Preparation Of Iron-based Metal Organic Framework Nanocomposites And Its Applications In Liver Cancer Therapy

Objective:Hepatocellular carcinoma(HCC)is one of the most common and deadly cancers worldwide.Current options for standard treatment of HCC include surgical resection,interventional therapy,radiotherapy,chemotherapy,targeted therapy and immunotherapy.However,these existing treatments have a narrow indication window and are prone to relapse after treatment.Therefore,it is extremely urgent to develop new and effective treatment methods.Ferroptosis,as a new type of cell death,has been paid more and more attention.It is a procedural cell death mode that depends on the existence of iron.It has been found that ferroptosis can significantly inhibit the proliferation of liver cancer cells.Therefore,it is of great clinical significance to develop new ferroptosis inducers for liver cancer cells.With the development of nanomedicine,the types of ferroptosis inducers are expanded,which provides a new choice for developing new ferroptosis inducers for liver cancer cells.However,the clinical efficacy of pure nanomaterials is insufficient,and its passive targeting efficiency is low.Therefore,in this study,iron-based metal organic framework nanoparticles MIL-101(Fe)NPs were used as carriers,and MIL-101(Fe)@Sor NPs was constructed by loading ferroptosis inducer(Sorafenib,Sor)of liver cancer cells with its high drug loading capacity.In the process of tumor treatment,it was administered with tumor targeting peptide(iRGD)in the form of uncoupling to study its targeting,therapeutic effect and biological safety in the treatment liver cancerMethods:1.Iron-based metal organic framework nanoparticles MIL-101(Fe)NPs were prepared by hydrothermal synthesis method.Scanning electron microscopy(SEM)and transmission electron microscopy(TEM)were used to observe the morphology and size of the prepared nanoparticles.The elemental composition was analyzed by SEM mapping and scanning electron microscopy x-ray diffraction(EDS).The ability of magnetic resonance imaging in vitro was detected by 3.0 T magnetic resonance scanner.MIL-101(Fe)@Sor NPs was synthesized by loading Sorafenib with mechanical stirring method,and the drug loading rate and encapsulation rate of Sorafenib were calculated,and the human body environment with different PH values was simulated to observe the drug release performance in vitro under different environments.The ability of MIL-101(Fe)NPs to catalyze peroxidase in vitro was tested by using 3,3',5,5'-tetramethylbenzidine(TMB)as substrate.2.MTT assay was used to detect the different concentrations of MIL-101(Fe)NPs on the survival rate of HepG2 cells,so as to determine the safe dose range of MIL-101(Fe)NPs in cell experment.MTT method was used to detect the effect of different concentrations of diluted H₂O₂ on the survival rate of HepG2 cells,so as to determine the safe dose of MIL-101(Fe)NPs in cell experment.The ability of MIL-101(Fe)NPs to catalyze peroxidase in HepG2 cells was detected by DCFH-DA method.The uptake of MIL-101(Fe)NPs by HepG2 cells under the help of iRGD peptide was detected by magnetic resonance imaging.At the same Sorafenib concentration,the different groups of Sor,MIL-101(Fe)@Sor NPs and MIL-101(Fe)@Sor NPs+iRGD on the survival rate of HepG2 cells were observed by MTT method.The morphological changes of HepG2 cells treated with MIL-101(Fe)@Sor NPs were observed under microscope.In the presence of ferroptosis inhibitors(Fer-1 and DFOM),the different groups of MIL-101(Fe),Sor,MIL-101(Fe)@Sor NPs and MIL-101(Fe)@Sor NPs+iRGD on the survival rate of HepG2 cells were detected by MTT assay.To test the changes level of MDA and GSH in HepG2 cells treated with different groups of MIL-101(Fe),Sor,MIL-101(Fe)@Sor NPs and MIL-101(Fe)@Sor NPs+iRGD were detected.The expression level of protein GPX-4 in HepG2 cells treated with different groups of MIL-101(Fe),Sor,MIL-101(Fe)@Sor NPs and MIL-101(Fe)@Sor NPs+iRGD was detected.Ferroptosis indicator BODIPY-C11was used to monitor the changes of LPO content in HepG2 cells after incubated with different groups of MIL-101(Fe),Sor,MIL-101(Fe)@Sor NPs and MIL-101(Fe)@Sor NPs+iRGD respectively3.Firstly,the biological safety of MIL-101(Fe)was verified in KM mice.MIL-101(Fe)of 25 mg/kg,50 mg/kg and 100 mg/kg was injected into the mice,and observed for 15days after treatment,during which the weight changes of mice were monitored.Finally,after treatment,the heart,liver,spleen,lung and kidney were taken for H&E staining.The mouse model of transplanted liver H22 hepatoma cells was constructed,and the tumor targeting of iRGD was verified by evans blue staining,prussian blue staining and magnetic resonance imaging.MIL-101(Fe)@Sor was injected into mice,and blood samples were collected to monitor the iron content in serum at 0.5 h,1 h,6 h,12 h,24 h,48 h and 72h after treatment.H22 hepatoma-bearing mice were randomly divided into groups and treated with normal saline,MIL-101(Fe),Sorafenib,MIL-101(Fe)@Sor and MIL-101(Fe)@Sor+iRGD,once every three days for 7 times,the dose of Sorafenib and iRGD was 5 mg/kg and 4 mg/kg.The tumor volume was measured.After treatment,the heart,liver,spleen,lung,kidney and tumor were taken for H&E staining,blood samples were prepared for blood routine examination and blood biochemical examination,and the tumor was taken for immunohistochemical staining and immunofluorescence staining of GPX-4.Resulus:1.MIL-101(Fe)nanoparticles were successfully prepared by hydrothermal synthesis method.their appearance is octahedral structure,with a diameter of about 200 nm,which is composed of iron,chlorine,oxygen and carbon.It have T2-weighted magnetic resonance imaging ability and hydrogen peroxide catalytic ability,high specific surface area and good crystallinity.2.Sorafenib was successfully loaded onto MIL-101(Fe)by mechanical stirring method,and MIL-101(Fe)@Sor was prepared.The drug loading rate was about 12%.In the phosphate system with PH=5.5,the release amount of Sorafenib increased continuously,and the highest release amount was about 45%.3.The results of MTT assay showed that when the concentration of MIL-101(Fe)was between 1.56?g and 200?g,the survival rate of HepG2 cells was over 85%,Hemolysis experiment showed that MIL-101(Fe)did not cause hemolysis of red blood cells.4.The results of scanning electron microscope showed that MIL-101(Fe)@Sor could be successfully endocytosed by HepG2 cells,and MIL-101(Fe)could catalyze the reaction in HepG2 cells.5.iRGD can promote the uptake of MIL-101(Fe)in HepG2 cells by the assay of Prussian blue staining and magnetic resonance scanning.6.MIL-101(Fe)@Sor combined with iRGD can significantly reduce the survival rate of HepG2 cells,promote the production of LPO and reduce the expression level of GPX-4 protein,which is statistically different from other groups.7.After injecting 100 mg/ml MIL-101(Fe)into mice via tail vein,it did not cause the weight loss of mice within the treatment time window.The results of H&E(hematoxylin and eosin)staining(heart,liver,spleen,lung and kidney)showed that there was no pathological changes,and blood routine and blood biochemistry were within normal range.8.By using Evans blue drug,Prussian blue staining of tumor and magnetic resonance scanning of mice,iRGD can promote tumor targeting and uptake of MIL-101(Fe)in H22hepatoma-bearing mice.9.In the anti-tumor experiment,MIL-101(Fe)@Sor combined with iRGD can significantly inhibit the tumor growth of H22 hepatoma-bearing mice,and the tumor volume grows slower than other groups,and the tumor weight is lower than other groups.H&E staining results showed that the necrosis degree of MIL-101(Fe)@Sor combined with iRGD treatment group was the most serious.Besides,Immunohistochemistry and immunofluorescence staining results of GPX-4 showed that MIL-101(Fe)@Sor combined with iRGD have the strongest inhibitory effect on the expression in all groups.10.In the biosafety experiment,MIL-101(Fe)@Sor combined with iRGD did not affect the weight gain of mice,and the H&E staining results showed which did not cause pathological changes to the heart,liver,spleen,lung and kidney of mice.the blood routine and blood biochemistry were within the normal range.However,Sorafenib group caused pathological changes in liver and lung of mice,and abnormal liver function.Conclusion:In this study,MIL-101(Fe)nano-materials were successfully synthesized,which have good drug-carrying ability,drug-releasing ability,magnetic resonance imaging ability and catalytic peroxidase activity.Cell experiments and animal experiments show that the nano-material has good biological safety,and can significantly inhibit tumor growth when combined with iRGD peptide.Further study showed that the composite material could induce ferroptosis of tumor and significantly inhibit the expression of GPX-4 protein.This experiment is based on the idea of ferroptosis treatment of tumor,and successfully applied nano-materials combined with existing clinical drugs in the treatment of liver cancer,which provides a new idea and direction for the future liver cancer treatment.