Construction Of Drug-loading Nanoparticles Based On ZIF-8 Framework And The Synergistic Combining Of Sonodynamic-chemotherapy Induced Pyroptosis In Gastric Cancer

Gastric cancer（GC）is the fifth most common cancer worldwide,the second most common cancer among all cancers,and the third most common cause of cancer-related death.The therapy for GC is a comprehensive treatment based on surgery,and chemotherapy is another important treatment method.However,because of the hypoxic microenvironment in most solid tumors,the sensitivity of tumor cells to chemotherapy drugs is restricted,resulting in the effects of chemotherapy drugs being weakened.Therefore,exploring new strategies for GC treatment is very important.With the continuous progress of nanotechnology,more and more nanocarriers are used in the research of medical treatment.Zeolitic imidazolate frameworks-8（ZIF-8）is one of the most representative zeolitic imidazolate frameworks,which has a strong drug-carrying capacity,high thermal and chemical stability,good biocompatibility,p H-responsive ability,and so on.Therefore,ZIF-8 is often used to load drugs or small molecules for cancer treatment research.Sonodynamic therapy（SDT）utilizes a combination of oxygen,low-frequency ultrasound（ultrasound,US）,and a sonosensitizer to generate reactive oxygen species（ROS）,high concentrations of which can kill tumor cells because it can not be cleared by cells immediately.Chlorin e6（Ce6）is a widely used sonosensitizer,which can release ROS under the stimulation of ultrasound and kill tumor cells.At the same time,the excessive accumulation of ROS in cells will aggravate hypoxia of tumor cell,which provides a choice for the activation of hypoxia-activated prodrugs.Tirapamine（TPZ）is a hypoxia-activated prodrug that is nontoxic in normoxia but is reduced to toxic free radicals in hypoxia and can be selectively toxic to hypoxic cells,thereby inducing cell DNA breakage.Therefore,this project used ZIF-8 nanocarriers loading with Ce6 and TPZ to release ROS and aggravated the hypoxia of GC cells to activate TPZ,further enhancing the killing effects on GC cells.Part 1:Construction,characterizations,and properties of ZIF-8 drug-loading nanoparticlesObjective:To construct drug-loading nanoparticles based on ZIF-8,and to detect its characteristics and properties.Methods:ZIF-8 loaded with Chlorin e6（Ce6）and Tirapamine（TPZ）was constructed by in-situ“one-pot”in methanol solution,and ZC（ZIF-8@Ce6）,ZT（ZIF-8@TPZ）and ZTC（ZIF-8@Ce6@TPZ）were constructed.Simultaneously,to modify the surface of ZTC with homologous tumor cell membranes,then ZTC@M（ZIF-8@Ce6@TPZ@Membrane）was constructed.The structure of the nanoparticles was detected by transmission electron microscopy（TEM）,and the particle size and Zeta potential were detected by the ZS90 Zeta Sizer.Absorption spectrum detection of nanoparticles was used to verify drugs were successfully loaded.Gel electrophoresis experiments were used to confirm ZTC@M was successfully wrapped with the cell membrane.Encapsulation efficiency was analyzed to evaluate the loading ability of nanoparticles.In vitro,drug release experiments under different p H conditions were performed to verify the p H responsiveness of nanoparticles.Flow cytometry and laser confocal phagocytosis assays were used to analyze the targeting ability of nanoparticles.1,3-Diphenylisobenzofuran（DPBF）was used to analyze the level of reactive oxygen species（ROS）.The biocompatibility of nano particles was analyzed by in vitro hemolysis experiments and in vivo safety experiments.Measurement data were presented as mean±standard deviation（Mean±SD）.Student’s t-test was used to compare the differences between two groups,and one-way analysis of variance（One-Way ANOVA）was used for pairwise comparisons between multiple groups.P<0.05 was considered statistically significant.Results:Transmission electron microscope（TEM）results showed that the ZTC@M nanoparticles were of uniform three-dimensional geometry.Zeta-sizer Nano analyzed the size of the nanoparticles to be about 253.7 nm,and the Zeta potential is-16.4 m V.The characteristic absorption peaks of Ce6 and TPZ were observed simultaneously in the absorption spectrum of ZTC@M,and the encapsulation efficiencies of Ce6 and TPZ were 77.57±0.48%,53.86±3.48%,respectively.Incubation for 6 h at p H 5.5 released approximately 61.95±1.40%of loaded TPZ and 73.57±2.01%of loaded Ce6 from the ZTC@M,and these values were much higher than those obtained at p H 7.4（TPZ 25.78±1.23%,Ce622.49±1.35%）.The results of flow cell experiments and laser confocal scanning microscopy（LCSM）presented that after the addition of cytomembrane of AGS,more ZTC@M nanoparticles could be uptaken by cells.After 10 min,the remaining DPBF in the DPBF+H2O group,DPBF+ZTC@M group,and DPBF+ZTC@M+ultrasound（US）group were about 94.20%,82.79%,and40.05%,respectively.The hemolysis assay showed that the hemolysis rates of the three groups with ZTC@M concentrations of 100,200 and 300μg/m L were0.78%,0.55%and 0.34%,respectively.After being injected with ZTC@M via the tail vein for 7 days and 30 days,there were no significant differences in white blood cell count,red blood cell count,hemoglobin,platelet value,aspartate aminotransferase,alanine aminotransferase,alkaline phosphatase,and blood urea nitrogen of mice among observation group and the control group were observed.No significant pathological changes by H&E staining in the main organs（heart,liver,spleen,lung,kidney）of the mice were observed.Conclusion:In this paper,ZIF-8 co-loaded with Chlorin e6（Ce6）and Tirapamine（TPZ）was successfully constructed by in-situ"one-pot"in methanol solution.Simultaneously,homologous tumor cell membranes were used to modify the surface of nanoparticles.The nan p H-responsive ability,targeting ability,good biocompatibility,and ROS release.oparticles have the characteristics of easy construction,Part 2 The evaluation of the antitumor effect of ZIF-8 drug-loading nanoparticles combined with sonodynamic therapy and chemotherapy Objective:To investigate the therapeutic effect of ZIF-8 drug-loading nanoparticles in combination with SDT and chemotherapy（ZTC@M+US）on gastric cancer.Methods:ZC,ZT,ZTC,ZTC@M nanoparticles were added to the cells,and the SDT treatment group was stimulated by ultrasound（3 min,1.0MHz,1.5W/cm2）,and the blank cell group was used as the control group.There were6 groups:CON,ZC,ZT,ZTC,ZTC@M and ZTC@M+US groups.The intracellular ROS levels in different groups were detected by 2’,7’-dichlorodihydrofluorescein diacetate（DCFH-DA）.Image-i T^TM green hypoxia probe,cellular immune Fluorescence and Western Blot（WB）were used to detect cell hypoxia.CCK-8,Calcein/PI and flow cytometry apoptosis experiment were used to evaluate the killing effects of nanoparticles on gastric cancer cells in vitro.The subcutaneous tumor model of nude mice was constructed,the body weight of nude mice and the volume of tumors were measured,the tumor inhibition rate was calculated,and the anti-tumor effect of ZTC@M+US was analyzed in vivo.Measurement data were expressed as mean±standard deviation（Mean±SD）.Student’s t-test was used to compare differences between two groups,and one-way analysis of variance（One-Way ANOVA）was used for pairwise comparisons between multiple groups.P<0.05was considered statistically significant.Results:The results of DCFH-DA detection and hypoxia detection showed that ZTC@M generated ROS in cells after US,and the accumulation of ROS aggravated intracellular hypoxia.The cytotoxicity results of CCK-8 showed that at Ce6 2μg/m L and TPZ 12μg/m L,the cell survival rates of ZC group,ZT group,ZTC group,ZTC@M group and ZTC@M+US group were 91.3%,91.91.3%,85.9%,71.2%,40.6%,which were higher than those in Ce6 1μg/m L,TPZ 6μg/m L cell viability.The results of Calcein/PI assay showed that the red fluorescence intensity quantification of dead cells in the CON,ZC,ZT,ZTC,ZTC@M and ZTC@M+US groups were 1.31±0.37%,4.51±0.64%,3.68±0.34%,7.42±0.37%,10.27±0.99%and 32.31±2.91%,respectively（P<0.05）.The results of the flow cytometry apoptosis experiment showed that the percentages of apoptotic cells in the CON,ZC,ZT,ZTC,ZTC@M and ZTC@M+US groups were about 8.13%,17.40%,15.49%.The results showed that Sonodynamic therapy（SDT）combined with chemotherapy enhanced the killing effect on gastric cancer cells.The results of in vivo animal experiments showed that the body weight of nude mice increased in different treatment groups,indicating that the nanoparticles had no obvious toxicity to the growth of nude mice.The volume of the transplanted tumor in the US group gradually decreased.the tumor inhibition rates in the ZC,ZT,ZTC,ZTC@M and ZTC@M+US groups to Con group were 10.7%,12.5%,16.1%,61.9%and 87.2%,indicating that SDT combined with chemotherapy can significantly enhance the inhibitory ability of tumor growth.Conclusion:ZIF-8 nanoparticles combined with SDT and chemotherapy can enhance the anti-tumor effect in vivo and in vitro.Part 3 The mechanism of ZIF-8 nanoparticles for synergistic sonodynamic therapy and chemotherapy against gastric cancerObjective:To explore the mechanisms of ZTC@M+US against gastric cancer.Methods:The effect of ZTC@M+US on the invasion and migration of gastric cancer cells were analyzed by transwell and wound healing experiments.The effect on gastric cancer cell proliferation was analyzed by Ed U assay.The effect on gastric cancer cell cycle was analyzed by flow cytometry.The morphology of AGS cells was analyzed by hase-contrast microscope and SEM.The expression of pyroptosis-related proteins and epithelial-mesenchymal transition（EMT）-related proteins were analyzed by WB.Results:1.ZTC@M+US induced pyroptosis of gastric cancer cells.Phase-contrast microscope observed that after ZTC@M+US treatment,the cells became larger,rounder and swollen,and there was no synaptic extension between cells.Scanning electron microscope results showed that after ZTC@M+US treatment,AGS cells were deformed and the cell membrane was destroyed,pits and holes of different sizes appeared on the cell surface,and the cells show the characteristic morphology of pyroptosis.Further,WB results showed that compared with the CON and ZTC@M groups,the pyroptosis-related proteins cleaved-caspase-1,NLRP3,ASC,mature IL-1βand GSDMD-N were significantly increased in the ZTC@M+US group（all P<0.05）.2.ZTC@M+US inhibited invasion and migration of AGS cells by inhibiting EMT.The results of transwell cell invasion assay showed that the number of cells passing through the chamber in CON,ZC,ZT,ZTC,ZTC@M and ZTC@M+US groups were 354.67±16.22,234.33±10.44,238.33±7.11,211±6.67,155.33±9.56 and 46.00±3.33,respectively（P<0.05）.The results of transwell cell migration assay showed that the number of cells passing through the chamber in the CON,ZC,ZT,ZTC,ZTC@M and ZTC@M+US groups were 407.00±5.33,238.30±8.89,265.00±3.33,and 175.70±0.89,92.33±5.56 and 33.00±4.00,respectively（P<0.05）.Wound healing results showed that the normalized scratch areas of the ZC,ZT,ZTC,ZTC@M and ZTC@M+US groups were 1.15times,1.09 times,1.26 times,1.35 times and 1.88 times than that of the CON group,respectively（P<0.05）.The results of Ed U experiment showed that the proportions of Ed U positive cells in the CON,ZC,ZT,ZTC,ZTC@M and ZTC@M+US groups were 37.16±6.86%,30.43±0.58%,31.60±3.09%,22.95±0.87%,17.88±1.54%and 5.34±1.42%,respectively（P<0.05）.The results of transwell assay,wound healing assay,and Ed U assay showed that ZTC@M+US could significantly inhibit the invasion,migration and proliferation of AGS.Flow cytometry experiments showed that ZTC@M+US treatment arrested the cell cycle in G0/G1 phase（P<0.0001）.The expression of EMT-related protein E-cadherin was significantly increased（P<0.0001）,while the expressions of N-cadherin,Vimentin and Snail were decreased（all P<0.05）.Conclusion:1.ZIF-8 nanoparticles combined with SDT and chemotherapy can induce pyroptosis of gastric cancer via NLRP3/Caspase-1/GSDMD pathway.2.ZIF-8 nanoparticles combined with SDT and chemotherapy can inhibit the invasion and migration of gastric cancer cells by inhibiting the epithelial-mesenchymal transition of gastric cancer cells.Part 4:Exploring the potential mechanism of nanoparticles synergizing SDT and chemotherapy against gastric cancer based on RNA-sequencing Objective:To further explore the potential mechanism of ZTC@M nano-drug-loaded particles in combination with SDT and TPZ therapy on gastric cancer.Methods:Gastric cancer AGS cells were treated with ZTC@M+US（TPZ 12μg/m L,Ce6 2μg/m L）,and untreated gastric cancer cells were used as a control to extract RNA from two groups of cells for high-throughput RNA sequencing.Using bioinformatics analysis,sample principal component analysis,heat map analysis,and cluster dendrogram analysis were performed on the sequencing results.Under the conditions of FDR<0.05 and|log2FC|>1,the differential genes were screened for differential analysis and enrichment analysis.The target gene was further screened for analysis.The TCGA database was screened for genes with high expression in gastric cancer（FDR<0.05,|log2FC|>1 with clinical data）,and the differential genes with low expression after ZTC@M+US treatment in the sequencing results were screened.Under the condition of|log2FC|>2,the high-expressed genes in the TCGA database were intersected with the genes whose expression was reduced after the nano-drug-loaded particles were treated,and the obtained genes were the key genes that may play an anti-cancer effect,and q RT-PCR was used to verify the m RNA expression levels of the genes.Results:1.Analysis of sequencing results.Principal component analysis,heat map analysis,and cluster tree analysis of the sequencing results showed that there were no obvious outliers between the CON group and the ZTC@M+US group,and the repeatability between the samples was good,which can be used for subsequent analysis.Under the condition of FDR<0.05 and|log2FC|>1,a total of 3008 differential genes were screened,including 1857 up-regulated genes and 1151 down-regulated genes.The results of GO enrichment analysis showed that the differential genes were mainly involved in the negative regulation of biological processes,cell adhesion,cell cycle,cell migration,cell proliferation,cell-cell adhesion,and other processes.The results of KEGG pathway enrichment analysis showed that the differential genes may be related to TGF-βsignaling pathway,MAPK signaling pathway,TNF signaling pathway,apoptosis,ferroptosis,p53 signaling pathway,necroptosis,HIF-1αsignaling pathway,cell cycle pathway and so on to regulate the biological behavior of gastric cancer cells.2.Bioinformatics analysis of target genes.A total of 7 target genes were screened,namely:HSD3B1,VRTN,CLDN2,NPFFR1,GAL3ST2,LGR5 and FGB.q RT-PCR results confirmed that the expressions of LGR5,GAL3ST2,VRTN,FGB,CLDN2,HSD3B1,and NPFFR1 were significantly decreased in the ZTC@M+US group compared with the CON group.Conclusion:1.The differential genes screened based on the sequencing results may be involved in biological processes such as cell differentiation,cell adhesion,cell cycle,and cell migration,and may be involved in TGF-βsignaling pathway,MAPK signaling pathway,TNF signaling pathway and other signaling pathways.Influence the biological behavior of gastric cancer.2.Nano-drug-loaded particles combined with SDT and chemotherapy significantly reduced the expression of HSD3B1,VRTN,CLDN2,NPFFR1,GAL3ST2,LGR5,and FGB.