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Amphiphilic block copolymers enhance the cellular uptake of DNA molecules through a facilitated plasma membrane transport.

Chèvre R, Le Bihan O, Beilvert F, Chatin B, Barteau B, Mével M, Lambert O, Pitard B - Nucleic Acids Res. (2010)

Bottom Line: We also showed that this improvement in transfection efficiency associated with the presence of Lutrol® was observed irrespective of the plasmid promoter.Considering the various steps that could be improved by Lutrol®, we concluded that the nucleic acids molecule internalization step is the most important barrier affected by Lutrol®.Our results strongly suggest that in vivo, Lutrol® improves transfection by a physicochemical mechanism, leading to cellular uptake enhancement through a direct delivery into the cytoplasm, and not via endosomal pathways.

View Article: PubMed Central - PubMed

Affiliation: INSERM, U915, l'institut du thorax, Nantes, F-44000, France.

ABSTRACT
Amphiphilic block copolymers have been developed recently for their efficient, in vivo transfection activities in various tissues. Surprisingly, we observed that amphiphilic block copolymers such as Lutrol® do not allow the transfection of cultured cells in vitro, suggesting that the cell environment is strongly involved in their mechanism of action. In an in vitro model mimicking the in vivo situation we showed that pre-treatment of cells with Lutrol®, prior to their incubation with DNA molecules in the presence of cationic lipid, resulted in higher levels of reporter gene expression. We also showed that this improvement in transfection efficiency associated with the presence of Lutrol® was observed irrespective of the plasmid promoter. Considering the various steps that could be improved by Lutrol®, we concluded that the nucleic acids molecule internalization step is the most important barrier affected by Lutrol®. Microscopic examination of transfected cells pre-treated with Lutrol® confirmed that more plasmid DNA copies were internalized. Absence of cationic lipid did not impair Lutrol®-mediated DNA internalization, but critically impaired endosomal escape. Our results strongly suggest that in vivo, Lutrol® improves transfection by a physicochemical mechanism, leading to cellular uptake enhancement through a direct delivery into the cytoplasm, and not via endosomal pathways.

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Effect of block copolymers on the percentage of transfected cells. C2C12 cells were transfected after Lutrol® treatment (gray bars) or without Lutrol® treatment (white bars). Treated cells were incubated with Lutrol® diluted at optimized concentration in culture medium for 2 h before transfection. One µg of GFP or luciferase encoding plasmid was complexed with DOSP/DOPE at a charge ratio of ±4.GFP expressing cells were counted by flow cytometry and a luciferase gene expression assay was performed 24 h after transfection.
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Figure 7: Effect of block copolymers on the percentage of transfected cells. C2C12 cells were transfected after Lutrol® treatment (gray bars) or without Lutrol® treatment (white bars). Treated cells were incubated with Lutrol® diluted at optimized concentration in culture medium for 2 h before transfection. One µg of GFP or luciferase encoding plasmid was complexed with DOSP/DOPE at a charge ratio of ±4.GFP expressing cells were counted by flow cytometry and a luciferase gene expression assay was performed 24 h after transfection.

Mentions: To confirm that block copolymers enhance transfection by DNA molecule internalization and/or endosomal escape, we measured the percentage of transfected cells by FACS analysis using plasmid DNA encoding GFP. FACS analysis showed a similar percentage of GFP-expressing cells in the presence or absence of Lutrol® pre-treatment (Figure 7). As a control, luciferase expression was strongly enhanced after pre-treatment with Lutrol® during the same experiment (Figure 7). These results were also in good agreement with the experiment using β-galactosidase as a reporter gene, as shown by the presence of the same number of blue cells (data not shown) irrespective of pre-treatment with Lutrol®. However, in these two experiments, we noticed that cells pre-treated with Lutrol® expressed the reporter gene at a higher level, as shown by the blue intensity of the cells (data not shown). The same observation was made in GFP-transfected cells pre-treated with Lutrol® (data not shown). These results strongly suggest that, even if the same number of cells were transfected in the presence of Lutrol®, the number of plasmids entering each cell would certainly be increased.Figure 7.


Amphiphilic block copolymers enhance the cellular uptake of DNA molecules through a facilitated plasma membrane transport.

Chèvre R, Le Bihan O, Beilvert F, Chatin B, Barteau B, Mével M, Lambert O, Pitard B - Nucleic Acids Res. (2010)

Effect of block copolymers on the percentage of transfected cells. C2C12 cells were transfected after Lutrol® treatment (gray bars) or without Lutrol® treatment (white bars). Treated cells were incubated with Lutrol® diluted at optimized concentration in culture medium for 2 h before transfection. One µg of GFP or luciferase encoding plasmid was complexed with DOSP/DOPE at a charge ratio of ±4.GFP expressing cells were counted by flow cytometry and a luciferase gene expression assay was performed 24 h after transfection.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3045598&req=5

Figure 7: Effect of block copolymers on the percentage of transfected cells. C2C12 cells were transfected after Lutrol® treatment (gray bars) or without Lutrol® treatment (white bars). Treated cells were incubated with Lutrol® diluted at optimized concentration in culture medium for 2 h before transfection. One µg of GFP or luciferase encoding plasmid was complexed with DOSP/DOPE at a charge ratio of ±4.GFP expressing cells were counted by flow cytometry and a luciferase gene expression assay was performed 24 h after transfection.
Mentions: To confirm that block copolymers enhance transfection by DNA molecule internalization and/or endosomal escape, we measured the percentage of transfected cells by FACS analysis using plasmid DNA encoding GFP. FACS analysis showed a similar percentage of GFP-expressing cells in the presence or absence of Lutrol® pre-treatment (Figure 7). As a control, luciferase expression was strongly enhanced after pre-treatment with Lutrol® during the same experiment (Figure 7). These results were also in good agreement with the experiment using β-galactosidase as a reporter gene, as shown by the presence of the same number of blue cells (data not shown) irrespective of pre-treatment with Lutrol®. However, in these two experiments, we noticed that cells pre-treated with Lutrol® expressed the reporter gene at a higher level, as shown by the blue intensity of the cells (data not shown). The same observation was made in GFP-transfected cells pre-treated with Lutrol® (data not shown). These results strongly suggest that, even if the same number of cells were transfected in the presence of Lutrol®, the number of plasmids entering each cell would certainly be increased.Figure 7.

Bottom Line: We also showed that this improvement in transfection efficiency associated with the presence of Lutrol® was observed irrespective of the plasmid promoter.Considering the various steps that could be improved by Lutrol®, we concluded that the nucleic acids molecule internalization step is the most important barrier affected by Lutrol®.Our results strongly suggest that in vivo, Lutrol® improves transfection by a physicochemical mechanism, leading to cellular uptake enhancement through a direct delivery into the cytoplasm, and not via endosomal pathways.

View Article: PubMed Central - PubMed

Affiliation: INSERM, U915, l'institut du thorax, Nantes, F-44000, France.

ABSTRACT
Amphiphilic block copolymers have been developed recently for their efficient, in vivo transfection activities in various tissues. Surprisingly, we observed that amphiphilic block copolymers such as Lutrol® do not allow the transfection of cultured cells in vitro, suggesting that the cell environment is strongly involved in their mechanism of action. In an in vitro model mimicking the in vivo situation we showed that pre-treatment of cells with Lutrol®, prior to their incubation with DNA molecules in the presence of cationic lipid, resulted in higher levels of reporter gene expression. We also showed that this improvement in transfection efficiency associated with the presence of Lutrol® was observed irrespective of the plasmid promoter. Considering the various steps that could be improved by Lutrol®, we concluded that the nucleic acids molecule internalization step is the most important barrier affected by Lutrol®. Microscopic examination of transfected cells pre-treated with Lutrol® confirmed that more plasmid DNA copies were internalized. Absence of cationic lipid did not impair Lutrol®-mediated DNA internalization, but critically impaired endosomal escape. Our results strongly suggest that in vivo, Lutrol® improves transfection by a physicochemical mechanism, leading to cellular uptake enhancement through a direct delivery into the cytoplasm, and not via endosomal pathways.

Show MeSH
Related in: MedlinePlus