Gene transfer with microbubble ultrasound and plasmid DNA into skeletal muscle of mice: comparison between commercially available microbubble contrast agents.

Purpose: To compare three commercial microbubble contrast agents (Optison, SonoVue, and Levovist) for their effect on gene delivery in skeletal muscle in conjunction with the use of therapeutic ultrasound.

Materials And Methods: The study was approved by the Animal Care and Use Committee. Plasmid DNA (10 microg) encoding green fluorescent protein (GFP) was mixed with microbubbles (or saline control) and injected into the tibialis anterior muscle of mice with and without adjunct ultrasound (1 MHz, 2 W/cm2, 30 seconds, 20% duty cycle). The efficiencies of GFP transgene expression were determined with four experimental conditions: (a) plasmid and saline as control (six mice), (b) plasmid and Optison (six mice), (c) plasmid and SonoVue (four mice), and (d) plasmid and Levovist (air based, four mice). The right legs were exposed to ultrasound, while the left legs were unexposed. Transfection efficiency was assessed by counting the number of GFP-positive fibers. Tissue damage was assessed by measuring the maximal-damage area on serial sections.

Results: When ultrasound was applied, both SonoVue and Optison significantly improved (P < .05) gene transfection efficiency. Optison was also effective (P < .05) even when no ultrasound was applied, which is consistent with previous studies. Levovist without ultrasound decreased the level of transfection (P < .05), with increased tissue damage.

Conclusion: Both non-air-based agents show promise in gene delivery in skeletal muscle with undetectable tissue damage. Enhanced gene transfer with additional ultrasound was achieved only with SonoVue.