Time Evolution of Ultrasmall Gold Nanoparticle–Protein Interactions

Abstract

To date, much effort has been devoted toward the study of protein corona formation onto large gold nanoparticles (GNPs). However, the protein corona concept breaks down for GNPs in the ultrasmall size regime (<3 nm), and, as a result, our understanding of ultrasmall GNP (usGNP)–protein interactions remains incomplete. Herein, we used anionic usGNPs and six different proteins as model systems to systematically investigate usGNP–protein interactions, with particular focus on the time evolution and long-term behavior of complex formation. The different proteins comprised chymotrypsin (Cht), trypsin (Try), thrombin (Thr), serum albumin (HSA), cytochrome c (Cyt c), and factor XII (FXII). We used a range of biochemical and biophysical methods to estimate binding affinities, determine the effects of usGNPs on protein structure and function, assess the reversibility of any protein structural and functional changes, and evaluate usGNP–protein complex stability. Among the main findings, we observed that prolonged (24 h)but not short-term (10 min)interactions between proteins and usGNPs permanently altered protein function, including enzyme activities (Try, Thr, and FXIIa), peroxidase-like activity (Cyt c), and ligand-binding properties (HSA). Remarkably, this occurred without any large-scale loss of the native global conformation, implying time-dependent effects of usGNPs on local protein conformation or dynamics. We also found that both short-(10 min) and long-term (24 h) interactions between proteins and usGNPs yielded short-lived complexes, i.e., there was no time-dependent “hardening” of the interactions at the binding interface as usually seen with large GNPs. The present study increases our fundamental understanding of nano-bio interactions in the ultrasmall size regime, which may assist the safe and effective translation of usGNPs into the clinic.

Stealth and Biocompatible Gold Nanoparticles through Surface Coating with a Zwitterionic Derivative of Glutathione

“…Blood compatibility is a critical requirement for NPs that are administered intravenously. − Importantly, we note that the absence of a hard protein corona on the NP surface does not in itself ensure blood compatibility. This is because transient NP interactions with proteins are capable of interfering with protein function and biochemical pathways, potentially triggering toxic responses. ,− ,− Moreover, NP interactions with the plasma membrane of blood cells can also perturb their normal functions. Therefore, a comprehensive evaluation of blood compatibility is necessary to assess potential risks associated with NP administration in vivo. ,− This assessment should include several complementary assays, including the hemolysis of red blood cells, platelet aggregation, leucocyte activation, and endothelial cell cytotoxicity.…”

“…This is because transient NP interactions with proteins are capable of interfering with protein function and biochemical pathways, potentially triggering toxic responses. 49 , 58 − 60 , 70 − 72 Moreover, NP interactions with the plasma membrane of blood cells can also perturb their normal functions. Therefore, a comprehensive evaluation of blood compatibility is necessary to assess potential risks associated with NP administration in vivo.…”

Protein structure–function continuum model: Emerging nexuses between specificity, evolution, and structure

“…Lately, there has been a vigorous renewal of interest in understanding protein interactions with surfaces of nanoparticles which has, again, importance in both in vivo and in vitro (Gupta & Roy, 2020; Mukherjee & Gupta, 2016a; Sousa et al, 2021). This also includes binding of IDRs to nanoparticles (Lima et al, 2023; Mahapatra et al, 2020; Munari et al, 2020). Viola et al reported that interaction of tau protein with ultrafine gold nanoparticles led to multimolecular assembly via hot spots of binding sites as in PPI interactions (Viola et al, 2022).…”

Kinetics and Timescales in Bio–Nano Interactions

“…Here, the analogy becomes even more pertinent due to the small size and high surface curvature of usNPs, which provide a limited binding interface for protein interactions. Hence, when proteins interact with usNPs, significant protein spreading and denaturation on the usNP surface are unlikely [99][100][101], and major lateral interactions between bound proteins are also improbable. In fact, usNPs with a diameter of 1.5 nm or less may be so small as to bind only to a single protein (Figure 6A) [95].…”