Laser induced stress wave-assisted gene transfection: improved transfection efficiency with cationic liposome-modified plasmid DNA

摘要

Although various methods for gene transfer have been investigated, a practical gene delivery system that fulfills the requirements for clinical application has not yet been developed. Gene transfer by the use of laser-induced stress waves (LISWs) is a physical method to facilitate gene transfer into cells with the effect of stress waves generated by irradiation an absorbing material with high-intensity laser pulses. This method has high spatial controllability and a potential for catheter-based gene transfer. We demonstrated selective high transfection efficiency in vivo. However, there remains a problem that transfection efficiency is limited to less than several percent in vitro. Thus we attempted to improve transfection efficiency by using plasmid DNA modified with cationic-liposome. Plasmid DNA coding for enhanced green fluorescent protein (EGFP) had been modified with Lipofectamine and it was added to a dish for NIH3T3 cell culture. A black rubber disk was placed on the upper side of the cells; the disk was irradiated with 532 nm, nanosecond laser pulses (spot diameter, 3 mm; fluence, 1.3 J/cm~2; number of pulses, 20). 24 hours after application of LISWs, transfection efficiency was evaluated with a fluorescence microscope, where efficiency was defined as the ratio of the number of cells emitting fluorescence to the total number of cells. At a DNA concentration of 7.8 μg/ml, transfection efficiency with naked plasmid DNA was as low as 0.05%, while it was increased to 23.7% by using plasmid DNA modified with Lipofectamine. Since both of the naked plasmid DNA and cell membranes have negative charge, plasmid DNA concentration around cells should be low. Since DNA-Lipofectamine complexes carry positive charge, density of plasmids existing around cells should be increased, resulting in much improved transfection efficiency.