J O U R N A L O F M A T E R I A L S S C I E N C E L E T T E R S 4 ( 1 9 8 5 ) 5 4 7 - 5 4 9

The degradation of a synthetic polyester by a lysomal enzyme

R. S M I T H , D. F. W I L L I A M S Department of Dental Sciences, School of Dental Surgery, University of Liverpool, Box 147, Liverpool, UK

High molecular weight synthetic polymers are widely used for implantation within the human body, both for the purpose of reconstructive surgery and controlled drug release. In some situations it is desirable that the polymers are resistant to degradative attack by the physiological environment since the implant has to remain in the tissues for the lifetime of the patient without undergoing structural changes or causing tissue irritation [1]. In others, the function of the implanted polymer may rely on controlled and sustained degradation [2]. In either case, the stability of the polymer within tissue fluids is of utmost importance. In recent years there has been considerable speculation over the role of enzymes in the degradation of synthetic polymers, but with very little supportive evidence. In this communication we describe an experiment which demonstrates that a synthetic polyester, poly (ethylene terephthalate) may be degraded by the activity of a lysomoal enzyme, esterase, in vitro.

There are several types of polyester used as bio- materials and these provide a range of behaviour with respect to environmental stability. At one extreme are the aliphatic polyesters such as poly(glycollic acid), poly(lactic acid)and polydi- oxanone, all totally resorbable and used as sutures and drug delivery vehicles [3-5] . At the other extreme are aromatic polyesters such as poly- ethylene terephthalate, which although hydrolysed at elevated temperatures, are normally considered stable under physiological conditions and are used, in fabric form, for arterial prostheses [6]. We have shown previously that the rate of hydrolysis of the aliphatic polyesters can be influenced by the presence of enzymes, the rate of loss of mechani- cal strength of sutures made from these materials varying in different enzymes solutions in vitro [7-10]. It is considerably more difficult to study minor degradative changes confined to the surface in nominally stable polymers such as the aromatic

polyesters, but such surface degradation could be extremely important in controlling tissue, and especially blood, compatibility.

We have, therefore, synthesized a radio-labelled aromatic polyester and studied it's susceptibility to degradation in vitro by an enzyme. Poly(ethyl- ene terephthalate) labelled with 14C was prepared from 14C-ethanediol and dimethylterephthalate as indicated in Fig. 1. The polymer was purified by continuous extraction with methanol and re- precipitated twice from TFA (trifluoroacetic acid). The specific activity of the labelledpolymer was 8.8/.tcig -1. The molecular weight M n was deter- mined to be 20 000 from measurements of intrin- sic viscosity [X], in a 0.5% solution of polymer in phenol/tetrachloroethane at 25 ° C and using the relationship [ 11 ]

[X] -- 0.0213Yrn°'s

A commercially available polyethylene terephtha- late (Melinar B90/7, ICI plc) reprecipitated from TFA was characterized for comparison and found to have an 3~ n value of 37 000. Infrared spectra of thin films cast from TFA solution and NMR spectra of solutions in deuterated TFA were obtained and shown to be identical for the syn- thesized labelled polymer and the Melinar.

The enzyme used was esterase (type I, from porcine liver, Sigma Chemical Company). The stability of this enzyme was measured by taking 0.2 cm 3 of the esterase suspension, mixing with 0.1M borate buffer at pH 8.0 and assaying before and after storage for 24 h at 37 ° C. The initial assay showed an activity of 320 units per 10 cm 3 , while after 24 h this was 250 units per 10 cm 3, one unit hydrolysing 1 ~tmole of ethyl butyrate per minute at pH 8.0 and 25 ° C.

14C-poly(ethylene terephthalate) (0 .1g )was mixed with 0.1M borate buffer at pH 8.0 con- taining 0.2 cm 3 esterase suspension. The mixture was stored at 37°C for 24 h and filtered. The

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14CH2 OH 14cNH 2 OH

COOCH 3

COOCH 3

CALCIUM C O O14CH~4CH2 OH ACETATE ~-- ~CO

O14CH~CH2 OH

ANTIMONY RIOXlDE

Figure 1 Synthesis of radiolabelled poly(ethylene terephthalate) from 14C- ethanediol and dimethylterephthalate.

radioactivity of the filtrate, in 1 ml aliquots, was determined using a Packard Tricarb 3000 liquid scintillation counter interfaced with an Apple computer for DPM (disintegrations per minute) conversion. Beckmann MP scintillation cocktail was used and a quench curve determined with quenched standards from Amersham International. The residual polymer was returned to fresh en- zyme solution after the filtration for a further 24 h, the process being repeated for a total of 15 day. Control experiments were performed in which the labelled polymer was mixed with the buffer but with the exclusion of the enzyme.

The results are given in Fig. 2. Standard devi- ations in the DPM figures were consistent in experimental and control groups throughout the tests and average -+ 2.5. The clear difference between the results of the test and control groups indicates that the radioactivity determined in the filtrate does not merely represent a leaching of residues but involves the release of fragments of degrading molecular chains. Using the sensitive radiolabelling technique, we have, therefore, been able to demonstrate that a lysosomal enzyme is able to cause degradation of the molecular structure of this synthetic poly(ethylene tereph-

4 0 0 -

3 0 0 -

D P M

2 0 0 -

1 0 0 -

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/ - - w i t h enzyme . . . . control

I I I I I I I I I I I I I 5 10 15

D A Y S

Figure 2 Radioactivity, measured as disintegrations per minute (DPM) released into esterase solution and control borate buffer, over each successive 24h periods from 14C- poly(ethylene terephthalate).

thalate). Enzymes are, of course, substrate specific and the effect of this esterase is consistent with its normal effect on the ester bond. The aromatic polyester used, however, has a different molecular structure to that of the polyesters normally encountered in physiological systems and these observations would suggest that enzymes may have a wider role in the degradation of implanted polymers than hitherto believed. We have now prepared a series of other labelled polymers, including acrylics, polyamides and polyurethanes to study their susceptibility to enzyme attack.

Acknowledgement This work was supported by a project grant from the Science and Engineering Research Council, Ref GRB34515.

References l. D. F. WILLIAMS, J. Mater. ScL 17 (1982) 1233. 2. D.K. GILDING, in "Biocompatibility of Clinical

Implant Materials", Vol. II, edited by D. F. Williams (CRC Press, Boca Raton, 1981) p. 209.

3. A.R. ANSCOMBE, N. HIRA and B. HUNT, Brit. J. Surg. 57 (1970) 917.

4. J .A. RAY, N. DODDI and D. RIGULA, Surg. GyneeoL Obstet. 153 (1981) 497.

5. C.G. PITT, T. A. MARKS and A. SCHNIDLER, in "Controlled Release of Bioactive Materials", edited by R. Baker (Academic Press, New York, 1980) p. 19.

6. M. KING, P. BLAIS, R. GUIDOIN, E. PROWSE, M. MARCOIS, C. GOSSELIN and H. P. NOEL, in "Biocompatibility of Clinical Implant Materials", Vol. II, edited by D. F. Williams (CRC Press, Boca Raton, 1981) p. 177.

7. D.F. WILLIAMS and E. J. MORT, Bioen. 1 (1977) 231.

8. D.F. WILLIAMS, Plast. Rubber. Mater. AppL 5 (1980) 179.

9. C.C. CHU and D. F. WILLIAMS, J. Biomed. Mater. Res. 17 (1983) 1029•

10. D. F. WILLIAMS, Eng. Med. 10 (1981) 5. 11. A. CONIX, Makromol. Chem. 26 (1958) 226.

Received 3 September and accepted 20 September 1984

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