Influence of ionisable lipid and sterol variations on lipid nanoparticle properties and performance

Abstract

Lipid nanoparticles (LNPs) have emerged as a vital delivery system for nucleic acids, particularly in the context of mRNA vaccines and therapeutics. This study evaluates the impact of various proprietary ionisable lipids on the physicochemical characteristics, encapsulation efficiency, and expression of mRNA within LNP formulations. We compared 11 novel ionisable lipids against the clinically used ALC-0315 in both in vitro and in vivo models. Alongside comprehensive physicochemical characterisation (including size, zeta potential, and polydispersity index), we uncovered significant variations in vitro in formulation performance influenced by lipid structure, where LNPs formulated with cone-shaped ionisable lipids exhibited markedly higher mRNA expression in HeLa cells compared to the control. In vivo assessments revealed distinct biodistribution patterns, with ALC-0315 formulations demonstrating preferential delivery to the liver, while alternative ionisable lipids shifted distribution toward the spleen, emphasising the role of lipid composition in therapeutic efficacy. Notably, some proprietary LNPs performed well in vitro but showed poor in vivo expression, especially via IV administration, underscoring the importance of delivery context and offering novel insight into route-independent formulation performance trends. However, the overall performance ranking was consistent across both in vivo administration routes, with the best- and worst-performing formulations maintaining their relative expression profiles. Our findings also indicate that while differences in sterol choice modulate LNP properties, the choice of ionisable lipids is crucial for optimising mRNA delivery. Furthermore, the study highlights the discrepancies between in vitro and in vivo results, emphasising the need for further research into the biological complexities of LNP behaviour. Collectively, this work offers new insight into structure–function relationships within LNP systems and provides valuable formulation strategies for next-generation RNA therapies.