Synthesis, Characterization, and Degradation Behavior of Amphiphilic

rate and the PHEA-rich degradation products are trendy to be soluble in water. ...... Chan Woo Park , Hyun Jin Lee , Hee-man Yang , Min-ah Woo , H...
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Biomacromolecules 2005, 6, 3449-3457

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Synthesis, Characterization, and Degradation Behavior of Amphiphilic Poly-r,β-[N-(2-hydroxyethyl)-L-aspartamide]-g-poly(E-caprolactone) Zhi-Mei Miao, Si-Xue Cheng, Xian-Zheng Zhang, and Ren-Xi Zhuo* Key Laboratory of Biomedical Polymers of Ministry of Education, Department of Chemistry, Wuhan University, Wuhan 430072, People’s Republic of China Received August 3, 2005; Revised Manuscript Received August 31, 2005

A series of biodegradable amphiphilic graft polymers were successfully synthesized by grafting poly(caprolactone) (PCL) sequences onto a water-soluble poly-R,β-[N-(2-hydroxyethyl)-L-aspartamide] (PHEA) backbone. The graft copolymers were prepared through the ring-opening polymerization of -caprolactone (CL) initiated by the macroinitiator PHEA with pendant hydroxyl groups without adding any catalyst. By controlling the feed ratio of the macroinitiator to the monomer, the copolymers with different branch lengths and properties can be obtained. The successful grafting of PCL sequences onto the PHEA backbone was verified by FTIR, 1H NMR, and combined size-exclusion chromatography and multiangle laser light scattering (SEC-MALLS) analysis. The hydrolytic degradation and enzymatic degradation of these graft copolymers were investigated. The results show the hydrolytic degradation rate increases with increasing content of hydrophilic PHEA backbone. While the enzymatic degradation rate is affected by two competitive factors, the catalytic effect of Pseudomonas cepacia lipase on the degradation of PCL branches and the hydrophilicity which depends on the copolymer composition. In situ observation of the degradation under polarizing light microscope (PLM) demonstrates the different degradation rates of different regions in the polymer samples. Introduction Biodegradable polyesters, especially polylactide (PLA), polyglycolide (PGA), poly(-caprolactone) (PCL), and their copolymers, have been extensively studied and widely applied in biomedical fields including drug delivery and tissue engineering, due to their good biocompatibility and biodegradability.1 To meet the increasing demands for better performances and satisfy the requirements of some specific applications, the properties of these materials can further be tailored by modifications including blending2 and copolymerization3,4 (i.e., to form block, graft, or star copolymers). Among these modifications, combining these hydrophobic polyester segments with hydrophilic segments through copolymerization can offer amphiphilic polymers, which are of special interests as biomaterials. These kinds of polymers may self-assemble into micelles which can encapsulate both hydrophobic and hydrophilic drugs and be used for the targeted drug delivery.5-7 Due to their balanced hydrophilic/ hydrophobic character, they can also improve the cell proliferation in tissue engineering.8 In the past decades, the amphiphilic block copolymers with hydrophilic PEG segments have been extensively investigated.9-11 Compared with the block copolymers, amphiphilic graft copolymers can easily form colloids within one or several polymer chains. In this work, we synthesized a series of novel amphiphilic poly-R,β-[N-(2-hydroxyethyl)-L-aspartamide]-g-poly(-caprolactone) (PHEA-g-PCL) copolymers with different composi* Corresponding author. Fax: +86-27-68754509. E-mail: pibmp@ public.wh.hb.cn.

tions and studied their properties including enzymatic degradation property. In the amphiphilic copolymers we synthesized, the hydrophobic side chains are PCL segments. Homopolymer PCL is a highly hydrophobic material with a slow hydrolytic degradation rate in the absence of enzyme and a fast enzymatic degradation rate in the presence of particular lipases, such as R. delemer lipase,12 Rhizopus arrhizus lipase,13 and Pseudomonas cepacia lipase.14 PCL is a promising candidate for drug delivery applications because of its high permeability to drugs at body temperature and good biocompatibility.15,16 The hydrophilic backbone is poly-R,β-[N-(2-hydroxyethyl)-L-aspartamide] (PHEA), which is a synthetic watersoluble polymer.17,18 PHEA is a nontoxic, nonantigenic, and nonteratogenic polymer, and its protein-like structure and multifunctional character endow it with interesting physicochemical properties. Its applications in the biomedical fields have been interestingly explored. For example, it has been investigated as a plasma expander16,17 and the carrier for incorporating drugs.18-20 By grafting hydrophilic poly(ethylene glycol) (PEG) or/and hydrophobic hexadecylalkyl groups onto the PHEA backbone, the resulting copolymers were also investigated as drug carriers.21,22 With hydroxyl functional groups, PHEA can initiate the ring-opening polymerization of some cyclic monomers, such as lactide, glycolide, and cyclic carbonates, to obtain graft copolymers.23 However, as far as we know, PHEA as a macromolecular initiator was seldom reported. In the present work, using PHEA as a macroinitiator to initiate the ring-opening

10.1021/bm050551n CCC: $30.25 © 2005 American Chemical Society Published on Web 10/18/2005

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polymerization of -caprolactone (CL), amphiphilic graft copolymers were obtained by one-step reaction conveniently. Because of the amphiphilicity of these polymers, they have promising applications in many biomedical fields. By varying the feed ratio of the macroinitiator PHEA to the monomer CL, graft copolymers with different hydrophobic branch lengths can be obtained. Thus, the adjustment of the physicochemical properties such as amphiphilicity and degradation property can be easily realized. Experimental Part Materials. -Caprolactone (Aldrich) was dried over CaH2 for 2 days and distilled under reduced pressure prior to use. L-Aspartic acid, phosphoric acid, dichloromethane, and diethyl ether (Shanghai Chemical Co. China) were of analytical grade and used as supplied. Ethanolamine (Shanghai Chemical Co) was distilled before use. N,N-Dimethylformamide (DMF) was purified by distillation over P2O5 and CaH2. Tetrahydrofuran (THF) was purified by distillation over sodium. Stannous octanoate Sn(Oct)2 (Aldrich) was purified by distillation under reduced pressure and then dissolved in dry toluene prior to use. Pseudomonas cepacia lipase was supplied by Fluka. Synthesis of PHEA-g-PCL Copolymers. PHEA was synthesized according to a published procedure.24 PHEA-gPCL copolymers were synthesized by the bulk ring-opening polymerization of CL using PHEA with pendant hydroxyl groups as a macroinitiator without adding any catalyst. The graft polymerization was carried out by the following procedure. PHEA and CL, with a specific feed ratio of the hydroxyl group in PHEA to the monomer CL (1/1, 1/4, and 1/10 for PHEA-g-PCL-1, PHEA-g-PCL-2, and PHEA-gPCL-3, respectively), were well mixed and transferred to a dry silanized glass flask with a magnetic stirring bar. The flask was evacuated, purged with argon three times, sealed, and then immersed in an oil bath at 200 °C for 5 min, allowing PHEA to become a viscous liquid. Then the reaction was carried out at 120 °C for a particular reaction time t. After graft polymerization, to obtain PHEA-g-PCL-1, the product was dissolved in distilled water, dialyzed using a dialysis membrane (MWCO 8000-12000) for 48 h, and then freeze-dried (Labconco). To obtain PHEA-g-PCL-2 and PHEA-g-PCL-3, the products were dissolved in dichloromethane and then precipitated in ether, subsequently isolated by centrifuging and dried under vacuum at 40 °C. For comparison, homopolymer PCL was synthesized by the ring-opening polymerization at 120 °C for 4 h using Sn(Oct)2 (0.1 mol %) as a catalyst. Sample PHEA-2 was obtained after heating the PHEA sample at 200 °C for 5 min followed by 120 °C for 48 h without adding CL to examine the molecular weight change of PHEA during the heating. Characterization of PHEA-g-PCL Copolymers. Fourier transform infrared (FTIR) spectra were obtained on a PerkinElmer-2 spectrometer. The samples were in KBr pellets. 1 H nuclear magnetic resonance (1H NMR) spectra were recorded on a Mercury VX-300 spectrometer (300 MHz)

Miao et al.

using D2O and CDCl3 as solvents and TMS as an internal standard. Combined size-exclusion chromatography and multiangle laser light scattering (SEC-MALLS) analysis was carried out to determine the molecular weights of graft copolymers. A dual detector system, consisting of a MALLS device (DAWN EOS, Wyatt Technology) and an interferometric refractometer (Optilab DSP, Wyatt Technology), was used. The SEC system (2690D, Waters) was equipped with a precolumn and two Shodex columns (K805 and K8025) when using CHCl3 as an eluent, and three Ultrahydrogel columns (2000, 250 and 120) when using 0.15 M NaCl/0.1 M phosphate buffer (pH 7.4) as an eluent. The polymer concentration was 3 mg/mL and the flow rate was 1 mL/ min. The MALLS detector was operated at a laser wavelength of 690.0 nm. Thermal properties of graft copolymers were measured by differential scanning calorimeter (DSC) (Perkin-Elmer Pyris1). PHEA-g-PCL copolymers and PCL were heated from -20 to +90 °C at a heating rate of 10 °C/min and then cooled to -20 °C at a cooling rate of 10 °C/min. PHEA was heated from -20 to +220 °C at a heating rate of 10 °C/min and then cooled to -20 °C at a cooling rate of 10 °C/min. Wide-angle X-ray diffraction (WAXD) analysis was conducted with a Shimadzu XRD-6000 diffractometer at 40 kV/30mA using Cu KR radiation. A continuous scan mode was used to collect data from 10° to 40° at a speed of 4°/min. Degradation Study on PHEA-g-PCL Copolymers. Weight Loss Measurement. PHEA-g-PCL-2 and PHEA-g-PCL-3 polymer films were obtained by compression molding at 60 °C, followed by cooling to room temperature with a cooling rate of 10 °C/min. The thickness of the samples was controlled at 0.5 mm, and the weight of each sample was about 10 mg. PCL film was prepared by a solvent-casting technique. The solution of PCL in chloroform was dropped on a glass substrate, evaporated at room temperature for 24 h, and then dried under vacuum for 24 h. The sample thickness was controlled at 0.5 mm, and the sample weight was about 10 mg. To determine the weight loss during hydrolytic degradation, each sample was incubated in a vial filled with 5 mL of phosphate buffer (pH 7.4) containing sodium azide (0.2 mg/mL). The vial was placed in a shaking water bath at 37.4 °C. At preset time intervals, the sample was taken out from the degradation medium, rinsed with distilled water, and subsequently dried under vacuum at room temperature to a constant weight before measurement. To determine the weight loss during enzymatic degradation, the sample was immersed in 5 mL of phosphate buffer (pH 7.4) containing Pseudomonas cepacia lipase (0.2 mg/mL) and sodium azide (0.2 mg/mL). The enzymatic degradation medium was changed every 48 h to restore the original level of enzymatic activity. Morphology Visualization by Scanning Electron Microscope (SEM). The surface morphologies of polymer films before and after degradation were observed by SEM (Hitachi X650, Japan). The sample films were prepared following

Amphiphilic PHEA-g-PCL

the same procedure for preparing the films used in weight loss measurement, and the hydrolytic and enzymatic degradation conditions were also the same. In Situ Degradation Study under Polarizing Light Microscope (PLM). About 1 mg of copolymer sample was sandwiched between two glass slides and placed on a heating stage (Linkam THMS-600). The sample was heated to 75 °C for 5 min to allow the copolymer to melt. Then the sample was quickly cooled to the isothermal crystallization temperature to carry out isothermal crystallization for 1 h. After removing the cover slide, a polymer thin film with a thickness about 10 µm was formed on a glass slide. To observe the morphology change of the polymer film during hydrolytic degradation, the polymer thin film on the glass slide was placed in a small glass vessel with a diameter of 2 cm and a height of 0.5 cm. Then 1.5 mL of phosphate buffer (pH 7.4) containing sodium azide (0.2 mg/mL) was added to the glass vessel. After that, the glass vessel was placed on the heating stage (Linkam THMS-600) under a polarizing light microscope (PLM) (Olympus BX51). The hydrolytic degradation of the polymer thin film was carried out at 37.4 °C, and the morphology of the thin film was observed by the PLM in situ. To observe the morphology change of the polymer film during enzymatic degradation, the polymer thin film on the glass slide was immersed in 1.5 mL of phosphate buffer (pH 7.4) containing Pseudomonas cepacia lipase (0.2 mg/mL) and sodium azide (0.2 mg/mL). The enzymatic degradation was carried out at 37.4 °C and visualized in situ using PLM.

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Figure 1. FTIR spectra of PHEA, PHEA-g-PCL copolymers, and PCL.

Results and Discussion Synthesis of PHEA-g-PCL Copolymers. A series of graft copolymers were prepared by the bulk ring-opening polymerization of CL using the hydroxyl groups in PHEA as initiators without adding any catalyst. The polymerization reactions were conducted under rigorously anhydrous conditions to avoid the initiation by water. In the presence of PHEA, the monomer CL can be efficiently initiated by the -OH groups and thus grafted onto the PHEA backbone. The successful grafting of PCL sequences onto PHEA was confirmed by FTIR and 1HNMR. A comparison experiment for the ring-opening polymerization of CL was carried out at 120 °C without adding PHEA and any catalyst. The result shows that no solid product can be obtained even after a 7-day reaction, which indicates the monomer CL is difficult to be polymerized in the absence of PHEA or other catalysts. Characterization of PHEA-g-PCL Copolymers. Figure 1 shows the FTIR spectra of the obtained polymers. Compared with PHEA, PHEA-g-PCL copolymers show a new peak at 1725 cm-1 corresponding to CdO stretching of PCL branches, and a comparison of PHEA-g-PCL copolymers with PCL clearly shows two new peaks appeared at 1660 and 1543 cm-1, which are ascribed to CdO stretching and N-H bending of the amide groups in PHEA. Since PHEA-g-PCL-1 can be dissolved in water, and PHEAg-PCL-2 and PHEA-g-PCL-3 were obtained by dissolving the polymerization products in dichloromethane for purifica-

Figure 2. 1H NMR spectra of PHEA, PHEA-g-PCL-1, and PCL. The solvent for PHEA and PHEA-g-PCL-1 was D2O, and the solvent for PCL was CDCl3.

tion, the appearance of these new peaks indicates that the PCL sequences were successfully introduced to the PHEA backbones. Figure 2 shows the 1H NMR spectra of PHEA, PCL, and the graft copolymer PHEA-g-PCL-1 as a typical example. In the spectrum of PHEA-g-PCL-1, the typical signals of PCL branches can be observed at 1.22 ppm (CH2CH2CH2CH2CH2), 1.46 ppm (CH2CH2CH2CH2CH2), 2.24 ppm

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Table 1. Synthesis and Molecular Weights of PHEA and PHEA-g-PCL Copolymers and PCLa

polymer PHEA PHEA-2 PHEA-g-PCL-1 PHEA-g-PCL-2 PHEA-g-PCL-3 PCL

feed ratio -OH/CL (mol/mol)

1/1 1/4 1/10 0/1

t (h) 48 24 48 48 4

yield (%) 95.1 27.8 73.8 85.8 90.0

HEA/CL in polymer (mol/mol)

1H NMR unreacted -OH groups in PHEA backbone (%)

1/0.2 1/4.9 1/15.7

100 100 86 55 45

mean number of CL units in each branch

1.6 10.8 28.5

SEC-MALLS

Mw (g/mol)

Mw/Mn

Rg (nm)

2.56 × 104 3.46 × 103 3.36 × 104 1.88 × 104 2.44 × 104 2.87 × 104

1.49 1.60 1.18 1.44 1.23 1.32

11.0 29.2 54.3 49.6 15.1

a In 1H NMR characterization, D O was used as a solvent for PHEA, and PHEA-g-PCL-1, and CDCl was used as a solvent for PHEA-g-PCL-2, 2 3 PHEA-g-PCL-3 and PCL. In SEC-MALLS analysis, 0.15 M NaCl/0.1 M phosphate buffer (pH 7.4) was used as an eluent for PHEA, PHEA-2, and PHEA-g-PCL-1, and CHCl3 was used as an eluent for PHEA-g-PCL-2, PHEA-g-PCL-3, and PCL.

(COCH2), and 3.96 ppm (CH2O), and the typical signals of PHEA backbones can be observed at 2.61 ppm (CHCH2CONH), 3.14 ppm (NHCH2CH2OH), 3.44 ppm (CH2CH2OH), and 4.50 ppm (NHCH(CO)CH2). Furthermore, two new signals appear at 3.32 ppm (CH2CH2OCO) and 4.02 ppm (CH2CH2OCO) in the spectrum of PHEA-g-PCL-1 because of the graft structure. The appearance of these new peaks further confirms the successful grafting. The molar ratio of HEA repeating units to CL repeating units in the graft copolymers can be calculated by comparing the integration values of peak a from HEA repeating units and peak g from CL repeating units. The HEA/CL ratios in graft copolymers are in good agreement with the -OH/CL feed ratios except for PHEA-g-PCL-1. The main reason for the difference between the HEA/CL ratio and the -OH/CL feed ratio for PHEA-g-PCL-1 can be attributed to the purification procedure to obtain the copolymer, which may remove the water insoluble copolymers with lower HEA/ CL ratios. From 1H NMR, we can also determine the percentage of the unreacted -OH groups in PHEA backbone by comparing the integration values of peak b with peak a in the PHEA backbone. Based on the HEA/CL ratio in the graft polymers and the percentage of unreacted -OH groups in the PHEA backbone, we can further estimate the mean number of CL units in each branch. The results are listed in Table 1. Clearly, with decreasing -OH/CL feed ratio, the percentage of the unreacted -OH groups in PHEA backbones decreases and the grafted chain length increases. The molecular weight and size of the polymers were determined by the combined SEC-MALLS analysis. As we can find from Table 1, the molecular weight Mw of PHEA-2 is much lower than PHEA, indicating that the thermal degradation occurs during the heating at 200 °C for 5 min and then 120 °C for 48 h. The degradation of the backbone ultimately leads to a lower Mw of the resulting graft polymer. Nevertheless, the Mw values of all graft polymers are still reasonably high, and all graft copolymers have unimodal molecular weight distributions and reasonably narrow polydispersities, ranging from 1.18 to 1.44. These results indicate that the graft polymerization is successful and that -OH groups in PHEA are effective propagation centers. As we know, in a dilute solution, the polymer architectures can be classified as rod, random coil, or other shapes, depending on the structure and the stiffness of the polymer chains. The root-mean-square radius of gyration (Rg) of a polymer is dependent on the molecular architecture and the average

Figure 3. WAXD patterns of PHEA, PHEA-g-PCL copolymers, and PCL.

molecular weight. From SEC-MALLS (Table 1), it can be found that both Mw and Rg of PHEA-g-PCL-1 are much higher than PHEA-2 although the content of PCL branches in the graft copolymer is low. This is due to the micellization of the amphiphilic PHEA-g-PCL-1 in water. Similar phenomenon was observed for another amphiphilic graft copolymer with PHEA as the backbone and poly(2,2dimethyltrimethylene carbonate) segments as hydrophobic branches in our pervious studies.23 Since the common suitable solvent for all graft polymers is not available, the SECMALLS measurements have to be carried out in different solvents. It should be noted that the Rg values are not comparable because the data were obtained in different solvents. Figure 3 shows WAXD patterns of PHEA, PCL, and PHEA-g-PCL copolymers. PHEA is an amorphous polymer. No crystal pattern can be observed for PHEA-g-PCL-1 because the grafted PCL branches are short. Both PHEAg-PCL-2 and PHEA-g-PCL-3 exhibit two characteristic peaks at 2θ ) 21.4° and 23.7° due to the crystallization of PCL branches. DSC was carried out to investigate the melting and crystallization behaviors of the graft polymers. The DSC thermograms are shown in Figure 4, and corresponding data are listed in Table 2. Being consistent with WXRD results, the DSC thermogram shows PHEA is an amorphous polymer without a melting peak. Similar to PHEA, no Tm can be observed for PHEA-g-PCL-1 because of its short PCL branches. For PHEA-g-PCL-2 and PHEA-g-PCL-3, obvious melting transition peaks are observed. In the graft copolymers, the PHEA component is amorphous, so it is assumed

Amphiphilic PHEA-g-PCL

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Figure 5. Weight loss of PHEA-g-PCL-2, PHEA-g-PCL-3, and PCL during hydrolytic degradation.

Figure 4. DSC thermograms of PHEA, PHEA-g-PCL copolymers, and PCL. Table 2. Thermal Properties of PHEA and PHEA-g-PCL Copolymers and PCLa polymer

Tm (°C)

Tc (°C)

∆H m (J/g)

∆Hc (J/g)

fw

Xc (%)

PHEA PHEA-g-PCL-1 PHEA-g-PCL-2 PHEA-g-PCL-3 PCL

50.8 52.0 60.6

15.4 16.2 29.8

20.8 45.3 85.1

-55.4 -60.0 -67.3

0.14 0.78 0.92 1

0 19.6 36.2 62.6

a T , T , ∆H , and ∆H were determined by DSC. f was calculated m c m c w from the composition of copolymers determined by 1H NMR (Table 1).

that the crystallinity is only from the PCL component. The crystallinity of the PCL component, Xc, can be calculated from the following equation: Xc ) ∆Hm/(∆Hmofw) × 100% where ∆Hm is the melting enthalpy measured by DSC, ∆Hmo (136 J/g) is the melting enthalpy for 100% crystalline PCL,25 and fw is the weight fraction of PCL component in the copolymers. As shown in Table 2, the crystallinity increases with an increase in the PCL segment length. Both DSC and WAXD results indicate that the thermal and crystallization properties of the graft polymers are strongly dependent on the composition of the copolymers. Hydrolytic Degradation of PHEA-g-PCL Copolymers. Homopolymer PCL is a highly hydrophobic material with a slow in vitro hydrolytic degradation rate. After incorporating PCL segments into hydrophilic PHEA backbones, the graft copolymers become amphiphilic. Since PHEA-g-PCL-1 is water-soluble, the in vitro degradation study was carried out only for PHEA-g-PCL-2 and PHEA-g-PCL-3 copolymers and PCL homopolymer. Among these three polymers, PHEA-g-PCL-2 is the most hydrophilic one, containing 17 mol % HEA repeating units, and PHEA-g-PCL-3 is less hydrophilic, and its HEA repeating unit content is 6 mol %. As depicted in Figure 5, during the whole degradation process, the weight loss of PHEA-g-PCL-2 is the highest because of its high content of hydrophilic PHEA backbone, which results in a lower crystallinity of the copolymer,

facilitates water absorption, and produces PHEA-rich degradation products that tend to be soluble in water. As expected, PHEA-g-PCL-3 shows a lower weight loss as compared with PHEA-g-PCL-2, and homopolymer PCL has the lowest weight loss. Clearly, the hydrolytic degradation rate increases with increasing content of PHEA backbone in the copolymer and increases with decreasing crystallinity of the copolymer. The surfaces of polymer samples before and after hydrolytic degradation were observed by SEM. As demonstrated in Figure 6, the SEM images are in accordance with the weight loss data. After hydrolytic degradation for 144 h (6 days), the surface of PHEA-g-PCL-2 becomes un-smooth and some domains between lamellae start to disappear. Compared with PHEA-g-PCL-2, the surface morphology change during degradation for PHEA-g-PCL-3 is slower. For PCL, no obvious changes can be detected after degradation for 144 h. To further investigate the degradation process, the in situ observation of morphology changes of polymer lamellar crystals during degradation was carried out under PLM. As shown in Figure 7, after degradation for 48 h, the surface roughness of PHEA-g-PCL-2 increases, which results in a clearer spherulitic morphology. For PHEA-g-PCL-3, the spherulite boundaries disappear, whereas for PCL, no obvious change can be detected after the short degradation time of 48 h. It should be noted that the samples for SEM and PLM characterizations were obtained in different ways for different experimental aims. The purpose of PLM observation is to visualize the in situ morphology changes of a single spherulite during degradation, whereas the SEM observation was performed to investigate the whole morphology changes of a sample film. To obtain clear PLM images and SEM images, we have to use the samples with different thicknesses. The samples for SEM observation are much thicker than that for PLM visualization. Since the sample thickness also affects the degradation rate, the morphologies obtained from SEM and PLM are not exactly the same even after the same degradation time. Enzymatic Degradation of PHEA-g-PCL Copolymers. Pseudomonas cepacia lipase is an enzyme having a significant effect on the degradation of PCL.14 In our study, enzymatic degradation for the copolymers was carried out

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Figure 6. SEM images of (a) PHEA-g-PCL-2, (b) PHEA-g-PCL-3, and (c) PCL before and after hydrolytic degradation for particular time intervals.

Figure 8. Weight loss of PHEA-g-PCL-2, PHEA-g-PCL-3, and PCL during enzymatic degradation.

Figure 7. PLM images of (a) PHEA-g-PCL-2, (b) PHEA-g-PCL-3, and (c) PCL before and after hydrolytic degradation for a particular time interval.

to investigate the effects of the graft structure and the existence of hydrophilic PHEA backbone on their enzymatic degradation. The weight loss values of copolymers and PCL are compared in Figure 8. In contrast to its slow hydrolytic

degradation, PCL shows the highest weight loss during enzymatic degradation because Pseudomonas cepacia lipase has a specific activity on PCL segment degradation. The weight loss of PHEA-g-PCL-2 is higher than that of PHEAg-PCL-3 during enzymatic degradation. This phenomenon is reasonable if we consider two competitive factors: the catalytic effect of Pseudomonas cepacia lipase on the degradation of PCL branches and the existence of hydrophilic PHEA main chain. Taking account of the catalytic effect of Pseudomonas cepacia lipase, a faster enzymatic degradation rate is expected for the sample with a higher PCL branch content. Whereas in view of the hydrophilic PHEA backbone, the high PHEA content leads to a faster water diffusion rate and the PHEA-rich degradation products are trendy to be soluble in water. Thus, the sample with higher PHEA content will degrade fast. As an overall result of these two competi-

Amphiphilic PHEA-g-PCL

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Figure 9. SEM images of (a) PHEA-g-PCL-2, (b) PHEA-g-PCL-3, and (c) PCL before and after enzymatic degradation for particular time intervals.

tive factors, our experiment shows PHEA-g-PCL-2 has a fast degradation rate because its high content of hydrophilic PHEA main chain plays a dominant role. SEM images are supportive to the weight loss data. As presented in Figure 9, after 48 h of enzymatic degradation, all three samples show obvious morphological changes. Pores appear on the PCL surface, and some domains between lamellae start to degrade for the graft copolymers. With a further increase in degradation time, a big pore is formed in the PCL sample, and three-dimensional structures appear in the surfaces of PHEA-g-PCL-2 and PHEA-g-PCL-3 as a result of the different degradation rates of different regions in the samples. As we know, compared with small molecules, the crystallinity of polymer materials is limited and amorphous regions exist beside the crystalline regions. Even inside crystalline regions, the thermodynamic stabilities of different domains may be different. When polymers crystallize, they can adopt a spherulitic morphology, where the lamellae grow outward radially.26 More detailed studies of polymeric systems showed there are two distinct types of lamellar crystals present in the spherulites. Dominant (leading) lamellae with a thicker appearance are identified as growing first to provide a skeleton for the spherulites. Subsidiary (in-filling) lamellae are seen as being rather thinner and having an ill-defined orientation, which grow after the dominant lamellae, filling in the spaces between the primary. Besides the presence of dominant and subsidiary crystals in spherulites, along single lamella the thermal stability is even different, which is due to an uneven distribution of segments with different crystallizibilities caused by different structure regularities along

polymer chains. In the beginning of crystallization, the chains/segments with higher crystallizibility crystallize first, and those chains/segments with lower crystallizibility are first rejected and accumulated in the front of crystal growth. Later on, as soon as these parts cannot diffuse away quickly enough, they have to crystallize to form less perfect crystals. These defective or less perfect crystals accumulate in certain locations.26 The in situ observation of enzymatic degradation of our graft copolymers and PCL under PLM clearly demonstrates the different stabilities of different domains in the samples. As shown in Figure 10a for PHEA-g-PCL-2, after 60 h degradation, only one spherulite remains in the view area under PLM because of the floating away of other spherulites due to the degradation and disappearance of spherulite boundaries. Also the spherulite becomes thinner and some domains between lamellae disappear. According to previous studies, the crystallinity of PCL increases during degradation because the degradation of amorphous regions occurs first.27 In our PLM images, the missing domains between lamellae may be ascribed to the amorphous regions or the less perfectly orientated subsidiary lamellae. For PHEA-g-PCL-3 with a slower degradation rate (Figure 10b), after degradation for 60 h, the disappearance of spherulite boundaries can be clearly observed, whereas the degradation of the regions between lamellae cannot be detected yet. The phenomenon is explainable. In the process of spherulite growth, the PHEArich parts with lower crystallizibility in the graft copolymers are pushed to the edges of spherulites and finally accumulate at the boundaries of spherulites. Once degradation takes place, these sections degrade and disappear first. For homo-

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Figure 10. PLM images of (a) PHEA-g-PCL-2, (b) PHEA-g-PCL-3, and (c) PCL before and after enzymatic degradation for particular time intervals.

polymer PCL with the highest enzymatic degradation rate, after degrading for 48 h, the degradation of domains between lamellae can be visualized. After 60 h, some locations, where defective or less perfect crystals accumulate, in single lamella disappear. However, the spherulite boundaries of PCL do not show a fast degradation rate, which is different from the copolymer samples. This is because the homopolymer only has one component and a uniform chain structure, which do not lead to obvious degradation rate difference between the spherulite boundaries and the inner parts of spherulites. Conclusions Biodegradable amphiphilic graft polymers were successfully synthesized by grafting PCL sequences onto a watersoluble PHEA backbone through the ring-opening polymerization of CL using PHEA bearing hydroxyl groups as a macroinitiator. By controlling the feed ratio of the macroinitiator to the monomer, the branch lengths of graft polymers can be conveniently adjusted, and graft polymers with different hydrophilicities and crystallinities can be obtained. The degradation study shows the hydrolytic degradation rate is mainly dependent on the hydrophilicity of the copolymers. And the enzymatic degradation rate is affected by two competitive factors, the catalytic effect of Pseudomonas cepacia lipase on the degradation of PCL branches and the hydrophilicity which depends on the copolymer composition. The different degradation rates of different regions in the polymer samples have been observed through SEM and in situ study under PLM. The amorphous regions and the less perfect subsidiary lamellae degrade first.

Acknowledgment. This research was supported by grants (20204010 and 20474046) from the National Natural Science Foundation of China. One of the authors, Si-Xue Cheng, is grateful to the Ministry of Education of China for the financial support of “Trans-Century Training Program Foundation for the Talents”. We thank Prof. Tian-Xi Liu at Fudan University and Prof. Guo-Ping Yan at Wuhan Institute of Technology for their assistance. The excellent technical assistance of Ms. Qing-Rong Wang for the SEC-MALLS measurement is gratefully acknowledged. References and Notes (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) (14)

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