Approach of mRNA Delivery by Using Nanoparticles to Recover Endocardial Notch Signaling

Notch signaling plays a significant role in cardiac development and morphogenesis, especially for cardiac chamber development. Failure of mechanotransduction of endocardial Notch signaling in ventricular chamber causes lack of trabeculation. Advent of nanoparticle (NP) technology offers a therapeutic approach of targeted delivery. Although delivering mRNA is an effective method, it is challenging due to stability and cell defense mechanisms. To overcome the limitations and effectively deliver mRNA to endocardium, we used Poly(lactic-co-glycolic acid) (PLGA) synthesized NPs, which have been used in various drug delivery systems due to its biocompatibility, efficient encapsulation of hydrophilic and hydrophobic materials, specific targeting effects, and lower payload concentration. We hypothesized our PLGA NPs are able to deliver NICD mRNA to target endocardium to overexpress Notch signaling in a Notch inhibited zebrafish animal model. Using double emulsion, we synthesized PLGA NPs at 55–65 kDa and performed in vitro test on HUVECs. 69.8 ± 4.3% of Rhodamine B was successfully loaded into the NPs, and after incubation with HUVECs the signal lasted for 28 days after burst release. Hemolysis and whole blood clotting assays proved that NPs did not interfere with the blood clotting process or lyse blood cells. We characterized mRNA incorporated PLGA nanoparticles and incubated with HUVECs after antibody conjugation. To test in vivo study, we injected gata1a morpholino oligomer (MO) to Tg(fli1:gfp) transgenic line to inhibit hematopoiesis, which reduces wall shear stress (WSS) of ventricular chamber leading lack of trabeculae. At 2 days post fertilization (dpf), NICD mRNA incorporated nanoparticles were injected into the circulatory system of gata1a MO injected zebrafish to rescue trabeculation. At 5 dpf, zebrafish hearts were harvested and analyzed via qRT-PCR to compare Notch related gene expression among control, gata1a MO injected, and rescue group. The NICD mRNA injected group successfully rescued the expression level of Notch components despite a lack of WSS and mechanosensitive Notch signaling via gata1a MO injection. This finding will provide an initial therapeutic approach of NICD mRNA for trabecular related congenital heart disease.