Enhancement of ultrasound-mediated gene transfection by membrane modification.
BACKGROUND
Ultrasound-mediated gene transfection (USMGT) with an echo contrast agent could be a new promising physical method of triggering localized gene delivery, but the effect is still modest. The aim of this study is to devise a method to improve efficiency of USMGT. We examined the effect of lidocaine and different temperatures on USMGT, each of which is a known membrane modifier, since the plasma membrane can be considered a site of action in USMGT.


METHODS
We observed the effect of lidocaine (0.01, 0.1 or 1.0 mM) and different temperatures (7, 20, 37, 42 or 44 degrees C) on USMGT (1 MHz, 3.6 W/cm(2) (I(SATA)) and 20 s exposure) in the presence of Levovist (10 mg/ml). At 20 h after sonication, transfection efficiency was evaluated by luciferase assay. Membrane fluidity was examined by fluorescence polarization measurement. Cavitational activity was measured by ESR spin trapping with 5,5-dimethyl-1-pyrroline N-oxide. The number of cells transfected with the GFP gene was counted under a fluorescence microscope.


RESULTS
Lidocaine (1 mM) and heat (42-44 degrees C) significantly increased luciferase expression approximately 18-fold and 19-fold higher than Levovist only. Both treatments were shown to increase membrane fluidity; in addition, heat enhanced a cavitational effect. It was confirmed by an experiment using the GFP gene that increase in luciferase expression was due to the increase in number of cells.


CONCLUSIONS
This enhancement could be useful for ultrasound-mediated gene therapy in the future since both treatments for membrane modification could be directly applied to the living body.
Nozaki
Tetsuo
T
Department of Urology, Faculty of Medicine, Toyama Medical and Pharmaceutical University, 2630 Sugitani, Toyama 930-0194, Japan.
Ogawa
Ryohei
R
Feril
Loreto B
LB
Kagiya
Go
G
Fuse
Hideki
H
Kondo
Takashi
T
D000779:Anesthetics, Local / Q000494:pharmacology; D018929:Cell Culture Techniques; D002462:Cell Membrane / Q000502:physiology; D003287:Contrast Media / Q000008:administration & dosage; D018014:Gene Transfer Techniques; D015316:Genetic Therapy / Q000379:methods*; D006801:Humans; D008012:Lidocaine / Q000494:pharmacology; D008560:Membrane Fluidity; D011134:Polysaccharides / Q000008:administration & dosage; D013696:Temperature; D014465:Ultrasonics
D000779:Anesthetics, Local; D003287:Contrast Media; D011134:Polysaccharides; C059891:SHU 508; D008012:Lidocaine