Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

A polymer nanoparticle with engineered affinity for a vascular endothelial growth factor (VEGF165)

Abstract

Protein affinity reagents are widely used in basic research, diagnostics and separations and for clinical applications, the most common of which are antibodies. However, they often suffer from high cost, and difficulties in their development, production and storage. Here we show that a synthetic polymer nanoparticle (NP) can be engineered to have many of the functions of a protein affinity reagent. Polymer NPs with nM affinity to a key vascular endothelial growth factor (VEGF165) inhibit binding of the signalling protein to its receptor VEGFR-2, preventing receptor phosphorylation and downstream VEGF165-dependent endothelial cell migration and invasion into the extracellular matrix. In addition, the NPs inhibit VEGF-mediated new blood vessel formation in Matrigel plugs in vivo. Importantly, the non-toxic NPs were not found to exhibit off-target activity. These results support the assertion that synthetic polymers offer a new paradigm in the search for abiotic protein affinity reagents by providing many of the functions of their protein counterparts.

This is a preview of subscription content

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Figure 1: The functional and sulfonate and sulfated monomers used for nanoparticle synthesis and a schematic showing the general synthesis of polymer NPs and their chemical composition.
Figure 2: Screening of polymer nanoparticles interacting with VEGF165.
Figure 3: In vitro VEGF-inhibition experiment and comparison with heparin.
Figure 4: Anti-angiogenic effect of NPs.
Figure 5: Schematic images of the interaction of heparins and VEGF165 with NPs of various compositions.

References

  1. 1

    Hardiman, G. Next-generation antibody discovery platforms. Proc. Natl Acad. Sci. USA 109, 18245–18246 (2012).

    CAS  Article  Google Scholar 

  2. 2

    Dubel, S., Stoevesandt, O., Taussig, M. J. & Hust, M. Generating recombinant antibodies to the complete human proteome. Trends Biotechnol. 28, 333–339 (2010).

    Article  Google Scholar 

  3. 3

    Lollo, B., Steele, F. & Gold, L. Beyond antibodies: new affinity reagents to unlock the proteome. Proteomics 14, 638–644 (2014).

    CAS  Article  Google Scholar 

  4. 4

    Cobaugh, C. W., Almagro, J. C., Pogson, M., Iverson, B. & Georgiou, G. Synthetic antibody libraries focused towards peptide ligands. J. Mol. Biol. 378, 622–633 (2008).

    CAS  Article  Google Scholar 

  5. 5

    Marx, V. Calling the next generation of affinity reagents. Nat. Methods 10, 829–833 (2013).

    CAS  Article  Google Scholar 

  6. 6

    Arkin, M. R. & Wells, J. A. Small-molecule inhibitors of protein–protein interactions progressing towards the dream. Nat. Rev. Drug Discov. 3, 301–317 (2004).

    CAS  Article  Google Scholar 

  7. 7

    Klinger, D. & Landfester, K. Stimuli-responsive microgels for the loading and release of functional compounds fundamental concepts and applications. Polymer 53, 5209–5231 (2012).

    CAS  Article  Google Scholar 

  8. 8

    Schild, H. G. Poly(N-isopropylacrylamide): experiment, theory and application. Prog. Polym. Sci. 17, 163–249 (1992).

    CAS  Article  Google Scholar 

  9. 9

    Uversky, V. N., Oldfield, C. J. & Dunker, A. K. Intrinsically disordered proteins in human diseases: introducing the D2 concept. Annu. Rev. Biophys. 37, 215–246 (2008).

    CAS  Article  Google Scholar 

  10. 10

    Rogers, J. M., Wong, C. T. & Clarke, J. Coupled folding and binding of the disordered protein PUMA does not require particular residual structure. J. Am. Chem. Soc. 136, 5197–5200 (2014).

    CAS  Article  Google Scholar 

  11. 11

    Hoshino, Y. et al. Affinity purification of multifunctional polymer nanoparticles. J. Am. Chem. Soc. 132, 13648–13650 (2010).

    CAS  Article  Google Scholar 

  12. 12

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

    CAS  Article  Google Scholar 

  13. 13

    Oh, Y. I., Sheng, G. J., Chang, S. K. & Hsieh-Wilson, L. C. Tailored glycopolymers as anticoagulant heparin mimetics. Angew. Chem. Int. Ed. 52, 11796–11799 (2013).

    CAS  Article  Google Scholar 

  14. 14

    Nguyen, T. H. et al. A heparin-mimicking polymer conjugate stabilizes basic fibroblast growth factor. Nat. Chem. 5, 221–227 (2013).

    CAS  Article  Google Scholar 

  15. 15

    Dernedde, J. et al. Dendritic polyglycerol sulfates as multivalent inhibitors of inflammation. Proc. Natl Acad. Sci. USA 107, 19679–19684 (2010).

    CAS  Article  Google Scholar 

  16. 16

    Hoshino, Y. et al. The rational design of a synthetic polymer nanoparticle that neutralizes a toxic peptide in vivo. Proc. Natl Acad. Sci. USA 109, 33–38 (2012).

    CAS  Article  Google Scholar 

  17. 17

    Yoshimatsu, K., Koide, H., Hoshino, Y. & Shea, K. J. Preparation of abiotic polymer nanoparticles for sequestration and neutralization of a target peptide toxin. Nat. Protoc. 10, 595–604 (2015).

    CAS  Article  Google Scholar 

  18. 18

    Koch, S. J., Renner, C., Xie, X. L. & Schrader, T. Tuning linear copolymers into protein-specific hosts. Angew. Chem. Int. Ed. 45, 6352–6355 (2006).

    CAS  Article  Google Scholar 

  19. 19

    Lee, S. H. et al. Engineered synthetic polymer nanoparticles as IgG affinity ligands. J. Am. Chem. Soc. 134, 15765–15772 (2012).

    CAS  Article  Google Scholar 

  20. 20

    Yoshimatsu, K. et al. Temperature-responsive "catch and release" of proteins by using multifunctional polymer-based nanoparticles. Angew. Chem. Int. Ed. 51, 2405–2408 (2012).

    CAS  Article  Google Scholar 

  21. 21

    Robinson, C. J., Mulloy, B., Gallagher, J. T. & Stringer, S. E. VEGF165-binding sites within heparan sulfate encompass two highly sulfated domains and can be liberated by K5 lyase. J. Biol. Chem. 281, 1731–1740 (2006).

    CAS  Article  Google Scholar 

  22. 22

    Brozzo, M. S. et al. Thermodynamic and structural description of allosterically regulated VEGFR-2 dimerization. Blood 119, 1781–1788 (2012).

    CAS  Article  Google Scholar 

  23. 23

    Hoshino, Y. et al. Design of synthetic polymer nanoparticles that capture and neutralize a toxic peptide. Small 5, 1562–1568 (2009).

    CAS  Article  Google Scholar 

  24. 24

    Hoshino, Y. et al. Recognition, neutralization, and clearance of target peptides in the bloodstream of living mice by molecularly imprinted polymer nanoparticles a plastic antibody. J. Am. Chem. Soc. 132, 6644–6645 (2010).

    CAS  Article  Google Scholar 

  25. 25

    Richard, B., Swanson, R. & Olson, S. T. The signature 3-O-sulfo group of the anticoagulant heparin sequence is critical for heparin binding to antithrombin but is not required for allosteric activation. J. Biol. Chem. 284, 27054–27064 (2009).

    CAS  Article  Google Scholar 

  26. 26

    Ye, S. et al. Structural basis for interaction of FGF-1, FGF-2, and FGF-7 with different heparan sulfate motifs. Biochemistry 40, 14429–14439 (2001).

    CAS  Article  Google Scholar 

  27. 27

    Guimond, S., Maccarana, M., Olwin, B. B., Lindahl, U. & Rapraeger, A. C. Activating and inhibitory heparin sequences for FGF-2 (basic FGF). Distinct requirements for FGF-1, FGF-2, and FGF-4. J. Biol. Chem. 268, 23906–23914 (1993).

    CAS  PubMed  Google Scholar 

  28. 28

    Manning, G. S. Counterion binding in polyelectrolyte theory. Acc. Chem. Res. 12, 443–449 (1979).

    CAS  Article  Google Scholar 

  29. 29

    Quesada-Perez, M., Callejas-Fernandez, J. & Hidalgo-Alvarez, R. An experimental test of the ion condensation theory for spherical colloidal particles. J. Colloid Interface Sci. 233, 280–285 (2001).

    CAS  Article  Google Scholar 

  30. 30

    Manning, G. S. The critical onset of counterion condensation: A survey of its experimental and theoretical basis. Ber. Bunsen. Phys. Chem. 100, 909–922 (1996).

    CAS  Article  Google Scholar 

  31. 31

    Popov, A. & Hoagland, D. A. Electrophoretic evidence for a new type of counterion condensation. J. Polym. Sci. Pol. Phys. 42, 3616–3627 (2004).

    CAS  Article  Google Scholar 

  32. 32

    Manning, G. S. Counterion condensation on charged spheres, cylinders, and planes. J. Phys. Chem. B 111, 8554–8559 (2007).

    CAS  Article  Google Scholar 

  33. 33

    Graells, J. et al. Overproduction of VEGF165 concomitantly expressed with its receptors promotes growth and survival of melanoma cells through MAPK and PI3K signaling. J. Invest. Dermatol. 123, 1151–1161 (2004).

    CAS  Article  Google Scholar 

  34. 34

    Munoz, E. M. & Linhardt, R. J. Heparin-binding domains in vascular biology. Arterioscl. Throm. Vas. 24, 1549–1557 (2004).

    CAS  Article  Google Scholar 

  35. 35

    Warkentin, T. E. et al. Heparin-induced thrombocytopenia in patients treated with low-molecular-weight heparin or unfractionated heparin. N. Engl. J. Med. 332, 1330–1336 (1995).

    CAS  Article  Google Scholar 

  36. 36

    Karamysheva, A. F. Mechanisms of angiogenesis. Biochemistry (Mosc.) 73, 751–762 (2008).

    CAS  Article  Google Scholar 

  37. 37

    Carmeliet, P. & Jain, R. K. Angiogenesis in cancer and other diseases. Nature 407, 249–257 (2000).

    CAS  Article  Google Scholar 

  38. 38

    Brekken, R. A. et al. Selective inhibition of vascular endothelial growth factor (VEGF) receptor 2 (KDR/Flk-1) activity by a monoclonal anti-VEGF antibody blocks tumor growth in mice. Cancer Res. 60, 5117–5124 (2000).

    CAS  PubMed  Google Scholar 

Download references

Acknowledgements

Financial support from the University of California Cancer Research Coordinating Committee and the National Science Foundation (DMR-1308363) is gratefully acknowledged. This research was also supported by a Grant-in-Aid for JSPS Postdoctoral Fellowships for Research Abroad (25-426), Grant-in-Aid for MEXT (23111716 and 25107726), Grant-in-Aid for Young Scientists (A) (23685027), The Kurata Memorial Hitachi Science and Technology Foundation and The Uehara Memorial Foundation. We thank T. Ozeki for help with QCM measurements.

Author information

Affiliations

Authors

Contributions

H.K., K.Y., K.J.S., Y.H., N.O. and Y.M. designed the research and H.K. and Y.H. designed the experiments. Y.H., Y.N., and Y.M. synthesized GlcNAc monomers. H.K, K.Y., A.W., S.-H.L. and Y.Y. performed the binding assay with QCM and synthesized NPs. Y.N. took TEM pictures of the NPs. H.K., A.O. and S.A. preformed the in vitro assay with supervision from N.O.; H.K., K.Y., Y.M., Y.H., N.O. and K.J.S. wrote the manuscript. All authors discussed the results and commented on the manuscript.

Corresponding authors

Correspondence to Naoto Oku, Yoshiko Miura or Kenneth J. Shea.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary information

Supplementary information (PDF 6226 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Koide, H., Yoshimatsu, K., Hoshino, Y. et al. A polymer nanoparticle with engineered affinity for a vascular endothelial growth factor (VEGF165). Nature Chem 9, 715–722 (2017). https://doi.org/10.1038/nchem.2749

Download citation

Further reading

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing