Affinity Constants of Bovine Serum Albumin for 5 nm Gold Nanoparticles (AuNPs) with ω-Functionalized Thiol Monolayers Determined by Fluorescence Spectroscopy

Abstract

A detailed understanding of the binding of serum proteins to small (d core <10 nm) nanoparticles (NPs) is essential for the mediation of protein corona formation in next generation nanotherapeutics. While a number of studies have investigated the details of protein adsorption on large functionalized NPs, small NPs (with a particle surface area comparable in size to the protein) have not received extensive study. This study determined the affinity constant (K a ) of BSA when binding to three different functionalized 5 nm gold nanoparticles (AuNPs). AuNPs were synthesized using three ω-functionalized thiols (mercaptoethoxy− ethoxy−ethanol (MEEE), mercaptohexanoic acid (MHA), and mercaptopentyltrimethylammonium chloride (MPTMA)), giving rise to particles with three different surface charges. The binding affinity of bovine serum albumin (BSA) to the different AuNP surfaces was investigated using UV−visible absorbance spectroscopy, dynamic light scattering (DLS), and fluorescence quenching titrations. Fluorescence titrations indicated that the affinity of BSA was actually highest for small AuNPs with a negative surface charge (MHA-AuNPs). Interestingly, the positively charged MPTMA-AuNPs showed the lowest K a for BSA, indicating that electrostatic interactions are likely not the primary driving force in binding of BSA to these small AuNPs. K a values at 25 °C for MHA, MEEE, and MPTMA-AuNPs were 5.2 ± 0.2 × 10 7 , 3.7 ± 0.2 × 10 7 , and 3.3 ± 0.16 × 10 7 M −1 in water, respectively. Fluorescence quenching titrations performed in 100 mM NaCl resulted in lower K a values for the charged AuNPs, while the K a value for the MEEE-AuNPs remained unchanged. Measurement of the hydrodynamic diameter (D h ) by dynamic light scattering (DLS) suggests that adsorption of 1−2 BSA molecules is sufficient to saturate the AuNP surface. DLS and negative-stain TEM images indicate that, despite the lower observed K a values, the binding of MPTMA-AuNPs to BSA likely induces significant protein misfolding and may lead to extensive BSA aggregation at specific BSA:AuNP molar ratios.