Cell Membrane-coated Oncolytic Adenovirus Targeted Therapy For Glioblastoma

Glioblastoma(GBM),one of the most malignant tumors,is characterized by infiltrative growth and short median survival.Gliomas are mostly intracranial in origin,and the presence of the blood-brain barrier(BBB)has greatly limited the traditional treatments for gliomas.The mature traditional treatment is a multimodal combination of surgical resection,supplemented by radiotherapy and chemotherapy to reduce the probability of tumor recurrence,but the prognosis is poor and there is currently no cure for glioma.Emerging biologic therapies,represented by lysing adenovirus,offer hope for a cure of glioma.However,tumor treatment by intratumoral injection of lysing adenovirus alone has limitations for hard-to-reach malignant tumors such as glioma due to the problems of preexisting immunity and insufficient targeting.Biofilms,as natural components of cells,have better biocompatibility than artificial materials and have been widely used in recent years for the study of nano-drug delivery systems.Cell membranes of different origins have different targeting effects,and neural stem cell membranes and tumor cell membranes have been shown to have the ability to penetrate the blood-brain barrier and tumor-targeting properties.Therefore,we propose the following conjectures:(1)lysoadenovirus is modified by cell membranes so that the viral particles are completely obscured by the membrane structure to prevent lysoadenovirus from being cleared by the body’s antiviral immunity during blood delivery;(2)lysoadenovirus modified by cell membranes can cross the blood-brain barrier and can target glioma cells.Based on the above conjectures,a cell membrane-modified lysing adenovirus(MAd)was successfully prepared by co-extrusion of neural stem cells,glioma cell membranes and lysing adenovirus.The modified viruses were identified by transmission electron microscopy,particle size potential analysis,SDS-PAGE,Dot Blot and ELISA.These results demonstrated that the physical features of the viruses were covered by the structural features of the cell membrane after cell membrane modification,and the membrane structure was not significantly disrupted,and more viruses were stably encapsulated by the membrane.After the identification was completed,the infectivity of M-Ad in normal and glioma cells was analyzed by flow cytometry and immunofluorescence.The results showed that both M-Ad were able to enhance the infectivity of glioma cells and human brain microvascular endothelial cells(h CMEC/D3),while decreasing the infectivity of HEK293 cells.Using a human glioma cell line(U87)to establish an intracranial tumorbearing mouse model,in vivo imaging analysis revealed that both M-Ad were able to better infect tumor cells and express red fluorescent protein by intratumoral injection compared to unmodified tumor lysing adenovirus.After intravenous injection,the neural stem cell membrane-modified viruses were able to better penetrate the bloodbrain barrier and infect tumor cells.Later,the paper analyzed the ability of M-Ad to escape preexisting immunity.MAd and unmodified lysoadenovirus were incubated with sera having different concentrations of adenovirus-neutralizing antibodies and infected HEK293 cells,and it was found that M-Ad was able to infect cells to some extent in the presence of neutralizing antibodies;by injecting unmodified lysoadenovirus and M-Ad in normal mice and model mice with preexisting immunity,respectively,it was found that unmodified lysoadenovirus was only able to infect cells in normal The unmodified lysing adenovirus was only observed in normal mice,while M-Ad was still detected in the model mice with preexisting immunity,further demonstrating the ability of M-Ad to escape from preexisting immunity.Finally,in order to enhance the tumor suppression effect we encapsulated the lysing adenovirus carrying TRAIL gene constructed in the laboratory before and evaluated its tumor suppression effect in vitro and in vivo.Cells were killed in two glioma cell lines using different concentrations of M-Ad,and the results of the CCK-8assay demonstrated that M-Ad had a better tumor-killing effect than unmodified lysing adenovirus.Based on the results of in vitro experiments,we evaluated the tumor suppressive effect of M-Ad in the U87 mouse in situ tumor model.The results showed that the neural stem cell-modified M-Ad could more effectively inhibit tumor cell growth and prolong the survival of mice.The results of H&E staining and immunofluorescence analysis demonstrated that M-Ad could promote apoptosis of glioma cells without any effect on normal tissues.In conclusion,the main objective of this thesis was to modify the lysing adenovirus using tumor cell and neural stem cell-derived biofilms to further enhance its targeting and killing effect on glioma cells.It was demonstrated that both cell-derived membrane structures could enhance the ability of lysoadenovirus to target and infect glioma cells while effectively evading preexisting immunity,and that the neural stem cell membrane modified lysoadenovirus could better penetrate the blood-brain barrier to target and kill glioma cells.This study provides a new idea and strategy for the targeted modification of lysoadenovirus and glioma treatment.