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.

Enhanced transscleral delivery using superficial ultrasound exposure and drug-loaded hydrogel.

This study employed superficial ultrasound exposure of good ocular safety and a drug-loaded hydrogel of long residence time to enable transscleral delivery. First, we designed an acoustic adaptor to limit the ultrasound exposure depth to 1.59 mm to protect the posterior eye segments. Then, we optimized the alginate/polyacrylamide ratio (3:7) of a dual-crosslinked hydrogel to enable ultrasound-triggered release of model drug (70-kDa fluorescein isothiocyanate-conjugated dextran). Using fluorescence imaging to quantify the drug release, we showed that the developed method resulted in enhanced transscleral delivery in both ex vivo porcine scleras (2.6-fold) and in vivo rabbit scleras (2.2-fold). We also demonstrated that the method increased the drug penetration depth to the whole thickness of the sclera. In particular, the drug release efficiency increased with increasing ultrasound exposure time (1 and 3 min) and intensity (8, 19, 36, and 61 mW/cm2). Using scanning electron microscopy, we revealed that ultrasound exposure resulted in rougher surfaces and microscale rupture of the hydrogel. Moreover, Masson staining of scleral slices showed that the integrity of the top scleral fibers was disturbed by ultrasound exposure, and this disturbance recovered 3 days later. Our work demonstrates that the developed method holds great potential for mediating ocular drug delivery.