Effect of ultrasound-targeted microbubble destruction on skeletal muscle cell reporter gene transfection by different routes of administration

Abstract

Objective To determine the effect of ultrasound-targeted microbubble destruction (UTMD) on muscle cell gene transfection in mice and to compare the reporter plasmid transfection efficiency by different routes of administration.Methods Mice were randomly divided into four groups:mice treated with plasmid injection alone (group P),injection with microbubble (group P+MB),injection with ultrasonographic radiation (group P+US) and UTMD plus ultrasonographic radiation (group P+UTMD) respectively.Plasmids encoded with green fluorescent protein (GFP) or red fluorescent protein (RFP) were treated with microbubbles (or saline control) and injected into the tibialis anterior muscle unilaterally and caudal vein respectively.This was followed by ultrasonographic radiation (3 MHz and 1.0 W/cm2 for 2 minutes) in subgroups.The mice were sacririced at day 7 prior to extraction of the liver,heart and skeletal muscle tissues for subsequent preparation of frozen sections.Laser confocal microscopy was employed to determine the efficiency of GFP and RFP gene transfection in skeletal muscle.Luciferase activity in the liver and heart was assayed for group P+UTMD.By using haematoxylin-eosin staining,the pathologic changes in the liver,heart and skeletal muscle were assayed for group P+UTMD.The transfection efficiency of liver,heart and skeletal muscle in group P+UTMD was compared between intramuscular and caudal vein injection respectively.Results Single plasmid injection resulted in a minority of cells showing RFP and GFP expression in skeletal muscle cells,thus yielding a rate of (1.83±1.21) ％ and (1.18± 0.25) ％,respectively.Croups P+MB and P+US had similar expression rates of RFP [(2.33±1.39) ％,(3.48±0.18) ％] and GFP [[(3.42±1.09％),(2.52±0.33)％)] when compared with group P.In contrast,a markedly higher (both P＜0.05) expression rate of RFP [(23.96±2.13) ％] and GFP [(25.69±1.98) ％] was found in group P+UTMD,which was partly featured by slight fluorescent signals in the liver and heart tissues.Haematoxylin-eosin staining did not show evidence of significant injury of UTMD on the liver,heart and skeletal muscle,as supported by the intact tissue and absence of infection,hemorrhage,edema or cellular death.The combination of local plasmid injection and UTMD significantly promoted local skeletal muscle gene transfection than caudal vein plasmid injection alone (P＜0.05).Conclusion The fact that UTMD may effectively promote gene translocation and that the combination with local plasmid injection remarkably facilitates local skeletal muscle gene transfection suggests a novel technology featured by high safety and efficiency for gene transfection. Key words: Ultrasound; Microbubble; Muscle, skeletal; Genes, reporter; Transfection; Injections, intravenous; Injections, intramuscular