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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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Related in: MedlinePlus

In vivo and in vitro transfection efficiency of amphiphilic block copolymers, cationic lipids and naked DNA. A luciferase gene expression assay was performed to compare the transfection efficiencies of an amphiphilic block copolymer, Lutrol® and cationic liposomes of DOSP–DOPE, either in vivo in mouse tibial anterior muscle 7 days after transfection (black bars) or in vitro in the C2C12 mouse muscle cell line 24 h after transfection (white bars). The luciferase gene-encoding plasmid was either naked or complexed with 3% Lutrol® or with DOSP–DOPE at a charge ratio of ±4. The amount of plasmid transfected in vitro and in vivo was, respectively, 1 and 10 µg.
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Figure 1: In vivo and in vitro transfection efficiency of amphiphilic block copolymers, cationic lipids and naked DNA. A luciferase gene expression assay was performed to compare the transfection efficiencies of an amphiphilic block copolymer, Lutrol® and cationic liposomes of DOSP–DOPE, either in vivo in mouse tibial anterior muscle 7 days after transfection (black bars) or in vitro in the C2C12 mouse muscle cell line 24 h after transfection (white bars). The luciferase gene-encoding plasmid was either naked or complexed with 3% Lutrol® or with DOSP–DOPE at a charge ratio of ±4. The amount of plasmid transfected in vitro and in vivo was, respectively, 1 and 10 µg.

Mentions: In order to assess the impact of the cell environment on transfection efficiency, plasmid DNA either naked or complexed either with Lutrol®, an amphiphilic block copolymer of 80% PEO, or with cationic liposomes of DOSP/DOPE, was injected into mouse tibial anterior muscle (Figure 1) or incubated, in vitro, with C2C12 mouse muscle cells (Figure 1). Luciferase reporter gene expression in C2C12 and in tibial anterior muscle was evaluated 24 h and 7 days after transfection, respectively. Injection of 10 µg plasmid DNA complexed with 3% Lutrol® led to high luciferase expression in mouse tibial anterior muscle, compared with the very low luciferase expression achieved with naked DNA or DOSP/DOPE–DNA lipoplexes. By contrast, transfection of cultured C2C12 with 1 µg of pCMV-Luciferase complexed with Lutrol® did not allow luciferase expression, whereas cationic liposomes of DOSP/DOPE led to a dramatic increase in luciferase expression. These results strongly suggest that the cell environment plays an important role in amphiphilic block copolymer and cationic liposome transfection efficiency.Figure 1.


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)

In vivo and in vitro transfection efficiency of amphiphilic block copolymers, cationic lipids and naked DNA. A luciferase gene expression assay was performed to compare the transfection efficiencies of an amphiphilic block copolymer, Lutrol® and cationic liposomes of DOSP–DOPE, either in vivo in mouse tibial anterior muscle 7 days after transfection (black bars) or in vitro in the C2C12 mouse muscle cell line 24 h after transfection (white bars). The luciferase gene-encoding plasmid was either naked or complexed with 3% Lutrol® or with DOSP–DOPE at a charge ratio of ±4. The amount of plasmid transfected in vitro and in vivo was, respectively, 1 and 10 µg.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 1: In vivo and in vitro transfection efficiency of amphiphilic block copolymers, cationic lipids and naked DNA. A luciferase gene expression assay was performed to compare the transfection efficiencies of an amphiphilic block copolymer, Lutrol® and cationic liposomes of DOSP–DOPE, either in vivo in mouse tibial anterior muscle 7 days after transfection (black bars) or in vitro in the C2C12 mouse muscle cell line 24 h after transfection (white bars). The luciferase gene-encoding plasmid was either naked or complexed with 3% Lutrol® or with DOSP–DOPE at a charge ratio of ±4. The amount of plasmid transfected in vitro and in vivo was, respectively, 1 and 10 µg.
Mentions: In order to assess the impact of the cell environment on transfection efficiency, plasmid DNA either naked or complexed either with Lutrol®, an amphiphilic block copolymer of 80% PEO, or with cationic liposomes of DOSP/DOPE, was injected into mouse tibial anterior muscle (Figure 1) or incubated, in vitro, with C2C12 mouse muscle cells (Figure 1). Luciferase reporter gene expression in C2C12 and in tibial anterior muscle was evaluated 24 h and 7 days after transfection, respectively. Injection of 10 µg plasmid DNA complexed with 3% Lutrol® led to high luciferase expression in mouse tibial anterior muscle, compared with the very low luciferase expression achieved with naked DNA or DOSP/DOPE–DNA lipoplexes. By contrast, transfection of cultured C2C12 with 1 µg of pCMV-Luciferase complexed with Lutrol® did not allow luciferase expression, whereas cationic liposomes of DOSP/DOPE led to a dramatic increase in luciferase expression. These results strongly suggest that the cell environment plays an important role in amphiphilic block copolymer and cationic liposome transfection efficiency.Figure 1.

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