Visualizing the Effect of Escape-Promoting Agents on Endosomal Escape of RNA Nanoparticles by Using High Time-Spatial Resolution Imaging

Abstract The therapeutic efficacy of RNA nano-drugs is severely hampered by the endosomal delivery bottleneck, wherein internalized cargo is trapped and degraded within the endolysosomal pathway. While promoting endosomal escape strategies have been extensively pursued, their efficiency remains limited; understanding the mechanism of promoting endosome escape is urgently necessary. Herein, confocal microscopy was utilized to visually characterize how canonical chemical agents (nigericin, chloroquine, and sucrose) and cell-penetrating peptides (TAT and F-TAT) facilitate endosomal escape, with particular focus on their modulatory impacts on endosomal morphology and intracellular cargo release. It is found that the chemical agents could enhance the escape probability by enlarging the early endosomes and lysosomes with pores on the endosomes, and the degree of endosomal swelling, rather than the mere number of pores, is the principal driver of differential escape efficiency. In contrast, cell-penetrating peptides TAT could significantly promote the escape efficiency even for the late endosome, without altering the morphology of endosomes. Furthermore, the fluorinated derivative F-TAT facilitated a more rapid endosomal escape, and the most escape can be completed within 30 s. High spatiotemporal resolution direct stochastic optical reconstruction microscopy (dSTORM) imaging further revealed the conformation of pores on the endosomes after treatment with chemical agents. Our findings provide crucial insights into the mechanism of promoting endosomal escape, establishing a correlative framework between agent-induced endosomal remodeling and escape outcomes. This work will offer guideline for achieving high efficiency of intracellular RNA release, thereby maximizing therapeutic potential.