Nuclear transport of oligonucleotides in HepG2-cells mediated by protaminesulfate and negatively charged liposomes

Purpose. The aim of this study was to characterize the intracellular fate a nd nuclear uptake kinetics of oligonucleotides (ON) that were complexed wit h protamine sulfate (PS) and negatively charged liposomes at different rati os of ON to PS. Methods. Double-fluorescence labelling of ON and liposomal lipid was applie d to simultaneously monitor the interaction as well as the individual fate of active agent and carrier upon intracellular delivery using confocal lase r scanning microscopy (CLSM). A DNA-analogue of a 68-mer intramolecular dou ble-stranded RNA:DNA-hybridoligo-nucleotide (chimeraplasts) with unmodified phosphate backbone was employed. This construct was condensed with PS and coated with a liposomal formulation (AVE(TM)-3 = artificial viral envelope) . Results. PS-ON complexes and AVE(TM)-3-coated complexes with a defined comp osition were very effective in nuclear transport of ON for a ON:PS charge r atio of 1:3. Nucleus:cytosol fluorescence ratios peaked at about 10 hrs and started to decrease again at 21 hrs. Conclusions. AVE(TM) associates with PS-condensed ON, and this complex is a ble to be taken up by cells and to deliver ON to the nucleus. PS-ON complex es are released from the liposomal formulation, mainly as an extranuclear e nzymatic degradation of the liposomal phospholipids. The results of the kin etic analysis can be used to optimize transfection protocols with ON in Hep G2 cells.