Synthesis and Characterization of Biodegradable Amphiphilic Triblock

Biomacromolecules 2005, 6, 1954-1960

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Synthesis and Characterization of Biodegradable Amphiphilic Triblock Copolymers Containing L-Glutamic Acid Units Huili Guan,†,‡ Zhigang Xie,†,‡ Peibiao Zhang,† Chao Deng,†,‡ Xuesi Chen,†,‡ and Xiabin Jing*†,‡ State Key Laboratory of Polymer Physics and Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, P. R. China, and Graduate School of Chinese Academy of Sciences, Beijing 100039, P. R. China Received December 16, 2004; Revised Manuscript Received March 3, 2005

A novel biodegradable amphiphilic triblock copolymer bearing pendant carboxyl groups PLGG-PEG-PLGG was successfully prepared by ring-opening copolymerization of L-lactide (LA) with (3s)-benzoxylcarbonylethyl-morpholine-2, 5-dione (BEMD) in the presence of dihydroxyl poly(ethylene glycol) (PEG) as a macroinitiator in bulk at 130 °C using SnOct2 as catalyst and by subsequent catalytic hydrogenation. The copolymer could form micelles in aqueous solution with the cmc dependent on the composition of the copolymer. The micelles exhibited a homogeneous spherical morphology and a unimodal size distribution. Their degradation rate in the presence of proteinase K was faster than that of PLA, and they showed a low degree of cytotoxicity to the articular cartilage cells. This biodegradable amphiphilic block copolymer with pendant carboxyl groups is capable of further modification and is expected to facilitate a variety of potential biomedical applications, such as drug carriers, tissue engineering, etc. Introduction Aliphatic polyesters such as poly(glycolic acid) (PGA), poly(lactic acid) (PLA), poly(-caprolactone) (PCL), and their copolymers are well-known as an important class of synthetic biodegradable polymers. Due to their intrinsic properties such as nonimmunogenicity, good biocompatibility, and biodegradability, they may be widely used in biomedical applications, including sutures, implants for bone fixation, scaffolds in tissue engineering, and carriers in drug delivery, etc.1-3 However, an important limitation in the use of these polyesters for biomedical applications is their lack of pendant functional groups to which bioactive molecules (drugs, recognition agents, adhesion promoters, or probes) can be covalently attached.4 Recently, more and more attention has been paid to the preparation of biodegradable polymers possessing functional or reactive groups such as amino, hydroxyl, carboxyl, thiol, etc. Several notable examples of functionalized polymers have been reported in the literature: (1) polyesters from cyclic diesters, such as poly(Rmalic acid) from malide dibenzyl ester5 and poly(glycolic acid-co-malic acid);6,7 (2) poly(ester amides) from morpholine-2,5-dione derivatives, including those containing carboxyl, amine, thiol, and hydroxyl groups;8-20 (3) polyesters based on four-, six-, or seven-membered lactones bearing carboxyl,21 and hydroxyl,22 etc. In addition, several linear functionalized polycarbonates have been prepared, including poly(5-methyl-5-benzyloxycarbonyl-1,3-dioxan-2-one),23 poly* To whom correspondence should be addressed. Telephone: +86-4315262112. Fax: +86-431-5685653. E-mail: [email protected]. † Changchun Institute of Applied Chemistry. ‡ Graduate School of Chinese Academy of Sciences.

carbonate synthesized by ROP of trimethylene carbonate (TMC) with 1,2-O-isopropylidene-D-xylofuranose-3, 5-cyclic carbonate,24 poly(carbonate-ester)s from glycerol, and lactic acid.25 It was also well-known that poly(ethylene glycol) (PEG) is an amphiphilic macromolecule that has found wide chemical, biomedical, and industrial applications because it has useful properties such as high solubility in both water and organic solvents, biocompatibility, ease in chemical modification, etc.26-30 Many studies focused on the amphiphilic block copolymers of PEG-polyesters in expectation of achieving unique properties and corresponding applications.31-35 However, very few polyesters modified by both PEG segment and functional groups were reported. In this paper, an ABA-type triblock copolymer PLGBGPEG-PLGBG consisting of PEG and poly{(lactic acid)-co[(glycolic acid)-alt-(γ-benzyl-L-glutamic acid)]} (PLGBG) was synthesized by ring-opening polymerization (ROP) of LA with morpholine-2,5-dione (BEMD) derived from γ-benzyl-L-glutamic acid in the presence of dihydroxyl PEG as a macroinitiator. After catalytic hydrogenation, the pendant ester groups were converted to carboxyl groups, leading to the formation of PLGG-PEG-PLGG with pendant functional groups. Several properties of PLGG-PEG-PLGG were examined, including the formation of micelles and critical micelle concentration (cmc), micelle morphology and size, enzymatic degradation rate, and in vitro cytotoxicity. Experimental Section Materials. Stannous octanoate (SnOct2) and dihydroxyl PEG with molecular weights of 2000 and 4600 were obtained from Aldrich. Prior to use, PEG was dried by an azeotropic

10.1021/bm0492069 CCC: $30.25 © 2005 American Chemical Society Published on Web 04/14/2005

Copolymers Containing L-Glutamic Acid Units

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Scheme 1. Synthesis of Monomer BEMD

distillation in benzene. Palladium on activated charcoal (Pd/ C, 10%) was received from Suzhou Xukou Chemical Corporation in China. L-Lactide (LA) was prepared in our own laboratory and recrystallized from ethyl acetate for three times before use. PLA was obtained by ROP of LA using SnOct2 as catalyst. Ethyl acetate, N,N-dimethylformamide (DMF), and chloroform were refluxed over CaH2 and distilled under argon. Benzene and toluene were dried and distilled in the presence of sodium immediately before use. (3s)-3-Benzoxylcarbonylethylmorpholine-2,5-dione (BEMD) was synthesized according to the procedure (Scheme 1) as described in the literature.16 Measurements. FT-IR spectra were recorded on a BioRad Win-IR instrument. 1H NMR spectra were measured by a Unity-300 NMR spectrometer at room temperature, with CDCl3 as solvent and TMS as internal reference. The GPC measurements were conducted at 35 °C with a Waters 410 GPC instrument equipped with two Waters Styragel columns (HT6E, HT3) and a differential refractometer detector. CHCl3 was used as eluent at a flow rate of 1.0 mL min-1. Synthesis of PLGBG-PEG-PLGBG. The ABA triblock copolymer PLGBG-PEG-PLGBG is prepared by ROP of both LA and BEMD with stannous octoate (SnOct2) as catalyst in the presence of dihydroxyl PEG as a macroinitiator. First, 0.0729 g of PEG and benzene were added to a polymerization flask equipped with a stirrer, a thermometer, and a Dean Stark trap. An azeotropic refluxing of the stirred benzene solution under argon was conducted for 30 min. The solution was allowed to cool to ambient temperature, and the residual benzene was evaporated under a reduced pressure. Next, 0.687 g of fresh LA and 0.105 g of BEMD were added together into the above polymerization flask, and the flask was argon-purged several times. To give the desired PEG/monomer/catalyst molar ratio, 0.24 mL of SnOct2 (3.05 × 10-2 mol L-1) in toluene was added by using a glass syringe. The samples were kept under high vacuum to remove toluene. Finally, the flask was sealed in a vacuum by flame-heating and gently heated until the sample inside was melted, and the reaction was conducted in a constant temperature oil bath of 130 °C for 48 h. The polymerization was stopped by removing the flask from the oil bath and cooled to room temperature. Purification was performed by dissolving the reaction mixture in a small amount of chloroform and pouring it into an excess of methanol with stirring. The copolymers were collected and dried in vacuo. Synthesis of PLGG-PEG-PLGG. The hydrogenation reaction was conducted by using a 250 mL Parr hydrogenator, equipped with a magnetic stirring bar and a programmable temperature controller. The copolymer (0.54 g) was dissolved in THF (24 mL). 10% Pd/C (0.1 g) suspension in methanol (8 mL) and the copolymer solution were added together into the hydrogenator. After purging with argon three times, the reaction mixture was stirred under

1.0 MPa hydrogen pressure at 50 °C for 48 h, and then, the catalyst was removed by filtration and the solution was concentrated by rotary evaporation. The final polymer PLGG-PEG-PLGG with pendant carboxyl groups was precipitated with an excess amount of diethyl ether and dried in vacuo at room temperature overnight. Preparation of Micelles from PLGG-PEG-PLGG. The synthesized PLGG-PEG-PLGG copolymer (100 mg) was dissolved in acetone (10 mL) in a 100-mL volumetric flask, and 40 mL of doubly distilled water was added with gentle agitation. The acetone was tardily removed at ambient temperature over 2 h by rotary evaporation to get the micelles. Micelle Characterization. The formation of the micelles was confirmed by a fluorescence technique, using pyrene as a probe. A predetermined amount of pyrene solution in acetone (0.1 mg L-1) was added into a series of volumetric flasks, and the acetone was evaporated completely. The solid pyrene left in each flask was in such an amount that when the flask was filled with a solution to the calibration mark, the pyrene concentration in the final solution was 6 × 10-7 mol L-1, equal to the saturation solubility of pyrene in water at 22 °C. The micelle solution and doubly distilled water were added into the volumetric flasks containing the solid pyrene consecutively. Desired copolymer concentration from 10-4 to 1.0 g L-1 was achieved by predetermining the volume of the copolymer solution. Steady-state fluorescence spectra were obtained by a Perkin-Elmer LS50B luminescence spectrometer. Square quartz cells of 1.0 × 1.0 cm were used. For fluorescence excitation spectra, the detection wavelength λem was set at 390 nm. The spectra were recorded with a scan rate of 240 nm min-1. Size distribution of the micelles was measured by dynamic light scattering (DLS) with a vertically polarized He-Ne laser (DAWN EOS, Wyatt technology) at a fixed scattering angle of 90° and at a constant temperature of 25 °C. A drop of micelle solution was deposited onto a silicon chip and air-dried before ESEM (environmental scanning electron microscope) observation with an XL 30 ESEM FEG scanning electron microscope (Micrion FEI PHILIPS). In Vitro Degradation of PLGG-PEG-PLGG. The PLGGPEG-PLGG copolymer was dissolved in CHCl3 at a concentration of 20 g L-1 and dried to form a copolymer film. For the degradation study, each film was placed in 30 mL of Tris-HCl buffer in a bottle containing proteinase K (9.6 µg mL-1) and then incubated at 37 °C. At specific time intervals, the specimen was taken out, washed with distilled water, dried, and weighed. The degree of degradation was estimated from 100(W0 - Wt)/W0 (%), where W0 and Wt were the weights of copolymer before and after degradation at time t, respectively. Cytotoxicity of PLGG-PEG-PLGG. Chondrocytes from human fetal arthrosis (HFA) were used to investigate

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Table 1. Results of Copolymerization of LA and BEMD in Bulka

copolymersb

mol. content of BEMD in feed (%)

mol. content of BEMD unit in copolymer (%)c

Mn (104)d

Mw/Mnd

yield (%)

PLGBG-PEG2000-PLGBG1 PLGBG-PEG2000-PLGBG2 PLGBG-PEG2000-PLGBG3 PLGBG-PEG2000-PLGBG4 PLGBG-PEG4600-PLGBG5 PLGBG-PEG4600-PLGBG6 PLGBG-PEG4600-PLGBG7 PLGBG-PEG4600-PLGBG8

2 4 8 15 2 4 8 15

1.1 1.7 4.2 7.3 1.4 2.3 5.3 7.4

1.97 1.52 1.04 0.76 2.51 1.52 1.27 0.80

1.81 1.57 1.44 1.46 1.36 1.42 1.35 1.55

71 64 54 47 75 65 56 50

a The copolymerization was carried out in bulk at 130 °C in the presence of PEG with 1/1000 of SnOct as catalyst. b 2000 and 4600 are the M of the 2 n dihydroxyl PEG used for copolymers 1-4 and 5-8, respectively. c Determined by 1H NMR (CDCl3). d Determined by GPC (CHCl3 as eluent).

Figure 2. 1H NMR spectrum of PLGG-PEG4600-PLGG7 and its peak assignments. Figure 1. 1H NMR spectrum of PLGBG-PEG4600-PLGBG7 and its peak assignments.

cytotoxicity of PLGG-PEG-PLGG. Chondrocytes were first isolated and purified from HFA and maintained in MEM medium containing 50 mg L-1 vitamin C and 10% calf serum, and the culture medium was replaced once every day. The PLGG-PEG-PLGG was dissolved in CHCl3, and the solution was added to the wells of a 6-well plate and then dried in a vacuum for 48 h to form a copolymer film in each well. Then the MEM medium was added into each well. The cells were seeded in each well with a cell density of 1 × 105 cells well-1 and were incubated at 37 °C in 5% CO2, and the growth medium was replaced with fresh one once every 24 h. The status of adhesion and growth of the cells was observed at 2 h, 24 h, and 5 d, respectively by an inverted fluorescence microscope (TE2000-U). Figure 3. FT-IR spectra of PLGBG-PEG4600-PLGBG7 and PLGGPEG4600-PLGG7.

Results and Discussion Synthesis of PLGBG-PEG-PLGBG. The copolymerization of BEMD and LA was carried out in the bulk at 130 °C with SnOct2 as catalyst (monomer/catalyst ) 1000) in the presence of anhydrous PEG in a polymerization flask. Here, the PEG played a role of macromolecular initiator because it contained two hydroxyl end-groups. The basic data of the

resultant copolymers were summarized in Table 1. It was found that the molar fraction of the BEMD unit in the copolymer was lower than that in the monomer feed, both molecular weight and overall yield tended to decrease with increasing BEMD content in the feed though the content of BEMD in the copolymer increased as its content increased in feed, and Mw/Mn for all copolymers is quite high. This


Figure 4. GPC traces of (a) PLGBG-PEG2000-PLGBG3, Mn ) 1.04 × 104, Mw/Mn ) 1.44; (b) PLGG-PEG2000 ) PLGG3, Mn ) 1.15 × 104, Mw/Mn ) 1.39; (c) PLGBG-PEG4600-PLGBG7, Mn ) 1.27 × 104, Mw/Mn ) 1.35; and (d) PLGG-PEG4600-PLGG7, Mn ) 1.36 × 104, Mw/Mn ) 1.31.

could be ascribed to lower reactivity of BEMD than that of LA, to competing chain transfer reactions, chain termination reactions, or depolymerization reactions caused by contaminants in the reaction mixture such as water.13 In addition, the yield in all polymerizations is in the range of 50∼70%, decreasing with increasing BEMD content in the monomer feed, probably due to the removal of low molecular weight products during the purification by methanol precipitation. The structure of the block copolymer was confirmed by 1H NMR and FT-IR spectra. The 1H NMR peaks marked with letters from a to i for PLGBG-PEG4600-PLGBG7 are assigned to the corresponding hydrogen atoms of the copolymer as shown in Figure 1. The peaks a at 7.34 ppm and b at 5.16 ppm are attributed to C6H5 and C6H5CH2 of the protecting group, respectively. The peaks c at 5.16 ppm and i at 1.58 ppm are attributed to CH and CH3 of lactic acid. The peak d at 4.88 ppm is attributed to CH2 of glycolic acid. The peaks f at 4.62 ppm and g + h at 2.49 ppm are attributed to CH and CH2-CH2-COOH of L-glutamate. The peak e at 3.65 ppm is attributed to CH2-CH2 of PEG. In the FT-IR spectra (Figure 3), the δCH characteristic peaks of the benzyl group Scheme 2. Synthesis of Block Copolymer PLGG-PEG-PLGG


appeared at 756 and 701 cm-1, indicating also the presence of BEMD residues in the copolymer. The gel permeation chromatography (GPC; Figure 4) trace of the block copolymer showed a unimodal peak. This further indicated that the copolymerization was completed successfully and there were no homopolymers in the reaction product. Synthesis of PLGG-PEG-PLGG. The benzyl protecting groups of the PLGBG-PEG-PLGBG were easily removed by catalytic hydrogenolysis over Pd/C (10%) in anhydrous THF/methanol. The copolymer was dissolved in THF with the help of heating and ultrasonic treatment. The polymer solution and the Pd/C suspension in anhydrous methanol were added together into the hydrogenator. The debenzylation reaction was performed under 1.0 MPa H2 atmosphere at 50 °C for 48 h. Almost all benzyl groups were removed, as evidenced by the absence of the resonances at 5.13 ppm (C6H5CH2) and 7.34 ppm (C6H5) in the 1H NMR spectra (Figure 2) and the disappearance of the δCH vibrations of the benzyl group at 756 and 701 cm-1 in the FTIR spectra (Figure 3). In addition, the OH stretching band centered at 3417 cm-1 was strengthened tremendously due to the formation of COOH groups. The GPC (Figure 4) traces of the deprotected copolymer PLGG-PEG2000-PLGG3 and PLGGPEG4600-PLGG7 (corresponding to PLGBG-PEG2000PLGBG3 and PLGBG-PEG4600-PLGBG7 in Table 1, respectively) also showed a unimodal shape, and moreover, the molecular weights of the deprotected copolymers were larger than those of protected ones, probably due to the intermolecular association caused by hydrogen bonding among the carboxyl groups of PLGG-PEG-PLGG. The same phenomenon was also reported in the literature.36,37 Characterization of PLGG-PEG-PLGG Micelles. Similar to conventional amphiphilic triblock copolymers, PLGGPEG-PLGG can self-assemble into micelles in aqueous solution. Here, micellization of PLGG-PEG2000-PLGG3 and PLGG-PEG4600-PLGG7 was studied as two representative

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Guan et al.

Scheme 3. Micelles Formed from PLGG-PEG-PLGG

examples. Scheme 3 illustrates the assumed structure of the micelles. Fluorescence probing technique was employed to confirm the formation of micelles. Figure 5 shows the excitation spectra of pyrene in the presence of various concentrations of PLGG-PEG-PLGG block copolymer. A red shift from 333 to 335.5 nm was observed with increasing concentration of PLGG-PEG-PLGG, indicating the formation of micelles because pyrene is preferentially partitioned into the hydrophobic core of the micelles with a change of the photophysical properties. As an example, Figure 6 shows a plot of the pyrene fluorescence intensity ratio I335.5/I333 versus the logarithm of PLGG-PEG4600-PLGG7 concentration. 333

Figure 7. ESEM image of PLGG-PEG4600-PLGG7 micelles.

Figure 5. Excitation spectra at λem ) 390 nm of pyrene in water solutions of various concentrations of PLGG-PEG4600-PLGG7.

Figure 8. Size distribution of the PLGG-PEG2000-PLGG3 (a) and PLGG-PEG4600-PLGG7 (b) micelles determined by DLS measurement.

Figure 6. Plot of the intensity ratio I335.5/I333 in pyrene excitation spectra vs log C for PLGG-PEG4600-PLGG7.

and 335.5 nm were chosen as the peak wavelengths of the (0, 0) band in the pyrene excitation spectra in the aqueous phase and in the entirely hydrophobic core of the polymeric micelle, respectively. The ratio I335.5/I333 was almost constant at low polymer concentrations, but from a certain concentra-



Figure 9. In vitro degradation of PLGG-PEG4600-PLGG6 (9) and PLA (2) in Tris-HCl buffer, pH: 8.2, protease K content: 9.6 µg/mL, 37 °C.

tion, it started to increase steadily and finally reached a plateau, indicating the incorporation of pyrene into the hydrophobic cores of the micelles. The apparent critical micelle concentration (cmc) was obtained from the intersection of the two tangent lines as shown in Figure 6. The cmc values of PLGG-PEG2000-PLGG3 and PLGG-PEG4600PLGG7 were 2.9 and 5.4 mg/L, respectively. The successful formation of PLGG-PEG-PLGG micelles was also confirmed by ESEM photographs as shown in Figure 7. The pictures clearly revealed a homogeneous spherical morphology of the self-assembled micelles. Their sizes were examined by dynamic light scattering. As shown in Figure 8, all micelle aggregates had a unimodal size distribution, and the mean diameters of PLGG-PEG2000PLGG3 and PLGG-PEG4600-PLGG7 are 69.5 and 93.9 nm, respectively. Moreover, above the cmc, the aggregates are quite stable upon dilution. In Vitro Degradation of PLGG-PEG-PLGG. The in vitro enzymatic degradation of PLGG-PEG4600-PLGG6 (corresponding to PLGBG-PEG4600-PLGBG6 in Table 1) as a representative sample was evaluated in the presence of proteinase K at 37 °C and compared with that of PLA in order to reveal the influence of the PEG segment and pendant carboxyl groups on that of PLGG-PEG-PLGG. The degree of degradation was estimated from (W0 - Wt)/W0, as shown in Figure 9. It was found that the degradation rate of the PLGG-PEG4600-PLGG6 is faster than that of PLA. When the degradation was performed over 11 h, the copolymer lost 43% of its weight whereas PLA lost only 25%. This was attributed mainly to the disruption of crystallinity by the incorporation of L-glutamate residues and the improved hydrophilicity caused by the PEG segment and the pendant carboxyl groups in the copolymer. In Vitro Cytotoxicity of PLGG-PEG-PLGG. In vitro cytotoxicity was evaluated qualitatively for PLGG-PEG4600PLGG7 as a representative sample. First, it was dissolved in CHCl3 and cast into films in the cultural wells. Second, the chondrocytes from HFA were seeded onto these polymer

Figure 10. Micrographs of HFA chondrocytes seeded on a PLGGPEG4600-PLGG7 film and incubated for 2 h (a); 24 h (b) and 5 d (c).

films. Third, the adhesion and growth behaviors of the cells were examined by optical microscope. Figure 10 shows the micrographs of the cartilage cells incubated for 2 h, 24 h, and 5 d. After 2 h, some cell spreading was seen on the polymer film. After 24 h, almost all cells got spread and began to grow. The cells continued to proliferate, and their density increased gradually and finally occupied the whole polymer surface in 5 d. These results preliminarily showed that this copolymer is a good compatible material suitable for biomedical applications such as drug delivery, tissue engineering, etc. Conclusion A series of biodegradable amphiphilic triblock copolymers bearing pendant carboxyl groups were successfully prepared by ROP of LA with BEMD in the presence of dihydroxyl PEG as a macroinitiator in bulk at 130 °C using SnOct2 as the catalyst and by subsequent catalytic hydrogenation. The

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copolymers could form micelles in aqueous solution with the cmc dependent on the composition of the copolymer. ESEM and DLS analysis of the micelles revealed homogeneous spherical morphology and a unimodal size distribution. In vitro degradation investigation in the presence of proteinase K showed that the degradation rate of PLGG-PEGPLGG was faster than that of PLA. The incubation of articular cartilage cells on the PLGG-PEG-PLGG surface showed a low degree of cytotoxicity. Presence of the pendant carboxyl groups on PLGG-PEGPLGG is expected to facilitate further modifications of the polymer, such as attaching drug molecules, short peptides and oligosaccharides onto the carboxyl groups. Especially, if lipophilic drug was conjugated to PLGG-PEG-PLGG, the resulting polymer-drug conjugate is amphiphilic and can selfassemble into nanometer micelles which have been considered as powerful nanocarriers in the dawning era of polymertherapeutics.38 Further investigation is in progress and will be reported elsewhere. Acknowledgment. The project was financially supported by the National Natural Science Foundation of China (Project No. 20274048 and 50373043), by the “863” Program (Project No. 2002AA326100) from the Ministry of Science and Technology of China, and by Chinese Academy of Sciences (Project No. KJCX2-SW-H07). References and Notes (1) Holland, S. J.; Tighe, B. J.; Gould, P. L. J. Controlled Release 1986, 4, 155-180. (2) Reed, A. M.; Gilding, D. K. Polymer 1981, 22, 494-498. (3) Gilding, D. K.; Reed, A. M. Polymer 1979, 20, 1459-1464. (4) In,t Veld, P. J. A.; Dijkstra, P. J.; Fijian, J. Macromol. Chem. 1992, 193, 2713-2730. (5) Ouchi, T.; Fujino, T. Makromol. Chem. 1989, 190, 1523-1530. (6) Kimura, Y.; Shirotani, K.; Yamane, H.; Kitao, T. Macromolecules 1988, 21, 3338-3340. (7) Kimura, Y.; Shirotani, K.; Yamane, H.; Kitao, T. Polymer 1993, 34, 1741-1748. (8) Feng, Y.; Knufermann, J.; Klee, D.; Ho¨cker, H. Macromol. Chem. Phys. 1999, 201, 1506-1514. (9) Feng, Y.; Klee, D.; Keul, H.; Ho¨cker, H. Macromol. Chem. Phys. 2000, 201, 2670-2675. (10) Feng, Y.; Knufermann, J.; Klee, D.; Ho¨cker, H. Macromol. Rapid Commun. 1999, 21, 88-90. (11) Shirahama, H.; Sanaka, A.; Yasuda, H. J. Polym. Sci., Part A: Polym. Chem. 2002, 40, 302-316. (12) Barrera, D. A.; Zylstra, E.; Lansbury, P. T.; Langer, R. J. Am. Chem. Soc. 1993, 115, 11010-11011.

Guan et al. (13) Barrera, D. A.; Zylstra, E.; Lansbury, P. T.; Langer, R. Macromolecules 1995, 28, 425-432. (14) Ouchi, T.; Nozaki, T.; Ishikawa, A.; Fujimoto, I.; Ohya, Y. J. Polym. Sci., Part A: Polym. Chem. 1997, 35, 377-383. (15) Wang, D.; Feng, X. Macromolecules 1997, 30, 5688-5692. (16) Deng, X.; Yao, J.; Yuan, M.; Li, X.; Xiong, C. Macromol. Chem. Phys. 2000, 201, 2371-2376. (17) Feng, Y.; Klee, D.; Ho¨cker, H. Macromol. Chem. Phys. 2002, 203, 819-824. (18) John, G.; Tsuda, S.; Morita, M. J. Polym. Sci., Part A: Polym. Chem. 1997, 35, 1901-1907. (19) John, G.; Morita, M. Macromol. Rapid Commun. 1999, 20, 265268. (20) John, G.; Morita, M. Macromolecules 1999, 32, 1853-1858. (21) (a) Cammas, S.; Guerin, Ph. Macromol. Symp. 2000, 153, 167-186. (b) Caron, A.; Braud, C.; Bunel, C.; Vert, M. Polymer 1990, 31, 1797-1802. (c) Trollsas, M.; Lee, V. Y.; Mecerreyes, D.; Lowenhielm, P.; Moller, M.; Miller, R. D.; Hedrick, J. L. Macromolecules 2000, 33, 4619-4627. (22) (a) Tian, D.; Dubois, P.; Grandfils, C.; Je´roˆme, R. Macromolecules 1997, 30, 406-409. (b) Tian, D.; Dubois, P.; Je´roˆme, R. Macromolecules 1997, 30, 1947-1954. (c) Tian, D.; Halleux, O.; Dubois, P.; Je´roˆme, R. Macromolecules 1998, 31, 924-927. (d) Stassin, F.; Halleux, O.; Dubois, Ph.; Detrembleur, C.; Lecomte, Ph.; Je´roˆme, R. Macromol. Symp. 2000, 153, 27-39. (23) Al-Azemi, T. F.; Bisht, K. S. Macromolecules 1999, 32, 65366540. (24) Shen, Y.; Chen, X.; Gross, R. A. Macromolecules 1999, 32, 38913897. (25) Ray, W. C.; Grinstaff, M. W. Macromolecules 2003, 36, 3557-3562. (26) Adams, M. L.; Andes, D. R.; Kwon, G. S. Biomacromolecules 2003, 4, 750-757. (27) Tang, Y.; Liu, S. Y.; Armes, S. P.; Billingham, N. C. Biomacromolecules 2003, 4, 1636-1645. (28) Herold, D. A.; Keil, K.; Bruns, D. E. Biochem. Pharmacol. 1989, 38, 73-76. (29) Richter, A. W.; Akerblom, E. Int. Arch. Allergy Appl. Immunol. 1983, 70, 124. (30) Handbook of Water-Soluble Gums and Resins; McGraw-Hill: New York, 1980; Chapter 18. (31) Luo, L.; Tam, J.; Maysinger, D.; Eisenberg, A. Bioconjugate Chem. 2002, 13, 1259-1265. (32) Jeong, B.; Kibbey, M. R.; Birnbaum, J. C.; Won, Y. Y.; Gutowska, A. Macromolecules 2000, 33, 8317-8322. (33) Rashkov, I.; Espartero, J. L.; Manolova, N.; Vert, M. Macromolecules 1996, 29, 57-62. (34) Youxin, L.; Kissel, T. J. Controlled Release 1993, 27, 247-257. (35) Kricheldorf, H. R.; Meier-Haack, J. Makromol. Chem. 1993, 194, 715-725. (36) Kimura, Y.; Shirotani, K.; Yamane, H.; Kitao, T. Polymer 1993, 34, 1741-1748. (37) He, B.; Bei, J.; Wang, S. Polymer 2003, 44, 989-994. (38) (a) Bae, Y.; Fukushima, S.; Harada, A.; Kataoka, K. Angew. Chem., Int. Ed. 2003, 42, 4640-4643. (b) Haag, R. Angew. Chem., Int. Ed. 2004, 43, 278-282.

BM0492069

Synthesis and Characterization of Biodegradable Amphiphilic Triblock

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