Nanoparticles have been extensively used as therapeutic agents, but nanoparticles are rapidly coated by proteins in biological fluids, forming a protein corona. This corona will affect the fate of the nanoparticle in vivo. Developing predictive models of bio-nano interactions is, therefore, of high interest. Here we present an in-situ study to understand the mechanism of corona formation on gold nanoparticles (AuNPs) in serum by tracking the behavior of bovine serum albumin (BSA) in complex protein mixtures. The binding of BSA on AuNPs is confirmed by ¹H NMR, UV-vis, and dynamic light scattering (DLS). Serum albumin was isotopically tagged at Lys residues using ¹³C formaldehyde as a methylating agent. A ¹H-¹³C HSQC NMR spectrum reveals that the adsorption of BSA increases linearly as we increase the AuNP concentration. This led us to investigate the binding capacity of BSA on AuNPs. The effects of other serum proteins such as Transferrin (Tr), Fibronectin (Fin), and Immunoglobulin G (IgG) were determined on the binding of BSA on AuNPs using NMR spectroscopy. The data reveals that Tr and Fin can compete with BSA to bind to AuNPs, whereas IgG does not displace BSA. DLS analysis on these samples suggests that IgG forms aggregates of AuNPs and BSA can be displaced by Tr and Fin. Similar results were found when different combinations of these proteins were mixed. In the presence of serum, it was determined that the binding of BSA decreases as the concentration of serum is increased. A mixture of BSA, Tr, Fin, and IgG provides similar displacement behavior as complete serum. These results indicate that serum proteins at high concentrations out-compete a tagged protein of interest in complex mixtures. Circular dichroism revealed that protein mixtures could alter the protein secondary structure in unpredictable ways in the presence of AuNPs. This work demonstrates that each serum component uniquely contributes to the corona. Moreover, it suggests that a simple mixture of four protein components may be sufficient to reproduce many aspects of adsorption in complete serum.

Tanveer Shaikh

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