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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 copolymer complexation on lipoplex transfection efficiency. CHO cells and heparan sulphate-deficient CHO-2241 cells (CHO HS−) were transfected with 1 µg luciferase-encoding plasmid complexed with DOSP/DOPE at a charge ratio of ±4. Lipoplexes were formulated with Lutrol® (gray bars) or without Lutrol® (white bars). After 24 h, the luciferase gene expression assay was performed.
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Figure 10: Effect of block copolymer complexation on lipoplex transfection efficiency. CHO cells and heparan sulphate-deficient CHO-2241 cells (CHO HS−) were transfected with 1 µg luciferase-encoding plasmid complexed with DOSP/DOPE at a charge ratio of ±4. Lipoplexes were formulated with Lutrol® (gray bars) or without Lutrol® (white bars). After 24 h, the luciferase gene expression assay was performed.

Mentions: As efficient, cationic vector-mediated transfection requires the condensation of DNA in positively charged particles, it has been inferred that anionic proteoglycans are potential receptors (26). Direct evidence for the involvement of heparan sulfate proteoglycans (HSPGs) in transfection has been obtained by several groups (27,28). Figure 10 shows clearly that transfection of heparan sulfate-deficient CHO (CHO HS−) cells by DOSP/DOPE–DNA lipoplexes was strongly decreased compared with that obtained in normal CHO cells. By contrast, CHO HS− transfected by lipoplexes in the presence of Lutrol® exhibited an enhanced luciferase expression. These data strongly suggest that Lutrol® enhanced the interaction of DNA particles with the cell membrane, improving their uptake.Figure 10.


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 copolymer complexation on lipoplex transfection efficiency. CHO cells and heparan sulphate-deficient CHO-2241 cells (CHO HS−) were transfected with 1 µg luciferase-encoding plasmid complexed with DOSP/DOPE at a charge ratio of ±4. Lipoplexes were formulated with Lutrol® (gray bars) or without Lutrol® (white bars). After 24 h, the luciferase gene expression assay was performed.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 10: Effect of block copolymer complexation on lipoplex transfection efficiency. CHO cells and heparan sulphate-deficient CHO-2241 cells (CHO HS−) were transfected with 1 µg luciferase-encoding plasmid complexed with DOSP/DOPE at a charge ratio of ±4. Lipoplexes were formulated with Lutrol® (gray bars) or without Lutrol® (white bars). After 24 h, the luciferase gene expression assay was performed.
Mentions: As efficient, cationic vector-mediated transfection requires the condensation of DNA in positively charged particles, it has been inferred that anionic proteoglycans are potential receptors (26). Direct evidence for the involvement of heparan sulfate proteoglycans (HSPGs) in transfection has been obtained by several groups (27,28). Figure 10 shows clearly that transfection of heparan sulfate-deficient CHO (CHO HS−) cells by DOSP/DOPE–DNA lipoplexes was strongly decreased compared with that obtained in normal CHO cells. By contrast, CHO HS− transfected by lipoplexes in the presence of Lutrol® exhibited an enhanced luciferase expression. These data strongly suggest that Lutrol® enhanced the interaction of DNA particles with the cell membrane, improving their uptake.Figure 10.

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