Design Of Membrane Fusogenic Liposomes For Rolling Circle Amplification Induced Gene Silicing In Live Cells

DNAzyme has attracted tremendous attentions due to its good selectivity,high catalytic efficiency and low immunogenicity.It can specifically recognize and cut target RNA,and thus regulate gene expression.DNAzyme nanoflowers,which contain repetitive sequence of DNAzyme produced by Rolling Circle Amplification(RCA),have been proved to be uptaken by cells and subsequently catalytically cleave target genes.However,the compact structure of DNAzyme nanoflowers impares the spatial structure of DNAzyme and thus limit their intracellular activity.In this thesis,a membrane fusogenic liposome was designed to deliver the RCA reactants to live MCF-7 cells with retained activities,enable the intracellular RCA reaction,and then induce EGR-1 gene siliencing by repeative DNAzyme sequence in RCA products.In the second chapter,the intracellular RCA reaction was determined by electroporation.Firstly,the effective occurrence of RCA reaction was characterized by fluorescence probe.Then,the RCA reactants was delivered to the cancer cells under the electroporation condition of 240 V,20 ms.The interaction between ss DNA synthesized by RCA reaction and DNA fluorescence probe releases fluorescence,which proves the effective occurrence of intracellular RCA reaction.In the third chapter,fusogenic liposomes were successfully prepared and characterized.The encapsulation efficiency of fusogenic liposomes on protein,DNA,and fluorescent dyes was also investigated.Then the interaction of fusogenic liposomes with cell membranes was investigated using super resolution imaging.It proved that fusogenic liposomes can achieve cytoplasmic delivery of encapsulated substances through fusion with the cell membrane.In the fourth chapter,fusogenic liposomes were used to deliver RCA reactants to the cytoplasm to induce the intracellular RCA reactions and the repeative EGR-1DNAzyme sequence was synthesized in RCA products.The gene silencing efficiency was improved about 20% compared with the control group,and the effect of gene silencing was further confirmed by immunofluorescence.In conclusion,a novel fusogenic liposome was designed to deliver the RCA reactants to live cancer cells with retained activity,enable the intracellular RCA reaction,and result in target gene silencing.The highlight of this system is to induce RCA reaction in live cells to achieve gene silencing by using fusogenic liposomes.By adjusting the sequence of DNAzyme,this method can be applied to gene therapy of various cancers,greatly broadening the application of RCA reaction.