Gene Transfection Mediated By Sonoporation And Its Immunization Application Against Hepatitis B

Compared with the traditional recombinant protein vaccine,the gene vaccine known as "third generation" vaccine can be developed and produced quickly within weeks after the gene sequence of the virus antigen protein has been published,and its highly simulated intracellular antigen expression mode of virus infection can induce humoral immunity and cellular immunity to achieve virus prevention and treatment.Among gene vaccine,DNA vaccines predominate in storage stability.However,the clinical transformation of DNA vaccines still faces the technical challenges of low gene transfection efficiency and low immunogenicity.Therefore,a novel technique for DNA vaccine in vivo transfection and immunity using ultrasound combined with cationic microbubbles-mediated sonoporation was proposed.Considering that ultrasound and microbubbles are the key to control the injury energy of cellular sonoporation.In this paper,the acoustic pressure parameters(0.13,0.28,0.43 and 0.58 MPa)and the charge properties(positive and neutral)of microbubbles were optimized based on the transfection efficiency of GFP reporter in vitro cell experiments.Subsequently,luciferase reporter gene was further transfected in vivo in the anterior tibial muscle of the posterior leg of mice.The expression of luciferase at different time points under different sound pressure parameters was imaged and quantitatively measured in a Living Images system.Lastly,we synthesized hepatitis B DNA vaccine based on the gene sequence of hepatitis B virus surface antigen HBsAg,and tested the feasibility of sonoporation mediated hepatitis B DNA vaccine to induce antibody production and immune cell activation in vivo.The results of this study showed that the transfection efficiency of green fluorescent protein reporter gene increased as ultrasonic acoustic pressure parameters increased,and the gene transfection effect of cationic microbubbles was better than that of neutral microbubbles.It was found that the cationic polymer PEI compressed gene plasmid and their cell transfection efficiency were superior to that of uncompressed plasmid.The expression efficiency of luciferase gene in vivo also increased with the increase of ultrasonic pressure.As the studies of the temporal distribution of gene expression in vivo indicated,luciferase expression in mice peaked on the 4th day and then decreased slowly to some extent.The expression of luciferase protein was detected during the observation period of 30 days,providing evidence for the expression of long-term and efficient expression of sonoporation-induced gene vaccine in vivo.The sonoporation technique successfully induced an immune response in some mice after delivery of hepatitis B DNA vaccine plasmid.HBsAg antibody production was detected in serum,indicating the feasibility of sonoporation-mediated DNA vaccine immunity.