LAUSR

The delivery efficiency of biologics via lipid nanoparticles (LNPs) is critically dependent on the incorporation of ionizable lipids. The subtle structural changes to these lipids led to dramatic variations in cytosolic delivery efficiency. However, beyond the pKa values of tertiary amines in ionizable lipids, our understanding of the structure-activity relationships (SARs) between ionizable lipid spatial conformations and their endosomal escape efficacies remains limited. Here, we constructed a library of 18 bioreducible ionizable lipids with varying numbers of alkyl tails, ranging from two to four. We found that three-tailed ionizable lipids exhibited robust endosomal disruption and cytosolic delivery efficiency compared to those of their counterparts with similar pKa values. Molecular dynamics simulations and 31P NMR spectroscopy show that the three-tailed ionizable lipid adopts a characteristic cone-shaped structure, which upon interaction with endosomal phospholipids promotes the formation of inverted hexagonal phases and facilitates endosomal membrane disruption. These results underscore that the number of alkyl tails in ionizable lipids must be precisely tuned, as excessive tail branching may not linearly correlate with improved endosomal disruption. Our research contributes to the mechanistic comprehension of ionizable lipids, highlighting that the spatial configuration serves as a critical design parameter governing intracellular biologic transport.