Recent in vivo studies have established ultrasmall (< 3 nm) gold nanoparticles coated with glutathione (AuGSH) as a promising platform for applications in nanomedicine. However, systematic in vitro investigations to gain a more fundamental understanding of the particles’ biointeractions are still lacking. Herein we examined the behavior of ultrasmall AuGSH in vitro, focusing on their ability to resist aggregation and adsorption from serum proteins. Despite having net negative charge, AuGSH particles were colloidally stable in biological media and able to resist binding from serum proteins, in agreement with the favorable bioresponses reported for AuGSH in vivo. However, our results revealed disparate behaviors depending on nanoparticle size: particles between 2 and 3 nm in core diameter were found to readily aggregate in biological media, whereas those strictly under 2 nm were exceptionally stable. Molecular dynamics simulations provided microscopic insight into interparticle interactions leading to aggregation and their sensitivity to the solution composition and particle size. These results have important implications, in that seemingly small variations in size can impact the biointeractions of ultrasmall AuGSH, and potentially of other ultrasmall nanoparticles as well.

Ultrasmall gold nanoparticles coated with a zwitterionic derivative of glutathione are shown to be stable against aggregation and nonspecific binding from serum proteins.

Ultrasmall gold nanoparticles (AuNPs) are an emerging class of nanomaterials exhibiting distinctive physicochemical, molecular, and in vivo properties. Recently, we showed that ultrasmall AuNPs encompassing a zwitterionic glutathione monoethyl ester surface coating (AuGSH zwt ) were highly resistant to aggregation and serum protein interactions. Herein, we performed a new set of biointeraction studies to gain a more fundamental understanding into the behavior of both pristine and peptide-functionalized AuGSH zwt in complex media. Using the model Strep-tag peptide (WSHPQFEK) as an integrated functional group, we established that AuGSH zwt could be conjugated with increasing numbers of Strep-tags by simple ligand exchange, which provides a generic approach for AuGSH zwt functionalization. It was found that the strep-tagged AuGSH zwt particles were highly resistant to nonspecific protein interactions and retained their targeting capability in biological fluid, displaying efficient binding to Streptactin receptors in nearly undiluted serum. However, AuGSH zwt functionalized with multiple Strep-tags displayed somewhat lower resistance to protein interactions and lower levels of binding to Streptactin than monofunctionalized AuGSH zwt under given conditions. These results underscore the need for optimizing ligand density onto the surface of ultrasmall AuNPs for improved performance. Collectively, our findings support ultrasmall AuGSH zwt as an attractive platform for engineering functional, protein-mimetic nanostructures capable of specific protein recognition within the complex biological milieu.

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