Study On Chelator-free Multinuclides Labeled Au NCs And Their Applications In Cerenkov Luminescence Induced Therapy

Cerenkov luminescence induced therapy（CLIT）can overcome the shortcoming that external light sources have low tissue penetration in conventional photodynamic therapy.But there remain two challenges,one is sequestration of nanocarriers by reticuloendothelial system in liver,spleen and bone marrow once delivered intravenously,which can cause severe damage to normal tissues and organs when radionuclides and photosensitizers are simultaneously loaded on the nanocarrier.The other is conventional radionuclides labeling methods using chelating agents may change the pharmacokinetics of nanocarrier,especially for ultra-small nanoparticles.Therefore,in this study,we will synthesize ultra-small Au nanoparticles with renal clearance and explore chelator-free mono-and dual-radionuclide labeling methods,based on which,we will develop CLIT system for photodynamic therapy of tumors.In this paper,gold nanoclusters with a size of about 2 nm were synthesized by reduction of HAu Cl₄ with glutathione.To prove the principle of chelator-free labeling of ⁶⁸Ga,¹⁷⁷Lu,⁶⁴Cu and ⁸⁹Sr,we first doped the“cold”Ga,Lu,Cu and Sr into the gold nanoclusters during the synthesis through co-reduction of the metal ions by glutathione.The successful synthesis of these nanoclusters was revealed by the characterization of their hydrodynamic diameter,UV-Vis absorption spectra and transmission electron microscope（TEM）.As-synthesized nanoclusters were stable for a long time in PBS solution and serum-containing DMEM and had a high metal-doping rate.Next,the radionuclides of ⁶⁸Ga,¹⁷⁷Lu and ⁸⁹Sr were labeled according to the“cold”method.The labeling efficiency was estimated by measuring the radioactivity of the labeled products and filtrate.The experimental results showed that the nuclides labeling method proposed in this paper could efficiently label ⁶⁸Ga,¹⁷⁷Lu and ⁸⁹Sr into the gold nanoclusters,and the labeling stability was high.Through the characterization of the labeled nanomaterials by hydrodynamic diameter,UV-Vis absorption spectra and TEM,it was proved that the radionuclides labeling did not affect the properties of the nanomaterials,and they could also maintain good stability in PBS solution and serum-containing DMEM.To prepare the CLIT system,a kind of photosensitizer-Ce6 molecules were efficiently coupled to the surface of nanomaterials by a simple method,and ⁶⁸Ga was labeled simutaneously to construct ultra-small gold nanomaterials that could be used for CLIT.The Cerenkov luminescence induced photodynamic property of the system was then investigated,which indicated that the system could produce singlet oxygen in water and ROS in the cells.The damage of ROS to the cell DNA double strands was also evaluated.The results showed that the system constructed in this paper had great potential for PDT and could be used for CLIT.The in vivo behavior of the system was next studied.The results showed that the system could be rapidly cleared by the kidney,had a short blood half-life（5.91 min）and had low accumulation in the liver and spleen,avoiding the toxicity of the nanomaterials to normal tissues and organs.At the same time,blood biochemistry and H&E staining analyses of main organs of mice revealed that the system didn’t have significant toxicity.This study demonstrated a universal,chelator-free labeling method for labeling ultra-small gold nanoparticles with ⁶⁸Ga,¹⁷⁷Lu and ⁸⁹Sr,and a facile way to prepare Cerenkov luminescence induced photodynamic therapy system.The in vitro studies revealed the potential application of the system in CLIT.Also,estimation of the in vivo behavior of the system indicated that it was efficiently renal clearable and accumulated less in the liver and spleen,which allowed it for in vivo applications.This study solved the problems currently facing Cerenkov luminescence induced therapy by proposing a chelator-free and multi-nuclides labeling method for gold nanoclusters.The CLIT system developed owned favorable in vivo behavior and was expected to solve the problem that traditional nanomaterials used for CLIT were toxic to normal tissues and organs.