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 sinusoidalendothelial cells (LSECs), six different ionizable lipids (241C10to 246C10) were synthesized by an epoxide ring-openingreaction with piperazine- or piperidine-containing amines.Biodistribution and gene regulation of various iLNPs wereassessed in vivo, and the results showed that the 246C10iLNPs (containing piperazine amine) had the highest luciferaseexpression in the liver. When further analyzing the246C10 iLNPs transfection efficiency in different types of livercells, it was found that tdTomato fluorescence was mainly concentratedin hepatocytes, not in LSECs. Figure 6f shows that 80%of hepatocytes are fluorescent, 40% of LSECs are fluorescent, and20% of Kupffer cells are fluorescent. Due to the mannose receptoron LSECs, mannose-PEG lipid was introduced into 246C10iLNPs to alter the distribution of iLNPs in different liver cells. Asshown in Figure 6g, tdTomato fluorescence distribution was 15%of hepatocytes, 70% of LSECs, and 15% of Kupffer cells, significantlyimproved the ability of iLNPs to actively target LSECs.In contrast, this work indirectly shows that the iLNPs with piperazinehead lipid are more able to deliver mRNA to the liver andtranslate the target protein than the iLNPs with piperidinehead lipid. It is worth mentioning that the preparation buffer of 246C10iLNPs could influence the encapsulation efficiency of mRNA. With the addition of sodium chloride in the citrate buffer, theencapsulation efficiency of CRISPR-Cas9 mRNA and sgRNAwas increased. These iLNPs were able to treat hemophilia safely, without causing hepatotoxicity, the immune response induced byCas9 and off-target editing.