Delivery Of Arsenic Trioxide By Mitochondrial-targeted Triphenylphosphine-coated Mesoporous Nano-zirconium Dioxide Combined With Microwave Therapy Improves The Efficacy Of Treatment Against Hepatocellular Carcinoma

Background:Malignant degree of liver cancer is high,insidious,high mortality,and is not sensitive to conventional chemoradiotherapy.As an effective broad-spectrum chemotherapy drug,arsenic trioxide can also reverse drug resistance to a certain extent,which provides a new possibility for intravenous chemotherapy of liver cancer.However,arsenic trioxide has a high toxicity and a narrow biosafety window.To reduce arsenic trioxide in liver cancer systemic vein chemotherapy side effects in combination with the characteristics of drug-loading nanoparticles platform is good,we need to develop a nanoparticles can be controlled release drug delivery system,in order to reach the arsenic trioxide in liver cancer local release regular time,to guarantee the curative effect of local at the same time,reduce the systemic side effects.Method:1.SiO₂ seeds were used as templates to synthesize mesoporous nano-zirconia particles,and their surface was modified by amination.Arsenic trioxide was loaded by electric charge,and mitochondrial targeting ligand triphenyl phosphine was loaded.Physical and chemical characterization,drug loading rate and drug release rate were also studied.2.In vitro safety:different concentrations of TPA@ZrO₂ were incubated with HepG2 liver cancer cells,and their cytotoxicity was detected by cck-8 method.Through co-incubation of different concentrations of tp@zro2 with 2%rabbit erythrocyte suspension,hemolysis and agglutination were observed,and the safety of intravascular injection was detected,so as to evaluate the safety of tp@zro2 in vitro.In vitro effectiveness:MWA+TPA@ZrO₂ incubation with HepG2 liver cancer cells,by CCK-8,Transwell,EDU,JC-1,Annexin V-FITC/PI,PI/RNase detection cell inhibition rate,mobility,proliferation rate,mitochondrial membrane potential changes,the change of apoptosis rate and cell cycle,in order for the microwave trigger（5 min,3.8 W）composite nanoparticles drug delivery system effectiveness evaluation on the treatment of liver cancer cells;3.The subcutaneous tumor model of mouse H22 hepatocellular carcinoma was constructed and its safety and effectiveness were evaluated.Security in the body:the mice tail intravenous 40 mg/kg（in arsenic trioxide,the dose of lethal dose）of TPA@Zr O₂,after the injection of 1 day,3 day and 7 days respectively to death three mice,draw blood for blood routine and blood biochemical examination,at the same time take its heart,liver,spleen,lung,kidney,HE staining,observe its necrosis and inflammatory change.Three blank control groups were killed at each time point for the same sampling and testing.The in vivo safety of the material was evaluated.Effectiveness in the body:the same mouse tail 40 mg/kg intravenous TPA@Zr O₂,after 6 hours of tumor local 3.8 W,5 minutes to trigger,after 1 day,3 days,7 days,days 14 and 28 measure the weight of mice and tumors had long,wide diameter,volume calculation tumors,with 28 days and put to death between groups（3 mice in each group）,remove the tumors had weighed,and HE staining and TUNEL apoptotic staining to observe the tumor necrosis and apoptosis.Combined treatment:the time of microwave treatment was extended to 30 minutes.After treatment,3 mice in each group were sacrificed,and the tumors were taken for HSP70 immunohistochemical staining and western-blotting protein quantification.The body weight,tumor volume and tumor weight（3 in each group）of mice were measured at the same time point,and the protein CyclinD,CD34,p53,VEGF and V-ATPase of tumor tissues were quantitatively analyzed,so as to briefly elaborate the mechanism of action of the combined therapy.Results:1.TPA@Zr O₂ was successfully prepared.It was a mesoporous shell structure with an average particle size of 140nm.2.High resolution mass spectrometry showed that there was an obvious absorption peak at 262,indicating that the loading of triphenylphosphine was successful.3.Fourier infrared broad spectrum showed obvious absorption peaks at 1400cm^-1and 2917cm^-1,which proved that the encapsulation of tetradecanol was successful.4.ICP-MS indicated that As accounted for 41.29%,and the drug loading rate of arsenic trioxide was 54.5%.5.The distribution of Zr,O,C,P,Si and As elements can be seen by SEM and EDS analysis.6.ICP-MS detected the release amount of drug after successive trigger and calculated the release rate,and the final release rate was about 36.67±1.32%.7.The concentration gradient of 0.01mM-2mM TPA@ZrO₂ was co-incubated with HepG2 hepatocellular carcinoma cell lines,and the cell survival rate was above90%with good cell safety.8.TPA@ZrO₂ with a concentration gradient of 0-5mM was co-incubated with rabbit erythrocyte suspension.No erythrocyte agglutination and hemolysis were found,which was safe for injection.9.MWA+TPA@ZrO₂ can significantly inhibit HepG2 cells,but there is no significant difference with PA@ZrO2 group,and there is significant difference with each control group;10.MWA+TPA@ZrO₂ significantly inhibited the migration of HepG2 cells compared to the PA@ZrO₂ group,p=0.022<0.5.11.MWA+TPA-ZrO2 significantly inhibited the proliferation of HepG2 cells compared with the PA@ZrO₂ group,p=0.014<0.5.12.MWA+TPA@ZrO₂ could effectively destroy the mitochondria of HepG2cells and reduce its membrane potential,which was significantly different from that of the PA@ZrO₂ group（p=0.027<0.5）.13.MWA+TPA@ZrO₂ had a significant difference in promoting apoptosis of HepG2 cells compared with the PA@ZrO₂ group,p=0.001<0.5.14.MWA+TPA@ZrO₂ could effectively inhibit cell cycle,which was significantly different from the group of PA@ZrO₂（p=0.049<0.5）.15.All the mice in the drug delivery system that received lethal dose of arsenic trioxide through the tail vein survived,and no significant abnormalities were found between the blood routine,blood biochemistry and HE staining of tissues and the control group.The subcutaneous tumor model of mice heated up rapidly and steadily under microwave heating.16.Within 28 days after treatment,the tumor volume of the mice decreased significantly,tumor weight decreased,and there was no significant difference in body weight.In addition,the tumor volume of the target group decreased continuously,while the tumor in the non-target group began to grow gradually after 14 days of gradual reduction.17.After 28 days of MWA+TPA@ZrO₂ treatment,tumor samples were collected in mice,and obvious necrosis and apoptosis were found,with a higher apoptosis rate in the targeted group（p=0.028）.18.After extending the time of microwave treatment to 30 minutes,that is,combined with microwave hyperthermia,the tumor shrinkage trend was more obvious.19.Immunohistochemistry of HSP70 protein suggested that there was a significant difference between the combined treatment group and the trigger group.The expression of HSP70 in the combined treatment group was increased.20.Western Blotting indicated that the protein expressions of HSP70,CD34,p53,CyclinD,VEGF and V-ATPase in the combined treatment group were significantly different from those in the trigger group,thus predicting and revealing the treatment mechanism of the combined treatment group.Conclusion:In this study,we successfully constructed a nano zirconia carrier with controlled release drug with mitochondrial target,and successfully loaded the highly toxic chemotherapy drug arsenic trioxide,after amination,namely tpp-pcm-ato@ZrO2,which has a good drug loading capacity.The drug delivery system has good biological safety,and can release arsenic trioxide after in vitro low-power microwave trigger,which can inhibit tumor growth and kill tumor cells.It was also found that combined with in vitro microwave thermotherapy,it can enhance the effect of chemotherapy and hyperthermia,and further improve the therapeutic effect.This study provides a new idea and method for the development of new controlled-release nano drugs and non-invasive chemotherapy for liver cancer.