Investigation of the Impact of Poly(Ethylene Glycol)-Modulation of Poly(B-Hydroxybutyrate) Syntheses on Cell Interactions of the Resulting Polymers

Abstract

Poly(-hydroxybutyrate), PHB is a bacterial polyester known for its excellent bone compatibility, however, the material lacks blood and tissue compatibility. Poly(ethylene glycol), PEG,-modulated fermentation of Alcaligenes latus and Azotobacter vinelandii UWD was employed to yield copolymers consisting of PHB and PEG that exhibit diminished cell-adhesion surface properties. PEGs with molecular weights of 3400, 2000, and 400 as well as diethylene glycol, DEG, and pentaerythritol ethoxylate, PEE, were used in a concentration of 2% (w/v) for amending the fermentation broths. This modulation of the fermentation conditions did not influence polymer yields. However, the resulting copolymers had drastically reduced molecular weights, 82% less for the DEG-amended fermentation of A. latus. The reduction in molecular weight was attributed to an end-capping reaction of the nascent PHB-chain with PEG and/or early chain termination by water facilitated by the presence of the highly hydrophilic PEG-molecules. The formation of a covalent linkage was proven unambiguously by H-NMR-spectroscopic methods only for the copolymers obtained in the DEG-modified fermentations of both strains. Cell growth experiments using SK-MEL 28 and MDA-MB 231 cells were used for the evaluation of polymer-cell interaction. Copolymer films obtained from PEG-modulated syntheses showed significantly less cell adhesion with reductions in cell adhesions; up to 74% less in the two-day experiments (MDA-MB 231 on the copolymer obtained in DEG-modified fermentation of A. latus) and 48% less in the seven-day experiments (SK-MEL 28 on the copolymer obtained in PEG 400-modified fermentation of A. vinelandii UWD). In the two-day experiments, no differences in the cellinteraction was observed between the polymers obtained from two different bacterial sources, the polymers differed in their long-term, seven-day, cell interaction with copolymers obtained from A. vinelandii UWD maintaining more effective cell repulsion.

Get full access to this article

View all access options for this article.

References

1. Marois, Z., Zhang, Z., Vert, M., Deng, X., Lenz, R.W. and Guidon, R. (1999). Hydrolytic and Enzymatic Incubation of Poly(hydroxyoctanoate): A Shortterm in vitro Study of Degradable Bacterial Polyesters , J. Biomater. Sci. Polym. Ed., 10(4): 483-483 .

2. Williams, S.F., Martin, D.P., Horowitz, D.M.Peoples, O.P. (1999). PHA Applications: Addressing the Price Performance Issue I. Tissue Engineering , Int. J. Biol. Macromolecules, 25: 111-111 .

3. Saito, T., Tomita, K., Juni, K. and Ooba, K. (1991). In vivo and in vitro Degradation of Poly(3-hydroxybutyrate) in Rat , Biomaterials, 12(3): 309-309 .

4. Gogolewski, S., Jovanovich, M., Perren, S.M., Dillon, J.G. and Hughes, M.K. (1993). Tissue-Response and in-Vivo Degradation of Selected Polyhydroxyacids – Polylactides (PLA), Poly(3-hydroxybutrate) (PHB), and Poly(3-hydroxybutyrate-Co-3-hydroxyvalerate) (PHB/VA) , Journal of Biomedical Materials Research, 27(9): 1135-1135 .

5. Reusch, R.N., Sparrow, A.W. and Gardiner, J. (1992). Transport of Poly-beta-hydroxybutyrate in Human Plasma , Biochim. Biophys. Acta, 1123(1): 33-33 .

6. Müller, H.M. and Seebach, D. (1993). Poly(hydroxy fatty acid esters), A Fifth Class of Physiologically Significant Organic Biopolymers? Angew. Chem. Int. Ed. Eng., 105: 477-477 .

7. Doyle, C., Tanner, E.T. and Bonfield, W. (1991). In vitro and in vivo Evaluation of Polyhydroxybutyrate and of Polyhydroxybutyrate Reinforced with Hydroxyapatite , Biomaterials, 12(9): 841-841 .

8. Holland, S.J., Yasin, M. and Tighe, B.J. (1990). Polymers for Biodegradable Medical Devices. VII. Hydroxybutrate-hydroxyvalerate Copolymers: Degradation of Copolymers and their Blends with Polysaccharides Under in vitro Physiological Conditions , Biomaterials, 11(3): 206-206 .

9. Yagmurlu, M.F., Korkusuz, F., Gürsel, I., Korkusuz P., Ö rs, Ü. and Hasirci, V. (1999). Sulbactam-cefoperazone Polyhydroxybutyrate-co-hydroxyvalerate (PHBV) Local Antibiotic Delivery System: In vivo Effectiveness and Biocompatibility in the Treatment of Implant-related Experimental Osteomyelitis , J. Biomed. Mater. Res., 46(4): 494-494 .

10.

10. Sendil D., Gursel, I., Wise, D.L. and Hasirci, V. (1999). Antibiotic Release from Biodegradable PHBV Microparticles , J. Controlled Release, 59(2): 207-207 .

11.

11. Embleton, J.K. and Tighe, B.J., Polymers for Biodegradable Medical Devices. X. (1993). Microencapsulation Studies: Control of Poly-hydroxybutyratehydroxyvalerate Microcapsules Porsity via Polycaprolactone Blending , J. Microencapsul., 10(3): 341-341 .

12.

12. Hazari, A., Johansson-Ruden, G., Junemo-Bostrom, K., Ljungberg, C., Terenghi, G., Green, C. and Wiberg, M. (1999). A New Resorbable Wraparound Implant as an Alternative Nerve Repair Technique , J. Hand. Surg. [Br.], 24(3): 291-291 .

13.

13. van der Giessen, W.J., Lincoff, A.M., Schwartz, R.S., van Beusekom, H.M., Serruys, P.W., Holmes Jr., D.R., Ellis, S.G. and Topol, E.J. (1996). Marked Inflammatory Sequelae to Implantation of Biodegradable and Nonbiodegradable Polymers in Porcine Coronary Arteries, Circulation , 94(7): 1690-1690 .

14.

14. Gabizon, A., Horowitz, A.T., Goren, D., Tzemach, D., Mandelbaum-Shavit, F., Qazen, M.M. and Zalipsky, S. (1999). Targeting Folate Receptor with Folate Linked to Extremities of Poly(ethylene glycol)-grafted Liposomes: in Vitro Studies , Bioconjug. Chem., 10: 289-289 .

15.

15. Kataoka, K. (1996). Targetable Polymeric Drugs. In: Park, K. (ed.), Controlled Drug Delivery: Challenges and Strategies, American Chemical Society, Washington, DC ,49-49.

16.

16. Scholz, C., Iijima, M., Nagasaki, Y. and Kataoka, K. (1998). Polymeric Micelles as Drug Delivery Systems: A Reactive Polymeric Micelle Carrying Aldehyde , Polym. Adv. Technol., 9: 1-1 .

17.

17. Zalipsky, S. and Harris, J.M. (1997). Poly(ethylene glycol) Chemistry and Biological Applications, In: Harris, J.M. and Zalipsky, S.(eds), ACS Symposium Series 680, 1-1.

18.

18. Desai, N.P. and Hubbell, J.A. (1991). Biological Responses to Polyethylene Oxide Modified Polyethylene Terephthalate Surfaces , Journal of Biomedical Materials Research, 25: 829-829 .

19.

19. Lee, J.H., Kopeckova, P., Kopecek, J. and Andrade, J.D. (1990). Surface Properties of Copolymers of Alkyl Methacrylates with Methoxy (polyethylene oxide) Methacrylates and their Application as Proteins-resistant Coatings , Biomaterials, 11: 455-455 .

20.

20. Kwon, G.S. and Kataoka, K. (1995). Block Copolymer Micelles as Long-Circulating Drug Vehicles , Adv. Drug Delivery Rev., 16: 295-295 .

21.

21. Bergstroem, K., Österberg, E., Holmberg, K., Hoffman, A.S., Schuman, T.P., Kozlowski, A. and Harris, M.J. (1994). Effects of Branching and Molecular Weight of Surface-bound Poly(ethylene oxide) on Protein Rejection , J. Biomater. Sci. Polymer Edn., 6(2): 123-123 .

22.

22. Jeon, S.I. and Andrade, J.D. (1991). Protein-Surface Interactions in the Presence of Polyethylene Oxide , J. Coll. Interface Sci., 142: 159-159 .

23.

23. Shi, F., Gross, R.A. and Rutherford, D.R. (1996). Microbial Polyester Synthesis: Effects of Poly(ethylene glycol) on Product Composition, Repeat Unit Sequence, and End Group Structure , Macromolecules, 29: 10-10 .

24.

24. Shi, F., Ashby, R. and Gross, R.A. (1996). Use of Poly(ethylene glycol)s to Regulate Poly(3-hydroxybutyrate) Molecular Weight during Alcaligenes eutrophus Cultivation , Macromolecules, 29: 7753-7753 .

25.

25. Ashby, R.D., Shi, F. and Gross, R.A. (1997). Use of Poly(ethylene glycol) to Control the End Group Structure and Molecular Weight of Poly (3-hydroxybutyrate) Formed by Alcaligenes latus DSM 1122 , Tetrahedron, 53(45): 15209-15209 .

26.

26. Page, W.J. and Cornish, A. (1993). Growth of Azotobacter vinelandii UWD in Fish Peptone Medium and Simplified Extraction of Poly- -Hydroxybutyrate , Appl. Environ. Microbiol., 59: 4236-4236 .

27.

27. Chen, G. and Page, W.J. (1997). Production of Poly- -hydroxybutyrate by Azotobacter vinelandii in a Two-stage Fermentation Process , Biotechnol. Tech., 11: 347-347 .

28.

28. Page, W.J. (1992). Suitability of Commercial Beet Molasses Fractions as Substrates for Polyhydroxyalkanoate Production by Azotobacter Vinelandii UWD , Biotechnol. Lett., 14(5): 385-385 .

29.

29. Cailleau, R., Young, R., Olive, M. and Reeves W.J., Jr.(1974). Breast Tumor Cell Lines From Pleural Effusions , J. Natl. Cancer Inst., 53: 661-661 .

30.

30. Carey, T.E., Takahashi, T., Resnick, L.A., Oettgen, H.F. and Old, L.J. (1976). Cell Surface Antigens of Human Malignant Melanoma: Mixed Hemadsorption Assays for Humoral Immunity to Cultured Autologus Melanoma Cells . Proc. Natl. Acad. Sci., 73(9): 3278-3278 .

31.

31. Ashby, R.D., Gross, R.A. and Shi, F.A. (1999). A Tunable Switch to Regulate the Synthesis of Low and High Molecular Weight Microbial Polyesters , Biotechnology and Bioengineering, 62: 106-106 .

32.

32. Shi, F., Scholz, C., Deng, F. and Gross, R. A. (1996). Hybrid Natural-Synthetic Materials: Microbial Polyester-Polyethylene Glycol Adducts by In Vivo Processing , Polymer Reprints, 39(2): 102–103 .

33.

33. Zanzig, J. and Scholz, C. (May 14, 2003). Effects of Poly(ethylene glycol) on the Production of Poly(-hydroxybutyrate) by Azotobacter Vinelandii UWD , J. Polym. Environ. accepted.