Synthesis and Characterization of Biodegradable Poly (ethylene

Biomacromolecules 2004, 5, 1810-1817

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Synthesis and Characterization of Biodegradable Poly(ethylene glycol)-block-poly(5-benzyloxy-trimethylene carbonate) Copolymers for Drug Delivery Faquan Zeng,† Jubo Liu,† and Christine Allen*,†,‡ Department of Pharmaceutical Sciences and Department of Chemistry, University of Toronto, 19 Russell Street, Toronto, Ontario, Canada M5S 2S2 Received March 18, 2004; Revised Manuscript Received June 3, 2004

Amphiphilic diblock copolymers with various block compositions were synthesized with monomethoxyterminated poly(ethylene glycol) (MePEG) as the hydrophilic block and poly(5-benzyloxy-trimethylene carbonate) (PBTMC) as the hydrophobic block. When the copolymerization was conducted using MePEG as a macroinitiator and stannous 2-ethylhexanoate (Sn(Oct)2) as a catalyst, the molecular weight of the second block was uncontrollable, and the method only afforded a mixture of homopolymer and copolymer with a broad molecular weight distribution. By contrast, the use of the triethylaluminum-MePEG initiator yielded block copolymers with controllable molecular weight and a more narrow molecular weight distribution than the copolymers obtained using Sn(Oct)2. GPC and 1H NMR studies confirmed that the macroinitiator was consumed and the copolymer composition was as predicted. Two of the newly synthesized MePEGb-PBTMC copolymers were evaluated in terms of properties primarily relating to their use in micellar drug delivery. MePEG-b-PBTMC micelles with a narrow monomodal size distribution were prepared using a high-pressure extrusion technique. The MePEG-b-PBTMC copolymers were also confirmed to be biodegradable and noncytotoxic. Introduction Amphiphilic diblock copolymers with poly(ethylene glycol) (PEG) as the hydrophilic block and a poly(ester),1-4 poly(amino acid)5,6 or poly(ether)7 as the hydrophobic block have been explored extensively for applications in drug delivery.8 In an aqueous medium, these copolymers selfassemble to form micelles that consist of a hydrophobic core surrounded by a hydrophilic shell. The hydrophobic core may serve as a nanoreservoir for loading hydrophobic drugs, while the hydrophilic shell provides a protective coating between the core and the external medium. Drug formulations based on block copolymer micelles have entered clinical trial development, whereas others are showing promise in preclinical evaluation.9-12 In several studies involving block copolymer micelles as drug delivery systems, it has become clear that polymerdrug compatibility is one of the key factors influencing the performance-related parameters of the delivery system.13-15 Specifically, the compatibility between the drug and the coreforming block of the copolymer has been found to affect drug-loading capacity, drug loading efficiency, stability of the formulation, and the release kinetic profile of the drug from the micelles. Since each drug is unique in terms of physical and chemical properties, it is unlikely that any one copolymer will serve as the ideal material for delivery of all * To whom correspondence should be addressed. Phone: (416) 9468594. Fax: (416) 978-8511. E-mail: [email protected]. † Department of Pharmaceutical Sciences. ‡ Department of Chemistry.

drugs. For this reason, there is a need to design and explore new biocompatible, biodegradable copolymers for use in drug delivery.15 To date, several of the block copolymers that have been explored for preparation of micellar delivery systems include a hydrophobic polyester block such as polycaprolactone1-3 or poly(D,L-lactide).1-3 We now propose the use of block copolymers containing a hydrophobic polycarbonate block for preparation of micellar delivery systems. To our knowledge, amphiphilic diblock copolymers containing a hydrophobic polycarbonate block remain relatively unexplored for applications in drug delivery. By contrast, polycarbonate polymers are approved for use in biomedical applications such as materials for surgical closures and dental void fillers. To this point, the polycarbonate that has been explored most extensively is poly(trimethylene carbonate) (PTMC). It has been found that homopolymer PTMC may be synthesized by solution or bulk polymerization methods with a variety of initiators.16,17 In addition, TMC has been copolymerized with glycolide, lactide, caprolactone and other carbonates.18-20 In recent years, a few groups have also reported on the synthesis of amphiphilic block copolymers containing polycarbonate blocks. For example, Wang et al. reported the synthesis of an ABA triblock copolymer with poly(ethylene glycol) as the central block and PTMC as the hydrophobic end blocks. This triblock copolymer was synthesized using PEG as the macroinitiator and stannous 2-ethylhexanoate (Sn(Oct)2) as the catalyst.21 Also, Kim et al. prepared poly(2-ethyl-2-oxazoline)-b-poly(trimethylene carbonate) with

10.1021/bm049836a CCC: $27.50 © 2004 American Chemical Society Published on Web 07/13/2004

MePEG-b-PBTMC Copolymers

poly(2-ethyl-2-oxazoline) as the macroinitiator and Sn(Oct)2 as the catalyst.22 However, studies in our laboratory revealed that this system, MePEG as the macroinitiator and Sn(Oct)2 as the catalyst, is selective. Our use of MePEG as a macroinitiator along with Sn(Oct)2 as the catalyst for the ring opening polymerization of 5-benzyloxy-trimethylene carbonate (BTMC) yielded a mixture of MePEG-b-PBTMC block copolymer and PBTMC homopolymer. In addition, the molecular weight distribution of the copolymer was relatively broad (Mw/Mn ) 1.5-1.6). For this reason, we explored the use of an aluminum-based initiator system for the synthesis of the MePEG-b-PBTMC copolymer. This paper reports the synthesis of amphiphilic diblock copolymers containing poly(5-benzyloxy-trimethylene carbonate) as the hydrophobic block. Specifically, monomethoxyterminated poly(ethylene glycol) (MePEG) was used as the hydrophilic block and macroinitiator for the polymerization of 5-benzyloxy-trimethylene carbonate, as the hydrophobic block. The ring opening polymerization of 5-benzyloxytrimethylene carbonate was performed in the presence of either triethylaluminum (Al(Et)3) as an initiator precursor or Sn(Oct)2 as catalyst. Gel permeation chromatography (GPC) and 1H NMR were used to confirm that the macroinitiator was consumed and the copolymer composition was as predicted. Several properties of the MePEG-b-PBTMC copolymer and micelles relating to use in drug delivery were examined including critical micelle concentration, micelle morphology, rate of copolymer degradation and in vitro cytotoxicity in two cell lines. Experimental Section Materials. The macroinitiator of monomethoxy-terminated poly(ethylene glycol) (MePEG, Mn) 2000, Mw/Mn) 1.06 as determined by SEC and Mn ) 5000, Mw/Mn) 1.06 as determined by SEC) was obtained from Aldrich and dried by azeodistillation of toluene. Al(Et)3 (1.9 M in toluene), Sn(Oct)2, (95 wt %) and 1,6-diphenyl-1,3,5-hexatriene (DPH) were purchased from Aldrich and used without further purification. Toluene was dried under calcium hydride and distilled before use while 1,1,2,2-tetrachloroethane was dried by molecular sieves. Other solvents such as hexane, ethyl ether, chloroform and acetone were used as received. Methods and Measurements Synthesis of BTMC. The monomer of 5-benzyloxytrimethylene carbonate (BTMC) was synthesized and purified using a procedure that had been described elsewhere.23 In brief, 10 g of 2-benzyloxy-1,3-propanediol (29.6 mmol), 6.7 g of ethyl chloroformate (74.0 mmol), and 80 mL of tetrahydrofuran (THF) were added to a two neck roundbottom flask and cooled to 0 °C. 6.2 g of triethylamine (74.0 mmol) was then added dropwise to this mixture at 0 °C over a 30 min period. The reaction mixture was then stirred for 2 h at room temperature. The precipitate that formed was filtered and the solvent removed. The crude product was recrystallized from ethyl acetate yielding 3.16 g of a white crystal (yield ) 51%). 1H NMR (CDCL3): 3.7 (d, 1H,

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-CH-O-CH2-Ph), 4.39 (m, 4H, -(CH2)2-CH-O-CH2Ph), 4.56 (s, -O-CH2-Ph) and 7.36 (m, 5H, -C6H5). Synthesis of MePEG-b-PBTMC. The diblock copolymers of MePEG-b-PBTMC were synthesized using either Al(Et)3 or Sn(Oct)2. For polymerization using Al(Et)3 as the initiator precursor: 0.1 g of MePEG (0.05 mmol, Mn ) 2000, Mw/Mn ) 1.06), 0.1 g of BTMC (0.481 mmol) were added to a round-bottom flask and dried twice by toluene azeodistillation. 2 mL of 1,1,2,2-tetrachloroethane (dried by molecular sieves) was also added to the flask. The reaction mixture was then heated to 110 °C in an oil bath and a stoichiometric amount of Al(Et)3, with respect to the MePEG in toluene (29 µL, 1.9 M), was added. The reaction mixture was stirred at this temperature for 5 h. The block copolymer was isolated by precipitation in cold hexane and then purified by dissolving the polymer in deionized water followed by extraction three times with chloroform. The aqueous emulsion layer was then dried by lyophilization. For polymerization using Sn(Oct)2 as the catalyst: 0.1 g of MePEG (0.05 mmol, Mn ) 2000, Mw/Mn ) 1.06) and 0.1 g of BTMC (0.481 mmol) were added to a round-bottom flask and dried twice by toluene azeodistillation. 2 mL of 1,1,2,2-tetrachloroethane dried by molecular sieves was then added to the flask. The temperature of the reaction mixture was raised to 150 °C and 5 mg of Sn(Oct)2 was added under a nitrogen atmosphere. The solution was stirred at this temperature for 30 h. The block copolymer was isolated by precipitation in cold hexane and dried under vacuum. Characterization of Block Copolymers. 1H NMR spectra were obtained on a Gemini 200 spectrometer (200 MHz for 1 H) in CDCl3 solvent. Chemical shifts were reported in ppm with CHCl3 as the internal standard. GPC measurements were carried out at room temperature using a Waters 590 liquid chromatography system equipped with three Waters Styragel HR 4E columns and a 410 differential refractometer detector. THF with 1% triethylamine was used as the solvent at a flow rate of 1.0 mL/min at 40 °C. Narrow polystyrene standards (Polysciences Inc., Warrington, PA) were used to generate a calibration curve. The data obtained were recorded and manipulated using the Windows-based Millenium 2.0 software package (Waters Inc., Milford, MA). Measurement of Critical Micelle Concentration. The critical micelle concentrations of MePEG-b-PBTMC copolymers were determined using an established fluorescencebased method.24 The copolymers studied were 2000-b-1900 (Table 1, entry 6) and 5000-b-4800 (Table 1, entry 10) (where for x-b-y, x denotes the molecular weight of MePEG and y the molecular weight for PBTMC). In short, a specific aliquot of MePEG-b-PBTMC dissolved in chloroform was added to glass vials such that the concentration of copolymer ranged from 0.1 to 200 mg/L. An aliquot of a DPH (1,6diphenyl-1,3,5-hexatriene) stock solution was then added to each vial such that the concentration of DPH was maintained at 1 mg/L in each solution. The solutions were vigorously stirred for 4 h and then thoroughly dried under nitrogen. The dried vials were heated to 60 °C, and 1 mL of double distilled water was slowly added into each vial. The solutions were equilibrated with stirring overnight at room temperature and fluorescence emission was measured at 430 nm (λex ) 350

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Table 1. Summary of the Results for the Synthesis of MePEG-b-PBTMC Copolymers Using Sn(Oct)2 as a Catalyst or Al(Et)3 as Initiator Precursor (Init-pre) under the Specified Conditions entry

macroinitiatora

catalyst or Init-pre

temp (°C)

solvent

[M]/[Cat or I]b

[ROH]/[Cat or I]c

1 2 3 4 5 6 7 8 9 10

MePEG2K MePEG2K MePEG2K MePEG2K MePEG2K MePEG2K MePEG2K MePEG2K MePEG2K MePEG5K

Sn(Oct)2 Sn(Oct)2 Al(Et)3 Al(Et)3 Al(Et)3 Al(Et)3 Al(Et)3 Al(Et)3 Al(Et)3 Al(Et)3

150 150 110 110 110 110 110 110 110 110

C2H2Cl4 C2H2Cl4 toluene C2H2Cl4 C2H2Cl4 C2H2Cl4 C2H2Cl4 C2H2Cl4 C2H2Cl4 C2H2Cl4

10 20 10 5 7.5 10 10 20 30 25

1:1 1:1 1:1 1:1 1:1 1:1 1:1 1:1 1:1 1:1

Mn,cald

Mn,GPCe

Mn,NMRf

1921 1438 3040 3600 4000 4080 6000 8000 10000

3390 4070 4380 4370 7400 9700 15700

Mw/Mn 1.55 1.66

2200 2900 3900 3800 6100 7950 9800

1.21 1.19 1.36 1.33 1.30 1.27 1.27

a MePEG2K, M ) 2000; MePEG5K, M ) 5000. b The mole ratio of monomer to Sn(Oct) or the mole ratio of monomer to Al(Et) . c The mole ratio n n 2 3 of MePEG to Sn(Oct)2 or the mole ratio of MePEG to Al(Et)3. d Mn,cal ) Mn,PEG + Mn,PBTMC; Mn,PBTMC is the calculated molecular weight of the PBTMC block based on the feed ratio of BTMC to MePEG. e Mn,GPC is the relative molecular weight with respect to poly(styrene) standards. f Mn,NMR ) Mn,PEG + Mn,PBTMC; Mn,PBTMC was calculated from 1H NMR analysis.

nm; Spectra GeminiXS dual-scanning microplate spectrofluorometer, Molecular devices, Sunnyvale, CA). Preparation of Micelles. Micelles were prepared using two methods, namely the dry-down method and the highpressure extrusion method. For the dry-down method, polymer stock solutions were prepared by dissolving the copolymer in DMF. The solutions were allowed to stir for 4 h and then thoroughly dried under nitrogen. The glass vials were left in a vacuum oven overnight prior to the addition of warmed PBS (phosphate buffer saline; 0.01M; 60 °C) to rehydrate the dried copolymer films. The solutions were vortexed and left to stir at room temperature for 72 h. For the high-pressure extrusion method, following the above outlined procedure the copolymer solutions were extruded 10 times using a 10 mL thermobarrel extruder (Northern Lipids, Vancouver, BC, Canada) fitted with a single 0.08 µM polycarbonate membrane (Whatman, Clifton, NJ). Determination of Micelle Morphology and Size. Dynamic Light Scattering. The hydrodynamic radius and the polydispersity of micelles were determined by dynamic light scattering (DLS) (DynaPro-MS/X; Protein Solutions Inc., Lakewood, NJ). The sample solutions were diluted in filtered double-distilled water prior to analysis. The instrument equipped with Dynamic V6.0. software calculates the hydrodynamic radius of particles as well as the polydispersity or size distribution of the sample. Transmission Electron Microscopy (TEM). The morphology of the micelles was analyzed by TEM with a Hitachi 7000 microscope operating at an acceleration voltage of 75 kV. The micellar solutions prepared by the extrusion method were diluted using PBS and mixed in a 1:1 ratio (v/v) with PTA (phosphotungstic acid). The samples were then deposited onto copper grids that had been precoated with carbon. Measurement of Cytotoxicity of Copolymers. SKOV-3 ovarian cancer cells and CHO K-1 fibroblast cells were maintained in RPMI 1640 and R-ΜΕΜ medium, respectively. The RPMI 1640 and R-ΜΕΜ media were both supplemented with 10% (v/v) heat-inactivated FBS and 1% (v/v) penicillin-streptomycin (100 U/mL penicillin G and 100 µg/mL streptomycin). Cells were allowed to grow in a monolayer in a tissue culture flask incubated at 37 °C in 5% CO2 and 90% relative humidity. The cells were first seeded in a 96 well plate with a cell density of 10 000 cells/

well. Following a 24 h incubation period, the growth medium was removed and replaced with 200 µL of fresh medium containing the appropriate amounts of copolymer (with n ) 3). The cells were then incubated for a further 24 h and cell viability was determined using the Celltiter 96 proliferation assay (Promega, Madison, WI). Specifically, 20 µL of working reagent (i.e. 20:1 (v:v) mixture of MTS (3-(4,5)dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt) and PMS (phenazine methosulfate) was added to each well, and the samples were incubated for 2 h with n ) 3 for each treatment. Cell viability was measured by optical absorbance at 490 nm using a SPECTRA MAX plus microplate reader (Molecular devices, Sunnyvale, CA). Evaluation of In Vitro Degradation of Copolymers. The block copolymers (Table 1, entry 6 and 10) were first dissolved in DMF at a concentration of 100 mg/mL and dried to form a copolymer film. The film was then rehydrated in PBS (pH ) 7.4) or PBS containing lysozyme (20 µg/mL) to give a final copolymer concentration of 10 mg/mL. The solutions were incubated at 37 °C, and aliquots (0.2 mL) were removed at specific time intervals. The aliquot was dried down and dissolved in THF for GPC analysis. Results and Discussion Synthesis and Characterization of MePEG-b-PBTMC Copolymers. Recently, Wang et al. reported on the synthesis of PBTMC homopolymer, with a glass transition temperature of 0 °C, and random copolymers containing 5-benzyloxytrimethylene carbonate using Sn(Oct)2 as a catalyst or aluminum alkoxide as an initiator.23,25 The resulting copolymers had relatively high molecular weights (i.e., Mn ) 14 500-32 400) with polydispersity ranging from 1.5-1.85. To this point, the synthesis of a block copolymer containing PBTMC has not been reported. Previous work had demonstrated that MePEG was capable of initiating polymerization of TMC to yield block copolymers when Sn(Oct)2 was used as the catalyst.20,22,26-27 Therefore, the block copolymerization of MePEG with BTMC was tried using MePEG as the macroinitiator and Sn(Oct)2 as the catalyst at 150 °C. GPC analysis of the



Scheme 1. Synthesis of Block Copolymer of Monomethoxy-terminated Poly(ethylene glycol)-block-poly(5-benzyloxy-trimethylene carbonate) in 1,1,2,2-tetrachloroethane Solvent at 110 °C

Figure 2. Dependence of molecular weight (Mn,GPC, Mn,NMR, and Mn,cal) and polydispersity on the feed ratio of monomer to initiator ([M]0/ [I]0 ) [BTMC]0/ [MePEG]0).

Figure 1. (A) GPC traces for (a) MePEG, Mn ) 2000, Mw/Mn ) 1.06; (b) Table 1, entry 6, Mn ) 3900, Mw/Mn ) 1.36; (c) Table 1, entry 8, Mn ) 6100, Mw/Mn ) 1.30; (d) Table 1, entry 9, Mn ) 7950, Mw/Mn ) 1.27. (B) GPC traces for: (a) Table 1, entry 6 from the crude reaction solution; (b) Table 1, entry 6 following the purification.

samples obtained revealed that this method resulted in PBTMC homopolymer as well as MePEG-b-PBTMC block copolymers (Table 1, entry 1 and 2) with a broad molecular weight distribution (Mw/Mn > 1.5). The PBTMC homopolymer is produced since Sn(Oct)2 also serves as an effective initiator for BTMC polymerization.23 Aluminum alkoxides have been shown to be effective initiators for ring opening polymerization of cyclic aliphatic

lactones and lactides.28 Albertsson et al. demonstrated that aluminum alkoxides are also effective initiators for polymerization of trimethylene carbonate to yield functionalized polymers.16,29 Thus, we selected Al(Et)3 to react with MePEG to form an initiator for the block copolymerization of BTMC. The synthesis of diblock copolymers of MePEGb-PBTMC using Al(Et)3 as the initiator precursor is outlined in Scheme 1. The MePEG was reacted with Al(Et)3 to give the monoalkoxide of aluminum, the macroinitiator. The macroinitiator was then used for the initiation of the polymerization of BTMC, at 110 °C, to yield the block copolymers. Table 1 summarizes the results for the block copolymerization of MePEG with BTMC. When toluene was used as the solvent and Al(Et)3 as the initiator precursor, the aluminum alkoxide did not initiate the block copolymerization of BTMC (Table 1, entry 3); however, the copolymerization proceeded smoothly when 1,1,2,2-tetrachloroethane was used as the solvent at 110 °C. Figure 1 includes the GPC traces for MePEG-b-PBTMC from the crude reaction mixture. From the GPC traces, it is evidenced that there is low molecular weight PBTMC present

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Figure 3.

1H

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NMR spectrum of the MePEG-b-PBTMC copolymer (Table 1, entry 10).

in trace amounts: shown as tailing to the right of the main peaks. The production of PBTMC may result from the reaction of Al(Et)3 with moisture present within the system yielding Al(Et)2OH which is also an effective initiator for BTMC polymerization. The trace amount of low molecular weight homopolymer, PBTMC, was removed by dissolving the polymer in water and extracting three times with chloroform. The GPC trace for the purified copolymer is shown in Figure 1B. As shown in Figure 2, the molecular weight of the copolymer was found to increase linearly with an increase in the ratio of monomer to macroinitiator. This indicates that the polymerization is controllable, and therefore, block copolymers with different target molecular weights for the PBTMC block can simply be prepared by varying the ratio of monomer to macroinitiator (Table 1 and Figure 2). The molecular weight measured by 1H NMR was in agreement with the theoretical or calculated molecular weight, whereas the molecular weight measured by GPC deviated from the theoretical molecular weight (Figure 2). The discrepancy between the theoretical molecular weight and that determined by GPC analysis may be attributed to the difference between the hydrodynamic volumes of the block copolymers and the polystyrene standards (Figure 2). The resulting MePEG-b-PBTMC copolymers had a more narrow molecular weight distribution (i.e., Mw/Mn ) 1.27-1.36) than the copolymers prepared using Sn(Oct)2 as the catalyst. Figure 3 includes the 1H NMR spectrum for the block copolymer (Table 1, entry 10). As shown, in addition to the signals corresponding to the MePEG unit (3.60 ppm, -OCH2-H2-), signals for the PBTMC unit appeared at 3.81 ppm (m, O-CH2-CH-CH2-O), 4.22 ppm (m, O-CH2-CH-CH2-O), 4.59 ppm (m, O-CH2-Ph), and 7.32 (m, O-CH2-Ph). The GPC and NMR spectra thus confirmed the formation of MePEG-b-PBTMC. The molec-

Figure 4. Semilogarithmic plot of the fluorescence emission intensity versus log of the copolymer concentration. The copolymers analyzed are MePEG-b-PBTMC (2000-b-1900, Table 1 entry 6 and 5000-b4800, Table 1, entry 10).

ular weight of the second block was calculated from the overall composition from NMR and the known molecular weight of the MePEG precursor (Table 1). Critical Micelle Concentration for MePEG-b-PBTMC Copolymers. The critical micelle concentrations of the MePEG-b-PBTMC copolymers (Table 1, entry 6 and 10) were measured using a fluorescence-based method that relies on DPH (1,6-diphenyl-1,3,5-hexatriene) as the fluorescent probe. Figure 4 includes the plots of emission intensity versus the logarithm of the copolymer concentration. The CMC values were found to be 8.06 mg/L (0.82 µM) and 4.09 mg/L (1.05 µM) for the 2000-b-1900 (Table 1, entry 6) and 5000-b-4800 (Table 1, entry 10) of MePEG-b-PBTMC copolymers, respectively. Therefore, the CMC value for the copolymer decreased with an increase in the molecular weight (block length) of the hydrophobic block. It is well established that the CMC of a copolymer is mostly determined by the length of the hydrophobic block rather than the length of the hydrophilic block.30 In this way, it was expected that the 5000-b-4800 copolymer would have a



Figure 5. Transmission electron micrograph of MePEG-b-PBTMC micelles in aqueous solution (copolymer employed is that in Table 1, entry 10).

lower CMC value than the 2000-b-1900 MePEG-b-PBTMC copolymer. The CMC value for the 5000-b-4800 MePEGb-PBTMC copolymer, 4.09 mg/L, is between the CMC values reported for MePEG-b-poly(β-benzyl-L-aspartate) (4840-b-3900), CMC ) 5 mg/L and PEG-b-polystyrene (6800-b-1700), CMC ) 2.8 mg/L.30,32 This was anticipated as the degree of hydrophobicity of the PBTMC block is between that of poly(β-benzyl-L-aspartate) and polystyrene. Morphology and Size of MePEG-b-PBTMC Micelles in Aqueous Solution. The size and morphology of the MePEG-b-PBTMC (5000-b-4800) micelles in PBS were examined by DLS and TEM. For these studies, the micelles were prepared by two methods, a traditional method known as the dry-down method, and a high-pressure extrusion technique. Analysis of the prepared samples by DLS revealed that the traditional method of preparation gave rise to populations of copolymer aggregates having a bimodal size distribution. Specifically, this included primary micelles of approximately 82 nm in diameter and secondary aggregates with a diameter of 635.6 nm. The formation of bimodal populations of aggregates from copolymers containing PEG as the hydrophilic block is quite common.6,31-33 The secondary aggregates are believed to arise due to interactions between the exposed hydrophobic cores of individual micelles. The formation of large secondary aggregates has been

prevented or reduced by use of copolymers having a narrow degree of polydispersity as well as by introduction of a slight surface charge on the micelles.34 In our studies, we found that the high-pressure extrusion method reduced the formation of secondary aggregates. DLS analyses of micelles formed by this method were found to have a diameter of 71 nm with little or no formation of secondary aggregates (e3 wt %). As shown in Figure 5, the micelles prepared by the high-pressure method have a spherical morphology with a diameter of approximately 45 nm. The diameter of micelles determined by DLS (i.e., 71 nm) represents the hydrodynamic diameter of the core and shell of the micelles. The diameter obtained by TEM (i.e., 45 nm) is that of the core of the micelle with a small contribution from the collapsed shell. The calculated fully stretched length for the hydrophobic PBTMC block with a molecular weight of 4800 g/mol is 20 nm. However, due to the polydispersity of the copolymer, which is largely owed to the distribution of hydrophobic block lengths, the fully stretched length for some hydrophobic blocks is greater than 20 nm. The diameter attributed to the PEG shell is 26 nm (i.e., 71-45 nm), that is, approximately 13 nm for the end-toend distance of each PEG chain. The calculated end-to-end distance of PEG chains with a molecular weight of 5000 g/mol is 40 nm by the zigzag model for a fully extended

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Figure 6. Cytotoxicity of the MePEG-b-PBTMC copolymers (MePEGb-PBTMC; 2000-b-1900 from Table 1, entry 6 and 5000-b-4800 from Table 1, entry 10). The cell viabilities (relative to controls) of both the CHO K-1 fibroblast cells (A), and SKOV-3 human ovarian cancer cells (B), are expressed as a function of the logarithm of the concentration of copolymer.

conformation, 20 nm by the meander model, and 3.7 nm by the random-coil model.34,35 Therefore, the PEG chains of the MePEG-b-PBTMC micelles in PBS solution are likely in a conformation that lies between the meander and the randomcoil models. In Vitro Cytotoxicity of the MePEG-b-PBTMC Copolymers. Several different groups have established the biocompatibility of block copolymers containing PEG as the hydrophilic block and a wide range of polymers as the hydrophobic block.1-8 The cytotoxicity of random copolymers containing PBTMC has also been studied with a very low degree of cytotoxicity reported.25 In our study, the cytotoxicity of two MePEG-b-PBTMC copolymers, namely 2000-b-1900 and 5000-b-4800 (Table 1, entry 6 and 10), were evaluated in both the fibroblast cell line CHO-K1 and the human ovarian cancer cell line SKOV-3. As shown in Figure 6, the incubation of both cell lines with the copolymers resulted in a low degree of cytotoxicity, with relative cell viabilities above 90% for all copolymer concentrations (up to 5000 mg/L). Even at the highest concentration of both block copolymers, there was no significant change in cell proliferation, relative to controls, following a 24 h incubation period. These results provide a preliminary indication that this copolymer is suitable for biomedical applications such as drug delivery. In Vitro Degradation of MePEG-b-PBTMC Copolymers. It has been shown that aliphatic polycarbonates are more resistant to hydrolysis than aliphatic polyesters.36,37 In

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Figure 7. In vitro degradation of MePEG-b-PBTMC in PBS solution (0.01M, pH ) 7.4) at 37 °C. The copolymer concentration was 10 mg/mL. (A) 2000-b-1900 from Table 1, entry 6. (B) 5000-b-4800 from Table 1, entry 10. The percent change in molecular weight of the copolymer is expressed as a function of time (t) based on GPC analysis.

addition, the degradation of some aliphatic polycarbonates by lipolytic enzymes has been found to proceed to a lesser extent than that for aliphatic polyesters (e.g., polycaprolactone).36 The in vitro degradation of two MePEG-b-PBTMC copolymers (i.e., samples listed as entry 6 and 10 in Table 1) were evaluated in PBS (pH ) 7.4), in the presence and absence of lysozyme at 37 °C. As shown in Figure 7, panels A and B, in the absence of lysozyme, only 5-10% of the copolymer was degraded over the seven day period, whereas in the presence of lysozyme, over 20-30% of the copolymer was degraded during this period. Conclusion Block copolymers of MePEG-b-PBTMC were synthesized using MePEG as the macroinitiator and Al(Et)3 as initiator precursor. The copolymers prepared by this method were found to have a controllable molecular weight and more narrow molecular weight distribution (Mw/Mn ) 1.27-1.36), when compared to copolymers synthesized with Sn(Oct)2 as catalyst. The critical micelle concentration of the block copolymers in aqueous solution ranged from 4.09 to 8.06 mg/L. TEM and DLS analysis of the MePEGb-PBTMC (5000-b-4800) micelles, prepared by a highpressure extrusion technique, revealed a monomodal size distribution for the micelles with an average diameter of 71 nm. These copolymers were found to be biodegradable and noncytotoxic as confirmed in SKOV-3 and CHO K-1 cells. These materials will now be explored for applications in drug delivery.


Acknowledgment. The authors thank the National Science and Engineering Research Council (NSERC) for supporting this research. We also thank Professor Sefton for kindly donating the CHO K-1 fibroblast cells and Professor Uetrecht for allowing us to use the fluorescence and UV/vis microplate readers. References and Notes (1) Kim, S. Y.; Shin, I. G.; Lee, Y. M.; Cho, C. S.; Sung, Y. K. J. Controlled Release 1998, 51, 13. (2) Benahmed, A.; Ranger, M.; Leroux, J. C. Pharm Res. 2001, 18, 323. (3) Burt, H. M.; Zhang, X. C.; Toleikis, P.; Embree, L.; Hunter, W. L. Colloids Surf. B: Biointerfaces 1999, 16, 161. (4) Li, J.; Li, X.; Ni, X.; Leong, K. W. Macromolecules 2003, 36, 2661. (5) Harada, A.; Kataoka, K. Macromolecules 1998, 31, 288. (6) La, S. B.; Okano, T.; Kataoka, K. J. Pharma. Sci. 1996, 85, 85. (7) Miller, D. W.; Batrakova, E. V.; Waltner, T. O.; Alakhov, V. Y.; Kabanov, A. V. Bioconjugate Chem. 1997, 8, 649. (8) Kwon, G. S. Crit. ReV. Therapeutic Drug Carrier Syst. 2003, 20 (5), 357. (9) Liu, J.; Allen, C. Drug DeliVery Companies Report; U.K. Autumn/ Winter, 2003, p 25. (10) Alakhov, V.; Klinski, E.; Li, S.; Pietrzynski, G.; Venne, A.; Batrakova, E.; Bronitch, T.; Kabanov, A. Colloids Surf. B: Biointerfaces 1999, 16, 113. (11) Nakanishi, T.; Fukushima, S.; Okamoto, K.; Suzuki, M. J. Controlled Release 2001, 74, 295. (12) Duncan, R. Nat. ReV. Drug DiscoVery 2003, 2, 347. (13) Lavasanifar, A.; Samuel, J.; Kwon, G. S. AdV. Drug DeliVery ReV. 2002, 54, 169. (14) Liggins, R. T.; Burt, H. M. AdV. Drug DeliVery ReV. 2002, 54, 191. (15) Liu, J.; Xiao, Y.; Allen, C. J. Pharm. Sci. 2004, 93, 132. (16) Albertsson, A.; Sjoling, M. J. Macromol. Sci., Pure Appl. Chem. 1992, A29, 43. (17) Kricheldorf, H.; Jenssen, J.; Kreiser-Saunders, I. Makromol. Chem. 1991, 192, 2391.

Biomacromolecules, Vol. 5, No. 5, 2004 1817 (18) Barrows, T. Clin. Mat. 1986, 1, 233. (19) Jia, Y. T.; Kim, H. Y.; Gong, J.; Lee, D. R.; Ding, B.; Bhattarai, N. Polym. Int. 2004, 53, 312. (20) Kim, J. H.; Lee, J. H. Polym. J. 2002, 34, 203. (21) Wang, H.; Dong, J.; Qiu, K. J. Polym. Sci. Part A: Polym. Chem. 1998, 36, 695. (22) Kim, C.; Lee, S.; Shin, J.; Yoon, J. Macromolecules 2000, 33, 7448. (23) Wang, X.; Zhuo, R.; Liu, L.; He, F.; Liu, G. J. Polym. Sci. Polym. Chem. 2002, 40, 70. (24) Zhang, X.; Jackson, J.; Burt, H. J. Biochem. Biophys. Methods 1996, 31, 145. (25) Wang, X.; Zhou, R.; Huang, S.; Liu, L.; He, F. Macromol. Chem. Phys. 2002, 203, 985. (26) Kim, J.; Lee, J. Macromol. Res. 2002, 10, 54. (27) Kim, J.; Lee, S.; Chung, D. Polym. J. 2000, 32, 1056. (28) Lofgren, A.; Albertsson, A.; Dubois, P.; Jerome, R. J. Macromol. Sci., ReV. Macromol. Chem. Phys. 1995, C35, 379. (29) Kenneth, C.; Richter, R.; Krichedorf, H.; Hedrick, J. Macromolecules 1997, 30, 6074. (30) (a) Allen, C.; Maysinger, D.; Eisenberg, A. Colloids Surf. B: Biointerfaces 1999, 16, 3. (b) Kwon, G.; Naito, M.; Yokoyama, M.; Okano, T.; Sakurai, Y.; Kataoka, K. Langmuir 1993, 9, 945. (31) Allen, C.; Yu, Y.; Maysinger, D.; Eisenberg, A. Bioconjugate Chem. 1998, 8, 564. (32) Wilhelm, M.; Zhao, C. L.; Wang, Y.; Xu, R.; Winnik, M. A.; Mura, J. L.; Riess, G. Macromolecules 1991, 24, 1033. (33) Xu, R.; Winnik, M. A.; Hallett, F. R.; Riess, G.; Croucher, M. D. Macromolecules 1991, 24, 87. (34) Yamamoto, Y.; Nagasaki, Y.; Kato, Y.; Sugiyama, Y.; Kataoka, K. J. Controlled Release 2001, 77, 27. (35) Tanford, C.; Nozaki, Y.; Rohde, M. F. J. Phys. Chem. 1977, 81, 1555-1560. (36) Tokiwa, Y. In Biopolymers; Matsumura, S., Steinbuchel, A., Eds.; Wiley-VCH: Weinheim, Germany, 2003; Vol. 9, p 417. (37) Acemoglu, M.; Bantle, S.; Mindt, T.; Nimmerfall, F. Macromolecules 1995, 28, 3030.

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