Rational Design And Antitumor Study Of Multifunctional Nano-drug Delivery System Based On ZIF-8 Mental-organic Framework

Lung cancer is one of the malignant tumors with the highest mortality rate in the world,and non-small cell lung cancer(NSCLC)is the most common type of lung cancer with the five-year survival rate of patients is less than 20%.Epidermal growth factor receptor(EGFR)tyrosine kinase inhibitor gefitinib has been used to treat NSCLC,but the efficacy of gefitinib has been compromised by the development of drug resistance.Among various emerging cancer therapies,small interfering RNA(siRNA)has emerged as a powerful tool for suppressing gene expression in gene therapy,which could silence the unusual therapeutic targets specifically.Although siRNA has shown great potential in the treatment of various difficult diseases,the lack of efficient and specific delivery methods is the main obstacle for the clinical treatment of siRNA.Metal-organic frameworks(MOFs)are porous nanomaterials which have large pore volume and high specific surface area to achieve high drug loading efficiency.At the same time,zeolite imidazolate framework-8(ZIF-8)is a MOF material with high biocompatibility and stability under normal physiological conditions,but could be degraded in the acidic environment of tumors.Therefore,it can be used as a nanocarrier for the delivery and responsive release of antitumor drugs.A multifunctional nano-drug delivery system based on ZIF-8 organometallic framework was constructed in this work.The EGFR inhibitor gefitinib and siRNA were loaded by in situ encapsulation.The surface of nanoparticles was modified with targeting EGFR aptamer through electrostatic adsorption,lead to the tumor-targeting effect of Apt/(siRNA+GEF)@ZIF-8 nanoparticles.A series of characterization work was carried out on the synthesized Apt/(siRNA+GEF)@ZIF-8 nanoparticles to investigate the morphology,particle size distribution,Zeta potential,element distribution,thermal stability and nuclease stability of the nanoparticles.The results of transmission electron microscopy(TEM)showed that the nanoparticles exhibited a regular octahedral shape with a uniform particle size of around 75 nm,which meets the requirements of EPR effect.The X-ray diffraction and thermal stability experiments also demonstrated that the nanoparticles had good crystalline structure and thermal stability.The loading efficiency of gefitinib and siRNA in Apt/(siRNA+GEF)@ZIF-8 nanoparticles was measured by UV spectrophotometry and fluorescence spectrophotometry with the loading efficiency was(40±3.72)%,and the encapsulation efficiency of EGFR siRNA was about(73±2.23)%.In vitro release experiments demonstrated that more gefitinib was released from the nanoparticles in PBS at p H 5.5 compared to PBS at p H 7.4 over the same time period,validating the p H-responsive release ability of the nanoparticles.In addition,agarose gel electrophoresis experiments verified that nanoparticles could effectively protect EGFR siRNA from nuclease degradation.In vitro cellular uptake experiments demonstrated that Apt/(siRNA+GEF)@ZIF-8 nanoparticles could be effectively internalized by A549 cells.The experimental results of MTT showed that the tumor inhibitory efficiency of Apt/(siRNA+GEF)@ZIF-8 nanoparticles on A549 and PC-9 cells was higher than that of gefitinib at the same concentration.The generation of reactive oxygen species(ROS)demonstrated that the high toxicity of nanoparticles against tumor cells could be attributed to the increased of intracellular ROS.The cytotoxicity of the nanoparticles was further investigated and the results of JC-1 staining,AO staining and live-dead staining experiments demonstrated that the Apt/(siRNA+GEF)@ZIF-8nanoparticles had a stronger ability to induce apoptosis in A549 cells compared to equivalent concentrations of gefitinib.Cell cycle apoptosis assay illustrated that the proportion of early apoptotic cells and late apoptotic cells in the Apt/(siRNA+GEF)@ZIF-8 nanoparticle administration group reached 21.58%.And the cells in G0/G1 phase increased to 81.89%.The results of fluorescent quantitative PCR experiments and western blot experiments showed that Apt/(siRNA+GEF)@ZIF-8 nanoparticles could effectively inhibit the expression of EGFR gene in A549 and PC-9 cells,as well as the expression of EGFR protein and its downstream signaling pathway ERK protein,which finally led to the apoptosis of tumor cells.The high biocompatibility and low toxicity of Apt/(siRNA+GEF)@ZIF-8nanoparticles were demonstrated by hemolysis assays and in vivo toxicity experiment,which meet the requirements of further nude mouse experiments.In vivo imaging experiments verified that the nanoparticles could effectively protected EGFR siRNA from nuclease degradation,while significantly prolonging the circulation time of the formulation and increasing drug accumulation at the tumor site.The in vivo anti-tumor ability of Apt/(siRNA+GEF)@ZIF-8 nanoparticles was investigated by establishing A549 xenograft tumor model.The experimental results showed that Apt/(siRNA+GEF)@ZIF-8 nanoparticles could inhibit tumor growth to the greatest extent,and exhibited the strongest ability to promote tumor cell apoptosis.The PCR analysis of tumor tissue also verified that Apt/(siRNA+GEF)@ZIF-8nanoparticles could inhibit the expression of EGFR gene in tumor tissue,thereby inhibiting tumor growth.In summary,we have constructed a nano-delivery system to achieve the targeted co-delivery of gefitinib and EGFR siRNA based on ZIF-8.A series of characterization works were performed on the synthesized nanoparticles,and the biosafety,in vivo and in vitro anti-tumor activity were also initially analyzed and discussed.The experimental results showed that Apt/(siRNA+GEF)@ZIF-8 nanoparticles were potential multifunctional drug delivery system,which was expected to overcome the drug resistance problem of gefitinib and achieve safe and efficient anti-tumor therapy.