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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 treatment period or (B) the transfection temperature. Cells were transfected with 1 µg luciferase encoding plasmid complexed with DOSP/DOPE at a charge ratio of ±4. Transfection was performed with Lutrol® treatment (gray bars) or without Lutrol® treatment (white bars). Luciferase reporter gene assay was performed 24 h after transfection. (A) Cells were treated with Lutrol® diluted at optimized concentration in culture medium for 2 h before or after addition of DOSP/DOPE–DNA lipoplexes. (B) Cells were incubated for 2 h at 37 or 4°C, just after the addition of DOSP/DOPE–DNA lipoplexes.
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Figure 5: Effect of amphiphilic block copolymers on in vitro transfection as a function of (A) the block copolymer treatment period or (B) the transfection temperature. Cells were transfected with 1 µg luciferase encoding plasmid complexed with DOSP/DOPE at a charge ratio of ±4. Transfection was performed with Lutrol® treatment (gray bars) or without Lutrol® treatment (white bars). Luciferase reporter gene assay was performed 24 h after transfection. (A) Cells were treated with Lutrol® diluted at optimized concentration in culture medium for 2 h before or after addition of DOSP/DOPE–DNA lipoplexes. (B) Cells were incubated for 2 h at 37 or 4°C, just after the addition of DOSP/DOPE–DNA lipoplexes.

Mentions: To investigate a possible role of amphiphilic block copolymers in the stimulation of transcription and translation, cells were treated with Lutrol® after the intracellular internalization of DNA molecules had occurred. Figure 5A shows that enhancement of luciferase expression was observed only when cells were pre-treated with Lutrol®, suggesting that Lutrol® did not promote reporter gene expression stimulation at the transcription or translation level, but rather stimulated steps involved in DNA internalization, endosomal escape or nuclear targeting.Figure 5.


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 treatment period or (B) the transfection temperature. Cells were transfected with 1 µg luciferase encoding plasmid complexed with DOSP/DOPE at a charge ratio of ±4. Transfection was performed with Lutrol® treatment (gray bars) or without Lutrol® treatment (white bars). Luciferase reporter gene assay was performed 24 h after transfection. (A) Cells were treated with Lutrol® diluted at optimized concentration in culture medium for 2 h before or after addition of DOSP/DOPE–DNA lipoplexes. (B) Cells were incubated for 2 h at 37 or 4°C, just after the addition of DOSP/DOPE–DNA lipoplexes.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 5: Effect of amphiphilic block copolymers on in vitro transfection as a function of (A) the block copolymer treatment period or (B) the transfection temperature. Cells were transfected with 1 µg luciferase encoding plasmid complexed with DOSP/DOPE at a charge ratio of ±4. Transfection was performed with Lutrol® treatment (gray bars) or without Lutrol® treatment (white bars). Luciferase reporter gene assay was performed 24 h after transfection. (A) Cells were treated with Lutrol® diluted at optimized concentration in culture medium for 2 h before or after addition of DOSP/DOPE–DNA lipoplexes. (B) Cells were incubated for 2 h at 37 or 4°C, just after the addition of DOSP/DOPE–DNA lipoplexes.
Mentions: To investigate a possible role of amphiphilic block copolymers in the stimulation of transcription and translation, cells were treated with Lutrol® after the intracellular internalization of DNA molecules had occurred. Figure 5A shows that enhancement of luciferase expression was observed only when cells were pre-treated with Lutrol®, suggesting that Lutrol® did not promote reporter gene expression stimulation at the transcription or translation level, but rather stimulated steps involved in DNA internalization, endosomal escape or nuclear targeting.Figure 5.

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