Study Of Enhancement Of Efficiency Of Gene Delivery Using Ultraosund Combined With Microbubbles

Based on the interactions among DNA/drugs and cells, Gene therapy realizes its treatment by changing the structure of cells themselves and genetic information, the key technique is to enhance the efficiency of drug carrier delivery. The transfection of viral vectors in gene therapy is sensitive to the human immune system and may be harm to normal cells. Thus, the urgent requirement is to develop non-viral vectors delivery, delivering DNA or drugs into target cells safely and effectively. As reported in vitro experiments, US exposure assisted by ultrasound contrast agents (UCAs) could transiently enhance the cell membrane permeability, which could make cell membrane temporarily "open" to facilitate the flux of foreign gene/drugs into cells. This biophysical process is called sonoporation. However, obstacles still remain to achieve controllable sonoporation outcome. The aim of this work is to optimize the effect of sonoporation. As a result, the physical mechanisms of enhancement of efficiency of Gene delivery using combination of ultrasound and microbubbles is focused on. In current experimental work, MCF-7 cells mixed with PEI:DNA complex and UCA microbubbles were exposed to 1-MHz US pulses with 20-cycle pulse and varied acoustic peak negative pressure (P-; 0 (sham),0.3,0.75,1.4,2.2 or 3.0 MPa), total treatment time (0,5,10,20,40 or 60 s), and pulse-repetition-frequency (PRF; 0,20,100,250,500, or 1000 Hz). Then, four series experiments were conducted:(1) the IC activities were detected using a PCD system and quantified as ICD; (2) the DNA transfection efficiency was evaluated with flow cytometry; (3) the cell viability was examined by PI dying then measured using flow cytometry; and (4) scan electron microscopy was used to investigate the sonoporation effects on the cell membrane. The results showed that:(1) the ICD generated during US-exposure could be affected by US parameters (e.g., P-, total treatment time, and PRF); (2) the pooled data analyses demonstrated that DNA transfection efficiency initially increased linearly with the increasing ICD, then it tended to saturate instead of trying to achieve a maximum value while the ICD kept going up; and (3) the measured ICD, sonoporation pore size, and cell viability exhibited high correlation among each other. All the results indicated that IC activity should play an important role in the US-mediated DNA transfection through sonoporation, and ICD could be used as an effective tool to monitor and control the US-mediated gene/drug delivery effect. While in current physical mechanisms work, the numerical calculus of the shear stress resulting from microstreaming near pulsating bubbles was carried on. Comparing with the experimental results, we found that the shear stress resulting from microstreaming near pulsating bubbles play an important role at the generation of sonoporation process.