Abstract 17672: Validation of Non-Invasive MicroRNA Delivery by Contrast-Ultrasound and Targeted Microbubbles
Background: Effective delivery strategies for microRNA (miR) are under investigation. Ultrasound targeted microbubble destruction (UMTD) is a method for non-invasive delivery of plasmid DNA; however, UTMD has not been used for microRNA (miR) delivery. Our goal was to evaluate the UTMD technique for microRNA delivery, in vitro and in vivo.
Methods: Cationic microbubbles were incubated with increasing concentrations (0, 1, 2.5, 5 μ g/ml) of miR-126. After incubation, conjugated microbubbles were assessed for size and amount of bound microRNA. The time course of circulation of conjugated and unconjugated miR-126 after intravenous injection in rats was assessed by qRT-PCR. UTMD of miR-126 (1x109 cationic microbubbles + 2.5 μ g of miR-126) (n=6) or scrambled miR (miR-SCR) (n=4) was performed in a subset of animals. Control animals (n=4) receiving no treatment. Tissue was harvested at various time points for analysis.
Results: Binding of miR-126 (2.5μ g) to microbubbles (1x109) saturated at approximately 120,000 miR-126 molecules per microbubble (n=6). After intravenous administration, greater concentrations of miR-126 were found in the plasma after injection of miR-126 conjugated microbubbles as compared to miR-126 alone, microbubbles alone or sham. PCR results for miR-126 levels after UTMD to normal distal hindlimb muscle showed an approximate 15-fold increase over control at 3h, with persistent expression up to day3 (n=3 per time point). MiR-126 targets (SPRED1, PIK3R2) were knocked down at 3h (SPRED1; 72±10% and PIK3R2 73±13%, p<0.05 v. Control), day1 (SPRED1; 48±5% and PIK3R2 44±11%, p<0.01 v. Control) and day3 (SPRED1; 69±13% and PIK3R2 80±20%, p<0.05 v. Control). MiR-SCR delivery had no effect on SPRED1 or PIK3R2 levels as compared to controls and there was no detectable transfection seen in remote organs.
Conclusion: MiR-126 had avid binding to cationic microbubbles, and led to prolonged circulation times of miR-126. UTMD of miR-126 shows robust transfection of normal hindlimb skeletal muscle, with significant downregulation of key targets, SPRED1 and PIK3R2. UTMD of miR is a promising in vivo miR delivery technique.
- © 2012 by American Heart Association, Inc.