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Injectable tissue integrating networks from recombinant polypeptides with tunable order

Nature Materials volume 17, pages 1154–1163 (2018)Cite this article

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An Author Correction to this article was published on 31 October 2018

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Abstract

Emergent properties of natural biomaterials result from the collective effects of nanoscale interactions among ordered and disordered domains. Here, using recombinant sequence design, we have created a set of partially ordered polypeptides to study emergent hierarchical structures by precisely encoding nanoscale order–disorder interactions. These materials, which combine the stimuli-responsiveness of disordered elastin-like polypeptides and the structural stability of polyalanine helices, are thermally responsive with tunable thermal hysteresis and the ability to reversibly form porous, viscoelastic networks above threshold temperatures. Through coarse-grain simulations, we show that hysteresis arises from physical crosslinking due to mesoscale phase separation of ordered and disordered domains. On injection of partially ordered polypeptides designed to transition at body temperature, they form stable, porous scaffolds that rapidly integrate into surrounding tissue with minimal inflammation and a high degree of vascularization. Sequence-level modulation of structural order and disorder is an untapped principle for the design of functional protein-based biomaterials.

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Fig. 1: Partially ordered polymer library and structural characterization
Fig. 2: Phase behaviour and tunable hysteresis
Fig. 3: Proposed mechanism for hysteresis
Fig. 4: Arrested phase separation into fractal networks
Fig. 5: Network stability and void volume
Fig. 6: In vivo stability and tissue incorporation of POPs

Data availability

The authors declare that all data supporting the findings of this study are available within the manuscript and its supplementary files and are available from the authors on reasonable request.

Change history

  • 31 October 2018

    In the version of this Article originally published, one of the authors’ names was incorrectly given as Jeffery Schaal; it should have been Jeffrey L. Schaal. This has been corrected in all versions of the Article.

References

  1. Keten, S., Xu, Z., Ihle, B. & Buehler, M. J. Nanoconfinement controls stiffness, strength and mechanical toughness of β-sheet crystals in silk. Nat. Mater. 9, 359–367 2010).

    Article  CAS  Google Scholar 

  2. Tamburro, A. M., Bochicchio, B. & Pepe, A. Dissection of human tropoelastin: exon-by-exon chemical synthesis and related conformational studies. Biochemistry 42, 13347–13362 2003).

    Article  CAS  Google Scholar 

  3. van der Lee, R.et al. Classification of intrinsically disordered regions and proteins. Chem. Rev. 114, 6589–6631 2014).

    Article  CAS  Google Scholar 

  4. Harmon, T. S., Holehouse, A. S., Rosen, M. K. & Pappu, R. V. Intrinsically disordered linkers determine the interplay between phase separation and gelation in multivalent proteins. eLife 6, e30294 (2017).

    Article  Google Scholar 

  5. McDaniel, J. R., Mackay, J. A., Quiroz, F. G. & Chilkoti, A. Recursive directional ligation by plasmid reconstruction allows rapid and seamless cloning of oligomeric genes. Biomacromolecules 11, 944–952 2010).

    Article  CAS  Google Scholar 

  6. Roberts, S., Dzuricky, M. & Chilkoti, A. Elastin-like polypeptides as models of intrinsically disordered proteins. FEBS Lett. 589, 2477–2486 (2015).

    Article  CAS  Google Scholar 

  7. McDaniel, J. R., Radford, D. C. & Chilkoti, A. A unified model for de novo design of elastin-like polypeptides with tunable inverse transition temperatures. Biomacromolecules 14, 2866–2872 (2013).

    Article  CAS  Google Scholar 

  8. Pometun, M. S., Chekmenev, E. Y. & Wittebort, R. J. Quantitative observation of backbone disorder in native elastin. J. Biol. Chem. 279, 7982–7987 (2004).

    Article  CAS  Google Scholar 

  9. MacEwan, S. R. & Chilkoti, A. Elastin-like polypeptides: biomedical applications of tunable biopolymers. Biopolymers 94, 60–77 (2010).

    Article  CAS  Google Scholar 

  10. Gosline, J. et al. Elastic proteins: biological roles and mechanical properties. Philos. Trans. R. Soc. B 357, 121–132 (2002).

    Article  CAS  Google Scholar 

  11. Lillie, M. A. & Gosline, J. M. The viscoelastic basis for the tensile strength of elastin. Int. J. Biol. Macromol. 30, 119–127 (2002).

    Article  CAS  Google Scholar 

  12. Miller, J. S., Kennedy, R. J. & Kemp, D. S. Solubilized, spaced polyalanines: A context-free system for determining amino acid α-helix propensities. J. Am. Chem. Soc. 124, 945–962 (2002).

    Article  CAS  Google Scholar 

  13. Chakrabartty, A. & Baldwin, R. Stability of α-Helices. Adv. Protein Chem. 46, 141–176 (1995).

  14. Bochicchio, B., Pepe, A. & Tamburro, A. M. Investigating by CD the molecular mechanism of elasticity of elastomeric proteins. Chirality. 20, 985–994 (2008).

    Article  CAS  Google Scholar 

  15. Sreerama, N. & Woody, R. W. Estimation of protein secondary structure from circular dichroism spectra: Comparison of CONTIN, SELCON, and CDSSTR methods with an expanded reference set. Anal. Biochem. 287, 252–260 (2000).

    Article  CAS  Google Scholar 

  16. Bernacki, J. P. & Murphy, R. M. Length-dependent aggregation of uninterrupted polyalanine peptides. Biochemistry 50, 9200–9211 (2011).

    Article  CAS  Google Scholar 

  17. Muñoz, V. & Serrano, L. Elucidating the folding problem of helical peptides using empirical parameters. Nat. Struct. Biol. 1, 399–409 (1994).

    Article  Google Scholar 

  18. Lacroix, E., Viguera, A. R. & Serrano, L. Elucidating the folding problem of α-helices: local motifs, long-range electrostatics, ionic-strength dependence and prediction of NMR parameters. J. Mol. Biol. 284, 173–191 (1998).

    Article  CAS  Google Scholar 

  19. Wright, E. R., McMillan, R. A., Cooper, A., Apkarian, R. P. & Conticello, V. P. Thermoplastic elastomer hydrogels via self-assembly of an elastin-mimetic triblock polypeptide. Adv. Funct. Mater. 12, 149–154 (2002).

    Article  CAS  Google Scholar 

  20. Glassman, M. J., Avery, R. K., Khademhosseini, A. & Olsen, B. D. Toughening of thermoresponsive arrested networks of elastin-like polypeptides to engineer cytocompatible tissue scaffolds. Biomacromolecules 17, 415–426 (2016).

    Article  CAS  Google Scholar 

  21. Reguera, J. et al. Thermal behavior and kinetic analysis of the chain unfolding and refolding and of the concomitant nonpolar solvation and desolvation of two elastin-like polymers. Macromolecules 36, 8470–8476 (2003).

    Article  CAS  Google Scholar 

  22. Cho, Y. et al. Hydrogen bonding of β-turn structure is stabilized in D2O. J. Am. Chem. Soc. 131, 15188–15193 (2009).

    Article  CAS  Google Scholar 

  23. Ding, F., Borreguero, J. M., Buldyrey, S. V., Stanley, H. E. & Dokholyan, N. V. Mechanism for the α-helix to β-hairpin transition. Proteins 53, 220–228 (2003).

    Article  CAS  Google Scholar 

  24. Urry, D. W. & Ji, T. H. Distortions in circular dichroism patterns of particulate (or membranous) systems. Arch. Biochem. Biophys. 128, 802–807 (1968).

    Article  CAS  Google Scholar 

  25. Urry, D. W., Starcher, B. & Partridge, S. M. Coacervation of solubilized elastin effects a notable conformational change. Nature 222, 795–796 (1969).

    Article  CAS  Google Scholar 

  26. Muiznieks, L. D., Jensen, S. A. & Weiss, A. S. Structural changes and facilitated association of tropoelastin. Arch. Biochem. Biophys. 410, 317–323 (2003).

    Article  CAS  Google Scholar 

  27. Yeo, G. C., Keeley, F. W. & Weiss, A. S. Coacervation of tropoelastin. Adv. Colloid. Interface Sci. 167, 94–103 (2011).

    Article  CAS  Google Scholar 

  28. Israelachvili, J. N., Mitchell, D. J. & Ninham, B. W. Theory of self-assembly of hydrocarbon amphiphiles into micelles and bilayers. J. Chem. Soc. Faraday Trans. I 72, 1525–1568 (1976).

    Article  CAS  Google Scholar 

  29. Dušek, K. Phase separation during the formation of three-dimensional polymers. J. Polym. Sci. B 3, 209–212 (1965).

    Article  Google Scholar 

  30. Cirulis, J. T., Keeley, F. W. & James, D. F. Viscoelastic properties and gelation of an elastin-like polypeptide. J. Rheol. 53, 1215 (2009).

    Article  CAS  Google Scholar 

  31. Tamburro, A. M., De Stradis, A. & D’Alessio, L. Fractal aspects of elastin supramolecular organization. J. Biomol. Struct. Dyn. 12, 1161–1172 (1995).

    Article  CAS  Google Scholar 

  32. Gustafsson, M. G. Nonlinear structured-illumination microscopy: wide-field fluorescence imaging with theoretically unlimited resolution. Proc. Natl Acad. Sci. USA 102, 13081–13086 (2005).

    Article  CAS  Google Scholar 

  33. Clarke, A. W. et al. Tropoelastin massively associates during coacervation to form quantized protein spheres. Biochemistry 45, 9989–9996 (2006).

    Article  CAS  Google Scholar 

  34. Caiazzo, M. et al. Defined three-dimensional microenvironments boost induction of pluripotency. Nat. Mater. 15, 344–352 (2016).

    Article  CAS  Google Scholar 

  35. Engler, A. J., Sen, S., Sweeney, H. L. & Discher, D. E. Matrix elasticity directs stem cell lineage specification. Cell 126, 677–689 (2006).

    Article  CAS  Google Scholar 

  36. DiMarco, R. L. & Heilshorn, S. C. Multifunctional materials through modular protein engineering. Adv. Mater. 24, 3923–3940 (2012).

    Article  CAS  Google Scholar 

  37. Fernandez-Colino, A., Arias, F. J., Alonso, M. & Rodriguez-Cabello, J. C. Amphiphilic elastin-like block co-recombinamers containing leucine zippers: Cooperative interplay between both domains results in injectable and stable hydrogels. Biomacromolecules 16, 3389–3398 (2015).

    Article  CAS  Google Scholar 

  38. Xu, C. & Kopecek, J. Genetically engineered block copolymers: influence of the length and structure of the coiled-coil blocks on hydrogel self-assembly. Pharm. Res. 25, 674–682 (2008).

    Article  CAS  Google Scholar 

  39. Wright, E. R. & Conticello, V. P. Self-assembly of block copolymers derived from elastin-mimetic polypeptide sequences. Adv. Drug Deliv. Rev. 54, 1057–1073 (2002).

  40. Martin, L., Castro, E., Ribeiro, A., Alonso, M. & Rodriguez-Cabello, J. C. Temperature-triggered self-assembly of elastin-like block co-recombinamers:the controlled formation of micelles and vesicles in an aqueous medium. Biomacromolecules 13, 293–298 (2012).

    Article  CAS  Google Scholar 

  41. MacEwan, S. R. et al. Phase behavior and self-assembly of perfectly sequence-defined and monodisperse multiblock copolypeptides. Biomacromolecules 18, 599–609 (2017).

    Article  CAS  Google Scholar 

  42. Xia, X. X., Xu, Q., Hu, X., Qin, G. & Kaplan, D. L. Tunable self-assembly of genetically engineered silk--elastin-like protein polymers. Biomacromolecules 12, 3844–3850 (2011).

    Article  CAS  Google Scholar 

  43. Luo, T. & Kiick, K. L. Noncovalent modulation of the inverse temperature transition and self-assembly of elastin-b-collagen-like peptide bioconjugates. J. Am. Chem. Soc. 137, 15362–15365 (2015).

    Article  CAS  Google Scholar 

  44. Ray, B., El Hasri, S. & Guenet, J. M. Effect of polymer tacticity on the molecular structure of polyelectrolyte/surfactant stoichiometric complexes in solutions and gels. Eur. Phys. J. E 11, 315–323 (2003).

    Article  CAS  Google Scholar 

  45. Ray, B. et al. Effect of tacticity of poly(N-isopropylacrylamide) on the phase separation temperature of its aqueous solutions. Polym. J. 37, 234–237 (2005).

    Article  CAS  Google Scholar 

  46. Amiram, M., Luginbuhl, K. M., Li, X., Feinglos, M. N. & Chilkoti, A. Injectable protease-operated depots of glucagon-like peptide-1 provide extended and tunable glucose control. Proc. Natl Acad. Sci. USA 110, 2792–2797 (2013).

    Article  CAS  Google Scholar 

  47. Nettles, D. L., Chilkoti, A. & Setton, L. A. Applications of elastin-like polypeptides in tissue engineering. Adv. Drug Deliv. Rev. 62, 1479–1485 (2010).

    Article  CAS  Google Scholar 

  48. Nettles, D. L. et al. In situ crosslinking elastin-like polypeptide gels for application to articular cartilage repair in a goat osteochondral defect model. Tissue Eng. Part A 14, 1133–1140 (2008).

    Article  CAS  Google Scholar 

  49. Ratner, B. D. A pore way to heal and regenerate: 21st century thinking on biocompatibility. Regen. Biomater. 3, 107–110 (2016).

    Article  CAS  Google Scholar 

  50. Sussman, E. M., Halpin, M. C., Muster, J., Moon, R. T. & Ratner, B. D. Porous implants modulate healing and induce shifts in local macrophage polarization in the foreign body reaction. Ann. Biomed. Eng. 42, 1508–1516 (2014).

    Article  Google Scholar 

  51. Meyer, D. E. & Chilkoti, A. Purification of recombinant proteins by fusion with thermally-responsive polypeptides. Nat. Biotechnol. 17, 1112–1115 (1999).

    Article  CAS  Google Scholar 

  52. Schneider, C. A., Rasband, W. S. & Eliceiri, K. W. NIH Image to ImageJ: 25 years of image analysis. Nat. Methods 9, 671–675 (2012).

    Article  CAS  Google Scholar 

  53. Karperien, A. FracLac for ImageJ, version 2.5 http://rsb.info.nih.gov/ij/plugins/fraclac/FLHelp/Introduction.html (1999–2013).

  54. Shao, L., Kner, P., Rego, E. H. & Gustafsson, M. G. Super-resolution 3D microscopy of live whole cells using structured illumination. Nat. Methods 8, 1044–1046 (2011).

    Article  CAS  Google Scholar 

  55. Gustafsson, M. G. et al. Three-dimensional resolution doubling in wide-field fluorescence microscopy by structured illumination. Biophys. J. 94, 4957–4970 (2008).

    Article  CAS  Google Scholar 

  56. Wood, W. G., Wachter, C. & Scriba, P. C. Experiences using chloramine-T and 1,3,4,6-tetrachloro-3-α,6-α-Diphenylglycoluril (Iodogen®) for radioiodination of materials for radioimmunoassay. J. Clin. Chem. Clin. Bio. 19, 1051–1056 (1981).

    CAS  Google Scholar 

  57. Diehl, K. H. et al. A good practice guide to the administration of substances and removal of blood, including routes and volumes. J. Appl. Toxicol. 21, 15–23 (2001).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank K. Wang for his invaluable help with SIM imaging and E. Betzig for use of his facilities at Janelia Farms for SIM. This work was funded by the NIH through grants GM061232 to A.C. and R01NS056114 to R.V.P., by the NSF through grants from the Research Triangle MRSEC (DMR-11-21107), NSF DMFREF (DMR-1729671) to A.C., MCB-1614766 to R.V.P., and through the Graduate Research Fellowship Program under grant no. 1106401 to S.R.

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Authors and Affiliations

  1. Department of Biomedical Engineering, Duke University, Durham, NC, USA

    Stefan Roberts, Jeffrey L. Schaal, Vincent Miao, Andrew Hunt, Yi Wen, Joel H. Collier & Ashutosh Chilkoti

  2. Department of Physics, Washington University in St. Louis, St. Louis, MO, USA

    Tyler S. Harmon

  3. Department of Biomedical Engineering and Center for Biological Systems Engineering , Washington University in St. Louis, St. Louis, MO, USA

    Tyler S. Harmon & Rohit V. Pappu

  4. Department of Biochemistry, Duke University, Durham, NC, USA

    Kan (Jonathan) Li & Terrence G. Oas

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Contributions

S.R. designed and performed experiments, analysed data and prepared the manuscript. T.S.H. designed and performed the coarse-grained simulations and co-developed the phenomenological model for hysteresis. J.L.S. designed, performed and analysed in vivo work. K.L. designed and performed structural characterization with NMR and CD. A.H. and V.M. constructed POPs and characterized their phase behaviour. V.M. also performed rheological experiments. Y.W. designed and aided in vivo experiments. T.O. provided guidance and analysed data for POP structural characterization. J.C. provided guidance for in vivo experiments. R.V.P. provided guidance, developed the conceptual framework for hysteresis, analysed results from the coarse-grained simulations, and contributed to preparing the manuscript. A.C. provided guidance, designed experiments and prepared the manuscript. All authors participated in discussion of the data and in editing and revising the manuscript.

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Correspondence to Ashutosh Chilkoti.

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Supplementary Methods and Discussion, Supplementary Figures 1–23, Supplementary Tables 1–4, Supplementary References 1–19

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Roberts, S., Harmon, T.S., Schaal, J. et al. Injectable tissue integrating networks from recombinant polypeptides with tunable order. Nature Mater 17, 1154–1163 (2018). https://doi.org/10.1038/s41563-018-0182-6

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