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Probing the in vitro mechanism of action of cationic lipid/DNA lipoplexes at a nanometric scale.

Le Bihan O, Chèvre R, Mornet S, Garnier B, Pitard B, Lambert O - Nucleic Acids Res. (2010)

Bottom Line: A better understanding of the identified key-steps, including endocytosis, endosomal escape and nuclear delivery is required for further developments to improve their efficacy.In addition, the formation of new multilamellar lipid assemblies was noted, which could result from the interaction between cationic lipids and cellular compounds.These results provide new insights into DNA transfer pathways and possible implications of cationic lipids in lipid metabolism.

View Article: PubMed Central - PubMed

Affiliation: CBMN UMR-CNRS 5248, Université Bordeaux, ENITAB, IECB, Avenue des Facultés, F-33405 Talence, France.

ABSTRACT
Cationic lipids are used for delivering nucleic acids (lipoplexes) into cells for both therapeutic and biological applications. A better understanding of the identified key-steps, including endocytosis, endosomal escape and nuclear delivery is required for further developments to improve their efficacy. Here, we developed a labelling protocol using aminated nanoparticles as markers for plasmid DNA to examine the intracellular route of lipoplexes in cell lines using transmission electron microscopy. Morphological changes of lipoplexes, membrane reorganizations and endosomal membrane ruptures were observed allowing the understanding of the lipoplex mechanism until the endosomal escape mediated by cationic lipids. The study carried out on two cationic lipids, bis(guanidinium)-tris(2-aminoethyl)amine-cholesterol (BGTC) and dioleyl succinyl paramomycin (DOSP), showed two pathways of endosomal escape that could explain their different transfection efficiencies. For BGTC, a partial or complete dissociation of DNA from cationic lipids occurred before endosomal escape while for DOSP, lipoplexes remained visible within ruptured vesicles suggesting a more direct pathway for DNA release and endosome escape. In addition, the formation of new multilamellar lipid assemblies was noted, which could result from the interaction between cationic lipids and cellular compounds. These results provide new insights into DNA transfer pathways and possible implications of cationic lipids in lipid metabolism.

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Gallery of endosomes that contain labelled DOSP lipoplexes with deep morphological modifications providing snapshots on the endosomal escape process. (A–C) Partially disrupted endosome containing native labelled lipoplexes (B and inset) with a 6.5-nm repeat distance and new 5.5-nm repeat lipid structures devoid of Nps (C and inset). DNA dissociation from DOSP and its release from the endosome seem concomitant and probably occur in a more direct way than with BGTC, for which an intermediate step was observed. (D–F) Totally disrupted endosome containing only Nps in luminal volume. At its boundary, 5.5 nm multilamellar planar structures (arrow heads in E) and budding structures (white arrows in F) are present. (G–I) ‘Ghost’ endosome without visible membrane. The presence of Nps (black arrows) and multilamellar vesicles (H) allows its identification. Note its location close to the nucleus (I). Asterisks indicate nuclear pores. Scale bars: 1 µm (A, D and G), 100 nm (B, C, E, F, H and I) and 20 nm (insets).
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Figure 7: Gallery of endosomes that contain labelled DOSP lipoplexes with deep morphological modifications providing snapshots on the endosomal escape process. (A–C) Partially disrupted endosome containing native labelled lipoplexes (B and inset) with a 6.5-nm repeat distance and new 5.5-nm repeat lipid structures devoid of Nps (C and inset). DNA dissociation from DOSP and its release from the endosome seem concomitant and probably occur in a more direct way than with BGTC, for which an intermediate step was observed. (D–F) Totally disrupted endosome containing only Nps in luminal volume. At its boundary, 5.5 nm multilamellar planar structures (arrow heads in E) and budding structures (white arrows in F) are present. (G–I) ‘Ghost’ endosome without visible membrane. The presence of Nps (black arrows) and multilamellar vesicles (H) allows its identification. Note its location close to the nucleus (I). Asterisks indicate nuclear pores. Scale bars: 1 µm (A, D and G), 100 nm (B, C, E, F, H and I) and 20 nm (insets).

Mentions: Figure 7 shows typical morphological changes of labelled DOSP lipoplexes at the level of the endocytic vesicle. Interestingly, vesicles appeared less dense than those in Figure 6C, and the endocytic membrane was missing over a long distance suggesting a release of material as observed for BGTC in Figure 4C. We did not, however, observe vesicles containing extensive membrane reorganization similar to that described in Figure 4A and B. These data suggested a more direct endosomal escape for DOSP than for BGTC. The endocytic vesicles contained Nps and lamellar structures located preferentially at their boundaries (Figure 7A, D and G). Two types of lamellar structures were observed, i.e. 6.5-nm repeat compact assemblies ascribed to lipoplexes (Figure 7B) and 5.5-nm multilamellar structures devoid of Nps, likely corresponding to newly-formed lipid stacks (Figure 7C–I). The 5.5-nm repeat assemblies had various shapes, i.e. long planar structures (Figure 7D and E), curved structures (Figure 7C) and elongated vesicles present at the vicinity of the endocytic vesicles (Figure 7E–H). The formation of the latter vesicles could arise from the budding of the planar structures as suggested by the curved shape (Figure 7C) and the protruding structure (Figure 7F). It is also important to note that these modifications occurred near the nuclear membrane dotted lines in Figure 7D and G), as noted with BGTC lipoplexes.Figure 7.


Probing the in vitro mechanism of action of cationic lipid/DNA lipoplexes at a nanometric scale.

Le Bihan O, Chèvre R, Mornet S, Garnier B, Pitard B, Lambert O - Nucleic Acids Res. (2010)

Gallery of endosomes that contain labelled DOSP lipoplexes with deep morphological modifications providing snapshots on the endosomal escape process. (A–C) Partially disrupted endosome containing native labelled lipoplexes (B and inset) with a 6.5-nm repeat distance and new 5.5-nm repeat lipid structures devoid of Nps (C and inset). DNA dissociation from DOSP and its release from the endosome seem concomitant and probably occur in a more direct way than with BGTC, for which an intermediate step was observed. (D–F) Totally disrupted endosome containing only Nps in luminal volume. At its boundary, 5.5 nm multilamellar planar structures (arrow heads in E) and budding structures (white arrows in F) are present. (G–I) ‘Ghost’ endosome without visible membrane. The presence of Nps (black arrows) and multilamellar vesicles (H) allows its identification. Note its location close to the nucleus (I). Asterisks indicate nuclear pores. Scale bars: 1 µm (A, D and G), 100 nm (B, C, E, F, H and I) and 20 nm (insets).
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Figure 7: Gallery of endosomes that contain labelled DOSP lipoplexes with deep morphological modifications providing snapshots on the endosomal escape process. (A–C) Partially disrupted endosome containing native labelled lipoplexes (B and inset) with a 6.5-nm repeat distance and new 5.5-nm repeat lipid structures devoid of Nps (C and inset). DNA dissociation from DOSP and its release from the endosome seem concomitant and probably occur in a more direct way than with BGTC, for which an intermediate step was observed. (D–F) Totally disrupted endosome containing only Nps in luminal volume. At its boundary, 5.5 nm multilamellar planar structures (arrow heads in E) and budding structures (white arrows in F) are present. (G–I) ‘Ghost’ endosome without visible membrane. The presence of Nps (black arrows) and multilamellar vesicles (H) allows its identification. Note its location close to the nucleus (I). Asterisks indicate nuclear pores. Scale bars: 1 µm (A, D and G), 100 nm (B, C, E, F, H and I) and 20 nm (insets).
Mentions: Figure 7 shows typical morphological changes of labelled DOSP lipoplexes at the level of the endocytic vesicle. Interestingly, vesicles appeared less dense than those in Figure 6C, and the endocytic membrane was missing over a long distance suggesting a release of material as observed for BGTC in Figure 4C. We did not, however, observe vesicles containing extensive membrane reorganization similar to that described in Figure 4A and B. These data suggested a more direct endosomal escape for DOSP than for BGTC. The endocytic vesicles contained Nps and lamellar structures located preferentially at their boundaries (Figure 7A, D and G). Two types of lamellar structures were observed, i.e. 6.5-nm repeat compact assemblies ascribed to lipoplexes (Figure 7B) and 5.5-nm multilamellar structures devoid of Nps, likely corresponding to newly-formed lipid stacks (Figure 7C–I). The 5.5-nm repeat assemblies had various shapes, i.e. long planar structures (Figure 7D and E), curved structures (Figure 7C) and elongated vesicles present at the vicinity of the endocytic vesicles (Figure 7E–H). The formation of the latter vesicles could arise from the budding of the planar structures as suggested by the curved shape (Figure 7C) and the protruding structure (Figure 7F). It is also important to note that these modifications occurred near the nuclear membrane dotted lines in Figure 7D and G), as noted with BGTC lipoplexes.Figure 7.

Bottom Line: A better understanding of the identified key-steps, including endocytosis, endosomal escape and nuclear delivery is required for further developments to improve their efficacy.In addition, the formation of new multilamellar lipid assemblies was noted, which could result from the interaction between cationic lipids and cellular compounds.These results provide new insights into DNA transfer pathways and possible implications of cationic lipids in lipid metabolism.

View Article: PubMed Central - PubMed

Affiliation: CBMN UMR-CNRS 5248, Université Bordeaux, ENITAB, IECB, Avenue des Facultés, F-33405 Talence, France.

ABSTRACT
Cationic lipids are used for delivering nucleic acids (lipoplexes) into cells for both therapeutic and biological applications. A better understanding of the identified key-steps, including endocytosis, endosomal escape and nuclear delivery is required for further developments to improve their efficacy. Here, we developed a labelling protocol using aminated nanoparticles as markers for plasmid DNA to examine the intracellular route of lipoplexes in cell lines using transmission electron microscopy. Morphological changes of lipoplexes, membrane reorganizations and endosomal membrane ruptures were observed allowing the understanding of the lipoplex mechanism until the endosomal escape mediated by cationic lipids. The study carried out on two cationic lipids, bis(guanidinium)-tris(2-aminoethyl)amine-cholesterol (BGTC) and dioleyl succinyl paramomycin (DOSP), showed two pathways of endosomal escape that could explain their different transfection efficiencies. For BGTC, a partial or complete dissociation of DNA from cationic lipids occurred before endosomal escape while for DOSP, lipoplexes remained visible within ruptured vesicles suggesting a more direct pathway for DNA release and endosome escape. In addition, the formation of new multilamellar lipid assemblies was noted, which could result from the interaction between cationic lipids and cellular compounds. These results provide new insights into DNA transfer pathways and possible implications of cationic lipids in lipid metabolism.

Show MeSH
Related in: MedlinePlus