Flow cytometry and live confocal analysis for the evaluation of the uptake and intracellular distribution of FITC-ODN into HaCaT cells.

ABSTRACT

In this study, the mechanism of the internalization and the cellular distribution of 59 fluorescein conjugated PS-ODN (FITC-ODN) after transfection with different mixed lipidic vesicles/oligo complexes (lipoplexes) have been investigated. Mixed lipidic vesicles were prepared with one of the most used cationic lipid (DOTAP) and different amounts of a cholic acid (UDCA) to release the oligo into HaCaT cells. Using flow cytometry, the cellular uptake of the oligo was studied with and without different inhibitors able to block selectively the different pathways involved in the internalization mechanism. The intracellular distribution of the oligo was analyzed by confocal laser scanning microscopy (CLSM), treating the cells with the lipoplexes and directly observing without any fixing procedure. To better carry out the colocalization studies, fluorescent-labeled markers, specific for the different cellular compartments, were coincubated with 59 fluorescein-conjugated 29-mer phosphorotioate oligonucleotide (FITC-ODN). The different lipidic vesicles affect the internalization mechanism of FITC-ODN. After using the inhibitors, the uptake of complexes involved a different internalization mechanism. The live CLSM analysis demonstrated that, after 1 hour from the complex incubation, the oligo was transferred into cells and localized into the endosomes; after 24 hours, the oligo was intracellularly localized close to the nuclear structure in a punctuate pattern. However, the results from fusion experiments showed also a binding of a quite low amount of oligo with the cell membranes.