Notch Intracellular Domain Plasmid Delivery via Poly(lactic-co-glycolic acid) Nanoparticles to Upregulate 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. Liposomes and retroviruses are most commonly used to deliver genetic material to cells. However, there are many drawbacks to these systems such as increased toxicity, nonspecific delivery, short half-life, and stability after formulation. We utilized the negatively charged and FDA approved polymer poly(lactic-co-glycolic acid) to encapsulate Notch Intracellular Domaincontaining plasmid in nanoparticles. In this study, we show that primary human umbilical vein endothelial cells readily uptake the nanoparticles with and without specific antibody targets. We demonstrated that our nanoparticles also are nontoxic, stable over time, and compatible with blood. We also determined that we can successfully transfect primary human umbilical vein endothelial cells (HUVECs) with our nanoparticles in static and dynamic environments. Lastly, we elucidated that our transfection upregulates the downstream genes of Notch signaling, indicating that the payload was viable and successfully altered the genetic downstream effects.

In vivo Evaluation of Non-viral NICD Plasmid-Loaded PLGA Nanoparticles in Developing Zebrafish to Improve Cardiac Functions

“…Additionally, we loaded NICD plasmid to our PLGA nanoparticles to upregulate Notch signaling for the zebrafish in vivo experiment. From our previous work, we demonstrated that Tie2+Tie1 antibody conjugated PLGA nanoparticles successfully upregulated Notch related genes within the in vitro flow channel with HUVECs (Messerschmidt et al, 2021b). We also optimized NICD loaded PLGA nanoparticle concentration, 5 nL of 25 µg/mL, and injected at 48 hpf of zebrafish through the CCV.…”

“…TetO-FUW-NICD was a gift from Rudolf Jaenisch (Addgene plasmid #61540) (Cassady et al, 2014). Poly(D, L-lactide-coglycolic acid) nanoparticles (PLGA, 50:50, 55-65 kDa, Akina Inc., West Lafayette, IN, United States) were fabricated by a standard double emulsion method as previously described (Messerschmidt et al, 2021b). In brief, 250 µg of plasmid was diluted in 5% glucose solution to 200 µL which was then emulsified into 0.5 mL of 5% (w/v) PLGA solution in chloroform using a probe sonicator at 40W energy output for 15 s to form a primary water/oil emulsion.…”

“…Before in vivo experiment, we have characterized the properties of PLGA nanoparticles and tested in vitro with HUVECs (Messerschmidt et al, 2021b). In addition, there were no significant changes in size of NICD plasmid loaded PLGA nanoparticles over time in both saline and cell growth media with serum proteins (Supplementary Figure 4).…”

“…However, the unbound nanoparticles flew through the rest of the zebrafish as fast as in vascular system due to rapid blood velocity and motion of the cardiac wall. Therefore, for effective targeting, the conjugation technique with antibodies would be more effective (Messerschmidt et al, 2021b).…”

“…In addition to the high fecundity and transparency, their small size allows for multi-well assays to be easily used. As an important approach to validate nanomedicine using zebrafish, delivery through nanomaterial-based systems, such as PLGA nanoparticles, has been shown to reduce the adverse effects as well as to control the release of therapeutics by prolonging the drug while circulating in cardiovascular system (Thorley et al, 2014;Blanco et al, 2015;Chang et al, 2015;Messerschmidt et al, 2021b). However, translation of nanomedicines into clinical use is still a long-term challenge.…”