Synthesis and Screening of Bispecific Linker‐based Combinatorial Libraries Identified Thiosuccinimide Lipids for mRNA Delivery

Abstract Ionizable lipids are essential for lipid nanoparticle‐mediated mRNA delivery. The discovery of novel ionizable lipids relies on combinatorial chemistry synthesis and screening. However, current libraries mainly focus on optimizing amine cores and hydrophobic tails, often neglecting linker optimization, which restricts novel ionizable lipid discovery. To address this, a one‐pot, two‐step, three‐component reaction based on N‐hydroxysuccinimide‐ester‐maleimide bispecific linkers is first developed for constructing combinatorial libraries. Two libraries containing 344 thiosuccinimide lipids are synthesized. Through in vitro and in vivo screening, it is discovered that thiosuccinimide lipids with different types of linkers exhibit different organ tropism. On this basis, T 3 A‐L5‐2C 10 derived from an alkyl chain‐based bispecific linker is identified as a lead lipid. When encapsulating mRNA encoding luciferase, T 3 A‐L5‐2C 10 LNP achieved 12‐, 9.8‐, and 44.7‐fold higher luciferase expression compared to the commercial lipids DLin‐MC3‐DMA, ALC‐0315, and Lipid 5, respectively. Furthermore, when co‐delivering Cas9 mRNA and sgRNA targeting proprotein convertase subtilisin/kexin type 9, T 3 A‐L5‐2C 10 LNP demonstrated 62‐fold and 33‐fold greater gene editing efficiency than DLin‐MC3‐DMA and ALC‐0315 LNPs, respectively. These results highlight the great promise of T 3 A‐L5‐2C 10 for the delivery of mRNA‐based therapeutics.