Triggerable ligand presentation using caged-RGD

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Lee, Ted
García, Andrés J.
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Cells rely on time-dependent binding and activation by the ECM to initiate downstream signal transduction. It is unknown whether adhesion to a ligand is required throughout various cell processes, or only during a specified time period ("temporal threshold”). Current approaches to ligand presentation often comprise of static, constant densities of ligands. In contrast, natural cell adhesive interactions with ECMs exhibit spatiotemporal patterns of binding and activation. Therefore, a key to future research in controlling cell-material interactions will be the development of materials that can respond to external stimuli. The objective of this project is to engineer biomaterials that present a UV-labile caged-Arginine-Glycine-Aspartic Acid (RGD) ligand and evaluate the effects on cell activities. RGD is the minimal adhesive sequence of fibronectin. By dynamically modulating adhesive ligand presentation, the effects of temporal control on cell processes can be elucidated. In this caged-peptide, a photo-labile group adjacent to the aspartic acid residue of RGD effectively “masks” a cyclo(RGDfk) peptide. Upon UV irradiation (360 nm), the caging group is released thereby restoring the adhesive activity of the peptide. By having unparalleled spatiotemporal control of RGD ligand presentation, we demonstrated two novel tools for discovery: 1) in vivo ligand presentation to probe downstream tissue behavior and cell infiltration to biomaterial implants, and 2) in vitro ligand presentation in situ using confocal-based live cell microscopy to investigate real-time vinculin recruitment and cell traction force generation. These studies represented the first demonstration of triggerable adhesive ligand presentation in vivo and demonstrated the utility of caged-compounds for probing specific receptor-ligand responses on highly defined PEG-based hydrogels. Triggerable in vitro ligand presentation, combined with traction force microscopy, demonstrated a new research tool for investigating focal adhesion formation and downstream force generation. Taken in whole, these results provide previously unknown insights into the importance of spatiotemporal control of adhesive ligands and created novel new research platforms for future discovery.
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