Connecting Protein Structure and Dynamics on Biomaterials with the Foreign Body Response

The harsh environment of the foreign body response (FBR) has the potential to negatively impact the implantations of biomaterials in the body. The FBR is initiated by inflammatory cells that recognize the material as foreign through surface-adsorbed proteins. When proteins interact with surfaces, they can unfold and expose epitopes that may be recognized by immune cells and trigger a series of reactions. Importantly, the presentation of unfolded proteins is directly influenced by the highly dynamic and heterogeneous behavior of proteins in near-surface environments, as well as by the physicochemical features of the underlying surface. Such behavior is the result of transient unfolding and refolding, rapid exchange of folded and unfolded protein molecules between the surface and the bulk solution, intermittent interfacial diffusion, and protein-protein associations. While these interfacial processes are likely involved in the FBR, both their characterization and respective roles in the FBR have been ignored due to the lack of experimental techniques to directly observe individual molecular processes. The work presented here aims to address this lack of fundamental understanding by applying novel single-molecule (SM) methods, which are uniquely sensitive to interfacial dynamics as well as protein and surface heterogeneities, to investigate the mechanisms that lead to the FBR. Specifically, we focused on tuning surface functionalization to reveal the connection between material properties, protein adsorption and stabilization, and ultimately cell response. Total internal reflection fluorescence microscopy (TIRFM) was combined with Förster resonance energy transfer (FRET) to independently dissect individual molecular processes, such as adsorption, desorption, diffusion, folding, unfolding, and binding. The studies were performed using recombinant fibronectin (FN) as a model protein, which was site-specifically labeled to undergo FRET. First, the effect of poly(ethylene glycol) (PEG) grafting density on protein adsorption and stabilization was studied. Furthermore, mapping accumulated probe trajectories (MAPT) with an environmentally sensitive molecule was used as a tool to identify local changes in brush hydrophobicity. Secondly, in order to understand the connection between surface properties, FN conformation (ligand), and integrins (cell receptors), a three-color FRET method was developed to track both protein conformation and ligand-receptor binding as a function of surface chemistry. Finally, the extent to which the addition of a zwitterionic polymer (poly(sulfobetaine)) to PEG can improve the stability of FN was explored. Altogether, the results obtained from these studies will shed light on the rational design of materials to mediate cell signaling in physiological and synthetic environments