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Intracellular distribution of oligonucleotides delivered by cationic liposomes: light and electron microscopic study
K Lappalainen1, R Miettinen, J Kellokoski
1MediCity Research Laboratory, University of Turku, Finland.
Abstract:
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. Although 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, an 1.0 microM) in CaSki cells. Two cationic lipids (10 microM) 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 microM 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.
Insights
Cationic liposomes deliver gene silencing oligonucleotides (ODNs) into cells mainly via endocytosis. The nuclear membrane limits ODN entry, but positively charged complexes facilitate faster nuclear transport, with some ODNs reaching the nucleoplasm.
Area of Science:
- Biochemistry
- Cell Biology
- Nanotechnology
Background:
- Oligonucleotides (ODNs) are crucial for gene expression control via anti-sense and anti-gene technologies.
- Cationic liposomes enhance cellular uptake of ODNs, but delivery mechanisms remain unclear.
- Ultrastructural studies on liposomal ODN delivery are scarce.
Purpose of the Study:
- To investigate the cellular uptake and intracellular distribution of liposomal ODNs at the ultrastructural level.
- To compare the efficiency of two cationic lipids, DOSPA and DDAB, in delivering ODNs into CaSki cells.
- To determine the influence of liposome type and complex charge on ODN intracellular trafficking.
Main Methods:
- Electron microscopy was used to visualize the uptake and distribution of digoxigenin-labeled ODNs complexed with liposomes.
- CaSki cells were treated with liposomal ODNs at varying concentrations (0.1, 0.2, 1.0 microM).
- Two liposome formulations, DOSPA/DOPE and DDAB/DOPE, were compared for ODN delivery efficiency.
Main Results:
- Endocytosis was identified as the primary route for cellular uptake of liposomal ODNs.
- Following endocytosis, ODNs were transported to the perinuclear region, with the nuclear membrane acting as a barrier.
- Positively charged oligo-DDAB/DOPE complexes (at 0.1 and 0.2 microM) showed faster perinuclear transport and some nuclear penetration.
- DOSPA/DOPE efficiently delivered ODNs to the cytosol, but no nuclear entry was observed.
Conclusions:
- The cellular uptake pathway for liposomal ODNs is predominantly endocytosis.
- The nuclear membrane restricts ODN entry into the nucleus.
- Both liposome composition and the net charge of the oligonucleotide-liposome complex significantly impact intracellular ODN distribution and potential nuclear targeting.