STUDY ON THE DEGRADATION OF POLYCAPROLACTONE FILMS IN PHOSPHATE BUFFER SOLUTION CONTAINING ASPERGILLUS SP. LIPASE

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1 STUDY ON THE DEGRADATION OF POLYCAPROLACTONE FILMS IN PHOSPHATE BUFFER SOLUTION CONTAINING ASPERGILLUS SP. LIPASE SOŇA HERMANOVÁ 1,2, JIŘINA OMELKOVÁ 3, STANISLAVA VOBĚRKOVÁ 4, RADKA BÁLKOVÁ 1,2, LUKÁŠ RICHTERA 1,2, LUDMILA MRAVCOVÁ 5 and JOSEF JANČÁŘ 1,2 1 Institute of Materials Chemistry, Faculty of Chemistry, Brno University of Technology, Purkyňova 118, Brno, Czech Republic (hermanova-s@fch.vutbr.cz) 2 CEITEC, Brno University of Technology, Technická 3058/10, Brno, Czech Republic 3 Institute of Food Science and Biotechnology, Faculty of Chemistry, Brno University of Technology, Purkyňova 118, Brno, Czech Republic 4 Institute of Chemistry and Biochemistry, Faculty of Agronomy, Mendel University in Brno, Zemědělská 1/1665, , Czech Republic 5 Institute of Chemistry and Technology of Environmental Protection, Faculty of Chemistry, Brno University of Technology, Purkyňova 118, Brno, Czech Republic Abstract: Poly(ε-caprolactone) films with Mn = 18 kda obtained by compression moulding (CM) and solution casting (SC) were subjected to Aspergillus oryzae (AO) lipase action in a phosphate buffer at ph 7 and at 37 C. The appearance of randomly oriented cracks on the surface of incubated PCL films accompanied by a decrease in weight-average molecular weight (M w ) by 10% was observed during a 42-day degradation period. Based on the increase in crystallinity, degradation proceeds in amorphous phase of aged films. The degradation proceeds somewhat faster in SC films probably due to better accessibility of ester bonds in amorphous phase of spherulites. Key words: poly(ε-caprolactone), DSC, CLSM, degradation, Aspergillus sp., lipase INTRODUCTION Poly(ε-caprolactone) (PCL), a representative of aliphatic polyesters, is one of the most promising biodegradable and biocompatible semicrystalline polymers (TOKIWA et al., 1990). PCL has been used for temporary therapeutic applications such as matrices of sustained drug delivery systems, surgical sutures, scaffolds for tissue regeneration, etc.. (COOMBES et al., 2004; WILLIAMSON and COOMBES, 2004). PCL becomes attractive material also in the field of environmental applications for a biodegradation which can occur due to the action of microorganisms frequently present in an ecosystem. PCL-based materials have been used as mulch films in agriculture or as packaging material (MATZINOS et al., 2002). Extensive studies have been carried out also on the enzymatic chain-cleavage degradation of PCL in vitro by the action of lipase-type enzymes (ZENG et al., 2004; HERZOG et al., 2006; SEKOSAN et al., 2010). Lipases (triacylglycerol hydrolases, EC ) are naturally occurring biocatalysts capable of hydrolysis of triacylglycerols into glycerol and fatty acids at a lipid-water interface (SHARMA et al., 2011). According to the results, the PCL-degrading microorganism, which produces different types of lipases and esterases, represents one of the most important factor influencing the PCL degradation even when the degradation rate is related to the PCLs initial crystallinity and molecular weight. This work is focused on the study of the degradation of medium-molecular weight PCL homopolymer (Mn = 18 kda) exposed to Aspergillus oryzae lipase in the phosphate buffer solution at 37 C. The 1 H NMR analyses of the studied PCL homopolymer confirmed presence of both hydroxylic and benzoxylic end groups. 193

2 The Aspergillus sp. lipases are known to possess several properties of immense industrial importance, such as their ph and temperature stability (CONTESINI et al., 2010) and a few reports on their promising polyester degradation capability have been published (ALBERTSSON et al., 1998; RENSTAD et al., 1998; ELDSATER et al., 2000; SANCHES et al., 2000; HOSHINO and ISONO 2002; LOVERA et al., 2007). The PCL samples were prepared in a form of films (Ø 1 cm, 100 µm) by solution casting (SC) and by compression moulding (CM) to study the effect of processing as well. PCL samples were weighted and analyzed by differential scanning calorimetry (DSC), gel permeation chromatography (GPC) and confocal laser scanning microscopy (CLSM) after predetermined time intervals to evaluate changes in PCL structure. MATERIALS AND METHODS Aspergillus sp. lipase assay Aspergillus oryzae lipase (WE-Nr ) was obtained from Mucos Pharma. Lipolytic activity was determined using UV/VIS HELLIOS spectrophotometer (DELTA Thermospectronic, England) using p-nitrophenyl-laurate (pnpl) as a substrate, which was dissolved in ethanol. The reaction mixture consisted of 0.25 ml enzyme solution (0.5 mg/ml), 3.25 ml of 0.05 M phosphate buffer (ph = 7) and 0.25 ml of 2.5 mm pnpl in ethanol. The hydrolytic reaction was carried out at 37 C for 30 min and afterwards, 0.5 ml of 0.1M Na 2 CO 3 was added to stop the reaction. Subsequently, the absorbance at 420 nm was recorded. One unit of lipase activity (U) was defined as the amount of enzyme that caused the release of 1 μmol of p-nitrophenol from pnpl in one min under the test conditions. Degradation study The degradation study was carried out in a phosphate buffer solution at ph = 7. PCL films were placed in a test tube containing 3 ml of 0.05 M phosphate buffer and a 0.5 mg/ml of Aspergillus oryzae lipase and then incubated at 37 C. The buffer/enzyme system was changed every 72 h in order to maintain the original level of enzymatic activity. After 14, 28 and 42 days of incubation, the films were removed, gently washed with distilled water and dried to the constant weight under vacuum. The control samples were treated in the same way; their incubation was carried out in a buffer without enzyme but sodium azide 0.02 % (w/w) was added to the solution to prevent a contamination. Tests DSC (Q 2000, TA Instruments) was used for determination of melting temperature and crystallinity of PCL samples weighted about 1.5 mg heated at 10 C/min to 100 C. GPC- Agilent Technologies 1100 Series device equipped with a refractive index (RI) detector, two PL gel mixed columns mm with particle size of 5 μm was used to determine molecular weights. CLSM (Lext OLS 3000, Olympus) was used to reveal (bio)degradation pattern of PCL samples surfaces. RESULTS AND DISCUSSION In this work, the degradation of poly(ε-caprolactone) (PCL) film as a lipophilic substrate by the action of Aspergillus oryzae (AO) lipase in a phosphate buffer solution (37 C, ph 7) was studied. The characteristics of PCL before and after the degradation test are summarized in Table 1. It was estimated that hydrolytic degradation of PCL did not occur in a significant extent because only negligible weight loss (<1 wt. %) of the control samples was measured even after 42 days. 194

3 The initial activity of AO lipase (determined spectrophotometrically using pnpl as a substrate) was U/mg of enzyme. Since the lipase under investigation retained only 20 % of its activity at 37 C and ph of 7 after 72 hours, the buffer/enzyme system was regularly changed every 72 h till the end of experiment (42 days). It is worth noting that the optimum ph is 7 and the temperature optimum is 37 C for AO lipase. The comparison of the obtained data with those reported in literature represents demanding task namely because of different lipase assays used. Nevertheless, the obtained data corresponds well with those reported by CARVALHO et al, They found that the optimal ph for lipase from Aspergillus oryzae (AC-122) was in the range of and the optimum temperature at C. Table 1. Characteristic data of PCL before and after degradation experiment. Sample* Period Weight loss M n (GPC) M w (GPC) M w / M n **W 1st c Tm days % [kda] [kda] [%] [ C] SC_PCL CM_PCL SC_14d SC_28d SC_42d CM_14d CM_28d CM_42d * SC solution casting, CM-compression moulding, PCL - poly(ε-caprolactone) ** W 1st c : crystallinity calculated from the first heating as a ratio of ΔHm/ΔHm 0, where ΔHm 0 of PCL is J/g 195 It can be seen from Table 1 that the weight loss of both PCL samples obtained by compression moulding (CM) and solution casting (SC) increases with degradation time up to 3.6 wt. % and 5.8 wt. %, respectively, and the weight-average molecular weight M w of both types of samples gradually decreased by 10% during 42 days in comparison with the original PCL film. These results lead to the idea that different processing of PCL films did not have significant effect on enzymatic degradation process but DSC data showed the difference after 42 days. The increase of crystallinity as well as of melting temperature during the degradation test points up the change of PCL matrix structure as a consequence of the AO lipase action. It can be assumed that the AO lipase preferentially attacked the amorphous phase where the macromolecules are loosely packed and hence more susceptible to degradation. Further, once the degradation initiates, the accumulation of water-soluble oligomeric species (the chain scission products of polymer) causes an osmotic inflow of water accelerating the degradation process. Consequently, pores formed during progressive degradation process make possible to release oligomers and monomers which lead to the weight loss of material (XU et al., 2006) and microscopic cracks start to be visible on the exposed sample surface. Microscopic cracks were observed both on degraded CM and SC films (Fig. 1) but not on control samples which, together with gradual weight loss, confirmed the occurrence of surface erosion. If one compares the data of CM and SC samples after 42 days, there is a big difference both in crystallinity and surface morphology. The reason is the most probably a little different initial structure and surface morphology of CM and SC films, which seemed to have significant influence on the AO lipase action. It is evident that AO lipase attacks amorphous phase but the degradation process seems to be selective due to the different surface of SC

films which is formed by spherulites while the surface of CM samples is amorphous.

crystallinity increase by approx. 25 % and somewhat higher weight loss.

CLSM micrographs showing the PCL surface 1) original CM film, 2) CM PCL after 42 days of degradation, 3) control CM PCL after

42 days in phosphate buffer solution CONCLUSION The study of enzymatic degradation of PCL film (Mn = 18 kda) by the action of

4 films which is formed by spherulites while the surface of CM samples is amorphous. The chain scission process in SC film is assumed to be easier in amorphous phase of spherulites as documented by significant crystallinity increase by approx. 25 % and somewhat higher weight loss. On the other hand, the degradation on CM surface seems to be hindered even if the surface is amorphous and the attack is flatness. Also only sporadic cleavage of bonds in amorphous surface can only lead to higher increase of crystallinity as compared to SC films. 196 Fig. 1. CLSM micrographs showing the PCL surface 1) original CM film, 2) CM PCL after 42 days of degradation, 3) control CM PCL after 42 days in phosphate buffer solution, 4) original SC film, 5) SC PCL after 42 days of degradation and 6) control SC PCL after 42 days in phosphate buffer solution CONCLUSION The study of enzymatic degradation of PCL film (Mn = 18 kda) by the action of Aspergillus oryzae (AO) lipase in phosphate buffer (ph= 7) at 37 C was performed in this work. Based on the results obtained we suggest that the enzymatic hydrolysis is a surface erosion process where the PCL chains are degraded initially by the action of AO lipase as seen from the drop in weight-average molecular weight M w. Subsequently, the surface erosion occurred because of the development of randomly oriented cracks on the surface of both PCL films obtained by compression moulding and by solution casting accompanied by the weight loss in comparison with the unchanged control samples.

5 ACKNOWLEDGEMENT 197 This work was supported by the Ministry of Education, Youth and Physical Training of the Czech Republic under the research project no. MSM REFERENCES: ALBERTSSON, A.C., ELDSATER, C., ERLANSSON, B., KARLSSON, S., RENSTAD, R.: Effect of processing additives on (bio)degradability of film-blown poly(ε-caprolactone). J. Appl. Polym. Sci., 70, 1998, CARVALHO, P.O., CONTESINI, F.J., BIZACO, R., MACEDO, G.A.: Kinetic properties and enantioselectivity of the lipases produced by four Aspergillus species. Food Biotechnol., 19, 2005, CONTESINI, F.J., LOPES, D.B., MACEDO, G.A., NASCIMENTO, M.G., CARVALHO, P.O.: Aspergillus sp. lipase: Potential biocatalyst for industrial use. J. Mol. Catal. B: Enzym., 67, 2010, COOMBES, A.G.A., RIZZI, S.C., WILLIAMSON, M., BARRALET, J.E., DOWNES, S., WALLACE, W.A.: Precipitation casting of polycaprolactone for applications in tissue engineering and drug delivery. Biomaterials, 25, 2004, ELDSATER, C., ERLANDSSON, B., RENDSTAD, R., ALBERTSSON, A.C., KARLSSON, S.: The biodegradation of amorphous and crystalline regions in film-blown poly(εcaprolactone). Polymer, 41, 2000, HERZOG, K., MULLER, R.J., DECKWER, W.D.: Mechanism and kinetics of the enzymatic hydrolysis of polyester nanoparticles by lipases. Polym. Degrad. Stab., 91, 2006, HOSHINO, A., ISONO, Y.: Degradation of aliphatic polyester films by commercially available lipases with special reference to rapid and complete degradation of poly(l-lactide) film by lipase derived from Alcaligenes sp.. Biodegradation, 13, 2002, LOVERA, D., MÁRQUEZ, L., BALSAMO, V., TADDEI, A., CASTELLI, C., MULLER, J.: Crystallization, morphology and enzymatic degradation of polyhydroxybutyrate/polycaprolactone (PHB/PCL) blends. Macromol. Chem. Phys., 208, 2007, MATZINOS, P., TSERKI, V., KONTOYIANNIS, A., PANAYITOU, C.: Processing and characterization of starch/polycaprolactone products. Polym. Degrad. Stab., 77, 2002, RENSTAD, R., KARLSSON, S., SANDGREN, A., ALBERTSSON, A.C.: Influence of Processing Additives on the Degradation of Melt-Pressed Films of Poly(ε-Caprolactone) and Poly(Lactic Acid). J. Environ. Polym. Degrad., 6, 1998, SANCHES, J.G., TSUCHII, A., TOKIWA, Y.: Degradation of polycaprolactone at 50 C by a thermotolerant Aspergillus sp.. Biotechnolog. Lett., 22, 2000, SEKOSAN, G., VASANTHAN, N.: Morphological changes of annealed poly-ε-caprolactone by enzymatic degradation with lipase. J. Polym. Sci. Part B Polym. Phys., 48, 2010, SHARMA, D., SHARMA, B., SHUKLA, A.K.: Biotechnological approach of microbial lipase, a review. Biotechnology, 10(1), 2011, TOKIWA, Y., ANDO, T., SUZUKI, T., TAKEDA, K. in GLASS, J.E., SWIFT, G. (Eds.).: Agriculture and Synthetic Polymers, Biodegradability and Utilization. ACS Symposium Series 433, Am. Chem. Soc., Washington, DC, 1990, WILLIAMSON, M.R., COOMBES, A.G.A.: Gravity spinning of polycaprolactone fibres for applications in tissue engineering. Biomaterials, 25, 2004,

6 ZENG, J., CHEN, X., LIANG, Q., XU, X., JING, X.: Enzymatic degradation of poly(llactide) and poly(ε-caprolactone) electrospun fibers. Macromol. Biosci., 4, 2004, XU, H.J., Sy, J.C., SHASTRI, V.P.: Towards developing surface eroding poly(a-hydroxy acids). Biomaterials, 27, 2006,

THE EFFECT OF PROCESSING OF POLYCAPROLACTONE FILMS ON DEGRADATION PROCESS INITIATED BY ASPERGILLUS ORYZAE LIPASE

THE EFFECT OF PROCESSING OF POLYCAPROLACTONE FILMS ON DEGRADATION PROCESS INITIATED BY ASPERGILLUS ORYZAE LIPASE International Journal of Polymer Anal. Charact., 17: 465 475, 2012 Copyright # Taylor & Francis Group, LLC ISSN: 1023-666X print=1563-5341 online DOI: 10.1080/1023666X.2012.696402 THE EFFECT OF PROCESSING