Temporal Patterning of Hydrogel Biochemical Environments to Study Cell-Matrix Interactions

Lambert, Catherine (McMaster University)
Wylie, Ryan G (McMaster University)

Introduction

The ECM plays a critical role in stem cell and cancer biology [1,2]. The ECM surrounding stem cells in vivo is not static, and the dynamic changes in the chemical and mechanical environment provide important regulation of cell fate [1]. Elucidating the role of cell-ECM interactions will allow us to develop novel regenerative therapies using stem cells and targeted cancer treatments. In an effort to understand features of the ECM that control cellular processes, we are designing ECM mimics from hydrogels where the biochemical environment can be tuned over time. This will allow us to recapitulate the dynamic in vivo extracellular environment, and better understand the role of cell-matrix interactions. To this end, we have development a method to reversibly immobilize biomolecules in hydrogels using the desthiobiotin-streptavidin physical interaction. Agarose polymers were covalently modified with desthiobiotin, which act as binding sites for streptavidin. To immobilize biomolecules, we synthesized peptide-streptavidin conjugates by covalently linking a bioactive peptide (GRGDS) to streptavidin. Therefore, adding peptide-streptavidin conjugates to desthiobiotin agarose gels results in peptide immobilization. Peptide-streptavidin conjugates were unbound from the hydrogel by adding biotin, which binds to streptavidin with greater affinity than desthiobiotin (KD 10-11M versus KD of 10-15M) [3]. The newly formed biotin-streptavidin-peptide complexes can then be washed from the hydrogel to once again yield agarose-desthiobiotin hydrogels, which can be re-modified with peptide-streptavidin conjugates indefinitely.

Materials and Methods

Agarose was modified with primary amines by reacting agarose polymers with carbonyl diimidazole followed ethylene diamine. Desthiobiotin was reacted with EDC and NHS to produce NHS-desthiobiotin, which was then reacted amino-agarose to yield agarose-desthiobiotin. Streptavidin-RGD was synthesized by reacting maleimide-Streptavidin with CGRGDS which was then fluorescently labelled with NHS-Alexa 488. Fluorescent streptavidin-488 and streptavidin-647 were purchased from Life Technologies (Burlington, ON). All polymers and proteins were purified by dialysis. The binding of fluorescently labelled streptavidin conjugates to agarose-desthiobitoin hydrogels was followed by tracking hydrogel fluorescence. Binding experiment were performed by adding solutions of fluorescent streptavidin (with or without conjugated RGD) to 0.7 wt. % agarose-desthiobiotin hydrogels. Excess streptavidin was washed away by soaking the gels in buffer. To unbind streptavidin, gels were soaked in buffers containing biotin. After a final washing step to remove free biotin, the agarose-desthiobiotin gels were once again chemically modified with different streptavidin molecules. Experiment were performed in PBS with 0.5 % bovine serum albumin or 1 % calf bovine serum.

Results

Repeated temporal patterning of streptavidin-488 in agarose-desthiobiotin hydrogels was achieved by varying the components of the buffer surrounding the gels. Agarose-desthiobiotin gels were soaked in solution of streptavidin-488. After binding and removal of excess streptavidin-488, we obtained chemically modified hydrogels (streptavidin bound) that are stable for over 20 days. To revert the hydrogels back to their original state (agarose-desthiobiotin), a biotin solution was added to unbind and wash away streptavidin-488 from the gels. This process was repeated to demonstrate the sequential patterning of streptavidin-488. Similarly, sequential temporal patterning of different chemical environments was demonstrated by first immobilizing and removing streptavidin 488 and then streptavidin 647. Agarose gels without desthiobiotin were used as controls. The density of biomolecules in the hydrogel can be controlled by mixing bioactive streptavidin conjugates with inactive streptavidin molecules during the immobilization step. Controlling the ratio of streptavidin-RGD-488 (bioactive) to streptavidin 647 (inactive) in solution during immobilization will determine the amount of streptavidin-RGD-488 bound to the hydrogel. Importantly, it was demonstrated that streptavidin-RGD is bioactive by performing fibroblast adhesions assays.

Discussion and Conclusion

Using the desthiobiotin-streptavidin patterning system we are able to both control the type of chemical environment and concentration of bioactive ligands in hydrogels over time. The strong (KD = 10-11M) but reversible interaction between desthiobiotin and streptavidin allows for the stable and reversible chemical modification of hydrogels. This method will allow us to tune the biochemical environment of hydrogels over long time periods to mimic the dynamic nature ECM. This system will be particularly useful to study cell-matrix interactions and control cellular activities for application in tissue engineering.

Acknowledgements

We are grateful to the McMaster University and the Natural Sciences and Engineering Research Council (NSERC) for partial funding of this research. We are also grateful to the NSERC USRA program(CL).

References

[1] Murphy, W. L., McDevitt, T. C. & Engler, A. J. Materials as stem cell regulators. Nature materials 13, 547-557 (2014) [2]Lu, P., Weaver, V. M. & Werb, Z. The extracellular matrix: a dynamic niche in cancer progression. The Journal of cell biology 196, 395-406 (2012) [3] Hirsch, J. D. et al. Easily reversible desthiobiotin binding to streptavidin, avidin, and other biotin-binding proteins: uses for protein labeling, detection, and isolation. Analytical biochemistry 308, 343-357 (2002)

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