Limits...
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.

Show MeSH

Related in: MedlinePlus

Cellular uptake of DNA in the presence or in the absence of Lutrol® pre-treatment. DNA molecules were labelled with YOYO-1 (one molecule every 150 base pair). Hela cells were transfected after 1 h 3% Lutrol® treatment or without Lutrol® treatment. One µg of labelled luciferase encoding plasmid was complexed with DOSP/DOPE at a charge ratio of ±4. Then, cells were analysed by FACS 24 h after transfection.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3045598&req=5

Figure 9: Cellular uptake of DNA in the presence or in the absence of Lutrol® pre-treatment. DNA molecules were labelled with YOYO-1 (one molecule every 150 base pair). Hela cells were transfected after 1 h 3% Lutrol® treatment or without Lutrol® treatment. One µg of labelled luciferase encoding plasmid was complexed with DOSP/DOPE at a charge ratio of ±4. Then, cells were analysed by FACS 24 h after transfection.

Mentions: Next, we investigated if the transfection enhancement observed after lutrol® pre-treatment was associated with an increase in lipoplex internalization. In order to detect DNA cellular uptake by TEM, lipoplexes were labelled with NPs. Cells were incubated for 2 h in the presence of labelled lipoplexes and then submitted to a fixation procedure. Labelled lipoplexes were observed in both untreated and treated cells and possessed similar morphological aspects indicating that Lutrol® treatment did not modify lipoplex structure (Figures 8C and F). To quantify the cellular uptake of lipoplexes in both conditions, we estimated the Np amount per cell by measuring the Np surface per cell surface (see ‘Materials and methods’ section). The analysis was performed on 158 cell sections from 13 grid squares. Our quantitative analysis revealed an increase of DOSP-lipoplex uptake by treated cells, as shown in Figure 8E and I. This result suggests that Lutrol® promoted an enhanced DNA internalization through the cell membrane. As a control, cells transfected with labelled and unlabelled lipoplexes showed similar luciferase activities (data not shown). Moreover, a strong transfection enhancement after cell treatment with Lutrol® was also observed with labelled lipoplexes (data not shown), indicating that Nps did not influence transfection efficiency, confirming findings of Le Bihan (25). In addition, for both untreated and treated cells, we also determined that the surface of cells containing Nps represented 30% of the total area of cells, which corresponds to the percentage of transfected cells as indicated by FACS analysis and microscopy on GFP transfected cells. We also analysed the lipoplexes internalization by means of YOYO-1-labelled DNA. Flow cytometry analysis showed that cells pre-treated with Lutrol® exhibit a 2-fold increase in fluorescence intensity compare to cells that were not pre-incubated with Lutrol® (Figure 9). Altogether, these results indicated clearly that Lutrol® enhanced the cellular uptake of nucleic acids.Figure 8.


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)

Cellular uptake of DNA in the presence or in the absence of Lutrol® pre-treatment. DNA molecules were labelled with YOYO-1 (one molecule every 150 base pair). Hela cells were transfected after 1 h 3% Lutrol® treatment or without Lutrol® treatment. One µg of labelled luciferase encoding plasmid was complexed with DOSP/DOPE at a charge ratio of ±4. Then, cells were analysed by FACS 24 h after transfection.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 9: Cellular uptake of DNA in the presence or in the absence of Lutrol® pre-treatment. DNA molecules were labelled with YOYO-1 (one molecule every 150 base pair). Hela cells were transfected after 1 h 3% Lutrol® treatment or without Lutrol® treatment. One µg of labelled luciferase encoding plasmid was complexed with DOSP/DOPE at a charge ratio of ±4. Then, cells were analysed by FACS 24 h after transfection.
Mentions: Next, we investigated if the transfection enhancement observed after lutrol® pre-treatment was associated with an increase in lipoplex internalization. In order to detect DNA cellular uptake by TEM, lipoplexes were labelled with NPs. Cells were incubated for 2 h in the presence of labelled lipoplexes and then submitted to a fixation procedure. Labelled lipoplexes were observed in both untreated and treated cells and possessed similar morphological aspects indicating that Lutrol® treatment did not modify lipoplex structure (Figures 8C and F). To quantify the cellular uptake of lipoplexes in both conditions, we estimated the Np amount per cell by measuring the Np surface per cell surface (see ‘Materials and methods’ section). The analysis was performed on 158 cell sections from 13 grid squares. Our quantitative analysis revealed an increase of DOSP-lipoplex uptake by treated cells, as shown in Figure 8E and I. This result suggests that Lutrol® promoted an enhanced DNA internalization through the cell membrane. As a control, cells transfected with labelled and unlabelled lipoplexes showed similar luciferase activities (data not shown). Moreover, a strong transfection enhancement after cell treatment with Lutrol® was also observed with labelled lipoplexes (data not shown), indicating that Nps did not influence transfection efficiency, confirming findings of Le Bihan (25). In addition, for both untreated and treated cells, we also determined that the surface of cells containing Nps represented 30% of the total area of cells, which corresponds to the percentage of transfected cells as indicated by FACS analysis and microscopy on GFP transfected cells. We also analysed the lipoplexes internalization by means of YOYO-1-labelled DNA. Flow cytometry analysis showed that cells pre-treated with Lutrol® exhibit a 2-fold increase in fluorescence intensity compare to cells that were not pre-incubated with Lutrol® (Figure 9). Altogether, these results indicated clearly that Lutrol® enhanced the cellular uptake of nucleic acids.Figure 8.

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