Bioeffects of Low-Frequency Ultrasonic Gene Delivery and Safety on Cell Membrane Permeability Control

Abstract

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.

In vitro ultrasound-mediated leakage from phospholipid vesicles

“…The results of [7] have shown that low frequency ultrasound , with intensities (most likely I SATA , spatial average temporal average intensity) between 0.2 W/cm 2 and 2.7 W/cm 2 , could be used to increase skin permeability. The outcome presented in [11] suggested that ultrasound of 35.1 kHz applied at pressure amplitudes between 67.1 kPa and 102.5 kPa could be effective in ultrasonic naked gene delivery while maintaining 90% cell viability. The experimental results published recently by the team of the University of Vermont suggested that sonoporation at 2 MHz carried out at 10% duty cycle and 80 W/cm 2 , spatial-peak, pulse-average (I sppa ) intensity could be successful in targeted drug delivery [12].…”

Gene therapy progress and prospects: Ultrasound for gene transfer

“…40,44,45 More controversial is the potential role of extra and/or intracellular free radical production produced during transient cavitation. 25,46,47 We have reported that the presence of free radical scavengers in the transfection medium has no effect on transgene expression following UEGT, suggesting that free radicals are not required for sonoporation. 46 In contrast, Juffermans et al 25 found that scavenging hydrogen peroxide (H 2 O 2 ) with extracellular catalase largely prevented the increased calcium permeability induced by low MI (0.1-0.5) ultrasound (1-3.6 MHz) in H9c2 rat cardiomyoblast cells.…”

DNA Acts as a Nucleation Site for Transient Cavitation in the Ultrasonic Nebulizer

“…These high frequency vibrations have proved to be a useful, noninvasive method by which genetic therapeutics can be delivered both in vivo and in vitro. [1][2][3][4][5][6] This approach has successfully been used to aerosolize complexed DNA for topical delivery to the lungs. 7,8 Additionally, ultrasound has been employed to increase cell membrane permeability which has resulted in an enhancement in transfection efficiency.…”