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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

Effect of amphiphilic block copolymers on in vitro transfection as a function of (A) the block copolymer molecular weight or (B) the plasmid promoter used. Cells were transfected with 1 µg luciferase encoding plasmid complexed with DOSP/DOPE at a charge ratio of ±4. Transfection was performed after block copolymer treatment (gray bars) or without block copolymer treatment (white bars). Luciferase reporter gene assay was performed 24 h after transfection. (A) Treated cells were incubated with F38, Lutrol® or F108 diluted at optimized concentration in culture medium for 2 h before transfection. (B) Cells were incubated with 3% Lutrol® diluted in culture medium for 2 h before transfection with two different plasmids with a CMV or SV40 promoter.
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Figure 4: Effect of amphiphilic block copolymers on in vitro transfection as a function of (A) the block copolymer molecular weight or (B) the plasmid promoter used. Cells were transfected with 1 µg luciferase encoding plasmid complexed with DOSP/DOPE at a charge ratio of ±4. Transfection was performed after block copolymer treatment (gray bars) or without block copolymer treatment (white bars). Luciferase reporter gene assay was performed 24 h after transfection. (A) Treated cells were incubated with F38, Lutrol® or F108 diluted at optimized concentration in culture medium for 2 h before transfection. (B) Cells were incubated with 3% Lutrol® diluted in culture medium for 2 h before transfection with two different plasmids with a CMV or SV40 promoter.

Mentions: Next, in order to validate our in vitro model and to provide an insight into the in vivo mechanism of action of block copolymers, we investigated the influence of the plasmid DNA promoter and the molecular weight of the block copolymer on transfection efficiency in cultured cells. Figure 4 shows that, as observed in vivo (Figure 2), pre-treatment of cultured cells with block copolymers of various molecular weights led to a similar increase in luciferase expression (Figure 4A). We also showed that either CMV or SV40 promoters led to similar reporter gene expression enhancement in cells pre-treated or not with Lutrol® (Figure 4B). These data strongly suggest that, as observed in vivo, Lutrol® and other 80% PEO block copolymers did not activate promoter-specific transcription factor signalling pathways, but increased gene expression by another mechanism.Figure 4.


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 amphiphilic block copolymers on in vitro transfection as a function of (A) the block copolymer molecular weight or (B) the plasmid promoter used. Cells were transfected with 1 µg luciferase encoding plasmid complexed with DOSP/DOPE at a charge ratio of ±4. Transfection was performed after block copolymer treatment (gray bars) or without block copolymer treatment (white bars). Luciferase reporter gene assay was performed 24 h after transfection. (A) Treated cells were incubated with F38, Lutrol® or F108 diluted at optimized concentration in culture medium for 2 h before transfection. (B) Cells were incubated with 3% Lutrol® diluted in culture medium for 2 h before transfection with two different plasmids with a CMV or SV40 promoter.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 4: Effect of amphiphilic block copolymers on in vitro transfection as a function of (A) the block copolymer molecular weight or (B) the plasmid promoter used. Cells were transfected with 1 µg luciferase encoding plasmid complexed with DOSP/DOPE at a charge ratio of ±4. Transfection was performed after block copolymer treatment (gray bars) or without block copolymer treatment (white bars). Luciferase reporter gene assay was performed 24 h after transfection. (A) Treated cells were incubated with F38, Lutrol® or F108 diluted at optimized concentration in culture medium for 2 h before transfection. (B) Cells were incubated with 3% Lutrol® diluted in culture medium for 2 h before transfection with two different plasmids with a CMV or SV40 promoter.
Mentions: Next, in order to validate our in vitro model and to provide an insight into the in vivo mechanism of action of block copolymers, we investigated the influence of the plasmid DNA promoter and the molecular weight of the block copolymer on transfection efficiency in cultured cells. Figure 4 shows that, as observed in vivo (Figure 2), pre-treatment of cultured cells with block copolymers of various molecular weights led to a similar increase in luciferase expression (Figure 4A). We also showed that either CMV or SV40 promoters led to similar reporter gene expression enhancement in cells pre-treated or not with Lutrol® (Figure 4B). These data strongly suggest that, as observed in vivo, Lutrol® and other 80% PEO block copolymers did not activate promoter-specific transcription factor signalling pathways, but increased gene expression by another mechanism.Figure 4.

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