Nanoparticle technology is increasingly being applied to solve problems in the materials and biomedical engineering fields. In these applications, engineered nanoparticles frequently encounter complex mixtures of proteins in a biological fluid, and these proteins will spontaneously adsorb onto the nanoparticle, potentially abolishing its intended function. This “corona” of adsorbed proteins is typically classified into hard and soft coronae. The hard corona is closest to the nanoparticle surface and contains tightly-bound proteins, whereas in the soft corona, proteins are weakly bound and exchangeable. Understanding the behavior of proteins in the hard and soft coronae could lead to engineered nanoparticles that are more resistant to fouling. However, this remains a significant challenge, as conventional methods for characterizing protein structure cannot be applied to nanoparticle-protein systems. Here, we use a combination of NMR-based approaches, including hydrogen deuterium exchange, reductive lysine methylation, relaxation, mutagenesis, and selective isotopic labeling, to identify the physical properties of the GB3 protein binding to gold nanoparticles. We observe that this protein has a preferred orientation in the hard corona, and that it retains a native-like structure. Binding kinetics are very fast, initially occurring in less than 60 s, but a second, slow transition is observed after the initial corona formation. This slower transition occurs over the span of several hours. GB3 binding can also be tracked in complex media, such as dilute fetal bovine serum, and in serum, the slower adsorption phase is abolished entirely. GB3 does not appear to participate in the nanoparticle/serum soft corona, but relaxation analysis identifies other proteins that do bind weakly. Protein-nanoparticle interactions are enormously complicated, especially in complex media, but this work establishes the utility of NMR for characterizing the physical properties of adsorption.

NMR-based approaches for monitoring protein binding and structure on nanoparticle surfaces

ACS Fall 2021

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.