Bioeffects of low-frequency ultrasonic gene delivery and safety on cell membrane permeability control.
OBJECTIVE
To develop a novel method of ultrasonic naked gene delivery (UNGD); to examine the relationship between optimal parameters of ultrasound exposure and cell membrane permeability, enzymes, and free radicals; and to find optimal control parameters that were realizable, reliable, and noncytotoxic for use in gene therapy.


METHODS
Suspensions of chicken, rabbit, and rat red blood cells and S180 cells were exposed to a calibrated ultrasonic field with different parameters in both the still and flowing states to obtain optimal parameters for UNGD. The optimal parameters then were used to implement UNGD. We examined morphologic characteristics, membrane permeability, enzymes, free radicals, naked gene expression efficiency, cell damage threshold, and cell viability by laser scanning confocal microscopy, fluorescent microscopy, flow cytometry, and spectrophotometry.


RESULTS
Green fluorescent protein (GFP) as a reporter was delivered into S180 cells under the optimal parameters without cell damage or cytotoxicity. The transfection rate (mean +/- SD) was approximately 35.83% +/- 2.53% (n = 6) in viable cells, and cell viability was 90.17% +/- 1.47% (n = 6). The intensity of GFP expression with UNGD showed a higher fluorescent peak over both an adeno-associated virus vector-GFP group and a control group (P < .001). Additionally, malondialdehyde, hydroxyl free radicals, alkaline phosphatase, and acid phosphatase displayed an S-shaped growth model (r = 0.98 +/- 0.01) in response to permeability and morphologic alteration.


CONCLUSIONS
Under optimal conditions, low-frequency ultrasound can safely deliver naked genes into cells without causing cell damage. The analytical results indicate that, except for subcavitation, free radical products are responsible for bioeffects in gene delivery. The constant E of energy deposition at 90% cell viability is the optimal control factor, and 80% viability represents the damage threshold. Optimal gene uptake by cells and safety depend on E. Constant E can be applied to control the gene delivery effect in combination with other parameters.
Wei
Wang
W
National Medical Instrument Special Laboratory, Life and Science Technological School, Xi'an Jiaotong University, Xi'an, China. wangwei@mail.lazmc.edu.cn
Zheng-zhong
Bian
B
Yong-jie
Wu
W
Qing-wu
Zhou
Z
Ya-lin
Miao
M
D000818:Animals; D004912:Erythrocytes; D015316:Genetic Therapy / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000235:genetics; D014162:Transfection / Q000379:methods*; D014465:Ultrasonics
D049452:Green Fluorescent Proteins