The Role And Mechanism Of A Dual-targeting Membrane Coated Nano-drug Delivery System In The Synergistic Treatment Of Osteosarcoma

Objective: Osteosarcoma(OS)is the most common primary malignant bone tumor.The survival rate of OS patients has remained stagnant for years,so it is urgent to develop other novel strategies for OS treatment.This study attempts to prepare a dual-targeting biomimetic nano-drug delivery system which exerts synergistical anti-tumor effect,and explore the mechanism of the killing effect and its regulation on the tumor microenvironment.Methods: Glucose oxidase(GOX)and tirapazamine(TPZ)were loaded on polydopamine(PDA)nanoparticles,a widely-used mesoporous nanocarrier with photothermal conversion ability,to obtain drug-loaded nanoparticles,named PGT.Cyclic-Arg-Gly-Asp(a circular targeting peptide sequence)modified tumor cell membranes(c RGD-M)were camouflaged on the surface of PGT by co-extrusion methods to obtain a dual-targeting membrane-coated nano-drug delivery system(PGT@c RGD-M).In order to detect the characterization of the synthesized materials,technologies like transmission electron microscopy were used.The photothermal conversion capabilities of materials can be confirmed by infrared laser emitters and infrared thermal imaging cameras in vivo and in vitro.SDS-PAGE can be used to detect the membrane protein retention after the extrusion procedures.Confocal microscopy and in vivo bioluminescence imaging system can be used to detect the targeting and penetration capabilities of materials on different types of cells or tissues in vivo and in vitro.Ultraviolet absorption spectroscopy can be conducted to detect the stability,loading content,drug release and pharmacokinetic curve of the materials.The biocompatibility of materials can be tested through red blood cell hemolysis test and staining of vital organs after materials administration.CCK-8,Live-Death staining,Transwell,tumor volume measurement,Ki67 and TUNEL staining can be performed to confirm the anti-tumor effect and mechanism of the materials.Finally,bioinformatic methods combined with immunohistochemistry,immunofluorescence staining can be used to explore the regulation of the nano-drug delivery system on the inhibitory tumor microenvironment.Results: Transmission electron microscopy and other characterization results showed that the membrane-coated drug-loaded nanoparticles were successfully prepared.Infrared imaging indicated that the drug-loaded system had satisfied photothermal conversion effect in vivo and in vitro after irradiated with 808 nm laser for 80 seconds,and stably reached 55℃which might promote the drug releasing and penetration.SDS-PAGE showed that the proteins on tumor cell membrane were completely preserved after extrusion,which made homologous adhesion between tumor cells possible.The confocal and in vivo imaging results showed that the materials could target the tumor cells and penetrate deeply into the tumor tissues.The ultraviolet spectrum indicated that the drug-loading system had good stability in vivo and in vitro,and there was no obvious degradation or aggregation within a week.Besides,the drug-loading capacity and pharmacokinetic characteristics of the nanoparticles could meet the anti-tumor needs and the drug release could be controlled by infrared light.The material had a half-life time around 1.8 hours in plasma,and there was no obvious tissue residue.No obvious hemolytic reaction was observed after the material was co-cultured with red blood cells,no abnormality was found in the HE staining of various organs after tail vein administration,indicating that its biocompatibility was good.In vivo and in vitro anti-tumor experiments showed that the nano-drug delivery system could efficiently inhibit the proliferation and migration of tumor cells as well as promote tumor cell apoptosis,thereby effectively devastated OS.According to the bioinformatic analysis,HIF-1α is associated with immunosuppressive tumor microenvironment,while PGT@c RGD-M could down-regulate the high expression of HIF-1α in hypoxic tumor tissue,which reversed the exhaustion of tumor-killing T cells and promoted macrophage polarization to modulate the suppressive tumor microenvironment.Conclusion: A infrared light-responded biomimetic nano-drug delivery system(PGT@c RGD-M)was successfully prepared,which could dual-target and penetrate OS tissues to exert sustainable anti-tumor cascade and remodel the tumor microenvironment.It may be a potential strategy for the OS treatment in the future.