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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.

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