A novel approach of low-frequency ultrasonic naked plasmid gene delivery and its assessment.
OBJECTIVE
To deliver the naked genes into cells through the bioeffects of cell membrane porous produced by low-frequency ultrasound (US) and to investigate the safety by determining the threshold of cell damage and membrane permeability.


METHODS
The suspension of red cells from chickens, rabbits, rats, and S180 cells was exposed to calibrated US field with different parameters in still and flowing state. Laser scanning confocal microscopy, fluorescent microscopy, scanning electron microscopy, flow cytometry and spectrophotometry were used to examine cell morphology, membrane permeability, enzymes, free radicals, naked gene expression efficiency, threshold of cell damage and cell viability.


RESULTS
The plasmid of green fluorescent protein (GFP) as a reporter gene was delivered into S180 cells under optimal conditions without cell damage and cytotoxicity. The transfection rate was (35.83 +/- 2.53)% (n = 6) in viable cells, and the cell viability was (90.17 +/- 1.47)% (n = 6). Also, malondialdehyde, hydroxyl free radical, alkaline phosphatase, and acid phosphatase showed a S-shaped growth model (r = 0.98 +/- 0.01) in response to the permeability change and alteration of cell morphology. The constant E of energy accumulation in US delivery at 90% cell viability was an optimal control factor, and at 80% cell viability was the damage threshold.


CONCLUSIONS
US under optimal conditions is a versatile gene therapy tool. The intensity of GFP expression in US group has a higher fluorescent peak than that in AVV-GFP group and control group (P < 0.001). The optimal gene uptakes, expression of gene and safety depend on E, which can be applied to control gene delivery efficiency in combination with other parameters. The results are helpful for development of a novel clinical naked gene therapeutic system and non-hyperthermia cancer therapeutic system.
Institute of Biomedical Informatics, Key Laboratory of Preclinical Study for New Traditional Chinese Medicine, Lanzhou University, Lanzhou 730000, Gansu, China. Wangwei@mail.lazmc.edu.cn
Wei
W
Wang
Zheng-Zhong
ZZ
Bian
Yong-Jie
YJ
Wu
Ya-Lin
YL
Miao
D000818:Animals; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure; D002470:Cell Survival / Q000235:genetics; D002478:Cells, Cultured; D002645:Chickens; D004249:DNA Damage / Q000235:genetics; D005609:Free Radicals / Q000378:metabolism; D017930:Genes, Reporter / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000235:genetics; D008315:Malondialdehyde / Q000378:metabolism; D010539:Permeability; D010957:Plasmids / Q000235:genetics*; D016062:Porosity; D011817:Rabbits; D051381:Rats; D018570:Risk Assessment; D014162:Transfection / Q000379:methods*; D014465:Ultrasonics
D005609:Free Radicals; D049452:Green Fluorescent Proteins; D008315:Malondialdehyde