Lipid Nanoparticle Surface Engineering with Heparosan Polysaccharides Enhances mRNA Efficacy via Reduced Endosomal Retention

Abstract In this study, we developed a library of mRNA lipid nanoparticles (LNPs) containing clinically used ionizable lipids. We surface-modified these nanoparticles with heparosan polysaccharides and systematically compared their performance with conventional poly(ethylene glycol) (PEG)-modified formulations. Our heparosan (HEP)-coated LNPs increased mRNA expression in dendritic and macrophage cell lines by up to ∼10-fold. Mechanistic studies using live-cell imaging and intracellular nanoparticle trafficking analysis revealed that HEP-modified LNPs exhibited reduced colocalization with late endosomal (Rab7+) and lysosomal (LAMP1+) compartments. These findings are consistent with enhanced endosomal escape of HEP-modified formulations. Repeated subcutaneous administrations of HEP-modified firefly luciferase mRNA-LNPs demonstrated sustained expression and distinct response dynamics in mice. We further observed significantly enhanced humoral anti-spike protein immune responses in mice after subcutaneous administration of HEP-modified LNPs containing SARS-CoV-2 spike S-2P mRNA. Histopathological evaluation and spleen immune cell profiling showed no observable toxicity or adverse effects. Overall, these results demonstrate the effects of HEP surface modification on mRNA LNP expression, intracellular trafficking, and in vivo activity, and support HEP-based surface engineering as a PEG-free approach for nucleic acid delivery and potential future applications in nanomedicine.