Incorporating immunostimulatory lipids into lipid nanoparticles: exploring expression and immune responses

Lipid nanoparticles (LNPs) are a well-established platform for mRNA vaccine delivery, offering efficient encapsulation and intracellular delivery. However, opportunities remain to understand how lipid composition influences immunogenicity. In this study, we investigated the incorporation of dimethyldioctadecylammonium bromide (DDAB), a permanently cationic lipid with reported immunostimulatory properties, into SM-102 LNPs. The fixed positive charge of DDAB at physiological pH can enhance cellular uptake, support local antigen expression, and promote immune activation, all features desirable for vaccine applications. LNPs were prepared with a fixed combined content of 50 mol% SM-102 and DDAB, blended at varying molar ratios (50:0 to 0:50), alongside 10 mol% DSPC, 38.5 mol% cholesterol, and 1.5 mol% DMG-PEG2000. Formulations were characterised for particle size, polydispersity index, zeta potential, and mRNA encapsulation efficiency. Physicochemical characterisation showed that increasing DDAB content led to a progressive increase in particle size while maintaining low polydispersity, neutral zeta potential, and high mRNA encapsulation efficiency across all formulations. Incorporation of ≥ 40% DDAB also altered the apparent pKa profile of the LNPs, consistent with an increasing dominance of permanent cationic charge. In vitro transfection assays in HEK293 cells demonstrated enhanced expression with DDAB-containing LNPs, with the 10% DDAB formulation achieving a six-fold increase in mRNA expression compared to SM-102-only LNPs. However, in vivo luciferase expression following intramuscular administration was significantly reduced in DDAB-LNPs compared to the SM-102 LNPs (p < 0.05). Immunisation studies showed that low-level DDAB substitution (10-30%) did not significantly alter antibody responses, while higher levels (≥40%) reduced immunogenicity, indicating no overall advantage to incorporating DDAB into SM-102 LNP vaccines.