Enhancement of gene delivery by ultrasound sonication on plasmid DNA prior to transfection

In this study, we investigated the gene delivery efficiency of ultrasound sonication on plasmid DNA prior to transfection. The transfection efficiencies of the normal pEGPF (concentration of 500ng/well), and the pEGPF applied ultrasound were evaluated by transfection experiment with lipofectamine™2000 (Invitrogen) to CT26 cell. The 4.2-kbp pEGFP-C1 (Roche Applied Science) were amplified in Escherichia coli and purified by Endo-free plasmid mega kit (QIAGEN Mega kit). CT26 cells were cultured in Dulbecco´s modified Eagle´s medium (DMEM, Gibco) containing antibiotics, supplemented with 10% fetal bovine serum (FBS, Gibco), at 37degree C in a 5% CO₂ atmosphere incubator for a day before transfection on a 12-well cell culture plate. Ultrasound was sonicated with a 12.5 mm diameter transducer to the pEGFP with concentration of 500ng/μl (frequency: 1.1 MHz, pressure: 300, 400, 700 KPa, period: 10 ms, duty cycle: 1%, exposure time: 30, 60s) prior to tranfection experiment. CT26 cells were harvested 24 hours after transfection and analyzed the count of pEGFP expressing cells using Flow cytometry (FACS caliber). The transfection efficiency of lipofectamine™ 2000 with normal pEGFP achieved an approximate 24.45%, whereas lipofectamine™ 2000 with pEGFP applied ultrasound achieved an approximate 38.81%, 41.86%, 38.67%, 34.80%, and 36.60% at each parameter of US1, US2, US3, US4, and US5 respectively. The transfection efficiency on pEGFP applied ultrasound was more effective than normal pEGFP. In this study, we believe that sonication on pEGFP prior to transfection induce increase of transfection efficiency. In several studies, specific formation of pEGFP such as a supercoil is considered to be more effective at transferring gene expression. In order to verify the formation change of plasmid DNA by ultrasound the electrophorsis was conducted with the denatured pEGFP. Because the normal pEGFP including large amount of supercoiled pEGFP, - s difficult to observe clear difference of formation change, the denaturated pEGFP heated at 94 degree C for 180 sec was used in electrophoresis experiment. The control of denaturated pEGFP and denaturated pEGFP applied ultrasound were analyzed by horizontal electrophoresis using 0.8% agarose gels containing ethidium bromide (0.5 ug/ml) in TAE buffer (40 mM Tris base, 20 mM acetic acid and 1 mM EDTA, pH 8.0). As a result of electrophoresis, increase of supercoil formation was observed at denaturated pEGFP lane applied ultrasound. We supposed that transfection efficiency might be increased due to repackaging pEGFP to smaller size by ultrasound.