Biomacromolecules 2002, 3, 525-530
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Enzymatic Degradation of Block Copolymers Prepared from E-Caprolactone and Poly(ethylene glycol) Suming Li,* Henri Garreau, Bernard Pauvert, Jonathan McGrath, Alexandra Toniolo, and Michel Vert Centre de Recherche sur les Biopolyme` res Artificiels, Faculte´ de Pharmacie, 15 avenue Charles Flahault, 34060 Montpellier Cedex 2, France Received December 5, 2001; Revised Manuscript Received February 1, 2002
Block copolymers were prepared by ring-opening polymerization of -caprolactone in the presence of monohydroxyl or dihydroxyl poly(ethylene glycol) (PEG), using Zn powder as catalyst. The resulting poly(-caprolactone) (PCL)-PEG diblock and PCL-PEG-PCL triblock copolymers were characterized by various analytical techniques such as NMR, size-exclusion chromatography, differential scanning calorimetry, and X-ray diffraction. Both copolymers were semicrystalline polymers, the crystalline structure being of the PCL type. Films were prepared by casting dichloromethane solutions of the polymers on a glass plate. Square samples with dimensions of 10 × 10 mm were allowed to degrade in a pH ) 7.0 phosphate buffer solution containing Pseudomonas lipase. Data showed that the introduction of PEG blocks did not decrease the degradation rate of poly(-caprolactone). Introduction Degradable polymers are of growing interest in the field of temporary therapeutic applications such as sustained drug delivery systems, surgical sutures, osteosynthetic devices, scaffolds for tissue repair and regeneration, etc.1-3 They have become also attractive in the field of environmental applications because of the problems related to plastic waste accumulation.4,5 In fact, degradable polymers can be used as mulch film in agriculture or as packaging material. Poly(-caprolactone) (PCL) is one of the most promising synthetic polymers that can degrade in an aqueous medium or in contact with microorganisms and thus can be used to make compostable polymeric devices.6-8 The enzymatic degradation of PCL polymers has also been investigated, especially in the presence of lipase-type enzymes.9-12 Three kinds of lipase were found to significantly accelerate the degradation of PCL, i.e., Rhizopus delemer lipase,9 Rhizopus arrhizus lipase,10 and Pseudomonas lipase.11,12 Highly crystalline PCL was reported to totally degrade in 4 days in the presence of Pseudomonas lipase,11,12 in contrast to hydrolytic degradation which lasts several years.1 In a previous paper,13 we considered the enzymatic degradation of PCL and its blends with poly(L-lactide) (PLLA) in the presence of Pseudomonas lipase or proteinase K, the latter being a protease capable of hydrolyzing ester bonds of PLLA. It was observed that the two faces of solution cast films showed different morphologies due to the solvent evaporation process. The lower face appeared more crystalline than the upper face because of the plasticizing effect of entrapped solvent which facilitated crystallization. The two polymers in the blends exhibited well-separated crystalline * Corresponding author (phone, 33-(0)467418266; fax, 33-(0)467520898; e-mail,
[email protected]).
domains. The selective degradation of PCL or PLLA components revealed the inner morphology of the blends where microsphere-like or isle-like patterns were observed.13 Moreover, the presence of PLLA strongly reduced the enzymatic degradation of PCL in the blends. PCL is a highly hydrophobic and crystalline polymer. These properties considerably restrained its potential applications. Introduction of hydrophilic polyether blocks into PCL chains is a means to enhance the hydrophilicity as compared with the parent homopolymer. Poly(ethylene glycol) (PEG) has been used to form various block copolymers with PCL.14-16 In this paper, we wish to report on the synthesis, characterization, and enzymatic biodegradation of PCL/PEG diblock and triblock copolymers with the aim of identifying the effect of PEG incorporation on the biodegradation characteristics of PCL. The copolymers were processed to films by solution casting. Biodegradation of the copolymers was carried out at 37 °C in a 0.05 M pH ) 7.0 phosphate buffer containing Pseudomonas lipase in comparison with a PCL homopolymer. Physicochemical property changes were discussed from results obtained by proton nuclear magnetic resonance (NMR), size-exclusion chromatography (SEC), differential scanning calorimetry (DSC), X-ray diffraction, and environmental scanning electron microscopy (ESEM). Experimental Section Materials. PCL homopolymer, -caprolactone, and dihydroxyl PEG with molecular weight of 4600 (PEG4600) were supplied by Aldrich and used as received. Monomethyl ether of PEG5000 and Pseudomonas lipase (40 U/mg) were purchased from Fluka. Zinc powder was obtained from Merck.
10.1021/bm010168s CCC: $22.00 © 2002 American Chemical Society Published on Web 04/04/2002
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Table 1. Molecular Characteristics of PCL/PEG Diblock and Triblock Copolymers acronym
type of PEG (M h n)
[CL]/[EO] (feed)
[CL]/[EO] (product)a
yield (%)
M h nb
M h nc
M h n/M h nc
PCL-PEG PCL-PEG-PCL
HO-PEG-OCH3 (5000) HO-PEG-OH (4600)
2.0 2.0
1.9 2.4
76.9 79.3
29200 32900
21000 28000
1.6 1.5
a The [CL]/[EO] molar ratio was determined by 1H NMR. b M hn ) M h n(PEG) + 114(M h n(PEG)/44)([CL]/[EO]). c Data obtained by SEC with respect to polystyrene standards.
Figure 1. SEC chromatograms of PCL-PEG and PEG5000.
Methods. PCL-PEG-PCL triblock copolymer was synthesized by bulk ring-opening polymerization of -caprolactone in the presence of dihydroxyl PEG4600, using zinc powder (0.05%) as catalyst. The caprolactone/ethylene oxide or [CL]/[EO] molar ratio was 2/1. Predetermined quantities of PEG (1.1 g), -caprolactone (5.82 g), and zinc (12.6 mg) were introduced into a flask. After degassing, PEG was melted at 100 °C and the flask was cooled in ice water. High vacuum (83%). Thermal properties of the various films were investigated by DSC in comparison with the corresponding PEG. The melting temperature (Tm) and melting enthalpy (∆H) of the various polymers are shown in Table 3. Tm of the two PEG polymers was in the range of 67-69 °C, i.e., slightly higher than that of PCL (65 °C). The melting enthalpy of PEG was
much higher than that of PCL. In the case of the copolymers, the melting peak resulted from the PCL phase as shown by X-ray diffraction (Figure 2), Tm and ∆H values being very close to those of the PCL homopolymer. The enzymatic degradation of the copolymer films was investigated in comparison with PCL. Weight loss data were collected after various degradation times. As shown in Figure 3, pure PCL degraded very fast. After 22 h, 67% of the initial weight was lost. After 46 h, weight loss attained 85%. PCLPEG-PCL showed initially a weight loss profile identical to that of PCL. However, its weight loss reached 97% after 46 h. PCL-PEG degraded initially more slowly than PCL and PCL-PEG-PCL. At the later stages, the rate of weight loss increased and reached 83% after 46 h. In the case of the control samples in the absence of enzyme, the three polymers showed negligible weight loss, thus excluding the contribution of hydrolytic degradation or solubilization of initially present PEG-rich chains. Table 4 shows the thermal property changes of PCL and the copolymers during degradation. The melting enthalpy of PCL largely increased from an initial 39 to 54 J/g after 22 h, and to 68.6 J/g after 54 h. Its Tm also slightly increased. The control sample showed similar increase of ∆H and Tm, indicating that PCL crystallized in the degradation medium at 37 °C, which is much higher than its Tg (-65 °C). This phenomenon can be explained by the fact that initial crystallization of PCL film was far from complete during the solvent evaporation process. ∆H and Tm of PCL-PEG greatly increased after 22 h due to crystallization. After 54 h, however, a decrease of ∆H and Tm was observed. It is of interest to note the presence of a double melting peak on the thermogram of PCL-PEG after 22 h. The control sample exhibited also a double melting peak. Similar changes were observed for PCL-PEG-PCL (Table 4). Therefore, it could be assumed that crystallization of the polymers occurred during degradation, but the enzymatic attack eroded both the amorphous and crystalline zones. It is of interest to compare our results with literature data.11,12 Gan et al. reported that crystallinity of PCL films decreased during enzymatic degradation as DSC curves showed a steady decrease of the total melting enthalpy and that X-ray diffraction spectra showed a decrease of diffraction peak area.11 Unfortunately, the authors did not mention the
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Table 4. Thermal Property Changes of PCL and PCL/PEG Copolymers with Degradation PCL
PCL-PEG
PCL-PEG-PCL
time (h)
Tm (°C)
∆H (J/g)
time (h)
Tm (°C)
∆H (J/g)
time (h)
Tm (°C)
∆H (J/g)
0 22 54 b
65 68 66 69
39 54 69 66
0 22 54 b
66 67, 69a 65 65, 68a
37 72 51 67
0 22 54 b
65 66 66 66
36 56 39 61
a
double melting peak. b Control.
Figure 4. (a) ESEM micrograph of the upper face of PCL-PEG-PCL film before degradation. (b) ESEM micrograph of the lower face of PCL-PEG-PCL film before degradation. (c) ESEM micrograph of the upper face of PCL-PEG-PCL film after 22 h of degradation by Pseudomonas lipase. (d) ESEM micrograph of the lower face of PCL-PEG-PCL film after 22 h of degradation by Pseudomonas lipase.
quantity of material used for these measurements or give the melting enthalpy (J/g) changes of the products. In fact, the decreases of peak areas observed on DSC and X-ray curves did not necessarily reflect a decrease of crystallinity as the weight loss of PCL films was very rapid during degradation. According to the weight loss curve reported by Gan et al.,11 weight loss reached nearly 90% after 35 h and 95% after 54 h. Furthermore, the initial weight of the films was about 10 mg only. Therefore, very little material remained for DSC and X-ray diffraction measurements. In consequence, the authors’ conclusion that crystallinity of PCL films decreased during enzymatic degradation seems questionable. Chemical composition changes of the copolymers were followed by 1H NMR. After 30 h in the presence of lipase, the [CL]/[EO] ratio of PCL-PEG increased from initially
1.9 to 2.3, while the [CL]/[EO] ratio of PCL-PEG-PCL remained unchanged (2.4). The relative stability of the chemical composition during degradation can be explained by the fact that, on one hand, only PCL segments are degraded but, on the other hand, soluble PEG or PEG-rich segments can escape from the bulk and dissolve in the degradation medium. Surface morphology changes were followed by ESEM, which is a technique of choice to monitor polymer degradation because it does not necessitate high vacuum or gold coating which could result in artifacts. Figure 4 shows the ESEM micrographs of PCL-PEG-PCL. The two faces of the film presented different morphologies as previously reported.13 The upper face, which was in contact with air during solvent evaporation, presented rather large spherulites of about 100 µm with clearly distinguishable boundaries
Enzymatic Degradation of PCL/PEG Block Copolymers
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Figure 5. (a) ESEM micrograph of the upper face of PCL-PEG film before degradation. (b) ESEM micrograph of the lower face of PCL-PEG film before degradation. (c) ESEM micrograph of the upper face of PCL-PEG film after 22 h of degradation by Pseudomonas lipase. (d) ESEM micrograph of the lower face of PCL-PEG film after 22 h of degradation by Pseudomonas lipase.
(Figure 4a), while the lower face showed smaller spherulites of about 50 µm (Figure 4b). After 22 h of degradation by lipase, the upper face appeared strongly eroded with a very rugged pattern (Figure 4c). Fibrillar and spongelike structures were observed. The lower face was largely degraded with a spongelike structure (Figure 4d). The boundaries between the spherulites disappeared. However, a number of more or less degraded spherulites were still present, indicating that all the spherulites were not degraded at the same rate. In the case of PCL-PEG, the upper face exhibited initially a number of pores and cavities, while the lower face was full of spherulites (Figure 5a and Figure 5b). After 22 h of degradation, both faces appeared eroded (Figure 5c and Figure 5d). However, PCL-PEG seemed less degraded than PCL-PEG-PCL from ESEM observations (Figure 4 and Figure 5). This is in agreement with weight loss data (Figure 3). The difference between the initial weight loss rates of PCL-PEG and PCL-PEG-PCL could be assigned to the different surface morphologies. Both faces of PCL-PEGPCL films were full of spherolites formed by PCL blocks, which were rapidly degraded by enzymes. Insofar as PCL is concerned, the upper face exhibited spherulites of 50100 µm (Figure 6a). After degradation, fibrillar structures were observed with clearly distinguishable boundaries as previously reported (Figure 6b).13
In a previous paper,13 we considered the enzymatic degradation of PCL/PLLA blend systems with the proportions of 100/0, 75/25, 50/50, 25/75, and 0/100, in the presence of Pseudomonas lipase or proteinase K. It was observed that the presence of semicrystalline PLLA strongly reduced the enzymatic degradation of PCL in the blends. This finding was assigned to the phase separation between both components, PLA microdomains preventing the PCL phase from further enzymatic degradation after initial surface attacks. In the cases of PCL/PEG block copolymers, however, it appears that the presence of PEG blocks does not alter the enzymatic degradation of PCL blocks. This finding could be attributed to the hydrophilicity of PEG blocks which allowed their solubilization once released from the PCL blocks. Conclusions PCL-PEG and PCL-PEG-PCL block copolymers were successfully synthesized by ring-opening polymerization of -caprolactone in the presence of monohydroxyl or dihydroxyl poly(ethylene glycol). The resulting PCL-PEG diblock and PCL-PEG-PCL triblock copolymers were semicrystalline polymers. Only the PCL component crystallized due to their high PCL content. Incorporation of
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the enzymatic degradation of PCL was not altered by the presence of PEG for both the diblock and triblock copolymers probably due to the hydrophilicity of PEG blocks. References and Notes
Figure 6. (a) ESEM micrograph of the upper face of PCL film before degradation. (b) ESEM micrograph of the upper face of PCL film after 22 h of degradation by Pseudomonas lipase.
PEG strongly increased the hydrophilicity of the copolymers as compared to a PCL homopolymer. On the other hand,
(1) Li, S.; Vert, M. Biodegradable polymers: polyesters. In The Encyclopedia of Controlled Drug DeliVery; Mathiowitz, E., Ed.; John Wiley & Sons: New York, 1999; pp 71-93. (2) Dunn, R. L. Clinical applications and update on the poly(R-hydroxy acids). In Biomedical Applications of Synthetic Biodegradable Polymers; Hollinger, J. O., Ed.; CRC Press: Boca Raton, FL, 1995; pp 17-31. (3) Li, S. J. Biomed. Mater. Res.: Appl. Biomat. 1999, 48, 342-353. (4) Mochizuki, M.; Mukai, K.; Yamada, K.; Ichise, N.; Murase, S.; Tokiwa, Y. Macromolecules 1997, 30, 7403-7407. (5) Mayer, J. M.; Kaplan, D. L. Trends Polym. Sci. 1994, 2, 227-235. (6) Jarrett, P.; Benedict, C.; Bell, J. P.; Cameron, J. A.; Huang, S. J. Mechanism of the biodegradation of polycaprolactone. In Polymers as Biomaterials; Shalaby, S. W., et al., Eds.; Plenum Press: New York, 1984; pp 181-192. (7) Lefebvre, F.; David, C.; Vander Wauven, C. Polym. Degrad. Stab. 1994, 45, 347-353. (8) Akahori, S. I.; Osawa, Z. Polym. Degrad. Stab. 1994, 45, 261-265. (9) Fukuzaki, H.; Yoshida, M.; Asano, M.; Kumakura, M. Polymer 1990, 31, 2006-2014. (10) Mochizuki, M.; Hirano, M.; Kanmuri, Y.; Kudo, K.; Tokiwa, Y. J. Appl. Polym. Sci. 1995, 55, 289-296. (11) Gan, Z.; Liang, Q.; Zhang, J.; Jing, X. Polym. Degrad. Stab. 1997, 56, 209-213. (12) Gan, Z.; YU, D.; Zhong Z.; Liang, Q.; Jing, X. Polymer 1999, 40, 2859-2862. (13) Liu, L.; Li, S.; Garreau, H.; Vert, M. Biomacromolecules 2000, 1, 350-359. (14) Cerrai, P.; Guerra, G. D.; Lelli, L.; Tricoli, M.; Sbarbati Del Guerra, R.; Casone, M. G.; Giusti, P. J. Mater. Sci.: Mater. Med. 1994, 5, 33-38. (15) Petrova, T.; Manolova, N.; Rashkov, I.; Li, S.; Vert, M. Polym. Int. 1998, 45, 419-426. (16) Li, S.; Garreau, H.; Vert, M.; Petrova, T.; Manolova, N.; Rashkov, I. J. Appl. Polym. Sci. 1998, 68, 989-998.
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