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Journal of Histochemistry and Cytochemistry, Vol. 45, 265-274, Copyright © 1997 by The Histochemical Society, Inc.

ARTICLE

Intracellular Distribution of Oligonucleotides Delivered by Cationic Liposomes: Light and Electron Microscopic Study1

Katriina Lappalainena, Riitta Miettinenb, Jari Kellokoskic, Ilpo Jääskeläinend, and Stina Syrjänena,e
a MediCity Research Laboratory, University of Turku, Turku, Finland
b Department of Neurology, University of Kuopio, Kuopio, Finland
c Department of Pathology, University of Kuopio, Kuopio, Finland
d Pharmaceutical Technology, University of Kuopio, Kuopio, Finland
e Department of Dentistry, University of Turku, Turku, Finland

Correspondence to: Katriina Lappalainen, Department of Pathology, University of Kuopio, PO Box 1627, 70211 Kuopio, Finland.

Synthesized oligonucleotides are used in anti-sense and anti-gene technology to control gene expression. Because cells do not easily take up oligonucleotides, cationic liposomes have been employed to facilitate their transport into cells. Athough cationic liposomes have been used in this way for several years, the precise mechanisms of the delivery of oligonucleotides into cells are not known. Because no earlier reports have been published on the liposomal delivery of oligonucleotides at the ultrastructural level, we performed a study, using electron microscopy, on the cellular uptake and intracellular distribution of liposomal digoxigenin-labeled oligodeoxynucleotides (ODNs) at several concentrations (0.1, 0.2, and 1.0 µM) in CaSki cells. Two cationic lipids (10 µM) were compared for transport efficiency: polycationic 2,3-dioleoyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminium trifluoroacetate (DOSPA) and monocationic dimethyl-dioctadecylammonium bromide (DDAB). Both liposomes contained dioleoyl-phosphatidylethanolamine (DOPE) as a helper lipid. Endocytosis was found to be the main pathway of cellular uptake of liposomal ODNs. After release from intracellular vesicles, ODNs were carried into the perinuclear area. The nuclear membrane was found to be a barrier against the penetration of ODNs delivered by liposomes into the nucleus. Release from vesicles and transport into the nuclear area was faster when the oligo-DDAB/DOPE complex had a positive net charge (0.1 and 0.2 µM ODN concentrations), and only under this condition were some ODNs found in nucleoplasm. Although DOSPA/DOPE could also efficiently deliver ODNs into the cytosol, no ODNs were found in nucleoplasm. These findings suggest that both the type of liposome and the charge of the oligo-liposome complex are important for determination of the intracellular distribution of ODNs. (J Histochem Cytochem 45:265-274, 1997)

Key Words: Oligodeoxynucleotides, Anti-sense, Cationic liposomes, Digoxigenin label, CaSki cells, Electron microscopy, Electron immunocytochemistry


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