Abstract 9675: Ultrasound and Microbubble-Targeted Delivery of Small Interfering RNA into Primary Endothelial Cells Is More Effective than Delivery of Plasmid DNA
Introduction Ultrasound and microbubble-targeted delivery (UMTD) is a promising non-viral technique for genetic-based therapy. Recently, we demonstrated that with UMTD employing low-intensity ultrasound small dextran molecules enter endothelial cells through transient pores, while the entry of larger dextrans was dependent upon endocytosis. Extrapolating these findings to UMTD of therapeutic compounds for effective genetic-based therapy, we hypothesized that UMTD employing low-intensity ultrasound is more effective when delivering small interfering RNA (siRNA) than plasmid DNA (pDNA).
Methods Red fluorescently-labeled siRNA against housekeeping gene GAPDH, pDNA encoding GAPDH, and red fluorescently-labeled pDNA encoding green fluorescent protein (GFP) were delivered into primary bovine aortic endothelial cells using ultrasound (1 MHz, 0.22 MPa) and SonoVue microbubbles. Cellular localization and GFP expression were determined using fluorescent microscopy. The effect of siRNA and pDNA delivery on GAPDH protein levels was determined by western blotting.
Results UMTD of siRNA transfected 97.9±1.5% of the cells, with labeled siRNA localized homogenously in the cytoplasm directly after ultrasound exposure. Whereas UMTD of pDNA resulted in only 43.0±4.2% transfected cells, with localization mainly in vesicular structures, co-localizing with endocytosis markers clathrin and caveolin. Delivery of siRNA decreased GAPDH protein levels to 24.3±7.9% of non-treated controls (p<0.01, n=5). In contrast, 24h after delivery of pDNA no increase in GAPDH protein levels could be detected. Transfection efficiency, verified with red fluorescently-labeled pDNA encoding GFP, showed that of the transfected cells only 2.0=0.7% expressed the transgene.
Conclusions The localization of small RNA and large DNA molecules after UMTD employing low-intensity ultrasound is an important determinant for the effectiveness of these two genetic-based technologies, as the target for siRNA is located in the cytosol, but for pDNA to be transcribed it needs be in the nucleus. In conclusion, this compartmentalization of therapeutic compounds should be taken into account when designing new genetic-based therapies with UMTD.
- © 2011 by American Heart Association, Inc.