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

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.

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.

Figure 2.

Physicochemical characterization of Np/pDNA interactions. (A) Agarose gel electrophoresis of Np/pDNA complexes prepared at the ratio (r) given on top. (B and C) After centrifugation, gel electrophoresis of the supernatant (B), and pDNA cosedimented with Np plotted in (C). (D) Influence of pH on Np/pDNA complexes at r = 5. (E) Influence of NaCl on Nps/pDNA complexes at r = 5. (F–H) TEM images of Np/pDNA complexes at r = 1, r = 5 and r = 10, respectively. (I) Influence of polyanion (dextran sulphate; 500 000 kDa) on complexes stability. Scale bar 200 nm.

Figure 3.

TEM images of labelled BGTC lipoplexes at early stages of the DNA transfer route. (A, B) Labelled lipoplexes interacting with the plasma membrane. Large view (A) and an enlarged area (B) marked with black square in (A). Multilamellar structures (shown in inset in B) are typical of lipoplex assemblies with a 6.5-nm spacing provided by the distance between peaks (white arrows) on Fourier transform image. (C and D) Labelled lipoplexes with their genuine morphologies internalized into the cell via an endocytic process. (D) Enlarged view of marked area with black square in (C). Black arrows indicate the endosomal membrane. Inset: Enlargement of a 6.5-nm multilamellar assembly. Note that Nps (black densities) are clearly identified as well as the lipid layer structures (inset). Scale bars 1 µm (A and C), 100 nm (B and D) and 50 nm (inset). Nu: Nucleus.

Figure 4.

Gallery of labelled BGTC lipoplexes exhibiting various morphological modifications within endosomal vesicles that correspond to steps closely related to endosomal escape. (A) Typical morphological changes of lipoplexes show membrane reorganization and a loss of 6.5 nm multilamellar assemblies, suggesting DNA dissociation from cationic lipids. Scale bar 500 nm. (B) Reorganized lipoplexes close to the nucleus and an enlarged view corresponding to dashed black square (aside panel). New multilamellar structures are present at the vesicle boundaries. Insets: Enlarged image and its Fourier transform revealing a 5.5-nm membrane spacing. White arrows indicate peaks on Fourier transform image. Scale bars 1 µm, 100 nm and 20 nm. (C) Morphologies of modified lipoplexes after endosomal membrane disruption (black arrows). The dashed black square delineates the enlarged lipid assembly (aside panel) composed of two repeat distances. Inset: Fourier transform image showing two sets of bright peaks corresponding to a distance of 5.5 and 3.6 nm, respectively. Scale bars 500 nm and 20 nm. (D) Another example of lipid assembly with two membrane motifs clearly visible in enlarged area (inset). Scale bars 100 nm and 20 nm. (E) Nps in the vicinity of the nucleus. Scale bar 1 µm. (F) Low and high magnification view of vesicles containing Nps and composed of multilayered membranes with a 3.6-nm spacing motif (inset). Scale bars 1 µm, 100 nm and 20 nm. Nu: Nucleus.

Figure 5.

Novel multilamellar lipid assemblies encountered inside (A and B) and outside (C and D) the cell, observed with BGTC cationic lipid. The enlarged views (B and D) correspond, respectively, to the marked areas (black squares). A 3.6-nm repeat motif is calculated from the peak distance on the enlarged image and its Fourier transform (inset). Scale bars 1 µm (A and C), 100 nm (B and D) and 20 nm (inset).

Figure 6.

Labelled DOSP lipoplexes during cellular uptake and early steps of intracellular trafficking. (A and B) Labelled lipoplexes bound to the plasma membrane. Large view (A) and an enlarged area (B) marked with a black square in (A). Typical lipoplexes with a 6.5-nm lamellar spacing. Back dots (white arrows) correspond to Nps. C and D) Labelled lipoplexes surrounded by endosomal membrane after their internalization into the cell. (D) Enlarged view (corresponding to a black square drawn in C) shows typical 6.5 nm multilamellar arrangements (clearly visible in inset in D). Black arrows indicate Nps. Scale bars 1 µm (A and C), 100 nm (B and D) and 20 nm (inset).

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).