Construction Of Anti-tumor Nano Drug Delivery System With Hypoxia Induced Chemotherapy/gene Therapy

In recent years,various nano therapeutic systems have been developed rapidly in the field of anti-tumor.However,a single treatment formula is difficult to achieve the desired therapeutic effect.In this project,we have constructed a nano-drug delivery system,which is based on hypoxia induced chemotherapy and supplemented by gene therapy.This kind of system effectively combined chemotherapy,gene therapy,hunger therapy and photothermal therapy to achieve ideal tumor treatment effects and provided a new strategy for malignant tumor treatment program.In this study,the anoxic-activated drug Tirazamine（TPZ）was loaded into the pores of mesoporous silica nanoparticles（MSNs）.Then the surface of MSNs was modified by amination,and HIF-1αsi RNA was further adsorbed on the surface by electrostatic adsorption.After the outer layer being coated with polydopamine（PDA）,glucose oxidase（GOx）was adsorbed on the surface of PDA coating to build TPZ@MSN/si RNA@PDA@GOx（MTRPG）nano drug carrier system.The results of chemical characterization and material performance experiments showed that the particle size was 280～300 nm,Zeta potential was-21.8±1.26 m V,PDI was 0.22±0.016 respectively.MTRPG was well dispersed in solution,the appearance was uniform and spherical,and the outer coating structure could be seen by TEM observation.MTRPG could load TPZ with high efficiency,and the encapsulation rate and the drug loading was 77.77±1.4%and 4.66±0.8%,respectively.The nano drug loading system showed good photothermal conversion performance and oxygen consumption capacity.In the drug release experiment,GOx in the outermost layer of MTRPG first played a role in consuming oxygen and producing gluconic acid,reducing the environmental p H value and promoting the degradation of PDA,and realized the sequential release of GOx and TPZ.Tumor cells could successfully ingest MTRPG nanoparticles according to the results of cell uptake experiments in vitro.The cytotoxicity test of CCK-8 and the staining test of living/dead cells confirmed that the nanoparticles MTRPG had a powerful killing effect on tumor cells in a concentration dependent manner,especially under hypoxic conditions.The results of[Ru（dpp）₃]Cl₂ oxygen indicator probe and reactive oxygen fluorescence staining showed that the content of oxygen in tumor cells decreased and the content of H₂O₂ increased significantly,which verified the role of MTRPG in hunger treatment through direct and indirect ways,and created an hypoxic microenvironment for subsequential activation of TPZ.Western blot analysis showed that MTRPG nanoparticles effectively down-regulated HIF-1α,which was increased due to the hypoxia environment induced by GOx and the rapidly growth of tumor cells,realizing the role of gene adjuvant therapy.After the establishment of the animal models,the tumor effects of the nano-particles were tested in vivo.The infrared thermal imaging results showed that MTRPG also possessed a good photothermal conversion effect in vivo,and could be gathered at the tumor site through passive targeting through EPR effect.Compared with negative control group,the anti-tumor rate of MTRPG+808 nm NIR treatment group reached 78.39±5.24%.The results of H&E,Ki-67,TUNEL staining and lipid peroxidation level detection further uncovered the anti-tumor mechanism of MTRPG was by inhibiting cell proliferation and promoting cell apoptosis.The results of ELISA showed that MTRPG effectively down-regulated hypoxia inducible factor-1α（HIF-1α）、vascular endothelial growth factor（VEGF）and endothelin-1（ET-1）gene expression.Body weight changes,pathological analysis of the main organ sections,organ index calculation,liver and kidney function indexes further proved that the MTRPG showed ideal biological safety in vivo.In conclusion,we constructed a multifunctional nano-drug delivery system on the basis of the sequential release of GOx and the anoxic activating drug TPZ.Both in vivo and in vitro experiments confirmed that MTRPG+808 nm NIR achieved effective synergy between multi-mode of chemotherapy,hunger therapy,photothermal therapy,and gene therapy and realized ideal cancer therapy effects.This study will provide basic experimental data and new ideas for the clinical treatment of malignant tumors.