Aptamer-functionalized Exosomes Targeting Enhances Tumor Inhibition And Radiosensitivity Of Glioma In Vitro

Glioblastoma(GBM)is the deadliest brain tumor with a 5-year mortality rate of over 90%.The high mortality rate of GBM is due to various limitations in its treatment,such as high mean age of onset,tumor location,and insufficient understanding of tumor pathophysiology.At present,the commonly used methods for the treatment of GBM are surgical resection,radiotherapy and chemotherapy.Epidermal growth factor receptor(EGFR)expression is very common in GBM,and patients with tumors characterized by EGFR overexpression generally have a lower survival rate.When EGFR amplification is detected,gene signatures associated with EGFR overexpression and other clinical features of GBM have been identified.EGFRvⅢ mutation is the most common type of EGFR mutation in GBM,and both EGFR overexpression and EGFRvⅢ mutation can enhance the tumorigenicity of glioblastoma.EGFRvⅢ is capable of autophosphorylation because of its ligand-independent tyrosine kinase activity.Studies have shown that EGFRvⅢ can also enhance the DNA repair capacity of tumor cells,thereby making tumor therapy resistant to radiotherapy.Aptamers are single-stranded nucleotides screened by systematic evolution of ligands by exponential enrichent technology(SELEX),which can bind to targets with high specificity,but have disadvantages such as being difficult to pass through biological barries.Exosomes are extracellular vesicles composed of complete membrane structures,which have good biocompatibility and are natural drug delivery vehicles,but they are not targeted.By combining exosomes with aptamers,the advantages of the two can be combined and the defects of the two can be complemented,so that the purpose of specific targeting and effective treatment of glioma can be achieved.In this project,we first extracted mesenchymal stem cells from mice with C57BL/6 background.After primary isolation,culture and passage purification,we observed the morphology of the cells,and then incubated the cells after P2 generation with flow antibodies like CD29,Sca-1,CD105,CD44,CD11b,CD34,CD45,CD31,la,and CD86 to identify immunophenotype and purity by flow cytometry.After culturing the mesenchymal stem cells,we used differential centrifugation combined with membrane affinity method to extract exosomes in the cell supernatant,and identified the extracted exosomes by combining electron microscope observation,nanoparticle tracking analyzer detection,and western blot method.After obtaining the mesenchymal stem cell-derived exosomes,we modified the 5’ end of the aptamer U2 targeting U87-EGFRvⅢ cells with a cholesterol group by using the principle that cholesterol has a high affinity for membranes.Cholesterol and U2 were supplemented with an intermediate linker,and the modified aptamer U2 was incubated with the extracted native exosomes for a certain period of time at room temperature to obtain the exosome-aptamer complex(Exo-U2),we then preformed experiments on Exo-U2 in vitro on tumor inhibition,radiosensitivity,and penetration of the blood-brain barrier.First,the green fluorescent dye PKH-67-labeled Exo-U2 with Cy5 red fluoresctnce was co-incubated with human glioma cell line U87-EGFRvⅢ,the cellular uptake of Exo-U2 was observed under a confocal microscope.Then we used the CCK-8 method and EdU method to explore the effect of Exo-U2 on the proliferation ability of U87-EGFRvⅢ cells.Transwell migration experiments were conducted to explore the effect of Exo-U2 on the migration ability of U87-EGFRvⅢ cells.Western blot was used to explore the molecular mechanism of Exo-U2’s inhibitory effect on the proliferation and migration of U87-EGFRvⅢcells.Colony formation assay were used to explore the effects of Exo-U2 combined with X-Ray irradiation on U87-EGFRvⅢ cells.And western blot was used to explore the molecular mechanism of the radiosensitization of Exo-U2 on U87-EGFRvⅢ cells.At the animal level,we injected Exo-U2 with Cy5 fluorescence into the tail vein of C57BL/6 mice,and observed the distribution of Exo-U2 in mice by in vivo imaging.Results:By observing the isolated and extracted mouse combact bone-derived mesenchymal stem cells under an inverted microscope and performing immunophenotype identification by flow cytometry,it can be concluded that the cells are adherent fibroblast-like cells with high positive expression mesenchymal cells markers CD29,CD44,CD 105,hematopoietic stem cell and MSC marker Sca-1,negative expressed hematopoietic marker CD34,CD45,CDllb,endothelial cell marker CD31,cell surface costimulating molecules Ia,CD86.After observing the morphology by electron microscope,detecting particle size by nanoparticle tracking analysis(NTA)technology and detecting protein markers by western blot,it was concluded that the extraxted exosomes were vesicle-like structures with a complete membrane structure,the particle size was about 120 nm,and the exosomes expressed CD63,CD81 and TSG101.Using an inverted laser confocal microscope,it was observed that the cells not only contained a large number of Exo-U2 with both green fluorescence,but also contained a large number of Exo-U2 with both green fluorescence and red fluorescence.CCK-8 and EdU experiments showed that Exo-U2 could inhibit the proliferation of U87-EGFRvⅢ cells.Through transwell migration experiments,it was found that Exo-U2 could reduce the relative migration rate of U87-EGFRvⅢ cells.Western blot results showed that Exo-U2 could reduce the expression of EGFRvⅢ phosphorylated proteins and AKT phosphorylated proteins in U87-EGFRvⅢ cells.The above results suggest that Exo-U2 has antitumor effect in vitro.Colony formation assays showed that Exo-U2 combined with X-Ray irradiation could reduce the number of colonies formed in U87-EGFRvⅢ cells.Western blot experiments showed that Exo-U2 could reduce the expression of ATM phosphorylated protein in U87-EGFRvⅢ cells.The above results suggested that Exo-U2 could increase the radiosensitivity of gliomas.In vivo imaging showed that the Exo-U2 could enter the brain,suggesting that Exo-U2 could penetrate the brain barrier.The results show that:(1)Mouse mesenchymal stem cell-derived exosomes have beed successfully obtained in this experiment.(2)We have connected both exosomes and aptamers,and Exo-U2 can be internalized by U87-EGFRvⅢ cells.(3)Exo-U2 has antitumor effect in vitro and can inhibit the proliferation and migration of U87-EGFRvⅢ cells.The possible mechanism is to reduce the expression of EGFRvⅢ phosphorylated proteins in cells,thereby affecting the downstream signaling pathway.(4)Exo-U2 can enhance the radiosensitivity of glioma and can be developed as a radiosensitizer for glioma.(5)Exo-U2 can enter the brain,suggesting that it may have the effect of penetrating the blood-brain barrier,laying a foundation for the next step in the research on the therapeutic effect of Exo-U2 in vivo.