Intracellular distribution and mechanism of delivery of oligonucleotides mediated by cationic lipids.
OBJECTIVE
To study the parameters influencing the intracellular trafficking of oligonucleotides delivered by cationic 1,2-dioleoyl-3-trimethy- lammonium-propane (DOTAP) lipids and to elucidate the mechanism of uptake.


METHODS
We have studied the intracellular localization of fluorescently labeled oligonucleotide (F-ODN) delivered by DOTAP using confocal microscopy and measured inhibition of luciferase synthesis. The delivery mechanism of ODN/DOTAP complexes was investigated using inhibitors of the endocytosis pathway.


RESULTS
F-ODN delivered by DOTAP liposomes redistribute from punctate cytoplasmic regions into the nucleus. The nuclear uptake of F-ODN depends on: charge ratio (+/-), time of incubation, temperature and presence of serum. A positively charged complex is required for enhanced uptake. The association of neutral lipids with DOTAP reduced the optimum charge ratio without altering the delivery efficiency. DOTAP lipids increased > 100 fold the antisense activity of a specific anti-luciferase ODN. Inhibitors of the endocytosis pathway show that the majority of F-ODN are introduced through an endocytic pathway mainly involving uncoated vesicles. Nuclear accumulation of oligonucleotides can be decreased by inhibitors of actin microfilaments, energy metabolism and proteins implicated in the fusion of endosomes. Nuclear uptake is independent of acidification of the endosomal vesicles and unaffected by inhibitors of microtubules.


CONCLUSIONS
Oligonucleotides are delivered by cationic lipids into the cytoplasm at an early stage of the endocytotic pathway which leads to a marked increase in antisense activity and oligonucleotide nuclear uptake.
University of California, School of Pharmacy, Department of Biopharmaceutical Sciences and Pharmaceutical Chemistry, San Francisço 94143-04461, USA.
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D001692:Biological Transport / Q000187:drug effects; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D002784:Cholesterol / Q000494:pharmacology; D003571:Cytochalasin B / Q000494:pharmacology; D003600:Cytosol / Q000378:metabolism; D004705:Endocytosis / Q000187:drug effects / Q000502:physiology; D005229:Fatty Acids, Monounsaturated / Q000737:chemistry* / Q000378:metabolism; D005456:Fluorescent Dyes / Q000737:chemistry* / Q000378:metabolism; D008081:Liposomes / Q000737:chemistry; D008156:Luciferases / Q000378:metabolism; D016376:Oligonucleotides, Antisense / Q000737:chemistry / Q000378:metabolism*; D010714:Phosphatidylethanolamines / Q000494:pharmacology; D000644:Quaternary Ammonium Compounds / Q000737:chemistry* / Q000378:metabolism
D005229:Fatty Acids, Monounsaturated; D005456:Fluorescent Dyes; D008081:Liposomes; D016376:Oligonucleotides, Antisense; D010714:Phosphatidylethanolamines; D000644:Quaternary Ammonium Compounds; D003571:Cytochalasin B; D002784:Cholesterol; D008156:Luciferases; C070046:1,2-dioleoyloxy-3-(trimethylammonium)propane