Pluronic block copolymers, a kind of non-ionic surfactant, also known as poloxamers, and ultrasound-targeted microbubble destruction have been respectively investigated as vectors for gene delivery in vitro and in vivo. However, they are limited for clinical application due to the relatively low transfer efficiency of each individual vector. In the present study, we explored if the combination of P85, a pluronic block copolymer, Optison, a microbubble contrast agent and ultrasound enhances the transfection of plasmid DNA in vivo using mouse skeletal muscle models. Plasmid encoding green fluorescent protein (GFP) was respectively conjugated with 0.05%P85, 10%Optison, or 0.05%P85 plus 10%Optison, and injected into mouse tibialis anterior (TA) muscles with or without ultrasound irradiation (1 MHz, 1 W/cm(2), 2 min and 20% duty cycle). Mice were sacrificed 1 week after injection. The TA muscles were collected and cryo-sectioned into a series of 7 mu m slices. To assess the efficiency of plasmid DNA transfection, tissue sections were counterstained with DAPI and scored by counting the number of GFP-positive fibers. Meanwhile the area of damaged muscles was measured based on the tissues stained with hematoxylin and eosin. Both P85 and Optison significantly enhanced the delivery of plasmid DNA in mouse TA skeletal muscles (P < 0.01 and P < 0.05 respectively, compared to saline control). In combination with Ultrasound irradiation, P85 (P < 0.01, compared to P85 alone) but not Optison (P > 0.05, compared to Optison alone) exerted a more pronounced effect on the transfection efficiency. Furthermore P85-induced gene delivery was higher than that by Optison regardless of the presence of ultrasound (P < 0.01). The highest transfection efficiency was observed when P85, Optison and ultrasound irradiation were administrated together (P < 0.01, compared to any other treatment in this study). The area of damaged muscles was enlarged by ultrasound irradiation in the presence of Optison microbubbles (P < 0.01, compared to those groups without ultrasound irradiation). These results suggest that P85, microbubbles and ultrasound irradiation synergistically enhance plasmid DNA delivery in mouse skeletal muscles in vivo. (C) 2010 Elsevier B.V. All rights reserved.