Bioconjugate Chem. 2005, 16, 397−405
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Synthesis and Evaluation of a Star Amphiphilic Block Copolymer from Poly(E-caprolactone) and Poly(ethylene glycol) as a Potential Drug Delivery Carrier Fei Wang,*,† Tatiana K. Bronich,‡ Alexander V. Kabanov,‡ R. David Rauh,† and Jacques Roovers§ EIC Laboratories, Inc., 111 Downey Street, Norwood, Massachusetts 02062, and Department of Pharmaceutical Science, University of Nebraska Medical Center, 986025 University Medical Center, Omaha, Nebraska 68198-6025. Received September 13, 2004; Revised Manuscript Received December 30, 2004
A star polymer composed of amphiphilic block copolymer arms has been synthesized and characterized. The core of the star polymer is polyamidoamine (PAMAM) dendrimer, the inner block in the arm is lipophilic poly(-caprolactone) (PCL), and the outer block in the arm is hydrophilic poly(ethylene glycol) (PEG). The star-PCL polymer was synthesized first by ring-opening polymerization of -caprolactone with a PAMAM-OH dendrimer as initiator. The PEG polymer was then attached to the PCL terminus by an ester-forming reaction. Characterization with SEC, 1H NMR, FTIR, TGA, and DSC confirmed the star structure of the polymers. The micelle formation of the star copolymer (star-PCL-PEG) was studied by fluorescence spectroscopy. Hydrophobic dyes and drugs can be encapsulated in the micelles. A loading capacity of up to 22% (w/w) was achieved with etoposide, a hydrophobic anticancer drug. A cytotoxicity assay demonstrated that the star-PCL-PEG copolymer is nontoxic in cell culture. This type of block copolymer can be used as a drug delivery carrier.
INTRODUCTION
Micelles formed from amphiphilic block copolymers have recently attracted significant attention in diverse fields of medicine and biology. In particular, polymeric micelles have been developed as drug and gene delivery systems (1-3) as well as carriers for various contrasting agents in diagnostic imaging applications (4). In an aqueous environment, the hydrophobic blocks of the copolymer are expected to segregate into the core of the micelle, whereas the hydrophilic blocks form the corona or outer shell. Such a core-shell architecture of the polymeric micelles is essential for their utility as novel functional materials for pharmaceutical applications. The hydrophobic micelle core serves as a microenvironment for the incorporation of various therapeutic compounds; the corona, or outer shell, serves as a stabilizing interface between the hydrophobic core and the external medium. As a result, polymeric micelles can be used as efficient containers for reagents with poor solubility and/or low stability in physiological environments (5, 6). Interest in polymeric micelles for drug delivery has increased rapidly since the late 1980s (7). Most of the work has focused on classical micelles formed by intermolecular aggregation of amphiphilic polymers as the drug delivery vehicle, and the advantages of using micelle structures as a drug delivery system have been demonstrated (8, 9). The major factors that influence the performance of polymeric micelles for drug delivery are loading capacity, release kinetics, circulation time, biodistribution, size, and stability (2). Micelle stability is particularly impor* Author to whom correspondence should be addressed [telephone (781) 769-9450; fax (781) 551-0283; e-mail
[email protected]]. † EIC Laboratories. ‡ University of Nebraska Medical Center. § Present address: 21 Wren Rd., Gloucester, ON K1J 7H5, Canada.
tant. Recent studies have shown that the in vivo antitumor activity of a drug incorporated into the polymer micelles is positively correlated with the stability of micelles in vitro (10). The formation of classical micelles is thermodynamically favorable only above a specific concentration of the amphiphilic molecules (critical micelle concentration, cmc). Above the cmc, micelles are in dynamic equilibrium with the free copolymer molecules (unimers) in solution, continuously breaking and reforming (11). When the concentration of the copolymer is below the cmc, micelles tend to disassemble. Such thermodynamic instability of micelles below the cmc is one of the concerns for their application in vivo. A delivery system is subject to a severe dilution upon intravenous injection into an animal or human subject. In the bloodstream, under dilution, micelles begin to disassemble, causing changes in micelle structure and size. Therefore, controlling the release rate of drugs is difficult. Sudden dissociation of micelles may cause serious toxicity problems due to potentially large fluctuations in drug concentrations. The problem associated with the classical micelle structure can be overcome by developing molecules in which the lipophilic components are covalently bound together within the micelle core. Core polymerization is an effective method to prevent dissociation of the block copolymer micelle. Kataoka’s group has successfully employed this idea (12). In their study, the micelles were prepared from an amphiphilic block copolymer in which the hydrophobic block contained a polymerizable end group. After micellation, the end groups on the hydrophobic block were polymerized to form a stable core for the star-shaped polymer structure. The resulting micelles showed fairly high stability and maintained small size. As anticipated, the core polymerized micelle showed excellent solubilization of rather large molecules such as taxol (13).
10.1021/bc049784m CCC: $30.25 © 2005 American Chemical Society Published on Web 02/23/2005
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Another approach developed recently by Uhrich et al. (14) with a three-arm star polymer composed of mucic acid substituted with fatty acid as the lipophilic inner block and with PEG as the hydrophilic outer block. This new type of molecule was capable of encapsulating a hydrophobic model drug in aqueous media. However, due to the structural constraints, the free volume of the hydrophobic core was limited, and only one or two drug molecules could be encapsulated in each micelle. A series of star block copolymers with the number of arms ranging from three to eight has also been synthesized (15). The arms were composed of block copolymer with PEG as the inner hydrophilic block and PCL as the outer hydrophobic block. The application of this type of copolymer as an injectable drug delivery system was reported (16). It was found that a reversible sol-gel transition process exists for this system, which is useful for drug delivery. However, such star copolymers do not form micelles in aqueous media because the hydrophilic block is located in the interior of the star. A recent paper (17) described the synthesis of a four-arm star block copolymer of PCL and PEG by the same route and similar chemistry as reported in this paper. Another paper (18) described the preparation of a four-arm star PCL-b-PEG polymer with diethylzinc catalyst. However, the molecular weight distribution of the block copolymer was unacceptably wide. Many studies have been carried out using dendrimers as drug delivery systems (19). Star polymers with a dendrimer as the hydrophobic core and multiple PEG chains as the hydrophilic arms have been synthesized and investigated as unimolecular micelles for drug delivery by Fre´chet and Kono (20, 21). It has been demonstrated that the micelles with larger dendrimer core have a higher encapsulation capability than those with smaller cores. However, due to the structural limitations involved in the synthesis of dendrimers of higher generation, and the relatively compact structure of the dendrimers, it is difficult to increase significantly the size of the hydrophobic dendritic core in the dendrimer-PEG star polymer. Therefore, such dendrimer systems have limitations in terms of drug-loading capacity and delivery of compounds of large size. In this paper, we describe the synthesis and micellar characterization of a novel amphiphilic star polymer. The core of this star polymer is a polyamidoamine (PAMAM) dendrimer; the inner block in the arm is lipophilic poly(-caprolactone) (PCL), and the outer block in the arm is hydrophilic poly(ethylene glycol) (PEG). First, a star PCL polymer was synthesized using the PAMAM-OH dendrimer as the initiator for ring-opening polymerization of -caprolactone. A PEG polymer was then attached to the terminal group of PCL by an ester-forming reaction. In this star polymer the arms consist of polymers that are biocompatible and biodegradable. The star structure of the polymers was confirmed by several physicochemical methods. To establish this system as a suitable drug carrier, the micellar properties of the star amphiphilic polymer in aqueous media were studied by fluorescence techniques and dynamic light scattering. The solubilization capacity of star-PCL-PEG micelles was evaluated using several highly hydrophobic compounds, such as pyrene and water-insoluble dye molecules. A series of entrapment experiments showed that various drugs could be incorporated in such micelles. These results indicate that the star-PCL-PEG micelle system is a promising drug carrier for the delivery of lipophilic drugs.
Wang et al. EXPERIMENTAL SECTION
Materials and Methods. PEG (Mn ) 5000) with methoxy and carboxymethyl terminal groups was used as received from Shearwater Polymers. 4-(Dimethylamino)pyridinium 4-toluenesulfonate (DPTS) was synthesized as described (22). Dichloromethane was dried by stirring over and distilling from P2O5. All other reagents and solvents were used as received from Aldrich Chemical. Indomethacin (IMC), doxorubicin hydrochloride (DOX), and etoposide were purchased from Sigma (St. Louis, MO) and were used without further purification. 1 H NMR spectra were recorded on a Hitachi R-1500 FT-NMR spectrometer at 60 MHz. Infrared spectra were recorded on a Nicolet 750 FTIR spectrometer. UV-vis spectra were recorded on a HP 8452A UV-vis diode array spectrophotometer. Size exclusion chromatography (SEC) was used to determine the molecular weights, polydispersity, and degree of branching of the synthesized polymers. SEC measurements were conducted using a Hewlett-Packard series 1050 HPLC equipped with one Polymer Laboratories PL gel 5µ mixed-D column and a Hewlett-Packard 1047A refractive index detector, Viscotek T60 light scattering (LS), and viscosity detectors. Chloroform was used as the eluting solvent at a flow rate of 1 mL/min. It was found that chloroform was the eluent of choice. The block copolymer is poorly soluble or is retained on the column as indicated by trailing elution profiles in other common SEC solvents. Data analysis was performed using Viscotek Trisec GPC software version 3.0. The thermal transitions of the polymers were determined using a Perkin-Elmer DSC7 differential scanning calorimeter under nitrogen purge. The samples were heated at a rate of 10 °C/min. The thermal stability of the prepared polymers was evaluated using a PerkinElmer TGA7 thermogravimetric analyzer under nitrogen purge with a heating rate of 20 °C/min. Synthesis of the Star-PCL. A 100 mL three-neck flask equipped with a condenser and an argon gas inlet was charged with PAMAM-OH dendrimer generation 2 with 16 terminal OH groups (20 wt % solution in methanol, 5.76 mL, 0.304 mmol). Methanol was then removed under vacuum. A predetermined amount of -caprolactone (14.42 g, 126 mmol) followed by 15 mL of benzene was added into the reaction flask. The mixture was heated to reflux using a Dean-Stark trap for 30 min, and benzene was distilled off. The heating of the reaction mixture to 140 °C resulted in the dissolution of the dendrimer in -caprolactone. Five milligrams (0.012 mmol) of tin(II) 2-ethylhexanoate [Sn(Oct)2] was then introduced into the reaction mixture followed by stirring at 115-120 °C for 20 h. The reaction mixture was cooled to room temperature, dissolved in THF, and precipitated into cold methanol. After drying, 15.4 g (100% yield) of a white solid product was obtained. The polymer was further purified by fractionation. Three grams of starPCL was dissolved in 60 mL of THF in a round-bottom flask, and methanol was added until the solution became cloudy. The solution was heated in a water bath at 60 °C and became clear. About 10 mL of methanol was added to reach a cloudy point, and the mixture was transferred into a hot separation funnel and allowed to stand at room temperature for 3 days. The polymer solution separated into two layers, and the top layer was removed without disturbing the lower layer. The lower layer was diluted with THF and precipitated into cold methanol; 1.47 g of polymer was obtained: 1H NMR (CDCl3) δ 4.07 (t, 2H, OCH2), 2.31 (t, 2H, COCH2), 1.1-2
Star Amphiphilic Block Copolymer as Drug Delivery Carrier
[m, 6H, (CH2)3]. The characteristic peaks of dendrimer and polymer were overlapping, and peak integration was inconclusive. Synthesis of the Linear-PCL. The linear PCL was synthesized in a similar procedure using -caprolactone (14.42 g, 126 mmol), ethylene glycol (0.098 g, 1.57 mmol), and a few drops of Sn(Oct)2. Isolation was conducted with the same procedure as described previously. Synthesis of the Star-PCL-PEG. Star-PCL (1.3 g, 0.0253 mmol), PEG (2.64 g, 0.527 mmol), and DPTS (0.155 g, 0.527 mmol) were dissolved in 80 mL of dry CH2Cl2 by stirring at room temperature. Then diisopropylcarbodiimide (DIPC) (0.2 g, 1.58 mmol) was added into the reaction mixture via syringe. The reaction mixture was stirred overnight at room temperature. An additional amount of DIPC (0.15 g) was added to the reaction mixture followed by stirring for 5 days. The reaction was stopped, and fractionation was used to purify the starPCL-PEG. Briefly, 20 mL of CH2Cl2 was added to dilute the product. Hexane was slowly added to the resulting solution at 45 °C until the appearance of cloudiness. Then the mixture was transferred into a hot separation funnel and was allowed to stand at room temperature. The top layer was removed, and the lower layer was diluted with CH2Cl2 and precipitated into hexane. Analysis of the polymer by 1H NMR indicated that the sample contained excess PEG and other impurities. The product was refractionated: the entire product (∼3.5 g) was dissolved in 100 mL of CH2Cl2 followed by the gradual addition of hexane until the mixture became cloudy. The resulting mixture was then heated in a 45 °C bath followed by the addition of more hexane until a stable cloudy point was reached and transferred into a hot separation funnel. The lower layer was diluted with CH2Cl2 and precipitated into hexane. After drying, 3.04 g (91% yield) of white solid product was obtained: 1H NMR (CDCl3) δ 4.07 (t, 2H, COOCH2), 3.64 (s, 18.8H, OCH2CH2O), 2.31 (t, 2H, COCH2), 1.1-1.9 [m, 6H, (CH2)3]. Preparation of Star-PCL-PEG Micelles. The starPCL-PEG polymer was dissolved in DMF at ∼100 mg/ mL and stirred overnight at room temperature. Water was added in a dropwise fashion to the star-PCL-PEG solution in DMF. The addition of water was continued until the desired water content was achieved (12.5-25 vol % of DMF in the final mixture). The obtained solution was stirred overnight and then dialyzed against Milli-Q distilled water using Membra-Cel MD-25-03.5 membrane tubing. The dialysate water was exchanged every hour for the first 4 h and then every 6 h for the next 12 h. The concentration of star-PCL-PEG solutions was determined by UV spectroscopy. The absorption spectrum of star-PCL-PEG in a DMF/water mixture (50 vol % DMF content) has a characteristic peak at 281.7 nm. The absorption maximum of star-PCL-PEG undergoes a bathochromic shift to 288.4 nm in pure aqueous solutions. A calibration curve of standard solutions of star-PCLPEG from 0.1 to 1.5 mg/mL was constructed by monitoring UV absorbance at 288.4 nm and used to determine the star-PCL-PEG concentration in aqueous solutions after dialysis (Y) 0.00006 + 0.1679 X, r2 ) 1.0). Micelle Characterization. Dynamic laser light scattering (DLS) measurements on the star-PCL-PEG micelles were carried out using a “ZetaPlus” zeta potential analyzer (Brookhaven Instrument Co.) equipped with the multiangle sizing option (BI-MAS) and with a 15 mW solid-state laser operated at a wavelength of 635 nm. All measurements were performed at 23 °C and at scattering angle of 90°. Software provided by the manufacturer was used to calculate the effective hydrodynamic diameters
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of the micelles. The apparent critical micelle concentration (Capp) was determined using pyrene as a fluorescent probe. Sample solutions were prepared by adding known amounts of pyrene in acetone to each of a series of empty vials. The amount of pyrene was chosen so as to give a pyrene concentration in the final solution of 6 × 10-7 M. After the evaporation of acetone, solutions of star-PCLPEG of various concentrations in phosphate buffer saline (10 mM, pH 7.4, 0.14 M NaCl) were added to the probe. Samples were equilibrated upon shaking overnight at the desired temperature. Steady-state fluorescence spectra were recorded using a Shimadzu RF5000U spectrofluorophotometer with bandwidths of 1.5 nm for excitation and 3 nm for emission, respectively. For fluorescence emission spectra, λex was 336 nm, and for excitation spectra, λem was 390 nm. Solubilization of Water-Insoluble Dyes. Oil Red O (ORO) and 1-(m-tolylazo)-2-naphthylamine (Yellow OB) were used as model hydrophobic molecules for solubilization experiments. Appropriate amounts of the copolymer solutions of various concentrations were added to the vials containing a given amount of solid dye and were equilibrated upon shaking for 2 days. Test solutions were centrifuged to remove the residual insoluble dye. The amount of dye entrapped in the micelles was determined by measuring the absorbance at 510 nm for ORO and at 450 nm for Yellow OB, respectively. Preparation of Drug-Loaded Micelles. Indomethacin (IMC) was incorporated into the copolymer micelles either by dialysis or by an oil/water emulsion method. (1) Dialysis Method. One hundred milligrams of IMC dissolved in 1 mL of DMF was added to 14 mg of polymer in 2 mL of a DMF/water mixture (50 vol % DMF). The solution was stirred for 4 h at room temperature and then dialyzed against 2 L of distilled water using MembraCel MD-25-03.5 membrane tubing. The precipitate of free IMC was removed by centrifugation at 15000 rpm for 30 min. (2) Oil/Water (O/W) Emulsion Method. One milligram of IMC dissolved in 100 µL of chloroform was added dropwise to 4 mL of aqueous dispersion of copolymer (7 mg) with vigorous stirring. The mixture was stirred in an open-air system to remove chloroform by evaporation and then stirred overnight. The precipitate of unbound IMC was removed by centrifugation at 15000 rpm for 30 min. The amount of IMC entrapped into the polymer micelles was determined by measuring the absorbance at 320 nm in ethanol/water (1:1) mixtures using calibration curves generated from standard solutions. Doxorubicin hydrochloride (DOX) loaded micelles were prepared according to a modified dialysis method. Specifically, 0.5 mL of aqueous star-PCL-PEG dispersion with a concentration of 15.3 mg/mL in dialysis tubing was placed in a beaker containing 100 mL of solution of DOX (0.1 mg/mL). The beaker was covered with Parafilm and wrapped in foil, and its content was allowed to equilibrate with continuous stirring for 24 h. At the end of the equilibration period, the sample in the dialysis tubing was placed into another beaker and dialyzed against 650 mL of a solution of DOX (0.46 µg/mL) for 24 h. Copolymer micelles loaded with DOX were further analyzed by SEC using a Shimadzu LC-10AT HPLC system equipped with a TSK-Gel G5000PWXL column (7.8 mm × 30 cm). NaCl (0.05 M) was used as the eluting solvent at a flow rate of 1.5 mL/min. Determination of the concentration of starPCL-PEG copolymer and DOX was done by measuring the absorbance at 288 and 485 nm, respectively. Etoposide was incorporated into the copolymer micelles according to two methods.
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Wang et al.
Scheme 1. Synthesis of Star-PCL-PEG Polymer
(1) Solid Extraction. A measured volume of a methanol solution of etoposide was added to a vial, and the solvent was removed under vacuum, leaving a solid film of etoposide. A polymer micelle solution was agitated with etoposide for 24 h. After filtration, the total etoposide concentration in the micelle solution was determined by UV spectroscopy (2) Solution Extraction. The stock solution of etoposide (19.2 mg/mL) in 1:1 (vol) acetonitrile/methanol was added slowly with agitation to the polymer solutions of various concentrations to obtain the final concentration of etoposide of 1.8 mg/mL (final concentrations of polymer of 3.7, 7.6, and 15.1 mg/mL). The initially clear solutions were shaken overnight at 27 °C, and the next day some precipitate was observed. The precipitate was removed by filtration, and the etoposide concentration in the solutions was determined by UV spectroscopy. The total etoposide concentration in the polymer micelle solution was determined by measuring the absorbance at 284 nm using calibration curves generated from known methanol solutions of etoposide. Cytotoxicity Assay. Porcine kidney epithelial cells (LLC-PK) were seeded into 96-well plates at a density of 5000 cells/well. Etoposide (0.95 mg/mL) was solubilized in the 7.6 mg/mL solution of polymer micelles as described above. Solutions were filtered through 22 µm filters, without loss of etoposide as determined by the absorbance intensity at 284 nm. The cells were exposed to etoposide alone, polymer micelles alone, or etoposide in polymer micelle compositions for 24 h, followed by washing with PBS and maintenance in the medium 199 for an additional 72 h. The cytotoxic effects were evaluated using a standard MTT assay (23). All experiments were performed in eight repeats. RESULTS AND DISCUSSION
Synthesis of Star-PCL-PEG Copolymer. The synthesis of the star amphiphilic polymer was carried out as outlined in Scheme 1. The synthesis of the star PCL polymer was conducted with the PAMAM-OH generation 2 dendrimer as the initiator for the ring-opening polymerization of -caprolactone with Sn(Oct)2 as the catalyst. The targeted molecular weight for each PCL arm is 3000 Da, which corresponds to a degree of polymerization of 26. Therefore, the feed molar ratio of -caprolactone to the hydroxyl groups of the dendrimer core was 26. The Sn(Oct)2 catalyst was added in a concentration of 1/200 relative to the amount of initiating hydroxyl groups, which is the optimum catalyst amount reported in the literature (24, 25). The reaction was carried out in neat -caprolactone at 115 °C for 20 h. In this way, the polymerization was well controlled, and the molecular weight of each arm in the product was determined by the monomer/initiator ratio. The star-PCL product was obtained in quantitative yield. SEC analysis indicated the existence of a small amount of low molecular weight fraction. Fractionation was used to purify the polymer. The prepared star-PCL
Figure 1. SEC chromatograms of linear-PCL, star-PCL, and star-PCL-PEG. Table 1. SEC Results for the Polymers linear-PCL star-PCL star-PCL-PEG theor mol wt wt-av mol wt, Mw no.-av mol wt, Mn polydispersity, Mw/Mn intrinsic viscosity (dL/g)
9200 13000 9500 1.37 0.362
51300 53300 49900 1.07 0.303
131300 133000 131000 1.02 0.489
has a molecular weight of ∼50000 Da and contains 16 terminal hydroxyl groups at the arm ends. To facilitate the star polymer structure characterization, a linear PCL polymer with a relatively broad molecular weight distribution was also synthesized according to the same method using ethylene glycol as the initiator. This polymer has been used as an auxiliary calibration tool of the SEC system. The next step was to introduce hydrophilic PEG into the star-PCL polymer. A commercial PEG polymer with one end capped by a methoxy group and the other end by a carboxymethyl group was used. The carboxylic acid group in the PEG was used to react with the hydroxyl terminal groups of the star-PCL polymer to form the ester linkage and produce the desired final star-PCL-PEG. The coupling reaction was accomplished in dichloromethane at room temperature in the presence of DPTS and using DIPC as the activating agent for the carboxylic group (22). The advantages of this method are that reactions are conducted under very mild conditions, there is no need for preactivation of the carboxylic acid, and the product is formed in high yield. After the reaction was complete, the product was purified twice by fractionation to remove excess PEG and other impurities. The yield of the final star-PCL-PEG polymer was 91%. Characterization of Star Polymers. The molecular weights of the synthesized polymers were determined by SEC and are summarized in Table 1. As can be seen, the molecular weight values of both star-PCL and star-PCLPEG polymers agreed well with the calculated values (Table 1). The polydispersity of these star polymers was sufficiently narrow (Mw/Mn < 1.07). The typical SEC chromatograms of the polymer samples are shown in Figure 1. The star-PCL sample is characterized by a narrower peak width when compared with linear PCL. This is consistent with the molecular weight distribution results for these two polymers. Comparison of the elution profiles for star-PCL and star-PCL-PEG samples revealed that star-PCL-PEG has a larger retention volume and a broader peak width compared with star-PCL. This is in apparent contradiction with the higher molecular weight and the narrow molecular
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Star Amphiphilic Block Copolymer as Drug Delivery Carrier
Table 3. Literature Experimental g′ Values f 8 12 16 18
polyisoprene in θ solvent
polyisoprene in good solvent
0.54 0.43
0.43 0.32
0.30
0.23
PEG polymer 0.46 0.26
Table 4. TGA and DSC Result of Polymers
Figure 2. Log-log plot of intrinsic viscosities against molecular weight for linear and star polymers.
linear-PCL star-PCL PEG star-PCL-PEG
Tdec (°C)
Tm (°C)
∆Hm (J/g)
283 266 260 362
57 54 62 57
68 65 164 82
Table 2. g′ Values Derived from Figure 2 star-PCL star-PCL-PEG
log[M]
g′ ) [η]star/[η]linear
4.60 4.70 4.80 5.06 5.113
0.32 0.298 0.285 0.259 0.271
weight distribution determined for this polymer. We believe that the broader peak width and slower elution of star-PCL-PEG from the SEC column are probably due to the presence of PEG blocks in the arms of star-PCLPEG copolymer. Indeed, PEG has a relatively high affinity to the column packing material, causing it to elute at higher retention times. Elution with other solvents (such as THF) showed severe trailing elution profiles, thus confirming the adsorptive interaction between the star-PCL-PEG copolymer and the column. However, the molecular weight determined from the analysis of data from the light scattering detector does not depend on the retention volume, and we are confident that the estimated molecular weight and polydispersity values for the star polymers are accurate. In addition to the molecular characteristics, the analysis of the SEC data also provided information on the intrinsic viscosity ([η]) of the dilute solutions of the polymers studied (Table 1). These data are presented in the form of the Mark-Houwink plot for linear and star polymers in Figure 2. The solution of the branched starPCL polymer had a lower [η] than the linear polymer, which has a lower molecular weight. This result is consistent with the more compact structure of the starshaped polymer. For star polymers and linear polymers with the same molecular weight, the ratio of their intrinsic viscosities, g′ ) [η]star/[η]linear, known as the shrinking factor, is a function of the number of arms (f) in the star polymer (26). Several theories have been developed to predict a relationship between g′ and f (26). The theoretically predicted values of g′ decrease with branching. Substantial experimental data have been collected for different molecular weights of star polymers in various solvent systems (26). The data of Figure 2 yield [η] ) 1.14 × 10-3 M0.628 dL/g for linear PCL. Here we use the extrapolated [η]linear values from this equation to obtain calculated g′ values for the star polymers. The ratios [η]star/[η]linear ) g′ are shown in Table 2 for different molecular weights of star-PCL and star-PCL-PEG. Experimental g′ values for three different model star polymer systems with various degrees of branching taken from the literature are summarized in Table 3 (26, 27). A comparison of the data in Tables 2 and 3 reveals that the g′ values calculated for star-PCL and star-PCL-PEG are close to the experimental g′ values for model star polymers with f ) 16. It is important to note that there are no data on g′ values for the PCL-based polymer
system. Also, one should be aware that the g′ parameter for the star-PCL-PEG polymer is derived as the ratio between the [η] of the star polymer with block copolymer arms and the [η] of a pure PCL linear polymer (extrapolated values from equation). Therefore, the comparison presented herein is based on different polymer systems, and it can be used only for qualitative consideration. Spectroscopic measurements also confirmed the structures of the star polymer products. In the 1H NMR spectrum of star-PCL, three major resonances attributed to the oxycarbonyl-1,5-pentamethylene repeat unit of a PCL [-O-CH2-(CH2)3-CH2-CO-]n are observed: the triplet peak at 4.07 ppm is assigned to methylene protons in the -OCH2- group; the triplet at 2.32 ppm is assigned to methylene protons adjacent to the carbonyl group; and the -(CH2)3- protons are shown as a multiple peak from 1.9 to 1.1 ppm. The dendrimer core contributes only minor peaks due to its relatively low content. In the 1H NMR spectrum of star-PCL-PEG, in addition to the peaks from PCL blocks, the single peak at 3.65 ppm is observed due to the methylene protons of oxyethylene units of PEG. The integrated peak area ratio of PEG and PCL for the purified star-PCL-PEG sample is consistent with the expected structure. The IR spectrum of the star-PCL had a band characteristic for the ester carbonyl at 1734 cm-1 and a band for hydroxyl at 3500 to 3200 cm-1. There is only one hydroxyl group per PCL chain, so the intensity of the hydroxyl band was low, reflecting the low content of -OH in the sample. In the IR spectrum of star-PCL-PEG, this -OH band has disappeared, confirming the conjugation of PCL and PEG blocks. A stronger band at 1112 cm-1 for C-O-C appeared, consistent with the addition of PEG ether units. Thermal Behavior. The thermal behavior of the synthesized copolymers as well as linear-PCL and PEG samples was investigated using thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC). The results are summarized in Table 4. It is seen that both the linear polymers and the star-PCL are characterized by similar relatively low thermal stabilities due to the chain terminal groups (-OH and -COOH), which are subject to rapid thermal degradation. In contrast, star-PCL-PEG has stable chain terminal groups (-OCH3), leading to a higher thermal stability. Therefore, the observed differences in the thermal stabilities of the synthesized polymers are consistent with their structures. The melting temperatures (Tm) and the heats of fusion (∆Hm) of these polymers were determined from DSC data. The melting behavior of star-PCL, characterized by lower Tm and ∆Hm values compared to those of linear-PCL, probably reflected the branched nature of the star-PCL. Indeed, star branched polymers contain more defects caused by the core, which cannot be incorporated
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Figure 3. DLS graph of the micelle size distribution of starPCL-PEG.
in the crystallites. The DSC thermogram of star-PCLPEG exhibited only a single melting peak characterized by Tm and ∆Hm values that were between those determined for star-PCL and PEG. Micellar Properties of Star-PCL-PEG. The starPCL-PEG polymer does not dissolve directly in water due to the hydrophobicity of the polycaprolactone coreforming block. For this reason, a dialysis method was employed to prepare polymeric micelles. The polymer was first dissolved in DMF, which is a good solvent for both PCL and PEG segments, and micellization was induced by the dropwise addition of water followed by dialysis. The micelle size distribution of a polymer solution with a concentration of ∼12 mg/mL was determined by DLS and is shown in Figure 3. It is a bimodal distribution with a smaller size component of ∼16.9 nm (80%) and a large size component of ∼75 nm (20%). These two components average to an overall effective hydrodynamic diameter of ∼20.1 nm and a polydispersity index of 0.18. From the data presented in Table 1, the viscosity radius of the starPCL-PEG can be calculated as Rv ) 10.1 nm. Thus, the diameter determined from viscosity is 20.2 nm. Generally, the diameters determined from viscosity and DLS measurements are identical for star polymers (26). Because the SEC is performed in chloroform, both PCL and PEG segments are expanded. The DLS experiment is performed in water, however, and the PCL segment is contracted. Therefore, the nonaggregated star polymer in water should have a diameter of
