Improving DNA vaccination performance through a new microbubble design and an optimized sonoporation protocol

As a non-viral transfection method, ultrasound and microbubble-induced sonoporation can achieve spatially targeted gene delivery with synergistic immunostimulatory effects. Here, we report for the first time the application of sonoporation for improving DNA vaccination performance. This study developed a new microbubble design with nanoscale DNA/PEI complexes loaded onto cationic microbubbles to attain significant increases in DNA-loading capacity (0.25 pg per microbubble) and in vitro transfection efficiency. Using live-cell imaging, we revealed the membrane perforation and cellular delivery characteristics of sonoporation. Using luciferase reporter gene for in vivo transfection, we showed that sonoporation increased the transfection efficiency by 40.9-fold when compared with intramuscular injection. Moreover, we comprehensively optimized the sonoporation protocol and further increased the transfection efficiency by 43.6-fold. Immunofluorescent staining results showed that sonoporation effectively activated the MHC-II⁺ immune cells. Using a hepatitis B DNA vaccine, sonoporation induced significantly higher serum antibody levels when compared with intramuscular injection, and the antibodies sustained for 56 weeks. In addition, we recorded the longest reported expression period (400 days) of the sonoporation-delivered gene. Whole genome resequencing confirmed that the gene with stable expression existed in an extrachromosomal state without integration. Our results demonstrated the potential of sonoporation for efficient and safe DNA vaccination.