Redefining LNP composition: phospholipid and sterol-driven modulation of mRNA expression and immune outcomes

Abstract

Ionisable lipids are essential components of lipid nanoparticles (LNPs), enabling nucleic acid encapsulation, cellular uptake, and endosomal escape. Helper lipids further modulate LNP stability, biodistribution, and intracellular trafficking. This study evaluated the in vitro and in vivo performance of LNPs incorporating different phospholipids (DSPC, DOPC, DOPE) and sterols (cholesterol, β-sitosterol), using HEK293 cells and murine models. LNPs were prepared via microfluidics at a fixed molar ratio (phospholipid : sterol/DOPE : SM-102 : PEG-lipid, 10 : 38.5 : 50 : 1.5 mol%). All formulations demonstrated comparable critical quality attributes, including particle size (80–120 nm), low polydispersity index (<0.2), near-neutral zeta potential, and high mRNA encapsulation efficiency (>95%). LNPs containing β-sitosterol exhibited significantly enhanced luciferase protein expression in vitro compared to the cholesterol-based control LNPs. In vivo, DSPC/cholesterol LNPs achieved the highest intramuscular luciferase expression, whereas DOPE-containing LNPs showed low expression. Immunisation studies showed that DOPE-containing LNPs generally enhanced total IgG and IgG1 responses, whereas IgG2a titres varied, with DOPC/DOPE highest and DSPC/DOPE lowest, indicating a disconnect between protein expression and immunogenicity. Ex vivo human whole blood assays revealed distinct cytokine profiles depending on sterol content. β-Sitosterol-incorporated LNPs induced elevated levels of TNF-α, GM-CSF, IL-8, IL-1β, IL-1RA, and IL-6, reflecting both pro- and anti-inflammatory activity, potentially via inflammasome activation. These findings demonstrate that phospholipid and sterol identity substantially influence both delivery efficiency and the quality of immune responses, emphasising the need to optimise the full lipid composition to tailor LNP performance for specific therapeutic applications.