Manganese Tungstate Nanorods Mediated Chemodynamic Therapy Combined With Radiotherapy For Diagnosis And Treatment Of Triple Negative Breast Cancer

Objective:Chemodynamic therapy(CDT),as one of the emerging tumor therapy methods,provides a new idea for the treatment of a variety of malignant tumors without specific therapeutic targets.Clinically,conventional treatment methods such as surgery,chemotherapy and radiotherapy are often used for triple-negative breast cancer without specific therapeutic targets.However,due to lack of target,the therapeutic effect of tumor treatment is often poor and the prognosis of advanced patients is poor.Chemodynamic therapy converts endogenous hydrogen peroxide which has low toxicity to hydroxyl radical(·OH)with higher toxicity via Fenton or Fenton-like reaction,then oxidative damage to intracellular biological macromolecules,and inducing the death of tumor cells,and no drug resistance.The concentration of hydrogen peroxide in the tumor environment is higher than the normal tissues,however,the uneven distribution of hydrogen peroxide in the tumor tissue still limits the therapeutic efficiency of CDT.Combined radiotherapy can improve the level of reactive oxygen species(mainly ·OH and H2O2)at the irradiated sites of tumors,so as to effectively kill tumor cells and inhibit tumor formation.The rapid development of nanotechnology provides a new therapeutic platform for the treatment of triple negative breast cancer.This paper aimed to design,synthesize and construct a new type of inorganic nanorods,namely,manganese tungstate nanorods(Mn WO4-PEG nanorods,MWP NRs),which are simple in synthesis,and reproducible,effective in tumor treatment,less toxic and side effects for investigating the effect of the combined treatment of CDT/RT mediated in a triple-negative breast cancer model.In addition,the potential mechanism of their combined treatment was explored,in order to provide new treatment ideas for clinically refractory triple-negative breast cancer.Methods:Synthesis,modification and characterization: 1.Manganese tungstate nanorods with stable and uniform morphology were synthesized by hydrothermal method using manganese chloride dihydrate and sodium tungstate as raw materials under p H near neutral conditions.MWP NRs were modified by layer-by-layer deposition.2.The morphology,crystal structure,element valence distribution and surface modification of the nanorods were characterized by transmission electron microscope(TEM),nano-size and Zeta potential analyzer(DLS),X-ray diffractometer(XRD),X-ray photoelectron spectroscopy(XPS).Evaluation the biological effects of MWP NRs in vitro: 1.Indirect detection of Fenton-like effect conditions of MWP NRs by peroxidase substrate methylene blue(MB)fading reaction and influencing factors for catalytic rate.2.MWP NRs catalyze hydrogen peroxide to generate hydroxyl radicals(·OH)under the corresponding conditions were detected by Electron Spin Resonance(ESR).3.The uptake of 4T1 cells and the subcellular structure distribution of MWP NRs were observed by bio-TEM.4.MTT cell proliferation assay investigated the toxic effects of MWP NRs on tumor cells and normal cells.5.Fluorescent probes and reactive oxygen species detection kits were used to detect the production of ROS by MWP NRs-mediated CDT/RT;6.Flow cytometry analyzed the apoptosis state of CDT/RT mediated by MWP NRs,and Western Blotting detected the expression changes of apoptosis-related proteins.7.CLSM monitored the phosphorylation of H2 AX,which is the most sensitive to DNA damage.q PCR and Western Blotting monitored the changes in the expression of key genes in the process of DNA double-strand break damage repair at different time points in the transcription and translation level.Analyze the changes of tumor cell cycle under different group treatment conditions by flow cytometry.Colony formation experiment was used to detect the effect of MWP NRs in mediating CDT combined with radio-sensitization to kill tumor cells,then calculating the sensitization enhancement ratio.To explore the ability of MWP NRs in the diagnosis and treatment of triple-negative breast cancer in vivo: 1.We constructed a mouse subcutaneous tumor model for triple-negative breast cancer by subcutaneous injection of 4T1 cells.2.The in vivo imaging system for small animals detected the distribution of MWP NRs in Balb/c tumor-bearing mice at different time points,and the retention of MWP NRs in major organs and tumors at 24 h.3.The ability of MWP NRs to enhance tumor tissue resolution was investigated by small animal MRI and the local signal intensity changes of tumors were recorded before and after MWP NRs administration.CT imaging technology was used to detect the imaging capabilities of prostheses containing MWP NRs and the corresponding clinical commonly used CT contrast agent(iohexol)of the same concentration in vitro,calculating the X-ray absorption coefficient of the two,monitoring the contrast for intensity change before and after intra-tumor administration,and conducting three-dimensional reconstruction.4.DCFH-DA probe detected the production of reactive oxygen species at the tumor site,and investigated the relationship between the ROS production site and the distribution of MWP NRs in the tumor.5.We carried out four-group treatment experiments,when the tumor volumes reached about100 mm3,the tumor-bearing mice were randomly divided into four groups: PBS group(control group);MWP NRs;X-ray group;MWP NRs + X-ray group.MWP NRs were administered via tail vein injection(50 mg/kg),and when MWP NRs peaked in tumor accumulation 24 hours after administration,the tumor sites of mice were locally irradiated with a small animal irradiator.By monitoring the tumor volume changes of mice during the experiment and recording the changes in mice body weight,the tumor suppression rates under variant treatments were compared with the control group via calculation at the end of the experiment,and the efficacy of MWP NRs,X-ray monotherapy and MWP NRs + X-ray-mediated CDT/RT combination therapy were investigated.The tumor tissue sections were used to detect tumor tissue morphology,nucleus distribution density and immunofluorescence staining to evaluate the intra-tumoral distribution of cell proliferation(Ki67),apoptosis(TUNEL)and cleaved Caspase-3,to explore the histopathological antitumor effects of different groups after treatments.6.The liver and kidney function and other indicators were investigated after treatment of different groups by blood biochemical experiments,and the status and function of the main organs after therapeutic treatments were evaluated.7.The biological safety of MWP NRs was investigated by hemolysis experiments and H&E staining of major organs’ sections.Results:Synthesis,modification and characterization: rod-shaped Mn WO4 was successfully synthesized by hydrothermal method,and its length was 60-80 nm and its diameter about 20 nm.The above results were observed by transmission electron microscopy.Mn WO4 nanorods were uniformly dispersed by layer-by-layer deposition of PAH,PAA and NH2-PEG with a hydrated particle size about 130 nm and a zeta potential of(-5.3±0.26)m V.In the range of p H 4.0~7.4,the morphology of manganese tungstate nanorods was stable.XRD result demonstrated clearly that Mn WO4 nanorods were successfully synthesized with the same crystal structure as the standard.Due to the presence of polyvalent Mn elements on the surface of MWP NRs,the nanorods had their own redox potential and could mediate Fenton-like reactions to achieve the purpose of chemodynamic therapy.Evaluation the biological effects of MWP NRs in vitro: 1.The results of MB ddegradation were consistent with the ESR results,and the catalytic efficiency of MWP NRs was concentration,time and temperature dependent.At the same time,the p H of the reaction system could significantly affect the catalytic efficiency of MWP NRs.It could be still catalytic in the lysosomal p H range(p H 4.0 ~ 6.0),however,the efficiency was low,and its catalytic effect could be improved under the neutral to weakly alkaline p H of tumor cytoplasm.2.ESR result demonstrated that MWP NRs could catalyze H2O2 to produce a large number of ·OH in the presence of HCO3-.3.Bio-TEM observed that4T1 cells uptook MWP NRs into the cell by endocytosis,and the endocytic vesicles containing MWP NRs bound to lysosomes,and some MWP NRs were released from lysosomes into the cytoplasm at 6 h.CLSM could also observe that the colocalization relationship between MWP NRs and lysosomes changed over time.4.MWP NRs had a IC50 of about 19.37 μg/m L for tumor cells 4T1 cells,while IC50 for normal cells HUVEC cells about 142.1 μg/m L.5.Laser confocal detection showed that MWP NRs-mediated CDT/RT combination therapy could produce higher levels of ROS.6.Flow cytometry results indicated that the combination therapy produced more 4T1 cells apoptosis than monotherapy,and WB demonstrated that after MWP NRs-mediated CDT/RT combination therapy,the expression of anti-apoptotic gene Bcl-2 decreased,the expression of pro-Caspase 3 decreased,and the expression of cleaved Caspase 3 as an apoptosis performer increased significantly.7.CLSM observed that the most sensitive H2 AX phosphorylation of DNA damage was significantly prolonged in the MWP NRs-mediated combination therapy group,while the expression of key genes Rad51 and53bp1 in the HR and NHEJ pathway for DNA damage repair decreased significantly at the transcription and translation level at the same time.8.After MWP NRs-mediated combination therapy,the cell cycle of 4T1 cells shifted to the G2/M phase,which was more sensitive to radiotherapy.9.Colony formation experiments showed that MWP NRs had obvious radiotherapeutic sensitization ability,and their sensitization enhancement ratio was about 1.45.To explore the ability of MWP NRs in the diagnosis and treatment of triple-negative breast cancer in vivo: 1.MWP NRs administration via tail vein were mainly distributed in the liver and tumor site in tumor-bearing mice,and the accumulation degree in the tumor site reached a peak 24 h after administration.2.The same concentration of MWP NRs prosthesis had better CT contrast effect than iohexol.Before and after intra-tumoral administration of MWP NRs,there was a significant contrast of signal intensity on CT/MRI imaging.3.DCFH-DA probe detected the production of ROS in tumor tissues,and some certain ROS had obvious colocalization relationship with Cy5.5-modified MWP NRs(Cy5.5-MWP NRs).4.MWP NRs-mediated CDT/RT combination therapy in group therapy could produce the best tumor suppression effect,the tumor suppression rate was about 80.40%,which was much higher than that of MWP NRs(26.44%)and X-ray(38.89%)in the single treatment group,and H&E staining of tissue sections of isolated tumors could also observe more sparsely arranged tumor cells.Immunofluorescence staining detected that the fluorescence intensity of Ki67,a cell proliferation index,was significantly reduced in the MWP NRs-mediated CDT/RT combination therapy group,while TUNEL as an apoptosis indicator was significantly increased,and the red fluorescence of cleaved Caspase 3 as an apoptosis actor was also significantly enhanced.5.There were no obvious abnormalities in the blood biochemical indexes such as ALT,AST,CRE and Urea of tumor-bearing mice after combined therapy mediated via MWP NRs,indicating that liver and kidney function were not affected.6.When MWP NRs reach 500 μg/m L,there was still no hemolysis.H&E staining of major organ sections of mice after combination treatment showed no obvious abnormalities,indicating that MWP NRs had a good biological safety.Conclusion:1.MWP NRs were obtained by hydrothermal synthesis and layer-by-layer deposition modification,which had a good crystal form,and a rod-like structure with uniform particle size,negative charge on the surface,uniform dispersion in different solvents for a long time,no obvious aggregation,and stable morphology under different p H conditions.XPS analysis showed that the surface distribution of MWP NRs included Mn(II),Mn(III)and Mn(IV),which provided a potential possibility for MWP NRs to mediate the Fenton-like effect.2.The biological effects of MWP NRs in vitro were as follows: The Fenton-like effect of MWP NRs was generated by the variable value of Mn ions on their surface,and the efficient Fenton-like effect depended on temperature,neutral and weak alkaline conditions under physiological conditions.MWP NRs could catalyze H2O2 to produce?OH.In addition,the catalytic rate of MWP NRs was concentration-dependent.MWP NRs entered 4T1 cells through endocytosis and were distributed in lysosomes,and with increasing time,they could escape from lysosomes to the cytoplasm for producing a Fenton-like effect.The toxic effect of MWP NRs on tumor cells was more pronounced,which was associated with the high expression of H2O2 levels in tumor cells.MWP NRs-mediated CDT/RT combination therapy could produce a large amount of ROS,inducing significant changes in the expression of apoptosis-related proteins in tumor cells,and promoting apoptosis in tumor cells.In addition,the cell cycle arrest caused by DNA damage through the CDT effect mediated by MWP NRs could make tumor cells more sensitive to radiotherapy during combination therapy,and the high atomic number W element contained in MWP NRs itself could also sensitize radiotherapy,so as to achieve a strong synergistic effect under combination therapy and induce apoptosis of a large number of tumor cells.3.In vivo experiments,the efficacy of CDT/RT combination therapy for TNBC mediated by MWP NRs was significantly better than that of chemodynamic therapy and radiotherapy alone.MWP NRs-mediated combination therapy effectively delayed tumor growth by causing ROS overload in the tumor,effectively inhibited tumor growth,and had no obvious side effects during treatments,which provided a new possibility for the clinical treatment of triple-negative breast cancer.