Thermally Responsive PM(EO)2MA Magnetic Microgels via Activators

Aug 7, 2009 - Activators generated by electron transfer atom transfer radical polymerization (AGET ATRP) of di(ethylene glycol) methyl ether methacryl...
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Chem. Mater. 2009, 21, 3965–3972 3965 DOI:10.1021/cm901143e

Thermally Responsive PM(EO)2MA Magnetic Microgels via Activators Generated by Electron Transfer Atom Transfer Radical Polymerization in Miniemulsion Hongchen Dong,† Venkat Mantha,‡ and Krzysztof Matyjaszewski*,† †

Center for Macromolecular Engineering, Department of Chemistry, Carnegie Mellon University, 4400 Fifth Avenue, Pittsburgh, Pennsylvania 15213, and ‡Department of Anesthesiology, Magee-Womens Hospital, 300 Halket Street, Pittsburgh, Pennsylvania 15213 Received April 24, 2009. Revised Manuscript Received July 17, 2009

Activators generated by electron transfer atom transfer radical polymerization (AGET ATRP) of di(ethylene glycol) methyl ether methacrylate (M(EO)2MA) was successfully conducted in miniemulsion at 65 °C. The reaction system was stable without diffusion of monomer and polymer into the aqueous phase because the monomer is water-insoluble and PM(EO)2MA becomes hydrophobic above 25 °C. The polymerization was well-controlled with a mild water-soluble reducing agent, hydrazine, yielding PM(EO)2MA homopolymer with narrow molecular weight distribution (Mw/Mn =1.2-1.6). Using this technique, well-defined PM(EO)2MA microgels were prepared with degradable disulfide cross-linker. The microgels became magnetic after physically loading oleic acid-coated Fe3O4 nanoparticles, which could not diffuse out of the microgels due to their hydrophobicity. Thermally responsive and drug loading-releasing behavior of the magnetic microgels was studied using Rhodamine B as a model for hydrophilic drugs. The drug releasing behavior can be well-controlled by both temperature and addition of reducing agent, indicating that the PM(EO)2MA magnetic microgels could find potential application for controlled targeted drug delivery. Introduction Stimuli-responsive polymer microgels show great potential in biomedical applications, such as controlled drug delivery systems.1-7 In addition to tunable chemical functionality and three-dimensional physical structure, high water content, and biocompatibility, the stimuliresponsive microgels exhibit a phase transition. Thus, abrupt volume changes can be triggered in response to small changes in external environmental parameters (e.g., pH, temperature, light, or electric field). Drug encapsulation and delivery can be efficiently controlled through reversible swelling-deswelling behavior triggered by external stimuli.8-11 During the past few years, incorpor*Corresponding author. E-mail: [email protected].

(1) Langer, R. Nature 1998, 392, 5–10. (2) Kost, J.; Langer, R. Adv. Drug Delivery Rev. 2001, 46, 125–148. (3) Peppas, N. A.; Hilt, J. Z.; Khademhosseini, A.; Langer, R. Adv. Mater. 2006, 18, 1345–1360. (4) Hoffman, A. S.; Stayton, P. S. Prog. Polym. Sci. 2007, 32, 922–932. (5) Nayak, S.; Lyon, L. A. Angew. Chem., Int. Ed. 2005, 44, 7686–7708. (6) Oh, J. K.; Drumright, R.; Siegwart, D. J.; Matyjaszewski, K. Prog. Polym. Sci. 2008, 33, 448–477. (7) Bajpai, A. K.; Shukla, S. K.; Bhanu, S.; Kankane, S. Prog. Polym. Sci. 2008, 33, 1088–1118. (8) Kiser, P. F.; Wilson, G.; Needham, D. Nature 1998, 394, 459–462. (9) Bromberg, L.; Temchenko, M.; Hatton, T. A. Langmuir 2003, 19, 8675–8684. (10) Nolan, C. M.; Serpe, M. J.; Lyon, L. A. Biomacromolecules 2004, 5, 1940–1946. (11) Serpe, M. J.; Yarmey, K. A.; Nolan, C. M.; Lyon, L. A. Biomacromolecules 2005, 6, 408–413. r 2009 American Chemical Society

ating magnetic nanoparticles into polymer microgels has received much attention, because it opens up potential applications for targeted drug delivery systems.12-23 The magnetic property can be used to guide the microgels to desired locations by an external magnetic field, so that drug transportation efficiency may be improved and toxic side effects reduced.24,25 Moreover, temperature-responsive polymer microgels containing magnetic nanoparticles are particularly interesting because the temperature (12) Kondo, A.; Fukuda, H. J. Ferment. Bioeng. 1997, 84, 337–341. (13) Xulu, P. M.; Filipcsei, G.; Zrinyi, M. Macromolecules 2000, 33, 1716–1719. (14) Elaissari, A.; Bourrel, V. J. Magn. Magn. Mater. 2001, 225, 151– 155. (15) Deng, Y.; Yang, W.; Wang, C.; Fu, S. Adv. Mater. 2003, 15, 1729– 1732. (16) Pich, A.; Bhattacharya, S.; Lu, Y.; Boyko, V.; Adler, H.-J. P. Langmuir 2004, 20, 10706–10711. (17) Zhang, J.; Xu, S.; Kumacheva, E. J. Am. Chem. Soc. 2004, 126, 7908–7914. (18) Suzuki, D.; Kawaguchi, H. Colloid Polym. Sci. 2006, 284, 1443– 1451. (19) Guo, J.; Yang, W.; Wang, C.; He, J.; Chen, J. Chem. Mater. 2006, 18, 5554–5562. (20) Rubio-Retama, J.; Zafeiropoulos, N. E.; Serafinelli, C.; RojasReyna, R.; Voit, B.; Lopez Cabarcos, E.; Stamm, M. Langmuir 2007, 23, 10280–10285. (21) Schmidt, A. M. Colloid Polym. Sci. 2007, 285, 953–966. (22) Wong, J. E.; Gaharwar, A. K.; Mueller-Schulte, D.; Bahadur, D.; Richtering, W. J. Magn. Magn. Mater. 2007, 311, 219–223. (23) Pyun, J. Polym. Rev. 2007, 47, 231–263. (24) Widder, K. J.; Senyei, A. E.; Scarpelli, D. G. Proc. Soc. Exp. Biol. Med. 1978, 158, 141–146. (25) Gupta, A. K.; Gupta, M. Biomaterials 2005, 26, 3995–4021.

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change can be generated by applying an external alternating current magnetic field onto magnetic nanoparticles. Therefore, a remote-controlled drug release might be achieved without an additional stimulus.26 Among a wide variety of magnetic nanoparticles, iron oxides are the most intensively studied because they are nontoxic toward humans. For instance, the use of superparamagnetic iron oxide nanoparticles as contrast agents in magnetic resonance imaging has been approved by the Food and Drug Administration (FDA).27,28 A number of suitable methods have been developed for the synthesis of superparamagnetic iron oxide nanoparticles with 10-20 nm diameters, which are well dispersed in organic solvents.29-31 To date, there are limited reports on temperature-responsive magnetic microgels, and in most cases, thermosensitive poly(N-isopropylacrylamide) (PNIPAM) homopolymer or its copolymers were studied.13-15,18-20,22 PNIPAM is biocompatible and exhibits a lower critical solution temperature around 32 °C.32 A variety of approaches have been reported on preparation of PNIPAM-based magnetic microgels, including surfactant-free polymerization using aqueous ferrofluids as reaction media,12,14 layer-by-layer assembly technique,20,22 and guided deposition of iron oxide in charged areas.17,18 Another kind of thermosensitive magnetic microgels based on poly(vinylcaprolactam) has also been synthesized. 16 The vinylcaprolactambased microgel with magnetite nanoparticles deposited inside exhibited a volume phase transition at around 28 °C. Poly(ethylene glycol) (PEG) is well-known as a noncharged, nontoxic, protein-resistant, and biocompatible polymer and has been widely applied in biomedical applications.33,34 Recent reports describe a new kind of thermally responsive polymer based on oligo(ethylene glycol) methyl ether methacrylate.35-42 Copolymerization of two types of oligo(ethylene glycol) macromonomers with different numbers of ethylene glycol moieties (26) Mueller-Schulte, D.; Schmitz-Rode, T. J. Magn. Magn. Mater. 2006, 302, 267–271. (27) Bulte, J. W. M.; Zhang, S. C.; Van Gelderen, P.; Herynek, V.; Jordan, E. K.; Duncan, I. D.; Frank, J. A. Proc. Natl. Acad. Sci. U. S.A. 1999, 96, 15256–15261. (28) Lattuada, M.; Hatton, T. A. Langmuir 2007, 23, 2158–2168. (29) Lu, A. H.; Salabas, E. L.; Schueth, F. Angew. Chem., Int. Ed. 2007, 46, 1222–1244. (30) Sun, S.; Zeng, H.; Robinson, D. B.; Raoux, S.; Rice, P. M.; Wang, S. X.; Li, G. J. Am. Chem. Soc. 2004, 126, 273–279. (31) Wang, X.; Zhuang, J.; Peng, Q.; Li, Y. Nature 2005, 437, 121–124. (32) Xia, Y.; Yin, X.; Burke, N. A. D.; Stoever, H. D. H. Macromolecules 2005, 38, 5937–5943. (33) Schutz, E. Arzneim. Forsch. 1953, 3, 451–456. (34) Abuchowski, A.; Van Es, T.; Palczuk, N. C.; Davis, F. F. J. Biol. Chem. 1977, 252, 3578–3581. (35) Han, S.; Hagiwara, M.; Ishizone, T. Macromolecules 2003, 36, 8312–8319. (36) Kitano, H.; Hirabayashi, T.; Gemmei-Ide, M.; Kyogoku, M. Macromol. Chem. Phys. 2004, 205, 1651–1659. (37) Ali, M. M.; Stover, H. D. H. Macromolecules 2004, 37, 5219–5227. (38) Zhao, B.; Li, D.; Hua, F.; Green, D. R. Macromolecules 2005, 38, 9509–9517. (39) Lutz, J.-F.; Hoth, A. Macromolecules 2006, 39, 893–896. (40) Lutz, J.-F.; Andrieu, J.; Uezguen, S.; Rudolph, C.; Agarwal, S. Macromolecules 2007, 40, 8540–8543. (41) Yamamoto, S.-I.; Pietrasik, J.; Matyjaszewski, K. J. Polym. Sci., Part A: Polym. Chem. 2007, 46, 194–202. (42) Yamamoto, S.-i.; Matyjaszewski, K. Polym. J. 2008, 40, 496–497.

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yields thermosensitive random copolymers with a tunable LCST between 25 and 90 °C.43,44 The oligo(ethylene glycol) methacrylate-based (co)polymers exhibit comparable thermoresponsive behaviors to PNIPAM,45 and moreover, excellent biocompatible and biorepellent properties,43,46 indicating that they have great potential for the next generation of smart biomaterials. Recently, thermally responsive magnetic nanoparticles were synthesized by grafting the oligo(ethylene glycol) methacrylate-based copolymer chains onto magnetite nanoparticles. The nanoparticles exhibited reversible and temperature-sensitive agglomeration within red blood cells, in addition to colloidal stability and biocompatibility.47 Microgels are generally prepared by heterogeneous polymerization of monomers in the presence of either difunctional or multifunctional cross-linkers.5,6,48-53 Activators generated by electron transfer atom transfer radical polymerization54-67 (AGET ATRP) in the (inverse) miniemulsion system has been developed and applied to prepare well-defined microgels with narrow size distribution, a high degree of chain end functionality, a uniform cross-linked network, and properties (43) Lutz, J.-F. J. Polym. Sci., Part A: Polym. Chem. 2008, 46, 3459–3470. (44) Yamamoto, S.-I.; Pietrasik, J.; Matyjaszewski, K. J. Polym. Sci., Part A: Polym. Chem. 2008, 46, 194–202. (45) Lutz, J.-F.; Akdemir, O.; Hoth, A. J. Am. Chem. Soc. 2006, 128, 13046–13047. (46) Siegwart, D. J.; Oh, J. K.; Gao, H.; Bencherif, S. A.; Perineau, F.; Bohaty, A. K.; Hollinger, J. O.; Matyjaszewski, K. Macromol. Chem. Phys. 2008, 209, 2179–2193. (47) Chanana, M.; Jahn, S.; Georgieva, R.; Lutz, J.-F.; Baumler, H.; Wang, D. Chem. Mater. 2009, 21, 1906–1914. (48) Li, K.; Stover, H. D. H. J. Polym. Sci., Part A: Polym. Chem. 1993, 31, 3257–3263. (49) Li, W.-H.; Li, K.; Stover, H. D. H. J. Polym. Sci., Part A: Polym. Chem. 1999, 37, 2295–2303. (50) Li, W.-H.; Stover, H. D. H. J. Polym. Sci., Part A: Polym. Chem. 1999, 37, 2899–2907. (51) Jones, C. D.; Lyon, L. A. Macromolecules 2000, 33, 8301–8306. (52) (a) Qiu, J.; Charleux, B.; Matyjaszewski, K. Prog. Polym. Sci. 2001, 26, 2083–2134. (b) Lowe, A. B.; McCormick, C. L. Prog. Polym. Sci. 2007, 32, 283–351. (53) Gao, H.; Matyjaszewski, K. Prog. Polym. Sci. 2009, 34, 317–350. (54) Xia, J.; Matyjaszewski, K. Macromolecules 1999, 32, 2434–2437. (55) Wang, J.-S.; Matyjaszewski, K. J. Am. Chem. Soc. 1995, 117, 5614– 5615. (56) (a) Matyjaszewski, K.; Xia, J. Chem. Rev. 2001, 101, 2921–2990. (b) Tsarevsky, N. V.; Matyjaszewski, K. Chem. Rev. 2007, 107, 2270–2299. (c) Matyjaszewski, K.; Tsarevsky, N. V. Nature Chem. 2009, 1, 276–288. (57) Braunecker, W. A.; Matyjaszewski, K. Prog. Polym. Sci. 2007, 32, 93–146. (58) Jakubowski, W.; Matyjaszewski, K. Macromolecules 2005, 38, 4139–4146. (59) Min, K.; Gao, H.; Matyjaszewski, K. J. Am. Chem. Soc. 2005, 127, 3825–3830. (60) Min, K.; Jakubowski, W.; Matyjaszewski, K. Macromol. Rapid Commun. 2006, 27, 982. (61) Hizal, G.; Tunca, U.; Aras, S.; Mert, H. J. Polym. Sci., Part A: Polym. Chem. 2006, 44, 77–87. (62) Oh, J. K.; Tang, C.; Gao, H.; Tsarevsky, N. V.; Matyjaszewski, K. J. Am. Chem. Soc. 2006, 128, 5578–5584. (63) Matyjaszewski, K.; Jakubowski, W.; Min, K.; Tang, W.; Huang, J.; Braunecker, W. A.; Tsarevsky, N. V. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 15309–15314. (64) Matyjaszewski, K.; Dong, H.; Jakubowski, W.; Pietrasik, J.; Kusumo, A. Langmuir 2007, 23, 4528–4531. (65) Stoffelbach, F.; Belardi, B.; Santos, J. M. R. C. A.; Tessier, L.; Matyjaszewski, K.; Charleux, B. Macromolecules 2007, 40, 8813–8816. (66) Stoffelbach, F.; Griffete, N.; Bui, C.; Charleux, B. Chem. Commun. 2008, 4807–4809. (67) Li, W.; Min, K.; Matyjaszewski, K.; Stoffelbach, F.; Charleux, B. Macromolecules 2008, 41, 6387–6392.

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Table 1. AGET ATRP of M(EO)2MA in Miniemulsiona entry

ligand

1 2 3

BPMODA dNbpy BPMODA

reducing agent ascorbic acid ascorbic acid hydrazine

time (h)

conv. (%)

Mn(theo)b

Mnc

Mw/Mnc

8.6 8.6 5.5

67.5 56.5 84.0

50 850 42 540 63 260

87 570 63 150 64 430

3.30 1.82 1.61

a M(EO)2MA/EBiB/CuBr2/BPMODA (or dNbpy)/reducing agent=400:1:0.5:0.5 (or 1):0.15 at 65 °C. V(anisole)/V(monomer)=0.3. Solid content= 20 wt %; Brij98=2.3 wt % of oil content; hexadecane=3.6 wt % of oil content. b Mn(theo)=([M(EO)2MA]0/[EBiB]0)  conversion. c Determined by GPC with THF as eluent, based on poly(methyl methacrylate) standards.

Scheme 1. PM(EO)2MA Magnetic Microgels for Controlled Targeted Drug Delivery

(i.e., swelling ratio, degradation behavior, and colloidal stability) superior to microgels from conventional free radical polymerization.46,62,68,69 These unique features suggest that the well-defined functional microgels have potential in drug delivery application. Herein, we utilized AGET ATRP in miniemulsion to synthesize novel thermally responsive poly(di(ethylene glycol) methyl ether methacrylate) (PM(EO)2MA) magnetic microgels. The thermally responsive and drug loading-releasing properties of the magnetic microgels were investigated. Experimental Section Materials. Di(ethylene glycol) methyl ether methacrylate (M(EO)2MA) (95%, Aldrich) was purified by passing through a column filled with basic alumina. Bis(2-pyridylmethyl)octadecylamine54 (BPMODA) and bis(2-methacryloyloxyethyl) disulfide ((MAOE)2S2)70 were synthesized according to the procedures previously published. Ethyl 2-bromoisobutyrate (EBiB, 98%, Aldrich), CuBr2 (99.999%, Aldrich), 4,40 -dinonyl2,20 -bipyridine (dNbpy, Aldrich), polyoxyethylene(20) oleyl ether (Brij 98, Aldrich), hexadecane (Aldrich), L-(þ)-ascorbic acid (Aldrich), hydrazine solution (35 wt % in H2O, Aldrich), tri(n-butyl)phosphine (Aldrich), Rhodamine B (Aldrich), glutathione reductase from wheat germ (type II, g0.08 units/mg protein, Aldrich), FeSO4 3 7H2O (>99%, Aldrich), FeCl3 3 6H2O (68) Oh, J. K.; Siegwart, D. J.; Lee, H.-i.; Sherwood, G.; Peteanu, L.; Hollinger, J. O.; Kataoka, K.; Matyjaszewski, K. J. Am. Chem. Soc. 2007, 129, 5939–5945. (69) Oh, J. K.; Siegwart, D. J.; Matyjaszewski, K. Biomacromolecules 2007, 8, 3326–3331. (70) Tsarevsky, N. V.; Matyjaszewski, K. Macromolecules 2005, 38, 3087–3092.

(97%, Aldrich), and oleic acid (65%-88%, Aldrich) were used as received. Synthesis of Linear PM(EO)2MA via AGET ATRP in Miniemulsion. In a typical AGET ATRP of M(EO)2MA in miniemulsion (Table 1, entry 3), a round-bottom flask was charged with CuBr2 (3.0 mg, 0.013 mmol), BPMODA (6.1 mg, 0.013 mmol), M(EO)2MA (2.0 g, 10.6 mmol), and anisole (0.6 mL). The resulting mixture was stirred at 65 °C for at least 2 h to dissolve the copper complex and then cooled to room temperature. The EBiB initiator (4.0 μL, 0.027 mmol) and hexadecane (95 μL) were added into the cold solution. Aqueous Brij 98 solution (10.32 mL, 5 mmol/L) was added to the organic solution before the mixture was subjected to sonication (Heat Systems Ultrasonics W-385 sonicator; output control set at 8 and duty cycle at 70% for 2.5 min). The resulting homogenized miniemulsion was transferred to a Schlenk flask and purged with nitrogen for 50 min. The flask was then immersed in an oil bath thermostatted at 65 °C. An aqueous solution of hydrazine (0.5 mL, 8.13 μmol/mL) was injected into the reaction to initiate the polymerization. Aliquots were taken at periodic intervals to measure the conversion gravimetrically and to examine the evolution of molecular weight. Synthesis of PM(EO)2MA Microgels via AGET ATRP in Miniemulsion. CuBr2 (3.0 mg, 0.013 mmol), BPMODA (6.1 mg, 0.013 mmol), M(EO)2MA (2.0 g, 10.6 mmol), and anisole (0.6 mL) were charged into a round-bottom flask. The resulting mixture was stirred at 65 °C for at least 2 h to dissolve the copper complex and then cooled to room temperature. The EBiB initiator (4.0 μL, 0.027 mmol), (MAOE)2S2 cross-linker (15.7 mg, 0.054 mmol), and hexadecane (95 μL) were added into the cold solution. Aqueous Brij 98 solution (10.32 mL, 5 mmol/L) was added to the organic solution before the mixture was subjected to sonication (Heat Systems Ultrasonics W-385 sonicator; output control set at 8 and duty cycle at 70% for 2.5 min). The resulting homogenized miniemulsion was transferred to a Schlenk flask and purged with nitrogen for 50 min. The flask was then immersed in an oil bath thermostatted at 65 °C. An aqueous solution of hydrazine (0.5 mL, 8.13 μmol/mL) was injected into the reaction to initiate the polymerization. After 19.0 h, the reaction was stopped by opening the flask and exposing the catalyst to air. The monomer conversion was 75.3%, determined by gravimetry. The cross-linked microgels were purified by consecutive centrifugation (10 000 rpm  20 min) and redispersed in tetrahydrofuran (THF). Finally, the microgels in THF were subjected to solvent exchange treatment to obtain a toluene suspension. Degradation of Microgels. The purified cross-linked microgels (0.1 g, ∼2 μmol disulfide moieties) were degraded into the corresponding linear polymers in the presence of excess tri (n-butyl)phosphine (10 μL, 40 μmol) in THF at room temperature overnight. An aliquot of the mixture was analyzed by GPC. Synthesis of Oleic Acid Coated Fe3O4 Nanoparticles (Fe3O4/ OA). Ferrous sulfate heptahydrate (FeSO4 3 7H2O, 2.35 g, 8.45 mmol) and ferric chloride hexahydrate (FeCl3 3 6H2O, 4.10 g) were added to a 100 mL flask. The flask was sealed with

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Figure 2. Evolution of molecular weight distribution during AGET ATRP of M(EO)2MA in miniemulsion (Table 1, entry 3).

Figure 1. Kinetic (a) and evolution of molecular weights and Mw/Mn (b) with monomer conversion in AGET ATRP of M(EO)2MA in miniemulsion (Table 1, entries 1-3).

a rubber septum and purged with nitrogen for 1 h. About 100 mL of N2-bubbled deionized water was then injected into the flask to dissolve the salts. The solution was vigorously stirred, followed by addition of ammonium hydroxide solution (25 mL, 28-30 wt %) quickly at room temperature. The solution color changed from orange to black, indicating formation of Fe3O4 precipitates. Oleic acid (1.5 mL) was then slowly injected under vigorous stirring into the dispersion at 80 °C over 1 h. The whole process was carried out under a nitrogen atmosphere. After that, the Fe3O4/OA nanoparticle water dispersion was mixed with toluene (100 mL). By adding a small amount of sodium chloride, Fe3O4/OA nanoparticles transferred into the toluene phase. Finally, the toluene dispersion was refluxed to remove most of the water under the nitrogen atmosphere, and the concentration of Fe3O4/OA was diluted with toluene to 17 mg/mL. Preparation of PM(EO)2 MA Magnetic Microgels. The PM(EO)2MA microgel toluene suspension (10.0 g, 17 mg/mL) was mixed with the Fe3O4/OA nanoparticle toluene suspension (10.0 g, 17 mg/mL) for 48 h at room temperature. The PM(EO)2MA microgels loaded with Fe3O4/OA nanoparticles were separated from free Fe3O4/OA nanoparticles by consecutive centrifugation (10 000 rpm  20 min) and redispersed in toluene until the supernatant was colorless. The microgels were black and well dispersed in toluene. To disperse the magnetic microgels into water, toluene was first exchanged to THF by centrifugation, and then the microgel THF dispersion was slowly dropped into a large amount of cold water in ice bath. The magnetic microgels were finally precipitated by heating water to 40 °C and collected by magnet. The precipitate was washed by 40 °C water several times to remove THF, redispersed into 0 °C water, and stored at 4 °C.

Loading and Releasing of Rhodamine B in Microgels. The magnetic microgels (16.0 mg) in water were mixed with Rhodamine B (0.4 mg) for 48 h at 4 °C. Excess Rhodamine B was removed by centrifugation (10 000 rpm for 20 min at 4 °C). The supernatant was removed, and the magnetic microgels were washed with deionized water. Using the Beer-Lambert equation with the predetermined extinction coefficient of Rhodamine B (ε = 15 330 M-1 cm-1 in water), the amount of the rhodamine dye in the supernatants was determined. The loading level of Rhodamine B into microgels was 1.0 wt %. After the purification, the magnetic microgels loaded with Rhodamine B were immediately dispersed in deionized 0 °C water (4 mg microgels/mL H2O) and evenly distributed into three dialysis tubes (MW cut off=1000, which could not allow diffusion of microgels and glutathione reductase). In one of the tubes, 5 mg of glutathione reductase was added to degrade the microgels. The tubes were separately incubated in 50 mL of deionized water at the designated temperature. Aliquots were taken at periodic intervals to determine by UV the amounts of Rhodamine B released outside the dialysis tubes. The water was replaced with 50 mL of fresh deionized water after each aliquot was taken. Analyses. Molecular weight and molecular weight distribution were determined by GPC, conducted with a Waters 515 pump and Waters 2414 differential refractometer using PSS columns (Styrogel 105, 103, 102 A˚) in THF as an eluent (35 °C, flow rate of 1 mL/min). Toluene was used as an internal standard. Linear poly(methyl methacrylate) standards were used for calibration. Particle size and size distribution were measured by dynamic light scattering (DLS) on a high performance particle sizer, model HP5001 from Malvern Instruments, Ltd. The sizes are expressed as Dav ( S (volume-average diameter ( standard deviation). Transmission electron microscopy (TEM) images were measured using a JEOL 200EX instrument operated at 200 kV. For TEM imaging, a drop of the microgel aqueous suspension was deposited on a carboncoated copper grid. A drop of phosphotungstic acid aqueous solution (1 wt %) was then added on the top. The same copper grid was also analyzed by scanning electron microscopy (SEM, Hitachi S-2460N). Thermogravimetric analysis (TGA) experiments were performed with samples placed in aluminum pans, using a Polymer Laboratories TG1000 instrument operating in the 20-580 °C temperature range, under nitrogen, at a heating rate of 20 °C/min. UV-vis spectra were recorded on a Varian Cary 5000 UV-vis-NIR spectrophotometer using a 1-cm-wide glass cuvette.

Results and Discussion Synthesis of PM(EO)2MA Linear Polymers and Microgels via AGET ATRP in Miniemulsion. Three model reactions of AGET ATRP of M(EO)2MA were conducted in a miniemulsion at 65 °C. Anisole was used as

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Figure 3. TEM (a) and SEM (b) images of PM(EO)2MA microgels stained with phosphotungstic acid.

a solvent to help form the hydrophobic phase. Commercially available Brij98 and hexadecane acted as surfactant and cosurfactant, respectively. The monomer M(EO)2MA is not soluble in the aqueous phase. Its homopolymer is water-soluble below 25 °C and becomes water-insoluble above 25 °C.44 Since the reaction is conducted at 65 °C, the monomer and formed polymer are stable in hydrophobic droplets without diffusion into water phase. DLS measurements indicated that the volumeaverage diameter of monomer droplets was 212 ( 4 nm with narrow distribution (entry 3, Table 1), and there was no change during the reaction. The reaction was initially conducted with ascorbic acid as a water-soluble reducing agent and BPMODA as a highly hydrophobic ligand (entry 1, Table 1). The kinetic plot of the reaction was almost linear, but at high monomer conversion the molecular weight distribution became broad, plausible because of radical coupling or other side reactions (Figure 1a,b). To maintain better control over polymerization, a less reactive ligand dNbpy was used (entry 2, Table 1). The reaction became much slower, but the coupling or side reaction was not suppressed at high monomer conversion. Therefore, a mild water-soluble reducing agent, hydrazine, was used (entry 3, Table 1).63 The reaction was much faster and well-controlled. The molecular weights obtained from GPC did not vary significantly from the theoretical values (Figure 1b). The GPC traces (Figure 2) were symmetric and smoothly shifted to high molecular weight but exhibited a slight shoulder at high molecular weight at 84% conversion. During the whole reaction, the molecular weight distribution remained under 1.6. PM(EO)2MA microgels were synthesized with bis(2methacryloyloxyethyl) disulfide ((MAOE)2S2) cross-linker under similar reaction conditions to entry 3 in Table 1. The molar ratio of [(MAOE)2S2]0/[M(EO)2MA]0/[EBiB]0 was 2:400:1. The polymerization was stopped at 75.3% monomer conversion. The cross-linked microgels were purified via centrifugation. TEM and SEM images of the microgels (Figure 3) demonstrated that PM(EO)2MA microgels with spherical shape were about 200 ( 100 nm. The disulfide cross-linker in the microgel was degraded by overnight treatment with tri(n-butyl)phosphine. The molecular weight of the degraded polymer chain was close to the theoretical value, approximately Mn =59 000 (Figure 4).

Figure 4. GPC trace of degraded PM(EO)2 MA microgel. The PM(EO)2MA microgel degraded in the presence of tri(n-butyl)phosphine in tetrahydrofuran for 19 h at room temperature and then was injected to GPC without further purification.

Figure 5. Change of PM(EO)2MA microgel hydrodynamic volume (a) and diameter (b) vs temperature in aqueous solution. Concentration of PM(EO)2MA microgels = 1 mg/mL. The volume-average hydrodynamic diameter of the microgels is determined by DLS.

Temperature Responsiveness of PM(EO)2MA Microgels. Temperature response of PM(EO)2MA microgels in aqueous suspension was characterized by DLS, as shown in Figure 5. By slowly increasing the temperature from 17 to 55 °C, hydrodynamic volume of the microgels gradually decreased to only 15 vol % of the original value at 17 °C and reached plateau at 36 °C. The volume change as a function of temperature was reversible. The volumeaverage hydrodynamic diameter of the microgels at 18 °C was 2.2 times of that at 50 °C, corresponding to over 10fold change in volume, due to the low cross-linking density (∼0.5%). At both temperatures, the size distribution

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Figure 6. TEM images of oleic-acid modified magnetite nanoparticles (a) and PM(EO)2MA magnetic microgels (b).

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Figure 8. Images of PM(EO)2MA magnetic microgel aqueous solution at 40 °C (a), in ice bath without (b) and with magnet bar (c). In (a), the picture was taken immediately after shaking the suspension to get a clear view of insoluble microgels.

Figure 7. TGA analysis of PM(EO)2MA magnetic microgels.

of the microgels was narrow. The volume transition temperature of the microgels was 25.1 °C, which is the same as the lower critical solution temperature (LCST) of linear PM(EO)2MA homopolymer (∼25 °C). Synthesis of PM(EO)2MA Magnetic Microgel. One of the requirements for magnetic microgels for targeted drug delivery application is homogeneous distribution of magnetic nanoparticles in a polymer network to ensure a uniform response to the external magnetic field. Additionally, they should be properly encapsulated to avoid any leakage. One approach to prepare magnetic polymer microgels is miniemulsion polymerization in the presence of magnetic nanoparticles. In this method, however, appropriate surface modification of iron oxides is necessary to make magnetic nanoparticles compatible with monomer and formed polymer. Otherwise pure polymer particles and/or free magnetic nanoparticles or aggregates will be produced because of heterogeneous distribution of iron oxide nanoparticles in monomer/polymer droplets.71,72 Another approach relies on the two-step swelling technique, in which magnetic nanoparticles are physically entrapped inside polymer network by simply mixing nanoparticles and microgels in solution. Each microgel can be loaded with equal amounts of iron oxide nanoparticles, but the free nanoparticles have to be removed by centrifugation. In this research, the most common hydrophobic magnetic nanoparticles, oleic acid-modified Fe3O4 (Fe3O4/OA) (71) Ramirez, L. P.; Landfester, K. Macromol. Chem. Phys. 2003, 204, 22–31. (72) Lu, S.; Forcada, J. J. Polym. Sci., Part A: Polym. Chem. 2006, 44, 4187–4203.

Figure 9. Volume change of PM(EO)2MA magnetic microgels vs temperature in aqueous solution. Concentration of the microgels=1 mg/mL.

nanoparticles were prepared, using the known coprecipitation process of ferric and ferrous salts in the presence of OA surfactant in a highly alkaline medium.73,74 The nanoparticle size was about 15 nm with narrow size distribution, as exhibited by the TEM image in Figure 6a. The highly hydrophobic Fe3O4/OA nanoparticles were well dispersed in most aromatic solvents, such as toluene, anisole, and dicholorobenzene, but form visible aggregates in M(EO)2MA monomer. Therefore, the two-step swelling method was adopted. The PM(EO)2MA microgels were physically loaded with Fe3O4/OA nanoparticles and dispersed into cold water, yielding a dark brown suspension. The TEM image (Figure 6b) indicated that the microgels encapsulated Fe3O4/OA nanoparticles, but their shape became irregular because of surfactant removal and centrifugation. The magnetic nanoparticle content in the microgels was determined by TGA to be about 12 wt % (Figure 7). The PM(EO)2MA microgels loaded with Fe3O4/OA nanoparticles were dispersed well in cold water (Figure 8b). There was no diffusion of free Fe3O4/OA nanoparticles from the microgels into water due to high hydrophobicity of the particles. Moreover, the Fe3O4/OA nanoparticles-loaded micogels could be attracted by external magnetic field, as shown in Figure 8c. In vivo (73) Massart, R. IEEE Trans. Magn. 1981, MAG-17, 1247–1248. (74) Montagne, F.; Mondain-Monval, O.; Pichot, C.; Mozzanega, H.; Elaissari, A. J. Magn. Magn. Mater. 2002, 250, 302–312.

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Figure 12. Rhodamine B releasing efficiency of the PM(EO)2MA magnetic microgels vs time at at 40 and 0 °C with and without glutathione reductase.

Figure 10. (a) UV-vis spectra of free Rhodamine B and Rhodamine Bcontaining PM(EO)2MA magnetic microgel in water. The later spectrum was shifted up to give a clear view. (b) Calibration curve of absorbance at 552 nm of Rhodamine B aqueous solution vs concentration.

Figure 11. Images of bare and Rhodamine B-loaded PM(EO)2MA magnetic microgel (labeled as RB) aqueous solutions under daylight and under 365 nm UV light irradiation.

magnetic behavior of the microgels is currently under investigation. Although the magnetic microgels are stable at 20 °C, they quickly precipitate from solution at 40 °C (Figures 8a). The temperature response of the Fe3O4/OA nanoparticles-loaded microgels was further investigated by DLS (Figure 9). The hydrodynamic volume of the magnetic microgels started decreasing at 18 °C and reached a plateau at 35 °C, as demonstrated by DLS. The final volume at high temperature was only 8% of the original value at low temperature. The thermally responsive behavior was very similar to the pure microgels, indicating that incorporation of the magnetic nanoparticles had little effect on thermal properties of the PM(EO)2MA microgels.

Model Drug Loading and Releasing. Rhodamine B was used as a model for hydrophilic drugs to understand the drug loading and releasing behavior of the PM(EO)2MA magnetic microgels. The microgels in water were mixed with Rhodamine B at 4 °C. Excess Rhodamine B was removed by centrifugation and washing with deionized water. The amounts of dyes in the supernatants and embedded in the magnetic microgels were calculated from absorbance at 552 nm and the calibration curve (Figure 10b). The loading level of Rhodamine B in the microgels was 1.0 wt %. The UV-vis spectrum of the Rhodamine B-containing magnetic microgels showed a similar characteristic absorption peak as free Rhodamine B (Figure 10a). The Rhodamine B-containing magnetic microgel aqueous solution exhibited pink color, and emitted red light under UV irradiation at 365 nm (Figure 11). In contrast, the bare magnetic microgels did not emit light under irradiation. The releasing behavior of Rhodamine B from the magnetic microgels was studied at 40 and 0 °C with and without glutathione reductase (mol wt ∼ 118 kDa), a tripeptide that can degrade disulfide-containing polymers. Glutathione reductase is found within cells at millimolar concentration and exhibits insignificant cytotoxicity at 10 mM concentration.68,75,76 The microgel suspension was kept in a dialysis tube (MWCO = 68 kDa), and the released Rhodamine B was collected from the supernatant outside the dialysis tube at varied times. Release of free Rhodamine B was much faster at 40 °C than at 0 °C (Figure 12). After 2 h at 40 °C, the releasing efficiency almost reached the maximum value of ∼80%. In contrast, less than 60% of Rhodamine B was released at 0 °C after 5 h. The faster releasing rate at high temperature can be explained by the volume contraction effect during shrinking of the microgels. The drug releasing efficiency can also be enhanced by addition of a reducing agent. The partially degraded microgels released about 75% of Rhodamine B after 5 h. These results (75) Carelli, S.; Ceriotti, A.; Cabibbo, A.; Fassina, G.; Ruvo, M.; Sitia, R. Science 1997, 277, 1681–1684. (76) Li, C.; Madsen, J.; Armes, S. P.; Lewis, A. L. Angew. Chem., Int. Ed. 2006, 45, 3510–3513.

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indicate that the drug releasing behavior of PM(EO)2MA magnetic microgels can be controlled by temperature and addition of a reducing agent. Conclusion AGET ATRP of M(EO)2MA was successfully conducted in miniemulsion at 65 °C. The reaction system was stable without diffusion of polymers into aqueous phase because PM(EO)2MA becomes hydrophobic above 25 °C. Using this technique, well-defined PM(EO)2MA microgels were prepared with degradable disulfide cross-linker. The microgels became magnetic after physical loading with Fe3O4/OA. Thermally responsive and drug loading-releasing behavior of the

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magnetic microgels were studied using Rhodamine B as a model for hydrophilic drugs. The drug releasing performance can be well controlled by temperature and addition of a reducing agent, indicating that the PM(EO)2MA magnetic microgels have potential in controlled targeted drug delivery. Acknowledgment. Financial support by the National Science Foundation under grants DMR 05-49353 and CTS 03-04568, UPMC Health System Competitive Medical Research Fund (CMRF), and the John & Nancy Harrison Legacy Graduate Fellowship is acknowledged. Helpful discussions with Patricia L. Golas, Dr. Jaroslav Mosnacek, and Dr. Ke Min are also acknowledged. The authors thank Dr. Joseph Suhan for TEM and SEM analysis.