Study On Ultrasound Combined With Biological Gas Vesicles Enhancing E-cadherin Gene Transfection In The Inhibition Of Invasion And Metastasis Of Glioma

Background and purposeGlioma is one of the most common malignant tumors in central nervous system.At present,surgical resection is the main therapy method,supplemented by radiotherapy and chemotherapy.However,the aggressive growth characteristics of glioma make it difficult to treat.Studies have shown that the aggressive behavior of glioma is closely related to EMT(epithelial-mesenchymal transition,EMT),and E-cadherin encoded by the CDH1 gene plays a key role in the process of EMT.In recent years,gene therapy has opened up a new way to conquer cancer.Among them,ultrasound microbubble-mediated gene transfection system has unique advantages such as safety,non-invasiveness and targeted release,which shows great application prospects in the field of gene therapy.This study is mainly based on the following four parts:(1)Construction of acoustic field-based conformal transfection system(AFCT);(2)Synthesis and characterization of a novel type of Halo(Halobacterium NRC-1,Halo)biological GVs(Gas Vesicles,GVs);(3)In vitro experiments on ultrasound combined with biological GVs enhancing E-cadherin gene transfection in the inhibition of invasion and metastasis of glioma;(4)In vivo experiments on ultrasound combined with biological GVs enhancing E-cadherin gene transfection in the inhibition of invasion and metastasis of glioma.Materials and methodsIn this study,we set up an AFCT system,optimized acoustic parameters,and explored its universality in different materials,different reporter gene fluorescent proteins and different cells.We extracted biological GVs from Halo by lysis and centrifugation.Then,we characterized their basic physical and chemical properties such as morphology,particle size distribution and surface charge properties;we evaluated their stability,ultrasound imaging effect,acoustic responsiveness,and cytotoxicity.In vitro studies have used ultrasound combined with GVs to produce cavitation effects to promote E-cadherin gene transfection.Western Blot experiment was used to detect the expression of molecular markers related to epithelial cells and mesenchymal cells of EMT.The wound healing test and the Transwell test were used to explore the effect of ultrasound combined with biological GVs enhancing E-cadherin gene transfection in the invasion and metastasis behavior of glioma.In vivo studies were conducted by constructing orthotopic glioma mice model to further observe the survival period,HE staining and immunofluorescence staining to evaluate the effect of E-cadherin expression on the survival,invasion and metastasis of glioma mice.ResultsTargeting the current low efficiency of ultrasound-mediated gene transfection,this study independently set up an AFCT system,which can significantly improve the efficiency of gene transfection.The biological GVs have the characteristics of small and uniform particle size(247.7 ±4.2nm),high stability,good biocompatibility and excellent acoustic response.Compared with the control group and the GP/E-cadherin group,the ultrasound combined with biological GVs gene transfection group(GP/E-cadherin+US)has the highest efficiency.Among them,the molecular markers of epithelial cells Ecadherin were significantly up-regulated,and the molecular markers of mesenchymal cells N-cadherin,Snail1 and Smad2 were significantly down-regulated.The results showed that the process of EMT was reversed.Most importantly,the GP/E-cadherin+US group can significantly inhibit the malignant biological behavior of glioma cell invasion and metastasis after up-regulating E-cadherin,and prolong the survival time of glioma mice(p<0.05).ConclusionsHalo biological GVs are ideal gene vector.Ultrasound combined with biological GVs transfection system can improve the efficiency of gene transfection.Ultrasound combined with biological GVs promoted the up-regulation of epithelial cell molecular marker E-cadherin,and the down-regulation of mesenchymal cell molecular markers Ncadherin,Snail1 and Smad2,which reverses the process of EMT.Furthermore,it can inhibit the invasion and metastasis of glioma,prolong the survival time and improve the prognosis of glioma mice.This study provides a new potential strategy for gliomas.