Flow cytometric and microscopic characterization of the uptake and distribution of phosphorothioate oligonucleotides...

Gene-specific inhibition by antisense oligonucleotides has been successful in a large number of systems. In an attempt to use this strategy for the modulation of skin disease-specific gene expression, we studied oligonucleotide uptake in cultured human keratinocytes. This study revealed a heterogeneous uptake of fluorescently labeled phosphorothioate oligonucleotides. Flow cytometric and microscopic analysis showed two fluorescent cell populations with differences in intensity: a 'bright' population of highly fluorescent small cells and a 'dim' population of less fluorescent but larger cells. The heterogeneity in uptake between these two populations was not a result of differences in cell cycle phases of the keratinocytes, as shown by flow cytometric sorting and measurements of relative DNA content. In both populations the oligonucleotides were transported intracellularly and were mainly located in the cytoplasm. A typically speckled localization pattern was demonstrated by confocal laser scanning microscopy. We used propidium iodide (PI) to assess viability, and showed that in nonviable (PI-permeable) keratinocytes the oligonucleotides accumulated in the nucleus. The use of a lipidfection reagent also changed the intracellular distribution of oligonucleotides from a punctate cytoplasmic pattern to an intense nuclear localization. The process of uptake by the viable keratinocytes was dependent on oligonucleotide concentration, incubation time and temperature. This study underlines the importance of kinetic studies on oligonucleotide uptake in human keratinocytes which must be considered when specific oligonucleotides are used against skin disease-specific genes.