C12 200 1 2 distearoyl sn glycero 3 phosphocholine dspc

12 siRNAs targeting mouse Eef2 gene sequence with the lowest off-target potential were designed as described previously^{33 (link)}. siRNAs were synthesized and chemical modifications (modified bases (2′OMe) and phosphorothioate linkages) were introduced to stabilize siRNA in-vivo reducing the off-target potential of the sense strand and minimize immune response (Supplementary Table S3). siRNAs screened in Hepa 1–6 cell line. Cells were transfected with siRNA using Lipofectamine RNAiMAX (Invitrogen). siRNA with the lowest IC50 (determined by western blot and qPCR) was selected for further in-vitro and in-vivo experiments. Selected siRNA was formulated into lipid nanoparticles. The water phase contained siRNA duplex and ethanol phases with lipids (C12-200, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC, Avanti Polar Lipids), cholesterol (Sigma), C14 PEG 2000 (Avanti Polar Lipids) at a 50:10:38.5:1.5 molar ratio) were mixed together in a microfluidic chip device. LNPs were dialyzed overnight against PBS. LNP sizes were measured by dynamic light scattering (ZetaSizer, Malvern Instruments). Mean diameter of the particles prepared for injections was about 90–120 nm.

Eight siRNAs targeting mouse HAS2 gene sequence with the lowest off-target potential were designed. siRNAs were synthesised and chemically modified (methylated pyrimidine nucleotides (2′OMe) and phosphorothioate linkages) to stabilise siRNA in vivo, reducing the off-target potential of the sense strand and minimise the immune response (Supplementary Table S1). NIH 3T3 cells were transfected with siRNAs using Lipofectamine RNAiMAX (Invitrogen) to test the knockdown efficiency by qPCR for each siRNA. The siRNA with the highest potency and the lowest IC50 was selected for further experiments. The selected siRNA was formulated into lipid nanoparticles (LNPs). Briefly, the water phase contained siRNA duplex and ethanol phase with lipids (C12-200, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC, Avanti Polar Lipids), cholesterol (Sigma), C14 PEG 2000 (Avanti Polar Lipids) at a 50:10:38.5:1.5 molar ratio) was mixed in a microfluidic PDMS chip. LNPs were dialysed overnight against PBS. LNP sizes were measured by dynamic light scattering (ZetaSizer, Malvern Instruments). The mean diameter of the particles prepared for injections was about 90–120 nm.