Synthesis and Characterization of Six-Arm Star Poly (Î´-valerolactone

Biomacromolecules 2005, 6, 2140-2149

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Synthesis and Characterization of Six-Arm Star Poly(δ-valerolactone)-block-Methoxy Poly(ethylene glycol) Copolymers Faquan Zeng,† Helen Lee,† Marc Chidiac,‡ and Christine Allen*,†,‡ Department of Pharmaceutical Sciences and Department of Chemical Engineering and Applied Chemistry, University of Toronto, 19 Russell St., Toronto, Ontario, Canada M5S 2S2 Received February 17, 2005; Revised Manuscript Received April 13, 2005

Novel amphiphilic six-arm star diblock copolymers based on biocompatible and biodegradable poly(δ-valerolactone) (PVL) and methoxy poly(ethylene glycol) (MePEG) were synthesized by a two-step process. First, the hydrophobic star-shaped PVL with hydroxyl terminated functional groups was synthesized using a multifunctional alcohol, dipentaerythritol (DPE), as the initiator and fumaric acid as the catalyst. The amphiphilic six-arm star copolymer of poly(δ-valerolactone)-b-methoxy poly(ethylene glycol), (PVL-bMePEG)6, was then synthesized by coupling the hydroxyl terminated six-arm PVL homopolymer with R-methoxy-ω-chloroformate-poly(ethylene glycol) (MePEG-COCl). 1H NMR and GPC analyses confirmed the successful synthesis of star-shaped copolymers with predicted compositions and narrow molecular weight distributions. DSC analysis revealed that the glass transition temperatures of the star PVL homopolymers with Mn between 5000 and 49 000 are not dependent on their molecular weights, whereas the melting temperatures of both the PVL homopolymers and the amphiphilic (PVL-b-MePEG)6 copolymers increase with an increase in the PVL molecular weight. Micelles were prepared from the (PVL-b-MePEG)6 copolymers via the dialysis method and found to have effective mean diameters ranging from 10 to 45 nm, depending on the copolymer composition. In addition, the (PVL-b-MePEG)6 copolymers having lower PVL content were found to form micelles with a narrow monomodal size distribution, whereas the copolymers having higher PVL content tended to form aggregates with a bimodal size distribution. The noncytotoxicity of the copolymers was also confirmed in CHO-K1 fibroblast cells using a cell viability assay, indicating that the (PVL-b-MePEG)6 copolymers are suitable for biomedical applications such as drug delivery. Introduction Block copolymers have been explored for use in a wide range of biomedical applications including fabrication and/ or coating of biomedical devices,1,2 tissue engineering,3-5 and drug delivery.6-9 Recently, amphiphilic block copolymers have been used extensively for drug formulation and delivery.10-12 In an aqueous medium, these amphiphilic copolymers self-assemble to form nanosized micelles that consist of a hydrophobic core surrounded by a hydrophilic shell. The hydrophilic shell acts as an interface between the micellar core and the surroundings, whereas the hydrophobic core may serve as a loading reservoir for lipophilic agents. To this point, mainly linear di- or triblock copolymers have been employed as the building blocks for these nanosized delivery systems, whereas amphiphilic copolymers having more complex architectures remain relatively unexplored. Recent advancements in synthetic methods have afforded controlled preparation of block copolymers having complex architectures such as dendrimer-like, graft, or star-shaped.13 These complex architectures have mostly been formed from * To whom all correspondence should be addressed. Phone: (416) 946-8594. Fax: (416) 978-8511. E-mail: [email protected]. † Department of Pharmaceutical Sciences. ‡ Department of Chemical Engineering and Applied Chemistry.

nonbiocompatible, nonbiodegradable materials and only in some cases have their solution properties been studied systematically. The two major strategies commonly employed for preparation of star-type block copolymers are the armfirst and core-first methods.14,15 The range of star-type homopolymers and copolymers synthesized have been formed from monomeric units such as styrene, styrene derivatives, methacrylates, vinyl ethers, carbonates, and aliphatic esters.16-20 To date, few studies have reported the synthesis of biocompatible and biodegradable star-shaped polymers. Three-arm star homopolymers and copolymers of -caprolactone (CL), D,L- or L-lactide (LA), and glycolide have been prepared using the core-first method with glycerol or trimethylolpropane as the multifunctional initiator and stannous (II) octoate (Sn(Oct)2) as the catalyst.21-23 Four-arm PCL and PLA have also been prepared by using pentaerythritol as the initiator and Sn(Oct)2 as the catalyst.21,24 Thus, the metal-based catalyst, Sn(Oct)2, has been commonly used for the preparation of star-shaped polyesters, as well as linear homopolyesters or diblock copolymers containing a polyester as the hydrophobic block.25 Recently, Sanda et al. reported the synthesis of three and four-arm star PCL using an alcohol as the initiator (i.e., trimethylolpropane or pentaerythritol) and an acid as the catalyst.26 Another study conducted by

10.1021/bm050124+ CCC: $30.25 © 2005 American Chemical Society Published on Web 05/07/2005

Synthesis of Six-Arm Star Polymers

Heise et al. includes the synthesis of dendrimer-like polymers by ring opening polymerization of 5,5-dimethyl-1,3-dioxane2-one in the presence of fumaric acid.17 To our knowledge, these remain the only two reports that describe the use of cationic polymerization with fumaric acid as catalyst for preparation of star-type polyesters and polycarbonates. Reports on the synthesis and properties of amphiphilic starshaped diblock copolymers of type (AB)n, where A is a biocompatible, biodegradable hydrophobic block and B is composed of poly(ethylene glycol) (PEG), are scarce.14,27 In this study, we report the synthesis of novel six-arm amphiphilic star block copolymers, (AB)6, containing MePEG as the hydrophilic block and poly(δ-valerolactone) (PVL) as the hydrophobic block. PVL is a hydrophobic, semicrystalline aliphatic polyester that is structurally similar to the aliphatic polyester poly(caprolactone) (PCL). Unlike PCL, PVL remains relatively unexplored as a material for use in biomedical applications.28 Several methods have been outlined for the synthesis of linear homoPVL;29 however, the synthesis of star-shaped PVL had not yet been reported. In the present study, dipentaerythritol was used as the multifunctional initiator for polymerization of VL to produce starshaped PVL. This ring opening polymerization was performed in bulk in the presence of fumaric acid as the catalyst. The amphiphilic star copolymers were then synthesized by coupling chloroformate activated MePEG to the star PVL homopolymer. This cationic polymerization method is particularly advantageous for preparation of materials for use in biomedical applications because of its metal-free nature. Gel permeation chromatography (GPC) and 1H NMR were used to determine the molecular weights and the compositions of the PVL homopolymers and the (PVL-b-MePEG)6 copolymers. The thermal properties and the micellization characteristics of the (PVL-b-MePEG)6 copolymers including critical micelle concentrations (CMC), size, and size distribution of micelles were also examined. The in vitro cytotoxicity of the copolymers was evaluated in CHO-K1 fibroblast cells in order to gain a preliminary indication of the biocompatibility of these materials. These studies reveal that the nanosized micelles formed from the star-shaped (PVL-bMePEG)6 copolymers are promising materials for applications in drug formulation and delivery. Experimental Section Materials. Dipentaerythritol and fumaric acid were purchased from Aldrich (Oakville, Ontario) and used without further purification. Monomethoxy-terminated poly(ethylene glycol) (MePEG, with Mn ) 2000, Mw/Mn ) 1.06 and Mn ) 5000, Mw/Mn ) 1.06 as determined by GPC) were dried by azeotropic distillation of toluene. δ-VL (99%) was purchased from Acros Organics (Fisher Scientific; Pittsburgh, PA). Toluene was dried under calcium hydride and distilled prior to use. Synthesis of Six-Arm PVL. A total of 0.254 g of dipentaerythritol (1.0 mmol) and 1.16 g of fumaric acid (10.0 mmol) were added to a flame dried round-bottom flask. The reactor was degassed using three vacuum-nitrogen cycles. A total of 5 g of degassed δ-VL (50.0 mmol) was

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then transferred to the reactor using a flame dried and nitrogen purged syringe. The reactor was then immersed into an oil bath that had been heated to a specific temperature. Following the required reaction time, the polymer was dissolved in chloroform and washed three times with a saturated aqueous solution of NaHCO3 in order to remove the fumaric acid. The chloroform layer was dried using NaSO4. Finally, the polymer was precipitated in ethyl ether and the precipitate was vacuum-dried for 24 h at room temperature. The degree of monomer conversion was determined gravimetrically and the yield was found to be 96% (4.8 g). Synthesis of MePEG-COCl. The chloroformate terminated methoxy poly(ethylene glycol) (MePEG-COCl) was synthesized by reacting MePEG with phosgene.30 Briefly, 50 mL of toluene and 6.5 mL of phosgene in toluene (20 wt % or 1.93 M and 12.5 mmol, respectively) were added to a solution of 5 g of MePEG (Mn ) 2000, 2.5 mmol) that was dried twice by toluene azeodistillation. The mixture was allowed to stir for 24 h at room temperature. The toluene and excess phosgene were then removed under vacuum. The MePEG-COCl product was obtained without further purification. 1H NMR assignments were (CDCl3, 200 MHz): 3.39 ppm (3H, s, O-CH3), 3.64 ppm (4H, b, O-CH2-CH2O), and 4.44 ppm (2H, t, CH2-O-CO-Cl). Synthesis of Amphiphilic (PVL-b-MePEG)6 Star Copolymers. The amphiphilic (PVL-b-MePEG)6 copolymers were synthesized by coupling MePEG-COCl to the six-arm star-shaped PVL at room temperature. In a typical procedure, star-shaped PVL (1.0 g, Mn ) 8 200, Mw/Mn ) 1.17, 0.13 mmol) that had been dried three times by azeotropic distillation of toluene was added to a round-bottom flask with MePEG-COCl (1.65 g, Mn ) 2 000, Mw/Mn ) 1.06, 0.82 mmol, 1.05 × 6 equiv.) and 10 mL of dichloromethane (DCM). A total of 64.9 mg of pyridine (0.82 mmol) dissolved in 2.0 mL of DCM was then added dropwise to the reaction mixture over a period of approximately 20 min. The reaction was then left to stir overnight at room temperature. The copolymers were first purified by precipitation in ethyl ether, followed by solvent fractionation using THF as the solvent and hexane as the nonsolvent. The final product was dried under vacuum and resulted in a yield of approximately 20-35% and purity greater than 94% as estimated from GPC analysis. 1H NMR assignments were (CDCl3, 200 MHz): 1.68 ppm (4H, b, CO-CH2-CH2-CH2-CH2-O), 2.37 ppm (2H, b, CO-CH2-CH2-CH2-CH2-O), 3.38 ppm (3H, s, CH3-O), 3.60-3.80 ppm (4H, b, O-CH2-CH2-O), and 4.08 ppm (2H, b, CO-CH2-CH2-CH2-CH2-O). Characterization of the Polymers. 1H NMR spectra of materials were obtained on a Gemini 200 spectrometer (200 MHz for 1H) in CDCl3 solvent. Chemical shifts were reported in ppm with CDCl3 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 as the detector. THF with 1% triethylamine was used as the solvent at a flow rate of 1.0 mL/min at 40 °C, and narrow polystyrene standards (Polysciences Inc.; Warrington, PA) were used for generation of the calibration

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Scheme 1. Synthesis of Six-Arm Core PVL Using Dipentaerythritol (DPE) as the Initiator and Fumaric Acid as the Catalyst in Bulk at 100 °C

curve. The data obtained were recorded and manipulated using the Windows-based Millenium 2.0 software package (Waters Inc.; Milford, MA). Differential scanning calorimetry (DSC) analysis was performed on a DSC Q100 (TA Instruments Inc.; New Castle, DE) under nitrogen at a heating rate of 5 °C per min. Wide-angle X-ray diffraction (WAXD) was carried out using a fully automated Siemens D5000 diffractometer system. The operating conditions during the experiment were 50 kV and 35 mA with Cu KR radiation at room temperature. The secondary beam was monochromatized by a Kevex SS detector. A step scan mode was used during data collection with step width of 0.02° 2θ and counting time of 1.5 s. The scanning range was set from 2.5° to 35.0° 2θ. Preparation of Copolymer Micelles. Micelles were prepared using a dialysis method.9 In short, 0.6 mL of doubledistilled water was added slowly to 0.4 mL of a copolymer solution in DMF to yield a final copolymer concentration of 1 wt %. The copolymer solution was left to stir overnight prior to dialysis against water for 24 h using Spectra/Por 7 Dialysis Membrane with a molecular weight cut off of 8000 (Spectrum Laboratories Inc.; Rancho Cominquez, CA). Determination of Critical Micelle Concentrations. The critical micelle concentrations of the star (PVL-b-MePEG)6 copolymers were measured by a fluorescence-based method.31 In short, an aliquot of a DPH (1,6-diphenyl-1,3,5-hexatriene) stock solution prepared in chloroform was added into individual glass vials such that the final concentration of DPH in each solution was 1 mg/L. Subsequently, the star (PVL-b-MePEG)6 copolymer stock solutions, also prepared in chloroform, were added to the DPH-containing vials resulting in copolymer concentrations that ranged from 0.125 to 625 mg/L. The samples were vortexed and dried thoroughly under nitrogen. The dried vials were then heated to 60 °C, and 1 mL of double distilled water was added to each vial. All hydrated samples were stirred in the dark for 2 h at 60 °C and then overnight at room temperature. The fluorescence emission of the samples was measured at 430 nm (λex ) 350 nm) using a dual-scanning microplate spectrofluorometer (Spectra GeminiXS, Molecular Devices; Sunnyvale, CA). Determination of Micelle Size and Size Distribution. The hydrodynamic diameters of micelles were determined by dynamic light scattering (DLS) at an angle of 90° and a temperature of 25 °C (90Plus Particle Size Analyzer, Brookhaven Instruments Corporation; Holtsville, NY). The sample solutions were diluted in double-distilled water and filtered prior to analysis (0.22 µm Millipore filter). The copolymer concentration for the solutions analyzed was approximately 2 mg/mL. BIC Particle Sizing software was used to determine the effective mean diameters of particles

by cumulant analysis. The software also determines the size distributions by multimodal size distribution analysis in which the data are fitted using the non-negatively constrained least squares (NNLS) algorithm. Cytotoxicity of Copolymers. CHO-K1 fibroblast cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM). The DMEM was 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 atmosphere and 90% relative humidity. The cells were first seeded in a 96-well plate with a cell density of 15 000 cells/well. Following a 24-hour incubation period the growth medium was removed and replaced with 150 µL of fresh medium that contained copolymers in concentrations ranging from 2 to 1000 mg/L (n ) 3). The cells were then incubated for a further 24 h, and the cell viability was measured using the MTT assay. Specifically, 10 µL of 5 mg/mL thiazolyl blue tetrazolium bromide solution (in 0.01 M sterilized PBS) was added to each well and the samples were incubated for 4 h. Following incubation, 100 µL of cell extraction buffer (20% w/v SDS in a solution of 47.5% v/v DMF, 47.5% v/v H2O, 2.5% v/v of 80% acetic acid, and 2.5% v/v of 1N HCl) was added to each well to dissolve the MTT crystals. Cell viability was measured by optical absorbance at 570 nm using a Spectra Max plus microplate reader (Molecular Devices; Sunnyvale, CA). Results and Discussion Synthesis and Characterization of Six-Arm PVL. As outlined in Scheme 1, the six-arm star-shaped PVL homopolymers were synthesized using dipentaerythritol as the initiator and fumaric acid as the catalyst in bulk. A summary of the conditions examined and the results obtained for the polymerization of VL is presented in Table 1. The polymerization was initially optimized in terms of both temperature and ratio of initiator to catalyst. At 60 and 70 °C (Table 1, samples 1-2), the rate of polymerization was found to be relatively slow when compared to polymerization under the same conditions at 100 °C (Table 1, sample 5). The resulting polymers were also found to have high molecular weights and the initiator efficiency was less than 0.3 (Table 1, samples 1-2). An increase in the reaction temperature to 100 °C resulted in an increase in the initiator efficiency to 0.90 (Table 1, sample 5). The ratio of initiator to catalyst, [DPE]0/[FA]0, was also demonstrated to affect the polymerization of VL as shown in Table 1. At an initiator-to-catalyst ratio of 1:1 (i.e., ratio of [OH]0 to [FA]0 of 1/0.167), the molecular weight of the



Table 1. Summary of the Synthesis Conditions Examined for Polymerization of VL to Produce Six-Arm PVL Homopolymers sample

[DPE]0/[FA]0a

temp (°C)

time (h)

Mn,cal

1 2 3 4 5 6 7 8 9 10 11

1:10 1:10 1:1 1:6 1:10 1:10 1:10 1:10 1:10 1:10 1:10

60 70 100 100 100 100 100 100 100 100 100

72 48 18 18 18 18 18 18 18 18 18

7000 7250 7100 7350 7400 4800 9400 18 000 28 800 49 000 89 000

Mn,NMRb

7350 7700 8200 4920 10 800

Mn,GPC

Mw/Mn

fc

27 500 26 700 9600 9700 10 600 6150 15 100 32 800 44 500 64 700 99 100

1.13 1.15 1.36 1.30 1.15 1.21 1.13 1.13 1.09 1.09 1.07

0.25 0.27 0.96 0.95 0.90 0.97 0.87

a The mole ratio of dipentaerythritol (initiator) to fumaric acid (catalyst). b For samples that have M g 10 000, M c n n,NMR is not available. f denotes the initiator efficiency (Mn,cal/Mn,NMR or Mn,cal/Mn,GPC for samples 1 and 2).

adjacent to the terminal hydroxyl group of PVL (CO-CH2CH2-CH2-CH2-OH). The results from NMR analysis confirmed the synthesis of star-shaped PVL homopolymer terminated with hydroxyl end groups.21 The degree of VL polymerization was estimated from the relative ratio of the peak area at δ ) 2.36 ppm (marked b) to that at δ ) 3.62 ppm (marked c). Specifically, the molecular weight was calculated from the relative ratio of the peak areas from the 1 H NMR spectra, using the following equation: Mn,NMR ) Figure 1. Dependence of molecular weight (Mn,GPC and Mn,cal) and polydispersity on the monomer to initiator feed ratio ([M]0/[I]0 ) [VL]0/[DPE]0) used for polymerization.

resulting polymer was found to be 9600 with a broad molecular weight distribution (Mw/Mn ) 1.36; Table 1, sample 3). However, polymerization with an initiator-tocatalyst ratio of 1:10 (i.e., ratio of [OH]0 to [FA]0 of 1/1.67) yielded a polymer with a molecular weight of 10 600 and a relatively narrow molecular weight distribution (Mw/Mn ) 1.15; Table 1, sample 5). The influence of the ratio of monomer to initiator (i.e., [VL]0/[DPE]0) on the Mn and Mw/Mn of PVL was also examined. As presented in Table 1 (samples 6-11) and Figure 1, an increase in the monomer-to-initiator ratio resulted in a linear increase in the molecular weight of the polymer and a decrease in the molecular weight distribution. These results indicate that the polymerization is controllable, and thus, star-shaped polymers with specific target molecular weights can be prepared by varying the monomer-to-initiator ratio. In fact, it was found that star PVL homopolymer with controlled molecular weights up to 99 100 and a relatively narrow molecular weight distribution (i.e., Mw/Mn ) 1.07) could be synthesized by this method. Figure 2 includes a typical 1H NMR spectrum (Table 1, sample 5) for the sixarm PVL homopolymers. In Figure 2, signal a at δ ) 1.69 ppm was assigned to the inner methylene protons (CO-CH2-CH2-CH2-CH2-O), whereas signal b at δ ) 2.36 ppm and signal d at δ ) 4.08 ppm were assigned to the methylene protons adjacent to the carbonyl (CO-CH2CH2-CH2-CH2-O) and the oxy (CO-CH2-CH2-CH2CH2-O) moieties of the ester group, respectively. Signal c at δ ) 3.62 ppm corresponds to the methylene protons

(bc) 6 × 100 + 254

where Mn,NMR is the molecular weight determined by H NMR, b and c are the peak areas of the signals labeled accordingly in Figure 2. The molecular weights calculated from the 1H NMR spectra (i.e., Mn,NMR in Table 1) were in agreement with the theoretical values (i.e., Mn,cal in Table 1). However, the molecular weights of the PVL homopolymers determined by GPC deviated from the theoretical values (Figure 1). The discrepancy between the theoretical molecular weights and the values determined by GPC analysis may be attributed to the difference between the hydrodynamic volumes of the star PVL homopolymers and the polystyrene standards. Synthesis of Six-Arm (PVL-b-MePEG)6 Copolymers with Carbonate Linkage. As shown in Scheme 2, the amphiphilic six-arm (PVL-b-MePEG)6 was synthesized by coupling the chloroformate terminated MePEG (MePEG-COCl) to the star-shaped PVL in dichloromethane at room temperature. The chloroformate terminated MePEG, shown in Scheme 2A, was synthesized by reacting MePEG with phosgene.30 In short, MePEG with Mn ) 2000 or 5 000 was dried by azeotropic distillation of toluene and then a 5 equiv. stoichiometric amount of phosgene in toluene (1.93 M) was added. The reaction was monitored by FT-IR to confirm that the hydroxyl groups of MePEG had been completely reacted and that the characteristic absorbance of the CdO group (in chloroformate) at 1777 cm-1 appeared. 1 H NMR analysis revealed that the degree of substitution of the MePEG terminal hydroxyl groups with chloroformate groups was over 98%. In the second step of the (PVL-b-MePEG)6 synthetic procedure (Scheme 2B), the hydroxyl terminated six-arm PVL was reacted with a slight excess of MePEG-COCl 1

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Figure 2.

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NMR spectrum of the star-shaped PVL homopolymer (Table 1, sample 5).

Scheme 2. Synthesis of Six-Arm Diblock Copolymers of (PVL-b-MePEG)6: (A) Synthesis of Chloroformate Terminated Methoxy Poly(ethylene glycol) (MePEG-COCl); (B) Synthesis of Six-Arm (PVL-b-MePEG)6 by Coupling MePEG-COCl to Six-Arm Star PVL

(1.05 equiv. to molar of OH in PVL) in the presence of dried pyridine. The unreacted or excess MePEG was then removed by solvent fractionation with a THF-hexane system (i.e., THF as solvent and hexane as nonsolvent). Figure 3, panels A and B, includes the 1H NMR spectra of the MePEG-COCl and the star block copolymer (Table 2, sample 13), respectively. As shown, the signal at δ ) 4.44 ppm (2H, t, CH2-O-CO-Cl) from MePEG-COCl (Figure 3A) was completely shifted to δ ) 4.08 ppm (Figure 3B) indicating the formation of a carbonate linkage between the MePEG and PVL blocks. The typical GPC traces for MePEG-COCl (trace a, Mn ) 2000), star-shaped PVL (trace b; Table 1, sample 5), and amphiphilic (PVL-b-MePEG)6 copolymers (trace c; Table 2, sample 13) are illustrated in Figure 4. As shown, the GPC trace of the crude product (e.g., Figure 4, trace c) revealed that the star-shaped PVL homopolymer was completely consumed during the coupling reaction. The small peak in the GPC trace of the crude product at approximately 23 min is likely due to the presence of a trace amount of bifunctional PEG (i.e., ClCO-PEG-COCl) which accounts for approximately 1-10% of the total amount of PEG

originally added for the coupling reaction.32 The molecular weight distributions of the star-shaped block copolymers were relatively narrow ranging from Mw/Mn ) 1.11 to 1.26 (Table 2, samples 12-16). The composition of the copolymers and their molecular weights were evaluated from the 1 H NMR spectra. The 1H NMR analysis was performed using the ratio of the peak area for the methylene proton from PVL at δ ) 2.37 ppm (i.e., CO-CH2-CH2-CH2-CH2-O) to the methylene proton from PEG at δ ) 3.60-3.80 ppm (i.e., O-CH2-CH2-O). As illustrated in the 1H NMR spectrum, the signal at δ ) 4.44 ppm for the methylene proton in the chloroformate group completely disappeared following the reaction. The coupling efficiency was found to be higher than 87% for core star PVL with molecular weights of less than 20 000 (Table 2, samples 12-15). The copolymer compositions determined by 1H NMR were in agreement with those predicted from the known compositions of the star-shaped PVL and the MePEG-COCl (Table 2, samples 12-15). The molecular weights of the amphiphilic block copolymers determined by GPC analysis were found to be an overestimate relative to the corresponding theoretical molecular weights. The discrepancy between the empirical



Figure 3. 1H NMR spectra of MePEG-COCl and amphiphilic star copolymer. (A) MePEG-COCl, Mn ) 2000, Mw/Mn ) 1.06; (B) Six-arm star (PVL-b-MePEG)6 (Table 2, sample 13).

and the theoretical values is once again attributed to the difference between the hydrodynamic volumes of the copolymers and the polystyrene standards. Thermal Properties and WAXD Analysis of StarShaped PVL Homopolymers and (PVL-b-MePEG)6 Copolymers. The thermal properties of star-shaped PVL homopolymers and (PVL-b-MePEG)6 copolymers are summarized in Table 3. DSC analysis of the PVL homopolymers revealed the existence of glass transition temperatures (Tg) and melting temperatures (Tm), indicating that the PVLs are semicrystalline polymers (Table 3, samples 6-10). The Tgs were found to range from -47.1 to -53.6 °C and showed no dependence on the molecular weight of the polymer. By contrast, the melting temperatures (Tm) were found to increase from 38 to 55 °C with an increase in the molecular

weight of the PVL homopolymers. As the PVL content of the star homopolymers increased, the Tm of the homopolymers increased to a value close to the reported value for the Tm (56 °C) of a linear PVL homopolymer.33 The Tms for the 2k-MePEG and 5k-MePEG series of amphiphilic copolymers were found to be approximately 49 °C and 52 °C, respectively. The thermal transitions (i.e., Tg and Tm) of the PVL blocks were not detected in the thermograms obtained for the star copolymers (Table 3, samples 12-16). These results are in agreement with those reported by Park et al. for the thermal analysis of star-shaped PLA-b-MePEG copolymers.23 Figure 5 includes results from wide-angle X-ray diffraction (WAXD) analysis of the polymers. Both the MePEG (2k-MePEG) and star PVL homopolymers (Table 1, samples

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Table 2. Summary of the Results for Synthesis of the Six-Arm (PVL-b-MePEG)6 Copolymers Prepared by the Coupling Reaction in Dichloromethane at 25 °C block copolymers sample

Mn,prepolymer

PEG2K PEG5K 12 13 14 15 16

2000 5000 4920 8200 10 800 18 000 28 800

Mn,PEGa

10 800 11 800 11 200 27 000 22 200

Mn,PVL

4920 8200 10 800 18 000 28 800

Mn,total

15 700 20 000 22 000 45 000 51 000

Mn,GPC

20 800 30 200 33 300 81 600 104 300

XPVL (wt %)

31.2 40.2 47.0 40.0 56.0

fb

Mw/Mn

0.90 0.98 0.93 0.87 0.74

1.06 1.06 1.18 1.17 1.11 1.26 1.19

a Samples 12-14 coupled with 2k-MePEG, samples 15-16 coupled with 5k-MePEG; M b n,PEG ) Mn,PVL/XPVL - Mn,PVL. f denotes the coupling efficiency (Mn,PEG/MPEG,cal).

Figure 4. GPC traces for trace (a) MePEG-COCl, Mn ) 2000, Mw/Mn ) 1.06; (b) Table 1, sample 5; Mn ) 8200, Mw/Mn ) 1.15; (c) Table 2, sample 13, Mn ) 20 000, Mw/Mn ) 1.17.

6-7) were found to be crystalline as diffraction patterns for each were observed. For the 2k-MePEG, the X-ray diffraction peaks appeared at 2θ ) 19.4° and 23.5°, whereas for the six-arm PVL homopolymers, peaks appeared at 2θ ) 21.6° and 24.3°. The diffraction patterns for the star-shaped copolymers were found to be dependent on the core molecular weight of PVL. The diffraction peaks for PVL in the block copolymers were not observed when the core PVL molecular weight was less than Mn ) 10 800 (Table 2, samples 12-14); in this case, only the peaks corresponding to MePEG were observed in the diffraction patterns. However, the peak at 2θ ) 21.6° was observed when the PVL core molecular weight was 18 000 or 28 800 (Table 2, samples 15-16) as shown in Figure 5. Furthermore, as the PVL block length was increased, the intensity of the peak at 2θ ) 21.6° also increased. These results confirm that the extra peak observed in the diffraction patterns for the (PVL-b-5k-MePEG)6 star-shaped copolymers may indeed be attributed to the PVL block and that the PVL block is crystalline. Critical Micelle Concentrations of Star-Shaped (PVLb-MePEG)6 Copolymers. Table 3 includes the measured values for the critical micelle concentrations (CMC) of the five star-shaped (PVL-b-MePEG)6 copolymers. Overall, the CMCs of the copolymers were found to decrease with an increase in the length of the PVL core block for both the 2k-MePEG (Table 3, samples 12-14) and the 5k-MePEG (Table 3, samples 15-16) series. These results are in agreement with studies previously performed on many

amphiphilic block copolymer systems where it has been shown that an increase in the length of the hydrophobic block, with a constant hydrophilic block length, results in a decrease in the CMC of the copolymer.11,34,35 The CMC values reported by Kim et al. for a series of PCL-b-MePEG star-shaped copolymers,14 were much lower (i.e., CMC ranged from 0.76 to 2.1 mg/L) than the values obtained for the (PVL-b-MePEG)6 copolymers (i.e., CMC ranges from 93.1 to 211 mg/L). The higher CMC values obtained for the PVL-based system, when compared to the PCL-based system, are attributed to the lower degree of hydrophobicity of the PVL block with respect to PCL. Furthermore, in the current studies, DPH was used as the fluorescence probe for determination of the CMC values, whereas in the studies performed by Kim et al., pyrene was employed. It has been shown that use of DPH as the fluorescence probe for CMC determination results in elevated values when compared to the values obtained with pyrene.35,36 The architecture of amphiphilic block copolymers is also known to influence their CMC.37,38 For example, the CMCs of linear 5k-b-5k and 2k-b-2k PVL-b-MePEG copolymers were found to be 35.3 and 23.3 mg/L, respectively (Lee et al., to be submitted), whereas the CMCs of the corresponding (4.8k-PVL-b-3.7k-MePEG)6 (i.e., Table 3, sample 16) and (1.8k-PVL-b-1.9k-MePEG)6 (i.e. Table 3, Sample 14) starshaped copolymers were 121 and 93.1 mg/L, respectively. The high values obtained for the CMC of the star-shaped copolymers may be attributed to the likely reduction in entropy that accompanies micellization of these materials. Therefore, it is assumed that, in comparison to linear diblock copolymers of the same composition (i.e., block lengths), it is energetically more difficult to organize star-shaped copolymers into a micelle structure. The significance of the entropic effects associated with micellization of diblock and star-block copolymers has also been considered by Huh et al. in a Brownian dynamics simulation study.24 Characterization of Micelles Formed from Star-Shaped (PVL-b-MePEG)6 in Aqueous Solution. The size and size distribution of micelles formed from the amphiphilic sixarm star block copolymers as determined by dynamic light scattering measurements are listed in Table 3. As expected, the 2k-MePEG series of copolymers (Table 3, samples 12-14), with their shorter arm-lengths, were found to form micelles with smaller effective mean diameters than those of micelles formed from the 5k-MePEG series (Table 3, samples 15-16). Interestingly, the star copolymers were



Table 3. Effect of Copolymer Composition on the Thermal Properties and CMC Values of the Copolymers, as Well as the Size and Size Distribution of the Micelles Produced samplea 5 6 7 8 9 10 12 13 14 15 16

Mn,Total

Mn,PVL

15 700 20 000 22 000 45 000 51 000

8200 4920 10 800 18 000 28 800 49 000 4920 8200 10 800 18 000 28 800

Mn,PEGb

10 800 11 800 11 200 27 000 22 200

XPVL (wt %)

Tg (°C)

Tm (°C)

CMC (mg/L)

diameter of populationsc (nm)

effective mean diameterd (nm)

-48.7 -53.6 -49.7 -47.1 -50.1 -49.1

44.6 38.3 46.9 52.2 53.4 55.2 49.0 49.4 48.5 52.1 53.1

171 105 93.1 211 121

10 31.2 10.3, 44.4 8.4, 43.1 26.5, 136.7

13 ( 3 30 ( 4 23 ( 1 41 ( 3 44 ( 0.2

31.2 40.2 47.0 40.0 56.0

a Sample numbers correspond to samples in Tables 1 and 2. Samples 12-14 and samples 15-16 denote copolymers composed of MePEG with M n ) 2000 and Mn ) 5000, respectively. b Mn,PEG was calculated from 1H NMR data. c Diameter of populations denotes the diameter of individual micelle d populations present in the sample. Effective mean diameter was determined by dynamic light scattering at 90° using cumulant analysis, in terms of normalized intensity.

Figure 5. X-ray diffraction patterns of MePEG, six-arm star PVL, and six-arm (PVL-b-MePEG)6 determined at room temperature. The numbers in the figure correspond to the sample numbers in Tables 1 and 2.

found to produce monomodal or bimodal distributions of micelles depending on both the length of the MePEG block and the length of the PVL core-forming blocks. Specifically, the (0.8k-PVL-b-1.8k-MePEG)6 (i.e., Table 3, sample 12) and the (1.4k-PVL-b-2.0k-MePEG)6 (i.e., Table 3, sample 13) copolymers formed micelles with a narrow monomodal size distribution and effective mean diameters of 13 and 30 nm, respectively. The more hydrophobic (1.8k-PVL-b1.9k-MePEG)6 (i.e., Table 3, sample 14) copolymer formed a bimodal size distribution of aggregates with a small population having a diameter of 10.3 nm and a large population having a diameter of 44.4 nm. Similarly, the (3.0k-PVL-b-4.5k-MePEG)6 (i.e., Table 3, sample 15) copolymer also resulted in a bimodal distribution with a small population having a diameter of 8.4 nm and a large population having a diameter of 43.1 nm. The (4.8k-PVLb-3.7k-MePEG)6 (i.e., Table 3, sample 16) star copolymers also resulted in a bimodal distribution; yet in this case, the abundance of the populations was approximately equal and had diameters of 26.5 nm (54% of the detected normalized intensity) and 136.7 nm (46% of the detected normalized intensity). Therefore, overall it was found that as the length of the core-forming hydrophobic block was increased, or equivalently as the hydrophobicity of the copolymer was increased, there was a tendency toward formation of ag-

gregates having a bimodal size distribution. It is postulated that the individual populations include small primary micelles and secondary aggregates of primary micelles. The aggregation of primary micelles may result from incomplete surface coverage of the PVL core by the hydrophilic shell.15 In Vitro Cytotoxicity of the Star-Shaped (PVL-bMePEG)6 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.39-42 To this point, there have been no reports on the in vitro cytotoxicity of PVL and its copolymers. In the present study, the cytotoxicities of the star-shaped (PVL-b-MePEG)6 copolymers (Table 3, samples 12-16) were evaluated in the fibroblast cell line CHO-K1. As shown in Figure 5, the incubation of cells with the copolymers resulted in a low degree of cytotoxicity, with relative cell viabilities above 80% for the copolymer concentrations examined (up to 1000 mg/L). Even at the highest copolymer concentration, there was no significant change in cell proliferation, relative to controls, following a 24-hour incubation period. There was only one exception where a cell viability of 61% was observed when the CHO-K1 fibroblast cells were incubated with the high molecular weight (3.0k-PVL-b-4.5k-MePEG)6 copolymer at a concentration of 1000 mg/L. The (PVL-b-MePEG)6 copolymers were expected to be noncytotoxic as MePEG has been shown to be biocompatible43,44 and PVL is similar in chemical structure to PCL which has also been demonstrated to be biocompatible.45 In addition, the nonlinear, star architecture of the copolymers may hinder their intercalation into cell membranes. It has been reported that some linear amphiphilic copolymers are capable of intercalating into cell membranes and causing an increase in membrane permeability.46,47 At high concentrations of amphiphiles, significant membrane perturbation can lead to cytotoxicity due to loss of membrane integrity.48 In this way, the lower cell viability associated with the (3.0k-PVL-b-4.5k-MePEG)6 copolymer at a concentration of 1000 mg/L was unexpected and requires further investigation. However, in the case of an intravenous drug formulation, the copolymer concentration following administration will

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Zeng et al.

Professor Sefton for kindly donating the CHO-K1 fibroblast cells, Professor Uetrecht for use of the fluorescence and UV/vis microplate readers, and Professor Cheng for use of the Particle Size Analyzer. References and Notes

Figure 6. In vitro cytotoxicity of the (PVL-b-MePEG)6 copolymers. The cell viabilities are reported relative to controls and are expressed as a function of the logarithm of the copolymer concentration in CHO-K1 fibroblast cells. (A) 2k-MePEG series six-arm star copolymers, and (B) 5k-MePEG series six-arm star copolymers.

be much lower than 1000 mg/L due to the dilution in the total blood volume;34 therefore, the lower cell viability observed at the highest copolymer concentration examined is not a significant concern. These results provide a preliminary indication of the biocompatibility of this copolymer and potential suitability for use in biomedical applications such as drug delivery. Conclusion Novel amphiphilic six-arm diblock copolymers of (PVL-b-MePEG)6 with a carbonate linkage between the hydrophilic and hydrophobic blocks were synthesized using a cationic polymerization method. The cationic method employed eliminated the need for the use of a metal-based catalyst which may in turn increase the suitability of these materials for use in biomedical applications. The noncytotoxicity of the copolymers was confirmed in fibroblast cells as a preliminary indication of the biocompatibility of the materials. In addition, the amphiphilic nature of the copolymers allowed for formation of nanosized aggregates in aqueous media. In this way, the star-shaped copolymers may be suitable for use in drug formulation and delivery. Acknowledgment. The authors acknowledge National Science and Engineering Research Council (NSERC) for funding this research. The authors are also grateful to

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BM050124+

Synthesis and Characterization of Six-Arm Star Poly (Î´-valerolactone

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