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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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Labelled lipoplexes with Nps. (A) Cryo images of pDNA interacting with Nps. (B) Labelled lipoplexes. Nps are incorporated into the lamellar assembly of pDNA/BGTC complexes. Note that the black dots within Nps correspond to small maghemite cores. Scale bar 50 nm.
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Figure 1: Labelled lipoplexes with Nps. (A) Cryo images of pDNA interacting with Nps. (B) Labelled lipoplexes. Nps are incorporated into the lamellar assembly of pDNA/BGTC complexes. Note that the black dots within Nps correspond to small maghemite cores. Scale bar 50 nm.

Mentions: The present work aims to understand at a nanometre scale the gene transfer mechanism, from its cellular uptake to its delivery into the nucleus, using Np as the imaging probe. For this purpose, we labelled lipoplexes with Nps producing a high electron-scattering density easily observable by TEM (Figure 1). The Np with a 16.4 ± 4.8 nm mean diameter was a core-shell, silica-based Np containing 7 nm diameter maghemite nanocrystal, unambiguously recognizable by TEM (Supplementary Figure S1). Zeta potential measurements on these aminated-modified Nps showed that the Np surfaces exhibited a high density of positive charges brought by ammonium groups of the polysiloxane films in neutral and acidic media (Supplementary Figure S2). Its aminated-modified surface allowed the formation of Np/pDNA complexes mediated by electrostatic interactions (Figure 1A). To analyse the mechanism of action of lipoplexes, we focused our study on two cationic lipids, both known to be efficient DNA carriers. BGTC was made of a cholesterol hydrophobic moiety attached to guanidinium group and DOSP was composed of two aliphatic chains linked to an aminoglycoside head group. Thus, Np/pDNA complexes were mixed with either BGTC–DOPE or DOSP–DOPE liposomes to formulate labelled lipoplexes. Cryo-TEM image of labelled BGTC-lipoplexes revealed the presence of randomly-distributed Nps within lipid/pDNA assemblies (Figure 1B). This indicated that labelled lipoplexes kept their typical multilamellar organization and that their edges remained accessible to cell interaction. Thus, we expected that this labelling process should not modify the physicochemical properties of the lipoplexes, nor the transfection efficiency.Figure 1.


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)

Labelled lipoplexes with Nps. (A) Cryo images of pDNA interacting with Nps. (B) Labelled lipoplexes. Nps are incorporated into the lamellar assembly of pDNA/BGTC complexes. Note that the black dots within Nps correspond to small maghemite cores. Scale bar 50 nm.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3045597&req=5

Figure 1: Labelled lipoplexes with Nps. (A) Cryo images of pDNA interacting with Nps. (B) Labelled lipoplexes. Nps are incorporated into the lamellar assembly of pDNA/BGTC complexes. Note that the black dots within Nps correspond to small maghemite cores. Scale bar 50 nm.
Mentions: The present work aims to understand at a nanometre scale the gene transfer mechanism, from its cellular uptake to its delivery into the nucleus, using Np as the imaging probe. For this purpose, we labelled lipoplexes with Nps producing a high electron-scattering density easily observable by TEM (Figure 1). The Np with a 16.4 ± 4.8 nm mean diameter was a core-shell, silica-based Np containing 7 nm diameter maghemite nanocrystal, unambiguously recognizable by TEM (Supplementary Figure S1). Zeta potential measurements on these aminated-modified Nps showed that the Np surfaces exhibited a high density of positive charges brought by ammonium groups of the polysiloxane films in neutral and acidic media (Supplementary Figure S2). Its aminated-modified surface allowed the formation of Np/pDNA complexes mediated by electrostatic interactions (Figure 1A). To analyse the mechanism of action of lipoplexes, we focused our study on two cationic lipids, both known to be efficient DNA carriers. BGTC was made of a cholesterol hydrophobic moiety attached to guanidinium group and DOSP was composed of two aliphatic chains linked to an aminoglycoside head group. Thus, Np/pDNA complexes were mixed with either BGTC–DOPE or DOSP–DOPE liposomes to formulate labelled lipoplexes. Cryo-TEM image of labelled BGTC-lipoplexes revealed the presence of randomly-distributed Nps within lipid/pDNA assemblies (Figure 1B). This indicated that labelled lipoplexes kept their typical multilamellar organization and that their edges remained accessible to cell interaction. Thus, we expected that this labelling process should not modify the physicochemical properties of the lipoplexes, nor the transfection efficiency.Figure 1.

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