Stabilization of Fibronectin by Random Copolymer Brushes Inhibits Macrophage Activation.

We show that protein unfolding on biomaterials may be dramatically reduced via tuning the chemical heterogeneity of the protein-material interface. Specifically, using dynamic single-molecule methods, we confirmed that the transient structure and dynamics of fibronectin (FN) may be mediated through varying the composition of random copolymer brushes. The brushes, which themselves represent an intriguing biomaterial, were composed of oligoethylene glycol and sulfobetaine methacrylate and presumably stabilized FN through partitioning and/or segregation of the copolymers. We further showed that, by controlling the transient structure and dynamics of FN, the secretion of TNF-α and IL-6 by RAW 264.7 was markedly diminished.

Grafting Density Impacts Local Nanoscale Hydrophobicity in Poly(ethylene glycol) Brushes.

Accumulated single-molecule observations of a fluorescent solvatochromic probe molecule were found to provide detailed local information about nanoscale hydrophobicity in polymer brushes. Using this approach, we showed that local hydrophobicity in poly(ethylene glycol) (PEG) brushes was spatially heterogeneous and increased with the surface grafting density of the polymer chains. These findings may provide an explanation for prior observations of the denaturation of surface-adsorbed proteins on PEG brushes with high grafting densities, which is believed to influence protein-mediated cell-surface interactions. Moreover, by employing the broad range of existing environmentally sensitive fluorophores, this approach may potentially be used to characterize nanoscale changes in a variety of physicochemical properties within polymeric materials.

Connecting Protein Conformation and Dynamics with Ligand-Receptor Binding Using Three-Color Förster Resonance Energy Transfer Tracking.

Specific binding between biomolecules, i.e., molecular recognition, controls virtually all biological processes including the interactions between cells and biointerfaces, both natural and synthetic. Such binding often relies on the conformation of biomacromolecules, which can be highly heterogeneous and sensitive to environmental perturbations, and therefore difficult to characterize and control. An approach is demonstrated here that directly connects the binding kinetics and stability of the protein receptor integrin αvß3 to the conformation of the ligand fibronectin (FN), which are believed to control cellular mechanosensing. Specifically, we investigated the influence of surface-adsorbed FN structure and dynamics on αvß3 binding using high-throughput single-molecule three-color Förster resonance energy transfer (FRET) tracking methods. By controlling FN structure and dynamics through tuning surface chemistry, we found that as the conformational and translational dynamics of FN increased, the rate of binding, particularly to folded FN, and stability of the bound FN-αvß3 complex decreased significantly. These findings highlight the importance of the conformational plasticity and accessibility of the arginine-glycine-aspartic acid (RGD) binding site in FN, which, in turn, mediates cell signaling in physiological and synthetic environments.

Dense Poly(ethylene glycol) Brushes Reduce Adsorption and Stabilize the Unfolded Conformation of Fibronectin.

Polymer brushes, in which polymers are end-tethered densely to a grafting surface, are commonly proposed for use as stealth coatings for various biomaterials. However, although their use has received considerable attention, a mechanistic understanding of the impact of brush properties on protein adsorption and unfolding remains elusive. We investigated the effect of the grafting density of poly(ethylene glycol) (PEG) brushes on the interactions of the brush with fibronectin (FN) using high-throughput single-molecule tracking methods, which directly measure protein adsorption and unfolding within the brush. We observed that, as grafting density increased, the rate of FN adsorption decreased; however, surface-adsorbed FN unfolded more readily, and unfolded molecules were retained on the surface for longer residence times relative to those of folded molecules. These results, which are critical for the rational design of PEG brushes, suggest that there is a critical balance between protein adsorption and conformation that underlies the utility of such brushes in physiological environments.