Multicompartment Micelles from pH-Responsive Miktoarm Star Block

May 13, 2009 - We describe the synthesis of pH-responsive miktoarm star block terpolymers μ-[polystyrene][poly(ethylene oxide)][poly(2-(dimethylamino...
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Multicompartment Micelles from pH-Responsive Miktoarm Star Block Terpolymers† Chun Liu,‡ Marc A. Hillmyer,*,‡ and Timothy P. Lodge*,‡,§ ‡

Department of Chemistry and §Department of Chemical Engineering and Materials Science, University of Minnesota, Minneapolis, Minnesota 55455-0431 Received March 9, 2009. Revised Manuscript Received April 13, 2009

We describe the synthesis of pH-responsive miktoarm star block terpolymers μ-[polystyrene][poly(ethylene oxide)] [poly(2-(dimethylamino)ethyl acrylate)] ( μ-SODA) using a combination of two successive living anionic polymerizations and one reversible addition-fragmentation chain-transfer polymerization. Poly[2-(dimethylamino)ethyl acrylate] (PDMAEA) is a weak polybase that is hydrophilic at low pH and hydrophobic at high pH because of the protonation of the dimethylamino functional group with decreasing pH. In addition, our results suggest that PDMAEA is immiscible with polystyrene (PS), a feature that is desirable for the formation of multicompartment micelles. Using a combination of dynamic light scattering and cryogenic transmission electron microscopy, we demonstrate that μ-SODA micelles formed in water evolve from mixed corona (PEO + PDMAEA corona; PS core) and predominantly spherical micelles to multicompartment (PEO corona; PS + PDMAEA core) micelles with increasing pH.

Introduction The concept of a multicompartment micelle, in which the solvophobic core is subdivided into distinct nanodomains, derives inspiration from eukaryotic cells that possess multiple distinct organelles within the cell membrane.1-3 Various applications for multicompartment micelles have been envisioned, foremost of which is in drug delivery.4 The discrete subdomains within such micellar cores can facilitate the concurrent storage and delivery of multiple incompatible payloads in a prescribed stoichiometric ratio, supporting the technological relevance of multicompartment micelles.5 The self-assembly of synthetic ABC block terpolymers with one solvophilic block and two incompatible solvophobic blocks provides a straightforward pathway for preparing multicompartment micelles. Most experimental work regarding multicompartment micelles has focused on linear block terpolymers6-11 that tend to form concentric core-shell-corona structures, regardless of the overall micelle shape.6,12 A related strategy is to combine AB micelles with CD diblocks in which † Part of the “Langmuir 25th Year: Molecular and macromolecular selfassemblies” special issue. *To whom correspondence should be addressed. E-mail: hillmyer@ umn.edu (M.A.H.), [email protected] (T.P.L.).

(1) Ringsdorf, H.; Lehmann, P.; Weberskirch, R. Book of Abstracts; 217th National Meeting of the American Chemical Society, Anaheim, CA, March 2125, 1999. (2) Laschewsky, A. Curr. Opin. Colloid Interface Sci. 2003, 8, 274. (3) Lutz, J. F.; Laschewsky, A. Macromol. Chem. Phys. 2005, 206, 813. (4) Kwon, G. S.; Forrest, M. L. Drug Dev. Res. 2006, 67, 15. (5) Lodge, T. P.; Rasdal, A.; Li, Z.; Hillmyer, M. A. J. Am. Chem. Soc. 2005, 127, 17608. (6) Zhou, Z.; Li, Z.; Ren, Y.; Hillmyer, M. A.; Lodge, T. P. J. Am. Chem. Soc. 2003, 125, 10182. (7) Pochan, D. J.; Chen, Z.; Cui, H.; Hales, K.; Qi, K.; Wooley, K. L. Science 2004, 306, 94. (8) Kubowicz, S.; Thunemann, A. F.; Weberskirch, R.; Mohwald, H. Langmuir 2005, 21, 7214. (9) Kubowicz, S.; Baussard, J. F.; Lutz, J. F.; Thunemann, A. F.; von Berlepsch, H; Laschewsky, A. Angew. Chem., Int. Ed. 2005, 44, 5262. (10) Thunemann, A. F.; Kubowicz, S.; von Berlepsch, H.; Mohwald, H. Langmuir 2006, 22, 2506. (11) Cui, H.; Chen, Z.; Zhong, S.; Wooley, K. L.; Pochan, D. J. Science 2007, 317, 647. (12) Lodge, T. P.; Hillmyer, M. A.; Zhou, Z.; Talmon, Y. Macromolecules 2004, 37, 6680.

13718 DOI: 10.1021/la900845u

electrostatic attractions between polyanionic B and polycationic C blocks form higher-order assemblies.13,14 In contrast, mikto (mixed)arm star block terpolymers can also form multicompartment micelles but suppress the formation of core-shell-corona micelles by the mandatory convergence of three immiscible blocks at a single juncture.15-20 Understanding the underlying principles that govern the self-assembly of ABC block terpolymers is of fundamental importance to the control and application of these hierarchically structured nanoparticles. Recently, we have systematically studied multicompartment micelles obtained from the aqueous self-assembly of μ-[poly(ethyl ethylene)][poly(ethylene oxide)][poly(perfluoropropylene oxide)] (μ-EOF) miktoarm star block terpolymers.15-18 These nanostructures consist of discrete poly(perfluoropropylene oxide) (PFPO) and poly(ethyl ethylene) (PEE) subdomains within the core, surrounded by a poly(ethylene oxide) (PEO) corona. The manipulation of μ-EOF multicompartment micelle structures was achieved in three different ways: copolymer composition, copolymer blending, and solvent selectivity. Analogous to micelles formed by diblock copolymers, where an evolution from vesicles to worms to spheres is seen with increasing length of the solvophilic block,21-23 the μ-EOF micelle morphology was transformed from nanostructured vesicles and polygonal bilayers to segmented or multicompartment worms and finally to “hamburger” micelles simply by increasing the volume ratio of the (13) Zhang, W.; Shi, L.; Miao, Z. J.; Wu, K.; An, Y. Macromol. Chem. Phys. 2005, 206, 2354. (14) Lutz, J. F.; Geffroy, S.; von Berlepsch, H.; Bottcher, C.; Garnier, S.; Laschewsky, A. Soft Matter 2007, 3, 694. (15) Li, Z.; Kesselman, E.; Talmon, Y.; Hillmyer, M. A.; Lodge, T. P. Science 2004, 306, 98. (16) Li, Z.; Hillmyer, M. A.; Lodge, T. P. Nano Lett. 2006, 6, 1245. (17) Li, Z.; Hillmyer, M. A.; Lodge, T. P. Langmuir 2006, 22, 9409. (18) Li, Z.; Hillmyer, M. A.; Lodge, T. P. Macromolecules 2006, 39, 765. (19) Saito, N.; Liu, C.; Lodge, T. P.; Hillmyer, M. A. Macromolecules 2008, 41, 8815. (20) Mao, J.; Ni, P.; Mai, Y.; Yan, D. Langmuir 2007, 23, 5127. (21) Won, Y. Y.; Brannan, A. K.; Davis, H. T.; Bates, F. S. J. Phys. Chem. B 2002, 106, 3354. (22) Jain, S.; Bates, F. A. Science 2003, 300, 460. (23) Zupancich, J. A.; Bates, F. S.; Hillmyer, M. A. Macromolecules 2006, 39, 4286.

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hydrophilic PEO block to the hydrophobic PFPO and PEE blocks.16,17 On the other hand, an increase in the amount of PFPO block relative to the amount of PEE block drives the micellar structure from hamburger micelles or segmented worms to “raspberry” micelles or multicompartment worms.17 In the blending method, well-defined hamburger micelles evolved from a binary mixture of segmented wormlike micelles formed by a μ-EOF terpolymer and spherical micelles formed by a PEE-b-PEO diblock copolymer via a collision/fusion/fission mechanism.18 Besides manipulating the terpolymer itself, solvent selectivity has been shown to be an efficient way to achieve various micellar morphologies from a single block copolymer in different selective solvents or their mixtures.24-27 By incorporating tetrahydrofuran (THF), a good solvent for PEE, into aqueous dispersions, the μ-EOF micellar structure evolved from multicompartment disks to core-shellcorona worms and spheres and finally to mixed corona (PEE + PEO) oblate ellipsoidal micelles with increasing THF content.28 In the above example, any agent stored in the PEE block in such a micelle will be released as it undergoes the transition from the core to the corona, thus this type of evolution from multicompartment micelles to mixed corona micelles could be useful for sequential drug delivery. However, the THF-water system is not suitable for biomedical applications; such an evolution in aqueous solution by a stimulus such as pH, temperature, or ionic strength would be desirable.29-31 Among these, a pH-triggered structural change can be realized using a polybase block that can be rendered hydrophilic by protonation at low pH and vice versa. This has been demonstrated, for example, in the pH-triggered reversible self-assembly of zwitterionic copolypeptides into vesicles and the release of encapsulated dye.32,33 Herein, we report the design and synthesis of a new pH-responsive miktoarm star block terpolymer μ-[polystyrene] [poly(ethylene oxide)][poly(2-(dimethylamino)ethyl acrylate)] (μ-SODA) via a combination of two successive living anionic polymerizations and one controlled reversible addition-fragmentation chain-transfer (RAFT) polymerization. Micelle structures obtained from μ-SODA in dilute aqueous buffer solutions were examined using dynamic light scattering and cryogenic transmission electron microscopy. Poly[2-(dimethylamino)ethyl acrylate] (PDMAEA) is a biocompatible weak polybase (pKb ∼6.5)34,35 and can switch from hydrophilic to hydrophobic with increasing pH or temperature.36,37 In addition, our results suggest that PDMAEA is immiscible with polystyrene (PS), a feature favorable for the formation of multicompartment micelles. Therefore, as the pH increases, μ-SODA micelles are expected to evolve from mixed corona (PEO + PDMAEA (24) Lodge, T. P.; Bang, J.; Li, Z.; Hillmyer, M. A.; Talmon, Y. Faraday Discuss. 2005, 128, 1. (25) Bang, J.; Jain, S.; Li, Z.; Lodge, T. P.; Pedersen, J. S.; Kesselman, E.; Talmon, Y. Macromolecules 2006, 39, 1199. (26) Abbas, S.; Li, Z.; Hassan, H.; Lodge, T. P. Macromolecules 2007, 40, 4048. (27) Shen, H.; Eisenberg, A. J. Phys. Chem. B 1999, 103, 9473. (28) Liu, C.; Hillmyer, M. A.; Lodge, T. P. Langmuir 2008, 24, 12001. (29) Shim, W. S.; Kim, S. W.; Choi, E. K.; Park, H. J.; Kim, J. S.; Lee, D. S. Macromol. Biosci. 2006, 6, 179. (30) Borchert, U.; Lipprandt, U.; Bilang, M.; Kimpfler, A.; Rank, A.; Peschka-Suss, R.; Schubert, R.; Lindner, P.; Forster, S. Langmuir 2006, 22, 5843. (31) Chen, S.; Li, Y.; Guo, C.; Wang, J.; Ma, J.; Liang, X.; Yang, L.; Liu, H. Langmuir 2007, 23, 12669. (32) Rodriguez-Hernandez, J.; Lecommandoux, S. J. Am. Chem. Soc. 2005, 127, 2026. (33) Bellomo, E. G.; Wyrsta, M. D.; Pakstis, L.; Pochan, D. J.; Deming, T. J. Nat. Mater. 2004, 3, 244. (34) Park, T. G.; Jeong, J. H.; Kim, S. W. Adv. Drug Delivery Rev. 2006, 58, 467. (35) Jeong, J. H.; Kim, S. W.; Park, T. G. Prog. Polym. Sci. 2007, 32, 1239. (36) Plamper, F. A.; Schmalz, A.; Ballauff, M.; Muller, A. H. J. Am. Chem. Soc. 2007, 129, 14538. (37) Plamper, F. A.; Ruppel, M.; Schmalz, A.; Borisov, O.; Ballauff, M.; Muller, A. H. Macromolecules 2007, 40, 8361.

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corona; PS core) micelles to multicompartment (PEO corona; PS + PDMAEA core) micelles. Furthermore, the transition of PDMAEA from the corona to the core decreases the overall volume ratio of the hydrophilic block(s) to the hydrophobic block(s), hence leading to micellar structural changes in a similar manner to the variation of the copolymer composition.

Experimental Section Materials. All commercial solvents and chemicals were used as received, with the following exceptions. Cyclohexane, tetrahydrofuran (THF), and methylene chloride (CH2Cl2) were purified by passing through an activated alumina column using a homebuilt solvent-purification line.38 (()-Oxirane-2-methanol (Aldrich, 96%) was distilled under vacuum prior to use. Styrene (Aldrich, 99%) was sequentially vacuum distilled from calcium hydride and dibutylmagnesium after being stirred in a sealed flask at 0 °C for 6 and 4 h, respectively. Ethylene oxide (Aldrich, 99.5%) was vacuum distilled from n-butylmagnesium chloride after being stirred in a sealed flask at 0 °C for 1 h. 2-(Dimethylamino)ethyl acrylate (DMAEA, Aldrich, 98%) was passed through a basic alumina column prior to use. R-Methoxy-ω-hydroxy PEO (Aldrich, 2 kDa) was purified by precipitation in petroleum ether followed by drying under vacuum at 90 °C overnight. 2,20 -Azobisisobutyronitrile (AIBN, Aldrich, 98%) was recrystallized three times from methanol. 2-Methoxymethoxymethyloxirane (MMO) was prepared by the Williamson ether synthesis using (()-oxirane-2-methanol and chloromethyl methyl ether with the latter being the limiting reagent, as reported elsewhere.39 The dark-red diphenylmethyl potassium initiator was prepared by reaction between potassium naphthalenide and diphenylmethane in dried THF prior to use.40 The chain-transfer agent for RAFT polymerization, S-1-dodecylS0 -(R,R0 -dimethyl-R00 -acetic acid)trithiocarbonate (CTA), was synthesized following a previous report.41 Preparation of μ-SODA. μ-SODA miktoarm star block terpolymers were prepared using a combination of two successive living anionic polymerizations and one RAFT polymerization (Scheme 1). A typical polymerization procedure is briefly described as follows; a detailed description of the equipment and procedure can be found elsewhere.39,42 The synthesis of heterobifunctionalized poly(styrene) (PS-MMO) was initiated by injecting s-butyllithium (5.4  10-3 mol) into a solution of styrene monomer (52.3 g, 0.50 mol) dissolved in 1 L of cyclohexane. The mixture turned dark red within 2 min, and the polymerization was allowed to proceed for 6 h at 40 °C under an Ar atmosphere, after which the reaction solution was cooled down to 0 °C and MMO (4.72 g, 0.04 mol) was added. The dark-red color faded within 1 min, and the mixture was stirred for an additional 2 h at 0 °C before being terminated by adding degassed methanol (5 mL). The product solution was dried on a rotary evaporator, redissolved in 200 mL of CH2Cl2, and precipitated in 2 L of methanol. PS-MMO was dried under vacuum at 50 °C for 2 days and at 110 °C for 14 h. The PS-MMO homopolymer then served as a macroinitiator for the preparation of the midhydroxyl-functionalized poly(styrene)-b-poly(ethylene oxide) (μ-PS-PEO-OH) diblock copolymer. The PS-MMO/THF (10.0 g, 1  10-3 mol/500 mL) solution was titrated slowly by a solution of diphenylmethyl potassium until a light-pink color persisted for 20 min. The cold ethylene oxide monomer (6.0 g, 0.24 mol) was then added at 0 °C, leading to the immediate disappearance of the red color. The solution was slowly heated to 45 °C and stirred for 24 h. An additional 2 mL (38) Pangborn, A. B.; Giardello, M. A.; Grubbs, R. H.; Rosen, R. K.; Timmers, F. J. Organometallics 1996, 15, 1518. (39) Li, Z.; Hillmyer, M. A.; Lodge, T. P. Macromolecules 2004, 37, 8933. (40) Barker, M. C.; Vincent, B. Colloids Surf. 1984, 8, 289. (41) Lai, J. T.; Filla, D.; Shea, R. Macromolecules 2002, 35, 6754. (42) Ndoni, S.; Papadakis, C. M.; Bates, F. S.; Almdal, K. Rev. Sci. Instrum. 1995, 66, 1090.

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Liu et al. Scheme 1. Synthesis Route to μ-SODA Miktoarm Star Block Terpolymers

of diphenylmethyl potassium solution was injected into the solution to achieve the pink color before termination by ethyl bromide. An aliquot of the solution was taken for characterization. The remaining solution underwent hydrolysis to recover the midhydroxyl group by treating with 200 mL of HCl (2 mol/L) at 50 °C for 24 h.43 Most of the THF was removed using a rotary evaporator, and the remaining aqueous solution was extracted with 1 L of CH2Cl2. The aqueous phase was washed with 150 mL of CH2Cl2 three times. The combined CH2Cl2 solution was washed with 150 mL of a saturated sodium bicarbonate solution two times and 150 mL of deionized water four times. The organic phase was concentrated on a rotary evaporator and then precipitated in 2 L of petroleum ether. The resulting deprotected μ-PS-PEO-OH diblock copolymer was dried under vacuum at 60 °C overnight. The presence of the midhydroxyl group was confirmed using 1H NMR spectroscopy. μ-PS-PEO-OH (0.03 g) was dissolved in 0.75 mL of CDCl3, followed by the addition of 0.5 mL of trifluoroacetic anhydride. The mixture was stirred for 30 min before 1H NMR characterization. The PS-b-PEO macroinitiator ( μ-PS-PEO-CTA) for the preparation of μ-SODA via RAFT polymerization was synthesized as follows.44 CTA (1.0 g, 2.7  10-3 mol) was mixed with excess anhydrous oxalyl chloride in 10 mL of dry CH2Cl2 under an Ar purge and stirred at room temperature for 2 h, after which excess reagent and solvent were removed under vacuum. The viscous liquid was redissolved in 45 mL of CH2Cl2, followed by the addition of μ-PS-PEO-OH (4.5 g, 3  10-4 mol). The coupling reaction was allowed to proceed at room temperature for 24 h. μ-PS-PEO-CTA was purified by precipitation in petroleum ether four times to remove uncoupled CTA. Then, μ-PS-PEOCTA macroinitiator (1.0 g, 6.5  10-5 mol), DMAEA (3.7 g, 2.6  10-2 mol), and AIBN (2.2 mg, 1.3  10-5 mol) were dissolved in 5 mL of N,N-dimethylformamide (DMF) and purged with Ar for 30 min to remove air. RAFT polymerization was carried out at 80 °C under an Ar atmosphere for 4.5 h. After quenching in an ice bath, most of the unreacted DMAEA and DMF were removed under vacuum at 50 °C. The μ-SODA crude product was redissolved in 10 mL of CH2Cl2, precipitated in 100 mL of petroleum ether, and dried under vacuum at 50 °C for 2 days. The conversion of DMAEA monomer was estimated to be 35%.

Preparation of PEO-b-PDMAEA (ODA) and PS-bPDMAEA (SDA) Diblock Copolymers. Both ODA and SDA diblock copolymers were prepared via RAFT polymerization using R-methoxy-ω-hydroxy PEO and PS-MMO as the corresponding macroinitiators. Detailed synthesis routes and procedures can be found in the Supporting Information. Molecular Characterization. Size exclusion chromatography (SEC) was performed on a Waters 150C ALC/GPC equipped (43) Meyers, A. I.; Durandetta, J. L.; Munavu, R. J. Org. Chem. 1975, 40, 2025. (44) Rzayev, J.; Hillmyer, M. A. J. Am. Chem. Soc. 2005, 127, 13373.

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with three Phenomenex Phenogel columns of 103, 104, and 105 A˚ porosities, a Wyatt OPTILAB refractive index detector, and a Wyatt DAWN multiangle light-scattering detector. THF with 1 vol % N,N,N0 ,N0 -tetramethylethylenediamine was used as the mobile phase at a flow rate of 1 mL/min at 40 °C. 1H NMR spectra were recorded on a Varian Inova 500 MHz spectrometer at room temperature. All samples were dissolved in CDCl3. Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry was performed on a Bruker Reflex III instrument equipped with a 337 nm nitrogen laser, a delayed extraction Scout ion source, a high-resolution multichannel plate detector in reflection mode, and a 2 GHz digitizer. For the PS-MMO homopolymer, the matrix and cationizing agents were dithranol and silver trifluoroacetate, respectively. The sample solution consisted of 20 μL of dithranol (20 mg/mL in THF), 2 μL of silver trifluoroacetate (2 mg/mL in THF), and 10 μL of polymer solution (10 mg/mL in THF). Micelle Solution Preparation. μ-SODA micelle solutions were prepared using two dialysis methods: (Method one) μ-SODA was first dissolved in THF to make a 1 wt % solution and then dialyzed against deionized water for 3 days; the deionized water was changed twice a day. After dialysis, the pH of the solution was adjusted by adding an equal volume of buffer solution to make a 0.2-0.3 wt % dispersion. (Method two) μ-SODA was first dissolved in THF to make a 1 wt % solution and then directly dialyzed against buffer solution for 3 days; the buffer solution was changed twice a day. After dialysis, the ionic strength of the solution was adjusted by adding an equal volume of deionized water to make a 0.2-0.3 wt % dispersion. ODA micelle solutions were prepared by the direct dispersion of solid ODA samples into buffer solutions to make 1 wt % dispersions. All micelle solutions were continuously stirred in sealed vials at room temperature. Dynamic Light Scattering (DLS). Solutions were passed through 0.45 μm microfilters (Millipore) into 0.5-in.-diameter optical glass tubes that were scrupulously dusted. DLS measurements were carried out at 25 °C using a homebuilt photometer equipped with an electrically heated silicon oil bath, a Lexel 75 Ar+ laser operating at 488 nm, a Brookhaven BI-DS photomultiplier, and a Brookhaven BI-9000 correlator.45 The intensity autocorrelation functions, g2(t), were recorded at six angles ranging from 45 to 120° and accepted when the baseline differences (calculated vs measured) were less than 0.1%. A doubleexponential expression (eq 1) was used to fit the autocorrelation functions to extract the decay rates, Γ1 and Γ2. g2 ðtÞ -1 ¼ ½A1 expð -Γ1 tÞ þ A2 expð -Γ2 tÞ2

ð1Þ

(45) Pan, C.; Maurer, W.; Liu, Z.; Lodge, T. P.; Stepanek, P.; von Meerwall, E. D.; Watanabe, H. Macromolecules 1995, 28, 1643.

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After obtaining Γ1 and Γ2 at different scattering angles, two mutual diffusion coefficients Dm were determined by linearly fitting Γ1 and Γ2 versus q2, where q = (4πn/λ)sin(θ/2) and n, λ, and θ are the solvent refractive index, laser wavelength, and scattering angle, respectively. The hydrodynamic radius (Rh) was obtained from the Stokes-Einstein equation (eq 2), Rh ¼

kB T 6πηs Dm

ð2Þ

where kB, T, and ηs are the Boltzmann constant, absolute temperature, and solvent viscosity, respectively. Inverse Laplace transformations were also performed using constrained regularization program REPES to obtain the decay rate distributions, which reflect the size distribution of the micelles.46 Differential Scanning Calorimetry (DSC). DSC samples were prepared by sealing ∼10 mg of polymer in aluminum hermetic pans. DSC experiments were carried out using a TA Instruments Q1000 equipped with liquid nitrogen as a cooling system and He as a purge gas. To erase the temperature history, the samples were first equilibrated at 130 °C (well above the glasstransition temperature (Tg) of either component) for 2 min and then cooled to -80 °C and annealed for an additional 2 min. DSC scans were obtained by heating the samples from -80 to 130 °C at 10 °C/min, and Tg values were extracted using Universal Analysis 2000.

Cryogenic Transmission Electron Microscopy (CryoTEM). CryoTEM samples were prepared in a controlled environment vitrification system (CEVS), which was saturated with water vapor at room temperature.47 Typically, a drop of the micelle dispersion (5-10 μL) was loaded onto a lacey Formvar carbon-supported grid held by tweezers. The excess solution was blotted with a piece of filter paper to form thin films of 100-300 nm thickness in the holes. After allowing ca. 10 s to relieve the stress produced during the blotting, the sample was plunged into a reservoir of liquid ethane cooled by liquid nitrogen. The vitrified samples were then kept in liquid nitrogen until they were mounted on a cryogenic sample holder (Gatan 626) and examined with a JEOL1210 TEM operating at 120 kV and -178 °C. The images were recorded on a Gatan 724 multiscan CCD and processed with DigitalMicrographs version 3.3.1. Phase contrast enhancement was achieved at a nominal underfocus of 6-15 μm. The ramp-shaped optical density gradients in the background were digitally corrected.

Results Preparation of μ-SODA. The three-step synthesis route to μ-SODA miktoarm star block terpolymers is almost identical to that of μ-EOF except that the final coupling reaction is substituted by the RAFT polymerization (Scheme 1).39 Styrene was anionically polymerized in cyclohexane, and after complete consumption of the monomer, excess MMO coupling agent was added to terminate the polymerization, which yields a heterobifunctionalized PS (PS-MMO) with a narrow molecular weight distribution (PDI ≈ 1.03). The structure of mono-MMO end-capped PS was confirmed by the MALDI-TOF mass spectrum, which shows a set of PS-MMO peaks with excellent agreement between the experimental molecular weights and the calculated ones (Figure S1). In addition, the high level of regioselectivity of MMO addition to the propagating PS chain resulted in >95% addition at the least-substituted ring carbon, as supported by 1H NMR spectroscopy (Figure S2).39,48 (46) Jakes, J. Collect. Czech. Chem. Commun. 1995, 60, 1781. (47) Bellare, J. R.; Davis, H. T.; Scriven, L. E.; Talmon, Y. J. Electron Microsc. 1988, 10, 87. (48) Quirk, R. P.; Lizarraga, G. M. Macromolecules 1998, 31, 3424.

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Figure 1. SEC traces of (a) PS-MMO homopolymer precursor, (b) μ-PS-PEO-OH diblock copolymer precursor, and (c) μ-SODA(105-8) star block terpolymer.

The free hydroxyl group at the end of PS-MMO was converted into the corresponding potassium alkoxide by titration with diphenylmethyl potassium before the addition of ethylene oxide.40 The oxyanionic polymerization of ethylene oxide was carried out in THF at 45 °C for 24 h and terminated using ethyl bromide, which eliminated the possibility of having an additional hydroxyl group at the end of the PEO chain (Scheme 1). The methoxylmethyl protecting group then underwent hydrolysis upon treatment with a 2 M HCl solution to recover the midhydroxyl group. The successful deprotection of the methoxylmethyl group was confirmed by 1H NMR spectroscopy (Figures S3 and S4), which shows the disappearance of the characteristic resonance at 4.5 ppm (-O-CH2-OCH3) after hydrolysis. The presence of the midhydroxyl group was confirmed by treating μ-PS-PEO-OH with trifluoroacetic anhydride, which yields a resonance at 4.4 ppm (-O-CH2-OCOCF3, Figure S4).39 In addition, the SEC trace of μ-PS-PEO-OH has a single narrow peak with a lower elution volume than that of the PS-MMO precursor, consistent with no significant decomposition of diblock copolymer occurring during hydrolysis (Figure 1). The synthesis of μ-SODA was accomplished by the RAFT polymerization of DMAEA using μ-PS-PEO-CTA as the macroinitiator, which was prepared by the reaction between μ-PS-PEO-OH and the acid chloride end-functionalized CTA (Scheme 1). Compared with PS-MMO and μ-PS-PEO-OH, the SEC trace of μ-SODA exhibits a small shift to lower elution volume, which is due in part to the relatively strong attraction between the PDMAEA block and the column packing material (Figure 1)49 and in part to the star architecture. The formation of μ-SODA was further confirmed by the 1H NMR spectrum that shows three sets of characteristic resonances for PS, PEO, and PDMAEA, respectively (Figure 2). Taken together, the characterization results strongly support the formation of a new μ-ABC star block terpolymer containing one hydrophobic block (PS), one hydrophilic block (PEO), and one pH-responsive block (PDMAEA). Both ODA and SDA diblock copolymers were prepared by RAFT polymerization analogous to that of μ-SODA (Schemes S1 and S2), and their detailed characterization data are provided (49) Hoogeveen, N. G.; Cohen Stuart, M. A.; Fleer, G. J. Macromol. Chem. Phys. 1996, 197, 2553.

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Figure 2. 1H NMR spectrum of μ-SODA(10-5-8) star block terpolymer (*: CHCl3).

in Supporting Information (Figures S6-S9). The molecular parameters of the polymers employed in this study are summarized in Table 1. pH Response of the PDMAEA Block. To investigate the transition of PDMAEA from hydrophilic to hydrophobic, we performed DLS measurements on dilute dispersions of the ODA(2-14) diblock copolymer in buffer solutions. The expectation is that, as PDMAEA becomes hydrophobic with increasing pH, ODA will self-assemble to form micelles with a PDMAEA core and a PEO corona. Figure 3a compares the intensity autocorrelation functions g12(t) at different pH values, where the mean decay time reflects the average size of the aggregates. The higher pH values (8.1 and 9.0) lead to longer relaxation times, indicating the formation of ODA micelles. This is also supported by the increase in scattering intensity, which is proportional to the molecular weight of the aggregates, with pH (Figure 3a, inset). Furthermore, the evolution of the overall size distributions with pH is depicted in Figure 3b; two distinct Rh peaks centered around 2 and 30 nm are apparent. We assign the former one to the ODA unimer, which dominates at pH 2.6 and 5.2. (The weak peak above 10 nm is caused by the slight fluctuation in the baseline of the autocorrelation function due to the relative low scattering intensity.) Upon increasing the pH to 7.2, the solvent is no longer good for PDMAEA, resulting in the emergence of the ODA micelle peak centered around 30 nm. For pH g8.1, PDMAEA becomes so hydrophobic that most of the ODA unimers self-assemble into micelles. The presence of the residual unimer peak indicates that the solvent is still somewhat compatible with PDMAEA and thus the cores might be swollen by the solvent to some extent. Therefore, in the case of μ-SODA micelles in buffer solutions, the transition of PDMAEA from the corona to the core is expected to occur near pH 7, and PDMAEA becomes hydrophobic and locates in the micelle core at pH g8. 13722 DOI: 10.1021/la900845u

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Self-Assembly of μ-SODA in Buffer Solutions. DLS was performed to monitor the average size of the μ-SODA micelles in dilute buffer solutions after different time intervals to ensure the achievement of steady-state morphology. No temporal evolution occurred for μ-SODA(10-5-23) micelles regardless of the pH value or preparation method (Experimental Section); a gradual increase in micelle size was observed for μ-SODA(10-5-8) during the first 3 weeks (Figure S11). Therefore, all micelle solutions were stirred at room temperature for 3 weeks before carrying out any further measurements. Table 2 summarizes the double-exponential fitting results of μ-SODA micelles formed in buffer solutions as the pH was varied from 2.6 to 9.0. The evolution of the size distributions of these micelles obtained from REPES is provided in Figure S12, and no significant difference was observed between dialysis methods one and two (Figure 4). In this report, we will focus on the micelles obtained in pH 2.6 and 9.0 buffer solutions, where mixed corona and multicompartment micelles are expected to form, respectively, on the basis of the ODA results; it is interesting, however, that there is a maximum in Rh,2 at an intermediate pH for both μ-SODA(105-23) and μ-SODA(10-5-8) (Figure S12). The μ-SODA(10-5-23) micelles formed in pH 2.6 buffer solution exhibit two distinct Rh peaks at ca. 15 and 100 nm (Figure 4), consistent with the two Rh values obtained from the double-exponential fitting. As the pH increases to 9.2, these two peaks shift slightly to larger values and almost merge into one much broader distribution (Figure 4). A similar phenomenon was also observed for μ-SODA(10-5-8) micelles as shown in Figure S12b. Whereas DLS probes the ensemble behavior of bulk solutions, cryoTEM enables the direct visualization and determination of the detailed micelle structures, albeit on limited sample volumes. Typical cryoTEM images of μ-SODA(10-5-23) and μ-SODA(105-8) micelles at pH 2.6 and 9.0 prepared using dialysis method one are shown in Figure 5. At pH 2.6, μ-SODA(10-5-23) forms mostly mixed corona (PEO + PDMAEA) spherical micelles with a core radius of ca. 5 nm and a few mixed corona wormlike micelles with a length of ca. 100 nm (Figure 5a). These observations correspond well with the two distinct peaks seen in the Rh distribution at pH 2.6 (Figure 4). Upon increasing the pH to 9.2, μ-SODA(10-5-23) micelles evolve to mostly wormlike micelles with varied lengths (Figure 5b) that match the broad Rh distribution fairly well (Figure 4). The micellar cores possess alternating black and gray subdomains (Figure 5e). We assign the dark domains to the PS and the light domains to the PDMAEA as a result of the swelling of the PDMAEA core by the solvent as suggested by the DLS results of the ODA system, thus resulting in a significantly lower electron density difference with the vitrified solvent. Combined with the observation of two distinct Tg’s from the DSC traces of SDA diblock copolymers that indicate microphase segregation (Figure S10 and Table S1), it is evident that μ-SODA(10-5-23) forms multicompartment (PS + PDMAEA) wormlike micelles at pH 9.0. A similar evolution of the micelle structure was also identified for either μ-SODA(10-5-8) micelles prepared via dialysis method one (Figure 5c,d) or μ-SODA(10-5-23) micelles prepared via dialysis method two (Figure 6), suggesting that the micellization is dominated by the PS block, which would be relatively insensitive to pH.

Discussion The formation of well-defined μ-SODA star block terpolymers can be attributed to the use of the MMO coupling agent that contains one active and one dormant hydroxyl group, allowing Langmuir 2009, 25(24), 13718–13725

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Article Table 1. Molecular Parameters of μ-SODA Star Triblock and SDA and ODA Diblock Copolymers

sample IDa

NPSb

NPEOc

NPDMAEAc

wPSd

wPEOd

wPDMAEAd

Mn(kDa)e

PDIf

ODA(2-14) 50 96 0.14 0.86 15.9 1.21 SDA(10-9) 97 64 0.52 0.48 19.4 1.05 SDA(10-23) 97 159 0.31 0.69 33.0 1.13 μ-SODA(10-5-8) 97 122 53 0.44 0.23 0.33 22.6 1.04 μ-SODA(10-5-23) 97 122 160 0.26 0.14 0.60 38.3 1.10 a The numbers in parentheses denote the molecular weight of each block in kDa. b Number-average degree of polymerization calculated using MALDI-TOF. c Calculated by 1H NMR spectroscopy based on known NPS. d The weight fractions were calculated using the molecular weights from MALDI-TOF and 1H NMR spectra. e The sum of each block determined from MALDI-TOF and 1H NMR spectra. f PDI was obtained from SEC traces.

Table 2. μ-SODA Micelle Sizes from Double-Exponential Fits terpolymer

pH

Rh,1 (nm)

Rh,2 (nm)

μ-SODA(10-5-23)

2.6 5.2 7.2 8.1 9.0

16 28 29 20 19

100 215 339 116 124

μ-SODA(10-5-8)

2.6 5.2 7.2 8.1 9.0

23 44 33 31 29

140 317 236 242 186

Figure 3. DLS results obtained from dilute dispersions (1 wt %) of ODA(2-14) in buffer solutions: (a) Intensity autocorrelation functions. (Inset) scattering intensities. (b) Rh distributions. The scattering angle is 90°.

the stepwise polymerization of the PEO and PDMAEA block, respectively.19,39 The RAFT polymerization employed here is amenable to many families of monomers, and thus the synthesis route is applicable to other star block terpolymers containing RAFT-grown polymers such as a poly(acrylate) and poly(vinyl pyridine). At low pH, both terpolymers described here form predominantly spherical micelles with PS cores, as expected from the fact that the combined corona blocks are significantly larger than the core block. The DLS results suggest that under these conditions μ-SODA(10-5-23) forms smaller micelles than μ-SODA(10-5-8) (16 nm vs 23 nm), which may be attributed to a smaller aggregation number enforced by a more congested corona. The evolution of μ-SODA mixed corona (PEO + PDMAEA corona, PS core) micelles to multicompartment (PEO corona, PS + PDMAEA core) micelles with increasing pH (Figures 4-6) is controlled by the degree of protonation of PDMAEA (pKb ∼6.5), which in turn Langmuir 2009, 25(24), 13718–13725

Figure 4. Rh distributions obtained from dilute dispersions (0.3 wt %) of μ-SODA(10-5-23) in buffer solutions using REPES. The scattering angle is 90°.

determines its hydrophilicity.36,37 The PDMAEA block is deprotonated and hence relatively hydrophobic at pH ∼9.0-9.2, leading to the formation of multicompartment micelles. In this case, the combined volume fraction of the core blocks is larger than the corona, consistent with the transition to more extended, wormlike micelles. Although the cryoTEM images are not favored by a huge contrast between PS and PDMAEA, it appears that the two components form alternating domains along the core of the worm. This is similar to the results presented previously for μ-EOF polymers;15-17 however, in that case the DOI: 10.1021/la900845u

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Figure 6. CryoTEM images obtained from dilute dispersions (0.2 wt %) of μ-SODA(10-5-23) in buffer solutions prepared via dialysis method two: (a) pH 2.6 and (b) pH 9.2. The scale bars indicate 100 nm.

The formation of multicompartment micelles requires a strong effective repulsion between each block such that they segregate into distinct nanodomains.9,15,19 The critical value NχODT for the order-disorder transition (ODT) to occur in the bulk is 10.5 for a symmetric block copolymer.50 For PS and PDMAEA, the Flory-Huggins interaction parameter (χPS-PDMAEA) is roughly estimated to be 0.07 at 25 °C,51-53 and thus χNPS-PDMAEA is 10.8 and 18.5 for μ-SODA(10-5-8) and μ-SODA(10-5-23), respectively. However, any swelling of the PDMAEA core by water at pH ∼9.0-9.2 will also play a role in the determination of the effective χPS-PDMAEA. Because PS is very hydrophobic, even a slight swelling of the PDMAEA core should significantly increase the effective χPS-PDMAEA. Consequently, we conclude that the effective χNPS-PDMAEA is large enough to induce the microphase separation within the micellar core. This conclusion is supported by the DSC results on the dry SDA(10-9) and SDA(10-23) samples (Figure S10 and Table S1) as well as by the cryoTEM images (Figures 5b,d and 6b). Figure 5. CryoTEM images obtained from dilute dispersions (0.2-0.3 wt %) in buffer solutions prepared via dialysis method one: (a) μ-SODA(10-5-23) at pH 2.6, (b) μ-SODA(10-5-23) at pH 9.0, (c) μ-SODA(10-5-8) at pH 2.6, (d) μ-SODA(10-5-8) at pH 9.0, and (e) an expanded view of μ-SODA(10-5-23) multicompartment wormlike micelles shown in panel b. The scale bars indicate 100 nm for a-d and 50 nm for e.

larger contrast between PEE and PFPO blocks endowed the images with greater structural resolution. Also, the larger interfacial tension between the two hydrophobic blocks imposed an essentially flat interface between PEE and PFPO domains. A further, potentially important difference in the current system is the glassy nature of the PS cores. This may restrict micellar structural rearrangements. For example, the cryoTEM images at high pH suggest that the PS cores are “bridged” by overlapping PDMAEA patches to form micellar strings rather than true wormlike micelles. We also note that longer strings or worms are evident in the cryoTEM images than are implied by the DLS results. We speculate that the filtration step prior to DLS measurements may have broken some of the longer strings; the cryoTEM samples were not filtered. At intermediate pH (5.2-8.1), the partial protonation of the PDMAEA (the estimated degrees of protonation are 0.67 and 0.20 for pH ∼7.2 and 8.1, respectively) apparently induces some intermicellar attraction, as inferred from the larger hydrodynamic radii (Table 2). We speculate that this might involve the formation of hydrogen bonds between the protonated and free dimethylamino groups, but a full discussion of this pH regime is beyond the scope of this report. 13724 DOI: 10.1021/la900845u

Summary A new miktoarm star terpolymer system has been developed involving one hydrophobic block (PS), one hydrophilic block (PEO), and one pH-sensitive polybase (PDMAEA). The synthetic protocol to achieve well-defined μ-SODA terpolymers involved two successive anionic polymerizations and one RAFT polymerization. The micellization properties of two μ-SODA samples in water were studied by DLS and cryoTEM as a function of pH. Upon dispersion in water at low pH, where the PDMAEA block is fully protonated, the micelles are predominantly spherical, with a mixed corona of the two hydrophilic blocks. Conversely, at higher pH an increasing proportion of extended micellar structures is seen in which PS cores are bridged by overlapping PDMAEA brushes. Conceivably, the glassy nature of the PS domains inhibits full structural rearrangement to a segmented wormlike micelle. Nevertheless, measurements on two bulk SDA diblocks indicate that the two hydrophobic blocks should form well-segregated domains. These results indicate that the introduction of a pH-sensitive block into a miktoarm star framework provides a convenient experimental handle to tune multicompartment micellar morphology; it will be of interest to compare these results with similar pH-responsive miktoarm star terpolymers with rubbery core blocks. (50) Leibler, L. Macromolecules 1980, 13, 1602. (51) Russell, T. P.; Hjelm, R. P.; Seeger, P. A. Macromolecules 1990, 23, 890. (52) Russell, T. P. Macromolecules 1993, 26, 5819. (53) Costa, A. C.; Geoghegan, M.; Vlcek, P.; Composto, R. J. Macromolecules 2003, 36, 9897.

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Acknowledgment. This work was supported by the MRSEC program of the National Science Foundation under awards DMR0212302 and DMR-0819885 at the University of Minnesota. Parts of this work were carried out in the University of Minnesota I.T. Characterization Facility, which received partial support from NSF through the NNIN program. We thank Dr. Aggeliki I. Triftaridou for helpful synthetic input during the initial stages of this project.

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Article

Supporting Information Available: Complementary MALDI-TOF, SEC, and 1H NMR results of μ-SODA block terpolymers and ODA and SDA diblock copolymers. DSC results of SDA diblock copolymers and PS/PDMAEA blends. DLS results of μ-SODA micelles in buffer solutions. This material is available free of charge via the Internet at http://pubs.acs.org.

DOI: 10.1021/la900845u

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