original vesicles made by methods A and B. By comparison, and as described earlier, the average wall thickness of the vesicles prepared by either drying under ambient conditions (method A) or by direct freeze-drying (method B) was 40 ( 4 nm, as illustrated in Figure 3c. Figures 3d-g show typical TEM images of the other PAA47-bPS434 polymer vesicles after similar freeze-drying and resuspension in water. As can be clearly seen from these figures, freezedrying and resuspension in water of these polymer vesicles resulted in an increase in the average wall thickness by about 60%. The wall thickness of the vesicles in Figures 3e-g is 70 ( 4 nm compared to 46 ( 4 nm (Figure 3h) for the vesicles made by either method A or method B (Experimental Section). EM samples made by direct drying of the vesicle solutions under vacuum (method D, Experimental Section) showed a vesicle appearance similar to that of the vesicles made by the freezedrying and subsequent resuspension. Most of the vesicles made by the direct drying of the vesicles solution under vacuum, i.e. without freezing, appeared to have an unindented nearly round Langmuir 2010, 26(13), 10513–10523

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Figure 3. Typical TEM images of PAA34-b-PS324 (a, b) and PAA47-b-PS434 (d, e, f, g) vesicles after freeze-drying and resuspension in water (method C of the Experimental Section). TEM images c and h represent the controls PAA34-b-PS324 and PAA47-b-PS434 vesicles, respectively, prepared under ambient conditions (method A of the Experimental Section).

shape and unusually thick walls (Figures S6 and S7, Supporting Information). To investigate further the reason behind the sharp increase in the wall thicknesses of vesicles from both materials, as well the absence of the indentations, tilt experiments were conducted as illustrated in Figure 4. Figure 4a shows typical PAA34-b-PS324 vesicles after freeze-drying and subsequent resuspension in water. A series of increasing magnifications from 11.5K to 43K is shown in successive panels in Figure 4a. Low-magnification images containing a few hundred vesicles were used in the statistical calculations, whereas the higher-magnification images were used for the tilt experiments. As can be clearly seen from these images, the vast majority of the vesicles tend to have an apparently unindented nearly round appearance but thicker than usual walls (∼75 nm). Intentionally, the word round is used instead of “spherical” in the latter sentence, as well as in previous paragraphs, due to the fact that the vesicles appear spherical in traditional TEM images; however, they turn out not to be, as we will discuss below. A single area (a small part of the highlighted square in the first image of Figure 4a) was chosen for tilt studies due to the presence of one bilamellar vesicle (marked by an arrow in the first image of series Figure 4a). The existence of a visibly distinguishable single vesicle among many other vesicles is, in part, an excellent tool for the tilt experiments and can easily be used as a reference point. Figures 4b shows a part of the same labeled area seen in Figure 4a (within the marked square) after magnification and tilting the grid in both directions (i.e., clockwise and counterclockwise) by 40° and 60°. In the present case, positive tilting represents clockwise tilting, whereas negative tilting represents counterclockwise tilting. Only minor differences were observed when the grid holding the vesicles was tilted by up to þ30° (data not shown). However, when the surface was tilted to between 40° and 60°, the vesicle walls on the right side appeared darker, while those on the opposite side of the vesicles became lighter. It is worth mentioning, in this respect, that the intensification in the darkness of the vesicles walls on one side, as clearly observed by TEM, is an indication of a sharp increase in the distance traversed by the electrons through the wall material on that side of the vesicles. The substantial increase in the darkness of the vesicle Langmuir 2010, 26(13), 10513–10523

walls on one side after tilting is an indication that the electron beam, which has a fixed bombardment angle of 90° to the surface prior to tilting, has to penetrate more of the PS material on one side compared to the other at the new tilt angle. The more PS the electron beam has to traverse, the darker the region appears on the micrograph. Therefore, positive tilting of the grid resulted in an increase in the darkness of these structures on the side which moves down, while the electron density is simultaneously decreased on the opposite side of the vesicles, i.e., the side which moves up. By contrast, as expected, when the surface of the vesicles was negatively tilted (Figure 4b), behavior opposite to that seen for positive tilting was observed. Hence, negative tilting resulted in an increase in the thickness traversed by electrons on the left side of the vesicles, i.e., the side which moved down, while the length of the electron path on the right side of the vesicles, i.e., the side which moved up, was decreased. More tilting images of various magnifications can be seen in Figure S8 (Supporting Information). Figure 4c shows the distribution of vesicle wall thickness of the two types of vesicles: normal vesicles with the classical indentation and vesicles which appear more nearly circular without indentations. This histogram (divided into two subhistograms) was obtained from statistics accumulated from ca. 400 normal vesicles as well as from round vesicles having ca. double the wall thickness. It is obvious from this histogram that there are two distinct vesicle populations. One of them consists of normal vesicles (left subhistogram) obtained from images such those in Figures 2a,b with an average wall thickness of ca. 44 nm. The other vesicle population (right subhistogram) represents the apparently round vesicles with ca. double the wall thickness, obtained from images such as those in Figure 3a. The combined subhistograms (i.e., normal vesicles and vesicles with double the wall thickness) for PAA47-bPS434 were shown to behave similarly to that for PAA34-b-PS324; however, the gap in between the two populations was narrower in PAA47-b-PS434 (Figure S9, Supporting Information) than that measured for PAA34-b-PS324 vesicles (Figure 4c). Based on the electron density behavior of the tilted vesicles in Figure 4b and Figure S8 (Supporting Information), a fully collapsed structure or “kippah” is proposed for this unique morphology (Figure 5). In the course of this paper, we retain the use of DOI: 10.1021/la1004837

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Figure 4. A series of TEM images of vesicles with apparently double the wall thicknesses at various magnifications (a). (b) TEM images of a magnified area (labeled within the boundaries of a square in the first image of series a) after clockwise (þ) and counterclockwise (-) tilting. (c) Histogram showing the wall thickness distribution of PAA34-b-PS324 copolymer vesicles having both the normal wall thicknesses (left subhistogram) and the apparently double wall thicknesses (right subhistogram).

the word “vesicle” after “kippah” to emphasize the source of this structure; however, it is very important to remember that the new structure cannot be considered a real vesicle since the internal volume has collapsed completely. To our knowledge, tilting of specimens in EM to determine the geometric shape of polymer vesicles was not used in the past, although it is a well-known technique. The absence of tilt studies on vesicles is not surprising, since the tilting of a sphere does not provide additional insights. Figure 5a shows a cartoon illustrating the 3D geometry of a typical kippah vesicle after tilting by -60° (i.e., counterclockwise). In this cartoon, the short “hairs” in the interior and in the exterior of the vesicle represent the PAA corona chains of the vesicle. It is worth mentioning, in this respect, that while the cartoon shows a flat surface around the circumference on the open side, one would expect it to have a hemitoroidal shape. Figure 5b shows a real TEM image of a kippah vesicle after surface tilting by -60° (i.e., counterclockwise). As can be seen clearly from Figure 5b, counterclockwise tilting resulted, in this specific vesicle, in an increase in the darkness on the side that moves up (right side of image), while 10518 DOI: 10.1021/la1004837

drastically decreasing the darkness on the side that moves down (left side of image). Figure 5c represents the darkness as a function of the radial distance (d) from the outer left edge of the vesicle. The curve in Figure 5c was calculated from a cross-section model of the kippah vesicle having 500 nm in diameter and 70 nm in wall thickness. The cross section was tilted by -60° (i.e., counterclockwise), as in the micrograph of this sample and the darkness, which is being represented here by the thickness of the PS wall at a certain d value, was measured using SigmaScan 5.0 software. Knowing the diameter and wall thickness, which were used for calibration, it was easy to measure the vertical thickness at selected distances d from the outer left edge of the kippah vesicle. It is important to note that the edge-to-edge distances after tilting in either direction is smaller compared to the same edge-to-edge distance prior to tilting. The greater the tilt angle, the smaller the distance between the two edges. As can be seen from Figure 5c, the darkness increases slightly when moving from the outer left edge by ca. 50 nm. Between 50 and 200 nm, the darkness does not change greatly and a plateau is obtained. At some point, the Langmuir 2010, 26(13), 10513–10523

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Figure 5. Schematic representation of a typical kippah vesicle (initially open-side-up) after counterclockwise tilting by -60° (a). (b) A real

TEM image of a kippah vesicle at -60° tilt (i.e., counterclockwise). The graph in (c) describes the darkness (i.e., the distance traversed by the electron beam in its passage through the vesicle wall) as a function of the radial distance (d) from the outer left edge of the vesicle (nm). The curve in (c) was measured from a cross-section model of the kippah vesicle with 500 nm in diameter and 70 nm in wall thickness. The thicknesses of the PS wall (which is proportional to the darkness) at various d values were measured using SigmaScan 5.0 software. It is important to note that the edge-to-edge distance after tilting by -60°, as in the above example, is smaller than the same edge-to-edge distance before tilting.

darkness increases sharply again to reach its maximum at ca. 300 nm. Finally, the darkness drops sharply toward the right edge of the vesicle. Figure S10 illustrates the difference in the tilting patterns between large kippah vesicles and small spherical vesicles (i.e., not kippah). The large vesicles (i.e., ∼ 500 nm) were prepared from the PAA34-b-PS324 block copolymer, while the small vesicles (∼120 nm) were prepared from PAA47-b-PS307 block copolymer. Both copolymer vesicle samples were freeze-dried, resuspended in water separately, and subsequently mixed together at a 1/1 (w/w) ratio. As can be clearly seen from Figure S10, at zero tilting, the degree of darkness is the same around the circumference of the vesicles. The vesicles appear circular in that there is no change in the degree of darkness as one traverses the vesicle region between the walls. However, when the grid was tilted in either direction (þ42° or -42°), the darkness of the vesicle middle increases on the side that moves up, but only in large vesicles. By contrast, the shape or the darkness of the small vesicle wall was not changed upon tilting, which indicates that the small vesicles are, indeed, spherical. To understand better the differences in the profile of darkness of an identical kippah vesicle but in varying alignments to the surface, Figure 6 represents the predicted pattern of darkness, as a function of the radial distance from the outer left edge of the kippah vesicles, in six different scenarios. For example, Figures 6b,e represent two different alignments (i.e., orientations) where the kippah vesicle is lying on the surface either with the hemisphere up or open-side-down (Figure 6b) or with the hemisphere down or open-side-up (Figure 6e). In these two alignments, the TEM images of the kippah vesicles are expected to reflect a circular geometry, i.e., in a similar fashion to the classical spherical vesicles, but with thicker than usual walls. The curve under each TEM image represents the darkness of the vesicle image as a function of the radial distance from the outer left edge of the kippah vesicle (see Figure S11 for calculations). As can be easily seen from both curves under Figures 6b,e, the intensity of the darkness for the kippah vesicle image increases drastically when moving right from the outer left edge of the kippah vesicle, where it reaches its peak on the inner vesicle wall (i.e., d = σw). Afterward, the intensity of the darkness drops gradually, reaching its minimum at the center of the vesicle (d = Rv), where it reflects the electron stopping power of two wall thicknesses. Next, the intensity of the darkness increases once again to reach its second Langmuir 2010, 26(13), 10513–10523

peak on the inner side of the vesicle wall on the opposite side of the vesicle (d = 2Rv - σw). Finally, the intensity of the darkness decreases sharply once again toward the outer edge of the vesicle on the opposite side. Classical vesicles, i.e. not kippah, showed similar symmetrical curve as in the kippah one; however, the gap between the darkness of the maxima and the minima is deeper in the classical vesicle than in that of the kippah vesicle (Figure S13, Supporting Information). To simplify the calculations, the classical vesicle was assumed to have similar diameter (i.e., 500 nm), but with half the size of the wall thickness (i.e., 35 nm). The equations for the darkness curve of the classical vesicle are slightly different from those of the kippah vesicle and are summarized below (Figure S12 of the Supporting Information). Figures 6a,f show two different scenarios where the kippah vesicles are on opposite alignments relative to the surface (i.e., open-side-down and openside-up) after tilting in the both directions by -60° (Figure 6a) and þ60° (Figure 6f). As can seen from these images (Figures 6a, b), both scenarios are expected to yield similar intensification in the darkness on the left side of the vesicle. Figures 6c,d, on the other hand, describe the other two scenarios where the kippah vesicles showed, after tilting, the intensification in the darkness on the right side of the vesicle. In these scenarios, the kippah vesicle is either aligned open-side-down and tilted to the right side (Figure 6c) or aligned open-side-up and tilted to the left side (Figure 6d). On the basis of the above facts, we can conclude that it is impossible to determine the alignment of the kippah vesicle without tilting. However, it becomes possible to determine the kippah alignment simply after a single tilting in either of the two directions (i.e., clockwise or counterclockwise). For example, if a kippah vesicle is tilted to the right side (clockwise) and shows an increase in the darkness on the right side (i.e., the side that moves down), then it is evident that the vesicle is aligned on the surface open-side-down. However, if a similar kippah vesicle is tilted to the right side and shows an increase in the darkness on the opposite side (i.e., the side that moves up), then we can determine that the kippah vesicle is aligned open-side-up. Table 1 (Supporting Information) summarizes the different alignment scenarios as functions of tilting direction and side of darkness intensification. On the basis of the ideas presented above (Table 1 of the Supporting Information), it is obvious that the kippah vesicles in the TEM images of Figure 4b are lying on the surface open-side-down DOI: 10.1021/la1004837

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Figure 6. Cartoons representing the TEM projections of kippah vesicles in the open-side-down (or hemisphere-up) alignment (images a-c) and open-side-up (or hemisphere-down) alignment (images d-f) at three different tilt angles: -60°, 0°, and þ60°. The graph under each TEM projection represents the darkness (or thickness of the PS material traversed by the electron beam) as a function of the radial distance from the outer left edge of the kippah vesicles (d, nm). The graphs under images b and e were calculated theoretically as described under Figure S14 of the Supporting Information, whereas the graphs under images a, c, e, and f were determined using a cross-section model of the kippah vesicle having 500 nm diameter and 70 nm wall thickness. Please note in the schematic drawings that the electron beam is directed from the top to the bottom of the page, while in the electron micrographs it is directed from above to below the plane of the page.

or hemisphere-up. Clockwise tilting resulted in the intensification of darkness on the side that moves down (i.e., right side), while counterclockwise tilting resulted in the intensification of darkness on the side that moves up (i.e., left side). Figure 7 summarizes several experiments aiming to determine the alignment of the kippah vesicle on different TEM grids. Figure 7a illustrates a typical vesicle open-side-down (a cartoon illustration is attached under each TEM). Figure 7b, on the other hand, shows an opposite alignment where the vesicle is aligned open-side-up. Intensive investigations revealed that, in ca. 80% of the cases, 10520 DOI: 10.1021/la1004837

isolated kippah vesicles tend to have an open-side-down alignment (i.e., similar to Figure 7a); however, open-side-up alignment (i.e., similar to Figure 7b) was also seen. Figure 7c shows a situation where many kippah vesicles are lying together in a similar open-side-down alignment. The TEM grid, in this specific example, was pretreated with poly(L-lysine) to increase polymer adhesion due to the strong electrostatic interaction between the negatively charged PAA chains and the poly(L-lysine) segments. Pretreatment of EM grids with poly(L-lysine) is a well-known technique and is commonly used in electron microscopy to Langmuir 2010, 26(13), 10513–10523

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Figure 7. TEM images of PAA34-b-PS324 kippah vesicles before and after tilting in both directions. Two distinct kippah vesicles with openside-down and open-side-up alignment are shown in (a) and (b), respectively. (c) Many kippah vesicles lying on the surface with their openside-down. The surface of the grid in image c was cationized by pretreatment of the grid with poly(L-lysine). (d) Two vesicles of opposite alignment. (e) A kippah vesicle lying inside a bigger kippah vesicle (marked with an arrow). (f) TEM image of nested kippah vesicles after staining with an excess of cesium hydroxide (10 mol per 1 mol of PAA repeat units).

increase surface adhesion, especially for negatively charged particles.60 Surprisingly, a similar open-side-down alignment was obtained with negatively charged surface EM grids. To apply a negative charge to the EM grid, a 1 wt % solution of PAA100-bPS100 block copolymer in THF was added to carbon-coated grids followed by evaporation under ambient conditions. This forms a uniform PS-b-PAA film where the PAA can be easily reswollen upon wetting (i.e., sample deposition). The reason for either the open-side-up or the open-side-down is not clear; however, it is believed to be influenced by the surface charge of both the vesicles and the EM specimen grid. For more details on the effect of surface charge on the alignment of the vesicles, see Figures S14 and S15 and the subsequent explanations in the Supporting Information. Figure 7d shows a rare TEM image where two kippah vesicles are aligned opposite to each other. The slightly larger vesicle, i.e. the one above, was shown to have an open-side-down alignment, while the smaller vesicle (i.e., the one in below) showed an open-side-up alignment. Without tilting, the two vesicles in the TEM appear similar; however, it becomes clear after tilting in both directions that they are indeed in an opposite alignment. Figure 7e shows an additional rare case where a small kippah vesicle is seen lying inside a large kippah vesicle. The darkness in the open cavity of the small vesicle (highlighted with an arrow) is clearly more intense than the darkness of the cavity of the large

vesicle (i.e., outside the cavity of the small vesicle but within the boundaries of the cavity of the large vesicle). This is caused by the fact that the electron beam in the open cavity of the small vesicle is traversing both the PS wall of both the small and large vesicles combined. To confirm this rare condition of kippah-in-kippah vesicle, the sample was stained with a large excess of CsOH (10 mol/mol of PAA repeat units) prior to the deposition on the grid. Cesium is a heavy metal (atomic number 55) and is widely used in staining of acidic components in electron microscopy.61 For PAA-b-PS vesicles, the formation of cesium acrylate leads to a substantial increase in the darkness of PAA-rich areas. Therefore, the darkness, which is proportional to the electron density, will increase both in the cavity of the kippah vesicles where most of the cesium ions will be concentrated in the inside (i.e., open side) and in the outside (hemisphere). By contrast, the vesicle wall will appear lighter because the PS is not stained by cesium ions. Figure 7f shows a typical kippah-in-kippah vesicle pair after staining with cesium hydroxide. The wall thickness of the outer kippah vesicle was found to be 82 nm, while it was 76 nm for the inner kippah vesicle. The combined thickness of both walls was measured to be 166 nm, which leaves an ∼8 nm gap between the two vesicles (easily seen as a fine black line denoting the collapsed corona). As described above, the inner side of the vesicle appears much darker due to the high density of the cesium acrylate ions in

(60) Ung, T.; Giersig, M.; Dunstan, D.; Mulvaney, P. Spectroelectrochemistry of colloidal silver. Langmuir 1997, 13 (6), 1773-1782.

(61) Baalousha, M.; Motelica-Heino, M.; Galaup, S.; Le Coustumer, P. Supramolecular structure of humic acids by TEM with improved sample preparation and staining. Microsc. Res. Tech. 2005, 66 (6), 299-306.

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this section of the vesicle. Clockwise tilting of a similar kippahin-kippah structure resulted in fading of the light (i.e., vesicle wall) on the side that moves down (Figure S16, Supporting Information). The latter suggests that the vesicles in this specific structure are aligned open-side-up (see cartoon next to Figure S16b, Supporting Information). In another rare situation, a small truly spherical vesicle was seen lying in the inner cavity of a large kippah vesicle (Figure S17, Supporting Information). These two unique structures, i.e. kippah and normal vesicle lying inside a bigger kippah, were rarely observed but are being brought to the reader’s attention as an additional proof for the unique kippah structure. More TEM images of kippah vesicles stained with CsOH can be found in Figure S18 (Supporting Information). Unlike kippah vesicles after staining with CsOH, normal vesicles (with or without slight indentation) showed a lighter interior compared to the surroundings (marked with arrows in Figure S18 of the Supporting Information). CsOH is very polar and is not expected to diffuse appreciably to the interior of the vesicles due to the hydrophobic nature as well as to the high glass transition temperature of the PS polymer. Obviously, the formation of the kippah is strongly influenced by the method of preparation, as described earlier. When the vesicles were allowed to dry on the grid, either by drying under ambient conditions (method A) or by direct freeze-drying (method B), normal vesicles (i.e., not kippah) with the classical indentation pattern were the only structures to be observed. It should be noted that in very rare cases, which account for less than 1% of the total number of aggregates, isolated kippah vesicles were also obtained using these two methods. Kippah vesicles, on the other hand, are obtained only by freeze-drying and subsequent rehydration in water (method C) or by direct drying under vacuum (method D) where no freezing is involved. At this time, the reason why under some preparation conditions the vesicular structure is either maintained (methods A and B) or why it completely collapses into a kippah (methods C and D) is not fully understood; however, a very speculative explanation can be offered. Crucial to the explanation is the suggested relation between the presence of water and vesicle wall flexibility. In the presence of water, the PAA is hydrated and thus provides a soft layer in contact with the PS. Presumably, only a relatively thin layer of the PS is affected by thin soft layer, but the total PS thickness is only ca. 40 nm, so the effect on the flexibility of the whole wall can be appreciable. By contrast, in the absence of water, where the Tg of the PAA is similar to that of the PS, one would expect the PS wall to be much less flexible. Freeze-drying is very efficient in removing water because of the crystallization of the water, with the PAA chains remaining isolated. No PAA/ water layer is formed. By contrast, drying under ambient conditions (without freezing) results in the formation of a PAA/water layer, the Tg of which progressively increases as the water content decreases. Thus, it is relatively difficult to remove all the water, and the wall could retain some flexibility. In method A, where the vesicles are dried under ambient conditions of room temperature and atmospheric pressure, the vesicle wall is believed to possess some degree of flexibility, which can be ascribed to the presence of residual of water in the PAA. This coherent PAA/water layer is thus believed to maintain some flexibility of the PS vesicle wall. Given the fact that the process of water evaporation under ambient conditions is very slow, no appreciable pressure gradient is formed between the two sides of the vesicles during this process. This absence of a pressure gradient prevents the vesicle from collapsing even if the PS wall has some flexibility. Method D (drying under vacuum), on the other hand, results in the formation of kippah vesicles. As in 10522 DOI: 10.1021/la1004837

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method A, the vesicle wall is believed to maintain some flexibility because of the presence of some residual water. However, in contrast to method A, method D involves the use of high vacuum. While under vacuum, no pressure gradient is present since the vacuum is maintained for a considerable period, and the air from the inside has had a chance to diffuse out. Upon readmission of air, however, a drastic pressure gradient develops (atmospheric pressure outside, vacuum inside), and the vesicle collapses because the wall is still flexible. Unlike methods A and D, method B (i.e., freeze-drying) involves the sublimation of water molecules under high vacuum and relatively low temperatures (close to the freezing point of water). In freeze-drying, no coherent PAA/water layer is present on the PS surface and water removal is most efficient. The effective removal of water under the freeze-drying conditions assures that the Tg of PAA layer is high (ca. 100 °C), which keeps the flexibility of the PS wall very low. Readmission of air to the freeze-dried vesicles will yield a pressure gradient, as in method D; however, since the vesicles wall in this case has a very low flexibility, the pressure gradient does not collapse the vesicle. Thus, no kippah is formed. Method C involves the resuspension of the freeze-dried (“normal”) vesicles in water and yields kippah vesicles. It is likely that the air pressure in the interior and exterior of the vesicles equalizes quickly upon exposure to air at the end of the freezedrying process. Since the vesicles prepared by freeze-drying do not yield a kippah (as in method B), it is likely that complete vesicular collapse in this procedure occurs only upon the readmission of water. When water is added to the freeze-dried vesicles, the wall flexibility increases as a result of the rapid rehydration of the PAA in the internal and external corona, since water diffuses to the inside of the vesicle as well. As a result of PAA wetting in the inside, and given the fact that at this stage the inner part of the vesicle is still largely occupied by air, we suggest that the high interfacial energy of the PAA/water layer vs air in the interior62 drives the system to minimize the interface; coupled with now increased flexibility of the PS wall after rehydration of the PAA, the system has the opportunity to decrease the interfacial area by eliminating the hollow space inside, i.e., by collapsing to a kippah. It should be noted that no water/air interfacial tension is present in the exterior because the PAA corona chains are immersed in continuum of water, and no interface exists between in air and water as is present in the interior. We should stress again that the above explanation is speculative. However, and given the fact that the preparation of the kippah is completely reproducible, an explanation, even if speculative, might be of interest. In conclusion, we investigated the effect of the method of drying on the appearance of large PAA-b-PS-based vesicles in TEM. Vesicles dried either under ambient conditions or by freezedrying were seen to yield “normal” structures with the classical indentation pattern. However, when freeze-dried vesicles were subsequently rehydrated in water, a kippah-like structure was formed. The kippah structure, which can be defined as a vesicle that has completely collapsed to a hollow hemisphere, was confirmed by means of specimens tilting in TEM. The kippah vesicles were also obtained by direct drying of the vesicle solution in vacuum. To our knowledge, a complete and controlled collapse of a polymeric vesicle has not been reported in the literature. The cause of the kippah vesicle formation is not yet completely understood for all methods of preparation; however, it is postulated to be strongly influenced by one or more of the following parameters: the relative flexibility of the vesicle wall, pressure (62) Ishimuro, Y.; Ueberreiter, K. The Surface-Tension of Poly(Acrylic Acid) in Sodium-Chloride Solutions. Colloid Polym. Sci. 1980, 258 (9), 1052-1054.

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gradient, and interfacial tension. Since this study is the first of its kind, more studies are needed to understand better the process of vesicle collapse. We also show the usefulness of surface tilt in analyzing of the 3D structure of the kippah type, a method that can be also applied successfully to other deformed vesicular systems. It is also shown that merely a near-round appearance of vesicles in TEM does not necessarily mean that the aggregates are spherical. The first indication that these unique vesicles were “unusual” was their wall thickness, which was twice as large as in the “normal” vesicles. In addition, specimen tilting was found to allow the unambiguous determination of the orientation of the kippah on the surface (i.e., open-side-up or open-side-down).

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Acknowledgment. We thank the Natural Science and Engineering Research Council of Canada (NSERC) for the support of this work. The authors thank Mr. George Rizis for the help in the TEM experiments in the early stages of the study, Dr. Yiyong Mai for the help in the calculations, and Dr. Shaoyong Yu for the valuable discussions.

Supporting Information Available: Extra TEM images and descriptive calculations for the curve in Figure 5. This material is available free of charge via the Internet at http://pubs.acs.org.

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