Gemini Cationic Lipid-Type Nanovectors Suitable for the Transfection of Therapeutic Plasmid DNA Encoding for Pro-Inflammatory Cytokine Interleukin-12

Figures

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  Scheme 1. Molecular structure of (a) GCL1, (b) GCL2 and (c) GCL3. The molecular structure of the zwitterionic lipid DOPE is shown in (d).

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  Figure 1. (a) Plot of the ζ-potential as a function of the mL/mpDNA mass ratio for the optimum formulations of the GCL/DOPE-pCMV-Luc lipoplexes: GCL1/DOPE-pCMV-Luc (purple line), GCL2/DOPE-pCMV-Luc (pink line) and GCL3/DOPE-pCMV-Luc (blue line). (b) Schematic drawing of the charge inversion process.

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  Figure 2. Agarose gel electrophoresis experiments of GCL/DOPE-pCMV-Luc lipoplexes at selected optimal conditions of mL/mpDNA mass ratios and/or ρeff. Panel (a) shows the compaction assays, where free pDNA (lane 1) was used as the control. Panel (b) presents the protection assays of pDNA against degradation by DNase I, where free pDNA (lane 1) and free pDNA incubated with DNase I (lane 2) were used as controls. For both agarose electrophoresis experiments: (i) and (iv) correspond to the GCL1/DOPE-pCMV-Luc system, (ii) and (v) correspond to the GCL2/DOPE-pCMV-Luc system, and (iii) and (vi) correspond to the GCL3/DOPE-pCMV-Luc system.

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  Figure 3. Selection of cryo-TEM micrographs of GCL1/DOPE-pCMV-Luc lipoplexes formed at ρeff = 5. The scale bars of the micrographs are 200 nm (a), 50 nm (b) and 500 nm (c). Panel (d) shows a scheme of the Lα multilamellar phase observed in the micrographs.

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  Figure 4. Relative percentage of the proteins found in the protein corona (PC) of the GCL/DOPE-pCMV-Luc lipoplexes at ρeff = 5, classified by (a) molecular weight, MW in kDa; (b) isoelectric point, pI; and (c) physiological function.

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  Figure 5. Percentage of cell viability of COS-7 (a) and HeLa (b) cell lines in the presence of the optimized formulations of the GCL/DOPE-pCMV-Luc lipoplexes formed at ρeff = 5 (unstriped bars) and 10 (striped bars). The lipoplexes were incubated with cells for 48 h. The cell viability (%) was normalized to that obtained for untreated cells. The blue bar corresponds to commercial Lipo2000*, used here as positive control. Data represent the mean of three wells and errors are within ±5%.

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  Figure 6. Transfection efficiency levels of therapeutic gene expressed as IL-12 (ng/mL) in COS-7 (a) and HeLa (b) cell lines in the presence of GCL/DOPE-pCMV-IL12 lipoplexes formed at ρeff = 5 (unstriped bars) and 10 (striped bars). The lipoplexes were incubated with cells for 48 h. The blue bar corresponds to commercial Lipo2000*, used here as positive control. The data represent the mean of three wells, and errors are within ±5%.