Photoreversible patterning of hydrogel biomaterials with site-specifically-modified proteins

Abstract

Event Abstract Back to Event Photoreversible patterning of hydrogel biomaterials with site-specifically-modified proteins Jared A. Shadish1, Gabrielle M. Benuska2 and Cole Deforest1, 2, 3, 4* 1 University of Washington, Chemical Engineering, United States 2 University of Washington, Bioengineering, United States 3 University of Washington, Institute for Stem Cell and Regenerative Medicine, United States 4 University of Washington, Molecular Engineering & Sciences Institute, United States Introduction: As pericellular reconfigurations govern many important biological processes, in vitro culture platforms that recapitulate such dynamic phenomena would be invaluable for fundamental studies in stem cell biology, as well as in the eventual engineering of functional human tissue[1]. Preliminary efforts have exploited photochemical reactions to tether peptides spatially within hydrogels[2],[3]. While such approaches have proven successful in directing 3D cell physiology, the realized biological control has been confined to relatively simple cellular functions (e.g., adhesion, proliferation). To govern more complex decisions of fate, a system that enables dynamic presentation of full-length proteins would be of great interest[4]. However, proteins are commonly recognized to be as delicate as they are powerful; careful consideration must be given to the immobilization chemistry and precise site of protein tethering to ensure sustained stability and activity. Here we present a robust synthetic strategy enabling the user-defined immobilization and subsequent release of proteins in a site-specific manner within a 3D cell culture platform, thereby preserving protein function to modulate intricate cellular behavior including stem cell differentiation, protein secretion, and cell-cell interactions. Materials: Small molecule precursors were synthesized, purified by HPLC, and characterized by 1H and 13C NMR. Recombinant proteins were expressed in E. coli and isolated by 6xHis purification prior to LC/MS. Methods: Chemoenzymatic strategies were used to introduce photoreleasable aldehydes site-specifically onto recombinant proteins. Cell-laden hydrogels were formed upon reaction of a PEG tetra(cyclooctyne), a di-azide-containing MMP-degradable polypeptide, as well as a photocaged alkoxyamine. A photomediated oxime ligation was exploited to photolithographically pattern aldehyde-containing proteins into gels (λ = 365 nm, Fig. 1). Tethered proteins could be removed upon a second masked UV light exposure. Fig. 1. Hydrogel biomaterials are reversibly photofunctionalized with site-specifically-modified proteins. Results and Discussion: Proteins site-specifically modified to contain a single photoreleasable aldehyde were expressed recombinantly and purified with good yield (~20 mg/L). High cell viability was observed for gel formation, protein photoimmobilization, and protein photorelease. Micron-scale patterning of full-length proteins was obtained (Fig. 2). Patterning could be repeated to introduce/remove many proteins within the same system. By selectively photocoupling vitronectin (65 kDa) into a cell-laden hydrogel network, user-directed morphological, migratory, and differentiation changes were induced within, and confined to, the patterned regions for hMSCs. Fig. 2. Site-specifically modified fluorescent proteins are reversibly patterned in/out of the gel to ultimately regulate changes in 3D cell function. Conclusion: We have developed a synthetic approach enabling reversible hydrogel biofunctionalization using site-specifically modified proteins in the presence of cells. A material that affords this level of spatial and biomolecular control should provide useful in assaying stem cell decisions of fate.