Boosting LNP Performance: Higher Concentrations of Lipid Mixtures Improve In Vivo Gene Expression and Storage Stability

<b>Background</b>: An efficient formulation of lipid nanoparticles (LNPs) is often considered crucial in the successful development of nucleic acid therapeutics. This study explores the impact of varying the lipid and payload concentrations as starting materials on key LNP properties. <b>Results</b>: The outcomes of the study revealed that the desired particle properties could be retained even at a starting lipid mixture concentration of 70 mg/mL. Particle size remained largely unchanged despite changes in lipid mixture concentration, with polydispersity index values below 0.2. CryoTEM analysis revealed that LNPs prepared using higher lipid mixture concentrations were more uniform and more abundant in solid core morphologies. Buffer composition was shown to influence the LNP particle size, surface charge, and gene expression, as well as storage stability. In vivo studies in mice showed enhanced gene expression and biodistribution for LNPs formulated at higher lipid and RNA concentrations, with LNPs in Tris-sucrose eliciting superior gene expression compared to LNPs in PBS. <b>Conclusions</b>: This study demonstrated that intensified mixing processes based on confined jet-impingement allow the use of elevated starting material concentrations in LNP formulations, resulting in improved biological performance and stability of mRNA-LNPs, as well as enhanced scalability and throughput.