Redefining LNP Composition: Phospholipid and Sterol-Driven Modulation of mRNA Expression and Immune Outcomes

Abstract

Ionisable lipids are essential components of lipid nanoparticles (LNPs), influencing nucleic acid encapsulation, cellular uptake, and endosomal escape. Helper and co-helper lipids further modulate LNP stability, biodistribution, and intracellular trafficking. This study evaluates the in vitro and in vivo performance of LNPs incorporating various helper lipids and sterol components, using HEK293 cells and murine models. LNPs were prepared via microfluidics at a fixed molar ratio (helper lipid:sterol:SM-102:PEG-lipid, 10:38.5:50:1.5 mol%). All formulations demonstrated comparable critical quality attributes, including particle size (80–110 nm), low polydispersity index (<0.2), near-neutral zeta potential, and high mRNA encapsulation efficiency (>90%). LNPs containing β-sitosterol exhibited significantly enhanced luciferase protein expression both in vitro and in vivo compared to cholesterol-based controls and promoted Th1-biased immune responses. DOPE-containing LNPs exhibited high in vitro protein expression; however, they demonstrated low in vivo expression, yet elicited the highest total IgG titres, suggesting 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. By demonstrating that phospholipid and sterol identity significantly influence both delivery efficiency and immune activation, this work highlights that rational LNP design must consider the full lipid composition, not just the ionisable lipid, to optimise efficacy and tailor immunological outcomes for specific therapeutic applications.