Spleen-targeted mRNA delivery via long-chain PEGylated lipids at low molar ratio enhances antitumor immunity against melanoma

Lipid nanoparticles (LNPs) are pivotal for mRNA delivery, yet predominant hepatic accumulation limits their therapeutic application in immunologically active sites like the spleen. Engineering LNPs for splenic mRNA delivery is therefore essential for advancing mRNA-based immunotherapies. Here, we rationally designed a series of LNPs by modulating the alkyl chain length of PEGylated lipids (fast-shedding C14 DMG-PEG vs. slow-shedding C18 DSG-PEG) and their molar ratios (0.75% and 1.5%). This systematic variation precisely controlled nanoparticle physicochemical properties and protein corona composition. Among these formulations, DSG-PEG LNPs at 0.75% mol exhibited the largest size (∼170 nm by DLS; ∼60 nm core by cryo-EM) and a distinct protein corona. Notably, this DSG-PEG 0.75% mol formulation demonstrated significantly enhanced selective spleen accumulation and efficient mRNA delivery <i>in vivo</i>. Furthermore, the optimized LNP potently activated dendritic cells, expanded antigen-specific CD4⁺ T cell populations, and significantly inhibited tumor growth in a B16-ovalbumin (OVA) melanoma model. Collectively, our results establish a rational strategy via precise PEGylated lipid engineering to redirect LNP biodistribution from the liver to the spleen. These findings validate the spleen as a critical target for mRNA vaccines and provide a versatile platform for next-generation cancer immunotherapies.