From in vitro to in vivo: The Dominant role of PEG-Lipids in LNP performance.

Lipid nanoparticles (LNPs) are widely employed for delivering nucleic acids, such as mRNA, in both vaccines and therapeutic applications. These LNPs generally include an ionisable lipid (typically ∼ 50 %) to facilitate nucleic acid encapsulation and a PEG-lipid (typically ∼ 1.5 %) to enhance nanoparticle stability. To examine how the choice of PEG-lipid impacts LNP performance, we investigated the physicochemical characteristics and potency of LNPs prepared using two PEG-lipids with different acyl chain lengths: DMG-PEG 2000 and DSG-PEG 2000, containing 14 and 18 carbon tail lengths, respectively. These were combined with three commonly used ionisable lipids (ALC-0315, DLin-MC3 and SM-102). We evaluated the efficacy of these LNPs both in vitro (HeLa cells) and in vivo in mice after intramuscular (IM), subcutaneous (SC), and intravenous (IV) administration. In vitro studies showed that all LNP formulations primarily enter cells via clathrin-mediated endocytosis. Irrespective of the choice of ionisable lipid, DMG-PEG LNPs demonstrated higher in vitro mRNA transfection efficacy than DSG-PEG LNPs. These in vitro results aligned with the in vivo outcomes across all routes of administration tested. Our findings emphasise that despite the low percentage content of PEG-lipid, its selection critically influences LNP efficacy across different administration routes, with DMG-PEG-based LNPs outperforming DSG-PEG LNPs, regardless of the ionisable lipid used.