Tracking the Endosomal Escape of Nanoparticles in Live Cells Using a Triplex‐Forming Oligonucleotide

Nanoparticle-mediated intracellular delivery of oligonucleotides is a complex phenomenon that depends on the architecture and the intracellular trafficking of the engineered nanoparticles. Unravelling the molecular arrangements of oligonucleotides within the nanoparticles as well as their intracellular behavior are essential for designing effective nucleic acid delivery systems. Herein, a simple and general strategy for probing the endosomal escape of nanoparticles carrying oligonucleotides in live cells is reported. A triplex-forming oligonucleotide probe is designed to target the transcription factor, kappa-light-chain-enhancer of activated B cells (NF-kappa B), in the cytosol of cells and to transduce the binding into a fluorescent Forster resonance energy transfer (FRET) signal. The combined use of the triplex-forming oligonucleotide probe and super-resolution microscopy enables the elucidation of the morphology, intracellular localization, and endosomal escape of the oligonucleotide-loaded nanoparticles on a molecular level and with nanoscale resolution. The co-delivery of the FRET probe and mRNA in cells via lipid- and polymer- based nanoparticles allow simultaneous correlation of the endosomal escape properties of nanoparticles and gene expression efficiency.A general strategy for tracking the endosomal escape of nanoparticles in cells is reported. A triplex-forming oligonucleotide is designed to target the transcription factor, NF-kappa B, in the cytosol of live cells and to transduce the recognition of the protein into a FRET signal. Oligonucleotide and super-resolution imaging enables probing of the intracellular pathways and endosomal escape of cationic lipid and phytoglycogen nanoparticles, in real-time and at the nanoscale.image