Optimization of ultrasound and microbubbles targeted gene delivery to cultured primary endothelial cells.
Ultrasound and microbubbles targeted gene delivery (UMTGD) is a promising technique for local gene delivery. As the endothelium is a primary target for systemic UMTGD, this study aimed at establishing the optimal parameters of UMTGD to primary endothelial cells. For this, an in vitro ultrasound (US) setup was employed in which individual UMTGD parameters were systematically optimized. The criteria for the final optimized protocol were: (1) relative high reporter gene expression levels, restricted to the US exposed area and (2) induction of not more than 5% cell death. US frequency and timing of medium replacement had a strong effect on UMTGD efficiency. Furthermore, US intensity, DNA concentration and total duration of US all affected UMTGD efficiency. Optimal targeted gene delivery to primary endothelial cells can be accomplished with Sonovue microbubbles, using 20 microg/ml plasmid DNA, a 1 MHz US exposure of Ispta 0.10 W/cm(2) for 30 s with immediate medium change after UMTGD. This optimized protocol resulted in both an increase in the number of transfected cells (more than three fold) and increased levels of transgene expression per cell (170%).
Department of Clinical Pharmacology, Groningen Institute for Drug Exploration (GUIDE), University Medical Center Groningen, University of Groningen, Groningen, The Netherlands. b.d.m.meijering@med.umcg.nl
Bernadet D M
BD
Meijering
Robert H
RH
Henning
Wiek H
WH
Van Gilst
Ivana
I
Gavrilovic
Annemieke
A
Van Wamel
Leo E
LE
Deelman
D000818:Animals; D002417:Cattle; D002478:Cells, Cultured; D004247:DNA / Q000008:administration & dosage; D042783:Endothelial Cells / Q000378:metabolism*; D018014:Gene Transfer Techniques; D015316:Genetic Therapy; D045423:Microbubbles; D013535:Suspensions; D014465:Ultrasonics
D013535:Suspensions; D004247:DNA