In Silico Engineering of Stable siRNA Lipid Nanoparticles: Exploring the Impact of Ionizable Lipid Concentrations for Enhanced Formulation Stability

Abstract

Lipid nanoparticles (LNPs) are crucial in advancing the delivery of RNA-based therapeutics within the domain of gene therapy. A comprehensive understanding of their formation and stability is critical for optimizing the clinical efficacy of LNPs. This study systematically investigates the influence of concentration variations of positive and neutral ionizable lipids - specifically, 2-[2,2-bis[(9Z,12Z)-octadeca-9,12-dienyl]-1,3-dioxolan-4-yl]-N,N-dimethylethanamine (DLinKC2-DMA) and 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) - along with cholesterol and polyethylene glycol, on the formation of LNPs and encapsulation of small interfering RNA (siRNA). Utilizing coarse-grained classical molecular dynamics (MD) simulations with a system size matching experimental range (approximately 0.6 million beads), we conduct a comparative analysis and offer mechanistic insights into siRNA formulation within LNPs containing positive and neutral DLinKC2-DMA. We found that the LNPs with positive ionizable lipids encapsulate more than twice the siRNA compared to the LNPs with neutral ionizable lipids. In addition to the formation of LNPs, our study extends to the forces governing siRNA escape from LNPs, employing steered molecular dynamics simulations. The force experienced by siRNA to cross the LNP lipid layer containing positive ionizable lipids was 400kJ/mol/nm more than that of neutral ionizable lipids, suggesting the encapsulation is more favorable with positive ionisable lipids.