Effect of ultrasonic parameters on gene transfection efficiency and cell viability of the multifunctional microbubble in vitro

Studies have reported that microbubbles combined with focused ultrasound (FUS) can increase the membrane permeability of tumor cells by triggering membrane perforation (sonoporation) to improve brain tumor-targeted gene delivery. However, few studies focused on how to increase the efficiency of gene delivery to brain tumor cells with minor cell damage. The aim of this in vitro study was to find optimal parameters for efficient gene transfer into glioma cells using the VEGFR2-targeted cationic microbubbles gene vector (MB) for future brain tumor-targeted gene delivery. Transfection efficiency and cell viability were used to determine the optimal transfection parameters. Various MB concentrations (2 × 107 to 16 × 107/mL), post-transfection incubation times (1–3 days), acoustic pressures (0–1400 kPa), duty cycles (0.5–5%), pulse repetition frequencies (PRF) (0.5–2000 Hz) and cycle numbers (25–10000) were investigated. The results showed that the MB concentration of 4 × 107 MB/ml and the incubation time of 2 days were optimal conditions for maximum gene transfection efficiency and minimum cell damage. In addition, gene transfection efficiency and cell viability depended strongly on the acoustic exposure conditions. The optimal FUS parameters include an acoustic pressure of 700 kPa, a duty cycle of 5%, a PRF of 5Hz, and a cycle number of 10000. These findings support those MB concentrations and FUS parameters of the MB-mediated gene delivery system influence gene transfer efficiency and cell viability. They also describe our novel development of multifunctional MB for FUS-triggered gene delivery in brain tumor cells.