549. Ultrasound with Microbubbles Enhances Gene Expression of Plasmid DNA Delivered through Portal Vein Injection (2006)

[Background and Aim] Ultrasound has been shown to promote gene delivery in skin, skeletal and cardiac muscles, etc. Recently, we have also shown that ultrasound facilitated gene delivery of human factor IX plasmid to mouse liver by intrahepatic injection (Miao et al., Hum Gen Ther 16:893, 2005). The present study was carried out to further optimize and better characterize the application of ultrasound in gene delivery to liver under various conditions of ultrasound and microbubble use, and through portal vein injection instead of intrahepatic injection. [Methods] 50 μg of luciferase plasmid pGL3 was administered to C57/BL6 mice through intrahepatic or portal vein injection with or without ultrasound. Ultrasound with or without 10% microbubbles (Optison), as well as ultrasound of different parameters including acoustic pressure, pulse length, pulse repetition frequency, and timing of exposures were investigated. At least 3 mice per group were included in each experiment. Livers were harvested 24 h after experimental treatment. Luciferase activity was measured to determine the transfection efficiency. [Results] 1. Ultrasound mediated an average of 56-fold increase in luciferase activity only when microbubbles were present. Ultrasound without microbubbles, or microbubbles alone did not mediate any detectable enhancement of gene delivery. 2. Gene delivery via portal vein was enhanced by ultrasound dose-dependently with increasing peak negative acoustic pressure over the range of 0–4.5 MPa at 1.17 MHz, with a peak found at 3 MPa. However, no effect was observed at 0.25 MPa, an acoustic pressure of diagnostic ultrasound reported to be effective in gene delivery to other cell types in vitro or in vivo. 3. Portal vein injection resulted in similar yet more consistent gene expression levels compared with those by direct intrahepatic injection in the left lobe of the liver. Moreover, gene expression was also observed in the other lobes. Thus, portal vein is a better controlled and more efficient route of ultrasound- facilitated gene delivery into the liver. 4. Increasing pulse length (cycles) while decreasing pulse repetition frequency (Hz) to maintain a constant energy exposure at 4.3 MPa peak negative pressure did not improve transfection efficiency. 5. Extending treatment with ultrasound pre- or post-injection of plasmid DNA did not enhance gene delivery efficiency. [Conclusion] Coupled with microbubbles, ultrasound can efficiently and dose-dependently enhance gene expression from plasmid DNA delivered via portal vein injection.These results are consistent with a mechanism involving inertial cavitation. [Background and Aim] Ultrasound has been shown to promote gene delivery in skin, skeletal and cardiac muscles, etc. Recently, we have also shown that ultrasound facilitated gene delivery of human factor IX plasmid to mouse liver by intrahepatic injection (Miao et al., Hum Gen Ther 16:893, 2005). The present study was carried out to further optimize and better characterize the application of ultrasound in gene delivery to liver under various conditions of ultrasound and microbubble use, and through portal vein injection instead of intrahepatic injection. [Methods] 50 μg of luciferase plasmid pGL3 was administered to C57/BL6 mice through intrahepatic or portal vein injection with or without ultrasound. Ultrasound with or without 10% microbubbles (Optison), as well as ultrasound of different parameters including acoustic pressure, pulse length, pulse repetition frequency, and timing of exposures were investigated. At least 3 mice per group were included in each experiment. Livers were harvested 24 h after experimental treatment. Luciferase activity was measured to determine the transfection efficiency. [Results] 1. Ultrasound mediated an average of 56-fold increase in luciferase activity only when microbubbles were present. Ultrasound without microbubbles, or microbubbles alone did not mediate any detectable enhancement of gene delivery. 2. Gene delivery via portal vein was enhanced by ultrasound dose-dependently with increasing peak negative acoustic pressure over the range of 0–4.5 MPa at 1.17 MHz, with a peak found at 3 MPa. However, no effect was observed at 0.25 MPa, an acoustic pressure of diagnostic ultrasound reported to be effective in gene delivery to other cell types in vitro or in vivo. 3. Portal vein injection resulted in similar yet more consistent gene expression levels compared with those by direct intrahepatic injection in the left lobe of the liver. Moreover, gene expression was also observed in the other lobes. Thus, portal vein is a better controlled and more efficient route of ultrasound- facilitated gene delivery into the liver. 4. Increasing pulse length (cycles) while decreasing pulse repetition frequency (Hz) to maintain a constant energy exposure at 4.3 MPa peak negative pressure did not improve transfection efficiency. 5. Extending treatment with ultrasound pre- or post-injection of plasmid DNA did not enhance gene delivery efficiency. [Conclusion] Coupled with microbubbles, ultrasound can efficiently and dose-dependently enhance gene expression from plasmid DNA delivered via portal vein injection. These results are consistent with a mechanism involving inertial cavitation.