mRNA Lipoplexes with Cationic and Ionizable α-Amino-lipophosphonates: Membrane Fusion, Transfection, mRNA Translation and Conformation

Figures

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  Figure 1. Molecular structure of lipids used in this work (A) cationic lipids and (B) co-lipids.

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  Figure 2. Agarose gel electrophoresis of LXs. mRNA was complexed with 2-based liposomes at mRNA/liposomes charge ratios of 1/0.5 (a), 1/1 (b) and 1/2, (c). Lane 1 was mRNA in the absence of liposomes. Then, LX were analyzed on a 0.6% agarose gel. Gel running buffer was TAE (40 mM Tris-acetate, 1 mM EDTA). mRNA gel was stained with ethidium bromide (EtBr) at a concentration of 0.5 μg/mL. mRNA fluorescence was revealed under UV lamp.

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  Figure 3. Transfection efficiency of mRNA lipoplexes. DC 2.4 cells were transfected for 4 h with lipoplexes made eGFP mRNA at (black bars) 1/0.5, (grey bars 1/1) and (white bars) 1/2 mRNA/L charge ratio. The expression of eGFP was measured 48 h post-transfection by flow cytometry. (A,D) Transfected cell (%) corresponds to the percentage of eGFP positive cells; (B,E) MFI is the mean of the fluorescence intensity related to the amount of eGFP produced by the cell; (C,F) % * MFI is the global transfection efficiency (i.e., the product of the number of EGFP positive cells by MFI). Values are means ± SD of three different experiments.

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  Figure 4. Confocal microscopy analysis of upon transfection mRNA-LX. DC2.4 cells were transfected for 2 h with 2.5 μg of fluorescein labelled IVT mRNA complexed with the indicated liposomes at mRNA/L 1/0.5. In 3b/DOPE and 2/DOPE LX, the lipid molar ratio was 2/1. Scale bars = 25 µm.

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  Figure 5. mRNA release from LX. mRNA was first complexed with (black bar) 2/DOPE (lipid molar ratio 2/1) or (white bar) 3b/DOPE (lipid molar ratio 2/1) liposomes at various mRNA/L ratio. Then, LX were incubated in the absence or the presence of 90 µM dextran sulphate of 500 kDa (DS) for 1 h before electrophoresis on a 0.6% agarose gel. Gel running buffer was TAE (40 mM Tris-acetate, 1 mM EDTA) and mRNA gel was stained with ethidium bromide (0.5 μg/mL). mRNA fluorescence was revealed under UV lamp and quantified by image J. The percentage of mRNA release was determined from the intensity of mRNA that migrated after DS treatment of LX and the intensity of the same amount of free mRNA.

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  Figure 6. In vitro transcription of mRNA-Luc LX. mRNA-Luc (0.2 μg) (black bar) free or complexed with the indicated liposomes at various mRNA/L ratio was incubated (white bar) in the absence or (hacked bar) the presence of 90 µM DS for 1 h. For 2/1 and 3b/1 LXs the lipid molar ratio was 1/1; for 2/DOPE and 3b/DOPE LX it was 2/1 and it was 1/1 for DOTAP/DOPE. Next, samples were mixed with the TNT Quick Coupled Transcription/Translation Systems Kit (Promega). After 1 h 30 min incubation at 30 °C, the luciferase activity was measured and expressed as relative light unit (RLU).

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  Figure 7. Circular dichroism of LX. (A) (a, blue) mRNA. mRNA complexed with 2/DOPE (lipid molar ratio 2/1) liposomes at mRNA/L ratio 1/0.5 (b, red), 1/1 (c, green) and 1/2 (d, purple). (B) (a, blue) pDNA. pDNA complexed with 2/DOPE (lipid molar ratio 2/1) liposomes at DNA/L ratio 1/1.5 (b, red)1/2, (c, green).

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  Figure 8. Comparison between the transfection efficiency of mRNA and DNA LXs. DC2.4 cells were transfected with 5 μg of (black bar) mRNA-EGFP or (white bar) pEGFP complexed with the indicated liposomes at indicated mRNA/L or DNA/L ratio. In 3b/DOPE and 2/DOPE LX, the lipid molar ratio was 2/1. % * MFI is the global transfection (i.e., the product of the number of EGFP positive cells by MFI). The final values are means ± SD of three different experiments.