The Development of an AF4 Characterization Pipeline for Polymeric Nanoparicle-Protein Interactions

Abstract

The centrifugation-wash method is currently the standard technique used for the recovery of nanoparticle-protein complexes from protein-containing serum. However, this technique is disruptive, and leads to changes in nanoparticle parameters and the surface-bound protein composition. In this study, we explore the use of field flow fractionation (FFF) for the development of a robust pipeline for the characterization of nanoparticle parameters following protein corona formation under physiologically-relevant conditions. Model surface-modified polystyrene latex nanoparticles were treated with protein-containing medium mimicking cell-culture conditions under physiologically-relevant conditions. Following incubation, nanoparticle-protein samples were then fractionated using the FFF system and characterized using in-line detectors. Conventional nanoparticle-protein studies typically use the highly invasive centrifugation-wash method which leads to changes in measured particle parameters. Our studies show that AF4 allows for high resolution and characterization of in-situ nanoparticle-protein complexes. This work will allow for the development of a more harmonized approach for characterizing nanoparticle-protein interactions under biologically-relevant conditions.