Self-Assembled Micelles from an Amphiphilic Hyperbranched

Apr 12, 2010 - Ilya E. Nifant'ev , Andrey V. Shlyakhtin , Vladimir V. Bagrov , Pavel D. Komarov , Alexander N. Tavtorkin , Mikhail E. Minyaev , Maxim ...
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Self-Assembled Micelles from an Amphiphilic Hyperbranched Copolymer with Polyphosphate Arms for Drug Delivery Jinyao Liu, Wei Huang,* Yan Pang, Xinyuan Zhu, Yongfeng Zhou, and Deyue Yan* School of Chemistry and Chemical Engineering, State Key Laboratory of Metal Matrix Composites, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai 200240, PR China Received February 16, 2010. Revised Manuscript Received March 31, 2010 A novel type of amphiphilic hyperbranched multiarm copolymer [H40-star-(PLA-b-PEP-OH)] was synthesized through a two-step ring-opening polymerization (ROP) procedure and applied to drug delivery. First, Boltorn H40 was used as macroinitiator for the ROP of L-lactide to form the intermediate (H40-star-PLA-OH). Then, the ROP of ethyl ethylene phosphate was further initiated to produce H40-star-(PLA-b-PEP-OH). The resulting hyperbranched multiarm copolymers were characterized by 1H, 13C, and 31P NMR, GPC, and FTIR spectra. Benefiting from the amphiphilic structure, H40-star-(PLA-b-PEP-OH) was able to self-assemble into micelles in water with an average diameter of 130 nm. In vitro evaluation of these micelles demonstrated their excellent biocompatibility and efficient cellular uptake by methyl tetrazolium assay, flow cytometry, and confocal laser scanning microscopy measurements. Doxorubicinloaded micelles were investigated for the proliferation inhibition of a Hela human cervical carcinoma cell line, and the Doxorubicin dose required for 50% cellular growth inhibition was found to be 1 μg/mL. These results indicate that H40-star-(PLA-b-PEP-OH) micelles can be used as safe, promising drug-delivery systems.

Introduction During the past two decades, various polymeric micelles have been developed for potential application as a delivery vehicle for small-molecule drugs.1-3 Amphiphilic copolymers can selfassemble into micelles in aqueous solution through the hydrophobic interactions among the core-forming segments. The hydrophobic inner core serves as a container for hydrophobic drugs, and the outer shell composed of hydrophilic polymers maintains a hydration barrier to provide colloidal stability.4-10 These micelles possess several unique features, such as enhancing the aqueous solubility of drugs, prolonging the circulation time, improving the preferential accumulation at tumor sites by the enhanced permeability and retention (EPR) effect, and reducing systemic side effects.11,12 However, the conventional micelles from linear amphiphilic copolymers suffer from instability in vivo once the concentration of the copolymer falls below the critical micelle concentration in the bloodstream.13 The resulting disassembly of micelles leads to a burst release of loaded drugs, which may cause serious toxicity problems because of the potentially large fluctuations in drug concentrations.14 Benefiting from their unique core-shell structure, hyperbranched multiarm copolymers have been prepared as unimolecular micelles to overcome the disadvantages of classical micelles *Corresponding authors. E-mail: [email protected], [email protected].

(1) Duncan, R. Nat. Rev. Drug Discovery 2003, 2, 347. (2) Lukyanov, A. N.; Torchilin, V. P. Adv. Drug Delivery Rev. 2004, 56, 1273. (3) Kabanov, A. V. Adv. Drug Delivery Rev. 2006, 58, 1597. (4) Zhang, L.; Eisenberg, A. Science 1995, 268, 1728. (5) Zhang, L.; Yu, K.; Eisenberg, A. Science 1996, 272, 1777. (6) Zhang, L.; Eisenberg, A. Polym. Adv. Technol. 1998, 9, 677. (7) van Hest, J. C. M.; Delnoye, D. A. P.; Baars, M. W. P. L.; van Genderen, M. H. P.; Meijer, E. W. Science 1995, 268, 1592. (8) Cho, B. K.; Jain, A.; Nieberle, J.; Mahajan, S.; Wiesner, U.; Gruner, S. M.; T€urk, S.; R€ader, H. J. Macromolecules 2004, 37, 4227. (9) Zhang, L.; Eisenberg, A. J. Am. Chem. Soc. 1996, 118, 3168. (10) Zhou, Y. F.; Yan, D. Y. Chem. Commun. 2009, 1172. (11) Kataoka, K.; Harada, A.; Nagasaki, Y. Adv. Drug Delivery Rev. 2001, 47, 113. (12) Torchilin, V. P. Adv. Drug Delivery Rev. 2006, 58, 1532. (13) Wang, F.; Bronich, T. K.; Kabanov, A. V.; Rauh, R. D.; Roovers, J. Bioconjugate Chem. 2005, 16, 397. (14) Lawrence, M. Chem. Soc. Rev. 1994, 23, 417.

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in recent years.15-20 The unimolecular micelle does not disassemble in the dilute solution and is stable to environmental changes in vivo. Besides the good stability, the highly branched structure of hyperbranched multiarm copolymers can provide many nanocavities for drug encapsulation. Consequently, many double hydrophilic or amphiphilic hyperbranched multiarm copolymers have been prepared.21-37 Most of them have poly(ethylene glycol) (15) Kainthan, R. K.; Brooks, D. E. Bioconjugate Chem. 2008, 19, 2231. (16) Mugabe, C.; Hadaschik, B. A.; Kainthan, R. K.; Brooks, D. E.; So, A. I.; Gleave, M. E.; Burt, H. M. BJU Int. 2008, 103, 978. (17) Kontoyianni, C.; Sideratou, Z.; Theodossiou, T.; Tziveleka, L.; Tsiourvas, D.; Paleos, C. M. Macromol. Biosci. 2008, 8, 871. (18) Kitajyo, Y.; Kinugawa, Y.; Tamaki, M.; Kaga, H.; Kaneko, N.; Satoh, T.; Kakuchi, T. Macromolecules 2007, 40, 9313. (19) Ternat, C.; Ouali, L.; Sommer, H.; Fieber, W.; Velazco, M. I.; Plummer, C. J. G.; Kreutzer, G.; Klok, H.-A.; Ma˚nson, J. E.; Herrmann, A. Macromolecules 2008, 41, 7079. (20) Kainthan, R. K.; Mugabe, C.; Burt, H. M.; Brooks, D. E. Biomacromolecules 2008, 9, 886. (21) Tziveleka, L.; Kontoyianni, C.; Sideratou, Z.; Tsiourvas, D.; Paleos, C. M. Macromol. Biosci. 2006, 6, 161. (22) Du, W. J.; Xu, Z. Q.; Nystr€om, A. M.; Zhang, K.; Leonard, J. R.; Wooley, K. L. Bioconjugate Chem. 2008, 19, 2492. (23) Prabaharan, M.; Grailer, J. J.; Pilla, S.; Steeber, D. A.; Gong, S. Macromol. Biosci. 2009, 9, 515. (24) Zou, J.; Shi, W.; Wang, J.; Bo, J. Macromol. Biosci. 2005, 5, 662. (25) Xu, J.; Luo, S.; Shi, W.; Liu, S. Langmuir 2006, 22, 989. (26) Liu, J. Y.; Pang, Y.; Huang, W.; Zhu, X. Y.; Zhou, Y. F.; Yan, D. Y. Biomaterials 2010, 31, 1334. (27) Prabaharan, M.; Grailer, J. J.; Pilla, S.; Steeber, D. A.; Gong, S. Biomaterials 2009, 30, 3009. (28) Chen, S.; Zhang, X. Z.; Cheng, S. X.; Zhuo, R. X.; Gu, Z. W. Biomacromolecules 2008, 9, 2578. (29) Radowski, M. R.; Shukla, A.; von Berlepsch, H.; B€ottcher, C.; Pickaert, G.; Rehage, H.; Haag, R. Angew. Chem., Int. Ed. 2007, 46, 1265. (30) Klok, H.-A.; Rodrı´ guez-Hernandez, J. Macromolecules 2002, 35, 8718. (31) T€urk, H.; Shukla, A.; Rodrigues, P. C. A.; Rehage, H.; Haag, R. Chem.; Eur. J. 2007, 13, 4187. (32) Xu, S. J.; Luo, Y.; Haag, R. Macromol. Biosci. 2007, 7, 968. (33) Tian, H.; Chen, X.; Lin, H.; Deng, C.; Zhang, P.; Wei, Y.; Jing, X. Chem.; Eur. J. 2006, 12, 4305. (34) Tian, H.; Deng, C.; Lin, H.; Sun, J.; Deng, M.; Chen, X.; Jing, X. Biomaterials 2005, 26, 4209. (35) Sun, X. Y.; Zhou, Y. F.; Yan, D. Y. Sci. China, Ser. B: Chem. 2009, 52, 1703. (36) Hong, H. Y.; Mai, Y. Y.; Zhou, Y. F.; Yan, D. Y.; Chen, Y. J. Polym. Sci., Part A: Polym. Chem. 2008, 46, 668.

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(PEG) arms to endow themselves with hydrophilicity. However, PEG is biocompatible but not biodegradable. Therefore, hyperbranched multiarm copolymers composed of other appropriate hydrophilic arms with good biocompatibility, biodegradability, and facile structure tenability are very attractive for constructing promising drug-delivery systems. As an important class of eminent biomaterials, polyphosphates, have received a great deal of attention in the past few years owing to their good biocompatibility, biodegradability, and structural similarity to nucleic and teichoic acids.38-46 They can be degraded naturally into harmless low-molecular-weight products through hydrolysis or the enzymatic digestion of phosphate linkages under physiological conditions.47-49 More significantly, polyphosphates exhibit good flexibility in adjusting the pendant structures and their physicochemical properties on the basis of the convenient functionalization of pentavalent phosphorus.50,51 Consequently, hydrophilic polyphosphates may be an interesting alternative to the reported hydrophilic segments as the outer shell of micelles. Recently, many amphiphilic linear copolymers containing polyphosphates with various pendants have been produced and applied to therapeutic fields.52-57 To our knowledge, there have been no reports about using polyphosphate as hydrophilic arms to modify hyperbranched polyesters for drug delivery. In this article, a novel type of amphiphilic hyperbranched multiarm copolymer based on the H40 core, the poly(L-lactide) (PLA) inner shell, and the poly(ethyl ethylene phosphate) (PEP) outer shell was synthesized by the ring-opening polymerization of LA and EP. Both the core and the inner/outer shells are biocompatible and biodegradable. On the basis of the amphiphilic structure, H40-star-(PLA-b-PEP-OH) self-assembles into micelles in aqueous solution. Because the architectures of PLA and PEP blocks can be easily controlled during the ROP processes, the stability and drug-loading ability of the micelles can be improved by adjusting the hydrophobic/hydrophilic balance. Moreover, the large number of surface hydroxyl groups can be further modified by targeting and/or imaging ligands to construct drug-delivery systems with multiple functions. (37) Prabaharan, M.; Grailer, J. J.; Pilla, S.; Steeber, D. A.; Gong, S. Biomaterials 2009, 30, 5757. (38) Libiszowski, J.; Kaluzynski, K.; Penczek, S. J. Polym. Sci., Part A: Polym. Chem. 1978, 16, 1275. (39) Lapienis, G.; Penczek, S. J. Polym. Sci., Part A: Polym. Chem. 1977, 15, 371. (40) Lapienis, G.; Penczek, S. Macromolecules 1977, 10, 1301. (41) Pretula, J.; Kaluzynski, K.; Penczek, S. Macromolecules 1986, 19, 1797. (42) Xu, X. Y.; Yu, H.; Gao, S. J.; Mao, H. Q.; Leong, K. W.; Wang, S. Biomaterials 2002, 23, 3765. (43) Wen, J.; Kim, G. J. A.; Leong, K. W. J. Controlled Release 2003, 92, 39. (44) Wang, S.; Wan, A. C.; Xu, X.; Gao, S.; Mao, H. Q.; Leong, K. W.; Yu, H. Biomaterials 2001, 22, 1157. (45) Wen, J.; Mao, H. Q.; Li, W.; Lin, K. Y.; Leong, K. W. J. Pharm. Sci. 2004, 93, 2142. (46) Richards, M.; Dahiyat, B. I.; Arm, D. M.; Lin, S.; Leong, K. W. J. Polym. Sci., Part A: Polym. Chem. 1991, 29, 1157. (47) Wen, J.; Zhuo, R. X. Macromol. Rapid Commun. 1998, 19, 641. (48) Iwasaki, Y.; Wachiralarpphaithoon, C.; Akiyoshi, K. Macromolecules 2007, 40, 8136. (49) Wang, Y.; Tang, L.; Li, Y.; Wang, J. Biomacromolecules 2009, 10, 66. (50) Wang, J.; Mao, H. Q.; Leong, K. W. J. Am. Chem. Soc. 2001, 123, 9480. (51) Song, W.; Du, J.; Liu, N.; Dou, S.; Cheng, J.; Wang, J. Macromolecules 2008, 41, 6935. (52) Iwasaki, Y.; Akiyoshi, K. Biomacromolecules 2006, 7, 1433. (53) Sun, T. M.; Du, J. Z.; Yan, L. F.; Mao, H. Q.; Wang, J. Biomaterials 2008, 29, 4348. (54) Wang, Y.; Liu, X.; Sun, T.; Xiong, M.; Wang, J. J. Controlled Release 2008, 128, 32. (55) Wang, Y.; Tang, L.; Sun, T.; Li, C.; Xiong, M.; Wang, J. Biomacromolecules 2008, 9, 388. (56) Tang, L.; Wang, Y.; Li, Y.; Du, J.; Wang, J. Bioconjugate Chem. 2009, 20, 1095. (57) Cheng, J.; Ding, J. X.; Wang, Y. C.; Wang, J. Polymer 2008, 49, 4784.

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Experimental Section Materials. Tetrahydrofuran (THF) and toluene were dried by refluxing with a fresh sodium-benzophenone complex under N2 and distilled just before use. Triethylamine (TEA) was refluxed with phthalic anhydride, potassium hydroxide, and calcium hydride in turn and distilled just before use. CH2Cl2 was refluxed with calcium hydride and distilled just before use. PCl3 was distilled just before use. Ethylene glycol was purified by vacuum distillation. Ethanol was dried with molecular sieves. L-Lactide (LA) was purchased from Sigma and recrystallized from ethyl acetate before use. Boltorn H40 (H40) was obtained from Perstorp Polyols AB, Sweden. Tin(II) octoate (Sn(Oct)2) and 3-(4,5dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) were purchased from Sigma and used as received. Doxorubicin hydrochloride (DOX 3 HCl) was purchased from Beijing Huafeng United Technology Corporation and used as received. Clear polystyrene tissue-culture-treated 12-well and 96-well plates were obtained from Corning Costar. All other reagents and solvents were purchased from domestic suppliers and used as received. Measurements. Nuclear magnetic resonance (NMR) analyses were recorded on a Varian Mercury Plus 400 MHz spectrometer with deuterated chloroform (CDCl3) as the solvent. The numberaverage molecular weight (Mn), weight-average molecular weight (Mw), and polydispersity index (PDI) were measured by gel permeation chromatography (GPC). GPC was performed on a Perkin-Elmer series 200 system (10 μm PL gel 300  7.5 mm mixed-B and mixed-C columns, linear polystyrene calibration) equipped with a refractive index (RI) detector. DMF containing 0.01 mol/L lithium bromide was used as the mobile phase at a flow rate of 1 mL/min at 70 °C. Fourier transform infrared (FTIR) spectra were recorded on a Paragon 1000 instrument by the KBrsample-holder method. Synthesis of Ethyl Ethylene Phosphate. Ethyl ethylene phosphate (EP) was synthesized according to previous reports.58,59 To a mixture of PCl3 (413 g, 3.00 mol) and dry CH2Cl2 (350 mL), ethylene glycol (186 g, 3.00 mol) was added dropwise over a period of 5 h with vigorous magnetic stirring. Gaseous HCl was removed via N2 flow. After the complete addition of ethylene glycol, the solution was stirred at room temperature for another 0.5 h and the solvent was evaporated under reduced pressure. The residue was distilled under vacuum to give 2-chloro-1,3,2-dioxaphospholane. The oxidation of 2-chloro-1,3,2-dioxaphospholane was carried out by bubbling O2 through the toluene solution at 50 °C for 36 h. After the removal of toluene, the residue was distilled under vacuum to give a colorless liquid of 2-chloro-1,3,2-dioxaphospholane. A mixture of ethanol (9.20 g, 0.200 mol), dry TEA (20.2 g, 0.200 mol), and dry THF (250 mL) was cooled to 0 °C, and then 2-chloro-2-oxo1,3,2-dioxaphospholane (28.5 g, 0.200 mol) was added dropwise with stirring for 2 h. The resulting mixture was stirred at room temperature for another 2 h. The triethylamine hydrochloride was filtered off, and the filtrate was concentrated. The residue was distilled under vacuum to give EP, yield 82.6%. 1H NMR (CDCl3, ppm): 4.30-4.46 (4H, -OCH2CH2O-), 4.16-4.23 (2H, -OCH2CH3), 1.35 (3H, -OCH2CH3). 13C NMR (CDCl3, ppm): 66.16 (-OCH2CH2O-), 65.42 (-OCH2CH3), 16.47 (-OCH2CH3). 31 P NMR (CDCl3, ppm): 18.61. The 1H, 13C, and 31P NMR spectra of EP are shown in Supporting Information Figure S1. Purification of H40 by Fractional Precipitation. Ten grams of commercially available H40 was dissolved in 100 mL of acetone. One hundred milliliters of ethyl ether was slowly added to the polymer solution under vigorous stirring. After 12 h, the precipitates were filtered, washed twice with an acetone/ether mixture (v/v, 1:1), and finally dried under vacuum. Purified H40 was obtained in 42.5% yield. (58) Edmundson, R. S. Chem. Ind. 1962, 1828. (59) Liu, J. Y.; Huang, W.; Zhou, Y. F.; Yan, D. Y. Macromolecules 2009, 42, 4394.

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Synthesis of H40-star-PLA-OH. H40-star-PLA-OH was prepared by the ROP of LA using H40 as a macroinitiator and Sn(Oct)2 as a catalyst. In a typical polymerization procedure, H40 (250 mg, 5.65 mmol hydroxyl groups) was added to a 50 mL flask and dried under vacuum at 100 °C for 1 h. Then, LA (5.70 g, 39.6 mmol) and Sn(Oct)2 (16.2 mg, 0.040 mmol) were also added to the flask in a glovebox with a water content of less than 0.1 ppm. The reaction mixture was placed in an oil bath at 125 °C and stirred for 20 h. The crude H40-star-PLA-OH was diluted with THF and precipitated in cold diethyl ether to produce a white powder. Then it was purified by dissolving in THF, passing through a neutral alumina column, being concentrated, precipitating in diethyl ether, and finally drying under vacuum, yield 86.4%. Mn,GPC = 10.5  104 g/mol and Mw/Mn = 1.6 (Figure 4B). 1H NMR (CDCl3, ppm): 5.15 (-CCH(CH3)O-), 4.34 (-CCH(CH3)OH), 4.24 (-C(CH3)CH 2 O-), 1.56 (-CCH(CH 3 )O-), 1.24 (-C(CH 3 )CH 2 O-) (Figure 2B). Synthesis of H40-star-(PLA-b-PEP-OH). H40-star-(PLAb-PEP-OH) was prepared by the ROP of EP using H40-star-PLAOH as a macroinitiator and Sn(Oct)2 as a catalyst. In a typical polymerization procedure, H40-star-PLA-OH (250 mg, 0.152 mmol of hydroxyl groups) was added to a 50 mL flask and dried under vacuum at 100 °C for 1 h. Then, EP (1.39 g, 9.12 mmol) and Sn(Oct)2 (31 mg, 0.076 mmol) were also added to the flask in a glovebox with a water content of less than 0.1 ppm. The reaction mixture was dissolved in 10 mL of THF and placed in an oil bath at 25 °C for 40 min. The solution was concentrated, and the residue was precipitated into cold diethyl ether containing 10% methanol (v/v) twice. After filtration, H40-star-(PLA-b-PEPOH) was obtained by vacuum drying overnight, yield 34.5%. Mn,GPC = 15.0  104 g/mol and Mw/Mn = 2.2 (Figure 4C). 1H NMR (CDCl3, ppm): 5.15 (-CCH(CH3)O-), 4.24 (-OCH2CH2O-), 4.15 (-OCH2CH3), 3.76 (-OCH2CH2OH), 1.56 (-CCH(CH3)O-), 1.34 (-OCH2CH3) (Figure 2C). Preparation of Micelles. Micelles were prepared by a dialysis method. Briefly, 20 mg of H40-star-(PLA-b-PEP-OH) was dissolved in 2 mL of N,N-dimethylformamide (DMF) and stirred for 2 h at room temperature. Then, the polymer solution was added slowly to 5 mL of deionized water under vigorous stirring. Two hours later, the solution was transferred to dialysis membrane tubing (MWCO = 2000 g/mol) and dialyzed for 24 h to remove DMF against deionized water, during which the water was exchanged at appropriate intervals. Transmission Electron Microscopy Measurements. Transmission electron microscopy (TEM) studies were performed with a JEOL 2010 instrument operated at 200 kV. The samples were prepared by directly dropping micelle solutions onto carboncoated copper grids and drying at room temperature overnight without staining before measurement. Dynamic Light Scattering Measurements. Dynamic light scattering (DLS) measurements were performed in aqueous solution using a Malvern Zetasizer Nano S apparatus equipped with a 4.0 mW laser operating at λ = 633 nm. All samples of 1 mg/mL were measured at 20 °C and at a scattering angle of 173°. Preparation of DOX-Loaded Micelles. DOX-loaded micelles were prepared as follows. Briefly, 20 mg of H40-star-(PLAb-PEP-OH) was dissolved in 2 mL of DMF, followed by adding a predetermined amount of DOX 3 HCl and 2 mol equiv of TEA and stirring at room temperature for 2 h. Then, the mixture was added slowly to 5 mL of deionized water. After being stirred for an additional 2 h, the solution was dialyzed against deionized water for 24 h (MWCO = 2000 g/mol), during which the water was renewed every 4 h. To determine the total drug loading, the DOXloaded micelle solution was lyophilized and then dissolved in DMF. The UV absorbance at 500 nm was measured to determine the DOX concentration. Cell Culture. NIH 3T3 cells (a mouse embryonic fibroblast cell line) and Hela cells (a human cervical carcinoma cell line) were cultivated in DMEM (Dulbecco’s modified Eagle’s medium) Langmuir 2010, 26(13), 10585–10592

Article containing 10% FBS (fetal bovine serum) and antibiotics (50 units/mL penicillin and 50 units/mL streptomycin) at 37 °C in a humidified atmosphere containing 5% CO2. Relative Cytotoxicity of Micelles. The relative cytotoxicity of H40-star-(PLA-b-PEP-OH) micelles was estimated by an MTT viability assay and AO/EB double-staining methods against NIH 3T3 cells. In the MTT assay, NIH 3T3 cells were seeded into 96-well plates at 8  103 cells per well in 200 μL of medium. After 24 h of incubation, the culture medium was removed and replaced with 200 μL of a medium containing serial dilutions of micelles. The cells were grown for another 24 h. Then, 20 μL of a 5 mg/mL MTT assay stock solution in phosphate-buffered saline (PBS) was added to each well. After the cells were incubated for 4 h, the medium containing unreacted MTT was removed carefully. The obtained blue formazan crystals were dissolved in 200 μL/well dimethyl sulfoxide (DMSO), and the absorbance was measured in a Perkin-Elmer 1420 Multilabel counter at a wavelength of 490 nm. In AO/EB double staining, DNA-binding dyes AO and EB were used to detect the morphology of apoptotic and necrotic cells.60 NIH 3T3 cells were seeded in six-well plates at 5  105 cells per well in 1 mL of complete DMEM and cultured for 24 h, followed by removing the culture medium and adding 1 mL of a micelle solutions (in DMEM medium) with a concentration of 1 mg/mL. After 24 h of incubation, cells were rinsed with PBS twice and incubated with PBS containing AO (5 μg/mL) and EB (5 μg/mL) at 37 °C in 5% CO2 for 10 min. Live and dead cells were imaged by a Leica DM 4500B fluorescence microscope. Cellular Uptake of Micelles by Hela Cells. The cellular uptake experiments were performed with flow cytometry and confocal laser scanning microscopy (CLSM). For flow cytometry, Hela cells were seeded in six-well plates at 5  105 cells per well in 1 mL of complete DMEM and cultured for 24 h, followed by removing the culture medium and adding DOX-loaded micelle solutions (1 mL of the DMEM medium) at a concentration of 1 mg/mL for 0.5 and 3 h, respectively. Thereafter, the culture medium was removed and cells were washed with PBS three times and treated with trypsin. Then, 2 mL of PBS was added to each culture well, and the solutions were centrifugated for 5 min at 1000 rpm. After the removal of the supernatants, the cells were resuspended in 0.5 mL of PBS. Data for 1  104 gated events were collected, and analysis was performed by means of a BD FACSCalibur flow cytometer and CELLQuest software. In CLSM studies, Hela cells were seeded in six-well plates at 2  105 cells per well in 1 mL of complete DMEM and cultured for 24 h, followed by removing the culture medium and adding free DOX or DOX-loaded micelle solutions (1 mL of the DMEM medium) at a concentration of 1 mg/mL. After incubation at 37 °C for 0.5 h, the culture medium was removed and the cells were washed with PBS three times. Then, the cells were fixed with 4% formaldehyde for 30 min at room temperature, and the slides were rinsed with PBS three times. Finally, the cells were stained with 2-(4-amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI) for 10 min and the slides were rinsed with PBS three times. The slides were mounted and observed with a LSM 510META. Activity Analyses. The cytotoxicity of DOX-loaded micelles and free DOX against Hela cells was evaluated in vitro by MTT assay. Hela cells were seeded into 96-well plates at 8  103 cells per well in 200 μL of medium. After 24 h of incubation, the culture medium was removed and replaced with 200 μL of a medium containing serial dilutions of DOX-loaded micelles or free DOX. The cells were grown for another 48 h. Then, 20 μL of a 5 mg/mL MTT assay stock solution in PBS was added to each well. After the cells were incubated for 4 h, the medium containing unreacted MTT was removed carefully. The obtained blue formazan crystals were dissolved in 200 μL/well DMSO, and the absorbance was measured in a Perkin-Elmer 1420 Multilabel counter at a wavelength of 490 nm. (60) Cohen, J. J. Immunol. Today 1993, 14, 126.

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Scheme 1. Schematic Illustration of Synthesis and Self-Assembly of H40-star-(PLA-b-PEP-OH) for Drug Delivery

Figure 1.

13

C NMR spectra of H40 in DMSO-d6 (A) and H40star-PLA-OH in CDCl3 (B).

Synthesis and Characterization of H40-star-(PLA-bPEP-OH). The synthesis and self-assembly schematic illustration of H40-star-(PLA-b-PEP-OH) for drug delivery is shown in Scheme 1. H40 is a commercially available hyperbranched polyester with a large number of terminal hydroxyl groups and is often used as the core of amphiphilic hyperbranched multiarm copolymers.23-25,27,28 PLA is one of the frequently used biodegradable polymers in drug-delivery systems because of its high mechanical strength and excellent shaping and molding properties.61 Therefore, PLA blocks are selected to attach to the H40 core as the inner shell. Both the H40 core and PLA blocks form the hydrophobic segments in the self-assembled micelles. However, hydrophilic polyphosphate is used as the outer shell in amphiphilic copolymers because of its good biocompatibility, biodegradability, and pendant tenability.50,51 As is well known, ROP is an efficient method of synthesizing polymers with controlled architecture. Both aliphatic polyester and polyphosphate can be prepared through the ROP of cyclic monomers LA and EP, respectively, when hydroxyl groups and Sn(Oct)2 are present.62,63 In the first step, H40 was used as a macroinitiator to initiate the ROP of LA in the bulk at 125 °C for 20 h and produced the intermediate H40-star-PLA-OH. Then, H40-star-PLA-OH was further used as a macroinitiator to initiate the ROP of the second monomer EP in THF at 25 °C for 40 min to produce H40-star-(PLA-b-PEP-OH). All processes must be carried out under rigorously anhydrous conditions to avoid the formation of homopolymers initiated by water.

According to previous reports,64-66 the degree of branching (DB) of H40 is usually in the range of 0.31-0.38 with different molecular-weight fractions. In other words, the number of terminal hydroxyls may be lower than 30 for the low-molecular-weight fraction, but it is very close to 64 for the high-molecular-weight fraction. To control the chemical structure of the resulting polymers, commercial H40 was first purified by fractional precipitation. Then, the high-molecular-weight fraction was obtained and used as a macroinitiator. The 13C NMR spectrum of purified H40 is shown in Figure 1A. Here, its DB was 0.375, its degree of polymerization (DP) was 61, and its number-average molecular weight was 7300 g/mol by the method suggested by Zagar.67 Furthermore, the chemical shift of the quaternary carbon in macroinitiator H40 is also influenced by the degree of substitution of the two hydroxyl groups adjacent to it.28,68 The signal of the quaternary carbon appears at 50.6 ppm when the two hydroxyl groups are not substituted, at 48.8 ppm when one hydroxyl group is substituted, and at 46.8 ppm when both hydroxyl groups are substituted. The 13C NMR spectrum of H40-star-PLA-OH is shown in Figure 1B. The signals at 50.6 and 48.8 ppm disappeared, which confirms that all of the hydroxyl groups of H40 initiate the ROP of LA to produce H40-star-PLA-OH. The 1H NMR spectra of H40, H40-star-PLA-OH, and H40star-(PLA-b-PEP-OH) are shown in Figure 2. By comparing the 1 H NMR spectra of H40 (Figure 2A) and H40-star-PLA-OH (Figure 2B), the peaks at 1.56 and 5.15 ppm are ascribed to the protons of methyl and methine in PLA blocks, respectively. The weak peaks at 1.24 and 4.24 ppm are assigned to the protons of methyl and methylene groups of H40, respectively.23,27 These results also verify the formation of H40-star-PLA-OH. The Mn and DP of the PLA blocks were found to be about 2600 g/mol and

(61) Deng, C.; Chen, X. S.; Sun, J.; Lu, T. C.; Wang, W. S.; Jing, X. B. J. Polym. Sci., Part A: Polym. Chem. 2007, 45, 3218. (62) Yang, X.; Wang, Y.; Tang, L.; Xia, H.; Wang, J. J. Polym. Sci., Part A: Polym. Chem. 2008, 46, 6425. (63) Chen, V. J.; Ma, P. X. Biomaterials 2006, 27, 3708. (64) Magnusson, H.; Malmstroem, E.; Hult, A. Macromolecules 2000, 33, 3099.

(65) Hanselman, R.; Hoelter, D.; Frey, H. Macromolecules 1998, 31, 3790. (66) Ornatska, M.; Peleshanko, S.; Genson, K. L.; Rybak, B.; Bergman, K. N.; Tsukruk, V. V. J. Am. Chem. Soc. 2004, 126, 9675.   (67) Zagar, E.; Zigon, M. Macromolecules 2002, 35, 9913. (68) Claesson, H.; Malmstr€om, E.; Johansson, M.; Hult, A. Polymer 2002, 43, 3511.

Results and Discussion

10588 DOI: 10.1021/la1006988

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Figure 3.

31

P NMR spectrum of H40-star-(PLA-b-PEP-OH) in

CDCl3.

Figure 4. GPC curves of H40, H40-star-PLA-OH, and H40star-(PLA-b-PEP-OH).

Figure 2. 1H NMR spectra of H40 in DMSO-d6 (A), H40-star-PLAOH in CDCl3 (B), and H40-star-(PLA-b-PEP-OH) in CDCl3 (C).

18, respectively, by calculating the relative intensities of the peak at 4.34 ppm (the methine groups adjacent to the terminal hydroxyl groups in PLA blocks) and the peak at 5.15 ppm (the residual methine groups in PLA blocks). As expected, three new signals appear in the spectrum of H40-star-(PLA-b-PEP-OH) in Figure 2C. The peaks at 4.24, 4.15, and 1.34 ppm are attributed to the protons of -OCH2CH2O-, -OCH2CH3, and -OCH2CH3 in PEP blocks,55 respectively. However, the peak at 4.34 ppm (the methine groups adjacent to the terminal hydroxyl groups in PLA) also disappeared in Figure 2C, which clarified that all of the terminal hydroxyl groups in PLA initiated the ROP of EP to Langmuir 2010, 26(13), 10585–10592

form H40-star-(PLA-b-PEP-OH). Similarly, the Mn and DP of the PEP arms were found to be about 1300 g/mol and 9, respectively, by calculating the relative intensity of the signal at 3.76 ppm (methylene adjacent to the terminal hydroxyl groups in PEP blocks) and the signal at 4.24 ppm (residual methylene in PEP blocks). Furthermore, H40-star-(PLA-b-PEP-OH) was also characterized by 31P NMR and the corresponding spectrum was shown in Figure 3. The signal at -0.22 ppm indicates that phosphate units are introduced into the copolymer successfully and also verifies the chemical structure of H40-star-(PLA-b-PEP-OH). FTIR analyses provide additional information about H40-star(PLA-b-PEP-OH). The strong peak at 1754 cm-1 is a characteristic absorption of CdO stretching due to the presence of a hyperbranched polyester H40 core and PLA blocks. Absorptions at 1268 and 1180 cm-1 can be attributed to the asymmetrical and symmetrical PdO stretching in PEP blocks, respectively (Supporting Information Figure S2). The P-O-C stretching is also verified at 983 cm-1.26 The broad peak at around 3432 cm-1 confirms the existence of a large number of surface functional hydroxyl groups in the amphiphilic multiarm copolymer. The FTIR results further testify to the successful preparation of H40-star-(PLA-b-PEP-OH). In addition, the molecular weights and PDIs of these polymers were measured by GPC. The GPC profiles of H40, H40-starPLA-OH, and H40-star-(PLA-b-PEP-OH) are shown in Figure 4. All of them are relatively monomodal and symmetric and exhibit a clear shift toward the direction of high molecular weight in turn. The details are summarized in Table 1. Apparently, the molecular weights of H40-star-PLA-OH and H40-star-(PLA-b-PEP-OH) DOI: 10.1021/la1006988

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Liu et al. Table 1. Characterization of the Polymers sample

Mw (g/mol) Mn (g/mol) Mw/Mn Mn (g/mol) ( 104)a ( 104)a (PDI)a ( 104)b 0.5c 16.8 33.0

H40 H40-star-PLA-OH H40-star-(PLA-b-PEP-OH)

0.3c 10.5 15.0

1.8c 1.6 2.2

0.73 17.3 26.1

a Determined by GPC. b Determined by 1H NMR. c From the Perstorp data sheet.

Figure 5. DLS plot (A) and representative TEM microscope image (B) of H40-star-(PLA-b-PEP-OH) micelles.

are increased to 1.05  10 and 1.50  10 g/mol, respectively. In addition, the DPs of PLA and PEP blocks can be easily controlled by adjusting the molar ratio of LA/EP to hydroxyl groups in the feed during the ROP processes. The corresponding results are summarized in Supporting Information Table S1. Micellization of H40-star-(PLA-b-PEP-OH). Generally, an amphiphilic macromolecule can form micellar structures in water. The amphiphilic nature of H40-star-(PLA-b-PEP-OH) provides an opportunity to self-assemble into micelles in water. The proposed molecular packing model of H40-star-(PLA-b-PEP-OH) multimolecular micelles is illustrated in Scheme 1. In the selective solvent of water, H40-star-(PLA-b-PEP-OH) macromolecules spontaneously self-assemble into multimolecular micelles driven by the strong hydrophobic interactions of the H40 core with the PLA blocks, whereas the outer shell of hydrophilic polyphosphate maintains a hydration barrier to provide stability to the micelle. On the basis of our previous research, we find that these multimolecular micelles are a kind of multimicelle aggregate (MMA) with building blocks of small micelles or unimolecular micelles.10 The size of the micelle is an important parameter for drug delivery. Small (