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Sequential click reactions for synthesizing and patterning three-dimensional cell microenvironments

Nature Materials volume 8, pages 659–664 (2009)Cite this article

Abstract

Click chemistry provides extremely selective and orthogonal reactions that proceed with high efficiency and under a variety of mild conditions, the most common example being the copper(I)-catalysed reaction of azides with alkynes1,2. While the versatility of click reactions has been broadly exploited3,4,5, a major limitation is the intrinsic toxicity of the synthetic schemes and the inability to translate these approaches into biological applications. This manuscript introduces a robust synthetic strategy where macromolecular precursors react through a copper-free click chemistry6, allowing for the direct encapsulation of cells within click hydrogels for the first time. Subsequently, an orthogonal thiol–ene photocoupling chemistry is introduced that enables patterning of biological functionalities within the gel in real time and with micrometre-scale resolution. This material system enables us to tailor independently the biophysical and biochemical properties of the cell culture microenvironments in situ. This synthetic approach uniquely allows for the direct fabrication of biologically functionalized gels with ideal structures that can be photopatterned, and all in the presence of cells.

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Figure 1: Cytocompatible-click-hydrogel formation reaction and kinetics.
Figure 2: Cytocompatible, biochemical patterning within preformed click hydrogels.
Figure 3: Visualizing 3T3 collagenase activity through patterned detection peptide within 3D click hydrogels.
Figure 4: Effect of patterned RGD on 3T3 population within 3D click hydrogels.

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References

  1. Kolb, H. C., Finn, M. G. & Sharpless, K. B. Click chemistry: Diverse chemical function from a few good reactions. Angew. Chem. Int. Ed. 40, 2004–2021 (2001).

    Article  CAS  Google Scholar 

  2. Huisgen, R. 1.3-Dipolare Cycloadditionen—Ruckschau und Ausblick. Angew. Chem. Int. Ed. 75, 604–637 (1963).

    Article  CAS  Google Scholar 

  3. Moses, J. E. & Moorhouse, A. D. The growing applications of click chemistry. Chem. Soc. Rev. 36, 1249–1262 (2007).

    Article  CAS  Google Scholar 

  4. Kolb, H. C. & Sharpless, K. B. The growing impact of click chemistry on drug discovery. Drug Discov. Today 8, 1128–1137 (2003).

    Article  CAS  Google Scholar 

  5. Hawker, C. J. & Wooley, K. L. The convergence of synthetic organic and polymer chemistries. Science 309, 1200–1205 (2005).

    Article  CAS  Google Scholar 

  6. Baskin, J. M. et al. Copper-free click chemistry for dynamic in vivo imaging. Proc. Natl Acad. Sci. USA 104, 16793–16797 (2007).

    Article  CAS  Google Scholar 

  7. Srinivasan, R., Li, J., Ng, S. L., Kalesh, K. A. & Yao, S. Q. Methods of using click chemistry in the discovery of enzyme inhibitors. Nature Protocols 2, 2655–2664 (2007).

    Article  CAS  Google Scholar 

  8. Evans, M. J., Saghatelian, A., Sorensen, E. J. & Cravatt, B. F. Target discovery in small-molecule cell-based screens by in situ proteome reactivity profiling. Nature Biotech. 23, 1303–1307 (2005).

    Article  CAS  Google Scholar 

  9. Franc, G. & Kakkar, A. Dendrimer design using Cu–I-catalyzed alkyne-azide ‘click-chemistry’. Chem. Commun. 5267–5276 (2008).

  10. Malkoch, M. et al. Synthesis of well-defined hydrogel networks using click chemistry. Chem. Commun. 2774–2776 (2006).

  11. Polizzotti, B. D., Fairbanks, B. D. & Anseth, K. S. Three-dimensional biochemical patterning of click-based composite hydrogels via thiolene photopolymerization. Biomacromolecules 9, 1084–1087 (2008).

    Article  CAS  Google Scholar 

  12. Tornoe, C. W., Christensen, C. & Meldal, M. Peptidotriazoles on solid phase: [1,2,3]-triazoles by regiospecific copper(I)-catalyzed 1,3-dipolar cycloadditions of terminal alkynes to azides. J. Org. Chem. 67, 3057–3064 (2002).

    Article  CAS  Google Scholar 

  13. Franke, R., Doll, C. & Eichler, J. Peptide ligation through click chemistry for the generation of assembled and scaffolded peptides. Tetrahedron Lett. 46, 4479–4482 (2005).

    Article  CAS  Google Scholar 

  14. Prescher, J. A. & Bertozzi, C. R. Chemistry in living systems. Nature Chem. Biol. 1, 13–21 (2005).

    Article  CAS  Google Scholar 

  15. Chen, J. K. Fish ’n clicks. Nature Chem. Biol. 4, 391–392 (2008).

    Article  CAS  Google Scholar 

  16. Agard, N. J., Prescher, J. A. & Bertozzi, C. R. A strain-promoted [3+2] azide–alkyne cycloaddition for covalent modification of biomolecules in living systems. J. Am. Chem. Soc. 126, 15046–15047 (2004).

    Article  CAS  Google Scholar 

  17. Laughlin, S. T., Baskin, J. M., Amacher, S. L. & Bertozzi, C. R. In vivo imaging of membrane-associated glycans in developing zebrafish. Science 320, 664–667 (2008).

    Article  CAS  Google Scholar 

  18. Dondoni, A. The emergence of thiol–ene coupling as a click process for materials and bioorganic chemistry. Angew. Chem. Int. Ed. 47, 8995–8997 (2008).

    Article  CAS  Google Scholar 

  19. Hoyle, C. E., Lee, T. Y. & Roper, T. Thiol–enes: Chemistry of the past with promise for the future. J. Polym. Sci. A 42, 5301–5338 (2004).

    Article  CAS  Google Scholar 

  20. Khire, V. S., Benoit, D. S. W., Anseth, K. S. & Bowman, C. N. Ultrathin gradient films using thiol–ene polymerizations. J. Polym. Sci. A 44, 7027–7039 (2006).

    Article  CAS  Google Scholar 

  21. Killops, K. L., Campos, L. M. & Hawker, C. J. Robust, efficient, and orthogonal synthesis of dendrimers via thiol–ene ‘click’ chemistry. J. Am. Chem. Soc. 130, 5062–5064 (2008).

    Article  CAS  Google Scholar 

  22. Langer, R. & Vacanti, J. P. Tissue engineering. Science 260, 920–926 (1993).

    Article  CAS  Google Scholar 

  23. Cushing, M. C. & Anseth, K. S. Hydrogel cell cultures. Science 316, 1133–1134 (2007).

    Article  CAS  Google Scholar 

  24. Anseth, K. S. A biologist looks to ‘click chemistry’ for better three-dimensional tissue models. Nature 453, 5 (2008).

    Article  CAS  Google Scholar 

  25. Codelli, J. A., Baskin, J. M., Agard, N. J. & Bertozzi, C. R. Second-generation difluorinated cyclooctynes for copper-free click chemistry. J. Am. Chem. Soc. 130, 11486–11493 (2008).

    Article  CAS  Google Scholar 

  26. Nagase, H. & Fields, G. B. Human matrix metalloproteinase specificity studies using collagen sequence-based synthetic peptides. Biopolymers 40, 399–416 (1996).

    Article  CAS  Google Scholar 

  27. Lutolf, M. P. et al. Synthetic matrix metalloproteinase-sensitive hydrogels for the conduction of tissue regeneration: Engineering cell-invasion characteristics. Proc. Natl Acad. Sci. USA 100, 5413–5418 (2003).

    Article  CAS  Google Scholar 

  28. Lutolf, M. P. & Hubbell, J. A. Synthetic biomaterials as instructive extracellular microenvironments for morphogenesis in tissue engineering. Nature Biotech. 23, 47–55 (2005).

    Article  CAS  Google Scholar 

  29. Kunz, H. & Unverzagt, C. The allyloxycarbonyl (aloc) moiety—conversion of an unsuitable into a valuable amino protecting group for peptide-synthesis. Angew. Chem. Int. Ed. 23, 436–437 (1984).

    Article  Google Scholar 

  30. Lee, T. Y., Smith, Z., Reddy, S. K., Cramer, N. B. & Bowman, C. N. Thiol–allyl ether–methacrylate ternary systems. Polymerization mechanism. Macromolecules 40, 1466–1472 (2007).

    Article  CAS  Google Scholar 

  31. Mather, B. D., Viswanathan, K., Miller, K. M. & Long, T. E. Michael addition reactions in macromolecular design for emerging technologies. Prog. Polym. Sci. 31, 487–531 (2006).

    Article  CAS  Google Scholar 

  32. Cramer, N. B., Reddy, S. K., O’Brien, A. K. & Bowman, C. N. Thiol–ene photopolymerization mechanism and rate limiting step changes for various vinyl functional group chemistries. Macromolecules 36, 7964–7969 (2003).

    Article  CAS  Google Scholar 

  33. Ruoslahti, E. & Pierschbacher, M. D. New perspectives in cell-adhesion—RGD and integrins. Science 238, 491–497 (1987).

    Article  CAS  Google Scholar 

  34. Burdick, J. A. & Anseth, K. S. Photoencapsulation of osteoblasts in injectable RGD-modified PEG hydrogels for bone tissue engineering. Biomaterials 23, 4315–4323 (2002).

    Article  CAS  Google Scholar 

  35. Hahn, M. S., Miller, J. S. & West, J. L. Three-dimensional biochemical and biomechanical patterning of hydrogels for guiding cell behaviour. Adv. Mater. 18, 2679–2684 (2006).

    Article  CAS  Google Scholar 

  36. Lee, S. H., Moon, J. J. & West, J. L. Three-dimensional micropatterning of bioactive hydrogels via two-photon laser scanning photolithography for guided 3D cell migration. Biomaterials 29, 2962–2968 (2008).

    Article  CAS  Google Scholar 

  37. Luo, Y. & Shoichet, M. S. A photolabile hydrogel for guided three-dimensional cell growth and migration. Nature Mater. 3, 249–253 (2004).

    Article  CAS  Google Scholar 

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Acknowledgements

The authors would like to thank C. Bertozzi and J. Baskin for their initial donation of DIFO3, R. Shoemaker for his assistance with magic-angle spinning NMR, A. Kloxin and B. Fairbanks for their discussions on photopatterning, A. Aimetti for communication on peptide work and C. Kloxin for his critiques in the preparation of this manuscript. Thanks are also given to HHMI and the Janelia Farm Research Campus for support in the use of their two-photon confocal microscope. Fellowship assistance to C.A.D. was awarded by the US Department of Education’s Graduate Assistantships in Areas of National Need program and the National Institutes of Health (T32 GM-065103).

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Authors and Affiliations

  1. Department of Chemical and Biological Engineering,

    Cole A. DeForest, Brian D. Polizzotti & Kristi S. Anseth

  2. Howard Hughes Medical Institute, University of Colorado, UCB Box 424 Boulder, Colorado 80309-0424, USA

    Brian D. Polizzotti & Kristi S. Anseth

Authors
  1. Cole A. DeForest
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Contributions

B.D.P., C.A.D. and K.S.A. developed the material concept, C.A.D., B.D.P. and K.S.A. designed the experiments, C.A.D. carried out the experiments, and C.A.D. and K.S.A. composed the manuscript.

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Correspondence to Kristi S. Anseth.

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DeForest, C., Polizzotti, B. & Anseth, K. Sequential click reactions for synthesizing and patterning three-dimensional cell microenvironments. Nature Mater 8, 659–664 (2009). https://doi.org/10.1038/nmat2473

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