Preparation Of Synergistic Anti-Tumor Targeting Nanoparticles Based On Photothermal/Multi-Enzyme Cascade Reaction And Its Cell Biological Evaluation

The design of nanomaterials that can be treated tumors precisely and efficiently therapy without affecting normal tissue function is one of the core objectives in the field of tumor therapy,which has been a hot topic of research in the biomedical field in recent years.With intensive research of tumor cells therapy,it has been found that the surfaces of the material is coated with biologically active tumor cell membranes can confer active targeting capabilities to the material and avoid its clearance in the circulation.It has been shown that elevated reactive oxygen species（ROS）in tumor cells can increase the cell cycle to inhibit tumor growth and induce apoptosis.The use of highly active natural enzymes to generate large amounts of ROS within tumor cells to kill tumor cells for enzyme cascade kinetic therapy has been promising applications.Glucose oxidase（GOx）achieves starvation therapy by catalyzing the reaction of glucose and oxygen in the tumor cell interior to produce gluconate and H₂O₂,which deplete intracellular glucose.However,GOx is difficult to kill cancer cells through a single starvation therapy in a hypoxic tumor microenvironment（TME）.The catalase（CAT）can provide O₂ for GOx catalytic reactions in the TME.Furthermore,the horseradish peroxidase（HRP）can catalyze the removal of reaction products of GOx.The mix systems of the three enzymes not only accelerates the reaction rate,but also provides a large amount of ROS for killing tumor cells.Photothermal therapy,a tumor killing modality,has received much attention in recent years,which can be killed tumor cells with the local high temperature generated by the infrared thermal effect.At the same time,local warming can promote the catalytic efficiency of enzymes,and further enhance the ROS effect.Thus,by opportunity for precise and efficient anti-tumor treatment can be enhanced by supering all three effects onto the same nano platform to achieve synergistic tumor kulling.Therefore,we prepare nanoparticles AuNFs-GCH@CM with photothermal/multi-enzyme cascade synergistic therapeutic and active targeting of source cancer cells,then evaluates the cell biology of this composite targeting nanoparticle in this thesis.Fistly,We prepared gold nanoflowers（AuNFs）with a branching structure by seed growth method,and used AuNFs as the core to load multiple natural active enzymes GOx,CAT and HRP.Then,4TI tumor cell membranes（CM）were coated on the surface of AuNFs-GCH to prepare the multifunctional composite gold nanoparticles AuNFs-GCH@CM.The composite nanoparticles were characterized by transmission electron microscopy（TEM）and laser particle size measurement and the diameter of AuNFs-GCH@CM ranged from 200 to 260 nm with uniform size and good characteristics.The in vitro kit chemical assay was used to verify that the enzyme modification has still reserved multi-enzyme cascade activity after being grafting to the gold surface.The results of the optimized photothermal conversion performance experiments showed that the system temperature could reach 46.7℃ when the solution concentration of AuNFs-GCH@CM was 200 ppm and the laser irradiation intensity was 1.5 W/cm²,after 300 s of irradiation.The maximum temperature was maintained between 43⁴7 ℃ during the five repeated heating and cooling cycles.The results show that the composite nanoparticles AuNFs-GCH@CM have good photothermal conversion ability and photothermal stability.In vitro cell biological evaluation showed that the prepared composite nanoparticles AuNFsGCH@CM had good cytocompatibility and cancer cytotoxicity.It was confirmed by CCK-8 assay that the cell viability of the material remained above 90 % after 12 h co-incubation with normal mouse fibroblasts（L-929）.In contrast,after 12 h coincubation with tumor cells（4T1）,cell viability was only 40 %.In particularly,the viability of 4T1 cells was below 20% under laser irradiation.The production of ROS by AuNFs-GCH@CM in tumor cells was confirmed by fluorescent labelling method to be able to achieve apoptosis-inducing effect,and more ROS were produced under laser irradiation.In conclusion,the composite targeting nanoparticles AuNFs-GCH@CM combine multiple advantages of high cytocompatibility,active targeting,photothermal properties and multi-enzyme cascade activity.The novel multifunctional composite nanoparticles provide ideas and fundamental research data for the development of anti-tumor nanomaterials.