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Micellization Phenomena of Biodegradable Amphiphilic Triblock Copolymers Consisting of Poly(β-hydroxyalkanoic acid) and Poly(ethylene oxide) Jun Li,*,†,‡ Xiping Ni,† Xu Li,† Ngee Koon Tan,‡ Chwee Teck Lim,‡ Seeram Ramakrishna,‡ and Kam W. Leong§ Institute of Materials Research and Engineering, National University of Singapore, 3 Research Link, Singapore 117602, Singapore, Division of Bioengineering and Nanoscience & Nanotechnology Initiative, Faculty of Engineering, National University of Singapore, 9 Engineering Drive 1, Singapore 117576, Singapore, and Department of Biomedical Engineering, Johns Hopkins University, Baltimore, Maryland 21205 Received June 9, 2005. In Final Form: July 21, 2005 This paper reports the studies on micelle formation of new biodegradable amphiphilic poly(ethylene oxide)-poly[(R)-3-hydroxybutyrate]-poly(ethylene oxide) (PEO-PHB-PEO) triblock copolymer with various PHB and PEO block lengths in aqueous solution. Transmission electron microscopy showed that the micelles took an approximately spherical shape with the surrounding diffuse outer shell formed by hydrophilic PEO blocks. The size distribution of the micelles formed by one triblock copolymer was demonstrated by dynamic light scattering technique. The critical micellization phenomena of the copolymers were extensively studied using the pyrene fluorescence dye absorption technique, and the (0,0) band changes of pyrene excitation spectra were used as a probe for the studies. For the copolymers studied in this report, the critical micelle concentrations ranged from 1.3 × 10-5 to 1.1 × 10-3 g/mL. For the same PEO block length of 5000, the critical micelle concentrations decreased with an increase in PHB block length, and the change was more significant in the short PHB range. It was found that the micelle formation of the biodegradable amphiphilic triblock copolymers consisting of poly(β-hydroxyalkanoic acid) and PEO was relatively temperature-insensitive, which is quite different from their counterparts consisting of poly(R-hydroxyalkanoic acid) and PEO.
Introduction Amphiphilic triblock copolymers can self-assemble to form micelles in aqueous medium, containing dense cores of the insoluble blocks, surrounded by diffuse outer shells (coronas) formed by the soluble blocks.1-4 Recently, amphiphilic triblock copolymers consisting of poly(ethylene oxide) and poly(R-hydroxyalkanoic acid) such as poly(L-lactic acid) (PLLA) and poly(glycolic acid) (PGA) have attracted special attention in biomaterials research because of their self-assembly behavior in aqueous medium as well as their biodegradability and biocompatibility.4-9 For example, biodegradable amphiphilic triblock copolymers are used as micellar micro- or nanocontainers to incorporate drugs noncovalently, forming colloidal carrier * Corresponding author. Tel: +65-6874-8376. Fax: +65-68747273. E-mail:
[email protected];
[email protected]. † Institute of Materials Research and Engineering. ‡ Faculty of Engineering. § Johns Hopkins University. (1) Astafieva, I.; Zhong, X. F.; Eisenberg, A. Macromolecules 1993, 26, 7339-7352. (2) Wilhelm, M.; Zhao, C.; Wang, Y.; Xu, R.; Winnik, M. A.; Mura, J. L.; Riess, G.; Croucher, M. D. Macromolecules 1991, 24, 1033-1040. (3) Winnik, F. M.; Davidson, A. R.; Hamer, G. K.; Kitano, H. Macromolecules 1992, 25, 1876-1880. (4) Alexandridis, P.; Lindman, B. Amphiphilic Block Copolymers: Self-Assembly and Application; Elsevier: Amsterdam, 2000. (5) Kataoka, K.; Harada, A.; Nagasaki, Y. Adv. Drug Delivery Rev. 2001, 47, 113-131. (6) Jeong, B.; Kim, S. W.; Bae, Y. H. Adv. Drug Delivery Rev. 2002, 54, 37-51. (7) Kissel, T.; Li, Y.; Unger, F. Adv. Drug Delivery Rev. 2002, 54, 99-134. (8) Jeong, B.; Bae, Y. H.; Lee, D. S.; Kim, S. W. Nature 1997, 388, 860-862. (9) Jeong, B.; Bae, Y. H.; Kim, S. W. Colloids Surf. B 1999, 16, 185193.
systems for drug delivery.4 The micelle formation or gelation of triblock copolymers consisting of poly(ethylene oxide) and poly(R-hydroxyalkanoic acid) have been found to be temperature-sensitive, i.e., the hydrophobic polyester segments aggregate at elevated temperature but tend to dissociate at low temperature,8,9 which is similar to the intensively studied poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (PEO-PPO-PEO, Pluronic) triblock copolymers.10,11 Poly[(R)-3-hydroxybutyrate] (PHB) and related poly(β-hydroxyalkanoic acid) are a class of natural biopolyesters produced by many microorganisms as intracellular carbon and energy storage material.12 Being biocompatible and biodegradable, PHB is attractive in various biomedical applications. It has been a challenge to synthesize PHB with well-defined structures and chain lengths.13-17 Although block copolymers of PHB with other polyesters or polyurethanes have been synthesized to modify the mechanical properties of PHB,18-20 there had been no (10) Alexandridis, P.; Hatton, T. A. Colloids Surf. 1995, 96, 1-46. (11) Bromberg, L. E.; Ron, E. S. Adv. Drug Delivery Rev. 1998, 31, 197-221. (12) Doi, Y. Microbial Polyesters; VCH Publisher: New York, 1990. (13) Hirt, T. D.; Neuenschwander, P.; Suter, U. W. Macromol. Chem. Phys. 1996, 197, 1609-1614. (14) Seebach, D.; Fritz, M. G. Int. J. Biol. Macromol. 1999, 25, 217236. (15) Rueping, M.; Dietrich, A.; Buschmann, V.; Fritz, M. G.; Sauer, M.; Seebach, D. Macromolecules 2001, 34, 7042-7048. (16) Li, J.; Uzawa, J.; Doi, Y. Bull. Chem. Soc. Jpn. 1997, 70, 18871893. (17) Li, J.; Uzawa, J.; Doi, Y. Bull. Chem. Soc. Jpn. 1998, 71, 16831689. (18) Hirt, T. D.; Neuenschwander, P.; Suter, U. W. Macromol. Chem. Phys. 1996, 197, 4253-4268. (19) Reeve, M. S.; Mccarthy, S. P.; Gross, R. A. Macromolecules 1993, 26, 888-894.
10.1021/la0515266 CCC: $30.25 © 2005 American Chemical Society Published on Web 08/19/2005
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Figure 1. Synthesis of the PEO-PHB-PEO triblock copolymers.
attempt to or success in synthesizing amphiphilic triblock copolymers containing PHB and PEO, until we recently reported the successful synthesis of new PEO-PHB-PEO triblock copolymers starting from high molecular weight natural isotactic PHB (Figure 1).21 The new ABA triblock copolymer of PEO with PHB as the middle block may have novel properties, because the isotactic PHB is highly crystalline and hydrophobic.12 Moreover, PHB has lower in vivo degradation rate than PLLA and many other biopolyesters.22 Therefore, the potentially lower biodegradation rate of such PEO-PHB-PEO triblock copolymers would be advantageous in biomedical or environmental applications, where higher stability is desired. On the other hand, one of the most characteristic features of amphiphilic triblock copolymers, consisting of hydrophilic PEO and a hydrophobic middle block, is to form micelles in aqueous solution.10,23,24 As a new class of biodegradable amphiphilic triblock copolymer, the micelle formation properties of PEO-PHB-PEO triblock copolymers are of special interest with respect to their applications as biomaterials. Herein, we have prepared a series of water-soluble PEO-PHB-PEO triblock copolymers and studied the micelle formation of the copolymers. We have found that the new triblock copolymers have a strong tendency toward micelle formation in aqueous solution, with very small critical micelle concentration (cmc) values, as compared with their counterparts consisting of poly(R-hydroxyalkanoic acids) such as PLLA or PGA. Experimental Section Materials. Natural source poly[(R)-3-hydroxybutyrate] (PHB) was purchased from Aldrich. The PHB sample was purified by dissolving in chloroform followed by filtration and precipitation in petroleum ether before use. The Mn and Mw of the purified PHB are 8.7 × 104 and 2.3 × 105, respectively. Methoxypoly(ethylene oxide) monopropionic acid (methoxy-PEO-acid) with molecular weight of ca. 5000 was purchased from Shearwater Polymers, Inc. Its Mn and Mw were found to be 4740 and 4880, respectively. Methoxypoly(ethylene oxide) with one hydroxyl end with molecular weight of ca. 2000 was purchased from Sigma. Its Mn and Mw were found to be 1820 and 1870, respectively. Methoxy-PEO-acid prepolymers with Mn of 1820 were prepared by reaction of the methoxy-PEO with succinic anhydride in the presence of DMAP and triethylamine in 1,4-dioxane as reported previously.25 Bis(2-methoxyethyl)ether (Diglyme, 99%), ethylene glycol (99%), dibutyltin dilaurate (95%), 1,3-N,N′-dicyclohexyl(20) Andrade, A. P.; Neuenschwander, P.; Hany, R.; Egli, T.; Witholt, B.; Li, Z. Macromolecules 2002, 35, 4946-4950. (21) Li, J.; Li, X.; Ni, X.; Leong, K. W. Macromolecules 2003, 36, 2661-2667. (22) Gogolewski, S.; Jovanovic, M.; Perren, S. M.; Dillon, J. G.; Hughes, M. K. J. Biomed. Mater. Res. 1993, 27, 1135-1148. (23) Tuzar, Z.; Kratochivil, P. In Surface and Colloid Science; Matijivic, E., Ed.; Plenum Press: New York, 1993; Vol. 15. (24) Wanka, G.; Hoffmann, H.; Ulbricht, W. Macromolecules 1994, 27, 4145-4159. (25) Bae, Y. H.; Huh, K. M.; Kim, Y.; Park, K. J. Controlled Release 2000, 64, 3-13.
Li et al. carbodiimide (DCC, 99%), 4-(dimethylamino)pyridine (DMAP, 99%), succinic anhydride (97%), and triethylamine (99%) were obtained from Aldrich. Diglyme was dried with molecular sieves, and methylene chloride was distilled from CaH2 before use. The PEO-PHB-PEO triblock copolymer samples were synthesized according to our previous report (Figure 1).21 First, the telechelic hydroxylated PHB (PHB-diol) prepolymer with low molecular weights was prepared by transesterification from the natural PHB and diethylene glycol with dibutyltin dilaurate as catalyst in diglyme, as reported previously (yield, 80%).13 The PHB-diol was then allowed to react with methoxy-PEO-monocarboxylic acid with molecular weight of 2000 or 5000, using 1,3-N,N′-dicyclohexylcarbodiimide (DCC) in the presence of 4-(dimethylamino)pyridine (DMAP) to give the PEO-PHB-PEO triblock copolymers. Since the reaction is moisture sensitive, it was carried out in dried methylene chloride under nitrogen atmosphere. The target triblock copolymer was isolated and purified from the reaction mixture by repeated precipitation and fractionation. The GPC chromatogram showed that the purified triblock copolymer has a unimodal molecular weight distribution. Four triblock copolymer samples were synthesized in this work (Table 1). As a typical example, the synthesis procedures of PEO-PHBPEO (5000-3820-5000) are described as follows. The PHB-diol (0.38 g, 1.2 × 10-4 mol, Mn ) 3220), M-PEO-A (1.42 g, 3.0 × 10-4 mol, Mn ) 4740), and DMAP (12 mg, 9.8 × 10-5 mol) were dried in a 50-mL two-neck flask under vacuum at 60 °C (oil bath) overnight. Anhydrous methylene chloride (25-30 mL) was added to the flask and then was removed by distillation (oil bath, 75 °C), to remove any trace water in the system. When the flask cooled, DCC (0.098 g, 4.7 × 10-4 mol) dissolved in 4 mL of anhydrous methylene chloride was added and the mixture stirred overnight at room temperature under nitrogen. Precipitated dicyclohexylurea (DCU) was removed by filtration. The polymer was precipitated from diethyl ether (two times). The desired triblock copolymer product, redissolved in methanol or chloroform, was further purified by fractionation. Yield: 0.75 g, 56%. GPC (THF): Mn ) 12 720, Mw ) 13 770, Mw/Mn ) 1.08. Tm ) 54 °C (for PEO block) and 140 °C (for PHB block). 1H NMR (400 MHz, CDCl3): δ 5.29 (m, mechine H of PHB block), 4.32 (s, -COOCH2CH2COO-), 3.68 (s, -CH2OCH2- of PEO block), 3.42 (s, -OCH3 end group), 2.48-2.67 (m, methylene H of PHB block), 1.31 (d, methyl H of PHB block). IR (KBr): 2886, 1723, 1456, 1380, 1280, 1111, 1061, 962, 842, 516 cm-1. Polymer Characterization. Gel permeation chromatography (GPC) analysis was carried out with a Shimadzu SCL-10A and LC-8A system equipped with two Phenogel 5 µm, 50 and 1000 Å columns (size: 300 × 4.6 mm) in series and a Shimadzu RID-10A refractive index detector. THF was used as eluent at a flow rate of 0.30 mL/min at 40 °C. Monodispersed poly(ethylene glycol) standards were used to obtain a calibration curve. The 1H NMR spectra were recorded on a Bruker AV-400 NMR spectrometer at 400 MHz at room temperature. The 1H NMR measurements were carried out with an acquisition time of 3.2 s, a pulse repetition time of 2.0 s, a 30° pulse width, 5208-Hz spectral width, and 32K data points. Chemical shift was referred to the solvent peaks (δ ) 7.3 ppm for CHCl3). Transmission Electron Microscopy (TEM). The morphological examination of the copolymer micelles was performed using a Philips CM300 high-resolution transmission electron microscope operating at an acceleration voltage of 100 kV. A drop of PEO-PHB-PEO triblock copolymer aqueous solution (0.5-1 mg/mL) containing 0.1 wt % phosphotungstic acid (PTA) was deposited onto a 200 mesh copper grid coated with carbon. Excessive solution was removed with a Kimwipes delicate wipe. The shape and size of the micelles were directly determined from each transmission electron micrograph. Dynamic Light Scattering Measurements. The micellar size and size distribution were determined by dynamic light scattering (DLS) using a Coulter N4 Plus particle size analyzer. Each analysis lasted for 300 s and was performed at 23 °C with angle detection of 90°. The concentration of the polymer solution was 1 mg/mL. Fluorescence Spectroscopy. Steady-state fluorescence spectra were recorded on a Shimadzu RF-5301PC spectrofluorophotometer. Excitation spectra were monitored at 373 nm. The
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Table 1. Molecular Characteristics of the PEO-PHB-PEO Triblock Copolymers and the Data for Cmc Values and Micelle Sizes block copolymera
Mnb
Mwb
Mw/Mnb
PEO-PHB-PEO (2000-470-2000) PEO-PHB-PEO (5000-460-5000) PEO-PHB-PEO (5000-1750-5000) PEO-PHB-PEO (5000-3820-5000)
4500 9840 11270 12720
4730 10720 15250 13770
1.05 1.09 1.35 1.08
block length (Mn) PEOb PHBc 1820 4740 4830 4740
470 460 1750 3820
cmcd (g/mL)
micelle diameter (nm)
2.0 × 10-4 1.1 × 10-3 5.7 × 10-5 1.3 × 10-5
27 ( 4e 37 ( 8f 48 ( 7f
a The numbers in the parentheses show the indicative block length of each block in g/mol. b Determined by GPC. c Determined by combination of 1H NMR and GPC results. d Critical micelle concentration (cmc) in water determined by the pyrene probe technique at 23 °C. e Number average diameter of the micelles from AFM image. f Number average diameter of the micelles from TEM image.
Figure 2. Micelles observed by TEM for PEO-PHB-PEO (5000-3820-5000). slit widths for both excitation and emission sides were maintained at 1.5 nm. Sample solutions were prepared by dissolving a predetermined amount of block copolymer in an aqueous pyrene solution of known concentration, and the solutions were allowed to stand for 1 day for equilibration. The concentration of pyrene was kept at 6.0 × 10-7 M.
Results and Discussion The molecular characteristics of the triblock copolymers used in this study, including their molecular weight, molecular weight distribution, and compositions (lengths of PEO and PHB blocks), determined by combination of GPC and 1H NMR, are listed in Table 1. All the copolymers listed in Table 1 are soluble in water, and their micelle solutions were prepared by directly dissolving the copolymers in water. TEM was used to confirm the micelle formation. As a typical example, Figure 2 shows the TEM image of micelles formed by PEO-PHB-PEO (50003820-5000). It can be seen that the micelles take an approximately spherical shape, and most of the micelles have diameters in the range of 30-70 nm in the dried state. By close observation of the TEM image, for each micelle a bright region is found to surround a dark region. The bright region should correspond to the diffuse outer shell formed by the PEO block, while the dark region is the dense core of the micelle formed by the hydrophobic PHB block. The particle size distribution of the micelles was further determined by dynamic light scattering using a Coulter N4 Plus particle size analyzer. As shown in Figure 3, sizes of the micelles formed by PEO-PHBPEO (5000-3820-5000) are in the range of 20-200 nm, which is consistent with the TEM results, considering that the dynamic light scattering data reflect the micelle sizes in solution. A comparison of the micelle sizes in the dry state is given in Table 1. The average micelle diameters for PEO-PHB-PEO triblock copolymers of different block lengths range from 27 to 48 nm. Fluorescence probe technique is a powerful tool to study micellar properties of amphiphilic block copolymers.1,2
Figure 3. Particle size distribution of micelles formed by PEOPHB-PEO (5000-3820-5000).
Mostly, pyrene was chosen as the fluorescence probe because of its photophysical and other properties. With increasing copolymer concentration in an aqueous solution of pyrene, several significant changes in the fluorescence spectra, both emission and excitation, can be observed, with the onset of micellization of the block copolymer systems. There are an increase in the quantum yield of the fluorescence, a change in the vibrational fine structure of the emission spectra, and a shift of the (0,0) absorption band from about 334 to 337 nm in the excitation spectra.1 Theses changes are caused by the transfer of pyrene molecules from water environment to the hydrophobic micellar cores and thus provide information on the location of the pyrene probe in the system. According to the literature,1,2 the (0,0) band change of pyrene is more sensitive to the critical micellization concentration than lifetime measurements or fluorescence emission changes. In addition, the concentration dependence of the excitation spectra changes is more sensitive to a true onset of aggregation. Therefore, in this study, the critical micelle concentration (cmc) values of the PEO-PHB-PEO triblock copolymers in aqueous solution were determined using the fluorescence excitation spectra of the pyrene probe. Figure 4 shows the excitation spectra for pyrene in water at various concentrations of PEO-PHB-PEO (50003820-5000). With an increase in the copolymer concentration, a red shift of the (0,0) absorption band from 334 to 337 nm was observed. Figure 5 shows the intensity ratio of I337/I334 of pyrene excitation spectra as a function of the logarithm of copolymer concentrations for all four
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Figure 4. Steady-state fluorescence excitation spectra monitored at 373 nm for the pyrene probe in an aqueous solution of PEO-PHB-PEO (5000-3820-5000) at various concentrations at 23 °C. The concentration of pyrene is 6.0 × 10-7 M.
Figure 5. Plots of the I337/I334 ratio of pyrene excitation spectra in water as a function of PEO-PHB-PEO triblock copolymer concentration at 23 °C.
PEO-PB-PEO triblock copolymers. The I337/I334 vs log C plots present a sigmoid curve. A negligible change of intensity ratio of I337/I334 was observed at low concentration range for each triblock copolymer. With an increase in the copolymer concentration, the intensity ratio exhibited a substantial increase at a certain concentration, reflecting the incorporation of pyrene into the hydrophobic core region of the micelles. Therefore, the cmc values were determined form the crossover point at the low concentration range in Figure 5, and the results are listed in Table 1. The very low cmc values, as low as 1.3 × 10-5 g/mL for PEO-PHB-PEO (5000-3820-5000), indicate a very strong tendency of the triblock copolymers toward formation of micelles in aqueous solution. Generally, the cmc values for PEO-PHB-PEO triblock copolymers are much smaller than their counterparts consisting of poly(R-hydroxyalkanoic acids) such as poly(lactic acid) (PLA), poly(glycolic acid) (PGA), or copolymer of PLA and PGA (PLGA). For example, PEO-PLGAPEO (750-2370-750) has cmc values ranging from 1.2 × 10-4 to 1.4 × 10-4 g/mL at 20-30 °C.9 For PEO-PLGAPEO (750-2370-750), its PLGA block length is comparable with the PHB block lengths of PEO-PHB-PEO (5000-1750-5000) and PEO-PHB-PEO (5000-38205000), while its PEO block length is much shorter. Theoretically, the cmc of a PEO-PLGA-PEO triblock copolymer with the PLGA block length of 2370 and PEO
Li et al.
Figure 6. The cmc dependence on hydrophobic PHB block length for PEO-PHB-PEO triblock copolymers with PEO block length of 5000.
Figure 7. Plots of the I337/I334 ratio of pyrene excitation spectra in water as a function of copolymer concentration at different temperatures for PEO-PHB-PEO (5000-3820-5000).
block length of 5000 should be much larger than 10-4 g/mL, although the data are not available in the literature. Our results have shown that the cmc values of PEO-PHBPEO (5000-1750-5000) and PEO-PHB-PEO (50003820-5000) are within the scale of 10-5 g/mL (Table 1). Figure 6 plots the cmc values of the PEO-PHB-PEO triblock copolymers (PEO block length 5000) as a function of the length of the hydrophobic PHB block. It can be seen that in the shorter PHB block range (460-1750) the cmc values deceased more significantly than those in the longer PHB block range (1750-3820). The cmc values decreased by 19 times from PHB block length 460 to 1750, while only by 4.4 times from 1750 to 3820. The trend of cmc changes depending on hydrophobic block length for PEOPHB-PEO triblock copolymers is quite similar to that for PEO-polystyrene-PEO (PEO-PS-PEO) triblock copolymers.1,2 As pointed out in the previous reports,1,2 the dependence of cmc on the hydrophobic block length is generally stronger than that of the hydrophilic block length in an amphiphilic triblock copolymer system such as PEO-PS-PEO. We also observed a cmc decrease only by 5.5 times with a significant increase in PEO block from 2000 to 5000 for PEO-PHB-PEO triblock copolymer with PHB block length of about 500 (Table 1). Figure 7 shows the plots of the I337/I334 ratio as a function of copolymer concentration at different temperatures for PEO-PHB-PEO (5000-3820-5000). All the plots present a similar sigmoid curve, passing through the same point at copolymer concentration of 3.2 × 10-5 g/mL. The three
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curves also showed their first inflection at a copolymer concentration of 1.3 × 10-5 g/mL, which is the onset of the micellization of the copolymer. There were no changes for the cmc values at different temperatures from 23 to 45 °C. Therefore, the cmc of the PEO-PHB-PEO triblock copolymer is relatively temperature-insensitive. We found that all four triblock copolymer samples studied in this work showed the same temperature insensitivity. This is quite different from amphiphilic triblock copolymers consisting of PLLA or PGA as middle hydrophobic block, which are usually thermosensitive, whereas the I337/I334 ratio vs temperature plot presents a sigmoid curve and gives the critical micellization temperature at the first inflection in the sigmoid curve.7-9 It is thought that PHB has higher hydrophobicity than poly(R-hydroxyalkanoic acids) such as PLA and PGA, thus the tendency of selfassembly of PHB segments in the block copolymers is much stronger and not dependent on the temperature change, which is more similar to the cases of PEO-PS-PEO triblock copolymer systems.
mers studied in this report, the critical micelle concentrations ranged from 1.3 × 10-5 to 1.1 × 10-3 g/mL. For the same PEO block length of 5000, the cmc values decreased with an increase in PHB block length, and the change was more significant in the shorter PHB block range. The cmc was also found to be relatively temperature-insensitive, suggesting that the micelle formation of PEO-PHB-PEO triblock copolymers is similar to triblock copolymer systems such as PEO-PS-PEO, rather than to amphiphilic triblock copolymers consisting of poly(R-hydroxyalkanoic acids) such as PLA and PGA as the middle hydrophobic block. Although PHB and PLLA both belong to biodegradable biopolyesters, the PEO-PHBPEO triblock copolymers have quite different characteristics in micelle formation as compared with their counterparts with PLLA or PGA as the middle hydrophobic block. The new biodegradable PEO-PHB-PEO triblock copolymers with very low cmc values and temperatureinsensitive cmc may find promising biomedical applications as stable drug carriers under very diluted conditions.
Conclusions
Acknowledgment. The authors acknowledge the financial support from Singapore’s Agency for Science, Technology and Research (A*STAR) and National University of Singapore.
The micellization phenomena in new biodegradable amphiphilic PEO-PHB-PEO triblock copolymers were studied. The formation of the micelles by the copolymers was confirmed by TEM, which showed that the micelles took an approximately spherical morphology. The size distribution of the micelles was demonstrated using dynamic light scattering measurement. For micelles formed by PEO-PHB-PEO (5000-3820-5000) copolymer, the diameters were in the range of 20-200 nm in the solution, and the average diameter was 48 ( 7 nm in the dry state. For studying the critical micellization phenomena of the copolymers, the pyrene fluorescence probe technique using the (0,0) band changes of the pyrene excitation spectra was chosen because it was reported to be the most sensitive to the critical micellization concentration of amphiphilic block copolymers. For the copoly-
Supporting Information Available: Preparation of PHB-diol prepolymer, TEM images of micelles formed from PEOPHB-PEO (5000-1750-5000), and AFM image of micelles formed from PEO-PHB-PEO (2000-470-2000). This material is available free of charge via the Internet at http://pubs.acs.org. Note Added after ASAP Publication. This article was released ASAP on August 19, 2005. In the Results and Discussion section, paragraph 5, sentence 5, hydrophobic was changed to hydrophilic. The correct version posted on August 22, 2005. LA0515266
