Study On Reversing Tumor Drug Resistance Based On Nanoenzyme-Natural Enzyme Depleting Cell Membrane Cholesterol

At present,tumor multidrug resistance is still the main problem to overcome.The problems of difficult uptake and easy excretion of chemotherapy drugs in drug-resistant cells lead to the failure of drugs to exert their original efficacy,and the anti-tumor efficiency is greatly reduced.At present,the common means to overcome drug resistance is to inhibit the activity of drug transporter P-glycoprotein（P-gp）,reduce drug pumping or through chemical drug combination therapy,ignoring the drug intake difficult and slow in resistant cells.The main reason for the difficult uptake of drug-resistant cells is the change of physiological structure of cell membrane.Compared with sensitive cells,the membrane of drug-resistant cells contains a large amount of lipid substances such as cholesterol and sphingolipids.The structure of cholesterol determines its structural rigidity.Therefore,the increase of cholesterol content in cell membrane will lead to the enhancement of mechanical rigidity of the cell membrane,the decrease of fluidity,the drug is not easy to be uptaken by drug-resistant cells,and the curative effect is reduced.In addition,cholesterol-rich cell membranes are more suitable for P-gp targeting,and drugs are more likely to be captured and pumped out by P-gp.In order to overcome the problems of difficult uptake and easy excretion of drugs in drug-resistant cells,increase intracellular drug concentration and reverse multi-drug resistance of tumor,this topic to build a kind of nanometer catalyst based on cascade catalytic cholesterol depletion,the natural enzyme cholesterol oxidase（COD）catalyzes the consumption of cholesterol,and the reaction product H₂O₂is further catalyzed by nano enzyme（NH₂-MIL-88B,MOF）to generate hydroxyl radical（·OH）with killing effect.The DOX@MOF-COD nanoparticles are wrapped in the chondroitin sulfate gel shell（CS shell）with both CD44 targeting and apoptosis effect,delivering them to the target with high efficiency.In this study,peroxidase properties,response characteristics and the ability to reverse drug resistance of tumor were investigated.The specific research is as follows:1、Construction and characterization of DOX@MOF–COD@CSFirstly,MOF was synthesized with ferric chloride hexahydrate and 2-amino terephthalic acid as raw material,Through amide reaction,the carboxyl group of COD is connected with the amino group of MOF,and centrifuge to remove the precipitate at the bottom of the supernatant to obtain MOF-COD,DOX solution and MOF-COD solution are mixed and stirred to obtain DOX@MOF-COD nanoparticles,chondroitin sulfate and cystamine dihydrochloride are cross-linked by amide reaction to form a CS shell with disulfide bonds.Drop the CS shell solution into DOX@MOF-COD solution and obtain DOX@MOF-COD@CS nanoparticles by ultrasound（Power 100 W,time10 min）.Transmission Electron Microscopy（TEM）and Dynamic laser light scattering（DLS）showed that DOX@MOF-COD@CS is a spindle shape with obvious roundness and the particle size is about 266.7±8.6 nm and a potential of-30.77±0.6 m V.In addition,the specific surface area analyzer（BET）,thermogravimetric analysis（TGA）and X-ray diffraction（XRD）analysis showed the successful preparation of MOF.Fourier infrared（FT-IR）spectra and TEM proved the successful preparation of DOX@MOF-COD@CS.In vitro hemolysis experiments and stability experiments show that the delivery system has good safety and stability.2、Research on drug release characteristicsThe introduction of disulfide bonds by CS shell can respond to the GSH（2-10m M）fracture in tumor cells and release DOX.In vitro drug release studies showed that when GSH=10 m M and p H=6.8 for 10 h,the drug release rate of DOX@MOF-COD@CS was significantly higher than that in the normal environment,indicating that DOX@MOF-COD@CS nanoparticles could respond to GSH lysis and release DOX in the tumor environment.3、Research on the catalytic properties of enzymeThis paper systematically studied the peroxidase-like properties of nanoenzyme MOF.MOF could catalyze H₂O₂to generate·OH in a weakly acidic environment.Based on this reaction mechanism,the UV spectra of oxidation products with tetramethylbenzidine（TMB）,o-phenylenediamine（OPD）proved that MOF has enzyme like activity.The TMB experiment of MOF-COD proved that COD could oxidize cholesterol to H₂O₂without adding H₂O₂,and continue to be catalyzed by MOF to generate·OH.The above catalytic experiments showed that MOF had enzyme-like properties,and MOF-COD could cascade catalyze cholesterol to generate·OH,laying a foundation for the study on the reversal of drug resistance at cell level in vitro.4、DOX@MOF-COD@CS nanoparticles in vitro reversal of drug resistanceThe drug-resistant breast cancer MCF-7/ADR cells were used as the model in vitro.Laser confocal（CLSM）and flow cytometry detection results showed that compared with free DOX,the fluorescence intensity of DOX@MOF-COD@CS was significantly stronger.The cytotoxicity of nanoparticles was detected by SRB method.The IC₅₀ value of free DOX was 83.51μg·m L^-1,and the IC₅₀ value of the treated with nanoparticles was significantly decreased,which was 11.07μg·m L^-1 the decrease rate was about 86.7%.The results of cholesterol quantitative determination showed that the cholesterol content was significantly reduced.The fluidity of cell membrane was further investigated by fluorescence spectrum,and the results showed that the fluidity of cell membrane was enhanced.Flow cytometry measured the efflux of MCF-7/ADR cells.The results showed that nanoparticles intracellular DOX fluorescence intensity was significantly higher than that of free DOX 8 h after the uptake was terminated,which could inhibit the efflux of cell.CLSM and flow cytometry showed that the nanoparticles could destroy the structure of lipid rafts and reduce the green fluorescence intensity.5、In vivo studiesUsing nude mice bearing MCF-7/ADR tumors as a model,and in vivo imaging results showed that the system had good targeting ability.The blood test index,organ index and body weight of nude mice showed that the system had good safety,and the tumor inhibition rate was up to 92.17%.