Biomacromolecules 2003, 4, 1487-1490
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Communications New Amphiphilic Poly(2-ethyl-2-oxazoline)/ Poly(L-lactide) Triblock Copolymers Chau-Hui Wang and Ging-Ho Hsiue* Department of Chemical Engineering, National Tsing Hua University, Hsinchu, 300 Taiwan, ROC Received June 17, 2003; Revised Manuscript Received August 27, 2003
A new series of cationic, thermo-sensitive, and biodegradable poly(L-lactide)-poly(2-ethyl-2-oxazoline)poly(L-lactide) (PLLA-PEOz-PLLA) triblock copolymers were synthesized by ring-opening polymerization. With increasing molecular weight and crystallinity of hydrophobic PLLA blocks, the critical micellization concentrations (CMC) occurred at lower concentration. The PLLA-PEOz-PLLA aqueous solution was transparent at room temperature. Heating the solution resulted in precipitations, which were caused by the combination of dehydration of water around PEOz and the aggregations of PLLA segments. Acid/base titration profiles indicated that PLLA-PEOz-PLLA were protonated at neutral and acidic conditions. Considerable buffering capacity was found over the entire pH range. The specific PLLA-PEOz-PLLA triblock copolymers with thermal- and pH-sensitive properties can be tailored by varying the compositions and can be applied as controlled release carries for biomedical applications. In the past few decades, triblock copolymers played an important role in the drug delivery systems as delivery matrixes. Hydrogels of triblock copolymers such as poly(oxyethylene-b-oxypropylene-b-oxyethylene) (Pluronic) have been widely investigated in ophthalmic, subcutaneous, and topical administrations.1-4 In addition to Pluronic, Kim and co-workers synthesized thermoplastic biodegradable hydrogels (TBH) composed of hydrophilic poly(ethylene glycol) (PEG) and hydrophobic, biodegradable poly(lactic acid-coglycolic acid).5-8 These gelling polymers exhibited sol-gel transition between ambient and body temperature, suggesting the promising candidates as the injectable drug carriers. For the extended or controlled release of drugs, several approaches have been investigated in designing triblock copolymers, including the use of polyelectrolytes. Poly(2ethyl-2-oxazoline) (PEOz) is a water-soluble polyelectrolyte that has as low toxicity and higher hydrophilicity as that of poly(ethylene glycol).9 Polymer complexes composed of PEOz and poly(methacrylic acid) have been designed for the on-off insulin-released matrixes.10 In a previous publication, we reported the temperature- and pH-sensitive hydrogels consisting of PEOz and poly(D,L-lactide) (PLA).11 The PEOz based hydrogels showed swelling/deswelling behaviors in response to temperature and pH changes. Herein, a new amphiphilic triblock copolymer based on hydrophilic PEOz and hydrophobic poly(L-lactide) (PLLA) was designed for intelligent drug carriers. The resulting PLLA-PEOz-PLLA * To whom correspondence should be addressed. E-mail. ghhsiue@ che.nthu.edu.tw.
copolymer can be expected to behave as a thermoplastic, biocompatible, biodegradable, temperature- and pH-sensitive copolymer. A typical synthesis of a PLLA-PEOz-PLLA triblock copolymer was carried out as follows (Scheme 1). First, R,ωdihydroxypoly(2-ethyl-2-oxazoline) (HO-PEOz-OH) with different molecular weights was synthesized by cationic ringopening polymerization of 2-ethyl-2-oxazoline (EOz, Aldrich) using 1,4-dibromo-2-butene (Aldrich) as initiator. A solution of 1,4-dibromo-2-butene (420 mg) and EOz (9.8 g) in dry acetonitrile (30 mL) was heated to 100 °C and stirred for 20 h under nitrogen atmosphere. After cooling to room temperature, the resulting product was added to 0.1 N of methanolic KOH, and filtered through the silica gel.12 This obtained HO-PEOz-OH was purified by precipitation in diethyl ether and vacuum-dried for 24 h. Then L-lactide (0.582 g, Aldrich) and HO-PEOz-OH (2 g) were polymerized with stannous octoate (30 mg, Sigma) in dry chlorobenzene (20 mL) at 140 °C for 20 h under nitrogen atmosphere. After being allowed to cool, the product was filtered with a syringe filter (0.22 µm) and followed by precipitation in diethyl ether. The yielded copolymers were vacuum-dried for 24 h. 1H NMR (CDCl3): δ 1.07 (broad, N(COCH2CH3)CH2CH2), 2.10-2.40 (overlapping 2 × broad, N(COCH2CH3)CH2CH2), 3.40 (broad, N(COCH2CH3)CH2CH2), 1.53 (d, COCH(CH3)O), 5.11 (q, COCH(CH3)O). The cationic living polymerization of 2-ethyl-2-oxazoline provided a facile way to synthesize the narrow molecular weight distribution of HO-PEOz-OH. The polydispersity index of PEOz-5K and PEOz-10K determined by gel
10.1021/bm034190s CCC: $25.00 © 2003 American Chemical Society Published on Web 09/18/2003
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Communications
Scheme 1. Reaction Scheme for the Synthesis of PLLA-PEOz-PLLA Triblock Copolymers
Table 1. Molecular Weights and Compositions of PEOzs and PLLA-PEOz-PLLA Triblock Copolymers polymer code
LLA/EOza
DPPEOz
0.05 0.33 0.05 0.33 0.78
56 100 56 56 100 100 100
PEOz-5K PEOz-10K ABA-5K05 ABA-5K33 ABA-10K05 ABA-10K33 ABA-10K78
DPPLLA
Mnb
3 19 5 33 78
5600 10000 6000 8400 10700 14800 21300
a Estimated by 1H NMR; LLA/EOz ) DP b PLLA/DPPEOz. Estimated by GPC.
Table 2. Properties of PEOz Homopolymers and PLLA-PEOz-PLLA Triblock Copolymersa
polymer code
Tg (°C)
PEOz-5K PEOz-10K ABA-5K05 ABA-5K33 ABA-10K05 ABA-10K33 ABA-10K78
54.0 54.4 31.3 38.4 40.4 57.4 59.2
a
Tm (°C)
141.7 135.8, 146.5 141.2, 156.8
CMC (mg/mL)
0.86 0.35 0.7 0.2 N.D.
phase transition temp (°C)
p Ka
33 N.D. 38 N.D. N.D.
7.1 7.1 8.1 N.D. 8.1 N.D. N.D.
N.D. ) not determined.
permeation chromatography (GPC) was 1.2 and 1.1, respectively. The triblock copolymer exhibited only a single peak in the gel permeation chromatograms, which demonstrated the absence of homopolymer in the product. The LLA/EOz ratio was calculated by comparing the integral peak area of the methyl groups of PEOz and PLLA.13 Table 1 summarizes the compositions of copolymers estimated by GPC and 1H NMR. The properties of PEOz homopolymers and PLLAPEOz-PLLA triblock copolymers were listed in Table 2. The thermal behavior observed by DSC indicated that PEOz homopolymers had no melting endotherm but displayed a glass transition in the vicinity of 54 °C. The copolymers with a rather short PLLA segment (e.g., ABA-5K05 and ABA10K05) reflected the lower Tg as that of PEOz. This observation revealed that, as small amounts of PLLA were incorporated in the PEOz, the bulk became irregular to a certain degree. In the case of ABA-5K33, ABA-10K33, and ABA-10K78, which have a long chain length of PLLA, they exhibited melting peaks ranging from 141 to 156 °C. When the LLA/EOz ratio of copolymer was higher than 0.33, the
Figure 1. Determination of CMC of PLLA-PEOz-PLLA triblock copolymers by dye solubilization methods.
PLLA block was long enough to crystallize. Tg and Tm were increased as the PLLA chain length was increased. The critical micellization concentrations (CMC) of copolymers were determined employing a dye solubilization method.14 PLLA-PEOz-PLLA (100 mg) was dissolved in 10 mL of THF and added dropwise with 10 mL of Milli-Q water. The THF was then removed with a rotary evaporator at 25 °C. For the measurements of CMC, the concentration of the sample solution was adjusted ranging from 0.01 to 10 mg/mL. A total of 20 µL of 0.4 mM 1,6-diphenyl-1,3,5hexatriene (DPH, Aldrich) methanol solution was added to 2 mL of sample solution and equilibrated in dark for 3 h. The absorption spectra of these solutions were recorded from 300 to 500 nm using a UV/vis spectrometer (Lambda 2S, Perkin-Elmer). Absorbance at 356 nm was plotted against logarithmic concentration, and the crossing point of the extrapolated two straight lines was defined as the CMC. PLLA-PEOz-PLLA is an amphiphilic copolymer because water is a poor solvent for hydrophobic PLLA, whereas it is a good solvent for hydrophilic PEOz. The solubility of PLLA-PEOz-PLLA in water greatly depends on the molar ratio of PLLA and PEOz. Figure 1 shows the correlation between intensities at 356 nm and the logarithmic concentration of copolymers. The CMC value was obtained from the first inflection of the intensity versus log concentration sigmoidal curve.14 DPH is a hydrophobic dye, which has a characteristic absorbance at 356 nm. As copolymers formed micelles, the DPH was preferred to partition into the core of the micelles, thus increasing the absorbance. The CMC values of PLLA-PEOz-PLLA as a function of DPPEOz and DPPLLA are shown in Figure 2. On the left part of Figure 2, a number of LLA units reflected significantly effects on CMC value. Like the copolymer with the same hydrophilic segment, a higher DPPLLA resulted in lower CMC value. It is worth noting that the log CMC values for the copolymers solutions decreased in a roughly linear fashion as a function of DPPLLA (R2 > 0.99), without regarding the number of EOz units. On the other hand, the CMC values of copolymers that have a constant LLA/EOz ratio but varying molecular weight decrease gradually with increasing DPPEOz. It can be concluded that for a given LLA/EOz ratio PLLA-PEOz-
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Figure 4. Acid/base titration profile of PEOz homopolymers and PLLA-PEOz-PLLA triblock copolymers. Figure 2. CMC of PLLA-PEOz-PLLA triblock copolymers as a function of DPPEOz and DPPLLA.
Figure 3. Temperature dependence of optical transmittance changes for the copolymers in aqueous solutions.
PLLA with a higher molecular weight forms micelles more readily. The hydrophilic segment (DPPEOz) exhibited a smaller effect in the micellization process. The optical transmittance of aqueous triblock copolymer solutions was monitored at 500 nm by means of a UV/vis spectrophotometer (Lambda 2S, Perkim Elmer).15 The samples (2 wt %) were placed in Teflon-stopped quartz cuvettes. The transmittance of the copolymer solution at 20 °C was set on 100% and the temperature range employed was 20-45 °C. At least 15 min was allowed for the sample temperature equilibrium. The phase transition temperature is defined as the temperature at 50% of transmittance for each polymer solution. The changes of temperature-dependent transmittance in copolymer aqueous solutions are shown in Figure 3. The aqueous solutions of ABA-5K05 and ABA-10K05 were transparent at lower temperatures, whereas the transmittance of the solution changed drastically from transparent to turbid above characteristic temperatures. The lower critical solution temperature (LCST) of PEOz homopolymer ranged from 87 to 101 °C, depending on the polymeric concentration.16 The precipitation temperature decreased markedly (33 and 38 °C for ABA-5K05 and ABA-10K05, respectively)
after introducing hydrophobic PLLA into PEOz. This temperature-induced phase transition was due to the aggregation of PLLA segments, which might associate with the LCST of PEOz in water.17 The PEOz blocks became hydrophobic after elevating the temperature, leading to a change in the conformation of “flower” like micelles and forming the weak gels.18 In addition, the copolymer composition of PLLAPEOz-PLLA also affected the phase separation behavior. The precipitation was observed at a higher temperature as the molecular weight of PEOz and PLLA were increased. The acid/base titration was made with a standardized 0.01 N HCl solution and back-titrated with 0.02 N KOH. For each experiment, 500 mg of polymer was dissolved in 40 mL of 0.01 N HCl solution. The pH was measured using a SUNTEX sp-2200 pH meter. The acid/base titration profiles together with the first derivates of PEOz and PLLA-PEOzPLLA copolymers are shown in Figure 4. The titration curve trend turns nearly vertical suggesting little buffering capacity of PEOz-10K and PEOz-5K. Unlike PEOz homopolymers, PLLA-PEOz-PLLA triblock copolymers showed considerable buffer capacity over almost the entire pH range. The PLLA-PEOz-PLLA has a pKa around 8.1, which is higher than that of PEOz homopolymers (pKa ) 7.1). This behavior is related to the intermolecular hydrogen bonding between protonated nitrogen and the carbonyl group of PEOz, which reduces hydrogen exchange rate.11 The introduction of a PLLA segment on PEOz created the hydrophobic domain in aqueous solution, thus suppressing the forming of hydrogen bonding considerably. The conventional pH-sensitive polymers work limitedly in acidic pH and alkaline conditions. The results of acid/base titration indicate that PLLA-PEOzPLLA partially protonates and becomes a polycation in the neutral pH range. It is useful in designing carriers for controlled release and gene delivery (the endosomal disruption capability accompanied with the buffering capacity). However, the degree of ionization is known to affect extensively the solution behavior of thermosensitive polymers.19,20 Detailed studies on the relationship between the phase transition temperature and the degree of ionization are under investigation. In conclusion, a series of PLLA-PEOz-PLLA triblock copolymers have been prepared for stimuli-responsive drug
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carriers. The micelles/gels based on PLLA-PEOz-PLLA are expected to bind with anionic drugs, thus extending the release time until the degradation of polymers are occurred. The preliminary studies indicated that PLLA-PEOz-PLLA formed micelles in water at room temperature, while the solution experienced a sol to gel transition as the temperature increased. Hence, it is convenient to incorporate the polymer solution with drugs before administration. PLLA is a widely investigated biodegradable polymer in the field of biomaterials. The addition of a PEOz segment on PLLA provided more extensive applications because of its thermoand pH-sensitivity. The tailored PLLA-PEOz-PLLA triblock copolymer is believed to be a good candidate in the application of drug delivery. Acknowledgment. The author thanks the National Science Council of Republic of China for financial support of this work (NSC 91-2320-B-007-001-Y). References and Notes (1) Schmolka, I. R. J. Biomed. Mater. Res. 1972, 6, 571. (2) Miller, S. C.; Donovan, M. D. Int. J. Pharm. 1982, 12, 147. (3) Morikawa, K.; Okada, F.; Hosokawa, M.; Kobayashi, H. Cancer Res. 1987, 47, 37. (4) Miyazaki, S.; Takeuchi, S.; Yokouchi, C.; Takada, M. Chem. Pharm. Bull. 1984, 32, 4205.
Communications (5) Jeong, B.; Bae, Y. H.; Lee, D. S.; Kim, S. W. Nature 1997, 388, 860. (6) Jeong, B.; Choi, Y. K.; Bae, Y. H., Zentner, G.; Kim, S. W. J. Controlled Release 1999, 62, 109. (7) Jeong, B.; Bae, Y. H.; Kim, S. W. Macromolecules 1999, 32, 7064. (8) Jeong, B.; Bae, Y. H.; Kim, S. W. J. Controlled Release 2000, 63, 155. (9) Zalipsky, S.; Hansen, C. B.; Oaks, J. M.; Allen, T. M. J. Pharm. Sci. 1996, 85, 133. (10) Kwon, I. C.; Bae, Y. H.; Kim, S. W. Nature 1991, 354, 291. (11) Wang, C. H.; Hsiue, G. H. J. Polym. Sci. Part A: Polym. Chem. 2002, 40, 1112. (12) Lee, S. C.; Chang, Y.; Yoon, J. S.; Kim, C.; Kwon, I. C.; Kim, Y. H.; Jeong, S. Y. Macromolecules 1999, 32, 1847. (13) Jule, E.; Nagasaki, Y.; Kataoka, K. Bioconjugate Chem. 2003, 14, 177. (14) Alexandridis, P.; Holzwarth, J. F.; Hatton, T. A. Macromolecules 1994, 27, 2414. (15) Shibanuma, T.; Aoki, T., Sanui, K.; Ogata, N.; Kikuchi, A.; Sakurai, Y.; Okano, T. Macromolecules 2000, 33, 444. (16) Kim, C.; Lee, S. C.; Kang, S. W.; Kwon, I. C.; Jeong, S. Y. J. Polym. Sci. Part B: Polym. Phys. 2000, 38, 2400. (17) Lin, P.; Clash, C.; Pearce, E. M.; Kwei, T. K. J. Polym. Sci. Part B: Polym. Phys. 1988, 26, 603. (18) Ma, Y.; Tang, Y.; Billingham, N. C.; Armes, S. P. Biomacromolecules 2003, 4, 864. (19) Feil, H.; Bae, Y. H.; Feijen, J.; Kim, S. W. Macromolecules 1993, 26, 2496. (20) Chen, G. H.; Hoffman, A. S. Macromol. Chem. Phys. 1995, 196, 1251.
BM034190S
