School of Biomedical Engineering, Shenzhen University Medical School, Shenzhen University, Shenzhen 518055, Guangdong, China; National-regional Key Technology Engineering Laboratory for Medical Ultrasound, Shenzhen University, Shenzhen 518055, Guangdong, China.
School of Basic Medical Sciences, Shenzhen University Medical School, Shenzhen University, Shenzhen 518055, Guangdong, China.
Ultrason Sonochem. 2023 Dec;101:106685. doi: 10.1016/j.ultsonch.2023.106685. Epub 2023 Nov 7.
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.
作为一种非病毒转染方法，超声和微泡诱导的声孔可以实现具有协同免疫刺激作用的空间靶向基因传递。在这里，我们首次报告了声孔法在提高 DNA 疫苗性能方面的应用。本研究开发了一种新型微泡设计，将纳米级 DNA/PEI 复合物负载在阳离子微泡上，从而显著提高 DNA 装载能力（每个微泡 0.25pg）和体外转染效率。通过活细胞成像，我们揭示了声孔的膜穿孔和细胞递药特性。使用荧光素酶报告基因进行体内转染，我们发现与肌肉内注射相比，声孔可将转染效率提高 40.9 倍。此外，我们全面优化了声孔方案，进一步将转染效率提高了 43.6 倍。免疫荧光染色结果表明，声孔有效地激活了 MHC-II 免疫细胞。使用乙型肝炎 DNA 疫苗，与肌肉内注射相比，声孔诱导的血清抗体水平显著升高，并且抗体持续 56 周。此外，我们记录了基因传递的最长报道表达期（400 天）。全基因组重测序证实，具有稳定表达的基因以染色体外状态存在，没有整合。我们的结果表明，声孔法在高效和安全的 DNA 疫苗接种方面具有潜力。
