LIPID A6

– Ionizable lipid A6 (di(dec-3-yn-1-yl) 9-((4-(dimethylamino) butanoyl)oxy) heptadecanedioate) was developed by modifying the backbone structure of Dlin-MC3-DMA through introducing alkynyl and ester groups into the lipid tails. Alkyne lipid A6 demonstrated a significant improvement in transfection efficiency (~8.5, ~2.0, and ~2.5-fold higher than the original MC3, C12-200 and cKK-E12 containing LNPs, respectively).The performance of the A6 coprepared into iLNPs with other amine-containing lipid materials (cKKE12) was evaluated, and the molar ratio of the formulation was changed to 35:16.0:46.5:2.5 (A6/cKKE12: DOPE: Chol: PEG). The results showed that these formulations synergistically facilitated more effective mRNA delivery and improved tolerability following single and repeated dosing. It was confirmed that albumin-associated macrophage phagocytosis and endocytosis is an apolipoprotein-independent cellular internalization pathway of iLNPs in the liver. According to the fusion kinetics, the lipids with highfrequency tail protrusion and high lateral diffusion coefficient can perturb and trigger membrane sprouting, which in turn promotes membrane fusion. The application of iLNPs with those lipids (such as A6) may be an effective method to further enhance the release of mRNA in vivo.