BRIEF REPORT |
Correspondence to: Regina Reszka, AG Drug Targeting, Max-Delbrück Center for Molecular Medicine, Robert-Rössle-Str. 10, 12092 Berlin, Germany. E-mail: reszka@mdc-berlin.de
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Summary |
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We present a simple method based on transmission electron microscopy that allows investigation of the early steps of polyplex-mediated transfection without the use of labeled DNA. The ultrastructural analysis showed internalization of 0.21-µm aggregates composed of 3050-nm subunits. In addition, new details of the internalization process were revealed, suggesting an unspecific cell entry mechanism of large DNA aggregates.
(J Histochem Cytochem 51:12371240, 2003)
Key Words: gene transfer, polyplexes, transfection mechanism, NLS
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Introduction |
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THE DEVELOPMENT of novel non-viral gene delivery systems will depend on a better understanding of the gene transfer mechanism. Although many investigations point to an endocytic pathway of DNA complexes, the major factors influencing each of the different steps remain unclear. In the case of cationic polymer/DNA complexes (polyplexes), critical issues concern the physicochemical properties of transfection active complexes as well as the internalization process. It is not clear whether DNA complexes interact with cell surface molecules or specific receptors and how internalization is regulated. Here we used a morphological approach to define ultrastructural properties of DNA complexes during the transfection process and to gain more insight into the internalization mechanism.
Peptide-mediated gene transfer was used as a model system to transfect human cancer cells in vitro (6.5 kb) (
We examined the peptide/DNA complexes after their formation in solution using transmission electron microscopy. Complexes were stained with uranyl acetate on a formvar-coated grid (
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The complexes used in this study have a positive potential (data not shown) and therefore can potentially interact with serum proteins. Hence, we investigated the morphology of the complexes that sedimented onto the cell surface in vitro by scanning electron microscopy. RPMI medium (900 µl) containing 10% FCS, 100 µM chloroquine, and 4 mM CaCl2 was added to the complexes, transferred onto 4 x 105 human colon carcinoma cells HCT 116, and incubated at 37C. Chloroquine was used to facilitate endosomal escape of endocytosed DNA complexes. CaCl2 was added as an enhancing factor of in vitro transfection systems (
At 30 min after transfection, the cells were fixed with 2% glutaraldehyde (Serva; Heidelberg, Germany) in 0.1 M phosphate buffer for 20 min. Then the samples were washed twice in PBS and dehydrated in a graded series of acetone (25, 50, 70, 90, 100%) before being critical point-dried. The samples were then coated with a 17-nm gold/palladium layer (80 sec, 40 mA) in an SCD 050 sputter coater (Balzers) and examined in a JEOL 25SIII scanning microscope operated at 25 kV. The peptide/DNA complexes observed on the cell surface (Fig 1D and Fig 1E) shared the same size and morphology as those detected in solution (Fig 1A). This indicates that the serum present in the transfection medium does not induce any apparent alterations of the aggregates.
Next we investigated the internalization of the complexes and their intracellular fate at the ultrastructural level. A simple method based on transmission electron microscopy was adapted from
Thirty minutes after transfection, dark stained aggregates were found close to the cell surface (Fig 2A) or located in plasma membrane invaginations (Fig 2C), suggesting an ongoing internalization. Aggregates were also found inside the intracellular compartments (Fig 2B and Fig 2D). Examination of 100 sections from three different experiments showed that the size of internalized aggregates varied from 0.2 µm to 1 µm. We cannot exclude the possibility that the fixation procedure used in this study induces aggregation of the complexes at the cell surface and also inside the endosomal compartment. Therefore, the actual size of the internalized material may be smaller and may originate from single or oligomeric DNA complexes. However, complexes that are not connected to large aggregates represent only a relatively small portion of the total complex population found in solution. Considering the relatively large amount of the internalized material, it is more likely that large aggregates are actually internalized. Therefore, we hypothesize that under the present conditions the tumor cells behave as non-professional phagocytes able to ingest large particles.
To demonstrate that the observed aggregates were in fact the DNA complexes prepared before transfection, the same experiments were performed using digoxigenin-labeled plasmid DNA (
The positive potential of the complexes also suggests their direct binding to the cell surface and subsequent cell entry via phagocytosis. According to the classical zipper-type phagocytosis model, one may expect extension of pseudopods along the particles, resulting in continuous contact between the particle and the cell membrane or the vacuole wall. However, at higher magnification only a few focal contact points were observed (Fig 3A and Fig 3B), and the internalized material was located in relatively spacious vacuoles shortly after engulfment (Fig 2D). Nevertheless, we cannot exclude a possible attachment to the surrounding glycocalyx, which is not visible using this technique. Additional experiments would be necessary to analyze the role of the glycocalyx during the internalization process. Receptor-mediated cell uptake involves the recruitment of cellular factors at the internalization site of the membrane. However, ultrastructural changes accompanying this process were not observed (Fig 3A and Fig 3B).
The intracellular pathway was further examined using both methods with labeled and unlabeled DNA (Fig 3C3E). At 4 hr after transfection, large vacuoles containing DNA were observed. The morphology of the DNA-containing structures suggests an ongoing degradation of a large aggregate (Fig 3C). Vacuoles with dark condensed structures were also observed (Fig 3D and Fig 3E). However, no DNA was identified within these structures (Fig 3D), suggesting an advanced degradation stage. This result also shows that the technique based on simple contrast staining is more suitable to study early steps of transfection before degradation of the DNA takes place. Taken together, these observations suggest a cell entry process by a nonspecific phagocytosis-like mechanism that leads to the fusion of phagosomes with the lysosomal compartment.
Chloroquine is used as an enhancing reagent in polyplex-mediated transfection, but its mechanism of action is not completely understood. It is believed to prevent acidification of endosomes or lysosomes, which inhibits the activation of degradative enzymes and reduces the degradation of endocytosed DNA complexes (
Calcium has been shown to enhance the efficiency of histone- and cationic peptide-based transfection systems, probably by inducing dissociation of the DNA complex in the endosome and also by facilitating its release into the cytoplasm (
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Footnotes |
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1 These authors contributed equally to this work.
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Acknowledgments |
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We are grateful to Peter Scherrer for reviewing the manuscript and to Marianne Vannauer for excellent technical assistance.
Received for publication September 24, 2002; accepted March 27, 2003.
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