mRNA-Based FRET-FLIM Imaging Platform for Quantifying Lipid Nanoparticle Endosomal Escape and Membrane Damage

Efficient endosomal escape and membrane damage are crucial for mRNA-lipid nanoparticle (LNPs) delivery but remain challenging to quantify. Here, we develop FRET-lifetime imaging for mRNA-LNP tracking (FLINT), an intramolecular FRET-FLIM platform to dynamically monitor mRNA release kinetics and endosomal damage in live cells. Capitalizing on the 50- to 100-fold higher concentration of glutathione (GSH) in the cytosol relative to endocytic vesicles, the FLINT employs disulfide-linked fluorophores conjugated to mRNA uridines, enabling precise tracking of cytosolic mRNA via GSH-activated signal amplification (20-50×). Using FLINT, we find that LNPs with escape-favorable lipid compositions (e.g., C12-200) exhibit rapid FRET signal diffusion, while uptake-driven LNPs (e.g., MC3) accumulate in endosomes with limited release. FLINT further reveals GSH influx during endosomal damage and correlates membrane rupture patterns with mRNA expression. Additionally, FLINT uncovers the influence of lipid organization within LNPs─in addition to endosomal membrane damages─on mRNA delivery efficiency. This FLINT platform decouples uptake, escape, and structural determinants of delivery efficiency, offering a transformative tool for LNP optimization and mechanistic studies both in vivo and in vitro.