Study On Synergistic Efficiency Enhancement Of Tumor Microwave Hyperthermia And Chemodynamic Therapy Based On Multifunctional Bimetallic Organic Framework Nanospheres

Objectives:Traditional microwave hyperthermia（MWTT）is an effective method for tumor treatment,but it lacks sensitivity and specificity.Therefore,precise and efficient tumor hyperthermia cannot be achieved,which affects its application in clinical treatment.The introduction of microwave sensitizer is used to solve the problems.At present,the most popular microwave sensitizer belongs to metal-organic framework nanostructures（MOF）,most of which belong to single metal-organic framework structures,and usually need to load ionic liquid to achieve significant microwave heating efficiency.At the same time,this new microwave hyperthermia treatment method is relatively simple,and relatively few combined with other treatment methods.Considering the internal complex tumor tissue microenvironment and high ratio of recurrence and metastasis,so we hope that we can be prepared a new kind of bimetallic organic framework structure.It has the characteristics of low toxicity,good biocompatibility,acid degradation and so on.At the same time,it can realize significant microwave hyperthermia mediated by microwave without using ionic liquid.As a new microwave sensitizer,it can cooperate with chemodynamic therapy to inhibit tumor,which is obviously a very challenging task.Methods:（1）Synthesis and characterization of FCMP nanospheresIn our experiment,first,ferric chloride,copper nitrate and two organic bridging ligands（terephthalic acid and 2-aminophenyldicarboxylic acid）were added to N-N dimethylformamide（DMF）solvent,then ultrasonic cleaning and magnetic stirring for several minutes,then the obtained mixture was put into a reaction kettle containing polytetrafluoroethylene and reacted for 12 hours in an oven containing 120 degrees Celsius.FeCuMOF（FCM）,a biodegradable bimetallic organic framework,was obtained by centrifugation and washing,and was modified by PEG.FeCuMOF@PEG（FCMP）was finally obtained.Then the morphology and dispersion of FCM and FCMP nanospheres were observed by TEM and SEM.The particle size and potential changes of nanoparticles before and after loading PEG were verified respectively.Energy spectrum and Mapping diagram are used to analyze the distribution of elements in the structure and the specific proportion of the content;Fourier transform infrared spectroscopy（FT-IR）is used to determine the internal functional groups.FCM and FCMP are preliminarily characterized by the above methods,which proves that the synthesis of materials is successful.（2）Property verification of FCMP nanospheresFirst of all,we conducted microwave heating experiments.Different concentrations of FCMP solution were irradiated by microwave for different time to test its heating performance.Secondly,the ability of FCMP to scavenge glutathione（GSH）was further studied by changing the concentration range and time.In order to further clarify the difference of hydroxyl radical generation between the bimetallic organic framework FeCuMOF（FCM）and the single metal organic framework Fe MOF（FM）and Cu MOF（CM）,that is,the difference of dynamic therapy efficacy,TMB experiment was conducted.Subsequently,in order to simulate the change of the material itself in the acidic environment of the tumor,we put the nanospheres in acidic and neutral PBS environment respectively,and observed the degradation under transmission electron microscope after reacting for different time.Finally,we performed the verification of in vivo and in vitro magnetic resonance T₂imaging.（3）In vitro and in vivo safety evaluation of FCMP nanospheresAt the in vitro cell level,three kinds of cells were co-incubated with different concentrations of FCMP solution,respectively,and MTT cell proliferation assay was used to further verify the effect of different concentrations of materials on cell survival rate,so as to reflect the in vitro toxicity.In addition,FCMP solutions of different concentrations were injected into the tail vein of mice,and then the weight changes of the mice were recorded every day.14 days later,the blood of the mice’s eyeballs were collected for blood routine and blood routine tests.The important organs of the mice were collected and stained with H&E to observe the changes of the tissue,and the toxic and side effects were verified from the perspective of in vivo experiments.（4）Tumor inhibition experiment of FCMP nanospheres at the cellular levelWe used MTT method to carry out tumor inhibition experiment.The above TMB results show that FCMP can significantly catalyze hydrogen peroxide to produce rich hydroxyl radicals,thus enhancing the efficacy of dynamic therapy.Secondly,FCMP can achieve significant microwave hyperthermia with different microwave irradiation time.Finally,microwave hyperthermia combined with chemodynamic therapy can inhibit the tumor.We continued with a validation trial of tumor suppression efficacy.The inhibitory effect of FCMP nanoparticles combined with microwave irradiation on tumor was demonstrated in vitro by flow cytometry,cell migration and EDU cell proliferation.（5）Inhibition of tumor by FCMP nanospheres in tumor-bearing miceCombined with the results of cell-level tumor inhibition experiment,we continued the tumor inhibition experiment in tumor-bearing mouse model to further prove the tumor inhibition effect of FCMP combined with microwave.We established a tumor-bearing mouse model of liver cancer by subcutaneously injecting H22 cells.Seven days later,FCMP solution was injected through the tail vein,grouped according to the experiment,microwave irradiation or non-irradiation treatment.Subsequently,the weight of the mice,the size and volume of the tumors,and the survival of the mice were recorded.After 14 days,the tumor tissues and important organs of the mice were taken,photographed,weighed,and H&E stained.Finally,a Tunel fluorescent staining experiment was performed on the tumor tissue to verify the efficacy of inhibiting tumors.Results:1.We successfully synthesized FCM nanospheres by hydrothermal method,and modified their surfaces with PEG,and finally obtained a novel FCMP bimetallic organic framework.FCMP nanospheres have uniform size,good dispersion performance and uniform element distribution.2.Under microwave irradiation,with the increase of FCMP concentration and the prolonging of reaction time,the FCMP heating capacity becomes more significant.Similarly,with the increase of mass and reaction time gradient,the performance of FCMP to remove GSH gradually improved.3.Through the TMB experiment,the efficacy of the single metal organic frame structure and the new bimetal organic frame structure in dynamic therapy was compared.The results show that the ability of bimetallic organic framework FCM to generate hydroxyl radicals is 2.17 times and5.12 times that of monometallic organic framework FM and CM,respectively,which further demonstrates that FCM has significant advantages in dynamic therapy efficacy.4.The results of acid degradation showed that the structure of FCMP nano-spheres was destroyed with the increase of reaction time.When the reaction time reached 24 hours,the original structure of FCMP disappeared completely.However,under the condition of neutral PBS,there is basically no obvious change in its structure.5.Magnetic resonance imaging showed that the T₂signal of FCMP decreased with the increase of concentration in vitro.In vivo experiments showed that within 6 hours after the injection of FCMP,magnetic resonance signal at the tumor site decreased gradually with the increase of the injection time,indicating that FCMP has imaging function in vivo and in vitro.6.The results of in vitro and in vivo safety evaluation showed that FCMP had no obvious toxic side effects in vitro and in vivo.7.The cytotoxicity of MTT,flow apoptosis,EDU and Transwell assay showed that FCMP had obvious tumor inhibition effect.It can inhibit cell migration,proliferation and promote cell apoptosis.8.The results of tumor therapy in tumor-bearing mice showed that the tumor volume of mice increased significantly in the control group,while the tumor volume of mice in the FCMP+MW group did not increase,and some of them showed a slight trend of decrease.H&E staining results of tumor tissues proved that FCMP+MW had significant tumor inhibition effect.Tunel fluorescence staining of tumor tissue further proved that FCMP+MW group had significant apoptosis of tumor cells.Conclusions:FCMP,as a novel iron-copper based bimetallic nano-enzyme,catalyzes hydrogen peroxide and produces a large number of hydroxyl radicals and reactive oxygen species（ROS）.In the tumor microenvironment,FCMP can consume glutathione（GSH）and prevent the ROS generated from being eliminated,further enhancing the efficacy of dynamic therapy.Secondly,under low power microwave irradiation,FCMP nanospheres can also achieve significant microwave heating performance without loading ionic liquid,and reduce the complexity of the synthesis process.Thirdly,FCMP nanospheres have good biodegradability and biocompatibility,which makes them have good characteristics of biological metabolism and non-toxic accumulation.Finally,FCMP can achieve in vivo and in vitro magnetic resonance imaging functions,providing the possibility of further magnetic resonance mediated tumor therapy.FCMP particles have successfully realized the combination of microwave thermotherapy and chemodynamic therapy（CDT）as well as real-time MR-mediated T₂imaging mode.It provides a breakthrough for the integrated diagnosis and treatment of liver cancer and a new reference direction for the development of new microwave sensitizer.