Protein-capped Ultra-small Gold Nanoclusters For NIR-Ⅱ Imaging And Photodynamic Therapy

With controllable size,high specific surface area,tunable optical properties,excellent stability and good biocompatibility,gold nanoclusters（Au NCs）are widely applied for biomedical field,such as biolabeling,biosensing,biological imaging and disease treatment.It is reported that thiol compounds,peptides,polymers and proteins as ligands are required to stabilize Au NCs.Using thiol compounds as capping agents and sodium borohydride as reducing agent,the reduction of Au³⁺to Au⁺and Au to form stable Au_n（SR）_m is one of the most effective methods for the synthesis of Au NCs.Fine-tuning different ligands can achieve fluorescence emission of Au NCs at different spectral regions,but the quantum yields drop sharply as the wavelengths experience red-shift.Especially when the emission wavelengths shift in the near infrared range,the quantum yieds are relatively low（<0.1%）.Recently,biomolecules such as peptides and proteins have been used to induce the nucleation and growth of Au NCs.For example,bovine serum albumin（BSA）,as an abundant plasma protein with good stability,biological safety and low cost,has become a model protein for the preparation of BSA-Au NCs（BSA-protected Au NCs）.The cysteine and histidine residues in BSA molecules can coordinate with Au³⁺,while the tyrosine residues reduce Au³⁺to form BSA-stabilized Au NCs,which are more stable in quenching and photobleaching than Au NCs with small molecules as ligands,and have a640 nm red emission（QY≈6%）.However,there are no reports on synthesis and application of NIR-II BSA-Au NCs.Hence,this paper uses BSA as the template and combines different mild reducing agents to construct a new type of stable ultra-small BSA-Au NCs with red-shift of emission spectrum to the NIR-II window（1000 nm～1700nm）to enhance fluorescence penetration depth,can be used in NIR-II fluorescence imaging and photodynamic therapy（PDT）.This paper is mainly divided into the following three chapters:（1）Preparation and characterization of NIR-II BSA-Au NCs;（2）Intracellular photodynamic therapy of BSA-Au NCs;（3）In vivo imaging and photodynamic therapy of BSA-Au NCs.This research shows using BSA as the protective ligand and sodium hydroxide to adjust the p H value of solution system,simultaneously introducing an exogenous reducing agent（sodium borohydride）,ultra-small BSA-Au NCs（～2 nm,Au（I）≈43.27%）were prepapred by optimizing synthesis parameters based on single factor experiments and orthogonal design.BSA-Au NCs produced a maximum emission wavelength red-shifted from reported 640 nm to 915 nm and trailed to the NIR-II region with a shoulder peak at 1025nm.BSA-Au NCs with impressively high quantum yield of 1.1%in aqueous phase maintained high stability in different environments.Reactive oxygen species（ROS）were generated under the 808 nm laser,which caused the characteristic peak of DPBF to decrease significantly.At the cellular level,BSA-Au NCs had minimal toxic to 4T1,U87-MG and L02 cells,with cell survival rates higher than～80%,having good biocompatibility.Intracellular fluorescence imaging showed that a large number of BSA-Au NCs were endocytosed into tumor cells.Under laser irradiation,BSA-Au NCs produced high levels of ROS in the cells,resulting in a significant decrease of cell survival rate and achieving photodynamic therapy of tumor cells.At the animal level,BSA-Au NCs have high-resolution NIR-II subcutaneous or in situ 4T1 tumor imaging,vascular imaging,and lymphatic imaging.BSA-Au NCs show significant killing effect on tumor cell and inhibit the growth of tumor tissues in vivo treatment,and can be used for NIR-II fluorescence imaging-guided tumor treatment.In summary,we have realized a simple,repeatable,green synthesis method to prepare new ultra-small BSA-Au NCs,which have bright NIR-II fluorescence,photodynamic effects,good biocompatibility and stability.As a safe and stable protein-protected gold nanocluster theranostic platform,BSA-Au NCs have potential for cancer targeted imaging and imaging-guided photodynamics therapy in the NIR-II window and provide new opportunities for protein-protected clusters.