A novel approach to quantitative ultrasonic naked gene delivery and its non-invasive assessment.
The purpose of this study was to investigate practical, safe, easy-to-use, non-cytotoxic, and reliable parameters to apply to an ultrasound (US) naked gene therapy system. The ultrasound pressure at the point of cell exposure was measured using a calibrated hydrophone and the intensity calculated. An acoustic power meter calibrated using a hydrophone was used to measure the power of the transducer. Four cell types were exposed to US with different exposure times and intensities. Fluorescent microscopy, spectrophotometry, scanning electron microscope, laser scanning confocal microscopy, flow cytometry and histogram analysis were used to evaluate the results of the study. The plasmid of green fluorescent protein (GFP) served as the reporter gene. The energy accumulation E in US gene delivery for 90% cell survival was defined as the optimal parameters (E=3.56+/-0.06), and at 80% cell survival was defined as the damage threshold (E=59.67+/-3.54). US safely delivered GFP into S180 cells (35.1 kHz) at these optimal parameters without obvious damage or cytotoxity in vitro. Exposed cell function was proved normal in vivo. The transfection rate was 35.83+/-2.53% (n=6) in viable cells, corresponding to 90.17+/-1.47% (n=6) cell viability. The intensity of GFP expression showed a higher fluorescent peak in the group of adeno-associated virus GFP vector (AVV-GFP) than in the control group (P<0.001). The effect of US gene delivery and cell viability correlated as a fifth order polynomial with US intensity and exposure time. With optimal parameters, US can safely deliver naked a gene into a cell without damage to cell function. Both optimal uptake and expression of gene depend on the energy E at 90% cell survival. E can be applied as a control factor for bioeffects when combined with other parameters. Stable caviation results in optimal parameters for gene delivery and the transient caviation may cause cell damage, which will bring about a sharp rise of permeabilization. The results may be applied to the development of a novel clinical gene therapeutic system.
Wei
Wang
W
Life and Science Technological School of Xi'an Jiaotong University, Xi'an, Sha'an xi 710049, China. kinggreat@163.com
Zhengzhong
Bian
B
Yongjie
Wu
W
Lafeng
Yan
Y
Yalin
Miao
M
D000818:Animals; D002645:Chickens; D017930:Genes, Reporter; D015316:Genetic Therapy / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000235:genetics; D010957:Plasmids; D051381:Rats; D014162:Transfection / Q000379:methods*; D014465:Ultrasonics
D049452:Green Fluorescent Proteins