A Study of Micro-bubble Enhanced Ultrasound Gene Induction

Recently, a development of the ultrasound gene induction system, called Sonoporation has been investigated. It is known that micro-bubbles can help gene transfection. It is thought that genes are inducted into cells by collapses of cavitation-bubble or micro-bubble. However, the mechanism and optimal induction condition have not been clarified in detail. In this research, we improve gene induction rate by forming DNA/Block copolymer micelle. By forming micelle, DNA is compacted and the stability of DNA is improved. Therefore, it is expected that DNA become able to pass through a hole on cells easily and the expression ability of genes is advanced. Ultrasonic plane wave is exposured from PZT transducer. frequency is 2MHz and Duty Cycle is 10% (40/360). Mouse fibroblast cell line (NIH3T3) is cultured on the bottom of 24-well plate. We add plasmid DNA and Micro-bubble to culture solution and then exposure ultrasound from above the cells. We use GFP plasmid as reporter gene, and Sonazoid® as micro-bubble. Micelle is formed by combining DNA and block copolymer. Block copolymer is composed of polyethyleneglycol-group and poly-lysine. Each naked DNA and polymer micelle is added to culture medium with microbubble, and then exposure ultrasound. Experimental conditions are set as follows: plasmid density is 15

μ

g/ml, number density of micro-bubble is 1.7×105 count/mm3, ultrasound intensity is 10.2 W/cm2, ultrasound exposure time is 60 seconds, and sample number is 12. As a result, Gene induction ratio is doubled by forming polymer micelle (from about 1% to about 1.7%). Therefore, the availability of forming polymer micelle is confirmed.