August 20, 2020 — JASE
Background
Definity nanodroplets (DND), formulated from commercial Definity microbubbles (DMB), can extravasate through endothelial barriers and be reactivated to microbubbles by transthoracic ultrasound. The exact location of the droplets within the extravascular space is unknown, but acoustic reactivation (ArA) may be a method of targeting intracellular mediators of scar formation. We hypothesized that ArA may enhance targeted intramyocardial delivery of inhibitory RNA to S100A4 (S100A4-shRNA), a known mediator in cardiac fibrosis.
Methods
Plasmids containing S100A4-shRNA coadministered with DND (DND-P) were formulated from microbubbles (DMBs). They were then infused continuously through an in vitro flow system at 37 Celsius during diagnostic ultrasound transducer (S5-1, iE33, 1.3-MHz center frequency) insonation at different mechanical indices (MI, Fig. 1A) through a three centimeter tissue standoff to determine ArA threshold. Flow cytometry (FC), immunofluorescence (IF) and RT-PCR were conducted to test the binding and transfection efficacy of S100A4-shRNA in vitro (293T cells) and in vivo with six pigs undergoing 90 minutes of ischemia and reperfusion (I/R). Acoustic intensity of background subtracted images was analyzed by QLAB software.
Results
DND (84 ± 38 nm mean diameter) had a zeta potential of -11 ± 1 mV before, and -17 ± 3 mV after plasmid binding (P = 0.027). In vitro real-time imaging revealed that DND-P had virtually no AI at an MI of 0.2, but 35 ± 2 dB at 1.4 MI (P < 0.001, Fig. 1B). Furthermore, at high MI settings, transfection efficiency of DND-P improved by RT-PCR and IF, when compared to DMB-P. In pigs, contrast enhancement was observed at 14 days both in the infarct zone, as well as peri-infarct and normal zone of all pigs (Fig.1C). Using intermittent transthoracic high MI (≥1.0) for ArA , we observed transfection of
S100A4-shRNA in the DND-P treated pigs (n = 3) by RT-PCR and IF when compared to the controls (n = 3, Fig. 1D). Transmission electron microscopy confirmed droplet presence within myocytes.
Conclusion
Targeted delivery of S100A4-shRNA using acoustically activated DND-P within myocytes inhibits S100A4 expression, and is a myocyte-targeted therapeutic strategy to prevent cardiac fibrosis.
Authors: Lijun Qian1, Feng Xie2, Ping Zeng2, John Lof2, Di Xu1, Thomas Porter2
1 The First Affiliated Hospital of Nanjing Medical University, Nanjing, China
2 University of Nebraska Medical Center, Omaha, NE
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