Nuclear transport of oligonucleotides in HepG2-cells mediated by protamine sulfate and negatively charged liposomes.
OBJECTIVE
The aim of this study was to characterize the intracellular fate and nuclear uptake kinetics of oligonucleotides (ON) that were complexed with protamine sulfate (PS) and negatively charged liposomes at different ratios of ON to PS.


METHODS
Double-fluorescence labelling of ON and liposomal lipid was applied to simultaneously monitor the interaction as well as the individual fate of active agent and carrier upon intracellular delivery using confocal laser scanning microscopy (CLSM). A DNA-analogue of a 68-mer intramolecular double-stranded RNA:DNA-hybridoligonucleotide (chimeraplasts) with unmodified phosphate backbone was employed. This construct was condensed with PS and coated with a liposomal formulation (AVE-3 = artificial viral envelope).


RESULTS
PS-ON complexes and AVE -3-coated complexes with a defined composition were very effective in nuclear transport of ON for a ON:PS charge ratio of 1:3. Nucleus:cytosol fluorescence ratios peaked at about 10 hrs and started to decrease again at 21 hrs.


CONCLUSIONS
AVE associates with PS-condensed ON, and this complex is able to be taken up by cells and to deliver ON to the nucleus. PS-ON complexes are released from the liposomal formulation, mainly as an extranuclear enzymatic degradation of the liposomal phospholipids. The results of the kinetic analysis can be used to optimize transfection protocols with ON in HepG2 cells.
Welz
C
C
Department of Pharmaceutics and Biopharmacy, Philipps-University Marburg, Germany.
Neuhuber
W
W
Schreier
H
H
Metzler
M
M
Repp
R
R
Rascher
W
W
Fahr
A
A
D000838:Anions; D001692:Biological Transport; D002467:Cell Nucleus / Q000378:metabolism*; D004247:DNA / Q000008:administration & dosage / Q000737:chemistry / Q000493:pharmacokinetics; D006801:Humans; D008081:Liposomes; D018613:Microscopy, Confocal; D009841:Oligonucleotides / Q000008:administration & dosage* / Q000737:chemistry / Q000493:pharmacokinetics*; D011479:Protamines / Q000008:administration & dosage* / Q000737:chemistry / Q000493:pharmacokinetics*; D014407:Tumor Cells, Cultured
D000838:Anions; D008081:Liposomes; D009841:Oligonucleotides; D011479:Protamines; D004247:DNA