Hydrophobic Tail and Linker Diversification of PEG‐Lipids Unlocks Precise Muscle Tropism of mRNA‐Lipid Nanoparticles

ABSTRACT Intramuscular mRNA lipid nanoparticles (LNPs) often exhibit undesirable liver accumulation, compromising safety and efficacy. While optimization efforts focus on ionizable and helper lipids, the role of trace PEG‐lipids remains underexplored. To address this, we constructed a 45‐member PEG‐lipid library via Ugi/Passerini reactions, keeping the PEG chain constant at 2 kDa while systematically diversifying the hydrophobic tail and linker structures. High‐throughput screening identified Pr‐182‐BNT (P1B LNP) as a lead candidate. P1B LNPs enable highly efficient and selective mRNA delivery to skeletal muscle while drastically minimizing hepatic off‐targeting. This precise tropism originates from enhanced muscle cell uptake and optimized membrane interactions, driven by the lipid's unique branched, asymmetric tail. In Ai9 reporter mice, P1B LNPs drive potent muscle‐specific gene editing and reduce off‐target recombination. As an RSV mRNA vaccine, they elicit robust antigen‐specific IgG titers and expands polyfunctional CD8 + IFN‐γ + T cells, indicating a Th1‐skewed response. By re‐engineering only the trace PEG‐lipid, this work overcomes a key targeting limitation of classical LNPs and establishes a translatable platform for safer, more effective mRNA vaccines and therapeutics.