Optimizing C14120-based LNPs for in vitro and in vivo mRNA delivery

Lipid nanoparticles (LNPs) have proven to be an effective delivery system for RNA therapeutics. The chemical composition of LNPs determines their functional delivery efficiency and targeting properties, which vary between <i>in vitro</i> and <i>in vivo</i> contexts. Here, we have systematically characterized and compared 25 novel C14120-based LNP formulations for mRNA delivery <i>in vitro</i> and assessed <i>in vivo</i> mRNA expression and biodistribution using deep sequencing of DNA barcodes in a pooled LNP-mRNA library. <i>In vitro</i> experiments showed correlations of lipid composition with particle size and mRNA transfection efficiency in 4 different cell lines of distinct tissue and species origin. <i>In vivo</i> experiments employed a pooled LNP delivery of luciferase mRNA in combination with a multiplexed barcode system and identified LNP compositions with organ-specific targeting properties. Individual validation of three selected LNP candidates based on mRNA expression analysis confirmed high specificity for the lung-targeting candidate, lower specificity for the liver-targeting candidate, and inconclusive results for the spleen-targeting candidate. These findings identify LNP formulations with promising potential for <i>in vitro</i> and <i>in vivo</i> organ-targeted delivery.