Modulating the Nature of Ionizable Lipids and Number of Layers in Hyaluronan-Decorated Lipid Nanoparticles for In Vitro Delivery of RNAi.

Lipid nanoparticles (LNPs) have established their position as nonviral vectors for gene therapy. Tremendous efforts have been made to modulate the properties of LNPs to unleash their full clinical potential. Among the strategies being pursued, the layer-by-layer (LbL) technique has gained considerable attention in the biomedical field. Illuminated by our previous work, here we investigate if the LbL approach could be used to modify the LNP cores formulated with three different ionizable lipids: DODMA, MC3, and DODAP. Additionally, we wondered if more than three layers could be loaded onto LNPs without disrupting their gene transfection ability. Taking advantage of physicochemical analysis, as well as uptake and gene silencing studies, we demonstrate the feasibility of modifying the surface of LNPs with the LbL assembly. Precisely, we successfully modified three different LNPs using the layer-by-layer strategy which abrogated luciferase activity in vitro. Additionally, we constructed a 5×-layered HA-LNP containing the MC3 ionizable lipid which outperformed the 3×-layered counterpart in transfecting miRNA-181-5p to the pediatric GBM cell line, as a proof-of-concept in vitro experiment. The method used herein has been proven reproducible, of easy modification to adapt to different ionizable lipid-containing LNPs, and holds great potential for the translation of RNA-based therapeutic strategies.