Study On The Efficacy Of The Nano-immobilized Enzyme Synthesized By Biomimetic Mineralization For Synergistic Treatment Of Lung Cancer

BackgroundLung cancer(LC)is one of the most common malignant tumors in China.Its incidence ranks the first in male malignant tumors and the second in female malignant tumors,respectively,and its mortality ranks the first in both male and female malignant tumors.The 5-year relative survival rate is 18%.Histologically,non-small cell lung cancer(NSCLC)accounts for ahout 85%of all lung cancers.Due to the atypical early symptoms and limited examination methods,most patients are already in the advanced atage of cancer when they are diagnosed,and have lost the opportunity of radical surgery.Therefore,chemotherapy-based combination therapy is the main treatment method for advanced NSCLC.Traditional chemotherapy has poor targeting,high toxicity and side effects,and is prone to drug resistance,which not only greatly increases the difficulty of treatment,but also brings great pain to patients.Therefore,finding a new,effective and safe treatment method is the primary task in the field of malignant tumor treatment,including lung cancer.Nanoenzyme is a new type of mimetic enzyme,which not only has the efficient catalytic activity of traditional enzymes but also has the unique physical and chemical characteristics of nanomaterials.In this study,we incorporated glucose oxidase(GOx)and palladium(Pd)cubic nanoenzymes into zeolite imidazolate skeleton 8(ZIF-8)through biomimetic mineralization to form an effective ROS generator(GOx@Pd@ZIF-8).This generator exerts synergistic anticancer activity by blocking intracellular glucose metabolism and promoting ROS production.The study provides a novel anti-tumor strategy and shows promise for the treatment of solid cancers.ObjectivesThe biomimetic mineralization method was used to prepare nano-immobilized enzyme(GOx@Pd@ZIF-8),and its anti-tumor activity was systematacially evaluated through in vivo and in vitro experimental,aiming to provide a new strategy for the treatment of lung cancer.Methods1.The nano-immobilized enzyme was constructed by biomimetic mineralization,and the material properties were determined and characterized by scanning electron microscopy(SEM),transmission electron microscopy(TEM),energy dispersive spectrometry(EDS),inductively coupled plasma mass spectrometer(ICP-MS)and PANalytical B.V.Empyrean powder diffraction and hemolysis experiments.2.CCK8 assay,apoptosis and cycle assay,and cloning assay were used to verify the effects of nano-immobilized enzyme on the proliferation and apoptosis of lung cancer A549 cells;Migration experiments were carried out to verify the effects of nano-immobilized enzyme on the invasion ability of cells.ROS detection and endocytosis test were used to verify the action sites and related mechanism of the nano-immobilized enzyme3.BALB/c nude mice were treated by intratumoral injection of nano-immobilized enzyme to verify the anti-tumor properties and main tissue toxicity of nano-immobilized enzyme in vivo.4.The whole transcriptome sequencing technology(RNAseq)was used to detect and analyze the molecular mechanism of the action of nano-immobilized enzyme.Result1.System characterization.The nano-immobilized enzyme was successfully constructed.The SEM and TEM images showed that the nanometer bienzyme had irregular spherical shape.SEM results show that the average particle size is about 130 nm.PXRD showed that the structural integrity of ZIF-8 framework was well preserved in the presence of GOx and Pd nanosomes.The EDS and GOx@Pd@ZIF-8 profiles revealed the presence of Pd signaling in the complex,which confirmed the Pd nanoenzyme load in ZIF-8.The absorbance at 387 nm indicates that the load efficiency of GOx in ZIF-8 is close to 100%.By ICP-MS,the contents of Pd and Zn in GOx@Pd@ZIF-8 were 3.591 wt%and 18.996 wt%,respectively.The hemolysis experiment showed that the hemolysis rate was only 8%at 200μg/L.2.In vitro experiments.i)The relative viability of A549 cells treated with ZIF-8,Pd@ZIF-8,Gox@ZIF-8 and Gox@Pd@ZIF-8 at 110 μg/L for 24h was 96.8%,88.1%,64.2%and 5.6%,respectively.ii)The apoptosis rates of A549 cells were 8.09%,8.33%,8.81%and 18.26%after 24h treatment,and the induction of apoptosis was more obvious after 48h treatment.In live/death analysis,a stronger red fluorescence signal was detected in the GOX@Pd@ZIF-8 group compared to the control group.iii)The cell cycle detection showed that GOX@Pd@ZIF-8 could induce S phase arrest and realize S to G2 phase arrest transition over time.iv)The results of the clone formation assay showed that GOx@Pd@ZIF-8 significantly inhibited colony formation compared with ZIF-8 alone and the single enzyme group,with a formation rate of only 36.4%.v)After 6 hours of treatment,more FITC fluorescence signals can be detected in the cytoplasm of A549 cells;ROS detection experiments found that the GOx@Pd@ZIF-8 group showed stronger green fluorescence signals after 24 hours of treatment.3.In vivo experiments.i)Intratumoral injection of drugs does not affect the survival status of tumor-bearing mice,and the body weight is slightly increased than before;ii)The tumor volume of mice in the GOx@Pd@ZIF-8 group was significantly smaller than that of the other groups,and the difference was statistically significant(P<0.0189);iii)The Tunel staining fluorescence signal of GOx@Pd@ZIF-8 mouse tissue is stronger,and Ki67 expression is lower;iv)The cells in the HE staining of major tissues(heart,liver,spleen,lung,and kidney)showed normal tissue morphology,membrane interaction and nuclear morphology.4.RNAseq analysis.Most of the genes that map to GO items such as chromatin,adhesion junction,local adhesion,anchor junction and cell-substrate adhesion junction were down-regulated.KEGG analysis found that NF-kB was activated and the cell cycle was inhibited.ConclusionIn this study,a feasible and effective ROS generator(GOx@Pd@ZIF-8)was successfully constructed.The system characterization showed that the composite has the characteristics of irregular spherical structure,small particle size,complete structure,and high loading rate.A series of in vitro and in vivo experiments show that GOx@Pd@ZIF-8 can exert synergistic anti-tumor activity,and effectively inhibit the proliferation and invasion of A549 cells through intracellular ROS,and induce their apoptosis,but does not affect the main tissue functions.In addition,RNA-seq detection technology reveals the potential mechanism of the anti-tumor effect of the active molecule ROS at the molecular level,which is feasible and innovative.The current research provides an excellent,feasible and clinically applicable strategy for a safe and effective anti-tumor drug delivery system.