Construction Of Novel Ferritin-based Nanodrug And Preliminary Study On Cancer Treatment

Objective: Ferroptosis and autophagy are two forms of regulatory cell death that play important roles in cancer therapy.However,little is known about the combination effects of ferroptosis and autophagy in the treatment of cancer.In this study,we constructed a novel exogenous ferritin nanodrug,NFER,loaded with hydrophobic drugs Erastin and Rapamycin.NFER could induce cancer cell death through the dual pathways of autophagy and ferroptosis,which may provide a new strategy for cancer treatment.Methods: NFER nanodrug was prepared by phacoemulsification technique.The drug size,zeta potential,and in vitro stability of the drug in phosphate buffered saline(PBS)and water were detected by dynamic light scattering instrument.The content of iron in NFER is detected by ICP-MS.The loading efficiency and encapsulation efficiency of nanodrug were detected by high performance liquid chromatography(HPLC).BCA protein detection kit was used for detecting ferritin content in nanodrug.Rat adrenal gland pheochromocytoma poorly differentiated PC12 cells,mouse breast cancer 4T1 cells,mouse fibroblast L929 cells were cultured for cell uptake assay,cytotoxicity test,and the reversal effect of related inhibitory drugs.The degree of cell membrane lipid oxidation was observed by light microscopy,while the degree of intracellular membrane lipid oxidation and lipid peroxide was quantified by flow cytometry.The expression of glutathione peroxidase-4(GPX4)protein,autophagy-associated protein 7(Atg-7),trace related protein light chain(LC3),and ferritin heavy chain(FTH1)were detected by Western blot.The hemolysis test was used for detecting the biocompatibility of the nanodrug in the blood and provides a basis for subsequent in vivo experiments.A nude mouse model was constructed,intratumoral nanodrug uptake stay experiment and in vivo tumor regrowth inhibition experiment were performed.Results: NFER nanodrug was successfully prepared by phacoemulsification technique using polyethylene glycol 300(PEG 300)as a lyoprotectant.The average size of the nanodrug was 78.8 nm,and the zeta potential was-25.9 ± 3.3 mV,and the stability in vitro was good.The iron content was 2.47 mg/kg.With the increasing concentration of Erastin and Rapamycin,the nanodrug encapsulation efficiency and loading efficiency can increase.The highest drug loading efficiency of Erastin and Rapamycin were 0.35% and 5.6%,respectively,and the highest drug encapsulation rates were 35.75% and 78.78%,respectively,the content of ferritin was 7 mg/mL Release studies showed that the amount of Erastin and Rapamycin reached equilibrium after 24 h and 36 h,respectively,with a maximum of more than 30%.In vitro cell experiments demonstrated the cell uptake of NFER was as high as 53 times that of the control group.The combination of ferritin,Erastin and Rapamycin had higher cytotoxicity than each single drug,and NFER nanodrug can cause 20% of the effect of cell killing at lower concentrations.When an autophagy inhibitor or a ferrotopsis inhibitor was added to the cells cultured with NFER,the cell death rates were remarkably lowered.Which means the cells simultaneously experienced autophagy and ferrotopsis induced by the nanodrug.Treatment of cells with NFER resulted in the increase of LC3-II relative to LC3-I,as well as accumulation of Atg-7,which means that the cells experienced significant autophagic death.At the same time,the increase of FTH1 indicated that NFER induced autophagy,which in turn leads to the degradation of ferritin.In addition,significant lipid peroxidation and accumulation of reactive oxygen species(ROS)existed in cells and in the cell membrane,as well as inactivation of GPX4 prior to cell death,meaning that ferrotopsis was also evident in cell death.The results of in vivo tests illustrated that NFER can stay in the tumor for a long time and has a significant inhibitory effect on tumors regrowth.Conclusions: The nanodrug NFER has been successfully prepared,which has excellent properties.Cell and tumor death induced by NFER was a dual strategy of autophagy/ferroptosis,which provided a new method for cancer therapy.