Dense Poly(ethylene glycol) Brushes Reduce Adsorption and Stabilize the Unfolded Conformation of Fibronectin David Fauló n Marruecos, Mark Kastantin, † Daniel K. Schwartz,* and Joel L. Kaar* Department of Chemical and Biological Engineering, University of Colorado, Boulder, Colorado 80309, United States * S Supporting Information ABSTRACT: 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. ■ INTRODUCTION The use of polymer brushes as coatings to improve the biocompatibility of materials in physiological environments or in contact with proteins (e.g., biomaterials for tissue engineer- ing and drug delivery, medical devices, biosensors, contact lenses, food packaging materials) has received considerable attention. 1-7 Such coatings consist of a densely packed layer of polymer chains that are terminally anchored to a surface and may be prepared by a “grafting to” or “grafting from” approach. 8-11 The grafted polymer layer provides a steric barrier, shielding the surface from protein adsorption while simultaneously minimizing protein denaturation at the solution-solid interface. Due to its apparent protein resistance, poly(ethylene glycol) (PEG) is the most extensively studied material for the development of polymer brush coatings. 6 Despite a widespread interest in applications of PEG brushes, a mechanistic understanding of the connection between brush properties (e.g., grafting density and brush thickness) and nonspecific protein adsorption and denaturation remains elusive. Previous studies using macroscopic ensemble-averaging methods have shown a nonmonotonic trend of protein accumulation with grafting density, where accumulation decreases at low grafting densities (with increasing grafting density) and then increases at very high grafting densities (with increasing grafting density). This behavior is hypothesized to be due to an increase in PEG hydration with grafting density up to an optimal point, after which hydration of the brush decreases. 12-17 Although this hypothesis is plausible, it neglects alternative hypotheses, including a potential indirect effect where protein accumulation is influenced by the impact of grafting density on protein conformation. For example, it is plausible that, at high grafting density, the rate of protein adsorption is actually decreased, but that an increase in unfolding of adsorbed proteins leads to increased surface residence time. Accordingly, increased protein accumulation may result from differences in the surface residence time of folded and unfolded protein molecules rather than an increase in the rate of protein adsorption. Moreover, increased unfolding at high PEG grafting density may also lead to surface aggregation, further increasing the surface residence time of molecules on the brush. Ultimately, this gap in understanding prohibits the rational design of PEG brushes for improved biocompatibility and thus limits the utility of PEG brushes as protein-resistant coatings. To fully elucidate the link between protein accumulation and grafting density for PEG brushes, new methods to directly correlate protein adsorption and conformation on brush- containing surfaces are required. 18 Typical ensemble-averaging methods relate protein adsorption and conformation indirectly, by correlating changes in average protein conformation with the total amount of adsorbed protein. 19,20 Such indirect measurements of protein conformation are necessary due to challenges in measuring protein structure at solid-liquid interfaces. Because changes in protein conformation are Received: December 8, 2015 Revised: February 10, 2016 Article pubs.acs.org/Biomac © XXXX American Chemical Society A DOI: 10.1021/acs.biomac.5b01657 Biomacromolecules XXXX, XXX, XXX-XXX