Lipid Composition Modulates Organ-Specific Reactogenicity of Lipid Nanoparticles After Two Administrations

Background: Lipid nanoparticles (LNPs) can activate innate immunity, but the factors shaping repeat-dose reactogenicity remain unclear. We compared systemic, local, and organ-specific reactions after intramuscular administrations of empty LNPs containing ionizable lipids used in mRNA vaccines. Methods: Female BALB/c mice received two intramuscular injections, 2 weeks apart, of LNP-4 (four components, ALC-0315 as the sole ionizable lipid; 59 nm) or LNP-5 (five components, equimolar ALC-0315/SM-102 at the same total ionizable lipid content; 86 nm). Body weight was monitored for 15 days. Blood biochemical parameters, injection-site histology, and morphological and molecular markers in the hypothalamus, liver, lungs, and spleen were assessed 24 h after the second injection by histology, immunohistochemistry (IHC), immunofluorescence (IF), Western blotting, and quantitative PCR (qPCR). Results: Both LNPs were followed by transient body-weight loss, moderate biochemical changes, and local reactogenicity. The strongest organ-specific responses occurred in the liver and hypothalamus. Both LNPs induced hepatic sinusoidal dilation and leukocyte infiltration, but LNP-4 was associated with more myeloid cell numbers and higher inflammatory marker expression. In the hypothalamus, LNP-4 produced broader changes in inflammatory and myeloid marker expression than LNP-5. No marked structural damage was observed in the examined organs. Conclusions: Repeated LNP administration was followed by systemic and local reactogenicity, and organ-specific changes differed between the formulations, possibly reflecting lipid composition, particle size, and lipid dose. Carrier composition should be considered in the design and safety evaluation of LNP-based vaccine delivery systems.