ARTICLE |
Correspondence to: Katriina Lappalainen, Department of Pathology, University of Kuopio, PO Box 1627, 70211 Kuopio, Finland.
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Summary |
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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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Introduction |
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Oligonucleotides have been used for several years to control gene expression. They have also been shown to have potential as therapeutic agents. Several clinical trials are already in progress on their effectiveness in the treatment of acute myelogenous leukemia, human immunodeficiency virus, cytomegalovirus, and human papilloma virus infections (
Oligonucleotide-based techniques involve several problems, especially those associated with the stability, affinity, specificity, and cellular delivery of the oligos (
As for oligonucleotides, cationic liposomes have been widely used because of their several advantages over conventional delivery methods, such as spontaneous complex formation with negatively charged nucleic acids (
Although cationic liposomes have been successfully used as oligonucleotide carriers, the cellular uptake mechanisms and intracellular fate of liposomal oligonucleotides are not fully understood. Results from our previous studies suggest that liposomal ODNs are transported into the nucleus (
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Materials and Methods |
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Cell Culture
Adherent CaSki cells (a human cervical cancer cell line known to contain 600 copies of human papilloma virus 16 DNA) (
ODNs and Liposomes
Phosphodiester oligodeoxynucleotides purchased from Genosys (Genosys Biotechnologies; Cambridge, UK) had digoxigenin (DIG-ODNs) incorporated into the 5' end during synthesis and were purified by HPLC. The (anti-sense) sequence of DIG-ODN (16-mer) was 5'GTGTATCTCCATGCAT3', which is complementary to the initiation site of the E7 oncogene of HPV 16 (from nt 562). The cellular uptake of a random sequence, 5'CATCTGTATTGGATCG3', was also investigated.
A commercial polycationic transfection reagent, LipofectAMINE (Gibco BRL) containing 2,3-dioleoyloxy-N-[2 (sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminium trifluoroacetate (DOSPA) and dioleoyl-phosphatidylethanolamine (DOPE) (3/1 w/w) or a monocationic liposomal preparation containing dimethyldioctadecylammonium bromide (DDAB) and DOPE (2/5 w/w) was used. The liposomes containing DDAB and DOPE were prepared as described by
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Incubation of DIG-ODNs and Cationic Liposomes in CaSki Cells
On the basis of our earlier studies (
Immunocytochemistry
Detection of DIG-labeled ODNs was based on a direct immunocytochemical technique using horseradish peroxidase-labeled sheep polyclonal antibody against digoxigenin (anti-DIG-POD).
Processing of CaSki Cells. After incubation with DIG-ODNs and cationic liposomes, CaSki cells were fixed in 3% paraformaldehyde (EM grade; TAAB, Berks, UK) and 0.5% glutaraldehyde (EM grade; TAAB) in 0.1 M phosphate buffer (PB, pH 7.4) for 15 min. After washings, the cells were treated with 1% NaBH4 for 5 min to reduce free aldehyde groups and double bonds. After extensive washings, CaSki cells were immersed in 25% sucrose in 0.05 M PB for 5 min and freeze-thawed three times using liquid nitrogen to increase the penetration of the antisera during immunostaining.
Immunostaining. The samples were washed three times using 0.05 M Tris-buffered saline (TBS, pH 7.4). At the first washing, 0.2% Triton X-100 in TBS was used for 5 min. After incubation in 10% normal goat serum (NGS, 15 min) in TBS and washing in 1% NGS in TBS, the samples were incubated with horseradish peroxidase-conjugated anti-DIG antibody Fab fragments (anti-DIG-POD, 1:500; Boehringer Mannheim, Pentzberg, Germany) in 1% NGS in TBS at 4°C overnight. The immunoperoxidase reaction was developed using 3,3'-diaminobenzidine hydrochloride (DAB) as chromogen.
Controls. Three controls were used: (a) cells without DIG-ODNs and liposomes; (b) cells with cationic liposomes, but without oligonucleotides; and (c) cells treated with liposomal DIG-ODNs, omitting anti-DIG incubation during immunostaining.
Sample Preparation for Light Microscopy.
After immunostaining, the immunoperoxidase reaction product was intensified using 0.02% OsO4'PS8 in PB for 4 min. The samples were dehydrated in ethanol and treated with xylene for 5 min before mounting in DePex (Gurr; BDH, Poole, UK). At the light microscopic level the experiments were repeated three times in duplicate.
Sample Preparation for Electron Microscopy (EM). After immunostaining, the samples were treated with 1% OsO4 in PB for 1 hr. After staining with 2% uranyl acetate and dehydration, the samples were embedded in Epon (Fluka; Buchs, Switzerland). Ultrathin sections were cut on copper grids and stained with uranyl acetate and lead citrate. Some sections were left unstained for detection of any immunoperoxidase reaction in the nucleus. The sections were analyzed using JEOL JEM-1200 EX transmission EM. The experiments were performed twice in duplicate for electron microscopy. Many sections were cut from the samples for screening the intracellular localization of immunopositivity.
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Results |
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Immunopositive Signals on Plasma Membrane After 5 Min of Incubation
On light microscopy, immunopositive signals for DIG-ODNs delivered by DDAB/DOPE were already seen on the plasma membrane and in the cytoplasm after 5 and 20 min (Figure 1B) of incubation, respectively. After 1.5 and 4 hr (Figure 1E) of incubation, immunopositivity for DIG-ODNs with DDAB/DOPE was localized to the perinuclear or nuclear area.
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The immunopositive signals were strongest after 1.0 µM DIG-ODN and DDAB/DOPE treatment. Signals from DIG-ODNs delivered by DDAB/DOPE were more intense earlier (5 min, 20 min, 1.5 hr) than those delivered by DOSPA/DOPE, as seen with CaSki cells after 20 min of incubation with liposomal DIG-ODNs in Figure 1B and Figure 1C. Most cells (over 95%) contained immunopositivity after 1.5-hr incubation of 1.0 µM DIG-ODN with DDAB/ DOPE (Figure 1E). However, some immunopositivity from DIG-ODNs delivered using DOSPA/DOPE was also seen in the cytoplasm and perinuclear (or nuclear) area after 20 min (Figure 1C) and 1.5 hr of incubation, respectively. The difference disappeared after 4 hr (Figure 1E and Figure 1F), and after 24 hr the signals were slightly more intense from DIG-ODNs delivered using DOSPA/DOPE (Figure 1I) than from those delivered using DDAB/DOPE (Figure 1H). At ODN concentrations of 0.1 and 0.2 µM, only weak immunopositivity was seen with DOSPA/DOPE in CaSki cells, whereas intense immunoreactivity was seen with DDAB/DOPE. When cationic liposomes were not used, DIG-ODNs showed only weak immunoreactivity at all concentrations and observation times. Immunopositivity for liposomal random DIG-ODN was similar to that for liposomal anti-sense DIG-ODN.
Immunoreactivity in Intracellular Vesicles
At the ultrastructural level, liposomal DIG-ODNs were taken up into small intracellular vesicles, which were sometimes fused into large multivesicular bodies in the cytoplasm (Figure 2C-E). In addition, immunopositive plasmalemmal vesicles, open at the cell surface, could often be observed (Figure 2E). Irrespective of the oligo concentration or the type of liposome, the oligo-liposome complexes were similarly internalized. In the cytosol, liposomes were localized in the vicinity of immunoreactive signals for DIG-ODNs (Figure 2F). Interestingly, the plasma membrane and the envelopes of the intracellular vesicles frequently showed high immunoreactivity (Figure 2C-E). Although light microscopy showed possible weak immunoreactivity in a few CaSki cells treated with DIG-ODNs without liposomes (Figure 1D), no immunopositive signals were detected at the ultrastructural level. By contrast, cells treated with liposomes but without DIG-ODNs (Figure 1G) or cells without anti-DIG incubation were found immunonegative on light and electron microscopy.
Differences in Intracellular Distribution
After 1.5 hr of incubation of CaSki cells treated with DIG-ODNs at a concentration of 1.0 µM and DDAB/DOPE (when the oligo-lipid complex had a negative net charge; Table 1), most immunoreactive signals were still located in the intracellular vesicles (Figure 2E). With a longer incubation time (4 hr), most immunopositivity was found in the cytosol (Figure 2F). Although immunopositivity was detected in the perinuclear area, no immunoreactivity was seen within the nucleus (Figure 2F). After 24 hr of incubation, high immunoreactivity was still detected in the cytosol and nuclear envelope, but no immunopositivity was seen in the nucleoplasm (Figure 2G). The possible positive signals from the immunoperoxidase reaction in the nucleus had to be confirmed using ultrathin sections unstained by uranyl acetate and lead citrate (Figure 2H), since the chromatin of the nucleus is intensively stained with these agents (Figure 2A), hindering identification of any immunoperoxidase reaction product in the nucleus.
CaSki cells treated with DIG-ODNs at concentrations of 0.1 or 0.2 µM and DDAB/DOPE (when the oligo-lipid complexes were positive; Table 1) showed immunopositivity in their cytosol and perinuclear areas after only 1.5 hr of incubation (Figure 2I). The important finding was that although most immunopositivity was located on the nuclear envelope, some was also seen in the nucleoplasm after 4 hr of incubation (Figure 2J). After 24 hr of incubation, staining was less intense and no immunopositivity was seen in the nucleoplasm (Figure 2K and Figure 2L). Treatment of cells with ODNs (0.1, 0.2 or 1.0 µM) and DOSPA/DOPE (when the oligo-lipid complexes were positive; Table 1) did not produce any immunoreactivity in the nucleoplasm at any observation time. By contrast, immunoreactivity in the cytosol and nuclear membrane weakened with decreasing oligo concentrations and was similar to that in the DDAB/DOPE delivery system.
General Morphological Observations
In light microscopy there were no differences in cellular uptake between random and anti-sense oligos, regardless of the carrier system or oligo concentration used. At the EM level, random and anti-sense oligos were compared at the concentration of 1.0 µM after 1.5-hr incubation and no differences were observed. More foaming (bubbling) in the cytosol (Figure 1G) and blebbing of the plasma membrane (Figure 1F) occurred in cells treated with cationic liposomes than in control cells. However, cell membranes and nuclei remained intact after liposome treatment. The changes were more frequent with DOSPA/DOPE than DDAB/DOPE. Liposomes and liposomal DIG-ODNs also caused a slight reduction in cell growth after 24-hr incubation. No effects on cell growth or cell shape could be observed after DIG-ODN treatment without liposomes. Shrunken and rounded cells phagocytosed by adjacent cells (indicating possible apoptosis) were observed both in the control samples and in the samples treated with liposomes and DIG-ODNs. These changes were observed both in light and electron microscopy and are in agreement with the finding of
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Discussion |
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The most important findings of the present study were as follows: (a) liposomal ODNs are efficiently taken up by endocytosis irrespective of the charge of the ODN/liposome complex; (b) the charge of the ODN-DDAB/DOPE complex and the type of liposome affected intracellular distribution; and (c) the nuclear envelope was a barrier against the penetration of ODNs delivered by liposomes into the nucleus of CaSki cells.
Our study showed that both negatively and positively charged oligo-liposome complexes were rapidly and effectively taken up during endocytosis, released into the cytosol, and transported into the perinuclear area. Both polycationic and monocationic liposomes transferred ODNs along the endocytotic pathway. This is in agreement with recent studies in which the main route of liposomal DNA was found to be endocytosis and thereafter its release into cytosol (
In the presence of DDAB/DOPE, more ODNs were seen inside CaSki cells when ODNs were used at the highest concentration (1.0 µM). By contrast, at lower concentrations (0.1 and 0.2 µM), ODNs were released from intracellular vesicles into the cytosol and carried more rapidly into the perinuclear area. This suggests that ODNs are released more rapidly from intracellular vesicles into the cytosol if the oligo-liposome complex has a positive net charge, as shown with model membranes (
ODNs were delivered more rapidly into cells with DDAB/DOPE than with DOSPA/DOPE. At lower ODN concentrations (0.1 and 0.2 µM), DDAB/DOPE de-livered ODNs more effectively than DOSPA/DOPE did, although no remarkable differences occurred at higher concentration. Because our immmunocytochemical method is only semiquantitative, no firm conclusions can be drawn about the amount of intracellular ODNs. One explanation for the difference in delivery effectiveness may be that the amount of the helper lipid DOPE was lower in the DOSPA/DOPE preparation than in the DDAB/DOPE preparation. According to our measurements, particles were smaller in DOSPA/DOPE than DDAB/DOPE. The small particle size (<5 nm) has been reported to correspond with micelles rather than with liposomes (
Our EM study showed no ODNs inside CaSki cells incubated without cationic liposomes. However, the weak immunoreactivity in CaSki cells detected in light microscopy after incubation with ODNs but without liposomes suggests that some non-liposomal ODNs can be taken up by CaSki cells. Recently, a study at the ultrastructural level demonstrated non-liposomal ODNs in intracellular vesicles, indicating that the cellular uptake of ODNs also depends on endocytosis (
Using EM, our study is the first to show that the nuclear envelope can form a barrier against the penetration of liposomal ODNs into the nucleus of CaSki cells. This might be explained by the persisting association of ODNs with liposomes after release into the cytosol (
Only a few studies have investigated the nuclear localization of ODNs at the EM level (
Because the intracellular distribution may vary according to the cell type, each delivery system should be tested in the cells that would be the target cells in vivo. The CaSki cells used in the present study are known to contain human papillomavirus (HPV) DNA. Several molecular biological and epidemiological data have, in turn, provided evidence for the role of HPV infection in cervical cancer (
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Footnotes |
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1 Supported by a research contract from the Medical Research Council of the Academy of Finland, by the Finnish Cancer Fund, Research and Science Foundation of Farmos, and by the Finnish Cultural Foundation.
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