INTRACELLULAR-DISTRIBUTION OF OLIGONUCLEOTIDES DELIVERED BY CATIONIC LIPOSOMES - LIGHT AND ELECTRON-MICROSCOPIC STUDY

Synthesized oligonucleotides are used in anti-sense and anti-gene tech nology 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 repo rts have been published on the liposomal delivery of oligonucleotides at the ultrastructural level, we performed a study, using electron mic roscopy, on the cellular uptake and intracellular distribution of lipo somal digoxigenin-labeled oligodeoxynucleotides (ODNs) at several conc entrations (0.1, 0.2, and 1.0 mu M) in CaSki cells. Two cationic lipid s (10 mu M) were compared for transport efficiency: polycationic ineca rboxamido)ethyl]-N,N-dimethyl-1-propanaminium trifluoroacetate (DOSPA) and monocationic dimethyl-dioctadecylammonium bromide (DDAB). Both li posomes contained dioteoyl-phosphatidylethanolamine (DOPE) as a helper lipid. Endocytosis was found to be the main pathway of cellular uptak e of liposomal ODNs. After release from intracellular vesicles, ODNs w ere 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 nuclea r area was faster when the oligo-DDAB/DOPE complex had a positive net charge (0.1 and 0.2 mu M ODN concentrations), and only under this cond ition were some ODNs found in nucleoplasm. Although DOSPA/DOPE could a lso efficiently deliver ODNs into the cytosol, no ODNs were found in n ucleoplasm. These findings suggest that both the type of liposome and the charge of the oligo-liposome complex are important for determinati on of the intracellular distribution of ODNs.