Formation of and Coalescence from the Inclusion Complex of a

Characterization of 75:25 Poly(l-lactide-co-ε-caprolactone) Thin Films for the Endoluminal Delivery of Adipose-Derived Stem Cells to Abdominal Aortic...
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Biomacromolecules 2002, 3, 201-207

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Formation of and Coalescence from the Inclusion Complex of a Biodegradable Block Copolymer and r-Cyclodextrin. 2:† A Novel Way To Regulate the Biodegradation Behavior of Biodegradable Block Copolymers Xintao Shuai, Min Wei, Francis E. Porbeni, Todd A. Bullions, and Alan E. Tonelli* Fiber and Polymer Science Program, College of Textiles, North Carolina State University, Raleigh, North Carolina 27695-8301 Received September 18, 2001; Revised Manuscript Received November 2, 2001

A biodegradable block copolymer (PCL-b-PLLA, Mn ) 1.72 × 104, Mw/Mn ) 1.37) of poly(-caprolactone) (PCL) and poly(L-lactide) (PLLA) with very low crystallinity was obtained by forming the inclusion complex between R-cyclodextrin molecules and PCL-b-PLLA followed by coalescence of the guest polymer chains. Films of the as-synthesized and coalesced copolymer samples, PCL and PLLA homopolymers of approximately the same chain lengths as the corresponding blocks of PCL-b-PLLA, and a physical blend of PCL/PLLA homopolymers with the same molar composition as PCL-b-PLLA were prepared by meltcompression molding between Teflon plates. Subsequently, the in Vitro biodegradation behavior of these films was studied in phosphate buffer solution containing lipase from Rhizopus arrhizus, by means of ultraviolet spectra, attenuated total reflectance FTIR spectra, differential scanning calorimetry, wide-angle X-ray diffraction measurements, and weight loss analysis. PCL segments were found to degrade much faster than PLLA segments, both in the pure state and in copolymer or blend samples. Consistent with our expectation, suppression of the phase separation, as well as a decrease of crystallinity, in the coalesced copolymer sample led to a much faster enzymatic degradation than that of either as-synthesized copolymer or the PCL/PLLA physical blend sample, especially during the early stages of biodegradation. Thus the biodegradation behavior of biodegradable block copolymers, which is of decisive importance in drug delivery and controlled release systems, may be regulated by the novel and convenient means recently reported by us.1 Introduction Poly(-caprolactone) (PCL) and poly(L-lactide) (PLLA) are two well-known representatives of hydrophobic, aliphatic polyesters that exhibit inherent biodegradability and biocompatibility. They have been intensively investigated2-5 due to their potential pharmaceutical, biomedical, and environmental applications. These two polyesters have different properties, such as morphology, mechanical strength, biodegradation rate, etc. PLLA has a high tensile strength and high melting point (ca. 160 °C), but low elongation at break due to the brittleness that results from its high crystallinity and a glass transition temperature above room temperature. On the contrary, PCL has high flexibility, but its tensile strength and melting point (ca. 60 °C) are low.6 Therefore, these two polyesters are found to fall short of the required properties for many applications when they are used individually. To gain biomaterials with optimized properties from PLLA and PCL, two different approaches, blending and block copolymerization, are usually adopted. However, PCL and PLLA have been found to be incompatible. Phase †

For part 1, see ref 1. * To whom correspondence should be addressed. TEL: +1-919-5156588. FAX: +1-919-515-6532. E-mail: [email protected].

separation exists in both blend7 and block copolymer8,9 systems, with high crystallinity for both segregated components. On the basis of the fact, discovered by Harada and many other researchers, that cyclodextrins (CDs) may form crystalline inclusion complexes (ICs) with various kinds of linear polymeric guests of either hydrophilic or hydrophobic natures,10-13 we reported very recently attempts to suppress the phase separation in immiscible polymer blends or block copolymer systems by first forming their inclusion complexes with CDs as the host, and then coalescing the guest polymers from their CD IC crystals by washing the ICs with hot water or with cold water in the presence of cyclodextrin-degrading enzymes. In this manner, we obtained two intimately mixed blends from poly(methyl methacrylate)/polycarbonate14 and PCL/PLLA15 pairs. Furthermore, we obtained a coalesced sample of PCL-b-PLLA with very low crystallinity.1 Admittedly, the morphology of biodegradable polymers greatly affects their material performance,16,17 especially their biodegradability and permeability, which are of decisive importance in the controlled release of drugs. Therefore, it is anticipated that the decrease of crystallinity in the PCLb-PLLA copolymer, which may be achieved through the novel method we have reported in detail,1 may also lead to

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remarkable changes in the biodegradation behavior. The objective of this work is to study the effect of this decreased crystallinity on the biodegradation of PCL-b-PLLA. In addition, biodegradation of PCL, PLLA, and PCL/PLLA physical blends were also carried out in order to fully understand the relationship between the biodegradation behavior and the phase structure, as well as the composition of polyesters consisting of PCL and PLLA, under specific enzymatic biodegradation conditions.

Shuai et al. Table 1. Molecular Characteristics of PCL, PLLA, and PCL-b-PLLA Diblock Copolymer copolymer PCL-b-PLLA PLLA PCL

DPPCLa DPPLLAa (nc) (mc) Mna × 10-3 Mnb × 10-3 Mw/Mnb 94 89

82 97

16.8 7.0 10.1

17.2

1.37

a Calculated from the integration of characteristic signals in 1H NMR spectra. b Determined by SEC/GPC measurements. c See ref 1.

1

Experimental Section Materials. -Caprolactone and L-lactide (both from Aldrich) were purified as described in the literature.8 Toluene (from Fisher) was dried over CaH2 and distilled under dry argon. Stannous(II) octoate (SnOct, from Aldrich), R-CD (from Cerestar Co.), and methanol (from Fisher) were used as received. 1-Dodecanol (from Aldrich) was dried under high vacuum for 24 h at 60 °C, and then it was further dried by azeotropic distillation in dry toluene. Syntheses of Homopolymers and Diblock Copolymer. Two homopolymers, PCL and PLLA, were synthesized at 115 °C by ring-opening polymerization of -caprolactone and L-lactide in toluene with 1-dodecanol and SnOct (ca. 0.1% of monomer in molar amount) as an initiator and a catalyst, respectively. The product was purified by precipitation in cold methanol and was finally dried for 48 h at 40 °C in a vacuum oven. Diblock copolymer PCL-b-PLLA, whose individual blocks have about the same length as the corresponding PCL and PLLA homopolymers synthesized above, was synthesized and purified as we reported recently.1 Preparation of Samples. The IC of R-CD and PCL-bPLLA and the coalesced copolymer sample were prepared by washing the IC with hot water, as we reported.1,15 Films for the biodegradation tests were prepared by melt compression molding between Teflon plates, at 80 °C for PCL and 180 °C for other samples. Enzymatic Degradation. Film samples with initial weights of ca. 20 mg and dimensions of ca. 10 × 10 mm were incubated at 37 °C in small bottles containing 2 mL of phosphate buffer (pH ) 7) and lipase from R. arrhizus (5000 units). After selected time intervals, films were removed from the enzymatic solution and then washed with distilled water and dried to constant weight in a vacuum oven at room temperature. The average weight loss of three independent specimens was calculated and taken as the weight loss value for each sample. Control tests were carried out at 37 °C in phosphate buffer (pH ) 7) in the absence of enzyme. The initial-stage biodegradation behavior of film samples (ca. 8 mg), in 2 mL of phosphate buffer containing 2500 units of lipase from R. arrhizus, was monitored continuously at 37 °C by recording the UV absorbance at 205 nm. Sample Characterization. Size exclusion/gel permeation chromatography (SEC/GPC) analysis was carried out using a Waters Styragel HR4 104 Å WAT044225 column with THF as eluent and PMMA standards (Waters and American Polymer Standards) for column calibration. The eluent was analyzed with a differential refractometer, model R401 (Waters), together with a model 730 data module (Waters).

H NMR spectra were recorded on a Bruker 300 MHz DPX spectrometer in CDCl3 at room temperature. Differential scanning calorimetry (DSC) measurements were performed at heating rates of 10 °C/min on a PerkinElmer differential scanning calorimeter (DSC-7) calibrated with indium. Film X-ray diffraction analyses were conducted with a Siemens type-F X-ray diffractometer (30 kV, 20mA) using Ni-filtered Cu KR radiation. The samples were fixed on aluminum frames and scanned from 5° to 40° (2θ) at a speed of 2θ ) 1.2°/min. The attenuated total reflectance (ATR) FTIR spectral studies were carried out on a Nicolet 510P FTIR spectrometer in the range between 4000 and 750 cm-1, with a resolution of 2 cm-1. The time-dependent biodegradation behavior of various samples was investigated with a Cary 3E UV-visible spectrophotometer. Results and Discussion Physical Properties of Samples. The molecular characteristics of PCL and PLLA homopolymers and diblock copolymer, detected by SEC/GPC and 1H NMR measurements, are listed in Table 1. The lengths of individual blocks of the copolymer are approximately the same as those of the corresponding homopolymers. When we coalesced the copolymer chains from the IC crystals of PCL-b-PLLA and R-cyclodextrin (R-CD), a drastic decrease of crystallinity was observed for the coalesced sample. The phase transition of this copolymer during the process of IC formation and polymer chain coalescence has already been discussed.1 We concluded that, during IC formation, copolymer chains of both identical and opposite orientations were isolated and distributed randomly into the IC channels constructed by R-CD molecules. Therefore, the chance for both PCL and PLLA blocks to self-aggregate during the coalescence process was largely reduced compared to the solidification of the block copolymer from solution. It is well-known that composition and morphology play crucial roles in regulating the biodegradation behavior of biodegradable polymers, and for semicrystalline polyesters, enzymatic or nonenzymatic hydrolysis occurs first in the amorphous regions rather than the crystalline regions.17,18 As anticipated from the reduced crystallinity, we expect that the biodegradability of the coalesced copolymer could be changed in comparison with that of the as-synthesized copolymer. For a better understanding of the relationship between the phase structures and the biodegradability, the

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Regulating Biodegradable Block Copolymers

Table 2. Thermal Properties of Films of Homopolymers and As-Synthesized and Coalesced Diblock Copolymer identity

Tm-PCL (°C)

∆HPCL (J/gsample)

Xc-PCLa (%)

PCL PLLA PCL/PLLA blend as-synthesized copolymer coalesced copolymer

63.9

95.6

68.8

62.1 59.7 63.4

57.3 42.2 22.5

64.4 47.4 25.3

a

Tm-PLLA (°C)

∆HPLLA (J/gsample)

Xc-PLLAa (%)

160.5 162.4 160.2 164.1

59.1 23.4 22.5 5.9

63.5 70 67.2 17.6

Xc values of blend, as-synthesized, and coalesced samples are calculated based on the copolymer composition (64% PCL fraction, in wt %).1

biodegradation of two homopolymers, PCL and PLLA, and a PCL/PLLA physical blend were also studied. The phase characters of these samples were investigated, by DSC and FTIR measurements. As we have reported before,1 the PCL carbonyl absorption band in all PCLcontaining samples can be well resolved into a crystalline absorption at 1726 cm-1 and a noncrystalline absorption at 1736 cm-1. This feature enables us to compare the phase structures of these samples by observing the relative intensities of crystalline versus noncrystalline absorptions. Upon copolymerization with PLLA, the crystallinity of the PCL blocks became lower than that of PCL in a physical blend having the same molar ratio of PCL and PLLA. However, a high crystallinity of the PCL component still existed in both as-synthesized copolymer and the physical blend.19 The PLLA carbonyl bands in all PLLA-containing samples are not well resolved into amorphous and crystalline absorptions, and therefore the phase structure of PLLA or PLLA blocks is not indicated by the FTIR spectra. Quantitative results consistent with FTIR results were obtained by the DSC measurements, as presented in Table 2. Initial-Stage Biodegradation Monitored by UV Measurements. The enzymatic hydrolysis of polyesters yields water-soluble hydroxy acids, which allows the biodegradation process of polyesters to be monitored continuously by measuring the concentration of released hydroxy acids via their UV absorption at 205 nm. The biodegradation behavior of all samples were thus investigated by continuously monitoring the UV absorbance at 205 nm over 18 h of biodegradation. No appreciable increase of absorption accompanying degradation could be detected for all control samples, indicating that no or negligible sample hydrolysis occurred in the absence of lipase. In the presence of enzyme, as shown in Figure 1, an induction period of 1-5 h was observed, during which no obvious increase of UV absorbance was detected for all samples. This period should correspond to a process of sample wetting, as well as simultaneous enzyme attachment, which may play a key role in the enzymatic hydrolysis of some polyesters.20 A linear increase of absorption with biodegradation time, following the period of enzyme attachment, was observed for all samples, as shown in Figure 1. Obviously PLLA hydrolyzed much more slowly than PCL, when the biodegradation was catalyzed by lipase from R. arrhizus. The biodegradation rate of as-synthesized copolymer does not show significant differences from that of the physical blend, and both samples degraded more slowly than PCL, but much faster than PLLA. The coalesced sample, on the contrary, behaved in a different way, i.e., it was hydrolyzed even faster than PCL. These results demonstrate that the phase structure plays a very important role in the initial stages of biodeg-

Figure 1. UV absorbance monitored continuously at 205 nm during the initial stages of enzymatic degradation (37 °C, pH ) 7.0) of coalesced PCL-b-PLLA (a), PCL (b), as-synthesized PCL-b- PLLA (c), PCL/PLLA blend (d), and PLLA (e).

Figure 2. UV absorbance detected at 205 nm during the 3-week enzymatic degradation (37 °C, pH ) 7.0) of PCL (a), coalesced PCLb-PLLA (b), as-synthesized PCL-b-PLLA (c), PCL/PLLA blend (d), and PLLA (e), in comparison with a PCL control hydrolyzed in the absence of enzyme (f).

radation. These results are also consistent with literature reports17,18 describing polyesters that degrade first from the amorphous regions. Formation of IC between the block copolymer and R-CD, followed by coalescing the copolymer chains together, resulted in a significantly decreased crystallinity of the copolymer. Therefore, a larger fraction of copolymer amorphous regions was involved in the initial-stage biodegradation process in the coalesced sample. Because PLLA inherently degrades much more slowly than PCL under the established biodegradation conditions, it is easy to understand why assynthesized copolymer and the PCL/PLLA physical blend degraded more slowly than PCL, even though both samples are characterized by strong phase separation and high crystallinity.

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Figure 3. Weight loss of PCL (a), coalesced PCL-b-PLLA (b), as synthesized PCL-b-PLLA (c), PCL/PLLA blend (d), and PLLA (e), during their 3-week enzymatic degradation (37 °C, pH ) 7.0).

Enzymatic Degradation during 3 Weeks. The long-term biodegradation behavior observed during 21 days was assessed by UV spectroscopy and weight loss measurements, as presented in Figures 2 and 3, respectively. Consistent with the initial-stage biodegradation behavior revealed by the continuous UV measurements, PCL degraded much faster than PLLA during the studied time period of biodegradation. Therefore, it is believed that lipase from R. arrhizus shows higher activity in catalyzing the hydrolysis of PCL at 37 °C. Although the as-synthesized PCL-b-PLLA film appears to degrade somewhat faster than the PCL/PLLA physical blend film, both degraded more slowly than PCL film. On the contrary, the coalesced copolymer sample behaved differently. In approximately the first 2 weeks, it degraded even faster than the PCL film. After that period, it degraded slower than the PCL film but still faster than either as-synthesized copolymer or physical blend films. In addition, the control samples did not show evidence of significant nonenzymatic degradation during the 3-week biodegradation period. These results demonstrate again that the biodegradation behavior of PCL-b-PLLA is closely related to its phase character. Although it contains the PLLA component, which is not very sensitive to enzymatic hydrolysis, the coalesced

Shuai et al.

sample is apparently very susceptible to enzymatic hydrolysis due to its very low crystallinity, at least in the early stages of biodegradation. In contrast, as-synthesized copolymer and the physical blend could not be expected to undergo enzymatic degradation faster than PCL film because both have very high crystallinity and they also contain the more slowly degrading PLLA. The reason why degradation of the coalesced sample slowed after about 2 weeks is discussed in a subsequent section describing the changes in phase structure during the degradation process. ATR FTIR measurements were employed to investigate the biodegradation behavior of both the surface and interior of the films. The ATR FTIR spectra recorded for assynthesized copolymer films after different degradation periods are shown in Figure 4. The copolymer is characterized by strong and distinct peaks at ∼1726 cm-1 (s, νCdO of PCL) and ∼1759 cm-1 (s, νCdO of PLLA). The relative intensity of these two peaks varies with biodegradation time. The νCdO of the PCL block shows a higher intensity than that of the PLLA block in the spectrum of the copolymer before biodegradation, while its intensity gradually becomes lower than that of the PLLA block. The same phenomenon has also been observed during the course of biodegradation of the coalesced copolymer film (Figure 5), as well as for the physical blend film. These results demonstrate again that PCL degrades much faster than PLLA. The ATR FTIR spectrum of the cross section was found to be very similar to that of the surface after the film enzymatically degraded for 1 week. Therefore, not only the surface but eventually the bulk interior of sample films also underwent enzymatic hydrolysis. Although the enzyme is apparently too large to penetrate the initial films, it may begin to enter the bulk phase during the course of the biodegradation process, since amorphous regions degrade very fast and will likely result in a porous structure. In addition, the enzyme may enter the bulk phase at the very beginning of biodegradation if the prepared film is not very “tight” (e.g., gas bubbles may have resulted as defects during molding).

Figure 4. ATR FTIR spectra of as-synthesized PCL-b-PLLA film after degrading for 0 days (a), 7 days (b), 14 days (c), and 21 days (d), in phosphate solution (37 °C, pH ) 7.0) in the presence of lipase from R. arrhizus.

Regulating Biodegradable Block Copolymers

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Figure 5. ATR FTIR spectra of coalesced PCL-b-PLLA film after degrading for 0 day (a), 7 days (b), 14 days (c), and 21 days (d), in phosphate solution (37 °C, pH ) 7.0) in the presence of lipase from Rhizopus arrhizus.

Figure 6. ATR FTIR spectra in the carbonyl region (s, vCdO) for coalesced PCL-b-PLLA film after degrading for 0 day (a), 3 days (b), 7 days (c), and 14 days (d), in phosphate solution (37 °C, pH ) 7.0) in the presence of lipase from R. arrhizus.

Nevertheless, bulk hydrolysis was also revealed in the nonenzymatic degradation of some polyesters.18,21,22 ATR FTIR (Figure 6) shows evidence that, during the early stages of degradation, the amorphous regions of PCL blocks in the coalesced sample film are much more susceptible to the enzymatic hydrolysis than the crystalline regions. It is clear that the absorption intensity of the amorphous PCL phase at 1736 cm-1 (νCdO) has been drastically reduced upon biodegradation. To gain deeper sight into the relationship between biodegradation and phase structure, the samples were analyzed by wide-angle X-ray diffraction (WAXD) and DSC measurements. As shown in Figure 7, very high crystallinity for both PCL and PLLA blocks existed in the as-synthesized

copolymer film during the biodegradation process. Therefore, it is easy to understand why the as-synthesized copolymer films degraded much more slowly than pure PCL film, especially since it contains PLLA which inherently degrades more slowly than PCL. The diffuse halo from amorphous scattering became weaker, while the diffraction from crystalline regions clearly became stronger during the degradation of the coalesced copolymer films, indicating a trend of increasing crystallinity. Obviously, the crystallinity of the film sample that degraded for 2 weeks is much higher than that of the initial coalesced copolymer film. We have mentioned that the coalesced copolymer film degraded faster than PCL film only during the first ca. 2 weeks, and after this period it degraded more slowly than

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Figure 7. X-ray diffraction patterns of as-synthesized PCL-b-PLLA films (a) and coalesced PCL-b-PLLA films (b), after various enzymatic degradation times. Table 3. Comparison of Thermal Properties of As-Synthesized Copolymer and Coalesced Sample Films, before and after Degradation for 2 Weeksa Tm-PLLA ∆HPLLA degradation Tm-PCL ∆HPCL identity

time (days)

(°C)

(J/gsample)

(°C)

(J/gsample)

as-synthesized copolymer coalesced sample

0 14 0 14

59.7 65.3 63.4 68.6

42.2 44.4 22.5 37.6

160.2 158.3 164.1 162.0

22.5 24.6 5.9 15.2

a

Results of first heating run DSC measurements.

PCL film. This result coincides well with the trend of crystallinity change in the coalesced copolymer film revealed by WAXD measurements. Apparently, an increased crystallinity induced by biodegradation has resulted in the decline of the biodegradation rate of the coalesced copolymer films. Since PCL degrades much faster than PLLA, it appears very reasonable that the coalesced diblock sample should degrade more slowly than PCL once its crystallinity reaches some level where the amorphous phase loses its decisive role in determining the overall biodegradation rate. This conclusion is also supported by the thermal properties presented in Table 3. For as-synthesized and coalesced copolymer samples, the melting temperatures (Tm) of PCL and PLLA blocks show opposite changes upon degradation. After degrading for 1 week, the Tm of PCL blocks increased, while the Tm of PLLA blocks decreased somewhat. Changes in the crystallinities for these samples during the biodegradation process are deducible by comparing the enthalpies of fusion (∆H) of samples collected at different degradation times, as displayed in Table 3 and Figure 8. In the first 2 weeks of degradation, the coalesced sample showed significant increase in ∆H for both PCL and PLLA blocks, indicating increased crystallinities. In contrast, crystallinity of the as-synthesized copolymer was initially very high and only slightly increased during the same degradation period. Therefore, the faster degradation of amorphous regions of the coalesced copolymer films has resulted in increasing crystallinity, which gradually turned the biodegradaion into a slower process.

Figure 8. Heating-run DSC thermograms of coalesced samples after degrading for 0 days (a), 3 days (b), 7 days (c), 14 days (d), and 21 days (e).

Conclusions PCL is found to degrade much faster than PLLA in phosphate buffer solution (37 °C, pH ) 7) containing lipase from R. arrhizus. The biodegradation processes of all samples studied in this paper may be described as fast hydrolysis of amorphous regions followed by slow erosion of crystalline regions. Therefore, the biodegradation behavior of these samples is crucially determined not only by their composition but also by their phase structures, and any changes occurring to them during the biodegradation process. By first forming the inclusion complex (IC) between R-cyclodextrin (R-CD) and PCL-b-PLLA, and then coalescing the guest polymer chains together, the coalesced sample obtained possesses very low crystallinity compared to the as-synthesized copolymer sample.1 As a result of this phase transition, the coalesced copolymer film behaved in a manner very different from that of either as-synthesized copolymer or the simple physical blend of PCL and PLLA homopolymers, in the biodegradation tests. Acknowledgment. We are grateful to the National Textile Center (U.S. Department of Commerce) for supporting this research. X.S. is grateful to ERC and the Technical Research Center of Physics and Chemistry of the Chinese Academy of Sciences for allowing him to visit with Professor A. E. Tonelli. References and Notes (1) Shuai, X.; Porbeni, F. E.; Wei, M., Shin, I. D.; Tonelli, A. E. Macromolecules 2001, 34, 7355. (2) (a) Kang, S.; Hsu, S. L.; Stidham, H. D.; Smith, P. B.; Leugers, M. A.; Yang X. Macromolecules 2001, 34, 3542. (b) Yang, X.; Kang, S.; Hsu, S. L.; Stidham, H. D.; Smith, P. B.; Leugers, A. Macromolecules 2001, 34, 5037. (3) Choi, I. S.; Langer, R. Macromolecules 2001, 34, 5363. (4) Shen, Y.; Shen, Z.; Zhang, Y.; Yao, K. Macromolecules 1996, 29, 8299. (5) Majerska, K.; Duda, A.; Penczek, S. Macromol. Rapid Commun. 2000, 21, 1327.

Regulating Biodegradable Block Copolymers (6) (a) Shuai, X.; He, Y.; Asakawa, N.; Inoue, Y. J. Appl. Polym. Sci. 2001, 81, 762. (b) Shuai, X.; He, Y.; Na, Y.; Inoue, Y. J. Appl. Polym. Sci. 2001, 80, 2600. (7) Yang, J.; Chen, H.; You, Y.; Hwang, L. Polym. J. 1997, 29, 657. (8) Veld, P.; Velner, E.; Witte, P.; Hamhuis, J.; Dijkstra, P.; Feijen, J. J. Polym. Sci., Part A: Polym. Chem. 1997, 35, 219. (9) (a) Kim, J. K.; Park, D. J.; Lee, M. S.; Ihn, K. J. Polymer 2001, 42, 7249. (b) Ye, W.; Du, F.; Jin, W.; Yang, J.; Xu. Y. React. Funct. Polym. 1997, 32, 161. (10) (a) Harada, A.; Li, J.; Kamachi, M. Nature 1994, 370, 126. (b) Harada A.; Suzuki, S.; Okada, M.; Kamachi, M. Macromolecules 1996, 29, 5611. (c) Harada, A.; Kamachi, M. Macromolecules 1990, 23, 2821. (d) Harada, A.; Nishiyama, T.; Kawaguchi, Y.; Okada, M.; Kamachi, M. Macromolecules 1997, 30, 7115. (11) Huh, K. M.; Ooya, T.; Sasaki, S.; Yui, N. Macromolecules 2001, 34, 2402. (12) Weickenmeir, M.; Wenz, G. Macromol. Rapid Commun. 1997, 18, 1109. (13) Li, J.; Yan, D. Macromolecules 2001, 34, 1542. (14) Wei, M.; Tonelli, A. E. Macromolecules 2001, 34, 4061.

Biomacromolecules, Vol. 3, No. 1, 2002 207 (15) Rusa, C. C.; Tonelli, A. E. Macromolecules 2000, 33, 5321. (16) Izumikawa, s.; Yoshioka, S.; Aso, Y.; Takeda, Y. J. Controlled Release 1991, 15, 133. (17) Petrova, T.; Mandova, N.; Rashkov, I.; Li, S.; Vert, M. Polym. Int. 1998, 45, 419. (18) Arvanitoyannis, I.; Nakayama, A.; Kawasaki, N.; Yamamoto, N. Polymer 1995, 36, 2271. (19) Detailed results and discussions will be presented in a forthcoming paper. (20) (a) He, Y.; Shuai, X.; Cao, A.; Kasuya, K.; Doi, Y.; Inoue, Y. Macromol. Rapid Commun. 2000, 21, 1277. (b) He, Y.; Shuai, X.; Cao, A.; Kasuya, K.; Doi, Y.; Inoue, Y. Polym. Degrad. Stab. 2001, 73, 193. (c) He, Y.; Shuai, X.; Kasuya, K.; Doi, Y.; Inoue, Y. Biomacromolecules 2001, 2, 1045. (21) Holland, S. J.; Tighe, B. J.; Gould, P. L. J. Controlled Release 1986, 4, 155. (22) Gan Z.; Yu, D.; Zhang, Z.; Liang, Q.; Jing, X. Polymer 1999, 40, 2859.

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