Real-time gene delivery vector tracking in the endo-lysosomal pathway of live cells

Using live‐cell confocal microscopy and particle tracking technology, the simultaneous transport of intracellular vesicles of the endo‐lysosomal pathway and nonviral polyethylenimine (PEI)/DNA nanocomplexes was investigated. Due to potential problems associated with the use of acid‐sensitive probes in combination with a gene vector that is hypothesized to buffer the pH of intracellular vesicles, the biological location of PEI/DNA gene vectors was revealed by probing their trafficking in cells expressing fluorescent versions of either early endosome antigen 1, a protein that localizes to early endosomes, or Niemann Pick C1, a protein that localizes to late endosomes and lysosomes. Studies directly show that PEI/DNA nanoparticles are actively transported within both early and late endosomes, and display similar overall transport rates in each. Additionally, gene vector transfer between endosomes is observed. Over time post‐transfection, gene vectors accumulate in late endosomes/lysosomes; however, real‐time escape of vectors from membrane‐bound vesicles is not observed. Microsc. Res. Tech., 2012. © 2011 Wiley Periodicals, Inc.