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The susceptibility of poly(ether)urethanes to enzymatic degradation after oxidative pretreatment
Polymer Degradation and Stability 67 (2000) 171±178
The susceptibility of poly(ether)urethanes to enzymatic degradation after oxidative pretreatment Shan-hui Hsu a,*, Tsung-bin Huang b a b
Department of Chemical Engineering, National Chung Hsing University, Taichung, Taiwan 40227, ROC Department of Biomedical Engineering, Chung Yuan Christian University, Chung Li, Taiwan 32023, ROC Received 14 January 1999; accepted 5 March 1999
Abstract Susceptibility of poly(ether)urethanes (PEU) to hydrolytic degradation by enzymes after a limited exposure to free radicals was investigated. Two PEU, one chain extended with 1,4-butanediol and the other chain extended with 2-butene-1,4-diol, were used as model polymers. Each material was subjected to treatments including oxidative, enzymatic (with papain), and oxidative followed by enzymatic treatment. The degradative eect on surfaces was examined by optical microscopy, attenuated total re¯ectance infrared spectroscopy as well as XPS. It was found that enzymatic degradation occurred only after certain amount of oxidative change had been initiated. This was probably associated with enzyme adsorption on surfaces pre-exposed to oxidation. The extent of overall degradation was strongly in¯uenced by the oxidation stability of the materials. PEU chain extended with 2-butene-1,4-diol was more resistant to oxidation, and hence less susceptible to enzymatic degradation. It was concluded that exposure of PEU to oxidative pretreatment increased its susceptibility to hydrolysis by enzymes, and that oxidation followed by enzymatic attack could be the biodegradation mechanism of PEU in vivo. # 1999 Elsevier Science Ltd. All rights reserved.
1. Introduction Poly(ether)urethanes (PEU) have received extensive attention in the ®eld of biomaterials due to their excellent biocompatibility. However, in vivo as well as in vitro experiments [1±13] have demonstrated that PEU is susceptible to biodegradation. A number of hypotheses including two chemical mechanisms, oxidation [1±6] and enzymatic hydrolysis [5±11], have been proposed to explain the nature of biodegradation. Theoretically, killing substances such as H2O2 and .OH produced by macrophages and other phagocytes during respiratory burst may initiate the oxidation by free radical reactions [2]; lysosomal enzymes that are released from in¯ammatory cells could catalyze the hydrolysis [14]. Treating PEU with oxidative reagents [1,3,5,6] did reproduce several interesting features similar to those observed in vivo [2,4,12,13]. On the other hand, treating PEU with dierent enzymes led to a variety of results [5±11]. Most studies demonstrated very small amounts of degrada* Corresponding author. Tel.: +886-4-287-3181; fax:+886-4-2854734. E-mail address: [email protected] (S.-H. Hsu).
tion [7±9,11]. So far, evidence was insucient to determine the favorable mechanism in vivo [13]. However, it has been suggested that biodegradation may be the result of a synergistic environment, with the process being enhanced by factors such as pH and material dissolution [10], as well as plasma 2-macroglobulin [15]. Hydroperoxide or free radicals has been proved to mediate enzymatic hydrolysis of proteins [16,17]. A limited pre-exposure to free radicals increases the susceptibility of bovine serum albumin to hydrolysis by trypsin signi®cantly [17]. Such mediation may be due to the modi®cation of protein structure by hydroperoxide or free radicals. It is thus logical to think that oxidative pretreatment may have the same in¯uence on the susceptibility of PEU to enzymatic attack, which was the hypothesis behind the current study. Commercial products of medical-grade PEU, such as Pellethane and Biomer, usually have a very wide range of molecular weight distribution and signi®cant lot-tolot variations. These situations could result in dissolution of low-molecular-weight species in physiological environments and thus could play a role in varying results of biostability [6]. To avoid such complications,
0141-3910/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved. PII: S0141-3910(99)00110-X
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in a previous study, we synthesized two PEU ourselves and demonstrated that when 2-butene-1,4-diol instead of 1,4-butanediol was used as the chain extender in synthesis, the oxidation of the PEU by free radicals was reduced [18]. Here in this study, these two model polymers (one chain extended with 1,4-butanediol and the other chain extended with 2-butene-1,4-diol) were used to test the hypothesis that oxidative treatment could make PEU susceptible to enzymatic hydrolysis, as mentioned above. They were pretreated with free radicals, but less drastically than the condition employed in the previous study. The materials after limited free radical treatment were then subjected to a solution containing hydrolytic enzyme papain at the pH optimum condition for one month at 37 C. Papain was used due to its similar speci®city to cathepsin B [7], a lysosomal enzyme released by macrophages during the foreign body reaction. The degradative eect on surfaces was characterized using re¯ected optical microscopy, attenuated total re¯ectance infrared spectroscopy (ATR-IR) and XPS. The degree of degradation was compared with that of samples treated with other controlled environments, to de®ne the synergistic role of free radicals in the enzymatic degradation. 2. Experimental 2.1. Synthesis of PEU Two kinds of linear PEU, one (PEU A) chain extended with 1,4-butanediol and the other (PEU B) chain extended with 2-butene-1,4-diol, were synthesized by the conventional two-stage polymerization method. In the ®rst stage, MDI and polytetramethylene oxide (PTMO, M.W.=2000) at a molar ratio of 2:1 were reacted to form the prepolymer. The mixture was stirred at 60 C under dry nitrogen for approximately 6 h, until the amount of NCO reached the theoretical value determined by the dibutylamine back-titration method [19]. The prepolymer was then converted into the ®nal product by further reaction with the chain extender in dehydrated dimethylformamide (DMF) (40% solution). The reaction proceeded at 60 C overnight until the isocyanate disappeared, which was monitored by infrared spectrometry [20]. The products were puri®ed by extraction in methanol and stored in granular form. 2.2. Film casting and characterization The polymer was dissolved in DMF (25%) again and cast to a thickness of 0.5 mm on Te¯on substrates. Films were peeled from the substrates after being dried in a vacuum oven at 60 C, and were cut into pieces with a size 15 cm. The molecular weights were measured by gel permeation chromatography (GPC, Waters 410;
1 ml/min THF used as solvent). The surface was characterized by re¯ected optical microscopy (Nikon, Universal Type 104), attenuated total re¯ectance infrared spectroscopy (ATR-IR Spectrometer, Bio-Rad FTS-7; Ge crystal with an incident angle 45 ), and XPS (ESCA210 Spectrometer, VG Scienti®c; power of analysis 216W with take o angle 45 , Mg as the metal target of X-ray tube). The chemical composition of C, O and N elements was calculated from the XPS survey spectra. To analyze the chemical bonding states of the carbon atoms (carbon functional groups), the high-resolution C1S spectra were deconvoluted and curve-®t by the built-in software. The surface texture was scanned by an atomic force microscope using tapping mode probe at a scan size 11 mm (AFM, NanoScope III, Digital Instrument). 2.3. In vitro degradation test and surface characterization 10% H2O2 and 0.05 M CoCl2 was used as our oxidative pretreatment. PEU samples were immersed at 37 C for 1 week. The solution was changed every 3 or 4 days. This treatment was less drastic than that previously used [18]. After the treatment, the specimens were removed and rinsed thoroughly in deionized water. The transparency of the samples did not show apparent change visually. The surface morphology was observed by re¯ected optical microscopy. The change in the functional groups on the surface was examined using ATRIR and XPS. Samples were then immersed in papain solution. The papain (Sigma) had an activity 28 U/mg, determined by hydrolysis of benzoyl-l-arginine ethyl ester (BAEE) [21]. The papain was prepared to a concentration of 140 U/ml in the enzyme activating agent containing 0.05 M Cystein-HCl, 0.01M EDTA and 610ÿ4 M 2mercaptoethanol, pH adjusted to 6.2 by NaOH [21]. Samples were treated in the papain solution at 37 C for 1 month. The solution was changed every 3 or 4 days. The samples were cleaned by repeated sonication in 1% Triton X-100 (Aldrich) and in deionized water before characterization [9]. In addition, samples without oxidative pretreatment were immersed in papain solution for 1 month as controls. Some samples with oxidative pretreatment were soaked in activating buer as another set of controls. The degradative eect was characterized by using methods described above. For papain adsorption study, the surface of samples (untreated or pretreated) was cleaned thoroughly with deionized water before adsorption. The samples were immersed in papain solution for 72 h. Then the solutions were removed, and the samples were rinsed carefully with activating buer followed by deionized water (three rinses in total), and freeze-dried immediately for AFM imaging.
S.-H. Hsu, T.-B. Huang / Polymer Degradation and Stability 67 (2000) 171±178
3. Results and discussion For convenience of data presentation, the two PEU are designated as PEU A (chain extended with 1,4butanediol), and PEU B (chain extended with 2-butene1,4-diol). The molecular weights of these two PEU were comparable and were shown in Table 1. After immersion in 10% H2O2 and 0.05 M CoCl2 at 37 C for 1 week, the surface of both PEU under microscope showed limited amount of pitting, especially for PEU B, where almost no pitting was observed at magni®cation 100. A comparison between PEU A and PEU B exposed to oxidation is shown in Fig. 1(a) and (b). Also included is the original surface [Fig. 1(c)]. After subsequent enzymatic treatment, both PEU showed signi®cant surface changes [Fig. 2(a) and (b)] over the two controls, i.e. samples subjected to enzyme treatment for one month without oxidative pretreatment [Fig. 2(c)], and those subjected to oxidation followed by soaking in activating buer for one month [Fig. 2(d)]. In PEU A, extensive surface pitting was found [Fig. 2(a)]. The ATR-IR spectra of the untreated, oxidation-pretreated and oxidation plus enzyme-treated PEU samples
173
Table 1 Molecular weights of PEU Polymer
Mw a
Mn b
Polydispersity
PEU A PEU B
130 00018 000 100 00010 000
44 0002000 38 0001000
2.90.5 2.60.3
a b
Mw ; the weight average molecular weight. Mn ; the number average molecular weight.
are shown in Fig. 3. For both PEU A and PEU B, the 1week oxidation pretreatment only caused limited amount of surface degradation, especially for PEU B, where no evident change in spectra was noticed. However, enzymatic post-treatment caused degradation in PEU A, as indicated by an increase near 3400 cmÿ1 (O± H), as well as a lower intensity ratio of the two carbonyl bands at 1732 cm and 1702 cmÿ1 in the spectrum. In literature, the ratio was observed to decrease with the implantation time in vivo [13], and such decrease was attributed to the gradual loss of PTMO soft segment from the surface to the surrounding. Since PTMO was quite resistant to the hydrolysis, it was less likely that enzyme could attack PTMO directly. Indeed, no change of IR spectra was found for samples treated with
Fig. 1. The surface morphology of pretreated materials, as viewed by the optical microscope (100): (a) PEU A, after oxidative treatment; (b) PEU B, after oxidative treatment; and (c) PEU B, untreated.
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S.-H. Hsu, T.-B. Huang / Polymer Degradation and Stability 67 (2000) 171±178
enzyme without oxidative pretreatment (data not shown). Therefore, we assumed that free radicals exposure during the oxidative pretreatment had initiated certain amount of degradation, by oxidizing some ether to ester linkages; these esters might be hydrolyzed by papain (i.e. also an esterase [21]) afterwards. On the other hand, we found the IR spectroscopy was not sensitive enough to support the point that ester was produced in the ®rst treatment, because as mentioned, there was no apparent change in spectra after oxidation. The bands at 1102 cmÿ1 (C±O±C in ether) and at 1080 cmÿ1 (C±O±C in ester) remained similar to the original. In a previous study, however, where a stronger oxidative treatment was used, ester formation after oxidation was con®rmed by ATR-IR spectra [18]. Since ATR-IR could not detect early oxidation on the surface, we sought evidence from XPS. The XPS results were shown in Table 2. These data were calculated by the built-in software from the survey spectra as well as the high-resolution C1S spectra. The high-resolution C1S spectra (Fig. 4) could be deconvoluted into two major peaks, at 284.6 eV (hydrocarbon) and 286.0 eV (ether, alcohol) [22], as well as a small but
broad peak at 288.4±289.4 eV. Since the peak at 286.0 eV is indicative of the soft segment, and the small broad tail near 288.4±289.4 eV is primarily related to the hard segment, their ratio represents the relative contribution from the soft and hard segments. It was thus apparent that the polyether soft segment was enriched at the surface of the untreated PEU. This was con®rmed by the very small surface N/C atomic ratio observed, as well as the O/C atomic ratio that was close to the value estimated stoichiometrically from PTMO structure. After oxidative treatment, the O/C atomic ratio increased for both PEU (more for PEU A), indicating oxidation (oxygen inclusion) of the surface. From the calculated percentages for 286.0 eV peak area listed in Table 2, a signi®cant loss of ether from the surface was noted for both PEU after oxidative treatment, more severe in PEU A. The center position of the broad tail could also re¯ect the relative contribution from ester or acid (288.3±288.5 eV), and carbamate (288.8±289.4 eV). Indeed, as evident from Table 2 (or Fig. 4), a center at 288.6 eV, i.e. closer to ester or acid instead of carbamate, was observed on the oxidatively-treated surface. This suggested that some ester (or acid) was generated
Fig. 2. The surface morphology of post-treated materials, as viewed by the optical microscope (100): (a) PEU A, oxidation followed by 1-month papain treatment; (b) PEU B, oxidation followed by 1-month papain treatment; (c) PEU A, 1-month papain treatment only, without pretreatment; and (d) PEU A, oxidation followed by 1-month buer treatment.
S.-H. Hsu, T.-B. Huang / Polymer Degradation and Stability 67 (2000) 171±178
after oxidation, most likely at the a-carbon site adjacent to the ether group [23] in the soft segment. For samples after both oxidative and enzymatic treatments, more changes in XPS spectra were observed. The O/C atomic ratio further increased, especially for PEU A (to 0.32). We attributed the increase to the hydrolysis (oxygen inclusion due to H2O). Besides, the 288.6 eV peak shifted to 288.3 eV, i.e. even more con-
175
tribution from ester or acid. In this case, it was probably from acid, the hydrolytic product of the ester groups previously generated in the oxidation process. Also, the percentage of the 286.0 eV peak bounced back a little, suggesting alcohol formation, i.e. another hydrolytic product. A further comparison between the data of PEU A and PEU B (Table 2) clearly demonstrated that amount of degradative changes after enzymatic treat-
Fig. 3. The ATR-IR spectra of PEU samples after dierent treatments: curves marked ``untreated'', the spectra of untreated samples; curves marked ``oxidative'', the spectra of oxidation-pretreated samples; and curves marked ``oxidative+enzymatic'', the spectra of samples after oxidation followed by 1-month papain treatment: (a) for PEU A; and (b) for PEU B.
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Table 2 ESCA results after varieties of treatment Substrates
PEU A, untreated PEU A, oxidative only PEU A, oxidative and enzymatic PEU B, untreated PEU B, oxidative only PEU B, Oxidative and enzymatic a b
O/C atomic ratio
N/C
Fraction of C functional groups from high-resolution C1S peaks
Measured
Stoichiometrica
Measured
284.6 eV (hydrocarbon) (%)
286.0 eV (ether, alcohol) (%)
288.3±288.5 eV (ester, acid)
0.24 0.28 0.32 0.25 0.27 0.29
0.24b ± ± 0.24b ± ±
0.02 0.04 0.05 0.02 0.02 0.03
53.7 65.4 60.2 56.7 64.0 62.8
45.5 29.6 32.8 42.3 33.7 34.0
0.8% (broad 288.4±289.4 eV) 5.0% (center @ 288.6 eV) 7.0% (center @ 288.3 eV) 1.0% (broad 288.4±289.4 eV) 2.3% (center @ 288.6 eV) 3.2% (center @ 288.3 eV)
288.8±289.4eV (carbamate)
Stoichiometric ratios calculated from the known structure. Value estimated from the ploy(tetramethylene oxide) soft segment.
ment was determined by the extent of degradation during the previous oxidative treatment. Thus it was concluded from the XPS results that papain caused hydrolytic degradation in PEU pretreated with hydroperoxide, and that the susceptibility of PEU to enzy-
Fig. 4. High-resolution C1S ESCA spectra for PEU A after various treatments.
matic attack was associated with the limited amount of ester formation on the surface after exposure to free radicals. AFM images for the surfaces are shown in Fig. 5, each comprising a three-dimensional image (left) and a two-dimensional image (right). Samples after oxidation treatment had a surface texture (wavy patterns) similar to the original [Fig. 5(a)]. However, big shallow craterlike holes may appear in some regions [darker areas in Fig. 5(b)]. We attributed these to the degraded areas, since the edges of these areas seemed to be mechanically softer (blurred streaks in the middle of Fig. 5(b) are caused by the probe tip motion). Following the papain adsorption test, the surface contour changed [Fig. 5(c), for samples without oxidation]. But the change was still small and the surface was relatively clean (note that the surface depth was only 6 nm). On the other hand, the surface pre-exposed to oxidation had many sphere-like nanoparticles attached to the surface [Fig. 5(d)], which were believed to be papain aggregates. Such adsorption may be caused by non-speci®c interaction. Since the isoelectric point (pI) of papain (9.6) [21] was above the pH (6.2) of the activating buer, the papain should be positively charged in the solution. We postulated that after oxidation treatment, the surface was negatively charged due to some bond cleavage, and thus attracted positively charged papain molecules to the surface. Such binding may enhance enzyme activity and facilitate the hydrolytic reaction. Because limited change on the surface after oxidation could signi®cantly alter the susceptibility of PEU to enzymatic degradation, the overall biostability of the PEU could be enhanced by improving its oxidation stability, such as in the case of PEU chain extended with 1,4-butene-2-diol. Finally, since oxidative and enzymatic treatments were synergistic, we speculate that oxidation followed by enzymatic degradation could be an important degradation mechanism in vivo as well.
S.-H. Hsu, T.-B. Huang / Polymer Degradation and Stability 67 (2000) 171±178
177
Fig. 5. AFM images of the surfaces for: (a) untreated PEU A, before papain adsorption; (b) PEU A after oxidative treatment; (c) untreated PEU A, after papain adsorption test; and (d) PEU A after oxidative treatment, with papain adsorbed.
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S.-H. Hsu, T.-B. Huang / Polymer Degradation and Stability 67 (2000) 171±178
4. Conclusion Exposure to oxidative pretreatment increased the susceptibility of PEU to enzymatic hydrolysis by papain. The increase may be related to interaction between papain molecules and the oxidized surface. PEU chain extended with 2-butene-1,4-diol was more resistant to oxidation, and hence less susceptible to enzymatic degradation. Oxidation followed by enzymatic (hydrolytic) degradation could be an important biodegradation mechanism. Improving oxidation stability could reduce the overall biodegradation and increase durability of PEU in biomedical applications. Acknowledgements This work was funded by the National Science Council, Taiwan (NSC83-0420-B-033-004-M8 and NSC87-2312-B-005-003-M08). References [1] Stokes K, Coury A, Urbanski P. J Biomater Appl 1987;1:411. [2] Zhao Q, Topham N, Anderson JM, Hiltner A, Lodoen GA, Payet CR. J Biomed Mater Res 1991;25:177. [3] Hergenrother RW, Wabers HD, Cooper SL. Biomaterials 1993;14:449. [4] Wu Y, Zhao Q, Anderson JM, Hiltner A, Lodoen GA, Payet CR. J Biomed Mater Res 1991;25:725.
[5] Ratner BD, Gladhill KW, Horbett TA. J Biomed Mater Res 1988;22:509. [6] Tyler BJ, Ratner BD. J Biomed Mater Res 1993;27:327. [7] Marchant RE, Zhao Q, Anderson JM, Hiltner A. Polymer 1987;28:2032. [8] Zhao Q, Marchant RE, Anderson JM, Hiltner A. Polymer 1987;28:2040. [9] Bouvier M, Chawla AS, Hinberg I. J Biomed Mater Res 1991;25:773. [10] Santerre JP, Labow RS, Adams GA. J Biomed Mater Res 1993;27:97. [11] Smith R, Williams DF, Oliver C. J Biomed Mater Res 1987;21:1149. [12] Xi T, Sato M, Nakamura A, Kawasaki Y, Umemura T, Tsuda M, Kurokawa Y. J Biomed Mater Res 1994;28:483. [13] Sato M, Xi T, Nakamura A, Kawasaki Y, Umemura T, Tsuda M, Kurokawa Y. J Biomed Mater Res 1995;29:1201. [14] Williams DF. Plast Rubber: Mater Appl 1980;5:179. [15] Zhao QH, McNally AK, Rubin KR, Renier M, Wu Y, RoseCaprara V, Anderson JM, Hiltner A, Urbanski P, Stokes KJ. J Biomed Mater Res 1993;27:379. [16] Dean RT, Thomas SM, Garner A. Biochem J 1986;240:489. [17] Hunt JV, Simpson JA, Dean RT. Biochem J 1988;250:87. [18] Hsu S, Chen J. Polym Degrad Stab 1999;65:341. [19] Hepburn C. Polyurethane elastomers. London: Applied Science Publishers, 1982. [20] David DJ, Staley HB, editors. Analytical chemistry of the polyurethane. New York: Wiley, 1969 pp. 357. [21] Arnon R. In: Perlman Ge, Lorand L, editors. Methods in enzymology, vol. XIX. Academic Press, 1970. p. 226. [22] Ratner BD, McElroy BJ. In: Gendreau RM, editor. Spectroscopy in the biomedical sciences. Boca Raton, FL: CRC Press, 1986. p. 107. [23] Takahara A, Coury AJ, Hergenrother RW, Cooper SL. J Biomed Mater Res 1991;25:341.
The susceptibility of poly(ether)urethanes to enzymatic degradation after oxidative pretreatment Shan-hui Hsu a,*, Tsung-bin Huang b a b
Department of Chemical Engineering, National Chung Hsing University, Taichung, Taiwan 40227, ROC Department of Biomedical Engineering, Chung Yuan Christian University, Chung Li, Taiwan 32023, ROC Received 14 January 1999; accepted 5 March 1999
Abstract Susceptibility of poly(ether)urethanes (PEU) to hydrolytic degradation by enzymes after a limited exposure to free radicals was investigated. Two PEU, one chain extended with 1,4-butanediol and the other chain extended with 2-butene-1,4-diol, were used as model polymers. Each material was subjected to treatments including oxidative, enzymatic (with papain), and oxidative followed by enzymatic treatment. The degradative eect on surfaces was examined by optical microscopy, attenuated total re¯ectance infrared spectroscopy as well as XPS. It was found that enzymatic degradation occurred only after certain amount of oxidative change had been initiated. This was probably associated with enzyme adsorption on surfaces pre-exposed to oxidation. The extent of overall degradation was strongly in¯uenced by the oxidation stability of the materials. PEU chain extended with 2-butene-1,4-diol was more resistant to oxidation, and hence less susceptible to enzymatic degradation. It was concluded that exposure of PEU to oxidative pretreatment increased its susceptibility to hydrolysis by enzymes, and that oxidation followed by enzymatic attack could be the biodegradation mechanism of PEU in vivo. # 1999 Elsevier Science Ltd. All rights reserved.
1. Introduction Poly(ether)urethanes (PEU) have received extensive attention in the ®eld of biomaterials due to their excellent biocompatibility. However, in vivo as well as in vitro experiments [1±13] have demonstrated that PEU is susceptible to biodegradation. A number of hypotheses including two chemical mechanisms, oxidation [1±6] and enzymatic hydrolysis [5±11], have been proposed to explain the nature of biodegradation. Theoretically, killing substances such as H2O2 and .OH produced by macrophages and other phagocytes during respiratory burst may initiate the oxidation by free radical reactions [2]; lysosomal enzymes that are released from in¯ammatory cells could catalyze the hydrolysis [14]. Treating PEU with oxidative reagents [1,3,5,6] did reproduce several interesting features similar to those observed in vivo [2,4,12,13]. On the other hand, treating PEU with dierent enzymes led to a variety of results [5±11]. Most studies demonstrated very small amounts of degrada* Corresponding author. Tel.: +886-4-287-3181; fax:+886-4-2854734. E-mail address: [email protected] (S.-H. Hsu).
tion [7±9,11]. So far, evidence was insucient to determine the favorable mechanism in vivo [13]. However, it has been suggested that biodegradation may be the result of a synergistic environment, with the process being enhanced by factors such as pH and material dissolution [10], as well as plasma 2-macroglobulin [15]. Hydroperoxide or free radicals has been proved to mediate enzymatic hydrolysis of proteins [16,17]. A limited pre-exposure to free radicals increases the susceptibility of bovine serum albumin to hydrolysis by trypsin signi®cantly [17]. Such mediation may be due to the modi®cation of protein structure by hydroperoxide or free radicals. It is thus logical to think that oxidative pretreatment may have the same in¯uence on the susceptibility of PEU to enzymatic attack, which was the hypothesis behind the current study. Commercial products of medical-grade PEU, such as Pellethane and Biomer, usually have a very wide range of molecular weight distribution and signi®cant lot-tolot variations. These situations could result in dissolution of low-molecular-weight species in physiological environments and thus could play a role in varying results of biostability [6]. To avoid such complications,
0141-3910/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved. PII: S0141-3910(99)00110-X
172
S.-H. Hsu, T.-B. Huang / Polymer Degradation and Stability 67 (2000) 171±178
in a previous study, we synthesized two PEU ourselves and demonstrated that when 2-butene-1,4-diol instead of 1,4-butanediol was used as the chain extender in synthesis, the oxidation of the PEU by free radicals was reduced [18]. Here in this study, these two model polymers (one chain extended with 1,4-butanediol and the other chain extended with 2-butene-1,4-diol) were used to test the hypothesis that oxidative treatment could make PEU susceptible to enzymatic hydrolysis, as mentioned above. They were pretreated with free radicals, but less drastically than the condition employed in the previous study. The materials after limited free radical treatment were then subjected to a solution containing hydrolytic enzyme papain at the pH optimum condition for one month at 37 C. Papain was used due to its similar speci®city to cathepsin B [7], a lysosomal enzyme released by macrophages during the foreign body reaction. The degradative eect on surfaces was characterized using re¯ected optical microscopy, attenuated total re¯ectance infrared spectroscopy (ATR-IR) and XPS. The degree of degradation was compared with that of samples treated with other controlled environments, to de®ne the synergistic role of free radicals in the enzymatic degradation. 2. Experimental 2.1. Synthesis of PEU Two kinds of linear PEU, one (PEU A) chain extended with 1,4-butanediol and the other (PEU B) chain extended with 2-butene-1,4-diol, were synthesized by the conventional two-stage polymerization method. In the ®rst stage, MDI and polytetramethylene oxide (PTMO, M.W.=2000) at a molar ratio of 2:1 were reacted to form the prepolymer. The mixture was stirred at 60 C under dry nitrogen for approximately 6 h, until the amount of NCO reached the theoretical value determined by the dibutylamine back-titration method [19]. The prepolymer was then converted into the ®nal product by further reaction with the chain extender in dehydrated dimethylformamide (DMF) (40% solution). The reaction proceeded at 60 C overnight until the isocyanate disappeared, which was monitored by infrared spectrometry [20]. The products were puri®ed by extraction in methanol and stored in granular form. 2.2. Film casting and characterization The polymer was dissolved in DMF (25%) again and cast to a thickness of 0.5 mm on Te¯on substrates. Films were peeled from the substrates after being dried in a vacuum oven at 60 C, and were cut into pieces with a size 15 cm. The molecular weights were measured by gel permeation chromatography (GPC, Waters 410;
1 ml/min THF used as solvent). The surface was characterized by re¯ected optical microscopy (Nikon, Universal Type 104), attenuated total re¯ectance infrared spectroscopy (ATR-IR Spectrometer, Bio-Rad FTS-7; Ge crystal with an incident angle 45 ), and XPS (ESCA210 Spectrometer, VG Scienti®c; power of analysis 216W with take o angle 45 , Mg as the metal target of X-ray tube). The chemical composition of C, O and N elements was calculated from the XPS survey spectra. To analyze the chemical bonding states of the carbon atoms (carbon functional groups), the high-resolution C1S spectra were deconvoluted and curve-®t by the built-in software. The surface texture was scanned by an atomic force microscope using tapping mode probe at a scan size 11 mm (AFM, NanoScope III, Digital Instrument). 2.3. In vitro degradation test and surface characterization 10% H2O2 and 0.05 M CoCl2 was used as our oxidative pretreatment. PEU samples were immersed at 37 C for 1 week. The solution was changed every 3 or 4 days. This treatment was less drastic than that previously used [18]. After the treatment, the specimens were removed and rinsed thoroughly in deionized water. The transparency of the samples did not show apparent change visually. The surface morphology was observed by re¯ected optical microscopy. The change in the functional groups on the surface was examined using ATRIR and XPS. Samples were then immersed in papain solution. The papain (Sigma) had an activity 28 U/mg, determined by hydrolysis of benzoyl-l-arginine ethyl ester (BAEE) [21]. The papain was prepared to a concentration of 140 U/ml in the enzyme activating agent containing 0.05 M Cystein-HCl, 0.01M EDTA and 610ÿ4 M 2mercaptoethanol, pH adjusted to 6.2 by NaOH [21]. Samples were treated in the papain solution at 37 C for 1 month. The solution was changed every 3 or 4 days. The samples were cleaned by repeated sonication in 1% Triton X-100 (Aldrich) and in deionized water before characterization [9]. In addition, samples without oxidative pretreatment were immersed in papain solution for 1 month as controls. Some samples with oxidative pretreatment were soaked in activating buer as another set of controls. The degradative eect was characterized by using methods described above. For papain adsorption study, the surface of samples (untreated or pretreated) was cleaned thoroughly with deionized water before adsorption. The samples were immersed in papain solution for 72 h. Then the solutions were removed, and the samples were rinsed carefully with activating buer followed by deionized water (three rinses in total), and freeze-dried immediately for AFM imaging.
S.-H. Hsu, T.-B. Huang / Polymer Degradation and Stability 67 (2000) 171±178
3. Results and discussion For convenience of data presentation, the two PEU are designated as PEU A (chain extended with 1,4butanediol), and PEU B (chain extended with 2-butene1,4-diol). The molecular weights of these two PEU were comparable and were shown in Table 1. After immersion in 10% H2O2 and 0.05 M CoCl2 at 37 C for 1 week, the surface of both PEU under microscope showed limited amount of pitting, especially for PEU B, where almost no pitting was observed at magni®cation 100. A comparison between PEU A and PEU B exposed to oxidation is shown in Fig. 1(a) and (b). Also included is the original surface [Fig. 1(c)]. After subsequent enzymatic treatment, both PEU showed signi®cant surface changes [Fig. 2(a) and (b)] over the two controls, i.e. samples subjected to enzyme treatment for one month without oxidative pretreatment [Fig. 2(c)], and those subjected to oxidation followed by soaking in activating buer for one month [Fig. 2(d)]. In PEU A, extensive surface pitting was found [Fig. 2(a)]. The ATR-IR spectra of the untreated, oxidation-pretreated and oxidation plus enzyme-treated PEU samples
173
Table 1 Molecular weights of PEU Polymer
Mw a
Mn b
Polydispersity
PEU A PEU B
130 00018 000 100 00010 000
44 0002000 38 0001000
2.90.5 2.60.3
a b
Mw ; the weight average molecular weight. Mn ; the number average molecular weight.
are shown in Fig. 3. For both PEU A and PEU B, the 1week oxidation pretreatment only caused limited amount of surface degradation, especially for PEU B, where no evident change in spectra was noticed. However, enzymatic post-treatment caused degradation in PEU A, as indicated by an increase near 3400 cmÿ1 (O± H), as well as a lower intensity ratio of the two carbonyl bands at 1732 cm and 1702 cmÿ1 in the spectrum. In literature, the ratio was observed to decrease with the implantation time in vivo [13], and such decrease was attributed to the gradual loss of PTMO soft segment from the surface to the surrounding. Since PTMO was quite resistant to the hydrolysis, it was less likely that enzyme could attack PTMO directly. Indeed, no change of IR spectra was found for samples treated with
Fig. 1. The surface morphology of pretreated materials, as viewed by the optical microscope (100): (a) PEU A, after oxidative treatment; (b) PEU B, after oxidative treatment; and (c) PEU B, untreated.
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enzyme without oxidative pretreatment (data not shown). Therefore, we assumed that free radicals exposure during the oxidative pretreatment had initiated certain amount of degradation, by oxidizing some ether to ester linkages; these esters might be hydrolyzed by papain (i.e. also an esterase [21]) afterwards. On the other hand, we found the IR spectroscopy was not sensitive enough to support the point that ester was produced in the ®rst treatment, because as mentioned, there was no apparent change in spectra after oxidation. The bands at 1102 cmÿ1 (C±O±C in ether) and at 1080 cmÿ1 (C±O±C in ester) remained similar to the original. In a previous study, however, where a stronger oxidative treatment was used, ester formation after oxidation was con®rmed by ATR-IR spectra [18]. Since ATR-IR could not detect early oxidation on the surface, we sought evidence from XPS. The XPS results were shown in Table 2. These data were calculated by the built-in software from the survey spectra as well as the high-resolution C1S spectra. The high-resolution C1S spectra (Fig. 4) could be deconvoluted into two major peaks, at 284.6 eV (hydrocarbon) and 286.0 eV (ether, alcohol) [22], as well as a small but
broad peak at 288.4±289.4 eV. Since the peak at 286.0 eV is indicative of the soft segment, and the small broad tail near 288.4±289.4 eV is primarily related to the hard segment, their ratio represents the relative contribution from the soft and hard segments. It was thus apparent that the polyether soft segment was enriched at the surface of the untreated PEU. This was con®rmed by the very small surface N/C atomic ratio observed, as well as the O/C atomic ratio that was close to the value estimated stoichiometrically from PTMO structure. After oxidative treatment, the O/C atomic ratio increased for both PEU (more for PEU A), indicating oxidation (oxygen inclusion) of the surface. From the calculated percentages for 286.0 eV peak area listed in Table 2, a signi®cant loss of ether from the surface was noted for both PEU after oxidative treatment, more severe in PEU A. The center position of the broad tail could also re¯ect the relative contribution from ester or acid (288.3±288.5 eV), and carbamate (288.8±289.4 eV). Indeed, as evident from Table 2 (or Fig. 4), a center at 288.6 eV, i.e. closer to ester or acid instead of carbamate, was observed on the oxidatively-treated surface. This suggested that some ester (or acid) was generated
Fig. 2. The surface morphology of post-treated materials, as viewed by the optical microscope (100): (a) PEU A, oxidation followed by 1-month papain treatment; (b) PEU B, oxidation followed by 1-month papain treatment; (c) PEU A, 1-month papain treatment only, without pretreatment; and (d) PEU A, oxidation followed by 1-month buer treatment.
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after oxidation, most likely at the a-carbon site adjacent to the ether group [23] in the soft segment. For samples after both oxidative and enzymatic treatments, more changes in XPS spectra were observed. The O/C atomic ratio further increased, especially for PEU A (to 0.32). We attributed the increase to the hydrolysis (oxygen inclusion due to H2O). Besides, the 288.6 eV peak shifted to 288.3 eV, i.e. even more con-
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tribution from ester or acid. In this case, it was probably from acid, the hydrolytic product of the ester groups previously generated in the oxidation process. Also, the percentage of the 286.0 eV peak bounced back a little, suggesting alcohol formation, i.e. another hydrolytic product. A further comparison between the data of PEU A and PEU B (Table 2) clearly demonstrated that amount of degradative changes after enzymatic treat-
Fig. 3. The ATR-IR spectra of PEU samples after dierent treatments: curves marked ``untreated'', the spectra of untreated samples; curves marked ``oxidative'', the spectra of oxidation-pretreated samples; and curves marked ``oxidative+enzymatic'', the spectra of samples after oxidation followed by 1-month papain treatment: (a) for PEU A; and (b) for PEU B.
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Table 2 ESCA results after varieties of treatment Substrates
PEU A, untreated PEU A, oxidative only PEU A, oxidative and enzymatic PEU B, untreated PEU B, oxidative only PEU B, Oxidative and enzymatic a b
O/C atomic ratio
N/C
Fraction of C functional groups from high-resolution C1S peaks
Measured
Stoichiometrica
Measured
284.6 eV (hydrocarbon) (%)
286.0 eV (ether, alcohol) (%)
288.3±288.5 eV (ester, acid)
0.24 0.28 0.32 0.25 0.27 0.29
0.24b ± ± 0.24b ± ±
0.02 0.04 0.05 0.02 0.02 0.03
53.7 65.4 60.2 56.7 64.0 62.8
45.5 29.6 32.8 42.3 33.7 34.0
0.8% (broad 288.4±289.4 eV) 5.0% (center @ 288.6 eV) 7.0% (center @ 288.3 eV) 1.0% (broad 288.4±289.4 eV) 2.3% (center @ 288.6 eV) 3.2% (center @ 288.3 eV)
288.8±289.4eV (carbamate)
Stoichiometric ratios calculated from the known structure. Value estimated from the ploy(tetramethylene oxide) soft segment.
ment was determined by the extent of degradation during the previous oxidative treatment. Thus it was concluded from the XPS results that papain caused hydrolytic degradation in PEU pretreated with hydroperoxide, and that the susceptibility of PEU to enzy-
Fig. 4. High-resolution C1S ESCA spectra for PEU A after various treatments.
matic attack was associated with the limited amount of ester formation on the surface after exposure to free radicals. AFM images for the surfaces are shown in Fig. 5, each comprising a three-dimensional image (left) and a two-dimensional image (right). Samples after oxidation treatment had a surface texture (wavy patterns) similar to the original [Fig. 5(a)]. However, big shallow craterlike holes may appear in some regions [darker areas in Fig. 5(b)]. We attributed these to the degraded areas, since the edges of these areas seemed to be mechanically softer (blurred streaks in the middle of Fig. 5(b) are caused by the probe tip motion). Following the papain adsorption test, the surface contour changed [Fig. 5(c), for samples without oxidation]. But the change was still small and the surface was relatively clean (note that the surface depth was only 6 nm). On the other hand, the surface pre-exposed to oxidation had many sphere-like nanoparticles attached to the surface [Fig. 5(d)], which were believed to be papain aggregates. Such adsorption may be caused by non-speci®c interaction. Since the isoelectric point (pI) of papain (9.6) [21] was above the pH (6.2) of the activating buer, the papain should be positively charged in the solution. We postulated that after oxidation treatment, the surface was negatively charged due to some bond cleavage, and thus attracted positively charged papain molecules to the surface. Such binding may enhance enzyme activity and facilitate the hydrolytic reaction. Because limited change on the surface after oxidation could signi®cantly alter the susceptibility of PEU to enzymatic degradation, the overall biostability of the PEU could be enhanced by improving its oxidation stability, such as in the case of PEU chain extended with 1,4-butene-2-diol. Finally, since oxidative and enzymatic treatments were synergistic, we speculate that oxidation followed by enzymatic degradation could be an important degradation mechanism in vivo as well.
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Fig. 5. AFM images of the surfaces for: (a) untreated PEU A, before papain adsorption; (b) PEU A after oxidative treatment; (c) untreated PEU A, after papain adsorption test; and (d) PEU A after oxidative treatment, with papain adsorbed.
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4. Conclusion Exposure to oxidative pretreatment increased the susceptibility of PEU to enzymatic hydrolysis by papain. The increase may be related to interaction between papain molecules and the oxidized surface. PEU chain extended with 2-butene-1,4-diol was more resistant to oxidation, and hence less susceptible to enzymatic degradation. Oxidation followed by enzymatic (hydrolytic) degradation could be an important biodegradation mechanism. Improving oxidation stability could reduce the overall biodegradation and increase durability of PEU in biomedical applications. Acknowledgements This work was funded by the National Science Council, Taiwan (NSC83-0420-B-033-004-M8 and NSC87-2312-B-005-003-M08). References [1] Stokes K, Coury A, Urbanski P. J Biomater Appl 1987;1:411. [2] Zhao Q, Topham N, Anderson JM, Hiltner A, Lodoen GA, Payet CR. J Biomed Mater Res 1991;25:177. [3] Hergenrother RW, Wabers HD, Cooper SL. Biomaterials 1993;14:449. [4] Wu Y, Zhao Q, Anderson JM, Hiltner A, Lodoen GA, Payet CR. J Biomed Mater Res 1991;25:725.
[5] Ratner BD, Gladhill KW, Horbett TA. J Biomed Mater Res 1988;22:509. [6] Tyler BJ, Ratner BD. J Biomed Mater Res 1993;27:327. [7] Marchant RE, Zhao Q, Anderson JM, Hiltner A. Polymer 1987;28:2032. [8] Zhao Q, Marchant RE, Anderson JM, Hiltner A. Polymer 1987;28:2040. [9] Bouvier M, Chawla AS, Hinberg I. J Biomed Mater Res 1991;25:773. [10] Santerre JP, Labow RS, Adams GA. J Biomed Mater Res 1993;27:97. [11] Smith R, Williams DF, Oliver C. J Biomed Mater Res 1987;21:1149. [12] Xi T, Sato M, Nakamura A, Kawasaki Y, Umemura T, Tsuda M, Kurokawa Y. J Biomed Mater Res 1994;28:483. [13] Sato M, Xi T, Nakamura A, Kawasaki Y, Umemura T, Tsuda M, Kurokawa Y. J Biomed Mater Res 1995;29:1201. [14] Williams DF. Plast Rubber: Mater Appl 1980;5:179. [15] Zhao QH, McNally AK, Rubin KR, Renier M, Wu Y, RoseCaprara V, Anderson JM, Hiltner A, Urbanski P, Stokes KJ. J Biomed Mater Res 1993;27:379. [16] Dean RT, Thomas SM, Garner A. Biochem J 1986;240:489. [17] Hunt JV, Simpson JA, Dean RT. Biochem J 1988;250:87. [18] Hsu S, Chen J. Polym Degrad Stab 1999;65:341. [19] Hepburn C. Polyurethane elastomers. London: Applied Science Publishers, 1982. [20] David DJ, Staley HB, editors. Analytical chemistry of the polyurethane. New York: Wiley, 1969 pp. 357. [21] Arnon R. In: Perlman Ge, Lorand L, editors. Methods in enzymology, vol. XIX. Academic Press, 1970. p. 226. [22] Ratner BD, McElroy BJ. In: Gendreau RM, editor. Spectroscopy in the biomedical sciences. Boca Raton, FL: CRC Press, 1986. p. 107. [23] Takahara A, Coury AJ, Hergenrother RW, Cooper SL. J Biomed Mater Res 1991;25:341.