<br/>Polyethylene glycol (PEG) hydrogels have emerged as promising cell culture platforms for disease modeling because their mechanical and biochemical properties can be tuned to mimic a tissue's extracellular matrix (ECM). To control their biochemical properties, biomolecules are covalently tethered directly to the hydrogel backbone. While this approach is common for bioactive peptides, covalent tethering of whole signaling proteins to hydrogels has remained impractical due to the low stability of these proteins under cell culture conditions. Thus, a protein tethering strategy that enables the user to replace previously tethered, spent protein with fresh protein is required. Herein, we describe a methodology that enables fully reversible and repeatable tethering of whole signaling proteins to hydrogels. Specifically, this approach is based on the photo-mediated thiol-ene reaction, which affords both spatial and temporal control over protein immobilization. While traditional thiol-ene reactions are irreversible, we developed an allyl sulfide chain-transfer agent that acts as a regenerative handle for thiol-ene reactions. Using this strategy, whole signaling proteins can tethered via the thiol-ene reaction at a range of physiologically relevant protein concentrations. We also demonstrate that these proteins can be released through a subsequent thiol-ene reaction of a small molecule thiol. Importantly, this process of protein tethering followed by release is repeatable through multiple interations demonstrating the regenerative nature of the allyl sulfide handle. Since this process is photomediated, the reaction can be performed with three-dimensional spatial fidelity enabling the assembly of complex protein patterns within hydrogels. Finally, we demonstrate this methodology can be performed with whole signaling proteins in the presence of cells. Briefly, transforming growth factor-β (TGF-β) was tethered to hydrogels in the presence of a TGF-β reporter cell line. We observed that TGF-β can be patterned and released from the hydrogels <i>in situ</i> and the cells respond to these changes in real time.

Development of a reversible and repeatable thiol-ene reaction for controlled presentation of signaling proteins in hydrogels

2019 Fall National Meeting

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