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Design of Core-Shell-Type Nanoparticles Carrying Stable Radicals in the Core Toru Yoshitomi,† Daisuke Miyamoto,†,‡,§ and Yukio Nagasaki*,†,‡,§,|,⊥ Graduate School of Pure and Applied Sciences, Tsukuba Research Center for Interdisciplinary Materials Science (TIMS), Center for Tsukuba Advanced Research Alliance (TARA), Master’s School of Medical Sciences, Graduate School of Comprehensive Human Sciences, University of Tsukuba, Tennodai, Tsukuba, 305-8577 Ibaraki, Japan, and Satellite Laboratory, International Center for Materials Nanoarchitectonics (MANA), National Institute of Materials Science (NIMS), Tennoudai 1-1-1, Tsukuba, 305-8573, Ibaraki Received November 7, 2008; Revised Manuscript Received December 27, 2008
Utilizing the self-assembled core-shell-type polymeric micelle technique, high-performance nanoparticles possessing stable radicals in the core and reactive groups on the periphery were prepared. The anionic ringopening polymerization of ethylene oxide (EO) was carried out using potassium 3,3-diethoxypropanolate as an initiator, followed by mesylation with methanesulfonyl chloride to obtain acetal-poly(ethylene glycol)methanesulfonate (acetal-PEG-Ms; 1). Compound 1 was reacted with potassium O-ethyldithiocarbonate, followed by treatment with n-propylamine to obtain heterobifunctional PEG derivatives containing both sulfanyl and acetal terminal groups (acetal-PEG-SH) (2) in a highly selective and quantitative manner. Poly(ethylene glycol)-blockpoly(chloromethylstyrene) (acetal-PEG-b-PCMS) (3) was synthesized by the free-radical telomerization of chloromethylstyrene (CMS) using 2 as a telogen. The chloromethyl groups in the PCMS segment of the block copolymer (3) were quantitatively converted to 2,2,6,6-tetramethylpiperidinyloxys (TEMPOs) via the amination of 3 with 4-amino-TEMPO to obtain acetal-PEG-b-PCMS containing TEMPO moieties (4). The obtained 4 formed core-shell-type nanoparticles in aqueous media when subjected to the dialysis method: the cumulant average diameter of the nanoparticles was about 40 nm, and the nanoparticles emitted intense electron paramagnetic resonance (EPR) signals. The TEMPO radicals in the core of the nanoparticles showed reduction resistance even in the presence of 3.5 mM ascorbic acid. This means that these nanoparticles are anticipated as high-performance bionanoparticles that can be used in vivo.
Introduction Recently, in vivo bioimaging has attracted much attention in biorelated science and technology fields such as biotechnology, medical therapy and diagnostics. Among these bioimaging systems, magnetic resonance imaging (MRI) is one of the most powerful tools for visualizing specific tissues, for example, cancer tissue, but it is not perfect. The intensity and resolution are dependent on the size of the magnetic field and the accumulation time. Therefore, an average MRI examination requires significantly long time.1,2 Numerous efforts have been made to improve both the sensitivity and the resolution.3,4 The 19 F probe is one of the ideas for the improvement of the performances because this type of probe emits no endogenous signal in vivo, has a high signal intensity, and so on.5-7 Electron paramagnetic resonance (EPR) is known to have a sensitivity approximately 103 times higher than that of nuclear magnetic resonance (NMR).8,9 Recently, the research on in vivo EPR imaging has been stepped up by several groups.10-12 EPR imaging is now being investigated using stable radical species such as 2,2,6,6-tetramethylpiperidinyloxy (TEMPO) as the * To whom correspondence should be addressed. E-mail: nagasaki@ nagalabo.jp. † Graduate School of Pure and Applied Sciences. ‡ TIMS. § TARA. | Master’s School of Medical Sciences, Graduate School of Comprehensive Human Sciences. ⊥ MANA and NIMS.
probe. Under the reducing conditions in vivo, however, such radical species are easily reduced and the EPR signals disappear rapidly. To solve these issues, we set out to create a novel nanoparticle which can be used as an EPR probe. Our aim is to create a nanoparticle which is stable under in vivo conditions (Figure 1). This paper describes the synthetic methodology for an amphiphilic block copolymer possessing stable radicals in the side chain of the hydrophobic segment. The preparation of these core-shell-type nanoparticles from the obtained block copolymers and their characterization in terms of the EPR effect were also carried out.
Experimental Section Materials. 3,3-Diethoxypropanol (Aldrich Chemical Co., Inc., U.S.A.), tetrahydrofuran (THF), benzene (reagent grade; Kanto Chemical Co., Inc., Tokyo, Japan), and ethylene oxide (EO; 100%; Sumitomo Seika Chemicals Co., Ltd., Hyogo, Japan) were purified conventionally.13 A THF solution of potassium naphthalene was prepared by a method described in the previous report.14 2,2′-Azobisisobutyronitrile (AIBN; Kanto Chemical Co., Inc., Tokyo, Japan) was purified by recrystallization from methanol. Chloromethylstyrene (CMS) was kindly provided by Seimi Chemical Co., Ltd. (Kanagawa, Japan) and purified on a silica gel column to remove the inhibitor, followed by vacuum distillation under a nitrogen atmosphere. 4-Amino-2,2,6,6-tetramethylpiperidinyloxy (4-amino-TEMPO), 4-hydroxy-TEMPO (TEMPOL) (Aldrich Chemical Co., Inc., U.S.A.), 2-propanol, diethyl ether, potassium O-ethyldithiocarbonate, methanesulfonyl chloride, triethy-
10.1021/bm801278n CCC: $40.75 2009 American Chemical Society Published on Web 02/03/2009
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Figure 1. Schematic illustration of the nanoparticle to be used as an EPR probe, prepared from acetal-PEG-b-PCMS containing TEMPO moieties (4).
Figure 2. SEC diagram (a) and 1H NMR spectrum (b) of acetal-PEG-b-PCMS (3); SEC apparatus, TOSOH HLC-8120GPC; detector, RI; column, TOSOH SUPER HZ4000 + 3000; solvent, THF (containing 2 wt % triethylamine); flow rate, 0.35 mL min-1; calibration, standard PEGs.
lamine, L(+)-ascorbic acid, dimethyl sulfoxide (DMSO), acetonitrile, and N,N-dimethylformamide (DMF; Kanto Chemical Co., Inc., Tokyo, Japan) were used without further purification. Synthesis of Acetal-PEG-b-PCMS (3). Acetal-PEG-SH (2) was prepared (Supporting Information, Figure S1) by a method described in our previous report.15 The acetal-PEG-SH (2) contained dimers (acetal-PEG-S-S-PEG-acetal) due to the oxidation of the sulfanyl groups. To reduce the disulfide of the dimers, the obtained polymer were treated with 1,4-dithiothreitol in THF. After 0.02 mmol (92 mg) of the obtained 2 (Mn ) 4600) was weighed into a flask, the inside of the reactor was degassed and filled with nitrogen gas. The degassingN2-purge cycle was repeated three times. A total of 1 mmol (0.138 mL) of CMS, 0.01 mmol of AIBN, and 1 mL of benzene were then added to the flask. Polymerization was conducted for 24 h at 60 °C in an oil bath. The reaction mixture was recovered by precipitation into 40 mL of hexane, followed by freeze-drying with benzene. The reacted polymer was washed with diethyl ether three times to eliminate the PCMS homopolymer, followed by freeze-drying with benzene. The yield of the obtained polymer (3) was 55.1% (134 mg). 1H NMR (CDCl3, Figure 2b): δ 1.20-2.00 (m, 11H, Ha + Hf + Hd), 3.71 (m, 418H, Hb + He), 4.40-4.70 (m, 45H, Hc + Hh), 6.2-7.2 (m, 4H, Hg). Preparation of Acetal-PEG-b-PCMS Containing TEMPO Moieties (4). After 40 mg (3.6 µmol) of the obtained acetal-PEG-bPCMS (3; Mn ) 11100) were weighed into a 10 mL flask, a DMSO solution (2 mL) of 4-amino-TEMPO (520 µmol, 88 mg) was added to the flask and stirred for 5 h at room temperature. The reacted polymer was recovered by precipitation into 10 mL of cold 2-propanol (-15 °C) and centrifugation for 30 min at 5000 rpm (4500 × g). To purify the obtained polymer, the precipitation-centrifugation cycle was repeated three times, followed by freeze-drying with benzene. The yield of the obtained copolymer (4) was 74.6% (44 mg). To confirm the removal
Figure 3. Time course of the decrease in the CH2Cl signal in the 1H NMR spectrum resulting from the amination of 3 with 4-amino-TEMPO in three solvents: DMSO (b); acetonitrile (0); CHCl3 (O). (Temperature, 23 °C; number of scans, 64.)
of the unreacted 4-amino-TEMPO, the EPR spectra of the fractions (every 1 min) were measured after the SEC fractionation of the obtained block copolymer (4) from the unreacted 4-amino-TEMPO. 1H NMR (CDCl3, Figure 4): δ 1.00-2.20 (m, 23H, Ha + Hd + Hf + Hi), 3.71 (m, 419H, Hb + He + Hh), 6.6-7.6 (m, 4H, Hg). Preparation of the Core-Shell-Type Nanoparticles from Copolymer (4). Polymeric nanoparticles were prepared from 4 by a procedure described previously.16 Briefly, 20 mg of 4 was dissolved in 2 mL of DMF, and the polymer solution was transferred into a preswollen membrane tube (Spectra/Por; molecular-weight cutoff size: 3500) and then dialyzed for 24 h against 2 L of water, which was changed after 2, 5, and 8 h. DLS measurement was carried out to
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Figure 4. 1H NMR spectrum of acetal-PEG-b-PCMS containing TEMPO moieties (4) in the presence of phenylhydrazine.
determine the diameter of the obtained particles (Figure 6). (Concentration of the formed nanoparticle, 64 µM; average diameter, ca. 40 nm; polydispersity factor, µ/Γ2 ) 0.228.) Reduction Resistance of the TEMPO Radicals in the Core of the Nanoparticles in the Presence of Ascorbic Acid. To evaluate the reduction resistance of the TEMPO radicals in the core of nanoparticles prepared from 4, 200 µM of the nanoparticle solution were prepared as a stock solution A after a formation of the nanoparticles by the dialysis method. A total of 240 µL of 6.4 mM solution of ascorbic acid in sodium-phosphate buffer (180 mM, pH 7.4) were added to stock solution A (200 µL). The mixture was transferred to a capillary tube immediately, followed by measurement of the EPR spectrum of the solution over time. The final concentrations of the nanoparticle and ascorbic acid were 90 µM and 3.5 mM, respectively. A free-TEMPOL solution with the same TEMPO concentration in the same buffer was used as a control. Measurements. Size exclusion chromatography (SEC) measurements were carried out using a TOSOH HLC-8120 equipped with TSK gel columns (Super HZ4000 and Super HZ3000), an internal refractive index (RI), and ultraviolet (UV) detectors. THF with 5% (v/v) triethylamine was used as the eluent at a flow rate of 0.35 mL min-1 at 40 °C. The 1H NMR spectra were obtained using chloroform-d on a JEOL EX270 spectrometer at 270 MHz. A light-scattering spectrophotometer (Nano ZS, ZEN3600, Malvern Instruments, Ltd., U.K.) equipped with a He-Ne laser that produces vertically polarized incident beams at a detection angle of 90° at 25 °C was used in the present study for the dynamic light-scattering (DLS) measurements. The EPR spectra were recorded at room temperature on a Bruker EMX-T EPR spectrometer operating at 9.7 GHz with a 100 kHz magnetic field modulation. The spectra were collected with the following parameters: sweep width, 500 G; microwave power, 0.633 mW; receiver gain, 5.02 × 104; time constant, 5.120 ms; conversion time, 10.240 ms.
Results and Discussion Synthesis of Acetal-PEG-b-PCMS (3). The recent progress of living polymerization systems now enables the synthesis of versatile types of block copolymer. Especially, several kinds of living radical polymerization techniques, such as atom transfer radical polymerization (ATRP), reversible addition fragmentation chain transfer (RAFT) polymerization, and nitroxidemediated radical polymerization (NMRP), have been established during the last two decades and are being applied to widely
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different monomers such as hydrophilic monomers and nonconjugate monomers.17,18 Even such versatile living radical polymerization techniques, however, cannot be used with all monomers. For example, ATRP cannot be applied to vinyl monomers containing halogen atoms. To synthesize block copolymers possessing PCMS as one of the segments, we employed the classical free-radical telomerization reaction. The sulfanyl group is known to have a very high chain transfer constant in free-radical polymerization reactions.19,20 If our synthesized acetal-PEG-SH (2) acts as a telogen without any side reaction, the design of novel polymers will become feasible. For example, if the telomerization of hydrophobic monomers can be done using 2, new types of amphiphilic block copolymer can be synthesized. For the demonstration of radical telomerization, CMS was employed because the anticipated 3 possesses a high potential for modifying the PCMS segment. The synthesis route to 3 is described in Scheme 1. Utilizing the obtained 2, the radical telomerization of CMS was examined. Though the methine of the acetal group is known to be highly reactive to radical attack,21,22 the radical telomerization proceeded smoothly without any gel formation. The SEC profile of the reaction mixture after polymerization, however, showed bimodal distribution (Figure 2a). To obtain information on the products, their 1 H NMR spectra were measured after the two peaks were separated by SEC fractionation. The 1H NMR analysis showed that the composition ratios of PEG to PCMS of the two fractions were almost the same. The Mn value of PCMS was determined by 1H NMR data based on the Mn of PEG (4600), which is 3300. From the SEC profile, however, the number-average molecular weight (Mn) of the first fraction was two times higher than that of second fraction. Based on the Mn value of PEG (2, 4600), as determined by SEC, the Mns of the PCMS segments for the two fractions were determined to be 3300 and 6600, respectively. The difference in molecular weight was the only difference between the two products, which means that the two products were formed via different termination reactions. In the telomerization reaction using sulfanyl compounds, the chain transfer reaction takes place prior to conventional termination reactions. In the present polymerization, however, both terminations by recombination reaction and chain transfer reaction by macromolecular telogen took place competitively.23-25 The large molecular weight of telogen, acetal-PEG-SH, might be effected by the chain transfer reaction. This may be the reason that two products having the different molecular weight were obtained. Thus, the products were a mixture of diblock and triblock copolymers possessing the same composition. The ratios of diblock to triblock copolymer were determined by the chromatogram shown in Figure 2a and were 59 and 41%, respectively. Synthesis of Acetal-PEG-b-PCMS Containing TEMPO Moieties (4). The chloromethyl groups in the PCMS segment of block copolymer 3 were converted to stable radicals via the amination of 3 with 4-amino-TEMPO. When 3 was mixed with 4-amino-TEMPO, the CH2Cl signal of the PCMS segment in the 1H NMR spectrum gradually decreased. Figure 3 shows the time course of the decrease in the CH2Cl signal in the 1H NMR spectrum. The ratios of the methylene of the chloromethyl group in the CMS unit to the methylene in the EO unit were monitored using the 1H NMR spectra (Supporting Information, Figure S2). Kawabe, et al. reported previously that the rate of amination reaction of PCMS was strongly dominated by reaction media.26 Solvents with the higher dielectric constant shows higher reactivity. Actually, DMSO was the most suitable solvent. In fact, the CH2Cl signal completely disappeared within 5 h at room temperature. To confirm the amination of 3 by 4-amino-TEMPO,
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Scheme 1
NMR analysis was carried out after the purification. The spectrum of the obtained polymer was broad and hard to characterize (data no shown), indicating the presence of paramagnetic radicals based on TEMPO. This is one of the proofs that amino-TEMPO reacted covalently with 3. After the TEMPO radicals were reduced by phenylhydrazine, the entire 1H NMR spectrum was observed, as shown in Figure 4. After the reducing reaction, the signal for the methylene protons of the chloromethyl group in the PCMS unit at 4.5 ppm completely disappeared. Note that all the peaks, including those of the end acetal methane proton at 4.6 ppm, remained intact. On the basis of these results, it was confirmed that the TEMPOs were successfully introduced into the polymer without any side reactions such as cross-linking reactions and other undesirable reaction took place during the amination reaction. The purification of 4 was carried out by SEC fractionation after crude polymer was obtained by precipitation. After all the fractions (every 1 min) were collected, they were analyzed by EPR measurement. Figure 5 shows the plots of the EPR intensities versus the elution time. After purification by column
Figure 5. EPR signal intensities of each fraction after the purified copolymer (4) were separated by SEC fractionation. Precipitation: never (b); once (black triangle in circular enclosure); twice (black circle in circular enclosure); three times (O).
fractionation, the EPR signal based on amino-TEMPO completely disappeared at around 20-27 min, confirming the complete removal of the unreacted 4-amino-TEMPO. Preparation of Core-Shell-Type Nanoparticles Containing Paramagnetic Electrons in the Core. For the preparation of self-assembled polymeric nanoparticles, the dialysis method was employed.27,28 Compound 4 was dissolved in a solvent suitable for both segments (DMF) and then dialyzed against water. The size of the obtained nanoparticles was estimated by DLS measurement. As shown in Figure 6, the nanoparticles thus obtained showed a unimodal distribution in the histogram analysis. The average diameter and polydispersity factor (µ2/ Γ2), as determined by the cumulant method, were about 40 nm and 0.228, respectively. The EPR signals of the obtained nanoparticles were then analyzed by EPR measurement. Figure 7a shows the EPR spectrum of the nanoparticles. Contrary to the free-TEMPOL signal shown in Figure 7b, which is a clear triplet signal due to an interaction between the 14N nuclei and the unpaired electron in the dilute solution, the nanoparticle signal broadened. The signal of TEMPO as a side chain of the polymer in good solvent such as chloroform became also broad due to the spin-spin interaction of TEMPO in the side chain (data not shown).29 However, the signal in aqueous media was
Figure 6. Size distribution of nanoparticles prepared from 4 analyzed by DLS.
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Figure 7. EPR spectra of nitroxyl radical of nanoparticles prepared from 4 (polymer concentration: 90 µM; (a)) and TEMPOL (concentration: 2.0 mM; (b)) in 100 mM of sodium-phosphate buffer at pH 7.4.
were less than 20 s and about 15 min, respectively. More than 45 times, high stability of the TEMPO radicals in nanoparticle system means that signal will be observed several hours and is promising as new stable radical carrier in vivo.
Conclusions
Figure 8. Time-course of EPR signal intensity of TEMPO in nanoparticle and TEMPOL in the presence of 3.5 mM ascorbic acid: the nanoparticle (2); TEMPOL (0); temperature, 23 °C.
much broader than that in chloroform. We interpreted that it is due to aggregated state of PCMS-TEMPO segment in the selfassembling structure. These results suggest that there were TEMPO radicals in the hydrophobic solid core of the nanoparticles. Reduction Resistance of TEMPO Radicals in the Core of the Nanoparticles in the Presence of Ascorbic Acid. It is known that most of parts in vivo are under the reduction environment, though they are not the same extent. For example, glutathione exists in mammalian cells at the millimolar level (0.5∼10 mM). Though the concentration of glutathione in the blood is 0.5 µM∼10 µM, it often contains large amount of ascorbic acid. Both glutathione and ascorbic acid exhibit a strong reducing capability in vivo. In the blood stream, a certain amount of ascorbic acid exists. Thus, TEMPO possessing a stable radical is known to be easily reduced to 2,2,6,6-tetramethylpiperidin1-ol under reducing in vivo conditions, and EPR signals rapidly disappear.30,31 If our designed nanoparticles can maintain their EPR signaling under reducing conditions for a long time due to the compartmentalization of the TEMPOs in the core of the nanoparticles, the design of novel EPR probes for long-term measurement will become feasible, especially in the blood stream. The reduction resistivity of TEMPO radical in the nanoparticle core was compared with a low molecular weight model (TEMPOL). Figure 8 shows time-course of EPR signal intensity of TEMPO in the nanoparticle and TEMPOL. When the free-TEMPOL solution was monitored in the presence of 3.5 mM ascorbic acid, the signal intensity rapidly decreased within a minute. On the contrary, the EPR signal intensity of our prepared nanoparticles decreased very slowly, indicating that the TEMPO radicals in our prepared nanoparticles resisted reduction even in the presence of 3.5 mM ascorbic acid. Actually, half-lives of TEMPOL and TEMPO in nanoparticle
In conclusion, acetal-PEG-b-PCMS (3) was successfully synthesized by the free-radical telomerization of CMS using acetal-PEG-SH (2) as a telogen, followed by the conversion of the chloromethyl groups in the PCMS segment of the block copolymer (3) to stable radicals via the amination of 3 with 4-amino-TEMPO in DMSO. High-performance nanoparticles possessing stable radicals in the core and acetal groups as a platform moiety to install ligand molecules on the periphery were prepared using acetal-PEG-b-PCMS containing TEMPO moieties (4), and they showed intense EPR signals. The TEMPO radicals in the nanoparticles showed reduction resistance even in the presence of 3.5 mM ascorbic acid, which means that these nanoparticles are anticipated as high-performance bionanoparticles, which can be used in vivo. Acknowledgment. This work was partly supported by the World Premier International Research Center Initiative (WPI Initiative) on Materials Nanoarchitronics of the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan. Supporting Information Available. Synthetic protocol of acetal-PEG-SH, with 1H NMR and SEC data are provided. As well, reaction profile of amination reaction of acetal-PEG-bPCMS with amino-TEMPO monitored by 1H NMR is described. This material is available free of charge via the Internet at http:// pubs.acs.org.
References and Notes (1) Hildebrandt, I. J.; Gambhir, S. S. Clin. Immunol. 2004, 111, 210– 224. (2) Barentsz, J.; Takahashi, S.; Oyen, W.; Mus, R.; Mulder, P. D.; Reznek, R.; Oudkerk, M.; Mali, W. J. Clin. Oncol. 2006, 24, 3234–3244. (3) Kumagai, M.; Imai, Y.; Nakamura, T.; Yamasaki, Y.; Sekino, M.; Ueno, S.; Hanaoka, K.; Kikuchi, K.; Nagano, T.; Kaneko, E.; Shimokado, K.; Kataoka, K. Colloids Surf., B 2007, 56, 174–181. (4) Nakamura, E.; Makino, K.; Okano, T.; Yamamoto, T.; Yokoyama, M. J. Controlled Release 2006, 114, 325–333. (5) Oishi, M.; Sumitani, S.; Nagasaki, Y. Bioconjugate Chem. 2007, 18, 1379–1382. (6) Higuchi, M.; Iwata, N.; Matsuba, Y.; Sato, K.; Sasamoto, K.; Saido, T. C. Nat. Neurosci. 2005, 8, 527–533. (7) Powell, K. T.; Cheng, C.; Wooley, K. L. Macromolecules 2007, 40, 4509–4515. (8) Berliner, L. J.; Khramtsov, V.; Fujii, H.; Clanton, T. L. Free Radical Biol. Med. 2001, 30, 489–499.
Design of Core-Shell-Type Nanoparticles (9) Inbaraj, J. J.; Cardon, T. B.; Laryukhin, M.; Grosser, S. M.; Lorigan, G. A. J. Am. Chem. Soc. 2006, 128, 9549–9554. (10) Utsumi, H.; Yamada, K.; Ichikawa, K.; Sakai, K.; Kinoshita, Y.; Matsumoto, S.; Nagai, M. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 1463–1468. (11) He, G.; Deng, Y.; Li, H.; Kuppusamy, P.; Zweier, J. L. Magn. Reson. Med. 2002, 47, 571–578. (12) Soule, B. P.; Hyodo, F.; Matsumoto, K.; Simone, N. L.; Cook, J. A.; Krishna, M. C.; Mitchell, J. B. Free Radical Biol. Med. 2007, 42, 1632–1650. (13) Perrin, D. D.; Armarego, W. L. F.; Perrin, D. R. Purification of Laboratory Chemicals; Pergamon: Oxford, 1980. (14) Scott, N. D.; Walker, J. F.; Hansley, V. L. J. Am. Chem. Soc. 1936, 58, 2442–2444. (15) Akiyama, Y.; Otsuka, H.; Nagasaki, Y.; Kato, M.; Kataoka, K. Bioconjugate Chem. 2000, 11, 947–950. (16) Scholz, C.; Iijima, M.; Nagasaki, Y.; Kataoka, K. Macromolecules 1995, 28, 7295–7297. (17) Braunecker, W. A.; Matyjaszewski, K. Prog. Polym. Sci. 2007, 32, 93–146. (18) Goto, A.; Fukuda, T. Prog. Polym. Sci. 2004, 29, 329–385. (19) Kerep, P.; Ritter, H. Macromol. Rapid Commun. 2007, 28, 759–766. (20) Lele, B. S.; Leroux, J.-C. Macromolecules 2002, 35, 6714–6723.
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(21) Emami, S. H.; Salovey, R.; Hogen-Esch, T. E. J. Polym. Sci., Part A: Polym. Chem. 2002, 40, 3021–3026. (22) Iijima, M.; Nagasaki, Y. J. Polym. Sci., Part A: Polym. Chem. 2006, 44, 1457–1469. (23) Bevington, J. C.; Melville, H. W.; Taylor, R. P. J. Polym. Sci. 1954, 12, 449–459. (24) Bevington, J. C. Proc. R. Soc. A 1957, 239, 420–432. (25) Zammit, M. D.; Davis, T. P.; Haddleton, D. M.; Suddaby, K. G. Macromolecules 1997, 30, 1915–1920. (26) Kawabe, H.; Yanagita, M. Bull. Chem. Soc. Jpn. 1973, 46, 38–42. (27) Iijima, M.; Nagasaki, Y.; Okada, T.; Kato, M.; Kataoka, K. Macromolecules 1999, 32, 1140–1146. (28) Nagasaki, Y.; Okada, T.; Scholz, C.; Iijima, M.; Kato, M.; Kataoka, K. Macromolecules 1998, 31, 1473–1479. (29) Yoshida, E.; Tanaka, T. Colloid Polym. Sci. 2006, 285, 135–144. (30) Saphier, O.; Silberstein, T.; Shames, A. I.; Likhtenshtein, G. I.; Maimon, E.; Mankuta, D.; Mazor, M.; Katz, M.; Meyerstein, D.; Meyerstein, N. Free Radical Res. 2003, 37, 301–308. (31) Bobko, A. A.; Kirilyuk, I. A.; Grigor’ev, I. A.; Zweier, J. L.; Khramtsov, V. V. Free Radical Biol. Med. 2007, 42, 404–412.
BM801278N
