Ultrasmall Gold Nanoparticles Coated with Zwitterionic Glutathione Monoethyl Ester: A Model Platform for the Incorporation of Functional Peptides

Abstract

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.

Biomolecular interactions of ultrasmall metallic nanoparticles and nanoclusters

“…1b). 82,83 Class 3 comprises NPs that incorporate functional ligands (e.g., peptides) for the specic recognition of proteins (Fig. 2b, panel iii), such as cell surface receptors in cancer cells.…”

“…reversing the sign of the binding signal Ds/Dn P . 83 At the inection pointin the absence of binding signalit is clear that tting different binding models will lead to poorly dened binding constants and stoichiometries. Whether or not such a "blind spot" is experimentally observed will depend on the mass and density of the usGNP and the mass and shape of the bound protein.…”

“…Native gel electrophoresis is one technique that is commonly employed to assess the behavior of usNPs in protein-rich biological media, such as human plasma (HP) or fetal bovine serum (FBS). 46,83,228,229 A typical experiment consists in running usGNPs diluted in buffer vs. HP or FBS side-by-side in a gel, then observing any differences in the electrophoretic mobility of the particles as a result of nonspecic protein binding (Fig. 4g and h).…”

“…4h). 83 However, there are important limitations to take into consideration in these types of measurements, such as that samples are not strictly maintained in situ due to the separation of bands, 'cage effects' of polymers on the NP interaction, 230 and the qualitative nature of the results interpretation.…”

“…If as little as 10% protein occupancy of usGNPs is detected, this will allow the detection and quantitation of transient interactions with K D in the low mM range. 83 One difficulty for the precise analysis is posed by long-range hydrodynamic interactions altering the sedimentation process of concentrated solutions. Recently, mean-eld corrections have been introduced that bring this solution regime in focus for quantitative analysis of weak interactions, 231 which we envision will be applicable to usGNP studies.…”