Biomacromolecules 2001, 2, 373-377
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Biodegradation of [3H]Poly(E-caprolactone) in the Presence of Active Sludge Extracts Ste´ phanie Ponsart, Jean Coudane,* Benjamin Saulnier, Jean-Louis Morgat, and Michel Vert Centre de Recherche sur les Biopolyme` res Artificiels, UMR CNRS 5473, University of Montpellier I, Faculty of Pharmacy,15, Av Charles Flahault, 34060 Montpellier Cedex 2, France Received April 19, 2001
Poly(-caprolactone), PCL, is a commercial biodegradable and biocompatible polyester that can be bioassimilated by outdoor microorganisms. For biomedical and environmental applications, monitoring the fate of degradation products in vivo or under environmental conditions is one of the critical steps to evaluate degradation characteristics. [3H] radiolabeling is the best method to monitor the fate of degradable polymer chains in contact with complex living systems and to show bioassimilation. Therefore, tritiated PCL was synthesized by chemical modification using anionic activation by reaction with lithium diisopropylamide. The resulting radioactive polymer was characterized and allowed to degrade at 37 °C under aerobic conditions in the presence of active sludge. Comparison was made with abiotic hydrolytic degradation in pH ) 7.4, 0.13 M phosphate buffer at 37 °C. Water-soluble degradation products were assessed by measuring radioactivity in the solution phase. It was shown that biodegradation of PCL started after a few hours and proceeded up to the ultimate stage over ca. 72 days, giving tritiated water (80-90%) and biomass. Radioactivity detection appeared much more sensitive than measurement of CO2 production or consumption to monitor degradation phenomena. In particular, it showed that the onset of biodegradation occurs earlier than that reported using respirometry. Introduction Presently, there is an increasing demand from the biomedical, pharmacological, and environmental sectors of human activities for degradable, biocompatible, and biofunctional polymeric compounds that can be degraded and ideally bioassimilated and mineralized when placed in contact with living systems.1-3 Aliphatic polyesters are among the most promising candidates to replace biostable plastics, especially regarding devices such as fibers and films. Among aliphatic polyesters, poly(lactic acid), PLA, poly(glycolic acid), PGA, poly(lactic acid-co-glycolic acid) PLAGA, poly(-caprolactone), PCL, poly(β-hydroxybutyric acid), PHB, poly(hydroxybutyric acid-co-hydroxyaleric acid), PHBHV, polydioxanone, PDS, poly(β-malic acid), PMLA, and poly(ortho esters), POE, have been extensively evaluated.4-13 Poly(-caprolactone) was used in the medical field to make drug delivery devices,14,15 because of its excellent permeability to drugs related to its low glass transition temperature. In the agricultural field, it has been used for growing and transplanting trees12 and as thin-walled tree seedling containers.13 PCL is also known to have good mechanical thermoplastic properties. Like many other polyesters PCL is sensitive to chemical hydrolysis, but the rate of hydrolysis is very slow, especially if one compares with other polyesters such as PLA and PLAGA.6,15 Despite the fact that many researchers investigated its interest for application as bio* To whom correspondence may be addressed. E-mail: JCOUDANE@ PHARMA.UNIV-MONTP1.FR.
medical device and drug delivery systems, whether PCL is biodegradable, i.e., degradable in vitro or in vivo by human free enzymes or by enzyme-producing cells, is still a matter of discussion. In contrast there is no doubt that PCL is biodegraded by microorganisms. The biodegradability of PCL has already been observed in the presence of many microorganisms, including some from compost of household refuse,16 from landfill or sewer sludge,17,18 from natural waters,5 and from pure cultures of fungus or yeast.16,17,19 So far the biodegradation of PCL was investigated by monitoring the biochemical oxygen demand (BOD) under aerobic conditions and the changes of physicochemical properties, molecular weight, and sample weight.5,17,20 Biodegradation was also deduced from the measurement of the amount of CO2 or CH4, released by microorganisms growing under aerobic or anaerobic conditions, respectively.5,13,17,20 For the balance of the carbon material, the biomass, the aqueous medium must be taken into account to characterize the biodegradability of the sample. The resulting data are sometimes difficult to interpret because of rather poor reproducibility.13,20,21 In this paper we wish to report the results of an attempt to revisit PCL degradation in aqueous medium containing active sludge under aerobic condition, by using a radiolabeled polymer and comparison with simple hydrolytic degradation. The tritiated PCL was synthesized according to a previously described method based on the chemical activation of PCL by formation of a copolycarbanion using lithium diisoprop-
10.1021/bm015549k CCC: $20.00 © 2001 American Chemical Society Published on Web 05/11/2001
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ylamide.22 For years radioactivity has been known as a powerful method to investigate the fate of macromolecules or small molecules in contact with living systems. To our knowledge, tritiated PCL was never used to investigate biodegradation by β-scintigraphy. Our results were compared with those obtained by respirometry as described in the literature.20 Experimental Section (A) Chemicals. PCL (Mn ) 53 700; Mw ) 80 000) was obtained from Aldrich (Milwaukee, WI), and LDA (lithium diisopropylamide, 2 M in THF/n-heptane) was purchased from ACROS Organics (Geel, Belgium). All these compounds were used as received. NH4Cl and MgSO4 from Prolabo (Paris, France), CH2Cl2 from Riedel de Hae¨n (Seelze, Germany), and CH3OH from Carlo Erba (Milano, Italy) were also used as received. THF from BDH Laboratory Supplies (Poole, England) was distilled on benzophenone/sodium until a deep blue color was obtained. HTO, specific activity: 3.7 GBq/g (100 mCi/g) was purchased from NEN Life Science Products (Boston, MA) and was used as received. (B) Methods. (1) Tritiation Procedure. A solution of PCL (5.7 g, 0.05 mol in monomer unit) dissolved in 200 mL of anhydrous THF was introduced into a 500-mL threenecked round-bottomed flask and kept under a flow of argon. The gas was dried by passing through three gas-drying units containing sodium hydroxide pellets, molecular sieves and silica gel, respectively. The solution was magnetically stirred and the temperature kept at -78 °C using a dry ice/acetone mixture. A commercial solution of LDA, 2 M in THF/nheptane (6.25 mL, 0.0125 mol: 0.25 equiv/monomeric unit) was introduced with a syringe through a septum. The mixture was kept at -78 °C under stirring for 30 min. A 250-µL portion of HTO in 5 mL of THF (0.0139 mol of HTO: 66.6 MBq/mmol ≡ 1.8 mCi/mmol) was introduced into the reaction flask through a septum, and the stirred reaction solution was kept for a further 30 min at -78 °C. After return to room temperature an aqueous solution of ammonium chloride (10 g of NH4Cl in 200 mL of water) was added to the stirred reaction mixture. The alkaline mixture (pH ≈ 9) was acidified using a 37% aqueous HCl solution to pH ≈ 7. The tritiated polymer was extracted twice with 100 mL of dichloromethane. The combined organic phases were then washed twice with 100 mL of distilled water and dried over anhydrous MgSO4. After filtration of the hydrated salt, the solvent was partially evaporated under reduced pressure and the polymer was precipitated by addition of methyl alcohol. The coacervate phase was washed with methyl alcohol until the washing solution was clear. The collected tritiated polymer was finally dried under vacuum for several hours. The specific radioactivity of the [3H] PCL was 152 µCi/g. (2) Preparation of PCL Films. Films were prepared by evaporation under vacuum of a solution composed of 100 mg of tritiated PCL in 2 mL of CHCl3. The thickness of the films was ca. 0.08 mm as measured using a micrometer, and the specific radioactivity was 15.2 µCi.
Ponsart et al.
(3) Abiotic Degradation. The powdered tritiated PCL (15.2 µCi/100 mg) was dispersed in 10 mL of phosphate buffer (0.13 M, pH ) 7.4, NaN3 ) 0.02%) and was stored in an oven at 37 °C. The radioactivity was assessed at selected times. Films of tritiated PCL (15.2 µCi/100 mg) were placed in 50 mL of phosphate buffer (0.13 M, pH ) 7.4, NaN3 ) 0.02%) and underwent the same treatment. (4) Biotic Degradation. (a) Medium. The 200 mL of inorganic basal medium used for degradation experiments consisted of 2.3 mM KH2PO4, 4.5 mM Na2HPO4, 18.7 mM NH4Cl, 4.15 mM MgSO4, 450 µM CaCl2, and 31 µM FeCl3. The final pH was 6.9. (b) Inoculum. The activated sludge was collected from the Cereire`de (France) sewage plant and processed according to literature.16 The concentration in dry activated sludge was 1.1 g/L. The activated sludge was kept stirring under an air flow overnight and added to the incubation medium to achieve a 0.275% (v/v) final concentration. (c) Incubation. The tritiated PCL films (15.2 µCi/100 mg) were placed in a round-bottom flask in the presence of 200 mL of incubation medium at 37 °C in aerobic condition. The radioactivity was assessed occasionally and capillary zone electrophoresis (CZE) was used to monitor the eventual presence of oligomers. (5) Radioactivity Measurement. The radioactivity of the degradation medium was determined using a Liquid Scintillation Analyzer, Tri-Carb 2100 TR Packard. Typically, 100 µL, in the case of biotic degradation, and 50 µL, in the case of abiotic degradation, were added to 7 mL of Ecolite and counted. The radioactivity of the residual biomass was determined with the same equipment, but the biomass was dissolved in 5 mL of Soluene 100 (Packard) maintained under stirring for a few hours. A 100-µL aliquot was then added to 5 mL of Hionic-Fluor and counted. (6) Capillary Zone Electrophoresis. Data were collected using a P/ACE 5000 Beckman instrument equipped with UV detection at 254 nm. A fused-silica capillary (50 µm i.d.) was used with reverse mode. The capillary was conditioned every day by successive rinsing, namely, 30 min with 1 M HCl, 1 min with water, 30 min with 1 M NaOH, and finally 5 min with p-anisate/TRIS buffer. Furthermore, the capillary was rinsed before each run, first 5 min with 0.1% DEAE dextran in buffer, then 0.5 min with water, and finally 3 min with p-anisate/TRIS buffer. The migration electrophoregrams were developed at 25 °C with an applied voltage of 25 kV. After the last run of the day, the capillary was rinsed 30 min with 1 M NaOH and then 5 min with water and finally stored wet. For assessments, samples were injected in the capillary by the hydrostatic pressure method (10 s). The effective electrophoretic mobility of each resolved oligomer was determined by mathematical treatment.24 Results and Discussion The follow-ups of abiotic and biotic degradations of tritiated PCL were based on measurements of the radioactivity present in the solution phase of the aging media. The
Polymer Biodegradation
Figure 1. Percentage of the radioactivity initially present in solid PCL that was released as soluble compounds in the phosphate buffer during abiotic hydrolytic degradation: O, powder; 9, film.
detection of water-soluble byproducts allowed us to determine the percentage of radioactivity that was released by the solid phase into the solution phase. Abiotic Degradation. The hydrolytic degradation was achieved on powdered tritiated PCL (152 µCi/g) dispersed in stirred phosphate buffer saline at 37 °C. The powder was used instead of a film in order to relatively increase the degradation rate, which is known to be slow. However we also performed the same experiment with a PCL film in order to compare film and powder behaviors during the first hours of hydrolytic degradation. Typical radioactivity release profiles are shown in Figure 1. For both forms, a small release of radioactivity (≈0.7% for a film and ≈3% for the powder) was observed in the very first hours. These releases were assigned to water-soluble radioactive small molecules initially present in the tritiated PCL. The difference between film and powder was attributed to the difference of processing. Films were prepared by solvent evaporation under vacuum, a method that dragged off residual radioactive volatiles. In the case of the powder form (Figure 1) there was almost no more loss of radioactive material for the next
Figure 2. Device for biotic degradation study.
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200-day period, in agreement with the well-known resistance of PCL to abiotic hydrolysis. For the film, the release of soluble radioactive compounds was not investigated longer than 24 h, a plateau being already reached at that time. Biotic Degradation. The setup shown in Figure 2 was used to monitor the biodegradation of tritiated PCL films (15.2 µCi/100 mg) that were placed inside a ring and introduced in a nylon bag to avoid mechanical erosion upon stirring. The loaded nylon bag was introduced in the reactor containing the degradation medium kept at 37 °C and flashed with air flow. The volatile products were condensed through a condenser kept at 5 °C and followed by two other successive condensing flasks maintained at 5 °C too, only one of these flasks being represented in Figure 2. The assay was triplicated under similar conditions. Quantification of the released radioactivity was simply achieved by withdrawing aliquots of the solution phase, without any filtration or purification. The radioactivity of the aliquots was counted using a Liquid Scintillation Analyzer. Figure 3 shows the percentage of the initial radioactivity found in the reaction medium for the three attempts. For the sake of comparison, the data obtained in the case of abiotic degradation of the PCL film were also plotted to compare the first hours of the radioactivity release. Figure 3 shows that 75-90% of the initial radioactivity was released in the solution within 45 days, depending on the run. There was no solid PCL remaining in the reaction medium after 72 days. Between 40 and 72 days the release leveled off at ca. 80-90%. The differences between the three tests were in the range of those reported in the literature with modified Sturm tests. According to BOD measurement a ca. 5-day lag period is usually observed when PCL is allowed to age in the presence of active sludge.20 According to data reported in Figure 3,
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Figure 3. Proportion of the radioactivity initially present in PCL films that was released as soluble compounds in the incubation medium during biotic degradation (triplicate experiment) (s) and during abiotic hydrolytic degradation (- - -).
3% and 10% of the initially present radioactivity were released after 22 h and 3 days, respectively. These values were well above the residual radioactivity generated in the solution phase during abiotic degradation of a similar radioactive PCL film (Figure 3 dotted line). This finding suggested that enzymatic degradation began soon after incubation started. Therefore radioactive detection appeared much more sensitive than BOD measurements. Moreover, water-soluble radioactive byproducts were formed immediately, showing that PCL started degrading from the very beginning, with a very short lag time, if there was any. Aliquots of the aging medium were collected between 16 and 65 days. They were analyzed by CZE using the reverse mode,23 and data were compared to the CZE electropherogram of PCL oligomers prepared by opening the lactone and by polycondensation.24 During the first 16-day incubation no water-soluble oligomer was detected. As no oligomer greater than tetramer of hydroxycaproı¨c acid is hydrosoluble, according to CZE,24 the radioactive species present in the medium were not PCL oligomers but more likely tritiated water. Only a very low proportion of radioactivity was found in the first condensing flask (
