Lipid Nanoparticle Preparation and Characterization

CNLs were prepared as described in Sakae et al. [30 (link)]. Briefly, dioleoylphosphatidylethanolamine, distearoylphosphatidylcholine, distearoylphosphatidylethanolamine-polyethylene glycol 2000, polyethylene glycol 750-N-octanoylsphingosine, and C₆-ceramide were mixed in chloroform, dried to a thin film under nitrogen, and then hydrated by the addition of sterilized 0.9% NaCl solution at 60 °C with sonicating and vortexing. Lipid solutions were then extruded at 60 °C by passing through 100 nm polycarbonate filters. Size (80 nM) and charge (−7 mV) were validated using Malvern Zetasizer Nano (Malvern Panalytical, Malvern, UK).