| Ferroptosis is a newly discovered non-apoptotic form of cell death in recent years,which has received increasing attentions in cancer therapy owing to its unique advantages.However,ferroptosis is governed by the efficiency of reactive oxygen species(ROS)production and the tumor cell antioxidant microenvironment that compromises therapeutic efficacy of ferroptosis.The Fenton reaction is an effective way to induce ferroptosis in tumor cells by reacting ferrous ions with hydrogen peroxide(H2O2)in tumor cells to generate reactive oxygen species(ROS),which in turn causes lipid peroxidation.However,in tumor cells,the antioxidants such as thioredoxin 1(Trx-1)and glutathione(GSH)are significantly upregulated by the innate cancer cellular redox homeostasis,severely restricting the ROS-based therapy and compromising the effect of Fenton reaction-induced ferroptosis against tumors.Therefore,it is of great significance to develop a strategy that can both achieve high-efficiency ROS production and modulate tumor cell antioxidant microenvironment to amplify ferroptosis.Recently,it has been shown that radiotherapy can induce ROS production and GSH depletion to increase the accumulation of lipid peroxidation.Therefore,radiotherapy combined with the Fenton reaction is more likely to cause lipid peroxidation and promote ferroptosis in tumor cells.Based on this background,a nanoparticle(HP NPs)was designed in combination with radiotherapy to regulate redox homeostasis to enhance ferroptosis in tumor cells.The nanomedicine was constructed by co-assembling hemin(Fenton’s reagent)and PX-12(Trx-1 inhibitor)with the assistance of human serum albumin(HSA)to achieve HP nanoparticles(HP NPs).Hemin converts H2O2 to ROS via Fenton reaction to induce ferroptosis while PX-12 effectively inhibits the activity of antioxidant Trx-1 to suppress ROS depletion,resulting in amplified ferroptosis.Combining radiotherapy with the nanomedicine,radiotherapy depletes the other key antioxidant glutathione and generates additional radiotherapy-induced ROS,further boosting the ferroptosis effect.The main relevant studies are as follows.Firstly.the physicochemical properties of the nanomedicine HP NPs were verified.The composition in the HP NPs was analyzed using an ultraviolet spectrophotometer(UV-Vis)and Fourier infrared spectroscopy(FT-IR),and the results indicated the successful preparation of nanodrug.The morphology of nanodrug was observed by transmission electron microscopy(TEM),and the HP NPs were with spherical morphology.The particle size of HP NPs was 141.7±2.3 nm and the zeta potential was-19.9±0.2 mV as determined by dynamic light scattering(DLS).Methylene blue(MB)degradation assay showed that HPNPs were able to produce ROS.The hemolysis rate of HP NPs was less than 5%,indicating that HP NPs have good biosafety and are suitable for intravenous injection.Next,B16F10 melanoma cells were used to study the in vitro anti-tumor properties of HP NPs.The cellular uptake assay showed that the uptake efficiency of HP NPs was much higher than that of the free drug and showed a time dependence.MTT assay showed that HP NPs combined with radiotherapy had a strong cell-killing effect and were able to cause ferroptosis in tumor cells.The reactive oxygen species(ROS)assay showed that HP NPs combined with radiotherapy had excellent ROS production performance.The GSH and Trx-1 assay showed that HP NPs combined with radiotherapy could reduce the content of antioxidant substances GSH and Trx-1 in tumor cells,decrease the consumption of ROS,and enhance the ability of ROS to cause lipid peroxidation.Malondialdehyde(MDA)assay,glutathione peroxide 4(GPX4)assay,and mitochondrial membrane potential(MMP)assay showed that HP NPs combined with radiotherapy could increase the level of MDA and decrease the activity of GPX4 and MMP to enhance ferroptosis in tumor cells.Finally,a C57BL/6 mouse model of B16F10 melanoma was constructed to verify the in vivo distribution and anti-tumor properties of HP NPs.In vivo imaging experiments in mice showed that HP NPs could effectively accumulate in tumor sites.Tumor suppression assays showed that HP NPs significantly inhibited tumor growth in combination with radiotherapy.The dissected tumors were subjected to Western Blot assay of GPX4,GSH assay,and MDA assay,and the results showed that HP NPs combined with radiotherapy could significantly induce ferroptosis.Alanine aminotransferase(ALT),creatinine(CREA)levels,and histological analysis initially showed that HP NPs combined with radiotherapy had a good biosafety profile.In summary,this project constructed a nanoparticle capable of regulating redox homeostasis with the simultaneous combined application of radiotherapy.This strategy can simultaneously ensure efficient ROS production and regulation of tumor cell antioxidant microenvironment,thereby enhancing efficacy of ferroptosis in tumor therapy.Our work offers an innovative approach to amplify ferroptosis sensitivity against tumors by simultaneously promoting ROS production and regulating redox homeostasis. |
