Enhancement of ultrasound-mediated gene transfection by membrane modification

Background: Ultrasound-mediated gene transfection (USMGT) with an echo contrast agent could be a new promising physical method of triggering localized gene delivery, but the effect is still modest. The aim of this study is to devise a method to improve efficiency of USMGT. We examined the effect of lidocaine and different temperatures on USMGT, each of which is a known membrane modifier, since the plasma membrane can be considered a site of action in USMGT. Methods: We observed the effect of lidocaine (0.01, 0.1 or 1.0 mM) and different temperatures (7, 20, 37, 42 or 44°C) on USMGT (1 MHz, 3.6 W/cm² (I_SATA) and 20 s exposure) in the presence of Levovist (10 mg/ml). At 20 h after sonication, transfection efficiency was evaluated by luciferase assay. Membrane fluidity was examined by fluorescence polarization measurement. Cavitational activity was measured by ESR spin trapping with 5,5-dimethyl-1-pyrroline N-oxide. The number of cells transfected with the GFP gene was counted under a fluorescence microscope. Results: Lidocaine (1 mM) and heat (42-44°c) significantly increased luciferase expression approximately 18-fold and 19-fold higher than Levovist only. Both treatments were shown to increase membrane fluidity; in addition, heat enhanced a cavitational effect. It was confirmed by an experiment using the GFP gene that increase in luciferase expression was due to the increase in number of cells. Conclusions: This enhancement could be useful for ultrasound-mediated gene therapy in the future since both treatments for membrane modification could be directly applied to the living body.