Investigation of protein corona formation on PEGylated gold nanoparticles via nano surface energy transfer

Protein corona, a protein-rich coating on nanoparticles, plays a critical role in controlling the interactions and behaviours of nanoparticles in biological systems. Despite significant progress, the mechanisms and effects of protein corona formation on PEGylated nanoparticles, crucial for the design of potential nanoscale therapeutics, are still not fully understood. The current methods for investigating protein coronas are mainly based on analysing the bound proteins using methods such as gel electrophoresis, mass spectroscopy and proteomics, but these methods do not provide direct affinity information. Herein, by harnessing the universal, strong fluorescence quenching properties of gold nanoparticles (GNPs), we have introduced a new GNP based nano surface energy transfer (NSET)-quenching method for studying protein corona formation on PEGylated GNPs. We quantified their apparent binding affinities with two model proteins, bovine serum albumin (BSA) and lysozyme (Lys), present in high and low abundance in blood plasma, respectively. We have revealed that, (1) GNP PEGylation cannot completely prevent the adsorption of BSA and Lys; (2) the apparent binding affinity of the proteins is enhanced with increasing GNP scaffold size; (3) the different terminal functional groups of PEG ligands on GNPs have no significant impact on their binding to BSA and Lys; (4) the high-abundant BSA binds more strongly than low abundance Lys toward PEGylated GNPs, indicating that BSA may form the main component of the protein corona formed on GNPs. Our results suggest that NSET quenching provides an effective, comparative, and assay-defined readout to study protein corona in dye-labelled protein and gold nanoparticle systems.