Fluorescence-Based Assay for the Investigation of Intracellular Trafficking of Gene Carriers

Endosomal escape is a major barrier to the nuclear delivery of DNA. Quantitative measurements of endosomal escape would help identify inefficiencies of current nonviral gene therapy agents and would allow for the development of rationally designed delivery vehicles. We investigated two commonly used, commercially available nonviral delivery systems: Lipofectamine 2000 (L2K) and polyethylenimine (PEI). Oligonucleotides were labeled with 7-nitro-2,1,3-benzoxadiazol-4-yl (NBD), a fluorophore which can be reduced by membrane impermeable sodium dithionite leading to elimination of its fluorescence signal. We investigated dithionite-induced fluorescence quenching of NBD-oligos packaged in L2K and PEI in three cell lines: HeLa, NIH3T3, and CHO-K1. It was found that CHO-K1 cells have the least background fluorescence at the NBD emission wavelength. The detergent TritonX100 was used to disrupt the cell membranes to show that NBD fluorescence could be quenched to background levels. The further development of this method would allow for a better understanding of the intracellular distribution of different nonviral delivery vehicles by selective permeabilization of intracellular vesicles.