Optimisation of ultrasound-mediated gene transfer (sonoporation) in skeletal muscle cells.
Ultrasound (US) is a promising tool for facilitating direct gene transfer to skeletal muscle, but no systematic optimisation study has been performed. We exposed H2K myoblast cells to US with varying intensity of exposure and duration to evaluate its effect on cell viability and transfection efficiency using as endpoints transfection rate, average fluorescence intensity (fluorescence normalised by the number of transfected cells) and overall expression (the product of transfection rate and average fluorescence intensity) as indices. Cell viability decreased with exposure time and intensity, consistent with previous findings. Optimal setting of US was observed at the range of 0.5 to 1 W cm(-2) with duration of 20 s, producing maximum efficiency (transfection = 4.5%) in gene transfection with minimum cell toxicity (cell viability = 83%). Higher intensity alone or in combination with low intensity and long duration did not improve cell viability and transfection. The increase of eGFP (enhanced green fluorescence protein) plasmid concentration up to 200 microg per mL was related to an increase in average fluorescence intensity and overall expression. However, transfection rate saturated when DNA concentration reached 50 microg per mL despite initial increase with DNA concentration. The average fluorescence intensity was linearly proportional to the logarithm of DNA concentration, suggesting a diffusion-based model for DNA uptake under sonoporation. We conclude that low-intensity US irradiation provides a safe and effective alternative for gene delivery.
Liang
Hai-Dong
HD
Ultrasound Group, Imaging Sciences Department, Clinical Sciences Centre, Imperial College, Hammersmith Hospital, London W12 0HS, UK. Haidong.liang@imperial.ac.uk
Lu
Qi Long
QL
Xue
Shao-An
SA
Halliwell
Michael
M
Kodama
Tetsuya
T
Cosgrove
David O
DO
Stauss
Hans J
HJ
Partridge
Terence A
TA
Blomley
Martin J K
MJ
D000818:Animals; D002470:Cell Survival / Q000235:genetics; D002478:Cells, Cultured; D004247:DNA / Q000032:analysis; D005434:Flow Cytometry / Q000379:methods; D018014:Gene Transfer Techniques; D049452:Green Fluorescent Proteins / Q000032:analysis; D051379:Mice; D018482:Muscle, Skeletal / Q000000981:diagnostic imaging / Q000502:physiology*; D010957:Plasmids / Q000235:genetics; D013997:Time Factors; D014162:Transfection / Q000379:methods; D014465:Ultrasonics; D014463:Ultrasonography
C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D004247:DNA