Poly(lactic-co-glycolic acid) Nanoparticle Delivery of Notch Intracellular Domain Plasmid to Restore Notch Signaling

Abstract

Notch signaling is a highly conserved signaling system that is required for embryonic development and regeneration of organs. When the signal is lost, maldevelopment occurs and leads to a lethal state. Delivering exogenous genetic materials encoding Notch into cells can reestablish downstream signaling and rescue cellular functions. Here, we utilized the negatively charged and FDA approved polymer poly(lactic-co-glycolic acid) to encapsulate Notch Intracellular Domain-containing plasmid in nanoparticles. We show that primary human umbilical vein endothelial cells (HUVECs) readily uptake the nanoparticles with and without specific antibody targets. We demonstrated in vitro that our nanoparticles are nontoxic, stable over time, and compatible with blood. We further demonstrated that HUVECs could be successfully transfected with these nanoparticles in static and dynamic environments. We elucidated that these nanoparticles could upregulate the downstream genes of Notch signaling, indicating that the payload was viable and successfully altered the genetic downstream effects. We further tested our optimized nanoparticle in vivo using a zebrafish model. We determined that our nanoparticles did not cause severe malformations in the developing embryos, and that they survived development. Additionally, we injected NICD-loaded and anti-Tie2+Tie1 conjugated nanoparticles into 2 days post fertilization zebrafish and show that Notch1b and its related genes are upregulated after 24 hours.