Dilution mode acts as a critical process parameter that reprograms lipid nanoparticle assembly and in vivo mRNA delivery.

Messenger RNA (mRNA)-loaded lipid nanoparticles (LNPs) are predominantly manufactured by microfluidic mixing, where extensive efforts have focused on optimizing upstream formulation and mixing parameters. In contrast, the influence of dilution methods on the final physicochemical properties and biological performance of mRNA/LNPs remains insufficiently understood. In this study, we systematically compared two representative downstream manufacturing methods, conventional off-line dilution (OD) and integrated in-line dilution (ID), using a design-of-experiments (DOE) strategy encompassing different flow rates (FR), flow-rate ratios (FRR), dilution ratios (DR). Across multiple formulations, ID consistently produced smaller nanoparticles with lower encapsulation efficiency and more negative surface charge than OD, whereas the dominant process parameter shifted from FRR under OD to DR under ID. These physicochemical differences were accompanied by distinct biological responses. ID-derived LNPs exhibited enhanced transfection in AML12 cells and increased hepatic mRNA expression following intramuscular administration, whereas OD-derived LNPs elicited stronger antigen-specific IFN-γ responses. Collectively, these findings suggest that dilution method continues to influence the maturation of mRNA/LNPs after microfluidic mixing and contributes to the establishment of their final physicochemical and biological characteristics.