Uptake and quantification of intracellular concentration of lipophilic pro-oligonucleotides in HeLa cells

Several lipophilic prodrugs of oligonucleotides (T-12 and T-20) bearing enzymolabile protecting groups and labeled with fluorescein were synthesized. Their cellular uptake was studied by three different approaches using fluorescence: fluorescence microscopy, flow cytometry and spectrofluorometry. The corresponding pro-oligonucleotides (pro-oligos) were rapidly and efficiently taken up by HeLa cells and were found homogeneously in the cytoplasm and in the nucleus. The uptake was proportional to their relative lipophilicity and likely proceeded through a passive diffusion mechanism. Uptake followed a dose-response curve. This pro-oligo approach led to a 2-log increase of uptake in comparison with a T-20 phosphorothioate oligonucleotide. Finally, an intracellular concentration of pro-oligo was estimated between 4 and 6 muM for an external concentration of 1 muM and up to 27 muM for an incubation at 10 muM.