Enhanced gene transduction into skeletal muscle of mice in vivo with pluronic block copolymers and ultrasound exposure.

The pluronic block copolymers are able to enhance the ultrasound-induced gene delivery in vitro. In the present study, the effects of pluronics on the efficiency of gene transfer into skeletal muscle in vivo under sonoporation were investigated. Plasmid DNA encoding green fluorescent protein (GFP) in combination with three different pluronics, F127, L61, and P85, was injected into the tibialis anterior (TA) muscle of mice with and without adjunct ultrasound (1 MHz, 3 W/cm(2) 1 min, 20% duty cycle). Mice were killed 1 week after injection. The TA muscles were removed and snap frozen immediately in isopentane cooled by liquid nitrogen and sections of 7 µm thick were cut. Transfection efficiency was assessed by counting the number of GFP-positive fibers under fluorescence microscopy, and tissue damage by hematoxylin and eosin staining. The results suggested that all three pluronics significantly enhanced transgene expression in skeletal muscle (P < 0.01), especially the P85 showed significantly higher efficiency than the other two pluronics (P < 0.05). Ultrasound synergistically enhanced the gene delivery efficiency with P85 (P < 0.01), but was unable to do so with F127 and L61 groups. In short, P85 displays significantly synergistic effect with ultrasound for enhancing plasmid DNA transduction in skeletal muscle of mice in vivo.

P85, Optison microbubbles and ultrasound cooperate in mediating plasmid DNA transfection in mouse skeletal muscles in vivo.

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 µm 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.