Experiment of nanobubbles-mediated plasmid green fluorescent protein(GFP) transfer into skeletal muscle in mice

Abstract

Objective To explore the effect of nanoscale bubbles transfering gene in skeletal muscle cells in mice.Methods Plasmid DNA encoding green fluorescent protein (GFP) was mixed with bubbles dissolved in saline and injected into the tibialis anterior (TA) muscle of C57B10 mice with and without ultrasound (US).The ultrasound frequency was 1 MHz and the pulse repetition frequency was 100 Hz with 20％ duty cycle.The spatial peak temporal peak intensity (ISPTP) power level was 2 W/cm2.The entire treatment period was 30 seconds.The efficiencies of GFP transgene expression were determined under different experinmental conditions.Mice were sacrificed 1 week after plasmid DNA injection.Fibres with fluorescence green signals were determined as GFP-positive fibres by fluorescence microscopy.Readout was performed on the section with the maximum number of transfected fibers.Results 1 )Albumin nanobubbles:in the conditon of with or without ultrasound,albumin nanobubbles had significantly increased gene expression compared with negative control (P ＜0.05),but significantly decreased gene expression compared with positive control ( P ＜ 0.05 ).2) Phospholipid nanobubbles:In the conditon of without ultrasound,there were no significant differences compared with negative and positive control ( P ＞0.05).In the conditon of with ultrasound,phospholipid nanobubbles had significantly increased gene expression compared with negative control ( P ＜0.05).No significant difference was observed between phospholipid nanobubbles and positive control ( P ＞ 0.05).Conclusions Nanobubbles could enhance gene transfection efficiency for skeletal muscle fibres in mice.Nanobubbles with perfluoropropane gas and albumin have potentiality in gene-enhanced transfection. Key words: Ultrasonography; Microbubbles ; Nanotechnology; Transfection