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Novel Thermo-Responsive Formation of a Hydrogel byStereo-Complexation between PLLA-PEG-PLLA andPDLA-PEG-PDLA Block Copolymers

Tomoko Fujiwara, Takashi Mukose, Tetsuji Yamaoka, Hideki Yamane, Shinichi Sakurai, Yoshiharu Kimura*

Department of Polymer Science and Engineering, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku,Kyoto 606-8585, JapanE-mail: ykimura@ipc.kit.ac.jp

IntroductionPolymer gels are divided by their crosslinking mechanisminto two classes – chemical and physical gels. The physi-cal gels are those crosslinked by physical bonds such ashydrogen, ionic, and coordinate bonds and hydrophobicinteraction. In an extreme case, helix formation inducesthe formation of physical gels, as observed for gelatinand agarose in a cooled aqueous medium,[1] whereashydration of poly(oxyethylene) that has been grafted ontoa substrate forms a gel.[2] Poly(N-isopropylacrylamide)forms a thermo-responsive hydrogel that undergoes areversible sol–gel transition due to the changes in hydro-phobic and hydrogen bonding states with the environ-mental temperature.[3, 4]

Recently, these polymer gels have been applied to thebiomedical field with remarkable advances in medicalscience and biotechnology.[5] The applications involve

cell culture, tissue engineering, drug delivery systems(DDS), medical sensor, and so on, for which the biocom-patibility, biodegradability and safety of the gels areextremely important as well as the physicochemical prop-erties. Because of this feature, biodegradable block copo-lymers of poly(l-lactide) (PLLA) and poly(ethylene gly-col) (PEG) have been synthesized for designing func-tional biomedical materials in the forms of hydrogel anddispersed particles. S. W. Kim et al. succeeded in prepar-ing injectable microparticles for DDS from a PEG-PLLA-PEG triblock copolymer.[6, 7] The aqueous disper-sion of the microparticles is characterized by the sol–geltransition occurring at around body temperature. On theother hand, we discovered an interesting band morphol-ogy formed from the nanoparticles of PLLA-PEG diblockand PLLA-PEG-PLLA triblock copolymers that wereplaced on a flat substrate surface.[8–10] We verified that

Communication: Gel formation was discovered in anaqueous mixture of enantiomeric triblock copolymers,PLLA-PEG-PLLA and PDLA-PEG-PDLA. This systemis characteristic in that an interesting sol–gel transitionwas induced by the stereo-complexation of the PLLA andPDLA segments of the block copolymers around 378C.The process of gel formation was clearly monitored by therheological change, and the responsibility of the stereo-complex formation for the gelation was confirmed bywide-angle X-ray scattering. The mechanism of this gelformation is discussed in relation to its potential applica-tions.

Macromol. Biosci. 2001, 1, No. 5 i WILEY-VCH Verlag GmbH, D-69451 Weinheim 2001 1616-5187/2001/0507–0204$17.50+.50/0

Figure 1. Photographs of an aqueous dispersion of PLLA-PEG-PLLA (a, b, c) and a mixed dispersion of PLLA-PEG-PLLA and PDLA-PEG-PDLA (d, e, f) at different tempera-tures. (a, d): at room temperature, (b, e): after heating at37 8C for 1 h, (c, f): after heating at 758C for 1 h.

Macromol. Biosci. 2001, 1, 204–208

Novel Thermo-Responsive Formation of a Hydrogel ... 205

their band morphology is directed by crystallization ofthe PLLA segments in which the PLLA chain takes adoubly twisted structure with the ordinary 10/3 helicalconformation preserved. The two-dimensional network ofthe PLLA-PEG-PLLA bands formed on the surface simu-lates well the structure of the tridimensional network sys-tem in a melt, a concentrated solution, or a hydrogel. It isalso known that the enantiomeric PLLA and poly(d-lac-tide) (PDLA) form a stereo-complex the melting tempera-ture of which (2308C) is approximately 508C higher thanthat of the respective homopolymers.[11–15] Hennink et al.recently applied this stereo-complexation to the formationof a self-assembled hydrogel from enantiomeric polylac-tide oligomers grafted to dextran.[16] Since this stereo-complexation is very strong, it may affect the aforemen-tioned network formation of the PLLA-PEG and PLLA-PEG-PLLA block copolymers, if their enantiomeric blockcopolymers are mixed and self-assembled.

Here, we study the stereocomplexation in an aqueousdispersion of the enantiomeric triblock copolymers,PLLA-PEG-PLLA and PDLA-PEG-PDLA, to discoveran interesting spontaneous gel formation. This system ischaracterized by an interesting temperature-dependentsol–gel transition that is induced around at 378C – thestereocomplexation of the PLLA and PDLA segments.The gel formation was successfully monitored by therheological change of an aqueous dispersion, and thestereocomplex formation was confirmed by wide-angleX-ray scattering (WAXS). Here, a novel mechanism ofgel formation and potential applications of this hydrogelare first discussed.

Experimental PartThe triblock copolymers PLLA-PEG-PLLA and PDLA-PEG-PDLA were prepared by the ring-opening copolymeri-zation of l-lactide (Purac Biochem, Netherlands) and d-lac-tide (Shimadzu Co. Ltd. Kyoto, Japan), respectively, in thepresence of PEG by the catalysis of stannous 4-ethylhexano-ate (10 mol-% relative to PEG).[8–10] The number averagemolecular weight (M

—n) of PEG (supplied by Aldrich) was

4600 Da, and its polydispersity in weight-(M—

w) /number-(M—

n)average molecular weight ratio was 1.06. Both block copoly-mers were obtained in high yield. The M

—n values and block

ratios of the block copolymers were determined by the1H NMR spectra (in CDCl3): d 1.56–1.6 (d, CH3 for the lac-tate unit), 3.6–3.7 (m, CH2 for the oxyethylene unit), 4.3–4.4 (m, COOCH2 for the oxymethylene connecting with thelactate sequence), and 5.1–5.2 (q, CH for the lactate unit).The polydispersity in M

—w/M

—n was measured by gel permea-

tion chromatography relative to the polystyrene standardswith CHCl3 as the eluent. The block copolymers thusobtained were characterized as follows:

PLLA-PEG-PLLA (95% yield): M—

n = 7200 Da; M—

w/M—

n =1.10; PLLA/PEG = 36/64 (wt/wt) in block ratio.

PDLA-PEG-PDLA (86% yield): M—

n = 6800 Da; M—

w/M—

n =1.12; PDLA/PEG = 32/68 (wt/wt) in block ratio.

One gram of the above block copolymer was dissolved in5 mL of tetrahydrofuran (THF) and added to 10 mL of waterat 08C and an ultrasonic wave applied. From the resultantdispersion, THF was thoroughly evaporated under reducedpressure at 108C. The polymer concentration in the aqueousdispersion finally obtained was adjusted to 10 wt.-%.

An equivalent volume of the aqueous dispersions ofPLLA-PEG-PLLA and PDLA-PEG-PDLA were mixed andheated to 378C or 758C to induce a spontaneous gelation.

International Advisory Board Member*

Prof. Dr. Yoshiharu Kimura was born in 1948 and graduated in 1971 from KyotoUniversity, with a B.S. degree in Chemistry. His research interest in polymerscience was extended through his study with Professors Takeo Saegusa and ShiroKobayashi in the Graduate School of Kyoto University. He finished his Ph.D. pro-gram in 1976. After doing postdoctoral work at the University of Iowa, USA, heserved as an Assistant Researcher at Shiga Prefectural Junior College, Hikone,Japan. In 1981, he was appointed Assistant Professor of Fiber Chemistry at KyotoInstitute of Technology. He became an Associate Professor of Polymer Science andEngineering in 1985, and Professor in 1990. He now holds the additional post of theDirector of the Cooperative Research Center of the same University. In 1998, Dr.Kimura received the Award of the Society of Fiber Science and Technology, Japanfor his research activity. He has published more than 200 papers and has been theauthor or co-author of several books. His current research interests include thedevelopment and biomedical application of biodegradable polymers and inorganicpolymers and also the control of polymer–cell interaction. He has been and is stillan active member of the editorial members of four journals and is now a member ofthe International Advisory Board of Macromolecular Bioscience.

* Members of the International Advisory Board will be introduced to the readers withtheir first manuscripts.

206 T. Fujiwara, T. Mukose, T. Yamaoka, H. Yamane, S. Sakurai, Y. Kimura

An aqueous dispersion of PLLA-PEG-PLLA was heatedlikewise as a control experiment. WAXS of the dispersionsand the gels obtained was measured by the 2d-WAXS tech-nique with a synchrotron X-ray at the BL-15A beamline (PF,Tsukuba, Japan) equipped with a 2d-CCD camera. The expo-sure time was typically ca. 40 s, and the scattering intensitywas measured in the Bragg angle 2h from 3 to 608. Amor-phous polyethylene (PET) film was used as the sampleholder.

The mixed dispersion was quickly transferred in a slit (slitsize: 1361060.25 mm3) installed on a rheometer (Rheolo-gel-4000, UBM Co. Ltd., Japan), which was operated by theslit-shear measuring mode at a frequency of 128 Hz. Thetemperature was raised from 12 to 808C at a heating rate of28C N min–1.

Results and DiscussionWe reported that PLLA-PEG-PLLA with a similar blocklength of PEG (4600 Da) and PLLA (4300 Da per block)can form nanoparticles in its diluted aqueous dispersionand that the particle size increases with increasing poly-mer concentration.[10] The block copolymers used in thisstudy consisted of significantly shorter PLLA blocks(1300 Da in M

—n) compared with the PEG block (4600 Da

in M—

n) in order to solubilize them in high concentration(10 wt.-%). The resultant dispersions of the both blockcopolymers, being transparent in appearance, were mixedand heated to examine the possible gelation.

Figure 1 shows the typical changes in appearance of adispersion of PLLA-PEG-PLLA (10 wt.-%) and a mixeddispersion of PLLA-PEG-PLLA and PDLA-PEG-PDLA(total 10 wt.-%) before and after heating to 37 and 758C.The single dispersion of PLLA-PEG-PLLA remainedfluid after heating to 378C (1b) and even 758C (1c) inspite of a slight turbidity at 758C. On the other hand, themixed dispersion became a transparent gel at 378C (1e)and a white gel at 758C (1f). These results reveal thetemperature-dependent sol–gel transformation of themixed dispersion at 378C.

Figure 2 shows the temperature-dependent rheologicalchange with the gelation of the mixed dispersion. A dra-matic increase in storage modulus (G9) is shown from 20to 378C followed by a large decrease in tan d. The cross-ing of the G9 and loss modulus (G99) curves is detectedaround 238C. This change corresponds to the crosslinkingreaction that leads to gel formation. Above 378C, G9 sig-nificantly fluctuates around 103 Pa, which is an ordinaryG9 level for physically crosslinked gels.[17] It starts tojump up again above 708C, corresponding to the turbidityof the gel. Since this turbid gel regained its transparencywhen cooled, this turbidity change is attributed to theclouding phenomenon resulting from the desolubilizationof nonionic surfactants (i. e., PEG). These data supportthe gel formation of the mixed dispersion at around378C. The hydrogel obtained here is quite different from

the one obtained from the block copolymer PEG-PLLA-PEG[6, 7] in terms of the microstructure and gelationmechanism; this is in spite of their similarities in terms ofthe block components and properties.

We confirmed the responsibility of the stereo-complexformation of the enantiomeric polylactide segments onthe gellation by WAXS. Figure 3 shows the WAXS pro-files for the samples shown in Figure 1. In the originalsingle dispersion (3a), a small diffraction peak is shown.This peak, being superimposed with a small diffractionfrom the PET supporting film at a 2h = 16.08 (PET filmin Figure 3), is attributed to a diffraction of the PLLA

Figure 1. Photographs of an aqueous dispersion of PLLA-PEG-PLLA (a, b, c) and a mixed dispersion of PLLA-PEG-PLLA and PDLA-PEG-PDLA (d, e, f) at different temperatures.(a, d): at room temperature, (b, e): after heating at 37 8C for 1 h,(c, f): after heating at 75 8C for 1 h.

Figure 2. Changes in G9 (9), G99 (F), and tan d (h) as a functionof the temperature for a mixed dispersion of PLLA-PEG-PLLAand PDLA-PEG-PDLA.

Novel Thermo-Responsive Formation of a Hydrogel ... 207

crystals. Therefore, a slight crystallization of PLLA canbe identified at room temperature. At 378C (3b), the dif-fractions at 2h = 16.78 and 19.38 are shown. These dif-fractions are ascribed to the (200) or (110) plane and the(203) or (113) plane of the hexagonal PLLA 10/3-crystallattice and are intensified at 758C (3c). These results sug-gest that the PLLA segments in the dispersion shouldcrystallize on heating. On the other hand, the mixed dis-persion shows two different reflections at 2h = 12.08 and21.68 in addition to the small diffractions of the PLLAhexagonal crystals. The formers are reasonably ascribedto the crystals of the stereocomplex of PLLA and PDLA.At 378C, the diffractions of the stereocomplex are veryweak (3e), and accordingly, both the PLLA and PDLAchains may be mixed into a complexation state prior tocrystallization. At 758C, the crystal growth of the stereo-complex is clearly shown (3f). The crystallinities, asjudged from the WAXS patterns, are almost identical forthe single and mixed dispersions at each temperature. It istherefore suggested that the gel formation in the mixeddispersion is closely related with the stereo-complexationof the enantiomeric polylactide segments.

In the dispersions of the block copolymers, the hydro-phobic PLLA and PDLA segments aggregate to form a

hydrophobic core region that is surrounded by the hydro-philic PEG segments. Therefore, the PLLA and PDLAsegments are isolated from each other when the sepa-rately prepared dispersions of PLLA-PEG-PLLA andPDLA-PEG-PDLA are mixed. When heated, the aggrega-tion of the PLLA and PDLA segments in the core regionis weakened to allow the mixing of segments outside thecore. The short block segments of PLLA and PDLA arefavorable for this chain scrambling and mixing. On thischain mixing, the stereo-complexation starts, and the sus-pended hydrophobic cores are wholly crosslinked witheach other at 378C. With increasing temperature, thecrosslinking state is changed by the reorganization of thehydrophobic cores and the increased crystallization of thestereo-complex. This process may be a cause of the fluc-tuation of the G9 curve in Figure2. Because the stereo-complexation is slow, the gelation of the mixed disper-sion is not fast, but finishes around body temperature(378C). This effect is convenient for the application ofthis gel-forming dispersion to biomedical devices.

In concusion, a novel thermoresponsive hydrogel for-mation was discovered in an aqueous mixture of enantio-meric triblock copolymers, PLLA-PEG-PLLA andPDLA-PEG-PDLA. The sol–gel transition was inducedby the stereo-complexation of their PLLA and PDLA seg-ments.

Acknowledgement: We acknowledge the support receivedthrough a Grant-in-aid for Scientific Research on Priority Areas,“Sustainable Biodegradable Plastics”, No. 11217210 from theMinistry of Education, Culture, Sports, Science, and Technol-ogy, Japan. The WAXS experiments were performed in thePhoton Factory with an approved number of 99G241.

Received: February 20, 2001Revised: April 27, 2001

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Figure 3. WAXS profiles of an aqueous dispersion of PLLA-PEG-PLLA (a, b, c) and a mixed dispersion of PLLA-PEG-PLLA and PDLA-PEG-PDLA (d, e, f) at different temperatures.(a, d): at room temperature, (b, e): after heating at 37 8C for 1 h,(c, f): after heating at 75 8C for 1 h.0101H : hexagonal crystals, 0101

S : crystals of the stereo-complex.

208 T. Fujiwara, T. Mukose, T. Yamaoka, H. Yamane, S. Sakurai, Y. Kimura

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