695. Low-Frequency Ultrasound Increases Non- Viral Lung Gene Transfer
Authors: Stefania Xenariou, Hai-Dong Liang, Uta Griesenbach, Jie Zhu, Raymond Farley, Lucinda Somerton, Charanjit Singh, Peter K. Jeffery, Ronald K. Scheule, Seng H. Cheng, Duncan M. Geddes, Martin Blomley, Eric W.F.W. Alton
Journal: Molecular Therapy (2006)

We have previously shown that high-frequency (1 MHz) ultrasound (U/S) increases naked plasmid DNA (pDNA) gene transfer in the mouse lung and that this effect requires microbubbles (Optison) to be present in the formulation. Here, we assessed low-frequency (30–35 kHz) U/S, in an attempt to further increase non- viral gene delivery to the lung in vivo. The potential advantages of lowering U/S frequency include: a) increased cavitation, the mechanism responsible for the formation of transient pores on cell membranes and b) enhanced penetration of U/S energy into the lung, by reducing wave scattering and heat losses. Cationic lipid GL67 (GL67)/pDNA (80 μg/100 μl), polyethylenimine (PEI)/pDNA (20 μg/100 μl) and naked pDNA (100 μg/100 μl) were delivered to Balb/ c mice via intranasal instillation. Low-frequency U/S (30–35 kHz, 0.07 or 0.1 Mpa, 10 min) was then applied onto the chest and back. Sonoporation did not enhance GL67/pDNA and PEI/pDNA gene transfer. However, U/S exposure at 0.1 Mpa, but not at 0.07 Mpa, increased naked pDNA transfection approximately 4-fold (pDNA: 5.9±1.2 RLU/mg protein, pDNA + 0.1 Mpa: 22.6±3.8 RLU/mg protein, p<0.05, n=7–8). Importantly, low-frequency U/S increased lung gene transfer in the absence of microbubbles. Addition of Optison did not further enhance this effect, due to its inhibitory effects on non-viral lung gene transfer (pDNA + Optison: 0.4±0.1 RLU/mg protein, pDNA + Optison + 0.1 Mpa: 2.5±0.7 RLU/mg protein, n=7–8). Haemorrhage, a common U/S-induced side-effect, was also assessed and increased with pressure in a dose-dependent manner. Interestingly, haemorrhage occurred throughout the lung, indicating that U/S may penetrate deep into the tissue. We have, thus, established for the first time that low-frequency sonoporation can enhance non-viral gene delivery to the mouse lung in vivo. We have previously shown that high-frequency (1 MHz) ultrasound (U/S) increases naked plasmid DNA (pDNA) gene transfer in the mouse lung and that this effect requires microbubbles (Optison) to be present in the formulation. Here, we assessed low-frequency (30–35 kHz) U/S, in an attempt to further increase non- viral gene delivery to the lung in vivo. The potential advantages of lowering U/S frequency include: a) increased cavitation, the mechanism responsible for the formation of transient pores on cell membranes and b) enhanced penetration of U/S energy into the lung, by reducing wave scattering and heat losses. Cationic lipid GL67 (GL67)/pDNA (80 μg/100 μl), polyethylenimine (PEI)/pDNA (20 μg/100 μl) and naked pDNA (100 μg/100 μl) were delivered to Balb/ c mice via intranasal instillation. Low-frequency U/S (30–35 kHz, 0.07 or 0.1 Mpa, 10 min) was then applied onto the chest and back. Sonoporation did not enhance GL67/pDNA and PEI/pDNA gene transfer. However, U/S exposure at 0.1 Mpa, but not at 0.07 Mpa, increased naked pDNA transfection approximately 4-fold (pDNA: 5.9±1.2 RLU/mg protein, pDNA + 0.1 Mpa: 22.6±3.8 RLU/mg protein, p<0.05, n=7–8). Importantly, low-frequency U/S increased lung gene transfer in the absence of microbubbles. Addition of Optison did not further enhance this effect, due to its inhibitory effects on non-viral lung gene transfer (pDNA + Optison: 0.4±0.1 RLU/mg protein, pDNA + Optison + 0.1 Mpa: 2.5±0.7 RLU/mg protein, n=7–8). Haemorrhage, a common U/S-induced side-effect, was also assessed and increased with pressure in a dose-dependent manner. Interestingly, haemorrhage occurred throughout the lung, indicating that U/S may penetrate deep into the tissue. We have, thus, established for the first time that low-frequency sonoporation can enhance non-viral gene delivery to the mouse lung in vivo.