Lipid nanoparticles and endosomal escape: Characterizing the escape compartment and standardizing models

The interest generated by lipid nanoparticles (LNPs) for the delivery of RNA-based therapeutics has undeniably surged since the COVID-19 pandemic, as evidenced by the growing number of dedicated clinical trials. However, so far, only a handful have made it into a clinical approval. While the development of RNA-LNP systems is hindered by multifactorial issues, including off-target biodistribution and reactogenicity, inefficient cytosolic delivery of RNA via endosomal escape remains the most formidable obstacle to their translational therapeutic success. Deciphering the endosomal pathway and the compartments involved in RNA escape to the cytosol will help to develop effective tools and strategies to enhance RNA delivery and expression. The overall objective of this review is to compare all the published data to offer an up-to-date understanding of LNP trafficking and escape, focusing on three intertwined criteria: the endocytic markers LNPs colocalize with along the endosomal pathway, the endocytic markers likely characterizing the escape compartment, and the time points associated with the studied events. We also highlight discrepancies across the reviewed models (cell types, time points, LNP formulations) and suggest guidelines for future studies to standardize models and harmonize conclusions in the field.