Laterally Nanostructured Vesicles, Polygonal Bilayer Sheets, and

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Laterally Nanostructured Vesicles, Polygonal Bilayer Sheets, and Segmented Wormlike Micelles

2006 Vol. 6, No. 6 1245-1249

Zhibo Li,† Marc A. Hillmyer,*,† and Timothy P. Lodge*,†,‡ Department of Chemistry and Department of Chemical Engineering and Materials Science, UniVersity of Minnesota, Minneapolis, Minnesota 55455 Received April 18, 2006

ABSTRACT We report the formation of vesicles with a laterally nanostructured membrane by self-assembly of “miktoarm” star terpolymers, that is, macromolecules with three chemically distinct arms. The lateral structure consists of approximately hexagonally packed fluorocarbon channels with 4.1 nm radii, immersed in a continuous, two-dimensional hydrocarbon bilayer. We also show that the assembly of these vesicles proceeds via metastable polygonal, faceted bilayer sheets.

Vesicles represent synthetic models for both simple cells and coatings of virus particles and offer tremendous potential in the delivery of therapeutic agents and as nanoreactors.1-3 Although readily formed from surfactants and phospholipids (“liposomes”), vesicles may also be prepared from macromolecular analogues, that is, amphiphilic block copolymers (“polymersomes”).4-6 The array of design variables that polymers afford offer unprecedented flexibility in tuning vesicle properties, including membrane thickness, curvature, mechanical strength, permeability, chemical robustness, and sensitivity to external stimuli.4-9 An intriguing but as yet elusive possibility is to control structure within the twodimensional membrane manifold itself; such in-plane patterning could provide a useful route to the controlled distribution of recognition elements on the outer surface, tunable permeation characteristics of the vesicle to multiple chemical agents, and inclusion of incompatible agents within laterally segregated compartments of the membrane. The structures described here represent a new example of an important, emerging class of self-assembled aggregates: multicompartment micelles.10-16 In such structures, the solvophobic domains are further divided into distinct nanoscopic compartments. Multicompartment micelles represent a significant step toward hierarchical self-assembly with multiple functions and designed architectural features on several length scales. The miktoarm star architecture provides a versatile and powerful route to multicompartment micelles.16-18 In our prototype system, one arm comprises poly(ethylene * Corresponding authors. E-mail: [email protected]; lodge@ chem.umn.edu. † Department of Chemistry. ‡ Department of Chemical Engineering and Materials Science. 10.1021/nl0608700 CCC: $33.50 Published on Web 05/19/2006

© 2006 American Chemical Society

oxide) (“O”), which confers water dispersability, colloidal stability, and biocompatibility. The second and third arms are formed from a hydrocarbon, polyethylethylene (“E”), and a perfluorinated polyether, polyperfluoropropylene oxide (“F”), thereby installing in one molecule the necessary threefold “philicity”. The strong effective pairwise repulsion between E and F guarantees segregation into discrete nanodomains within the hydrophobic cores even at modest molecular weights. Most importantly, the star architecture effectively suppresses the formation of concentric domains, for example, core/shell/corona micelles that are the default structure adopted by linear ABC triblock terpolymers.14,19-21 The convergence of three blocks at a common point constrains the resulting O, E, and F nanodomains to meet along a common curve in space. These polymers can form approximately spherical micelles with distinct F and E core domains, and segmented wormlike micelles, in which flat nanoscopic disks of E and F are stacked alternately along the cylinder axis, while protected by a common corona of well-solvated O chains.16 Here we explore the aqueous self-assembly of three new terpolymers, designated µ-EOF(1.4-5-2.5), µ-EOF(1.4-3-2.5), and µ-EOF(1.4-2-2.5); the numbers refer to the molecular weights of the E, O, and F blocks, respectively, in kg mol-1. These polymers have lower overall molecular weights, volume ratios of E/F, and for the latter two polymers, significantly lower proportions of O than those described before.16 The µ-EOF miktoarm star terpolymers were prepared and characterized as described previously.17 Aqueous solutions (1 wt %) were prepared by direct dispersion of the terpolymers in deionized water and stirred in a sealed vial at room temperature or 50 °C. CryoTEM

Figure 1. CryoTEM images of segmented wormlike micelles obtained from a 1 wt % aqueous solution of µ-EOF(1.4-5-2.5). The scale bar is 100 nm.

samples were prepared at room temperature by suspending 100-200-nm-thick menisci of the 1 wt % micelle solutions on a lacey supported TEM grid, which was then plunged rapidly into liquid ethane near its melting point (-183 °C). The resulting vitreous water film preserves the self-assembled nanostructures, which were then imaged in a JEOL 1210 TEM (120 keV) at -178 °C. No external staining was applied, and low doses and short exposure times were applied carefully to minimize electron beam damage. The cryoTEM images in Figures 1, S1, and S2 reveal that µ-EOF(1.4-5-2.5), with the largest hydrophilic block, forms a mixture of segmented worms and some discrete spherical micelles (the F domains appear darkest, whereas the E blocks appear gray; the solvated O corona is not directly visible). These results are consistent with those reported previously for higher-molar-mass terpolymers with comparable O fractions.16 In contrast, Figures 2, S3, and S4 show examples of the remarkable structures formed by µ-EOF(1.4-3-2.5). This terpolymer tends to form faceted or polygonal sheets, often approximately hexagonal in overall shape. These shapes are unprecedented in amphiphile self-assembly, as far as we are aware. Furthermore, each polygon has at least one vertex with a protruding segmented worm. These sheets are themselves bilayers, with O blocks protruding both above and below the membrane; the distinctly visible graininess arises from the lateral segregation of E and F domains. The Fourier transforms shown as insets to Figure 2b indicate a roughly hexagonal packing of the E and F domains. The E block occupies about 30% more volume than the F block (Table S1); we therefore deduce that the minority F domains are approximately cylindrical, with the E forming a continuous matrix, as illustrated in Figure 3b. In some cases (Figures 2e, 2f, S3g, and S3h), these sheets can be seen to fold over into “semi-vesicles” or bowls.22,23 In these instances as well, the protruding segmented worms are clearly evident. Presumably, confinement into the meniscus during cryoTEM sample preparation has collapsed the hemispherical bowls (Figure 3c), which exhibit an especially evident thick rim. Images of the aggregates formed by µ-EOF(1.4-2-2.5), a terpolymer with an even smaller O fraction, indicate a large proportion of fully formed vesicles, or nearly completely closed vesicles with protruding tails (Figures 4 and S5). Each complete vesicle has a surface feature, indicated by arrows, 1246

Figure 2. Nanostructured, polygonal bilayer sheets and semivesicles formed from µ-EOF(1.4-3-2.5). The Fourier transformation insets of image b were performed from a regime of 208 × 208 nm2 on the indicated hexagonally shaped bilayer sheets. The scale bar is 100 nm. Nano Lett., Vol. 6, No. 6, 2006

Figure 3. Schematic illustrations of (a) segmented wormlike micelles, (b) nanostructured polygonal bilayers, (c) hemispherical bowls with remnant tails, and (d) a nanostructured vesicle. The red, green, and blue colors represent E, F, and O blocks, respectively.

that we propose is a remnant of the final stages of the growth process. These structures represent the first bilayer vesicles of which we are aware in which the membrane itself has a regular lateral structure on the nanometer scale. Recently, an ABCA linear tetrablock copolymer has also been shown to form some kind of structured monolayer vesicle, by segregation of the hydrophobic B and C blocks within the membrane, but without the lateral regularity or the bilayer symmetry of this system.24 We propose that all of the pertinent aspects of these results can be interpreted reasonably within one framework. Because of the relatively long hydrophilic block, µ-EOF(1.4-5-2.5) forms stable segmented wormlike micelles (Figures 1 and 3a). Conversely, the existence of vesicles for µ-EOF(1.4-2-2.5) is consistent with the decreased interfacial curvature brought about by reducing the hydrophilic block length significantly (Figure 4). For µ-EOF(1.4-3-2.5), flat sheets and partial vesicles represent intermediate, possibly metastable structures that will ultimately grow into vesicles (Figure 2). We further propose that the segmented worms emanating from the polygons are in the process of feeding material into the lateral edges of the sheets. Eventually, the sheets grow large enough to fold over and form vesicles, as depicted in Figure 3d. There are several reasons to infer that the sheets shown in Figure 2 are formed by a growth process, rather than, for example, by melting of larger sheets or vesicles. First, it is kinetically much easier to assemble a segmented worm than a large sheet upon initial terpolymer dissolution. We have shown previously how segmented worms can grow from “polymerization” of elementary “hamburger” units comprising a central F disk, E layers on top and bottom, and O chains emanating from the circular junctions;16 a subsequent growth of sheets from worms can readily be envisioned. Second, the straight edges suggest a rather regular process, in contrast to “melting”; if the sheets were unstable, then they would fall apart randomly. Third, as a general rule the more regular Nano Lett., Vol. 6, No. 6, 2006

Figure 4. Nanostructured vesicles formed from µ-EOF(1.4-2-2.5) terpolymer. The scale bar is 100 nm. The arrows indicate the vestiges of the final stage of the growth process.

the sheet, the fewer the number of protruding worms. For example, the hexagonal sheets (e.g., in Figures 2a and b, and S3a-e) all have a single worm, suggestive of a slow, orderly growth process. In contrast, sheets in Figures 2d and S3f are more irregular and display multiple worms, consistent with simultaneous growth at multiple sites. Finally, the edges of the regular hexagons have regularly alternating E (gray) and F (dark) compartments. This is consistent with the proposed chain-packing motif; the bilayer edge has segmented half cylindrical geometry, the other half has been merged into the body of the bilayer as illustrated in Figure 3b. Furthermore, two hexagonal-shaped bilayers in Figure 2c are coalescing along one common edge, as noted with arrows, indicating that merging of two sheets (and therefore one sheet and one worm) is possible. From the transforms in Figure 2b we can obtain a characteristic domain spacing of 6.1 nm. Assuming a hexagonal lattice, and the known volume fraction of F relative to E, we can estimate the “aggregation number” of an F cylinder that transects the bilayer to be about 250. This compares very favorably with the aggregation number (ca. 240) per repeat unit of the segmented worm protruding from the corresponding bilayer (Figure 2b). This observation may also shed light on the detailed growth mechanism, discussed below. The sequential “feeding” of the segmented worms into the edges of a bilayer sheet also provides an explanation for the prevalence of flat edges in Figure 2. Generally, disklike bilayers are intermediate structures in the micelle-to-vesicle transition.25-28 In a typical bilayer membrane there is a huge energetic penalty for exposing the hydrophobic domains to the solvent. Consequently, it is unusual to observe such a sheet; any such sheet ought to be circular to minimize the exposed edge.29-34 (A counterexample is a “bicelle”, in which 1247

Figure 5. Schematic illustration of three possible mechanisms by which a segmented worm could feed material into a growing polygonal bilayer sheet: (a) direct flow of chains into the edges of the polygon; (b) fusion of a worm section along an existing facet; (c) deposition of new facet material by extrusion from a worm.

the edge of a lipid bilayer disk is stabilized by an added surfactant.35 It is worth emphasizing that in the current work there is only a single solute present, albeit one with a remarkable threefold philicity.) In this case, however, the edge is formed by a segmented worm, where the F and E blocks are almost fully stretched because of their remarkably large interfacial tension with water.36 Thus, each edge, stabilized by the protruding O blocks, is only slightly more energetically expensive than the body of the sheet. It is worth considering in more detail the process by which the worms transform into sheets. Three distinct possibilities present themselves, as illustrated in Figure 5 for a worm connected to a vertex of a hexagon. In Figure 5a, the individual terpolymers simply “flow” into the edges of the polygon. This is the presumed mode by which surfactants and lipids evolve from worms to sheets.34 However, because of the strong thermodynamic constraint to mixing E and F, this process seems rather improbable here because it would require temporary “dissolution” of the separate E and F nanodomains (which, as noted above, contain on the order of 250 molecules). A second possibility is the merging or wrapping of entire worm sections along a facet of the polygon, as sketched in Figure 5b. This process would preserve the local segregation of E and F, while also providing a clear mechanism for the preservation of regular facets. A third candidate is a deposition process in which the worm extrudes new sheet edge material (Figure 5c). In this scenario, the worm would move progressively along a facet, becoming shorter as more material was incorporated into the sheet. The growth of a new layer of nanodomains along one facet is probably nucleated from the existing vertex, entailing a slow secondary nucleation process appealingly reminiscent of the Hoffmann-Lauritzen mechanism37 for polymer crystal growth. This hypothesis would account for the fact that the images generally show worms 1248

protruding from vertexes, rather than in the act of fusing along a facet. Detailed computer simulation might offer one route to distinguishing among these mechanisms, but in the interim we are inclined to suspect the process illustrated in Figure 5c is the most prevalent. In the case of µ-EOF(1.4-2-2.5), we observe either whole vesicles or long worms, with no evidence of polygonal sheets (Figure 4). The shorter O chain in this terpolymer favors the flat surface relative to the cylinder, and thus the thermodynamic driving force to form vesicles is greater than that for µ-EOF(1.4-3-2.5). However, we suspect that there may also be an important kinetic factor involved. If a segmented worm fuses with an existing edge, as in Figures 5b or c, there will be corona-corona repulsion among the O blocks, before the O blocks on the existing edge are pushed to either the top or bottom surface of the bilayer. This provides a barrier to the growth of sheets, which should be substantially higher for µ-EOF(1.4-3-2.5) than for µ-EOF(1.4-2-2.5). Consequently, the growth process is substantially slower for µ-EOF(1.4-3-2.5), giving rise to the multitude of intermediate structures. In other words, the vesicles formed by µ-EOF(1.4-2-2.5) are nucleationlimited, whereas those formed by µ-EOF(1.4-3-2.5) are growth-limited. Therefore there are either “un-nucleated” segmented worms or complete vesicles for µ-EOF(1.4-2-2.5), but no intermediate forms (Figure 4), whereas for µ-EOF(1.4-3-2.5) sheets of all sizes are observed because growth is slower than nucleation. To explore this hypothesis further, we annealed solutions of all three copolymers at 50 °C for a number of days. The resulting images for µ-EOF(1.4-5-2.5) and µ-EOF(1.4-2-2.5) showed no essential change (Figures S2 and S5). However, those for µ-EOF(1.4-3-2.5) showed a marked increase in the number of completed vesicles, fully consistent with the overcoming of a barrier to growth (Figure S4). Finally, we consider the vesicle surface features and pendant worms apparent in Figures 4 and S5. Once the vesicle is fully formed by the process of consumption of worms, any remaining protrusion has no obvious low-energy conformation to adopt. Plausibly, such worms eventually cleave off, leaving behind excess material. In other cases the worm is still attached, as in Figure 4c and d. Because of the relative metastability of the edges of the bilayer sheets, it is even conceivable that the complete vesicles retain a hole in the membrane; this possibility will be explored in the future. In this letter we have disclosed at least two novel features of self-assembly, namely, bilayer membranes with a regular nanoscale lateral structure (in either vesicles or sheets) and regular polygonal bilayer sheets. The fact that wormlike micelles can evolve into vesicles by way of sheets is already documented in surfactants and surfactant mixtures, but in no instance has nanoscopic periodicity within the bilayer been reported nor have faceted bilayer polygons any precedent, as far as we are aware. Interestingly, pioneering studies of two- and three-tailed low molar mass surfactants with mixed fluorocarbon and hydrocarbon tails did report sheet and vesicle formation, but no nanoscale intramembrane segregaNano Lett., Vol. 6, No. 6, 2006

tion was described.38 Finally, the nanostructured vesicles introduced in this letter could have potential as drug delivery vehicles, nanoreactors, and protocells with tunable membrane permeabilities. Acknowledgment. This work was supported primarily by the MRSEC Program of the National Science Foundation under Award No. DMR-0212302. Supporting Information Available: Complementary cryoTEM images with larger fields of view for micelles (Figures S1-S5) and molecular parameters of µ-EOF terpolymers (Table S1 and Figure S6). This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Chen, I. A.; Salehi-Ashtiani, K.; Szostak, J. W. J. Am. Chem. Soc. 2005, 127, 13213. (2) Guo, X.; Szoka, F. C. Acc. Chem. Res. 2003, 36, 335. (3) Vriezema, D. M.; Comellas Aragones, M.; Elemans, J. A. A. W.; Cornelissen, J. J. L. M.; Rowan, A. E.; Nolte, R. J. M. Chem. ReV. 2005, 105, 1445. (4) Discher, B. M.; Won, Y.-Y.; Ege, D. S.; Lee, J. C.-M.; Bates, F. S.; Discher, D. E.; Hammer, D. A. Science 1999, 284, 1143. (5) Discher, D. E.; Eisenberg, A. Science 2002, 297, 967. (6) Fo¨rster, S. Polymer Vesicles. In Encyclopedia of Polymer Science and Technology; John Wiley & Sons: New York, 2005. (7) Bermudez, H.; Brannan, A. K.; Hammer, D. A.; Bates, F. S.; Discher, D. E. Macromolecules 2002, 35, 8203. (8) Bellomo, E. G.; Wyrsta, M. D.; Pakstis, L.; Pochan, D. J.; Deming, T. J. Nat. Mater. 2004, 3, 244. (9) Napoli, A.; Valentini, M.; Tirelli, N.; Mueller, M.; Hubbell, J. A. Nat. Mater. 2004, 3, 183. (10) Kujawa, P.; Goh, C. C. E.; Calvet, D.; Winnik, F. M. Macromolecules 2001, 34, 6387. (11) Sta¨hler, K.; Selb, J.; Candau, F. Langmuir 1999, 15, 7565. (12) Kotzev, A.; Laschewsky, A.; Adriaensens, P.; Gelan, J. Macromolecules 2002, 35, 1091. (13) Lutz, J.-F.; Laschewsky, A. Macromol. Chem. Phys. 2005, 206, 813. (14) Kubowicz, S.; Baussard, J.-F.; Lutz, J.-F.; Thu¨nemann, A. F.; von Berlepsch, H.; Laschewsky, A. Angew. Chem., Int. Ed. 2005, 44, 5262.

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