Preparation Of N-acetyl-L-cysteine-Protected Gold Nanoclusters And Application In Cell Imaging

Intracellular redox balance is a key factor to maintain cellular physiological stability.Glutathione（GSH）is an important component in maintaining this balance.Therefore,the determination of intracellular GSH levels is essential for assessing different cellular states,especially immune response,senescence,and tumor metastasis.At present,the probes for intracellular GSH are mainly small organic molecules,with potential issues including cytotoxicity and photobleaching.Aimed at this problem,this study intends to develop novel imaging probes on basis of thiolates protected metal nanoclusters（NCs）to explore the feasibility for intracellular GSH imaging.We have synthesized two kinds of fluorescent gold NCs,namely,AuNAC NCs and AuNAC（1:3）NCs,using N-acetyl-L-cysteine（NAC）as the protective agent.Silver ions may bind to AuNAC NCs to form AuNAC@Ag NCs,which shows an excellent fluorescent response to GSH,with their fluorescence peak intensity at 570nm increasing by about 20 times.Similar phenomenon is not present in the control group comprised of a mixture of AuNAC NCs with GSH,indicating that silver ions and GSH have a synergistic effect on fluorescence enhancement of AuNAC NCs.According to mass spectrometry analysis,we infer that silver ions can assist the ligand exchange between ligands and GSH on the surface of AuNAC NCs,resulting in the generation of new type of highly fluorescent NCs,i.e.,AuNAC@Ag GSH.Other common thiols,such as cysteine（Cys）,cysteamine（CyA）and NAC,cannot produce similar fluorescence enhancement,which shows good fluorescence enhancement selectivity by AuNAC@AgNCs.The cell viability assays show that the AuNAC@Ag NCs had almost no cytotoxicity.Compared with the AuNAC NCs control group and the GSH reductase inhibition group,the intracellular fluorescence of the AuNAC@Ag NCs can be enhanced significantly and thus the proposed NCs can be used for selective imaging of intracellular GSH.Unlike AuNAC NCs,AuNAC（1:3）NCs are gel-like,which spontaneously form linear cross-linked structures accompanied by aggregation-induced emission at 610nm.The gel structures are reversibly decomposed under neutral pH conditions and the fluorescence disappears.Gd ions can induce the formation of the cross-linking structure by NCs at neutral p H.Its fluorescence peak wavelength is blue-shifted to～580nm.Considering the CT features of gold in the complex gel and the T₁MRI enhancement by Gd ions,it is promising to prepare a novel fluorescent-CT-magnetic resonance multimodal imaging contrast agent on basis of the Gd ions doped AuNAC（1:3）NCs.Overall,using NAC as the protection molecules we can synthesize and assemble diverse NCs structures with unique luminescence properties and low cytotoxicity,allowing the accurate imaging of important intracellular biomolecules such as GSH.This study lays the foundation for the development of high-performance biomedical imaging preparations.