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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

Plasmid promoter influence on transfection efficiency of block copolymers in mouse tibial anterior muscle as a function of the block copolymer molecular weight. Shaved tibial anterior muscles of CD1 mice were injected with 10 µg plasmid encoding luciferase under control of the CMV promoter (black bars) or SV40 promoter (white bars). Before injection, plasmid was formulated with 1% F38, 3% Lutrol® or 1% F108 in tyrode. Luciferase gene expression assay was performed 3 days after injection.
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Figure 2: Plasmid promoter influence on transfection efficiency of block copolymers in mouse tibial anterior muscle as a function of the block copolymer molecular weight. Shaved tibial anterior muscles of CD1 mice were injected with 10 µg plasmid encoding luciferase under control of the CMV promoter (black bars) or SV40 promoter (white bars). Before injection, plasmid was formulated with 1% F38, 3% Lutrol® or 1% F108 in tyrode. Luciferase gene expression assay was performed 3 days after injection.

Mentions: Next, we investigated a possible role of amphiphilic block copolymers with the 80% PEO used in this study, in the activation of some transcription factors, as has been previously described for block copolymers containing 50% PEO (15,16). For this purpose, we compared transfection efficiencies in tibial anterior muscle using two different plasmids encoding luciferase, controlled either by the CMV or SV40 promoter. These two promoters contain different transcription factor binding sites (Table 1). The three polymers tested were all composed of 80% PEO but with various molecular weights, ranging from 4700 to 14 700 Da. Figure 2 shows that similar luciferase expression was obtained after intramuscular injection of both plasmids complexed with the various block copolymers.Figure 2.


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)

Plasmid promoter influence on transfection efficiency of block copolymers in mouse tibial anterior muscle as a function of the block copolymer molecular weight. Shaved tibial anterior muscles of CD1 mice were injected with 10 µg plasmid encoding luciferase under control of the CMV promoter (black bars) or SV40 promoter (white bars). Before injection, plasmid was formulated with 1% F38, 3% Lutrol® or 1% F108 in tyrode. Luciferase gene expression assay was performed 3 days after injection.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 2: Plasmid promoter influence on transfection efficiency of block copolymers in mouse tibial anterior muscle as a function of the block copolymer molecular weight. Shaved tibial anterior muscles of CD1 mice were injected with 10 µg plasmid encoding luciferase under control of the CMV promoter (black bars) or SV40 promoter (white bars). Before injection, plasmid was formulated with 1% F38, 3% Lutrol® or 1% F108 in tyrode. Luciferase gene expression assay was performed 3 days after injection.
Mentions: Next, we investigated a possible role of amphiphilic block copolymers with the 80% PEO used in this study, in the activation of some transcription factors, as has been previously described for block copolymers containing 50% PEO (15,16). For this purpose, we compared transfection efficiencies in tibial anterior muscle using two different plasmids encoding luciferase, controlled either by the CMV or SV40 promoter. These two promoters contain different transcription factor binding sites (Table 1). The three polymers tested were all composed of 80% PEO but with various molecular weights, ranging from 4700 to 14 700 Da. Figure 2 shows that similar luciferase expression was obtained after intramuscular injection of both plasmids complexed with the various block copolymers.Figure 2.

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