Article | Published:

A heparin-mimicking polymer conjugate stabilizes basic fibroblast growth factor

Nature Chemistry volume 5, pages 221227 (2013) | Download Citation

Abstract

Basic fibroblast growth factor (bFGF) is a protein that plays a crucial role in diverse cellular functions, from wound healing to bone regeneration. However, a major obstacle to the widespread application of bFGF is its inherent instability during storage and delivery. Here, we describe the stabilization of bFGF by covalent conjugation with a heparin-mimicking polymer, a copolymer consisting of styrene sulfonate units and methyl methacrylate units bearing poly(ethylene glycol) side chains. The bFGF conjugate of this polymer retained bioactivity after synthesis and was stable to a variety of environmentally and therapeutically relevant stressors—such as heat, mild and harsh acidic conditions, storage and proteolytic degradation—unlike native bFGF. Following the application of stress, the conjugate was also significantly more active than the control conjugate system in which the styrene sulfonate units were omitted from the polymer structure. This research has important implications for the clinical use of bFGF and for the stabilization of heparin-binding growth factors in general.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

References

  1. 1.

    , , & Alteration of immunological properties of bovine serum-albumin by covalent attachment of polyethylene-glycol. J. Biol. Chem. 252, 3578–3581 (1977).

  2. 2.

    The dawning era of polymer therapeutics. Nature Rev. Drug Discov. 2, 347–360 (2003).

  3. 3.

    , & FDA-approved poly(ethylene glycol)–protein conjugate drugs. Polym. Chem. 2, 1442–1448 (2011).

  4. 4.

    & Poly(zwitterionic)protein conjugates offer increased stability without sacrificing binding affinity or bioactivity. Nature Chem. 4, 59–63 (2012).

  5. 5.

    , & Trehalose glycopolymers for stabilization of protein conjugates to environmental stressors. J. Am. Chem. Soc. 134, 8474–8479 (2012).

  6. 6.

    & Heparin–protein interactions. Angew. Chem. Int. Ed. 41, 391–412 (2002).

  7. 7.

    & Molecular modeling of protein–glycosaminoglycan interactions. Arteriosclerosis 9, 21–32 (1989).

  8. 8.

    , , & Mesoderm induction in early xenopus embryos by heparin-binding growth-factors. Nature 326, 197–200 (1987).

  9. 9.

    & FGF and VEGF function in angiogenesis: signalling pathways, biological responses and therapeutic inhibition. Trends Pharmacol. Sci. 22, 201–207 (2001).

  10. 10.

    , , , & Accelerated tissue regeneration through incorporation of basic fibroblast growth factor-impregnated gelatin microspheres into artificial dermis. Biomaterials 21, 489–499 (2000).

  11. 11.

    , & Effects of basic fibroblasat growth-factor on bone-formation in vitro. J. Clin. Invest. 81, 1572–1577 (1988).

  12. 12.

    et al. Fibroblast growth factor (FGF)-2 and FGF receptor 3 are required for the development of the substantia nigra, and FGF-2 plays a crucial role for the rescue of dopaminergic neurons after 6-hydroxydopamine lesion. J. Neurosci. 27, 459–471 (2007).

  13. 13.

    et al. Basic fibroblast growth factor support of human embryonic stem cell self-renewal. Stem Cells 24, 568–574 (2006).

  14. 14.

    , , , & Growth factors and cytokines in wound healing. Wound Repair Regen. 16, 585–601 (2008).

  15. 15.

    , , , & Growth of normal human keratinocytes and fibroblasts in serum-free medium is stimulated by acidic and basic fibroblast growth-factor. J. Cell Physiol. 138, 511–518 (1989).

  16. 16.

    & Serum-free culture of normal human melanocytes—growth-kinetics and growth-factor requirements. J. Cell Physiol. 140, 565–576 (1989).

  17. 17.

    , , & Controlled and modulated release of basic fibroblast growth-factor. Biomaterials 12, 619–626 (1991).

  18. 18.

    , & The fate of intravenously administered bFGF and the effect of heparin. Growth Factors 1, 156–164 (1989).

  19. 19.

    et al. Effect of topical basic fibroblast growth-factor on the healing of chronic diabetic neuropathic ulcer of the foot—a pilot, randomized, double-blind, placebo-controlled study. Diabetes Care 18, 64–69 (1995).

  20. 20.

    & Heparin protects basic and acidic FGF from inactivation. J. Cell Physiol. 128, 475–484 (1986).

  21. 21.

    et al. in Comprehensive Biomaterials Vol. 4 (eds Ducheyne, P.) Ch. 420, 333–339 (Elsevier, 2011).

  22. 22.

    , & Inhibition of human-endothelial cell-proliferation by heparin and steroids. Cell Biol. Int. Rep. 12, 1037–1047 (1988).

  23. 23.

    & The effect of heparin on cell-proliferation and type-I collagen-synthesis by adult human dermal fibroblasts. Biochim. Biophys. Acta 1180, 225–230 (1993).

  24. 24.

    et al. Modulation of fibroblast growth factor-2 receptor binding, signaling, and mitogenic activity by heparin-mimicking polysulfonated compounds. Mol. Pharmacol. 56, 204–213 (1999).

  25. 25.

    , , , & A glycopolymer chaperone for fibroblast growth factor-2. Bioconj. Chem. 15, 145–151 (2004).

  26. 26.

    et al. Nanoscale growth factor patterns by immobilization on a heparin-mimicking polymer. J. Am. Chem. Soc. 130, 16585–16591 (2008).

  27. 27.

    et al. Combination of integrin-binding peptide and growth factor promotes cell adhesion on electron-beam-fabricated patterns. J. Am. Chem. Soc. 134, 247–255 (2012).

  28. 28.

    , , , & Site-directed PEGylation of human basic broblast growth factor. Protein Expr. Purif. 48, 24–27 (2006).

  29. 29.

    et al. Purication and modication by polyethylene glycol of a new human basic broblast growth factor mutant-hbFGFSer25,87,92. J. Chromatogr. A 1161, 51–55 (2007).

  30. 30.

    , & Covalently immobilized gradients of bFGF on hydrogel scaffolds for directed cell migration. Biomaterials 26, 3227–3234 (2005).

  31. 31.

    , & Poly(ethylene glycol) modification enhances penetration of fibroblast growth factor 2 to injured spinal cord tissue from an intrathecal delivery system. J. Control. Rel. 144, 25–31 (2010).

  32. 32.

    et al. Solid-phase N-terminus PEGylation of recombinant human fibroblast growth factor 2 on heparin-sepharose column. Bioconj. Chem. 23, 740–750 (2012).

  33. 33.

    Proteins as initiators of controlled radical polymerization: grafting-from via ATRP and RAFT. ACS Macro Lett. 1, 141–145 (2012).

  34. 34.

    & Comprehensive Polymer Science Vol. 9 (eds Matyjaszewski, K. & Möller, M.) Ch. 17, 317–337 (Elsevier, 2012).

  35. 35.

    , & Living radical polymerization by the RAFT process—a third update. Aust. J. Chem. 65, 985–1076 (2012).

  36. 36.

    , , , & Differences in cytotoxicity of poly(PEGA)s synthesized by reversible addition–fragmentation chain transfer polymerization. Chem. Commun. 3580–3582 (2009).

  37. 37.

    & Discovery of a sulfated tetrapeptide that binds to vascular endothelial growth factor. Acta Biomater. 1, 451–459 (2005).

  38. 38.

    et al. Synthesis of maleimide-end-functionalized star polymers and multimeric protein–polymer conjugates. Macromolecules 42, 8028–8033 (2009).

  39. 39.

    et al. Electrospray ionization mass spectrometry and ion mobility analysis of the 20S proteasome complex. J. Am. Soc. Mass Spectr. 16, 998–1008 (2005).

  40. 40.

    The significance of surface pH in chronic wounds. Wounds UK 3, 52–56 (2007).

  41. 41.

    et al. Structures of the tyrosine kinase domain of fibroblast growth factor receptor in complex with inhibitors. Science 276, 955–960 (1997).

  42. 42.

    et al. Crystal structure of an angiogenesis inhibitor bound to the FGF receptor tyrosine kinase domain. EMBO J. 17, 5896–5904 (1998).

  43. 43.

    , , , & The human fibroblast growth-factor receptor genes—a common structural arrangement underlies the mechanisms for generating receptor forms that differ in their 3rd immunoglobulin domain. Mol. Cell. Biol. 11, 4627–4634 (1991).

  44. 44.

    et al. Crystal structure of a ternary FGF–FGFR–heparin complex reveals a dual role for heparin in FGFR binding and dimerization. Mol. Cell 6, 743–750 (2000).

  45. 45.

    , , & Structural basis for FGF receptor dimerization and activation. Cell 98, 641–650 (1999).

  46. 46.

    & Ligand specificity and heparin dependence of fibroblast growth-factor receptor-1 and receptor-3. J. Biol. Chem. 267, 16305–16311 (1992).

  47. 47.

    The pharmacology of PEGylation: balancing PD with PK to generate novel therapeutics. J. Pharm. Sci. 97, 4167–4183 (2008).

  48. 48.

    & Polymer–drug conjugation, recent achievements and general strategies. J. Pharm. Sci. 32, 933–961 (2007).

  49. 49.

    , , , & Noncovalent PEGylation by polyanion complexation as a means to stabilize keratinocyte growth factor-2 (KGF-2). Biomacromolecules 12, 3880–3894 (2011).

  50. 50.

    , , & A polymer scaffold for protein oligomerization. J. Am. Chem. Soc. 126, 1608–1609 (2004).

Download references

Acknowledgements

This work was supported by the National Science Foundation (CHE-0809832) and the National Institutes of Health (R01 EB136774 to H.D.M.; R01 GM103479 to J.A.L.). The authors thank D. Ornitz (Washington University) for providing the engineered BaF3 cell line.

Author information

Affiliations

  1. Department of Chemistry and Biochemistry, University of California, Los Angeles, 607 Charles E. Young Drive East, Los Angeles, California 90095, USA

    • Thi H. Nguyen
    • , Sung-Hye Kim
    • , Caitlin G. Decker
    • , Darice Y. Wong
    • , Joseph A. Loo
    •  & Heather D. Maynard

Authors

  1. Search for Thi H. Nguyen in:

  2. Search for Sung-Hye Kim in:

  3. Search for Caitlin G. Decker in:

  4. Search for Darice Y. Wong in:

  5. Search for Joseph A. Loo in:

  6. Search for Heather D. Maynard in:

Contributions

T.H.N. prepared the polymers and the conjugates, performed the biological cell studies and the mass spectrometry measurements, devised some of the experiments, and prepared the manuscript. S.H.K. initiated the on-column conjugation technique and initiated cell experiments. C.G.D. helped with the polymer syntheses. D.Y.W. assisted with the cell studies and performed statistical analyses. J.A.L. advised on the mass spectrometry and ion mobility studies. H.D.M. devised the project and many of the experiments, supervised all experiments, and helped analyse the data. All authors assisted with editing the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Heather D. Maynard.

Supplementary information

PDF files

  1. 1.

    Supplementary information

    Supplementary information

About this article

Publication history

Received

Accepted

Published

DOI

https://doi.org/10.1038/nchem.1573

Further reading