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Block Copolymer Assemblies with Cross-Link Stabilization: From Single-Component Monolayers to Bilayer Blends with PEO-PLA† Fariyal Ahmed, Alina Hategan, Dennis E. Discher,* and Bohdana M. Discher Chemical and Biomolecular Engineering Department, Department of Bioengineering, and Department of Biochemistry and Biophysics, University of Pennsylvania, Philadelphia, Pennsylvania 19104 Received February 1, 2003. In Final Form: April 9, 2003 Amphiphilic diblock copolymers containing poly(ethylene oxide) (PEO) and saturated polybutadiene (PBD) hydrophobic chains have been cross-linked in both monolayer and bilayer assemblies. For monolayers, the Langmuir isotherm proves consistent with prior measures of density and compressibility for bilayer vesicles, and the monolayers can be macroscopically transferred as Langmuir-Blodgett films. Since the films are inverted (PBD oriented away from the slide) at the air-water interface, they spontaneously reorganize when immersed in water into wormlike micelles. However, this transition is quenched when the film is stabilized by free radical polymerization. Atomic force microscopy shows a stabilized but pitted film, which allows height measurements that prove consistent with a monolayer. With bilayer vesicles, we investigate several additional aspects of cross-linking. Spatial control of membrane cross-linking on the micrometer-scale is demonstrated by dynamic inflation-deflation of a membrane fixed to the end of a micropipet. Complete polymerization of the bilayer renders vesicles resilient to chloroform solubilization, whereas preblending with a non-cross-linkable diblock copolymer of PEO-polylactic acid, undermines vesicle stability in chloroform-water solutions. The results prove miscibility and stable integration of two disparate block copolymers in a membrane, as well as a first-order scheme for controlled release of encapsulants.
Introduction The idea of stabilizing amphiphile self-assemblies by polymerization was introduced at least 30 years ago for monolayers and some 10 years later for bilayer vesicles.1,2 The envisioned potential of these novel polymerized materials has since stimulated years of investigation and, of course, generated a substantial body of literature on both lamellar3-8 and nonlamellar phases.9,10 Today, stabilized assemblies generated from a broad spectrum of lipid and synthetic polymers or peptides are being considered for applications ranging from drug delivery carriers,6,11-15 nanotubes,16 biosensors,17 artificial virus,18,19 and cytoskeletal mimics.20 * To whom correspondence may be addressed. E-mail: discher@ seas.upenn.edu. † Part of the Langmuir special issue dedicated to David O’Brien. (1) Dorn, K.; Klingbiel, R. T.; Specht, D. P.; Tyminski, P. N.; Ringsdorf, H.; O’Brien, D. F. J. Am. Chem. Soc. 1984, 106, 1627-1633. (2) Regen, S. L.; Czech, B.; Singh, A. J. Am. Chem. Soc. 1980, 102, 6638-6640. (3) Meier, H.; Sprenger, I.; Barmann, M.; Sackmann, E. Macromolecules 1994, 27, 7581-7588. (4) Liu, S. C.; O’Brien, D. F. Macromolecules 1999, 32, 5519-5524. (5) Jung, M.; den Ouden, I.; Montoya-Goni, A.; Hubert, D. H. W.; Frederik, P. M.; van Herk, A. M.; German, A. L. Langmuir 2000, 16, 4185-4195. (6) Nardin, C.; Winterhalter, M.; Meier, W. Langmuir 2000, 16, 77087712. (7) Maskos, M.; Harris, J. R. Macromol. Rapid Commun. 2001, 22, 271-273. (8) Liu, S. C.; O’Brien, D. F. J. Am. Chem. Soc. 2002, 124, 60376042. (9) Jeong, S. W.; O’Brien, D. F.; Oradd, G.; Lindblom, G. Langmuir 2002, 18, 1073-1076. (10) Yang, D.; O’Brien, D. F.; Marder, S. R. J. Am. Chem. Soc. 2002, 124, 13388-13389. (11) Hotz, J.; Meier, W. Langmuir 1998, 14, 1031-1036. (12) Mueller, A.; Bondurant, B.; O’Brien, D. F. Macromolecules 2000, 33, 4799-4804. (13) Graff, A.; Winterhalter, M.; Meier, W. Langmuir 2001, 17, 919923.
Here we focus on the preparation and stabilization of lipid-like block copolymer assemblies, beginning with monolayer films. A considerable body of work already exists for lipid monolayers on solid substrates. Most recently, O’Brien and co-workers have investigated the influence of monomer concentration and the requirements on reactive moieties for stability of synthetic lipid assemblies on solid supports.4,17,21 Block copolymer monolayers have also been recently studied,22-24 and the mere use of polymers suggests a substantial advantage over cross-linkable lipids in that the number of cross-linkable moieties in a polymer can be extremely large. This broadens considerably the range of achievable membrane properties as well as, perhaps, applications. Poly(ethylene oxide)-polybutadiene (PEO-PBD) is the primary amphiphilic diblock copolymer studied here as it is known, if suitably designed, to provide control over spherical, rodlike, and vesicular architectures when added to water.7,25-27 This diblock copolymer is composed of a (14) Yang, W. Y.; Lee, Y. S. Langmuir 2002, 18, 6071-6074. (15) Kukula, H.; Schlaad, H.; Antonietti, M.; Forster, S. J. Am. Chem. Soc. 2002, 124, 1658-1663. (16) Karlsson, M.; Sott, K.; Cans, A. S.; Karlsson, A.; Karlsson, R.; Orwar, O. Langmuir 2001, 17, 6754-6758. (17) Ross, E. E.; Bondurant, B.; Spratt, T.; Conboy, J. C.; O’Brien, D. F.; Saavedra, S. S. Langmuir 2001, 17, 2305-2307. (18) Liu, J. Q.; Zhang, Q.; Remsen, E. E.; Wooley, K. L. Biomacromolecules 2001, 2, 362-368. (19) Gosselin, M. A.; Guo, W. J.; Lee, R. J. Bioconjugate Chem. 2002, 13, 1044-1053. (20) Stauch, O.; Uhlmann, T.; Frohlich, M.; Thomann, R.; El-Badry, M.; Kim, Y. K.; Schubert, R. Biomacromolecules 2002, 3, 324-332. (21) Sisson, T. M.; Lamparski, H. G.; Kolchens, S.; Elyadi, A.; O’Brien, D. F. Macromolecules 1996, 29, 8321-8329. (22) Goedel, W. A.; Heger, R. Langmuir 1998, 14, 3470-3474. (23) Xu, H.; Goedel, W. A. Langmuir 2002, 18, 2363-2367. (24) Bearinger, J. P.; Terrettaz, S.; Michel, R.; Tirelli, N.; Vogel, H.; Textor, M.; Hubbell, J. A. Nat. Mater. 2003, 2, 259-264. (25) Hillmyer, M. A.; Bates, F. S. Macromolecules 1996, 29, 69947002.
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hydrophilic poly(ethylene oxide) (PEO) chain and a crosslinkable, hydrophobic polybutadiene (PBD) chain.28 Here, we describe the compression of PEO-PBD monolayers and apply atomic force microscopy (AFM) imaging to glasssupported Blodgett films in order to elucidate molecular reorganization upon exposure to aqueous solution. Covalent cross-linking of PBD chains clearly stabilizes molecular-scale organization. Like the copolymer reorganization seen here, Yoo and co-workers29 have also predicted multiple modes of molecular rearrangement of rod-coil copolymers in response to different environments. To broaden the realm of molecular architectures, we have also stabilized novel blends of PEO-PBD with noncross-linkable diblocks of poly(ethylene oxide)-poly(lactic acid) (PEO-PLA) (OL1) in bilayer architectures. Our studies are motivated in part by the potential for controllably cross-linked bilayers as drug-delivery vehicles. Natural source liposome systems have long been developed and now commercialized for such purposes,30-32 but they are sometimes found lacking in stability, either in the body, on the shelf, or when large targeting proteins are attached. Poly(ethylene glycol) (PEG)-lipid monolayers have also been studied and again prove unstable with a low-pressure transition corresponding to desorption of PEG chains and at least one high-pressure transition which is said to be poorly understood.33 Previous polymerization studies of bilayer vesicles of the block copolymer34 used in our monolayer studies have already shown that giant, cross-linked copolymer vesicles are novel in their resistance to solvent and osmotic stresses as well as cyclic dehydration. Figure 1B is a sketch of a bilayer assembly with cross-linking of reactive (alkyl) side chains of PBD. The dynamics and mechanical stability of “microballoons” formed by spatially controlled cross-linking are briefly probed here with micropipet aspiration. Anticipating application of copolymer vesicles as degradable delivery agents, the hydrolyzable and non-crosslinkable diblock, PEO-PLA, has also been integrated into bilayer vesicles of PEO-PBD copolymer (Figure 1C). Previous publications35,36 have shown that PEO-PLA diblock copolymer has a propensity for forming micelles, and it has been postulated that release of entrapped solutes or drugs is facilitated by hydrolysis of ester linkages in the hydrophobic PLA block. Incorporation of PEO-PLA to our PEO-PBD bilayer vesicles is shown to effectively sensitize the solid polymer shell to solvent and facilitate release of small hydrophilic encapsulants. We thus explore both monolayer and free bilayer assemblies of diblock copolymers with a particular focus on cross-linking and controlled destabilization. The structure and dynamics of copolymer assembly and orientation in response to microenvironmental stimuli suggest po(26) Won, Y. Y.; Davis, H. T.; Bates, F. S. Science 1999, 283, 960963. (27) 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, 1143-1146. (28) Bermudez, H.; Brannan, A. K.; Hammer, D. A.; Bates, F. S.; Discher, D. E. Macromolecules 2002, 35, 8203-8208. (29) Tsukruk, V. V.; Genson, K.; Peleshanko, S.; Markutsya, S.; Lee, M.; Yoo, Y.-S. Langmuir 2003, 19, 495-499. (30) Bangham, A. D. Prog. Biophy. Mol. Biol. 1968, 18, 29. (31) Bangham, A. D. H., M.; Hill, N. G. Methods Membr. Biol. 1974, 11, 38. (32) Papahadjopoulos, D. Ann. N. Y. Acad. Sci. 1978, 308, 1. (33) Naumann, C. A.; Brooks, C. F.; Fuller, G. G.; Knoll, W.; Frank, C. W. Langmuir 1999, 15, 7752-7761. (34) Discher, B. M.; Bermudez, H.; Hammer, D. A.; Discher, D. E.; Won, Y. Y.; Bates, F. S. J. Phys. Chem. B 2002, 106, 2848-2854. (35) Gref, R.; Minamitake, Y.; Peracchia, M. T.; Trubetskoy, V.; Torchilin, V.; Langer, R. Science 1994, 263, 1600-1603. (36) Riley, T.; Govender, T.; Stolnik, S.; Xiong, C. D.; Garnett, M. C.; Illum, L.; Davis, S. S. Colloids Surf., B 1999, 16, 147-159.
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Figure 1. Schematics of diblock copolymer lamellar assemblies, both cross-linked and blended. (A) A monolayer assembly of amphiphilic block copolymer, PEO-PBD, at the air-water interface, which is subsequently transferred onto a glass slide and cross-linked by free radical polymerization of PBD in aqueous solution. (B) A bilayer membrane of PEO-PBD selfassembled in solution and again stabilized by free radical polymerization of the PBD chains. (C) Bilayer membrane of a percolating blend of the diblock copolymer PEO-PLA with PEO-PBD: PEO-PLA does not participate in free radical polymerization and thus dilutes the cross-linking density. Nonetheless, PEO-PLA chains remain stably integrated in the close-packed bilayer network of polymer chains. Note that the illustrations are not drawn to scale.
tential applications in biosensors, bioresponsive films, and drug delivery. Materials and Methods Copolymers and Chemicals. The diblock copolymers OB2, OB18, and OL1 were synthesized by standard anionic ring opening polymerization as described elsewhere.25,37,38 Stock solutions of OB2, OB18, and OL1 at 10 mg/mL were prepared in chloroform and stored at 4 °C. Chloroform, sucrose, dextrose, fluorescent tritc-dextran, and the cross-linking reagents were from Sigma. Table 1. Summarizes the Physical Properties of the Amphiphilic Diblock Copolymers designated name
copolymer formula
OB225 OB1825 OL138
EO26-BD46 EO80-BD125 EO43-LA44
Mn approximately fEO (kg/moL) d (nm) P.D.I. (v/v) 3.6 10.5 6.0
9.6 14.8 10.4
1.10 1.10 1.10
0.28 0.29 0.33
Preparation of Cross-Linked Vesicles. Preparation of OB2 and OL1/OB18 vesicles was accomplished by film rehydration (see Table 1). Briefly, block copolymers were solubilized in chloroform, and in the case of mixed compositions, the polymers were blended at the desired mole ratio. A uniform polymer film was prepared by coating the walls of a clean glass vial, purging under nitrogen gas and vacuum for 5-7 h. The film was then hydrated with an aqueous solution containing a buffer (PBS) or an encapsulant, in this instance 300 mOsm sucrose (340 g/mol) and 0.1 mg/mL fluorescent tritc-dextran (17 200 g/mol). Selfassembly of vesicles by spontaneous budding was promoted by overnight incubation in a 60 °C oven. The hydrophobic PBD core of the assembled polymersomes was cross-linked by free radical polymerization with K2S2O8 as the initiator, followed with Na2S2O5 and FeSO4 as the redox couple.26 To avoid imposing an external osmotic stress on the vesicles, all solutions were prepared at the same osmolarity as the aqueous solution used for (37) Hillmyer, M. A.; Bates, F. S.; Almdal, K.; Mortensen, K.; Ryan, A. J.; Fairclough, J. P. A. Science 1996, 271, 976-978. (38) Ahmed, F.; Omaswa, I.; Brannan, A.; Bates, F. S.; Discher, D. E. In preparation.
Copolymer Assemblies with Cross-Link Stabilization rehydration and K2S2O8, Na2S2O5, and FeSO4 were added at volume ratios of 50:25:1, respectively. Visual aids such as a color change in the reaction mixture (yellowish brown to clear) and formation of dents and wrinkles on the otherwise smooth vesicle surface confirmed the completion of a successful cross-linking reaction. Chloroform-Vesicle Dissolution Dynamics. The crosslinked vesicles were subjected to the organic chloroform phase in a sealed glass slide chamber. These chambers were prepared by sealing cover slips (18 × 18) onto a clean glass slide (3 × 1 × 1 mm; FisherBrand) via a para-film interface. Vesicles were diluted (20-fold) in an isotonic dextrose solution and loaded at one end of the chamber. Vesicle integrity, prior to addition of the organic solvent, was determined with a Nikon TE-300 inverted microscope in both bright field and phase contrast. Chloroform was then injected at the other end of the chamber to a final concentration of 20-26% v/v, and the chamber was sealed to minimize evaporation of both the aqueous and organic solvent. The effect of chloroform on the vesicles was monitored through a 40× objective lens and recorded through a CCD video camera mounted on the front port of the microscope. Glass Slide Preparation. Glass cover slips were cleaned in a KOH/ethanol bath (24 g of KOH, 25 g of H2O, 135 g of ethanol) for 20 min, rinsed thoroughly with distilled water, further incubated in distilled water for 2 h, and subsequently dried with hot air. Langmuir-Blodgett Monolayer Film Preparation. The monolayers were prepared using a Langmuir-Blodgett film balance (Lauda Filmbalance FW2, Sybron/Brinkmann, Westbury, NY) following a general procedure described elsewhere.39 Briefly, OB2 polymer was dissolved in chloroform at concentrations of 4 mg/mL and spread at the air-water interface. A 15 min time interval was introduced between the spreading and compression cycle, to ensure complete solvent evaporation. The monolayers were compressed at 3 Å2 molecule-1 min-1 to the desired surface pressure which was then held constant during the film transfer. The glass slide was first passed vertically into the subphase at a relatively fast speed (800 mm/min), to minimize detectable deposition of the polymer on the surface. The submerged slide was then withdrawn slowly through the surface at 3 mm/min and allowed to air-dry. The monolayer was finally cross-linked by submerging the polymer-slide composition into a freshly prepared solution of cross-linking reagents (see above for details) for approximately 10 min. AFM Imaging of the Monolayer Assembly. AFM imaging was performed on a BioScope atomic force microscope (Digital Instruments, Santa Barbara, CA), equipped with a 120 µm xy and 6 µm z scanner. Sharpened silicon nitride cantilevers (Microlevers, Park Cantilevers) with a spring constant of 100 pN/nm were used. Samples of OB2 polymer deposited on glass slides were imaged under liquid (water) in tapping mode with scan rates of 1-8 Hz. We used the smallest force necessary to obtain good image contrast, to induce minimal compression of the polymer film during imaging. The scanned and rescanned images proved stable and reproducible, meaning the polymer samples were not remodeled by the AFM tip. AFM imaging was carried out consistently in a liquid (water) phase, at ambient room temperature. All images were first-order flattened, and the height profiles were low pass filtered to eliminate noise. Images depicted in Figure 3 were amplitude tapping mode images and the subsections were the corresponding height measurements.
Results and Discussion Cross-Linking of Langmuir-Blodgett Monolayers. Compression of a film of OB2 diblock copolymer spread at the air-water interface gives the π-A isotherm (24 °C) of Figure 2A. The data indicate that the copolymer monolayer is relatively compressible until the area per molecule reaches about 200 Å2/molecule at a surface pressure near 20 mN/m. The surface pressure then increases steeply up to a collapse pressure of 52 mN/m. (39) Popovic, Z. D.; Kovacs, G. J.; Vincett, P. S.; Alegria, G.; Dutton, P. L. Chem. Phys. 1986, 110, 227-37.
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Figure 2. π-A isotherm and its derivative for the PEO-PBD diblock, OB2, at 24 °C. (A) The isotherm is labeled with solid arrows, which correspond to points at which the film was transferred to glass support (transfer 1 ) 11 mN/m and transfer 2 ) 25 mN/m). The dashed arrow indicates the surface pressure of a bilayer membrane vesicle. (Β) The relative expansivity κa ) - dπ/dR versus the relative change in area, R, where the reference area per copolymer, Ao, is taken to be that of copolymers in the bilayer vesicles. As indicated in the text, a value of κa ≈ 60 mN/m is very close to micropipet-based measurements on vesicles composed of the same copolymer.
Neither the isotherm nor its first derivative reveals any plateau or kink that would suggest a phase transition as seen with PEG-lipid monolayers.33 This absence of any transition is in good agreement with previous observations of smooth and continuous area expansion with temperature for vesicles made with a very similar copolymer.40 The interfacial tension at either side of the OB2 vesicle bilayers is estimated to be close to γ ≈ 30 mN/m,40 which corresponds to a surface pressure of about 40 mN/m, and a molecular area per copolymer close to Ao ≈ 1 nm2 (depicted by a dashed arrow). This value is very close to that estimated from Ao ) Mn/(FPBDd/2), where d (≈10 nm) is the vesicle bilayer core thickness, Mn the molecular weight per polymer, and FPBD ) 1 g/cm3. Defining R ) (A/Ao) - 1 as a relative area change and differentiating π versus R gives the relative expansivity κa ) -dπ/dR (Figure 2B). Near Ao, κa ≈ 60 mN/m ≈ 2γ as expected for one monolayer or leaflet of a self-assembled bilayer.41 The isotherm data therefore confirm that a copolymer monolayer compressed to π ≈ 40 mN/m is essentially half of a bilayer. Compressed films were transferred onto glass supports at the pressures indicated by the solid arrows in Figure 2: transfer 1, 350 Å2/molecule at 11 mN/m; transfer 2, 150 Å2/molecule at 25 mN/m. The surface pressure was (40) Lee, J. C. M.; Bermudez, H.; Discher, B. M.; Sheehan, M. A.; Won, Y. Y.; Bates, F. S.; Discher, D. E. Biotechnol. Bioeng. 2001, 73, 135-145. (41) Israelachvilli, J. N. Intermolecular and surface forces, 2nd ed.; Academic Press: London, San Diego, 1991.
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Figure 3. AFM tapping mode amplitude images of the PEOPBD diblock, OB2, transferred to a glass support and imaged in water (at a scan rate of 1 Hz). The films were transferred at surface pressures of either 11 mN/m (A) or 25 mN/m (B and C), and the images were collected either without cross-linking reagents (A and B) or after the film has been cross-linked during immersion (C). Scale bars are 1 µm. The subregion image in (B) is a rescan (at an 8 Hz scan rate) and confirms the stability of the topography at high resolution. Each image is accompanied, at right, by a height analysis plot of a line section indicated by the white dashed line.
kept constant through sustained compression during the transfer; the film area decrease proved to be twice the area of the submerged glass slide indicating that a macroscopic Langmuir-Blodgett (LB) film was very efficiently transferred onto both sides of a glass cover slip. The LB films were subsequently imaged under water by AFM42,43 (Figure 3). Before immersion in water, the hydrophilic PEO chains of the diblocks face glass and the PBD chains are exposed to air (Figure 1A), leading to an unfavorable PBD-water interface when submerged. Following submersion, the two films that were not cross-linked reorganized into elongated stripes (Figure 3A,B) with dimensions and area coverage suggestive of wormlike micelle formation. The lower density film (350 Å2/copolymer) rearranged into a low(42) Magonov, S. N.; Cleaveland, J.; Elings, V.; Denley, D.; Whangbo, M.-H. Surf. Sci. 1997, 389, 201-211. (43) Radmacher, M.; Tillmann, R. W.; Fritz, M.; Gaub, H. E. Science 1992, 257, 1900-1905.
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density micellar network (Figure 3A); the higher density film (150 Å2/OB2) showed highly aligned worms suggestive of a surface nematic order that has been reported previously in bulk with closely related copolymers (fEO ) 45-50%), which have a clear preference to form worm micelles26 (Figure 3B). Although the reason for surface reorganization into worm micelles as opposed to membranous sheets could be kinetic and is unclear, the average height of these newly assembled copolymer structures under AFM imaging is about 15 nm, depending on the choice of baseline (black insets in parts A and B of Figure 3). This 15 nm diameter for the surface worm micelles exceeds the hydrophobic core dimension, d (≈10 nm), for OB2 membranes assessed from cryo-TEM (transmission electron microscopy) measurements.28 However, if we assume that the packing densities of the PEO and PBD in the wormlike micelles are the same as the PBD in the membrane, then the diameters of the wormlike micelles estimated from adding a hydrated PEO volume fraction fEO28 (28%) is ∼14 nm. The cross-linkable, pendant double bond of the hydrophobic PBD block enables covalent stabilization of the formed structures. As such, if the copolymer is cross-linked at different times after assembly, it has the potential to freeze intermediates and thus provide novel insight into processes of molecular reorganization of the film. Quick immersion, for example, of the deposited film into an aqueous solution containing freshly prepared cross-linking reagents shows that the cross-linking reaction successfully competes with the assembly of wormlike micelles and leads to an intermediate morphology: a relatively intact but clearly imperfect monolayer (Figure 3C). The cross-linking reaction blocks formation of wormlike micelles by stabilizing patches of the copolymer on the glass support. The surface appears smoother than that without cross-linking, and the peaks in height of the non-cross-linked samples become plateaus of height ∼7 nm, which corresponds very well to a supported but inverted monolayer stabilized on glass. The results thus clearly demonstrate the broad potential of monolayer stabilization by cross-linking. The application of a largely hydrophobic PBD surface with pitted defects has yet to be explored, but we suggest thats because of the PEO-glass interfaceslarge patches of such a robust film might be lifted off the LB support and used as highly asymmetric, porous skins. Inflation-Deflation Dynamics of a Regionally Cross-Linked Membrane. Prior studies34 have shown that OB2 readily forms bilayer vesicles that can be crosslinked and toughened by orders of magnitude. We add to such characterizations here with a brief illustration of the crumpling dynamics of a regionally cross-linked membrane. Here we have used micropipet manipulation28,34 to both spatially control and characterize the system (Figure 4). An un-cross-linked OB2 polymersome is aspirated into a micropipet27,34 and cross-linking reagents (see Materials and Methods) were added to the solution exterior to the micropipet. The cross-linking solution was changed to an isotonic reagent-free solution after 15 min, which proved sufficient for reagent-mediated cross-linking of the membrane held outside the micropipet. The spherical membrane (Figure 4A) starts to visibly wrinkle as the cross-linking reagents permeate into the hydrophobic core of the membrane being aspirated into the micropipet (Figure 4B,C). However, the segment of the membrane inside the micropipet proved to be shielded from the cross-linking reagents and remained un-crosslinked and fluid. This portion of the vesicle membrane tore away with a step increase in suction pressure, while the outer shell remained fixed to the micropipet entrance.
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Figure 4. Video sequence of a locally cross-linked vesicle selfassembled from the PEO-PBD copolymer, OB2. The membrane external to the micropipet is cross-linked while the membrane held within the micropipet (Rp ) 3.5 µm) is shielded from the cross-linking reagents. This interior segment detaches when stressed and leaves a micrometer-size hole, but the “microballoon” remains afixed to the end of the micropipet. The spherical remnant of vesicle (A) is partially deflated (B and C) by a negative pressure to an almost entirely collapsed state (D). Inflation under positive pressure brings the vesicle back to its original, spherical shape, and the process proves completely reproducible (F, G, H, and I). The two cycles of inflationdeflation take approximately 5 s and clearly illustrate the ruffled crumpling expected of a solidly cross-linked membrane.
Figure 5. Phase contrast images of giant vesicles of the PEOPBD copolymer, OB18, either cross-linked or not, and then exposed to chloroform. Vesicles were injected into an aqueous chamber, and a drop of chloroform was carefully injected, leading to slow dissolution and diffusion of chloroform into the aqueous phase. Vesicles are initially phase dense because they are loaded with small molecular weight sucrose, then diluted in buffered dextrose (300 mOsm), and finally incubated in closed chambers at 25 °C with the chloroform droplet. (A, B) Pure cross-linked OB18 vesicles remain resistant to the organic solvent and retain both their encapsulant and morphology over time. (C, D) In contrast, non-cross-linked membranes of OB18 imbibe the chloroform, leading to visible inclusions in the hydrophobic cores of the vesicles. The vesicles eventually lose their encapsulated sucrose. Scale bars are 8 µm.
Subsequent pressure changes, both positive and negative, were then used to inflate and deflate the vesicle. The collapsed, aspirated vesicle (Figure 4D) is first reinflated to its original shape (Figure 4E), and this process of deflation-inflation was repeated several times, proving to be highly reproducible (Figure 4F-I). Similar to the crumpling transition long described by Nelson and others for tethered membranes,44 such controlled changes in the vesicle volume provide immediate insight into the effective rigidity of these solid membranes. Cross-Linking of Thicker Vesicle Bilayers. As specified in Table 1, the copolymer OB18 has similar proportions to OB2, but OB18 is almost 3-fold larger in molecular weight. This has been shown to self-assemble into a considerably thicker and tougher (even before crosslinking) vesicle membrane.34 Because of the extreme toughness of OB18 membranes, we expected cross-linking of OB18 membranes to proceed without the sort of membrane rendering often seen with thinner lipid size systems. Further facilitating polymerization, OB18 also has an average of 125 double bonds per molecule. Prior to initiating the cross-linking reaction, the vesicles appear smooth, spherical, and phase dark (Figure 5A). This phase contrast is due to an established difference in the refractive index of the encapsulated sucrose against the external, isotonic solution of dextrose. Following cross-linking, the vesicles appeared stiffer, corrugated and wrinkled (Figure 5A), with little to no change in phase contrast or overall diametersjust as seen with cross-linking of the giant OB2 vesicles.
Chloroform-OB18 Vesicle Dissolution Dynamics. Solubility is often used to challenge cross-linking, and so we examined the solubilizing effect of a very good solvent for these copolymersschloroformson the stability of pristine versus cross-linked OB18 polymersomes. In the latter case, the vesicles remain entirely resistant. CHCl3 appears unable to infiltrate the solid, hydrophobic PBD core (Figure 5A,B), and these fully cross-linked vesicles retain both their corrugated morphology and sucrose encapsulant for at least 1-2 h. In contrast, addition of chloroform to an un-cross-linked sample of OB18 vesicles leads to rapid accumulation of CHCl3 into the fluid hydrophobic core and visible nucleation of membrane inclusions at the edge of the vesicle (Figure 5C). Over time, CHCl3 diffusion into the growing accumulation probably splits the existing bilayer into stressed monolayers which not only allow release of encapsulant but also eventually drives the self-assembly of smaller, micrometer-size vesicles and probably emulsion drops (not shown). Similar processes of vesicle disassembly have been seen recently with electroporated liposomes which lose lipid and even appear to reassemble on time scales of milliseconds; additionally, detergent and solvent solubilization of liposomes also results in catastrophic loss of membrane integrity (over a few seconds to minutes).45 Dilutions in Cross-Link Density with OL1/OB18 Blends. The PEO-PLA diblock copolymer, OL1, makes vesicles on its own and is being pursued for applications in controlled release.38 The utility lies in the fact that PLA degrades hydrolytically over time.35 PLA is nonethe-
(44) Nelson, D.; Piram, T.; Weinberg, S. Statistical Mechanics of Membranes and Surfaces; World Scientific: Teaneck, NJ, 1989.
(45) Hansen, U. K.; Maire, M. I.; Moller, J. V. Biophys. J. 1998, 75, 2932-2946.
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Figure 6. Dynamics of encapsulant release from giant, crosslinked vesicles made of OB18 and OL1 blends. (A, B) Phase contrast video sequence of OL1:OB18 blends at a 25:75 molar ratio before and during chloroform solubilization. (C, D) Vesicles of 50:50 OL1:OB18 are less resistant to such solubilization with leakage of the encapsulants followed by a catastrophic loss in shell integrity. In all of the experiments the vesicles encapsulate sucrose and are diluted in buffered dextrose (300 mOsm) in closed chambers at 25 °C. Scale bars are 8 µm.
less recognized as hydrophobic despite its high oxygen content. To understand this further, especially in the limit of PLA chains of just a few kilodaltons, we sought to make and characterize partially cross-linked blends of OL1 and OB18. Note that both copolymers have an fEO typical of vesicle-forming amphiphiles46 but are otherwise mismatched in terms of Mn (by almost 2-fold) and membrane thickness d. Our previous study of block copolymer membrane blends focused, in contrast, on two very similar copolymers of PEO-PBD and the saturated homologue PEO-poly(ethylethylene) (PEO-PEE) designated as OE7. We ultimately show here that partially cross-linked OL1OB18 systems provide control over encapsulant release kinetics and also establish a foundation for hydrolytic poration mechanisms. OL1 and OB18 were dissolved in chloroform at desired molar ratios and then dried as thin films. Hydration in the presence of sucrose and fluorescent dextrans spontaneously generated giant vesicles, visible in both fluorescence and phase contrast microscopy. The homogeneity and stability of OL1 integration in the OB18 bilayer has been verified by fluorescent labeling of the hydrophobic PLA block with observation of a uniform edge brightness in the blended membranes.38 As with cross-linking of pure OB18 membranes, reagent addition to these blended vesicles did not perturb the system as there was no visible loss of either membrane integrity or encapsulant. However, addition of chloroform did lead to changes. An increased molar ratio of OL1 in the blends with OB18 should proportionally dilute the cross-link density. Figure 6A,B shows that even at cross-linking density of 75 mol%, the blended vesicles are perturbed by chloroform. Within a few minutes of solvent addition, the otherwise impenetrable polymerized shell is ridden with defects that propagate across the corrugated surface. Solubilization of the un-cross-linkable OL1 polymer leads to the progressive loss of both sucrose and larger dextran polymer, as well as “gravitation” of any small interior vesicles toward the edge of the membrane. The “empty”, insoluble (46) Discher, D. E.; Eisenberg, A. Science 2002, 297, 967-973.
Ahmed et al.
Figure 7. Chloroform-triggered release of encapsulated sucrose from blended vesicles of OL1:OB18 with cross-linked PBD. Pure OB18 vesicles (100% cross-linked), represented by the horizontal dotted line, show no loss of encapsulant for at least several hours. The exponential decays for 25 and 50 mol% OL1 blends, or 75 and 50% cross-linking, have release times given by t ) τrelease. The inset shows the first-order solubilization rate depends on the mole percent of non-cross-linkable OL1 blended into the membranes.
polymeric shell ultimately appears somewhat stabilized by chloroform inclusions at the edge of the vesicle. For 50 mol% blends, the loss of shell integrity is far more catastrophic, exhibiting faster encapsulant release dynamics. Within 2 min of the chloroform “attack”, the initially phase-dense and partially cross-linked vesicle collapses and swiftly disintegrates. Chloroform penetration and solubilization of the mobile OL1 chains in the bilayer result in a “peeling” away of the vesicle bilayer and accelerated release kinetics. The action of chloroform on solubilization of OL1 integrated into the polymer shell can clearly be used as a mechanism to create defects in the covalently cross-linked network. Physical insight into the dimensions of these defects is provided by the molecular size of its hydrophilic encapsulants.47 Vesicles release both sucrose and tritc-dextran (17 200 g/mol) with molecular radii of 0.9 and 2.6 ( 0.8 nm, respectively. Solubilization thus creates leaky perforations of at least 4 nm in radius. Finally, we confirmed that chloroform dissolution dynamics above are independent of trace amounts of cross-linking reagents present by repeating the experiments with washed cross-linked vesicles. Effect of Cross-Linking Dilutions on Encapsulant Release Kinetics. Whereas addition of chloroform to pure cross-linked OB18 polymersomes showed no leakage of encapsulant, blends with OL1 as a cross-link diluent show dramatic and systematic effects on encapsulant release kinetics (Figure 7). The effect is illustrated by intensity analyses of vesicle interiors, representing the percent retention of sucrose at any given time and calculated as
% encapsulant retained )
〈I〉 - 〈If〉 〈Io〉 - 〈If〉
× 100
where 〈I〉 is the average bright field intensity of the vesicle at any given time and 〈Io〉 and 〈If〉 are average intensities before induced leakage and after the addition of chloroform, respectively. At 50 mol% OL1, for example, chloroform triggers leakage with an apparently exponential (47) Hobbie, R. K. Intermediate physics for medicine and biology, 3rd ed.; Springer-Verlag: New York, 1997.
Copolymer Assemblies with Cross-Link Stabilization
Figure 8. (A) Percolation of PBD cross-linking by addition of OB18 to OL1 copolymers dictates encapsulant release times as well as membrane lysis kinetics. Partial cross-linking up to an apparent ∼40% OL1 results in destabilization of the vesicle bilayer relative to non-cross-linked pristine OB18 (open circle) and is manifested in faster encapsulant release kinetics (τrelease). At higher % cross-linkable copolymer, this destabilization “dip” is restored and imparts vesicle stability and delays release kinetics. The inset (B) shows similar trends for the dependence of membrane lysis tension, τlysis, on cross-linking efficiency of OB2 blended with the saturated analogue OE7.34
release profile, and a rate constant of 0.25 min-1; this is almost 2-fold faster than the leakage rate seen with 75% cross-link dilution. When plotted versus OL1 addition, leakage rates increase linearly as illustrated in the inset to Figure 7, which shows initial first-order rate kinetics for sucrose leakage (1/τrelease). Dilution of the cross-link density with OL1 can clearly be used as a mechanism to decrease membrane permeability and release molecular encapsulants (e.g., drugs) in their aqueous lumen. The effects are quantified in Figure 8 as a function of mole % cross-linkable, and the results show a highly nonlinear dependence for release. An initially sharp destabilization “dip” at a percolation of pc ≈ 10-40% indicates faster release kinetics than even “pure” OB18 vesicles. Vesicle stabilization occurs at p > 40%. The observed trend is consistent with previous work on the lower molecular weight OB2-OE7 system34 (reproduced in the inset) which shows that above a percolation point of pc ≈ 15-20%, the membrane is increasingly stabilized as bonding cross-links percolate throughout the membrane. The difference could reflect the fact that addition of 40% OL1 leads to integration of only about half as much OL1 into OB18 membranes, i.e., 20%. Alternatively, the difference could also reflect copolymer mismatches within the membrane and differences in membrane thickness. Nonetheless, below pc there is a 3-fold drop in the membrane lysis tension (τlysis) compared to the non-cross-linked vesicles because, we conjectured, polymerized chains demix within the membrane and create defects that are generally well known to increase permeability in phase-separated lipid membranes. This phenomenon is reflected here in the enhanced release kinetics exhibited by OL1-OB18 blended polymersomes. A last salient feature worth noting is that the effective release of small molecular weight hydrophilic encapsulants is conditional on solubilization of only a few OL1 chains and does not necessitate catastrophic lysis of the vesicle.
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Conclusions Parallel physicochemical characterizations of diblock copolymers in monolayers and bilayers demonstrate a broad ability to stabilize pure and blended lamellar architectures. The fluid monolayer was effectively transferred onto a solid support using the Langmuir-Blodgett technique, and subsequent immersion of the inverted films (with hydrophobic chains up) into an aqueous solution is shown to destabilize the film and trigger reorganization into surface bound worm micelles. This morphology is verified by AFM height measurements, which correlate well with cryo-TEM analysis of these diblock bilayers in aqueous solution. More rapid immersion of the copolymer film into a solution of cross-linking reagents clearly blocks such reorganization and demonstrates pitted plateaus of height consistent with a monolayer. Rapid diffusion of the initiating reagents into the exposed hydrophobic chains is thus effective in opposing the energetically favored micellar reorganization and generating a relatively uniform, solid cross-link network of polymer chains. Future interest may lie in whether integration of PEO-PLA chains into such films will provide dynamic control over monolayer porosity and surface properties useful to antifouling,24 bioscaffolds, and other applications. With bilayer polymersomes we have also studied crosslink stabilization and demonstrated an ability to locally shield, with a micropipet, the membrane from cross-linking reagents. Through local rupture, this generates micropores in giant polymer vesicles. Cycles of inflation-deflation may lead to deeper insights into “tethered” membranes, including crumpling transitions long theorized by Nelson and others.44 As expected for a fully cross-linked structure, the solid hydrophobic core is also clearly shown to be resistant to dissolution in chloroform. However, blending of non-cross-linkable PEO-PLA into the polymersome membranes effectively destabilizes partially cross-linked shells, which follow first-order release kinetics of loaded encapsulants upon chloroform dissolution. Previous work on more homogeneous blend systems has shown that the kinetics of encapsulant release from partially cross-linked shells is a highly nonlinear function of the mole percent of non-cross-linkable copolymer, and we have observed very similar phenomenon here with different copolymers. As before, we suppose that the crosslinks percolate laterally across the membrane, limited only by the density of reactive double bonds in the hydrophobic PBD chains. With the novel blends of PEOPLA described here, we are also finding that the release kinetics due to PLA hydrolysis is retarded considerably by cross-linking.38 The various physiochemical results visibly demonstrate not only PEO-PLA miscibility but also the structure-property relationships of cross-linkstabilized polymersomes. We believe that such studies carry the development of polymersomes forward as controlled release drug carriers with predictable and sitespecific release profiles of encapsulated drugs. Acknowledgment. We thank Frank Bates and his lab for generous provision of the block copolymers studied here as well as many stimulating discussions. LA034178L
