Illuminating endosomal escape of polymorphic lipid nanoparticles that boost mRNA delivery.

Lipid-based nanoparticles (LNPs) for the delivery of mRNA have jumped to the forefront of non-viral gene delivery. Despite this exciting development, poor endosomal escape after LNP cell entry remains an unsolved, rate-limiting bottleneck. Here we report the use of a galectin 8-GFP (Gal8-GFP) cell reporter system to visualize the endosomal escape capabilities of LNP-encapsulated mRNA. LNPs substituted with phytosterols in place of cholesterol exhibited various levels of Gal8 recruitment in the Gal8-GFP reporter system. In live-cell imaging, LNPs containing β-sitosterol (LNP-Sito) showed a 10-fold increase in detectable endosomal perturbation events when compared to the standard cholesterol LNPs (LNP-Chol), suggesting the superior capability of LNP-Sito to escape from endosomal entrapment. Trafficking studies of these LNPs showed strong localization with late endosomes. This highly sensitive and robust Gal8-GFP reporter system can be a valuable tool to elucidate intricacies of LNP trafficking and ephemeral endosomal escape events, enabling advancements in gene delivery.