siRNA Delivery via Lipid Nanoparticles

ON-TARGETplus Non-targeting Control Pool or ON-TARGETplus SMARTPool siRNAs targeting mouse Tlr4, Nr1h3, Nr1h2, Eif2ak3, or Ero1l were purchased from Horizon Discovery Ltd. (Cambridge, UK). All siRNAs were packaged in lipid nanoparticles (LNPs) and transfected into cells. Cultured cells were transfected with siRNA for 24 h and then treated with cholesterol or 7-KC 12 h after transfection was completed. siRNA-loaded LNPs were formulated as previously described^{53 (link)}. Briefly, 400 μL of lipid solution containing 1.875 mM ssPalmO-Phe (Product# COATSOME^® SS-OP, NOF CORPORATION, Tokyo, Japan), 450 μM polyethylene glycol-dimyristoylglycerol (Product# SUNBRIGHT^® GM-020, NOF CORPORATION), and 1.875 mM cholesterol (Sigma–Aldrich) in ethanol was prepared. Then, 10 μg of siRNA in 124 μL of 20 mM malic acid buffer (pH 3.0) was gradually added to the lipid solution under vigorous mixing. The mixed solution was diluted with 1 mL of malic acid buffer and then mixed vigorously with 3 mL of PBS. This diluted solution was subjected to ultrafiltration with Amicon Ultra-4 (Merck Millipore, Billerica, MA) two or more times. The encapsulation efficiency and recovery ratio of siRNA in this LNP solution were measured with a RiboGreen assay (Thermo Fisher Scientific Inc.) as previously described^{54 (link)}.