Journal of controlled release : official journal of the Controlled Release SocietySep 16, 2026

How dilution changes lipid nanoparticle formation and mRNA delivery in the body

mRNA Technology Weekly Brief ↗PubMed ↗DOI ↗

Updated Sep 17, 2026

Abstract

Integrated in-line dilution produced smaller nanoparticles with lower encapsulation efficiency and more negative surface charge compared to conventional off-line dilution.

Integrated in-line dilution consistently resulted in nanoparticles with distinct physicochemical properties compared to conventional off-line dilution.
Under integrated in-line dilution, the dominant process parameter shifted from flow-rate ratios to dilution ratios.
Lipid nanoparticles from integrated in-line dilution showed enhanced transfection in AML12 cells.
Lipid nanoparticles derived from integrated in-line dilution led to increased hepatic mRNA expression following intramuscular administration.
Conventional off-line dilution resulted in stronger antigen-specific immune responses compared to integrated in-line dilution.

Simplified

Messenger RNA (mRNA)-loaded lipid nanoparticles (LNPs) are predominantly manufactured by microfluidic mixing, where extensive efforts have focused on optimizing upstream formulations 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.

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How dilution changes lipid nanoparticle formation and mRNA delivery in the body

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