Thermo- and pH-Responsive BiodegradablePoly( 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-substitutedγ-glutamine) has been developed, which was synthesizedby the reaction of poly(γ-glutamic acid) with amino alcohols. Appropriate combinations of the amino alcoholsprovided 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 becomingincreasingly attractive for biotechnology and medicine.1

Among them, thermoresponsive polymers that show a lowercritical solution temperature (LCST) have widely beeninvestigated due to their potential applications such ascontrolled drug delivery, biomimetic actuators, chromato-graphic separations, gene-transfection agents, and immobi-lized biocatalysts.2 Poly(N-isopropylacrylamide) (PNIPAAm)is one of the most typical thermoresponsive polymers.PNIPAAm exhibits a rapid and reversible hydration-dehydration change in response to small temperature cyclesaround its LCST (32°C).3 Besides PNIPAAm, variousthermoresponsive polymers, typically, poly(vinyl methylether), poly(2-isopropyl-2-oxazoline), poly(N-vinylalkyl-amide)s, and poly(phosphazene)s, have been developed.4

However, most of them are nonbiodegradable, resulting inlimitation of their use in biomedical fields.

Recently, polymers and hydrogels responding to morethan one stimulus have received much attention owing totheir physiological and biological systems.5 They may beregarded as intelligent materials.5a,6 Thermo- and pH-responsive polymers consisting of thermoresponsive poly-meric units and acidic or basic polymer units, for example,poly(acrylic acid)-graft-PNIPAAm5a and poly(N-isobutyl-vinylamide-co-vinylamine),5ewere reported. Poly(N-acryloyl-N-propylpiperazine) sensitive to these double stimuli wasdeveloped.5c

Polypeptides and related artificial poly(amino acid)s havesignificantly become important because of their specificpropertiessbiodegradability, biocompatibility, etc.7 Veryrecently, we have developed new thermoresponsive polymersbased on biodegradable poly(amino acid)s; poly(N-substi-tutedR/â-asparagine)s, obtained by reaction of poly(succin-imide) with a mixture of 5-aminopentanol and 6-aminohex-anol, showed a clear LCST in water.8 Furthermore,thermoresponsive hydrogels based on these biodegradable

poly(amino acid)s were synthesized, which showed a ther-moresponsive release of a model drug.9

Poly(γ-glutamate) (1) is a bacterially synthesized biopoly-mer10,11 and has extensively been studied in terms of itssynthesis and biochemistry under various environmentalconditions. Poly(γ-glutamate) is well-known to be bio-degradable, and the hydrolase for1 was recently identified.12

The bacteria of the genusBacillus, typically Bacilluslicheniformis and Bacillus subtilis, are often used forproduction of1, from which1 freely diffused into the growthmedium. This study deals with synthesis and properties ofnovel thermo- and pH-responsive biodegradable poly(γ-glutamine)s (3) from 1 (Scheme 1).

Synthesis of3 was carried out by the reaction of1 withamino alcohol (2) in the presence ofN,N′-carbonyldiimida-zole (CDI)13 as dehydration agent in anhydrousN,N-dimethylformamide (DMF).14 At first, an equimolar amountof 5-aminopentanol (2b) and 6-aminohexanol (2c) was usedfor the modification of1, the combination of which wasreported to give poly(N-substitutedR/â-asparagine) withsharp thermoresponse.8 Poly(R-N-substitutedγ-glutamine)3 was obtained in 75% yield, and the structure was confirmedby 1H NMR (see Supporting Information).15 The introductionratio of the amino alcohols onto1 was 82% for the carboxylgroup, and the unit ratio of2b and2c in 3 was 58:42, whichis relatively close to the feed ratio. The polymer was soluble

* 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

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10.1021/bm034123y CCC: $25.00 © 2003 American Chemical SocietyPublished on Web 08/01/2003

in cold water and dimethyl sulfoxide, partly soluble in DMF,and insoluble in acetone, toluene, and tetrahydrofuran,whereas1 was insoluble in these solvents.

Figure 1 shows the temperature dependence of lighttransmittance of 1 wt % aqueous solution at 500 nm. Theturbidity change took place sharply in both heating andcooling processes. A similar hysteresis was observed inaqueous solutions of thermoresponsive poly(N-substitutedR/â-asparagine)s. The LCST, defined as 90% transmittanceof the polymer solution during the heating process, was 21°C.

Next, poly(R-N-substitutedγ-glutamine)s with differentcomposition of amino alcohols were synthesized (Table 1).In all cases examined, the introduction ratio of the aminoalcohols into1 was around 85%, and the unit ratio of theamino alcohols in3 was close to the feed ratio. For all ofthe samples, a clear phase transition was observed on theheating process (Figure 2). The LCST was in the range from21 to 50°C and increased with increasing hydrophilicity of

the amino alcohols used. These data indicate that the LCSTcould be controlled by the composition of the side chain in3.

Figure 3 shows temperature dependence of the size of3(sample D) in water, determined by dynamic light scattering.Below 20°C, the diameter was less than 15 nm; on the otherhand, the diameter became larger than 180 nm above 35°C.These data suggest that the phase transition of3 took placein water near LCST, yielding nanoparticles with diameterof ca. 200 nm.

The effect of pH on the phase transition behaviors of3(sample D) was examined (Figure 4). The LCST was foundto increase progressively with increasing pH of the medium.At pH 7, the phase transition was not sharp and the LCSTwas 38°C, an increase of 17°C over that of the polymersolution in distilled water (pH 4.5). In addition, no phasetransition was observed at pH 9.5, which may be due to theneutralization of the carboxylic acid moiety in3. The effectof pH on transmittance was examined at 40°C by usingsample D (Figure 5). The clear phase transition was observedaround pH 7. These data clearly indicate pH-responsiveproperty of3.

In conclusion, new double stimuli-responsive poly(R-N-substitutedγ-glutamine)3 was synthesized by the reactionof 1 and2. Appropriate combinations of the amino alcoholsprovided the biodegradable poly(amino acid) exhibiting asharp LCST in the range from 21 to 50°C in water.Furthermore, the phase transition temperature was highlysensitive to pH changes.

The present thermo- and pH-responsive poly(amino acid)possesses hydroxyl and carboxylic acid groups in the sidechain. Therefore, functional molecules such as drugs andprobes can be easily introduced, which may be useful for

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.

Table 1. Synthesis and LCST of 3a

polymer

entry feed of 2yield(%) code

introductionratio of 2b (%)

compositionratiob (%)

LCSTc

(°C)

1 2a/2b ) 25/75 63 A 87 2a/2b ) 23/77 502 2b 68 B 84 413 2b/2c ) 75/25 71 C 82 2b/2c ) 71/29 274 2b/2c ) 50/50 75 D 82 2b/2c ) 58/42 21

a Reaction conditions, see note 14. b Determined by 1H NMR. c Deter-mined by turbidity.

Figure 2. Effect of temperature on light transmittance of 1 wt %aqueous solution of 3 (samples A-D) on heating process. Themeasurement was performed at 500 nm at a heating rate of 1 °C/min.

Figure 3. Temperature dependence of diameter of 3 (sample D,0.3%) in water.

Figure 4. Effect of pH on phase transition behaviors of 1 wt %aqueous solution of 3 (sample D) on heating process. The measure-ment was performed at 500 nm at a heating rate of 1 °C/min.

Communications Biomacromolecules, Vol. 4, No. 5, 2003 1133

various applications such as drug delivery systems (DDS)and bioconjugation. Further studies on intelligent materialsbased on biodegradable poly(amino acid)s are under way inour laboratory.

Acknowledgment. This work was supported by Programfor Promotion of Basic Research Activities for InnovativeBioscience and by the 21st Century COE Program, COE fora United Approach to New Materials Science. We acknowl-edge the gift of poly(γ-glutamate) from Meiji Seika Kaisha,Ltd.

Supporting Information Available. 1H NMR spectra of1 and 3. This material is available free of charge via theInternet at http://pubs.acs.org.

References and Notes

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(b) Cicek, 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 Release1999, 59, 287. (e) Kurisawa,M.; Yokoyama, Y.; Okano, T.J. Controlled Release2000, 68, 1. (f)Tuncel, A.; Unsal, E.; Cicek, H.J. Appl. Polym. Sci.2000, 77, 3154.

(3) (a) Heskins, M.; Guillet, J. E.J. Macromol. Sci., Chem. Ed.1968,A2, 1441. (b) Chen, G.; Hoffman, A. S.Nature1995, 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.Macromolecules2002, 35, 3876.(d) Chang, Y.; Powell, E. S.; Allcock, H. R.; Park, S. M.; Kim, C.Macromolecules2003, 36, 2568.

(5) (a) Zhang, J.; Peppas, N. A.Macromolecules2000, 33, 102. (b) Gan,L. H.; Gan, Y. Y.; Deen, G. R.Macromolecules2000, 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.Macromolecules2001, 34, 8014. (e) Yin,X.; Stover, H. D. H.Macromolecules2002, 35, 10178.

(6) Hoffman, A. S.Artif. Organs1995, 19, 458.(7) (a) Erhan S. InDesk 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.Biomacromolecules2000, 1, 75. (c) He, L. M.; Neu, M. P.; Vanderberg, L. A.EnViron.Sci. Technol.2000, 34, 1694.

(11) Molecular weight of1 was 3× 105 to 12× 105 (data of the supplier).(12) King, E. C.; Blacker, A. J.; Bugg, T. D. H.Biomacromolecules2000,

1, 75.(13) Paul, R.; Anderson, G. W.J. Am. Chem. Soc.1960, 82, 4596.(14) A typical procedure of synthesis of3 was as follows. Under argon,

a mixture of1 (0.28 g, 2.2 mmol unit of monomer) and CDI (0.40g, 2.5 mmol) was stirred in 30 mL of anhydrous DMF at 0°C. After6 h,2 (4.4 mmol) was added to the mixture at 0°C, and the mixturewas further stirred at room temperature for 4 days. Then, the reactionmixture was poured into a large amount of acetone, and theprecipitates were collected by centrifugation. After being dried invacuo, the obtained polymer was dissolved in dilute HCl, and thesolution was subjected to purification by dialysis (cutoff molecularweight ) 2 × 103) three times. The remaining solution waslyophilized to give3.

(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

Figure 5. Effect of pH on transmittance (500 nm) of 1 wt % aqueoussolution of 3 (sample D).

1134 Biomacromolecules, Vol. 4, No. 5, 2003 Communications