title: Uptake and quantification of intracellular concentration of lipophilic pro-oligonucleotides in HeLa cells.
abstract: Several lipophilic prodrugs of oligonucleotides (T12 and T20) 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 prooligonucleotides (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 prooligo approach led to a 2-log increase of uptake in comparison with a T20 phosphorothioate oligonucleotide. Finally, an intracellular concentration of pro-oligo was estimated between 4 and 6 microM for an external concentration of 1 microM and up to 27 microM for an incubation at 10 microM.
authors.affiliation: Laboratoire de Chimie Organique Biomoléculaire de Synthèse, UMR 5625 CNRS-UM II, Université de Montpellier II, France.
authors.forename: Jean-Charles
authors.initials: JC
authors.lastname: Bologna
authors.forename: Eric
authors.initials: E
authors.lastname: Vivès
authors.forename: Jean-Louis
authors.initials: JL
authors.lastname: Imbach
authors.forename: François
authors.initials: F
authors.lastname: Morvan
mesh_terms: D002138:Calibration; D005434:Flow Cytometry; D006367:HeLa Cells; D006801:Humans; D008856:Microscopy, Fluorescence; D009841:Oligonucleotides / Q000032:analysis / Q000493:pharmacokinetics*; D011355:Prodrugs / Q000032:analysis / Q000493:pharmacokinetics*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D009841:Oligonucleotides; D011355:Prodrugs