MR Imaging Guided Nanomimetic Enzyme For Synergistic Ferroptosis-Starvation Therapy In The Triple-negative Breast Cancer

ObjectiveIn this study,we designed and prepared a type of nanomimetic enzymes Ava@SPIOs to enhance their tumor targeting ability and anti-tumor effects by modifying the properties of contrast agents,used the methods immunohistochemistry and Western blotting to investigate the effects and mechanisms of these nanomimetic enzymes on TNBC at the cellular and animal model levels,providing a new theoretical basis and clinical ideas for the integrated diagnosis and treatment of TNBC.Method1.Preparation and characterization of Ava@SPIOs nanomimetic enzymes:The morphology,particle size,structure and stability evaluation of the nanomimetic enzymes were characterized by transmission electron microscopy,nano-zs and vibrating sample magnetometer;the drug release properties of the nanomimetic enzymes were examined under different pH environments;the T₂ relaxation properties of SPIOs and Ava@SPIOs nanomimetic enzymes were also examined by magnetic resonance imaging.2.Evaluation of the anti-tumor efficacy of Ava@SPIOs nanomimetic enzymes in vitro:Human-derived TNBC MDA-MB-231 cell line was cultured in vitro;the anti-tumor effects of nanomimetic enzymes were analysed by cell survival,cell ROS level detection,Western blotting of GPX4,MDA level detection and so on.3.The anti-tumor effect and mechanism of Ava@SPIOs nanomimetic enzymes in vivo:Human-derived TNBC MDA-MB-231 nude mouse transplantation tumor model was constructed,and the MR imaging performance,intra-tumor concentration process and biological distribution characteristics in vivo of Ava@SPIOs nanomimetic enzymes were analyzed and visualized in real time and quantitatively by BOLD-MRI.The drug was further evaluated by H&E staining,Prussian blue staining,Ki-67,TUNEL,HIF-2α,CD31,GPX4 and ROS immunofluorescence histochemistry to assess the anti-tumor effect and mechanism of the drug in vivo.Result1.The average particle size of the synthesized Ava@SPIOs nanomimetic enzyme was 18 nm,and the zeta potential was-22 m V.The nanomimetic enzyme showed regular morphology,uniform size and good dispersion of round spherical nanoparticles under electron microscope;it had good pH-responsive release performance;it had good T₂ relaxation performance,and there was good correlation between the iron concentration in the nanosolution.The stability of the nanomimetic enzyme was confirmed in nude mouse serum.2.Under nutrient-deficient conditions,the survival rate of MDA-MB-231 cells under the action of SPIOs was significantly and gradually reduced;and at lower drug concentrations（SPIOs:30-50μg/m L）,the killing effect of SPIOs on tumor cells was statistically different（P<0.01）;under nutrient-deficient Under nutrient-deficient conditions,the expression of GPX4 was significantly down-regulated（P<0.01）and the levels of-OH（P<0.05）,ROS（P<0.01）and MDA（P<0.05）were significantly increased after the action of SPIOs on cells compared to the control group.3.The synthesized Ava@SPIOs nanomimetic enzymes were concentrated in the tumor tissue after tail vein injection into the human-derived TNBC nude mouse transplantation tumor model,and their concentration levels were significantly higher than those of SPIOs alone at the same time point,and the concentration of nanomimetic enzymes in the tumor reached the maximum at 12h after administration,while exhibiting Ava@SPIOs nanomimetic enzymes showed a significantly lower tumor volume than the control group and the SPIOs group alone（P<0.001）;the overall survival rate of the tumor model was significantly higher in the Ava@SPIOs nanomimetic enzyme treatment group than in the other treatment groups（P<0.001）;the treatment at the endpoint,the tumor tissues of the Ava@SPIOs nano-mimetic enzyme treatment group showed decreased proliferation and apoptosis marker Ki-67,increased TUNEL,decreased microvascular marker CD31,increased HIF-2α,decreased iron death marker GPX4 and increased ROS（P<0.01）.While the Ava@SPIOs nanomimetic enzyme group showed no significant abnormalities in heart,liver,spleen,lung and kidney H&E staining and blood biochemical parameters at the endpoint of treatment,and no significant differences in body weight and body temperature compared to basal levels and other treatment groups during the same period.ConclusionIn this study,the multifunctional nanomimetic enzyme Ava@SPIOs was successfully synthesized and characterized by applying magnetic resonance T₂-negative contrast agent SPIOs wrapped and loaded with the anti-angiogenesis inhibitor Avastin to construct a nanomimetic enzyme,which possesses high transverse relaxation efficiency and efficient at tumor tissue concentration effect from BOLD-MRI in vivo.The synergistic mechanism of ferroptosis and starvation therapy was also investigated in vitro and in vivo,which effectively improved the overall survival rate of TNBC tumor-bearing nude mouse models with good biosafety,providing a theoretical basis for the next application of the novel nanomimetic enzyme in the integration of TNBC diagnosis and treatment.