The Inhibitory Effect Of FeSe₂ Nanoparticles On The Activity Of Bladder Cancer Cells

Objective:Bladder cancer is a tumor with high recurrence and metastasis.For advanced bladder cancer,the existing drug methods such as chemotherapy and immunotherapy have the problems of weak tumor-targeting effect and large toxic and side effects,which limit the therapeutic effect.Nanomaterials have become one of the new directions of tumor therapy due to their large specific surface area,low immunogenicity,long circulation time in blood,and strong ability to penetrate tumor tissue.In this study,based on the characteristics that the levels of reactive oxygen species（ROS）、glutathione（GSH）in tumor cells are significantly higher than those in normal cells,a ferrous selenide nanoparticles（FeSe₂ NPs）that selectively acts on tumor cells was designed and synthesized.And the inhibitory effect and possible mechanism of FeSe₂on bladder cancer activity were studied in vivo and in vitro,so as to provide a new therapeutic strategy for improving the clinical treatment of bladder cancer.Methods:FeSe₂ NPs were synthesized by one-step solvothermal method with ferrous chloride and selenium powder using ethanolamine as solvent.The cytotoxicity of FeSe₂NPs was detected by thiazolyl blue（MTT）method,live-dead cell staining kit and apoptosis detection kit.The peroxidase（POD）activity of FeSe₂ NPs was detected by3,3’,5,5’-tetramethylbenzidine（TMB）method and terephthalic acid（TA）method.The changes of GSH content after co-incubation of FeSe₂ NPs and GSH were detected by5,5’-dithiobis-（2-nitrobenzoic acid）（DTNB）method.The level of ROS in T24 cells was detected by ROS detection kit.The effect of the ferroptosis inhibitor Ferrostatin-1 on FeSe₂ NPs-induced T24 cytotoxicity was detected by MTT assay.The expression level of glutathione peroxidase 4（GPX4）was detected by Western blotting（WB）.Balb/c nude mice were subcutaneously implanted into tumors to establish a mouse bladder cancer model.FeSe₂ NPs was injected intratumorally to observe its effect on bladder and bladder cancer cells in vivo.Hematoxylin-eosin staining（H&E）was performed on vital organs（liver,heart,spleen,lung and kidney）and liver and kidney-related serum biochemical indicator was to detected to evaluate the biosafety of the material.Results:We successfully constructed FeSe₂ nanoparticles with GSH consumption,ROS generation ability and high biosafety.It was found that FeSe₂ NPs could significantly inhibit the proliferation of bladder cancer cells T24 cells by MTT method,and the inhibition effect was significantly greater than that of normal bladder epithelial SV-HUC-1 cells.Then,it was found that FeSe₂ NPs has POD activity which could generate highly toxic hydroxyl radicals by TMB method and TA method.Further use of DTNB method and ROS detection kit found that FeSe₂ NPs can deplete GSH and increase the level of intracellular ROS,thereby inhibiting the proliferation of T24 cells.Ferrostatin-1,an inhibitor of ferroptosis,was found to rescue the inhibitory effect of FeSe₂ NPs on T24 cell proliferation by MTT assay,suggesting that ferroptosis may also play an important role in FeSe₂ NPs-induced T24 cell death.We then constructed a subcutaneous tumor-bearing mouse model of bladder cancer,and further confirmed that FeSe₂ NPs have obvious therapeutic effects on bladder cancer.It can inhibit the growth of subcutaneous tumors in mice without causing pathological changes in the liver,heart,spleen,lung,kidney and serum biochemical indexes of mice.Conclusion and significance:We successfully constructed a new type of FeSe₂ nanoparticles,which can significantly inhibit the activity of bladder cancer T24 cells through in vitro and in vivo experiments.The mechanism may be closely related to the fact that FeSe₂ NPs can deplete GSH and increase the level of intracellular ROS,resulting in a serious imbalance of intracellular redox levels.In addition,we also found that the ferroptosis pathway also plays an important role in FeSe2 NPs inhibiting the activity of bladder cancer cells.This study found that FeSe2 NPs can selectively inhibit the activity of bladder cancer cells,providing a new strategy for the discovery of new bladder cancer therapeutics.