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Reactive Block Copolymer Vesicles with an Epoxy Wall Hui Zhu, Qingchun Liu, and Yongming Chen* State Key Laboratory of Polymer Physics and Chemistry, Joint Laboratory of Polymer Science and Material, Institute of Chemistry, The Chinese Academy of Sciences, Beijing 100080, China ReceiVed March 21, 2006. In Final Form: October 12, 2006 Recently, block copolymer vesicles have attracted considerable attention because of their properties in encapsulation and release. To explore their applications in biorelated fields, functionalization of the polymer vesicle is necessary. Herein, a reactive unilamellar vesicle is reported by self-assembly of poly(ethylene oxide)-block-poly(glycidyl methacrylate) copolymer (PEO-b-PGMA) in solution. When water was added into the PEO-b-PGMA solution in THF, unilamellar vesicles were produced. If hydrophobic primary amine additives, such as hexamethylenediamine (HDA) and dodecylamine (DA), were introduced during block copolymer assembling, the vesicular morphology remained unchanged; instead, the amines reacted with the epoxys and the vesicles were fixed by cross-linking. Furthermore, when 3-aminopropyl trimethoxysilane (APS) was applied, the organic/inorganic hybrid vesicles were obtained, which were stable against the solvent change. Therefore, this research not only supplies a new way to fix the vesicular morphology but also a reactive vesicle scaffold for introducing functional species.
Introduction Polymer hollow nano- or microparticles have attracted much attention in recent years because they display unique properties of encapsulation and release that may have exciting prospects in pharmaceutics and biotechnologies.1-4 Diblock copolymers may self-assemble into bilayer vesicles spontaneously in a selective solvent for one segment.2,3 As compared to the vesicles of small molecular surfactants, the stability of block copolymer vesicles is rather high and critical aggregation concentration is very low. Furthermore, the vesicle structure and properties can be controlled during block copolymer synthesis. Therefore, a great potential for encapsulation of various guest species within their cavities and further release is expected.5,6 Eisenberg’s group has done extensive research on block copolymer aggregation in solution.7 The polymers they applied include poly(ethylene oxide)-block-polystyrene and poly(acrylic acid)-block-polystyrene, whose soluble segments are much shorter than the insoluble segments in aqueous solution. Highly asymmetric composition is a precondition for vesicle formation, and their self-assemblies are crew-cut. To explore vesicle application, the block copolymer bearing reactive groups along one segment is necessary to perform the functions such as fixation, stimulus-responsive function, and supporting functional groups.8-17 Although polymer vesicles have * Corresponding author. E-mail:
[email protected]. (1) Shi, X. Y.; Shen, M. W.; Mo¨hwald, H. Prog. Polym. Sci. 2004, 29, 987. (2) (a) Discher, D. E.; Eisenberg, A. Science 2002, 297, 967. (b) Antonietti, M.; Fo¨rster, S. AdV. Mater. 2003, 15, 1323. (c) Kita-Tokarczyk, K.; Grumelard, J.; Haefele, T.; Meier, W. Polymer 2005, 46, 3540. (3) Soo, P. L.; Eisenberg, A. J. Polym. Sci., Part B: Polym. Phys. 2004, 42, 923. (4) Chen, D. Y.; Jiang, M. Acc. Chem. Res. 2005, 38, 494. (5) Choucair, A.; Lim Soo, P.; Eisenberg, A. Langmuir 2005, 21, 9308. (6) Ranquin, A.; Verse´es, W.; Meier, W.; Steyaert, J.; van Gelder, P. Nano Lett. 2005, 5, 2220. (7) Cameron, N. S.; Corbierre, M. K.; Eisenberg, A. Can. J. Chem. 1999, 770, 1311. (8) (a) Wooley, K. L. J. Polym. Sci., Part A: Polym. Chem. 2000, 38, 1397. (b) Joralemon, M. J.; O’Reilly, R. K.; Hawker, C. J.; Wooley, K. L. J. Am. Chem. Soc. 2005, 127, 16892. (c) Harrisson, S.; Wooley, K. L. Chem. Commun. 2005, 3259. (d) Turner, J. L.; Becker, M. L.; Li, X.; Taylor, J.-S. A.; Wooley, K. L. Soft Matter 2005, 1, 69. (e) Cheng, C.; Qi, K.; Khoshdel, E.; Wooley, K. L. J. Am. Chem. Soc. 2006, 128, 6808. (9) (a) Ding, J.; Liu, G.; Yang, M. Polymer 1997, 38, 5497. (b) Ding, J.; Liu, G. Macromolecules 1997, 30, 655. (c) Ding, J.; Liu, G. J. Phys. Chem. B 1998, 102, 6107.
been obtained from many block copolymers, reactive vesicles that can be used to introduce functionalities are still very limited. Fixing the morphology of block copolymer aggregates by crosslinking has been made by using reactive block copolymers.8-12 The block copolymers that bear photodimerization active segments have been applied to fix the vesicles generated in organic solvent.9 Other examples include the fixation of vesicles through polymerization by using carbon-carbon double bonds of poly(ethylene oxide)-block-polybutadiene,10 by using methacrylic ends of a telechelic triblock copolymer,11 and by using the thiophene rings of a coil-rod block copolymer.12 Our group has prepared poly(ethylene oxide)-block-poly[3-(trimethoxysilyl)propyl methacrylate] (PEO-b-PTMSPMA) by controlled radical polymerization that is reactive to sol-gel reaction.13a This block copolymer self-assembled into vesicles in a solvent such as a mixture of methanol and water. When a small amount of triethylamine was added, the gelable segments in the bilayer wall were transformed into a cross-linked silica oxide network, which is rather stable.13b,c This strategy has been extended to fix the stimulus-responsive block copolymer vesicles.14 However, a new approach to produce block copolymer vesicles that may be used as a reactive scaffold for introducing functionality is still desperately needed especially for biorelated applications, which require biocompatible polymers and water as an assembling medium.15-19 (10) (a) Maskos, M.; Harris, J. R. Macromol. Rapid Commun. 2001, 22, 271. (b) Discher, B. M.; Bermudez, H.; Hammer, D. A.; Discher, D. E.; Won, Y. Y.; Bates, F. S. J. Phys. Chem. B 2002, 106, 2848. (11) Nardin, C.; Hirt, T.; Leukel, J.; Meier, W. Langmuir 2000, 16, 1035. (12) Vriezema, D. M.; Hoogboom, J.; Velonia, K.; Takazawa, K.; Christianen, P. C. M.; Maan, J. C.; Rowan, A. E.; Nolte, R. J. M. Angew. Chem., Int. Ed. 2003, 42, 772. (13) (a) Du, J. Z.; Chen, Y. M. Macromolecules 2004, 37, 6322. (b) Du, J. Z.; Chen, Y. M.; Zhang, Y. H.; Han, C. C.; Fischer, K.; Schmidt, M. J. Am. Chem. Soc. 2003, 125, 14710. (c) Du, J. Z.; Chen, Y. M. Macromolecules 2004, 37, 5710. (14) Du, J. Z.; Armes, S. P. J. Am. Chem. Soc. 2005, 127, 12800. (15) Checot, F.; Lecommandoux, S.; Gnanou, Y.; Klok, H. A. Angew. Chem., Int. Ed. 2002, 41, 1339. (16) Koide, A.; Kishimura, A.; Osada, K.; Jang, W. D.; Yamasaki, Y.; Kataoka, K. J. Am. Chem. Soc. 2006, 128, 5988. (17) Napoli, A.; Valentini, M.; Tirelli, N.; Mu¨ller, M.; Hubbell, J. A. Nat. Mater. 2004, 3, 183. (18) Bellomo, E.; Wyrsta, M. D.; Pakstis, L.; Pochan, D. J.; Deming, T. J. Nat. Mater. 2004, 3, 244.
10.1021/la060766y CCC: $37.00 © 2007 American Chemical Society Published on Web 11/17/2006
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Scheme 1. PEO-b-PGMA Block Copolymer Structure and Reactive Vesicles Thereof
Herein, we report a new type of reactive polymer vesicles produced from a reactive block copolymer bearing epoxy groups. The block copolymer displaying such a unique property is poly(ethylene oxide)-block-poly(glycidyl methacrylate) (PEO-bPGMA). It is known that the epoxy groups of PGMA are reactive for nucleophilic attack of various chemicals and they may be used to graft useful functional groups. We found that PEO-bPGMA aggregated into vesicles, which is remarkable. What is interesting is that additive hydrophobic primary amines could be enriched in the vesicle wall and reacted with the epoxys to fix the vesicles. Scheme 1 shows the block copolymer structure and its bilayer vesicles. As far as we know, this is the first example of block copolymer vesicles whose walls are composed of epoxy groups. Experimental Section Methods. TEM images were obtained using a JEM 100 instrument operated at 100 kV. 1H NMR spectra were recorded on a Bruker DMX300 spectrometer with CDCl3, THF-d8, or D2O as solvent at room temperature. Gel permeation chromatography (GPC) was performed by a set of a Waters 515 HPLC pump, a Waters 2414 differential refractometer, and three Waters Styragel columns (HT2, HT3, and HT4) using THF as eluent at a flow rate of 1.0 mL/min at 35 °C. Polystyrene standards were used for the calibration. Dynamic light scattering (DLS) was performed on a laser light scattering spectrometer (ALV/DLS/SLS-5022F) equipped with a multi-τ digital time correlator (ALV5000) and a cylindrical 22 mW UNIPHASE He-Ne laser (λ0 ) 632 nm). The temperature was kept at 293 K, and the scattering data were collected at 90°. Materials. Glycidyl methacrylate (GMA) (Aldrich) was purified by distillation under vacuum prior to use. Methoxy poly(ethylene oxide) (molecular mass is 1900, Alfa) was modified by 2-bromoisobutyryl bromide to prepare macroinitiator PEO-Br following a standard procedure.13a Hexamethylenediamine (HDA) (Shenyang Reagent Factory), dodecylamine (DA) (Beijing Chemical Works), and other chemicals were used as received. PEO-b-PGMA copolymers were synthesized by atom transfer radical polymerization (ATRP) of GMA initiated by PEO-Br (see Supporting Information). The block copolymer composition was determined through a 1H NMR spectrum by comparing the proton areas of two segments. Two block copolymers, PEO43-b-PGMA121 (Mw/Mn ) 1.30) and PEO43-b-PGMA241 (Mw/Mn ) 1.40), were thus synthesized and used for self-assembling studies. Vesicle Formation. A solution of PEO43-b-PGMA121 block copolymer in THF, 2 mg/mL, was prepared, and then water was added dropwise under stirring until the water content reached 57 wt %. Next, the solution was stirred for 1 day. A drop of solution was taken and dropped onto a copper grid with carbon film. The solvent was evaporated to dryness in air, and TEM analysis was performed. To make a light scattering analysis, a polymer solution in THF was prepared and filtered through a membrane with pore size of 0.2 µm. Next, the filtrated water was dropped to induce self-assembly, and the container was shaken prior to measuring. (19) (a) Meng, F.; Hiemstra, C.; Engbers, G. H. M.; Feijen, J. Macromolecules 2003, 36, 3004. (b) Che´cot, F.; Lecommandoux, S.; Gnanou, Y.; Klok, H. A. Angew. Chem., Int. Ed. 2002, 41, 1340. (c) Kukula, H.; Schlaad, H.; Antonietti, M.; Fo¨rster, S. J. Am. Chem. Soc. 2002, 124, 1658.
Figure 1. TEM images of PEO43-b-PGMA121 vesicles at different water content. (A) Cini ) 2 mg/mL, Cw ) 50 wt % and (B) Cini ) 2 mg/mL, Cw ) 57 wt %. Vesicle Formation with Amine Additive: A General Procedure. A solution containing PEO43-b-PGMA121 (10 mg) and DA (5.9 mg) in THF (2 mL) was prepared, and then water was added until the water content reached 57 wt %. After being stirred for 4 days, the vesicle solution was dialyzed against water for 7 days.
Results and Discussion Vesicle Formation. PEO-b-PGMA is an amphiphilic block copolymer. Its vesicle formation was performed in a selective solvent for PEO segment by introducing water into the block copolymer solution in THF, which is a common solvent of the PEO-b-PGMA. This is a typical procedure to generate block copolymer aggregates in a selective solvent. Typically, PEO43b-PGMA121 was dissolved into THF, and the initial concentration (Cini) of the copolymer was kept at 2 mg/mL. To this solution, water was added dropwise to induce aggregation. The solution became turbid when water content (Cw) reached ca. 44 wt %. At different Cw contents, a drop of turbid solution was taken and dropped onto a copper grid coated with carbon film to observe the morphology by TEM directly without staining. Figure 1 shows the aggregates at different water content when Cini ) 2 mg/mL. Vesicles were observed at low water content where the solution just became turbid. Figure 1A gives a giant vesicle, which is over 5 µm. When water content increased to Cw ) 57 wt %, as shown in Figure 1B, the vesicles in the range of 500-1000 nm were observed. However, it was hard to evaluate the wall thickness from the TEM image because the contrast of this sample was rather low. At higher water content, the amorphous aggregates
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Figure 2. TEM images of PEO43-b-PGMA241 vesicles at different water content. (A) Cini ) 5 mg/mL, Cw ) 44 wt % and (B) Cini ) 5 mg/mL, Cw ) 57 wt %.
appeared, although vesicles still could be found. We have changed the Cini to be in the range of 1-10 mg/mL, while the Cw was kept at 57 wt %. The same vesicles were obtained under these conditions applied. To check the influence of method of sample preparation, the block copolymer solution in THF was also dropped into water, and the vesicles were still produced. A different sample, PEO43-b-PGMA241, whose hydrophobic segment length is double that of the above sample, could aggregate into vesicles as well. Figure 2A illustrates the vesicles produced at relative low water content. The image contrast of this sample is relatively higher because the length of PGMA segment is longer, and the wall thickness was evaluated to be ca. 25 nm. This value agrees with the unilamellar structure of a block copolymer membrane. However, spherical micelles and short rods were observed at higher water content as indicated in Figure 2B. It is remarkable that PEO-b-PGMA self-assembled into novel vesicles with epoxy walls. Because PGMA is a hydrophobic polymer while PEO is hydrophilic, it is easy to understand that the PGMA segments aggregate into the core while PEO segments tether onto the inner and outer surfaces. This structure has been confirmed by NMR analysis in the latter section. Reactivity of Epoxy Walls - Vesicle Stabilization. To explore the reactivity of the PGMA wall, primary alkyl diamines, HDA and DA, were added during block copolymer assembling as the additives. It is noticed that the vesicular morphologies remained unchanged after the amines were introduced. Figure
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3A and B shows the TEM images of PEO43-b-PGMA121 vesicles in the presence of HAD and DA additives, respectively. The vesicle appearance seems to have been improved, and large vesicles, ca. 1000 nm, dominate. It is interesting then to explore the effect of amine additives on the vesicles. It is well known that the chemistry of epoxy resin involves the nucleophilic reaction between epoxy and amine to induce cross-linking. The primary amine may react with two epoxys to generate a tertiary amine. In the present system, the epoxys were grafted along the polymer chain, and the crosslinking reaction would occur if the reacted epoxys came from different copolymers. To check this point, a large amount of THF, a good solvent for the block copolymer precursor, was added to the vesicle solution after the assembly for several days. From TEM images shown in Figure 3C, the vesicles in the presence of DA still remained essentially unchanged. In contrast, when THF was also added to the vesicles without amine additives, the blue tint appearance disappeared immediately and no vesicles could be found. This result demonstrates that the long alkylamines have been enriched in the vesicle wall and reacted with the epoxys of the block copolymers. As a result, the vesicles were fixed by cross-linking reaction. Therefore, this can be a new approach to fix the polymer vesicles in addition to introducing the functionalities. In our previous research, PEO-b-PTMSPMA forms vesicles in a mixture of methanol and water; the gelation process that followed stabilizes the vesicle structure by using the trimethoxysilyl groups along PTMSPMA segments.13 Similar hybrid vesicles can be obtained by using APS as an additive during the PEO-b-PGMA block copolymer self-assembly. This compound bears two functional groups: one is primary amine, which is reactive to epoxy groups as has been already revealed, and one is trimethoxysilane, which undergoes hydrolysis and polycondensation to generate cross-linked silica oxide. APS was mixed with the block copolymer in THF solution, and then water was added to induce the self-organization. Because APS is a weak base, the gelation process would occur when water was introduced. As shown in Figure 4A, vesicles of high quality were obtained. Two reactions could have occurred in the vesicle wall. One is the reaction between primary amine and epoxy, and one is the sol-gel process originated from trimethoxysilyl groups. Both reactions would result in a cross-linking network in the vesicles. As shown in Figure 4B, the vesicular morphology remained when a large amount of THF was introduced, revealing the crosslinking reaction did occur. However, the reaction extent is difficult to evaluate in the solution. The amount of APS additive to the block copolymer has also been changed. At conditions of Cini ) 5 mg/mL and Cw ) 57 wt %, the concentration of APS in the final vesicle solution was changed from 0.035 to 0.58 wt %. Under these conditions applied, the vesicles were produced exclusively. Dynamic Light Scattering Analysis. It is known that the vesicles observed by TEM images have already collapsed. DLS analysis will give the vesicle size information in solution. Figure 5 shows the dynamic radius distribution of the PEO43-b-PGMA121 vesicles in the absence and presence of amine additives. All three traces display bimodal particle distribution. The radius of vesicles ranges from 200 to 1000 nm, which is broad and accords roughly with the size given the TEM image. Also, a small peak, which may be attributed to small vesicles, was observed. When two amines, DA and DHA, were presented, both vesicle size distributions became narrow apparently. This observation agrees
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Figure 3. TEM images of PEO43-b-PGMA121 vesicles in the presence of (A) HDA (0.091 wt %), (B) DA (0.14 wt %), and (C) the vesicles in (B) were treated with a large amount of THF. Conditions: Cini ) 5 mg/mL, Cw ) 57 wt %.
Figure 5. Dynamic radius distribution of PEO43-b-PGMA121 selfassembly analyzed by DLS in the absence and presence of polymer Cini ) 5 mg/mL, Cw ) 57 wt % (-, polymer; ‚ ‚ ‚, polymer+DA; - ‚ - ‚ -, polymer+DAH).
Figure 4. TEM images of (A) vesicles from PEO43-b-PGMA121 in the presence of APS (0.27 wt %) and (B) after a large amount of THF was added. Conditions: Cini ) 5 mg/mL, Cw ) 57 wt %.
with the results from Figure 3, and the reason may be due to the plasticization from alkylamine, which improved vesicle distribution. NMR Analysis. Therefore, the presence of the alkylamines may stabilize the vesicles as expected. Also, we have tried to monitor the reaction between the epoxy and APS by 1H NMR analysis. Figure 6A shows the spectrum of the PEO-b-PGMA precursor in THF-d8 with assignment. After APS was added for 30 and 60 min, no obvious difference was observed from the spectra of the mixture (Figure 6B and C). Only a small change at δ 2.62 ppm (in Figure 6C) can be noticed attributed to the
Figure 6. 1H NMR spectra of (A) PEO43-b-PGMA121 (10 mg) in THF-d8 (1 mL), addition of APS (11 mg) for (B) 30 min and (C) 60 min, and (D) introduction of D2O (1.2 mL).
reaction; however, this reaction seems very slow in THF. This is understandable because the concentrations of two components are very low and the reaction at room temperature should be a slow process. In fact, the reaction before assembling is not helpful for the self-assembly because the reaction will alter the block copolymer structure and the aggregation behavior that followed. As demonstrated in Figure 6D, after D2O was added, those
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resonances corresponding to the protons of the PGMA segment disappeared, while that of PEO protons remained unchanged. This proves that PGMA forms the core and PEO forms the corona. It is noteworthy that the peaks of APS protons were broadened. This phenomenon implies that the movement of APS molecules has been restricted by the block copolymer aggregates. Therefore, APS has been enriched into the block copolymer walls due to the hydrophobic interaction. Also, it is of note that the peaks of the methoxy protons at δ 3.54 ppm disappeared when water was added, indicating that the gelation reaction occurred. Next, the epoxy reaction rate in this case should be much faster because two components were in a state close to the bulk. Unfortunately, the degree of this reaction in the vesicles dispersed in water solution is difficult to determine.
Conclusion In conclusion, a new family of block copolymer vesicles bearing epoxys as their walls has been generated by self-assembling PEO-b-PGMA in a mixture of water and THF. Thus, produced unilamellar vesicles have a wall composed of PGMA segments, which are reactive to nucleophilic reaction. When primary alkyl
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diamine additives were introduced during block copolymer assembling, they reacted with the epoxys and cross-linked the vesicle wall. This finding supplies not only a new way of fixing the block copolymer vesicles but also a reactive scaffold of hollow structure to introduce functional and bioactive segments in situ. The presence of a PEO segment should be addressed because this polymer may resist adhesion of proteins and cells. The vesicle structure with PEO corona and aqueous assembling media would fit the further interest of biotechnology and pharmaceutics. Also, this block copolymer can be used to form a well-defined membrane on an interface by using epoxy for fixation and functionalization. Acknowledgment. Financial support from NSF China (50473056, 20625412 and 20534010) and the Chinese Academy of Sciences (KJCX2-SW-H07) is greatly acknowledged. Supporting Information Available: Synthesis of block copolymers. This material is available free of charge via the Internet at http://pubs.acs.org. LA060766Y
