Formulation and Characterization of siRNA-LNP

We used one small interfering RNA (siRNA) described by Leuschner et al.^{51 (link)} and in Supplementary Methods. siRNA was selected to avoid off-target activity based on several known criteria as described previously^{52 (link)}. siRNA modification with 2′-OMe pyrimidine nucleotides and 3′-internucleotide phosphorothioates should reduce immune response and increase stability in vivo. Potency of siRNA targeting CCR2 were studied by transfection in Hepa1–6 cells followed by qPCR analysis after 24 h. siRNA were formulated in lipid nanoparticles (LNPs), as previously described^{52 (link)}. Particle size measurements were performed using a Zetasizer Nano ZSP (Malvern Panalytical) according to the manufacturer’s protocol, siRNA entrapment efficiency was determined using the Quant-iT™ RiboGreen® reagent (R11491, Thermo Fisher Scientific) as described^{52 (link)}.