ARTICLE |
Correspondence to: An Cao, Laboratoire de Chimie Structurale et Spectroscopie Biomoléculaire, CNRS UMR 7033, UFR de Médecine, Université Paris XIII, 74 rue Marcel Cachin, F93017 Bobigny Cedex, France. E-mail: cao@smbh.univ-paris13.fr
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Summary |
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We investigated by transmission electron microscopy the cellular route in tumor MCF7 cells of DNA labeled with digoxigenin, carried by cationic liposomes (Lip+) prepared from TMAEC-Chol [3ß(N-(N',N',N'-trimethylaminoethane)-carbamoyl)cholesterol iodide] and TEAPC-Chol [3ß(N-(N',N',N'-triethylaminopropane)-carbamoyl)cholesterol iodide], two cholesterol-based cationic lipids containing a quaternary ammonium. In a previous work we showed the pathway of cationic lipid/plasmid complexes from the beginning of endocytosis until their entry into the perinuclear area. Beyond this limit, unlabeled exogenous plasmids cannot be distinguished with nuclear DNA. This work dealt with the cellular fate of cationic liposome-vectorized plasmids labeled with digoxigenin using an immunogold procedure. Early after the beginning of transfection (30 min, 1 hr, 5 hr), gold particles were observed only in the cytoplasm and in endosome-like vesicles, whereas after 24 hr gold particles were densely present in the nucleus. These results demonstrate the nuclear localization of plasmids vectorized by the cationic liposomes used. The results are discussed in comparison with transfection efficiency measurements.
(J Histochem Cytochem 50:983991, 2002)
Key Words: cationic lipids, plasmids, MCF7 cells, digoxigenin, immunogold electron, microscopy, gene transfer
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Introduction |
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NON-VIRAL CARRIERS for gene transfer, especially cationic lipids, are more and more often investigated (
There are an increasing number of works to study the cellular pathway of DNA/lipid complexes (
In this work we used liposomes prepared from two cholesterol-based cationic lipids, TEAPC-Chol [3ß (N-(N',N',N'-triethylaminopropane)-carbamoyl)cholesterol iodide] and TMAEC-Chol [3ß(N-(N',N',N'-trimethylaminoethane)-carbamoyl)cholesterol iodide]. The efficiency of these liposomes has been shown in a previous work (
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Materials and Methods |
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Materials
Dioleoyl phosphatidylethanolamine (DOPE) was furnished by Aventi-Polar Lipid and was used without other purification. Reagents were analytical grade and were purchased from Aldrich-Sigma (St Louis, MO), Carlo Erba (Milano, Italia), and Fluka (Buchs, Switzerland). The digoxigenin DNA labeling kit was purchased from Roche Diagnostics (Meylan, France). Ultrasmall gold-labeled anti-digoxigenin sheep antibody Fab fragments, acetylated BSA (bovine serum albumin) (BSA-C), and the silver enhancement reagent were from Aurion (Wageningen, The Netherlands).
Cationic Lipids
The cationic lipids TMAEC-Chol and TEAPC-Chol used for this work were synthesized and characterized by Fourier transform infrared spectroscopy. The synthesis of TMAEC-Chol has been previously described (
Liposome Preparation
The cationic lipids TMAEC-Chol or TEAPC-Chol and DOPE (1:1) were dissolved and mixed in chloroform and the solution was dried in a rotating evaporator. Solvent trace was removed under vacuum overnight. The following day, sterile water (Millipore; Bedford, MA) was added and the mixture was sonicated for 1 hr in cycles of 15 min (13 min followed by 2 min rest) to clarity. The liposome preparations were controlled by negative staining electron microscopy and their size was characterized by quasi-elastic light scattering (QLS) using an apparatus constructed by our laboratory. Polystyrene latex spheres of 109 nm in aqueous solution were used for the calibration.
Plasmid
The plasmid used for this work was a plasmid of 7.164 kb including the ß-galactosidase reporter gene sequence under the control of the promoter of cytomegalovirus CMV (pCMV-ß), furnished by Promega (Madison, WI) and propagated according to the standard method using a maxiprep kit purchased from Qiagen France (Courtaboeuf, France) and conditioned in TrisEDTA buffer, pH 8.
Preparation of Digoxigenin-labeled Plasmids
In a sterile vial, 0.4 µl of reactive digoxigenin (DIG-Chem-Link from Roche, Meylan, France) was added to 2 µg of pCMV-ß plasmid in a final volume of 20 µl. After incubation for 30 min at 50C in a waterbath, the reaction was stopped by adding 5 µl of the stop solution and the solution was stored at -20C before use.
Formation of LiposomePlasmid Complexes
Plasmids (1 µg) and liposomes with known concentrations were separately diluted in sterile water to have 10 µl each, and then mixed. The plasmidliposome complexes formed instantaneously and were used within 30 min for transfection.
Gel Electrophoresis
Control of Digoxigenin-labeled Plasmids.
One µg of unmodified or modified plasmid was loaded on an agarose gel (1%) containing ethidium bromide in 1 x TBE (Tris buffered EDTA) buffer and analyzed electrophoretically under an electric voltage of 100 V (about 400 mA).
Retardation Control of DNALiposome Complex Formation. Formation of complexes between plasmid DNA and cationic liposomes results in a retardation of DNA movement and can be monitored by a gel retardation control prepared at various cationic lipid/DNA molar charge ratios. After 15 min of incubation at room temperature (RT), aliquots of complexes were mixed with the loading buffer (Blue 6X loading dye; Promega) and analyzed electrophoretically under the same conditions.
Cell Culture
Established adherent MCF7 cells (human breast carcinoma cells) were grown on plastic ware at 37C in a humid atmosphere containing 5% CO2 in air. The culture medium DMEM was supplemented with fetal calf serum (10%), penicillin/streptomycin (50 U/ml), and glutamine (2 mM).
Observation of Labeled Plasmids by Immunogold Electron Microscopy
Cells dissociated from the wells of the plate were fixed at 4C for 20 min with 1% paraformaldehyde and 0.1% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4 (PB). After washing the cells were treated for 10 min with 0.05% NaBH4 and 0.1% glycine in PB, to block the free aldehyde groups and to permeabilize the cells. The cells were then rinsed in PB and pre-incubated in 0.2% acetylated BSA (BSA-C) in PB for 30 min. They were then incubated for 2 hr at RT with diluted ultrasmall gold-labeled anti-digoxigenin antibody Fab fragments in 0.2% BSA-C in PB. The cells were rinsed in 0.2% BSA-C in PB, then in PB, and refixed with 2.5% glutaraldehyde for 10 min. After washing in PB, the samples were postfixed in 0.5% OsO4 in PB for 20 min, washed in de-ionized water, and treated with the silver enhancement reagent. After washings in distilled water, samples were stained with 0.5% uranyl acetate in 50% ethanol, dehydrated in a graded series of alcohols, embedded in Epon, sectioned, and examined with a Philips EM300 transmission electron microscope operating at 60 kV.
Transfection Efficiency
The efficiency of the transfection in cells was estimated by the X-gal test. The presence of expressed ß-galactosidase was observed by the blue staining with X-gal reagent. For this test, a 4-well permanox Lab-Tek plate (Nunc) was seeded with 4.104 MCF7 cells/well. Plasmids pCMV-ß (0.5 or 1 µg) and cationic liposomes at the molar charge ratio X = 2 were complexed and incubated with cells for 48 hr at 37C in 5% CO2. After the incubation, cells were washed with PBS, fixed with paraformaldehyde (1%) and glutaraldehyde (0.02%) for 3 min before the X-gal reagent (1 mg/ml X-gal, 5 mM potassium ferrocyanide, 5 mM potassium ferricyanide, 2 mM MgCl2) was added. Microscopic observation was performed 6 hr later.
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Results |
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Characterization of Liposomes and Efficiency of the PlasmidLiposome Complex Formation
Characterization of Cationic Liposomes.
QLS results indicated a size distribution of liposomes prepared with TMAEC-Chol/DOPE (1:1) or TEAPC-Chol/DOPE in water with mean sizes around 124 nm and 104 nm, respectively, and a polydispersity factor of 0.33 and 0.23. These liposomes were very stable. TEM showed a thickness of about 3 nm for the liposomal membrane, indicating a single bilayer, and demonstrated that liposomes are unilamellar.
Control of Plasmids Labeled with Digoxigenin. Plasmids labeled with digoxigenin were controlled by gel electrophoresis in comparison with native plasmid, as indicated in Fig 1. In this figure there is no difference between the migration of digoxigenin-labeled plasmid (Fig 1, Lane 2) and the native plasmid (Fig 1, Lane 3), suggesting that the circular supercoiled state was preserved.
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Efficiency of the Formation of PlasmidLiposome Complexes
After mixing of the same amount of plasmids with various amounts of liposomes, the solution containing pure plasmids and the solution containing the complexes were exposed to electrophoresis for a gel retardation observation.
Complexing of plasmids pCMV-ß with TMAEC-Chol/DOPE liposomes is shown in Fig 2. Lane 1 indicates molecular weight markers. Free plasmids migrated in Lane 2, whereas Lanes 3, 4, and 5 indicate plasmids mixed with TMAEC-Chol/DOPE liposomes in various charge molar ratios, X = 0.5, 2, and 4, respectively. In Fig 2, Lane 3 there is a trace of plasmids not entirely retained by liposomes but there is no print of free plasmids in Lanes 4 and 5, indicating a complete complexing of plasmids with liposomes when the molar charge ratio of lip(+)/DNA is greater than 2.
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It was the same for pCMV-ß complexed with TEAPC-Chol/DOPE liposomes. As shown in Fig 2, Lanes 612 show plasmids when mixed with liposomes in various charge molar ratios 0.5, 1, 1.3, 1.5, 1.7, 2, and 4 in the absence of serum. In Lane 10 (X = 1.7) there is a trace of plasmids not entirely retained by liposomes but there is no print of free plasmids in Lanes 11 (X = 2) and 12 (X = 4), indicating a complete complexing of plasmids with liposomes when the molar charge ratio of lip(+)/DNA is greater than 1.7. DNA can be retained 100% when the molar charge ratio exceeds this value.
These results can be explained as follows. One nucleotide (with one negative charge) was bound to one cationic lipid molecule (with one positive charge) on the external surface of the liposomes and therefore twice the number cationic lipid molecules is necessary if the liposomal structure was conserved. This result clearly indicates the electrostatic behavior of the binding between DNA to TMAEC-DOPE liposomes. In all cases, the yield of the formation of liposomeDNA can reach 100% for plasmids when the molar charge ratio is appropriately chosen.
Delivery of Vectorized DNA into MCF7 Cells
Transfection Efficiency.
The transfection efficiency was estimated by X-gal (5-bromo-4-chloro-3-indolyl ß-D-galactopyranoside) test, yielding a blue staining of expressed ß-galactosidase as shown in Fig 3. A control was performed with untransfected cells (Fig 3A), showing no blue stains due to endogenous ß-galactosidase. The aspect is very different with cells transfected by plasmids, showing large blue stains. Cells transfected by unvectorized plasmids (Fig 3B) present practically no blue cells. Cells transfected with 0.5 and 1 µg pCMV-ß vectorized by liposomes are shown in Fig 3C and Fig 3D. The fraction of blue-stained cells was estimated by counting, with an efficiency of about 50%. This indicates not only the internalization of the gene but also the conservation of its functionality revealed by the expressed protein.
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Observation of Plasmids by Immunogold Labeling. To follow the route of plasmids, the latter were labeled with digoxigenin and detected by anti-digoxigenin Fab coupled to ultrasmall gold particles. The size of gold particles was increased by a silver enhancement reagent. Fig 4A and Fig 4B show controls by untransfected cells treated with silver alone or with antibodies followed by silver. The micrographs do not show any dark staining and clearly indicate that, in the absence of plasmids labeled with digoxigenin, there are no dark spots. Fig 4C shows a cell 24 hr after transfection with plasmid pCMV-ß unvectorized by liposomes. Few gold particles are observed in the cytoplasm, indicating the presence of rare plasmids.
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Fig 4D4F show cells exposed to plasmids for 15 min and 30 min. At 15 min after incubation of cells with the complexes, black gold particles were seen only at the cell surface or in the cytoplasm but no particles were observed in the nucleus (Fig 4D). At 30 min after incubation, as at 15 min, gold particles were observed only in the cytoplasm and not in the nucleus (Fig 4E and Fig 4F). Cell components lightly stained by uranyl acetate do not contribute to these very dark patterns.
The micrograph in Fig 5A of an MCF7 cell transfected for 1 hr shows many gold particles but only in the cytoplasm and in electron-dense vesicles. No gold particles were seen in the nucleus. This indicates that plasmids, or at least complexes of plasmidlipid, are already internalized in the cell. Gold particles in the dense vesicles may represent endosomes containing plasmidlipid complexes. In the micrographs showing cells after 5 hr (Fig 5B), particles are still present in the cytoplasm and a few begin to appear in the nucleus of some cells. For cells observed at 24 hr after transfection there are many more plasmids in the nucleus, although some gold particles still exist outside the nucleus (Fig 5C and Fig 5D). Occasionally, gold particles are still present in protrusions of the cell surface. Magnifications of some portions of the nuclei show several gold particles in the nucleolus and nucleus (Fig 5C and Fig 5D). After 24 hr of incubation many but not all cells have immunopositive nuclei.
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Liposomes prepared from cationic lipid TEAPC-Chol were also used to carry plasmids. As early as 6 hr after addition of complexes to MCF7 cells, plasmids were observed in the nucleus, as shown by a high-magnification field showing gold particles in nuclear compartments (Fig 6A). At 24 hr after incubation, low magnification of an entire transfected cell revealed gold labeling especially in the nucleus, as for TMAEC-Chol/DOPE liposomes (Fig 6B). This indicates the similar behavior of the series of cationic lipids containing a quaternary ammonium head group.
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Discussion |
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The most important result in this work is that, with the immunogold technique, digoxigenin-labeled plasmids transferred by cationic liposomes have been visualized in the nucleus of the transfected cell. Until now, digoxigenin was used only for labeling of oligonucleotides (
Several controls were performed to ensure the validity of this technique. First, the Dig label of plasmid did not modify the structural integrity, as assessed by electrophoresis. Second, no black staining was observed in cells when samples were submitted to the silver enhancement treatment alone. Third, cells not incubated with the Dig plasmidliposome complexes but treated with gold anti-Dig antibodies and silver showed no immunoreactivity in the cytoplasm or in the nucleus, indicating that there was no nonspecific binding in cell preparations. When the cells were incubated with the Dig plasmid without cationic liposomes as carriers and further treated by the anti-Dig antibodies and silver, there was no immunoreactivity in most cells. Occasionally in some cells, rare gold particles were observed as shown in Fig 4C. Therefore, with these negative controls the presence of dark particles ensures the presence of gold particles and of Dig-labeled plasmids when plasmids are transferred by cationic liposomes (Fig 5).
The kinetics of plasmids carried by TMAEC-Chol/DOPE liposomes was studied and showed that, after the cytoplasmic step, TEM can prove that DNA penetrates inside the nucleus, as revealed by immunogold labeling. The time course is respected: at 15 min after transfection only very few plasmid copies were observed in the cytoplasm, and then 24 hr later there are many more plasmid copies, especially in the nucleus. Since we know that previous works have demonstrated lipids only in the cytoplasm and not inside the nucleus, it cannot be doubted that DNA was dissociated from lipids.
To understand these results, it is useful to recall some results of our previous work concerning the ultrastructure of lipoplexes and their cellular pathway (
The passage into the nucleus is a necessary step for the observed expression of the gene. This passage across the nuclear envelope may be due to various mechanisms, such as mitosis (
It is worth noting that in previous works (
Finally, it is important to emphasize that high efficiency can be obtained in the complexing of DNA and TMAEC-Chol/DOPE or TEAPC-Chol/DOPE liposomes. This high efficiency is confirmed with the plasmid, as demonstrated by gel retardation, that complete complexing occurred when the molar ratio was higher than 1.7 or 2, depending on the lipid.
In conclusion, the results of this work show that the digoxigenin-labeled plasmid DNA immunogold procedure is a useful method to detect exogenous DNA in cells by transmission electron microscopy. With this technique, we showed that plasmids are efficiently carried into MCF7 cells by cationic liposomes prepared from TMAEC-Chol and TEAPC-Chol. The functionality of the reporter genes carried by these liposomes was preserved inside the nuclei, resulting in the observed expression of ß-galactosidase in accordance with the passage of plasmids into the nuclei.
Received for publication December 19, 2001; accepted February 6, 2002.
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