Probing the Mechanism of Antibody-Triggered Aggregation of Gold Nanoparticles

Abstract

The unique physicochemical properties of gold nanoparticles (AuNPs) provide many opportunities to develop novel biomedical technologies. The surface chemistry of AuNPs can be engineered to perform a variety of functions, including targeted binding, cellular uptake, or stealthlike properties through the immobilization of biomolecules, such as proteins. It is well established that proteins can spontaneously adsorb onto AuNPs, to form a stable and functional bioconjugate; however, the protein–AuNP interaction may result in the formation of less desirable protein–AuNP aggregates. Therefore, it is imperative to investigate the protein–AuNP interaction and elucidate the mechanism by which protein triggers AuNP aggregation. Herein, we systematically investigated the interaction of immunoglobulin G (IgG) antibody with citrate-capped AuNPs as a function of solution pH. We found that the addition of antibody triggers the aggregation of AuNPs for pH < 7.5, whereas a monolayer of antibody adsorbs onto the AuNP to form a stable bioconjugate when the antibody is added to AuNPs at pH ≥ 7.5. Our data identifies electrostatic bridging between the antibody and the negatively charged AuNPs as the mechanism by which aggregation occurs and rules out protein unfolding and surface charge depletion as potential causes. Furthermore, we found that the electrostatic bridging of AuNPs is reversible within the first few hours of interaction, but the protein–AuNP interactions strengthen over 24 h, after which the protein–AuNP aggregate is irreversibly formed. From this data, we developed a straightforward approach to acrylate the basic residues on the antibody to prevent protein-induced aggregation of AuNP over a wide pH range. The results of this study provide additional insight into antibody–nanoparticle interactions and provide a pathway to control the interaction with the potential to enhance the conjugate function.

High-Affinity Points of Interaction on Antibody Allow Synthesis of Stable and Highly Functional Antibody–Gold Nanoparticle Conjugates

“…In silico analysis confirmed that acrylated lysine residues substantially reduce the positive charge on a fully characterized IgG. 45 Antibody chemical modification was carried out experimentally using NSA and reduced DSP (referred to as DSP). We envisaged that acrolein and a thiol propionate group will be covalently bonded to primary amines of lysine residues upon reacting with NSA and reduced DSP, respectively (Scheme 1).…”

“…Thus, chemical modification of the surface accessible lysine residues, through an addition–elimination reaction, can mask the basic moiety and serve to modulate the protein charge. In silico analysis confirmed that acrylated lysine residues substantially reduce the positive charge on a fully characterized IgG …”

“…36 Our group has previously demonstrated that acrylating the amino group on lysine residues reduces the overall protein charge to improve conjugate stability at low pH by circumventing electrostatic bridging of the AuNPs. 44,45 Lastly, antibodies have been modified with Traut's reagent to introduce additional thiols and increase the affinity of the protein−AuNP interaction. 46,47 Thus, these limited reports support the exploration of biofunctionalization with chemically modified proteins as a means to enhance conjugate stability and function.…”

“…Capitalizing on these protein–AuNP interactions, Treuel et al chemically modified bovine serum albumin (BSA) to modulate protein charge, thereby altering BSA orientation on the AuNP surface and leading to differences in cellular uptake of the bioconjugate . Our group has previously demonstrated that acrylating the amino group on lysine residues reduces the overall protein charge to improve conjugate stability at low pH by circumventing electrostatic bridging of the AuNPs. , Lastly, antibodies have been modified with Traut’s reagent to introduce additional thiols and increase the affinity of the protein–AuNP interaction. , Thus, these limited reports support the exploration of biofunctionalization with chemically modified proteins as a means to enhance conjugate stability and function.…”

Smart Therapeutic Strategy of pH‐Responsive Gold Nanoparticles for Combating Multidrug Resistance

“…The possible explanation of this case is either owing to the NPs agglomeration in the salty environment or PBS coordinate with the active surface functional groups over time to achieve the nonspecific binding by which aggregation or increase in hydrodynamic diameter occurs. [ 37 ] On the contrary, in FBS a nonsignificant change in hydrodynamic of PEG‐Au‐DOX@BLM was observed as shown in Figure 5b. We thought that the presence of proteins in FBS solution probably could develop protein corona around the PEG‐Au‐DOX@BLM, and may decrease the surface free energy and hence preventing particle agglomeration.…”