Effect And Mechanism Of Carbon Dots Nanoparticles On Radiotherapy Sensitization And Iron Ion Detection In Lung Cancer Cells

Objective:Ferroptosis is a new type way of cell death in dependence of iron,which plays an important role in cancer therapy including radiation therapy.However,radiotherapy resistance is present with a single dose of radiation therapy.Nano combination therapy gradually becomes a new way to improve the shortcoming of cancer diagnosis and treatment.The purpose of this study is to construct an anti-tumor nano-carrier with fenton-reaction ability and an efficient iron ion sensor with carbon dots（CDs）as the core,which can be used to promote the anti-tumor effect of radiotherapy in tumor cells.Methods:Fe-doped carbon dots（Fe-CDs）and high-fluorescence carbon dots（N-CDs）were prepared by one-step hydrothermal method.Fe-CDs was modified by self-assembly technology to obtain Fe-doped carbon dots lipidosome（Fe-CDs-PEG）.The morphology and structure of CDs were characterized by electron microscopy,Zeta potential was used to detect potential changes,fluorescence spectrum was used to detect fluorescence properties of CDs,and dispersion in different solvents was used to detect stability.3,3′,5,5′-tetramethylbenzidine（TMB）colorimetry was used to detect the catalytic production of reactive oxygen species（ROS）by Fe-CDs-PEG.CCK-8 assay was used to detect the cytotoxicity of Fe-CDs-PEG and N-CDs in lung cancer cells.Fluorescence microscopy was used to detect the effect of cell uptake,Calcein-AM/PI staining was used to detect the level of live/dead cells,DCFH-DA staining was used to detect the level of intracellular ROS,and BODIPY C11 staining was used to detect the level of lipid peroxidation.The level of glutathione（GSH）in and out of cells after Fe-CDs-PEG or combined radiotherapy was measured using 5,5’-dithiobenzene（2-nitrobenzoic acid）（DTNB）.The expression of glutathione peroxidase-4（GPX4）,a key ferroptosis protein,was detected by western blot.The nude mice subcutaneous implantation model was constructed combining with intratoral administration and radiotherapy.Hematoxylin-eosin staining（H&E）was used to detect the biological toxicity of Fe-CDs-PEG in mice tissues.Results:Characterization experiments showed that Fe-CDs-PEG was nano-sized with rich carboxyl and hydroxyl groups and evenly dispersed with stable physical and chemical properties.It has the excellent catalytic ability of Fenton reaction of H₂O₂ to produce ROS generation.When co-incubated with GSH,Fe-CDs-PEG could consume GSH and had a significant killing effect on lung cancer cells without toxicity to normal cells.Fluorescence microscopic imaging results showed that Fe-CDs-PEG could be effectively taken up by cells.combining with Fe-CDs-PEG,radiotherapy had enhanced cell killing effect.Intracellular ROS micrographs showed that Fe-CDs-PEG enhanced the radiotherapy effect to kill cancer cells through the ROS reaction.After treatment with Fe-CDs-PEG,the lipid peroxidation and the GSH level were decreased,and the expression of GPX4,a key ferroptosis protein,was down-regulated.These results suggest that ferroptosis was more obvious after combined radiotherapy and Fe-CDs-PEG can enhance the sensitivity of radiotherapy.The nude mice subcutaneous implantation model showed that radiotherapy had obvious tumor suppressive effect when combination with Fe-CDs-PEG with little biological toxicity.Characterization experiments showed that the high fluorescence carbon dots（N-CDs）were spherical with abundant carboxyl and hydroxyl groups on the surface and good water solubility.Fluorescence characterization showed excellent and stable fluorescence performance.Fe³⁺response detection results showed that N-CDs had high sensitivity and selectivity for Fe³⁺detection,and the detection limit was as low as 0.079μM.Cell imaging results showed that there was obvious fluorescence images and fluorescence quenching after adding Fe³⁺when N-CDs were co-incubated with cells for 6 h.Conclusion:In this study,Fe-CDs-PEG and high-fluorescence nitrogen-doped carbon dots were successfully prepared.Fe-CDs-PEG lead to the occurrence of excess iron,iron metabolism disorder,GSH degradation,ROS accumulation,which leads to the accumulation of lipid peroxides on the cell membrane and activate the ferroptosis pathway to achieve the sensitization effect of radiotherapy in lung cancer cells.Highly fluorescent N-CDs showed high sensitivity and selectivity to Fe³⁺and intracellular multicolor imaging.This study about the carbon dots-based research tool provides a new idea for the integration of tumor diagnosis and treatment.