553. Development of Echo-Contrast Gas Entrapping Liposome as Gene and siRNA Delivery Tool
Authors: Yoichi Negishi, Tetsuya Fukuyama, Yoko Endo, Ryo Suzuki, Kumiko Tanaka, Kaori Sawamura, Tomoko Takizawa, Kazuo Maruyama, Yukihiko Aramaki
Journal: Molecular Therapy (2006)

Background: Recently, ultrasound (US) has emerged as a unique method for the delivery of genes into cells and tissues, thus placing it as promising non-viral approach for gene delivery in the clinical settings. Echo-contrast gas entrapping microbubbles and US have been investigated with a view to improving the transfection efficiency of non-viral delivery systems for gene by cavitation. However, conventional microbubbles (Levovist, Optison, etc.) had some problems in stability and targeting ability. To improve these problems, we have previously developed the polyethyleneglycol (PEG)-modified liposomes entrapping echo-contrast gas known as US imaging gas. We have called the liposomes "Bubble liposomes". Objects: To validate the ability of gene or siRNA delivery into culture cells by cavitation following the eruption of "Bubble liposomes" under US exposure. Materials and methods: A solution of bubble liposome entrapping with echo-contrast gas and luciferase expressing plasmid DNA (pCMV-GL3) or GL3Luc siRNA for luciferase was added into COS7 cells in vitro. Immediately, US irradiation was performed with Sonoporation System (KTAC-3000, Nepagene Co., Ltd.) at various US intensity and insonication duration (frequency: 2 MHz, intensity: 0.1-2.5 W/cm2, irradiation time: 10 s). Results: Two days after the transfection, the highly luciferase activity, 109 RLU/mg protein, was observed in COS7 cells in vitro without remarkable cytotoxicity. This gene expression could be achieved by the US irradiation for only 10 s. The US-mediated gene transfection efficiency with Bubble liposome was higher than that of Optison. In the intracellular delivery of siRNA, the expression level of luciferase in the GL3 Luc siRNA group was much lower than the negative siRNA group, when pCMV-GL3 and the GL3 Luc siRNA were co-transfected by bubble liposome utilized with US irradiation. Conclusions: Our experiments demonstrated that "Bubble liposomes" could cause cavitation with US irradiation, and subsequently deliver plasmid DNA into the cells as well as siRNA without time-consuming preparation of gene transfer. Thus, "Bubble liposomes" may be a very safe and efficient gene delivery tool not only for gene expression, but also for the delivery of siRNA. In vivo study is still underway, but we consider that this "Bubble liposomes" could be a feasible tool in the clinical field.Acknowledgements: This study was supported by Industrial Technology Research Grand Program (04A05010) in '04 from NEDO of Japan. Background: Recently, ultrasound (US) has emerged as a unique method for the delivery of genes into cells and tissues, thus placing it as promising non-viral approach for gene delivery in the clinical settings. Echo-contrast gas entrapping microbubbles and US have been investigated with a view to improving the transfection efficiency of non-viral delivery systems for gene by cavitation. However, conventional microbubbles (Levovist, Optison, etc.) had some problems in stability and targeting ability. To improve these problems, we have previously developed the polyethyleneglycol (PEG)-modified liposomes entrapping echo-contrast gas known as US imaging gas. We have called the liposomes "Bubble liposomes". Objects: To validate the ability of gene or siRNA delivery into culture cells by cavitation following the eruption of "Bubble liposomes" under US exposure. Materials and methods: A solution of bubble liposome entrapping with echo-contrast gas and luciferase expressing plasmid DNA (pCMV-GL3) or GL3Luc siRNA for luciferase was added into COS7 cells in vitro. Immediately, US irradiation was performed with Sonoporation System (KTAC-3000, Nepagene Co., Ltd.) at various US intensity and insonication duration (frequency: 2 MHz, intensity: 0.1-2.5 W/cm2, irradiation time: 10 s). Results: Two days after the transfection, the highly luciferase activity, 109 RLU/mg protein, was observed in COS7 cells in vitro without remarkable cytotoxicity. This gene expression could be achieved by the US irradiation for only 10 s. The US-mediated gene transfection efficiency with Bubble liposome was higher than that of Optison. In the intracellular delivery of siRNA, the expression level of luciferase in the GL3 Luc siRNA group was much lower than the negative siRNA group, when pCMV-GL3 and the GL3 Luc siRNA were co-transfected by bubble liposome utilized with US irradiation. Conclusions: Our experiments demonstrated that "Bubble liposomes" could cause cavitation with US irradiation, and subsequently deliver plasmid DNA into the cells as well as siRNA without time-consuming preparation of gene transfer. Thus, "Bubble liposomes" may be a very safe and efficient gene delivery tool not only for gene expression, but also for the delivery of siRNA. In vivo study is still underway, but we consider that this "Bubble liposomes" could be a feasible tool in the clinical field. Acknowledgements: This study was supported by Industrial Technology Research Grand Program (04A05010) in '04 from NEDO of Japan.