Enhanced gene delivery into skeletal muscles with ultrasound and microbubble techniques

Rationale and Objectives. This experiment was directed to explore the effects of ultrasound microbubbles on gene structure in vitro and green fluorescent protein (GFP) plasmid transfer into skeletal muscles in vivo. By establishing a rat ischemic hind limb model, the effects of ultrasound-mediated microbubble destruction on vascular endothelial growth factor (VEGF) gene transfection to skeletal muscles were also studied in vivo. Materials and Methods. Ultrasound irradiation was applied on the mixture of microbubbles and GFP plasmid in vitro. Gel electrophoresis was used to detect the effects of ultrasound and microbubbles on GFP plasmid. For in vivo experiments, ultrasound irradiation was applied on the hind limb after directly injecting microbubbles into the hind limb of Wistar rats. Directly after treatment, the skeletal muscles were harvested to observe the microstructure. We also studied the transfer rate of GFP plasmid DNA into the skeletal muscles of rats by applying ultrasound and microbubble technique. Furthermore, a naked VEGF plasmid was applied to study the feasibility of angiogenesis by using rats ischemia models. Results. Gel electrophoresis of plasmid DNA showed that there was no difference between the groups. By studying the hematoxylin and eosin stained pictures of the skeletal muscles, we found that ultrasound irradiation of skeletal muscle after injection of microbubbles could cause the exudation of the red blood cells, whereas it had no effects on the microstructure of muscle fibers. In vivo experiments showed that an ultrasound microbubble could enhance the transfer of plasmid DNA to the skeletal muscles. Conclusions. The ultrasound-mediated microbubble technique provides an effective noninvasive method for gene therapy.