Ultrasound enhancement of liposome-mediated cell transfection is caused bycavitation effects

Cationic liposomes (CL) are widely used vectors for gene transfer, Recently , ultrasound (US) was reported to enhance liposome-mediated gene transfer t o eucaryotic cells in culture. The present study was aimed at studying the effects of 2-MHz pulsed Doppler US on malignant brain tumor cells transfect ion by cationic liposome/plasmid-DNA complexes (lipoplexes). Cationic lipos omes consisting of DOSPA/DOPE were complexed with a plasmid carl ping the c DNA encoding green autofluorescent protein (EGFP), Rodent (9L) and canine ( J3T) glioma cells were exposed to pulsed US in the presence of EGFP-lipople xes. A diagnostic transcranial Doppler de,ice (MultiDop L) was used for ins onation for 30, 60, and 90 s at 2 MHz/0.5 W/cm(2). To eliminate US reflecti on and cavitation. a custom-made absorption chamber was designed, where US is applied through a water tank before interacting with the cells and is fu lly absorbed after passing through the cell layer. Expression of the marker gene EGFP was quantified by FACS analysis and intravital fluorescent micro scopy. Cell viability was accessed by Trypan Blue staining. US treatment of tumor cells on microplates for 60 s yielded a significant increase in tran sfection rates without damaging the cells, but 90-s treatment killed most o f the cells, In the absorption chamber, no significant effects of US on tra nsfection were noted. Additional experiments employed US contrast agent (Le vovist(R), Schering) which was able to significantly increase turner cell t ransfection rate by enhancing cavitation effects, and also severely damaged most cells when applied at a concentration of 200 mg/mL, In conclusion, ou r results support the assumption that US effects on lipoplex transfection r ates in brain tumor cells in culture are mediated by cavitation effects, (C ) 2000 World Federation for Ultrasound in Medicine & Biology.