246C10

246C10 is a synthesized ionizable lipid. 246C10 can be formulated into lipid nanoparticles (LNPs) with dioleoylphosphatidylethanolamine (DOPE), cholesterol, and C16-PEG2000 ceramide (PEG-lipid) as well as mRNA. The lipid nanoparticle formulations can be used for mRNA delivery. To obtain iLNPs that could specifically target liver sinusoidal endothelial cells (LSECs), six different ionizable lipids (241C10 to 246C10) were synthesized by an epoxide ring-opening reaction with piperazine- or piperidine-containing amines. Biodistribution and gene regulation of various iLNPs were assessed in vivo, and the results showed that the 246C10 iLNPs (containing piperazine amine) had the highest luciferase expression in the liver. When further analyzing the 246C10 iLNPs transfection efficiency in different types of liver cells, it was found that tdTomato fluorescence was mainly concentrated in hepatocytes, not in LSECs. Figure 6f shows that 80% of hepatocytes are fluorescent, 40% of LSECs are fluorescent, and 20% of Kupffer cells are fluorescent. Due to the mannose receptor on LSECs, mannose-PEG lipid was introduced into 246C10 iLNPs to alter the distribution of iLNPs in different liver cells. As shown in Figure 6g, tdTomato fluorescence distribution was 15% of hepatocytes, 70% of LSECs, and 15% of Kupffer cells, significantly improved the ability of iLNPs to actively target LSECs. In contrast, this work indirectly shows that the iLNPs with piperazine head lipid are more able to deliver mRNA to the liver and translate the target protein than the iLNPs with piperidine head lipid. It is worth mentioning that the preparation buffer of 246C10 iLNPs could influence the encapsulation efficiency of mRNA. With the addition of sodium chloride in the citrate buffer, the encapsulation efficiency of CRISPR-Cas9 mRNA and sgRNA was increased. These iLNPs were able to treat hemophilia safely, without causing hepatotoxicity, the immune response induced by Cas9 and off-target editing.