1132
Biomacromolecules 2003, 4, 1132-1134
Thermo- and pH-Responsive Biodegradable Poly(r-N-substituted γ-glutamine)s Yoichi Tachibana,† Motoichi Kurisawa,†,‡ Hiroshi Uyama,† and Shiro Kobayashi*,† Department of Materials Chemistry, Graduate School of Engineering, Kyoto University, Kyoto 615-8510, Japan, and Bio-oriented Technology Research Advancement Institution Received April 24, 2003; Revised Manuscript Received July 2, 2003
New double stimuli-responsive poly(R-N-substituted γ-glutamine) has been developed, which was synthesized by the reaction of poly(γ-glutamic acid) with amino alcohols. Appropriate combinations of the amino alcohols provided the biodegradable poly(amino acid) exhibiting a sharp lower critical solution temperature (LCST) in water. Furthermore, the phase transition temperature was highly sensitive to pH changes. Water-soluble stimulus-responsive polymers are becoming increasingly attractive for biotechnology and medicine.1 Among them, thermoresponsive polymers that show a lower critical solution temperature (LCST) have widely been investigated due to their potential applications such as controlled drug delivery, biomimetic actuators, chromatographic separations, gene-transfection agents, and immobilized biocatalysts.2 Poly(N-isopropylacrylamide) (PNIPAAm) is one of the most typical thermoresponsive polymers. PNIPAAm exhibits a rapid and reversible hydrationdehydration change in response to small temperature cycles around its LCST (32 °C).3 Besides PNIPAAm, various thermoresponsive polymers, typically, poly(vinyl methyl ether), poly(2-isopropyl-2-oxazoline), poly(N-vinylalkylamide)s, and poly(phosphazene)s, have been developed.4 However, most of them are nonbiodegradable, resulting in limitation of their use in biomedical fields. Recently, polymers and hydrogels responding to more than one stimulus have received much attention owing to their physiological and biological systems.5 They may be regarded as intelligent materials.5a,6 Thermo- and pHresponsive polymers consisting of thermoresponsive polymeric units and acidic or basic polymer units, for example, poly(acrylic acid)-graft-PNIPAAm5a and poly(N-isobutylvinylamide-co-vinylamine),5e were reported. Poly(N-acryloylN-propylpiperazine) sensitive to these double stimuli was developed.5c Polypeptides and related artificial poly(amino acid)s have significantly become important because of their specific propertiessbiodegradability, biocompatibility, etc.7 Very recently, we have developed new thermoresponsive polymers based on biodegradable poly(amino acid)s; poly(N-substituted R/β-asparagine)s, obtained by reaction of poly(succinimide) with a mixture of 5-aminopentanol and 6-aminohexanol, showed a clear LCST in water.8 Furthermore, thermoresponsive hydrogels based on these biodegradable * To whom correspondence should be addressed. Tel: +81-75-753-5608. Fax: +81-75-753-4911. E-mail: kobayasi@ mat.polym.kyoto-u.ac.jp. † Kyoto University. ‡ Bio-oriented Technology Research Advancement Institution.
Scheme 1
poly(amino acid)s were synthesized, which showed a thermoresponsive release of a model drug.9 Poly(γ-glutamate) (1) is a bacterially synthesized biopolymer10,11 and has extensively been studied in terms of its synthesis and biochemistry under various environmental conditions. Poly(γ-glutamate) is well-known to be biodegradable, and the hydrolase for 1 was recently identified.12 The bacteria of the genus Bacillus, typically Bacillus licheniformis and Bacillus subtilis, are often used for production of 1, from which 1 freely diffused into the growth medium. This study deals with synthesis and properties of novel thermo- and pH-responsive biodegradable poly(γglutamine)s (3) from 1 (Scheme 1). Synthesis of 3 was carried out by the reaction of 1 with amino alcohol (2) in the presence of N,N′-carbonyldiimidazole (CDI)13 as dehydration agent in anhydrous N,Ndimethylformamide (DMF).14 At first, an equimolar amount of 5-aminopentanol (2b) and 6-aminohexanol (2c) was used for the modification of 1, the combination of which was reported to give poly(N-substituted R/β-asparagine) with sharp thermoresponse.8 Poly(R-N-substituted γ-glutamine) 3 was obtained in 75% yield, and the structure was confirmed by 1H NMR (see Supporting Information).15 The introduction ratio of the amino alcohols onto 1 was 82% for the carboxyl group, and the unit ratio of 2b and 2c in 3 was 58:42, which is relatively close to the feed ratio. The polymer was soluble
10.1021/bm034123y CCC: $25.00 © 2003 American Chemical Society Published on Web 08/01/2003
Biomacromolecules, Vol. 4, No. 5, 2003 1133
Communications
Figure 1. Effect of temperature on light transmittance of 1 wt % aqueous solution of 3 (sample D) on heating and cooling processes. The measurement was performed at 500 nm at a rate of 1 °C/min.
Figure 3. Temperature dependence of diameter of 3 (sample D, 0.3%) in water.
Table 1. Synthesis and LCST of 3a polymer entry
feed of 2
1 2 3 4
2a/2b ) 25/75 2b 2b/2c ) 75/25 2b/2c ) 50/50
yield introduction (%) code ratio of 2b (%) 63 68 71 75
A B C D
87 84 82 82
composition ratiob (%)
LCSTc (°C)
2a/2b ) 23/77
50 41 27 21
2b/2c ) 71/29 2b/2c ) 58/42
a Reaction conditions, see note 14. b Determined by 1H NMR. c Determined by turbidity.
Figure 4. Effect of pH on phase transition behaviors of 1 wt % aqueous solution of 3 (sample D) on heating process. The measurement was performed at 500 nm at a heating rate of 1 °C/min.
Figure 2. Effect of temperature on light transmittance of 1 wt % aqueous solution of 3 (samples A-D) on heating process. The measurement was performed at 500 nm at a heating rate of 1 °C/ min.
in cold water and dimethyl sulfoxide, partly soluble in DMF, and insoluble in acetone, toluene, and tetrahydrofuran, whereas 1 was insoluble in these solvents. Figure 1 shows the temperature dependence of light transmittance of 1 wt % aqueous solution at 500 nm. The turbidity change took place sharply in both heating and cooling processes. A similar hysteresis was observed in aqueous solutions of thermoresponsive poly(N-substituted R/β-asparagine)s. The LCST, defined as 90% transmittance of the polymer solution during the heating process, was 21 °C. Next, poly(R-N-substituted γ-glutamine)s with different composition of amino alcohols were synthesized (Table 1). In all cases examined, the introduction ratio of the amino alcohols into 1 was around 85%, and the unit ratio of the amino alcohols in 3 was close to the feed ratio. For all of the samples, a clear phase transition was observed on the heating process (Figure 2). The LCST was in the range from 21 to 50 °C and increased with increasing hydrophilicity of
the amino alcohols used. These data indicate that the LCST could be controlled by the composition of the side chain in 3. Figure 3 shows temperature dependence of the size of 3 (sample D) in water, determined by dynamic light scattering. Below 20 °C, the diameter was less than 15 nm; on the other hand, the diameter became larger than 180 nm above 35 °C. These data suggest that the phase transition of 3 took place in water near LCST, yielding nanoparticles with diameter of ca. 200 nm. The effect of pH on the phase transition behaviors of 3 (sample D) was examined (Figure 4). The LCST was found to increase progressively with increasing pH of the medium. At pH 7, the phase transition was not sharp and the LCST was 38 °C, an increase of 17 °C over that of the polymer solution in distilled water (pH 4.5). In addition, no phase transition was observed at pH 9.5, which may be due to the neutralization of the carboxylic acid moiety in 3. The effect of pH on transmittance was examined at 40 °C by using sample D (Figure 5). The clear phase transition was observed around pH 7. These data clearly indicate pH-responsive property of 3. In conclusion, new double stimuli-responsive poly(R-Nsubstituted γ-glutamine) 3 was synthesized by the reaction of 1 and 2. Appropriate combinations of the amino alcohols provided the biodegradable poly(amino acid) exhibiting a sharp LCST in the range from 21 to 50 °C in water. Furthermore, the phase transition temperature was highly sensitive to pH changes. The present thermo- and pH-responsive poly(amino acid) possesses hydroxyl and carboxylic acid groups in the side chain. Therefore, functional molecules such as drugs and probes can be easily introduced, which may be useful for
1134
Biomacromolecules, Vol. 4, No. 5, 2003
Figure 5. Effect of pH on transmittance (500 nm) of 1 wt % aqueous solution of 3 (sample D).
various applications such as drug delivery systems (DDS) and bioconjugation. Further studies on intelligent materials based on biodegradable poly(amino acid)s are under way in our laboratory. Acknowledgment. This work was supported by Program for Promotion of Basic Research Activities for Innovative Bioscience and by the 21st Century COE Program, COE for a United Approach to New Materials Science. We acknowledge the gift of poly(γ-glutamate) from Meiji Seika Kaisha, Ltd. Supporting Information Available. 1H NMR spectra of 1 and 3. This material is available free of charge via the Internet at http://pubs.acs.org. References and Notes (1) Galaev, I. Y.; Mattiasson, B. Trends Biotechnol. 1999, 17, 335. (2) (a) Park, T. G.; Hoffman, A. S. J. Biomed. Mater. Res. 1990, 21, 24. (b) C¸ icek, H.; Tuncel, A. J. Polym. Sci., Polym. Chem. Ed. 1998, 36, 543. (c) Kurisawa, M.; Matsuno, Y.; Yui, N. Macromol. Chem. Phys. 1998, 199, 705. (d) Ramkissoon-Ganorkar, C.; Liu, F.; Baudys, M.; Kim, S. W. J. Controlled Release 1999, 59, 287. (e) Kurisawa, M.; Yokoyama, Y.; Okano, T. J. Controlled Release 2000, 68, 1. (f) Tuncel, A.; U ¨ nsal, E.; Cicek, H. J. Appl. Polym. Sci. 2000, 77, 3154.
Communications (3) (a) Heskins, M.; Guillet, J. E. J. Macromol. Sci., Chem. Ed. 1968, A2, 1441. (b) Chen, G.; Hoffman, A. S. Nature 1995, 373, 49. (c) Jeong, B.; Kim, S. W.; Bae, Y. H. AdV. Drug DeliVery ReV. 2002, 54, 37. (4) (a) Uyama, H.; Kobayashi, S. Chem. Lett. 1992, 1643. (b) Suda, K.; Wada, Y.; Kikunaga, Y.; Morishita, K.; Kishida, A.; Akashi, M. J. Polym. Sci., Part A: Polym. Chem. 1997, 35, 1763. (c) Lee, B. H.; Lee, Y. M.; Sohn, Y. S.; Song, S.-C. Macromolecules 2002, 35, 3876. (d) Chang, Y.; Powell, E. S.; Allcock, H. R.; Park, S. M.; Kim, C. Macromolecules 2003, 36, 2568. (5) (a) Zhang, J.; Peppas, N. A. Macromolecules 2000, 33, 102. (b) Gan, L. H.; Gan, Y. Y.; Deen, G. R. Macromolecules 2000, 33, 7893. (c) Bulmus, V.; Ding, Z.; Long, C. J.; Stayton, P. S.; Hoffman, A. S. Bioconjugate Chem. 2000, 11, 78. (d) Yamamoto, K.; Serizawa, T.; Muraoka, Y.; Akashi, M. Macromolecules 2001, 34, 8014. (e) Yin, X.; Sto¨ver, H. D. H. Macromolecules 2002, 35, 10178. (6) Hoffman, A. S. Artif. Organs 1995, 19, 458. (7) (a) Erhan S. In Desk Reference of Functional Polymers, Syntheses and Applications; Arshady, R., Ed.; American Chemical Society: Washington, DC, 1997; pp 261-270. (b) Kaplan, D. L., Ed. Biopolymers from Renewable Resources; Springer: Berlin, 1998. (8) Tachibana, Y.; Kurisawa, M.; Uyama, H.; Kakuchi, T.; Kobayashi, S. Chem. Commun. 2003, 106. (9) Tachibana, Y.; Kurisawa, M.; Uyama, H.; Kakuchi, T.; Kobayashi, S. Chem. Lett. 2003, 32, 374. (10) (a) Kubota, H.; Matsunobu, T.; Uotani, K.; Takebe, H.; Satoh, A.; Tanaka, T.; Taniguchi, M. Biosci., Biotech., Biochem. 1994, 57, 1212. (b) King, E. C.; Blacker, A. J.; Bugg, T. D. H. Biomacromolecules 2000, 1, 75. (c) He, L. M.; Neu, M. P.; Vanderberg, L. A. EnViron. Sci. Technol. 2000, 34, 1694. (11) Molecular weight of 1 was 3 × 105 to 12 × 105 (data of the supplier). (12) King, E. C.; Blacker, A. J.; Bugg, T. D. H. Biomacromolecules 2000, 1, 75. (13) Paul, R.; Anderson, G. W. J. Am. Chem. Soc. 1960, 82, 4596. (14) A typical procedure of synthesis of 3 was as follows. Under argon, a mixture of 1 (0.28 g, 2.2 mmol unit of monomer) and CDI (0.40 g, 2.5 mmol) was stirred in 30 mL of anhydrous DMF at 0 °C. After 6 h, 2 (4.4 mmol) was added to the mixture at 0 °C, and the mixture was further stirred at room temperature for 4 days. Then, the reaction mixture was poured into a large amount of acetone, and the precipitates were collected by centrifugation. After being dried in vacuo, the obtained polymer was dissolved in dilute HCl, and the solution was subjected to purification by dialysis (cutoff molecular weight ) 2 × 103) three times. The remaining solution was lyophilized to give 3. (15) 1H NMR (D2O): δ 4.04 (br, NHCHC(dO)), 3.40 (br, CH2OH), 3.03 (br, NHCH2), 2.21 (br, C(dO)CH2), 1.93, 1.78 (br, CHCH2CH2), 1.37, 1.17 (br, CH2CH2CH2).
BM034123Y
