Reversing Tumor Multidrug Resistance Via Biomimetic Nanoenzyme Reactor Based On Cholesterol Depletion Strategy

Multidrug resistance（MDR）is the main reason for tumor chemotherapy failure.It is also a big problem for cancer treatment.Although the combination of different drugs or using P-glycoprotein（P-gp）inhibitors can increase drug concentration in MDR cells and reduce drug efflux.The effect is still not ideal.An important reason is that the existing therapies ignore the pathological structure of drug-resistant cell membrane and treat the symptoms but not the root cause.Compared with the sensitive cancer cell membrane,the drug-resistant cancer cell membrane is rich in cholesterol and sphingolipid.The"rigidity"is enhanced but fluidity was reduced.As a result,it was hard for drugs get into the cells.Therefore,depleting cholesterol to reduce the rigidity of resistant cell membranes,breaking through the pathological barrier,and efficiently delivering chemotherapeutic drugs into the cell,was expected to"cure both the symptoms and the root causes"to improve the efficacy.In this study,we combined chemotherapeutic drugs doxorubicin（DOX）,natural enzymes cholesterol oxidase（COD）and nanoenzymes metal organic framework Zr MOF（Cu）with the lipid metabolism in resistant cancer cells,and the nanoparticles DOX@COD-MOF@CCM was constructed.After the nanoparticles were injected intravenously,it achieved"homing"targeting.After being internalized into the cell,the Zr MOF（Cu）nanoparticles as the carrier act as a peroxidase-like effect to catalyze the production of cholesterol by COD.The H₂O₂,which was further catalyzed into ROS,effectively reduced the cholesterol in the resistant cell membrane,so that the drug continuously enterd the cell,and the exhaustion of cholesterol would also decrease the lipid rafts,reduce the expression of P-gp,and block the efflux of drugs.In this study,we here provide a new strategy for reversing multidrug resistance.It was mainly researched from the following aspects in this study:（1）Construction and characterization of nanosystemsUsing zirconium tetrachloride and 2,2’-bipyridine-5,5’-dicarboxylic acid as raw materials,Zr MOF nanoparticles were synthesized by hydrothermal method,and further combined with copper dichloride sulotions to obtain Zr MOF（Cu）.Cholesterol oxidase（COD）was loaded on MOF through amide reaction and DOX was loaded in Zr MOF（Cu）framework by stirring.Then the formed nanoparticles are wrapped with cancer cell membrane to obtain DOX@COD-MOF@CCM.The results of FT-IR,UV-Visand SDS-PAGE protein analysis showed that the delivery system was successfully prepared.The results of particle size and potential analysis and transmission electron microscopy showed that the average particle size of the nano-delivery system was about 238.3±4.1 nm,the average zeta potential was 21.7±1.1 m V,and the drug loading is 20.4%,with suitable particle size and good dispersibility.（2）Studies on properties of DOX@COD-MOF@CCMThe catalysis of Zr MOF（Cu）on hydrogen peroxide in solution was investigated using the agent TMB,indicating that Zr MOF（Cu）had peroxidase activity.Secondly,the cascade catalytic activity of COD-MOF was investigated,and the results showed that the preparation could convert cholesterol into reactive oxygen species through the cascade of"natural enzymes"and"nanoenzymes".In addition,in vitro drug release studies have shown that the DOX@COD-MOF@CCM nanosystem has a cumulative release rate of 75.8%in a buffer solution that simulates the acidic environment of tumor cells within 48 hours,which is higher than 31.0%under neutral physiological conditions.Through the hemolysis experiment and the investigation of particle size changes,it was shown that the nano-delivery system had good stability and biocompatibility.（3）Study on reversing tumor multidrug resistance in vitro:Firstly,the uptake of DOX@COD-MOF@CCM by cells from seven different sources was investigated,and the results showed that the preparation had the ability of homologous targeting and immune escape.Then,the MCF-7/ADR cells were used as main models to study the uptake and accumulation of free DOX and DOX@COD-MOF@CCM over time.The results showed that both the drug uptake and accumulationin in the cells of DOX@COD-MOF@CCM were higher than free DOX.By using the SRB method,the drug resistance factor of MCF-7/ADR was determined to be 36.4.After DOX@COD-MOF@CCM treatment,the drug resistance factor was reduced by 90%,indicating that the nanosystem can successfully reverse multidrug resistance.In addition,the nanosystem could effectively reduce the cholesterol content in drug-resistant cells,thereby reducing the lipid rafts on the cell membrane,and increasing the fluidity of the cell membrane.The DCFH-DA probe was used to measure reactive oxygen species in cells,and the results showed that DOX@COD-MOF@CCM had the stronger ability to generate reactive oxygen species.The Annexin V-FITC/PI method was used to determine cell apoptosis and the results showed that the nano-delivery system could effectively induce 44%cell apoptosis after 24 hours.JC-1 probe detection of mitochondrial membrane potential showed that DOX@COD-MOF@CCM could activate the mitochondrial-mediated apoptosis pathway.（4）In vivo pharmacodynamic researchBALB/c female nude mice bearing MCF-7/ADR breast cancer were used as animal models.The results of in vivo small animal imaging experiments showed that the nano-delivery system had a better ability to target tumor sites.After the tail vein injection of DOX@COD-MOF@CCM for 14 days,the tumor inhibition rate of DOX@COD-MOF@CCM reached 94.4%,which had a good anti-tumor effect.In addition,the organ index and blood biochemical indicators showed that the nano-delivery system had biological safety.In summary,we successfully built a nano-delivery system named DOX@COD-MOF@CCM,which could convert cholesterol into reactive oxygen species through the cascade catalysis of natural enzymes and"nanoenzymes",and successfully reversed multi-drug resistance by"exhausting"cholesterol.