A Gemini Cationic Lipid with Histidine Residues as a Novel Lipid-Based Gene Nanocarrier: A Biophysical and Biochemical Study

Abstract

This work reports the synthesis of a novel gemini cationic lipid that incorporates two histidine-type head groups (C₃(C₁₆His)₂). Mixed with a helper lipid 1,2-dioleoyl-sn-glycero-3-phosphatidyl ethanol amine (DOPE), it was used to transfect three different types of plasmid DNA: one encoding the green fluorescence protein (pEGFP-C3), one encoding a luciferase (pCMV-Luc), and a therapeutic anti-tumoral agent encoding interleukin-12 (pCMV-IL12). Complementary biophysical experiments (zeta potential, gel electrophoresis, small-angle X-ray scattering (SAXS), and fluorescence anisotropy) and biological studies (FACS, luminometry, and cytotoxicity) of these C₃(C₁₆His)₂/DOPE-pDNA lipoplexes provided vast insight into their outcomes as gene carriers. They were found to efficiently compact and protect pDNA against DNase I degradation by forming nanoaggregates of 120⁻290 nm in size, which were further characterized as very fluidic lamellar structures based in a sandwich-type phase, with alternating layers of mixed lipids and an aqueous monolayer where the pDNA and counterions are located. The optimum formulations of these nanoaggregates were able to transfect the pDNAs into COS-7 and HeLa cells with high cell viability, comparable or superior to that of the standard Lipo2000*. The vast amount of information collected from the in vitro studies points to this histidine-based lipid nanocarrier as a potentially interesting candidate for future in vivo studies investigating specific gene therapies.

Keywords: biophysical characterization; cell viability; gemini cationic lipid with histidine residues; gene delivery; lipid-based gene nanocarrier; plasmid DNAs; transfection.

Scheme 1

Chemical procedure used to synthesize the gemini cationic lipid C₃(C₁₆His)₂ from N(α)-Cbz-histidine: (a) (CH₃)₂SO₄ in methanol, (b) hexadecyl amine, benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate (BOP), and 1,4-diazabicyclo[2.2.2]octane (DABCO) in dimethyl formamide (DMF), (c) hydrogenation, Pd/C in methanol, and (d) glutaric acid (HOOC-CH₂-CH₂-CH₂-COOH), BOP, and DABCO in DCM.

Figure 1

Plots of the zeta potential (ζ) against the m_L/m_DNA mass ratio in the C₃(C₁₆His)₂/DOPE-pDNA lipoplexes at various mole fractions of the C₃(C₁₆His)₂ cationic lipid in the C₃(C₁₆His)₂/DOPE mixed lipid (α = 0.2, 0.4, 0.5, 0.6 and 0.7; red, blue, pink, orange and green, respectively) and of the C₃(C₁₆His)₂/DOPE-ctDNA lipoplex at α = 0.5 (black). Inset: Electrophoresis agarose gel of the C₃(C₁₆His)₂/DOPE-pDNA lipoplexes at various m_L/m_DNA mass ratios and α = 0.2 and 0.5.

Figure 2

SAXS diffractograms of C₃(C₁₆His)₂/DOPE-pDNA lipoplexes at an effective charge ratio ρ_eff = 4 and several molar fractions of the C₃(C₁₆His)₂ cationic lipid in the C₃(C₁₆His)₂/DOPE mixed lipid (α).

Scheme 2

Lamellar structure of the C₃(C₁₆His)₂/DOPE-pDNA lipoplex showing (a) the gel phase, and (b) the fluid phase of the C₃(C₁₆His)₂/DOPE mixed lipid bilayer after reaching the gel-to-fluid transition temperature (T_m). In the figure, the interlayer periodic distance (d) results from the sum of the lipid bilayer (d_m) and aqueous monolayer (d_w) thicknesses.

Figure 3

Fluorescence anisotropy at 430 nm (r₄₃₀) of the DPH fluorescent probe against the temperature for the C₃(C₁₆His)₂/DOPE-pDNA lipoplexes at an effective charge ratio ρ_eff = 4 and two C₃(C₁₆His)₂ cationic lipid molar fractions (ρ = 0.2 in red and α = 0.5 in black) in the C₃(C₁₆His)₂/DOPE mixed lipid. The inset shows determination of the gel-to-fluid transition temperature (T_m) following the Phillips method. Errors are less than 3%.

Figure 4

pDNA protection assay against degradation by DNase I (gel electrophoresis experiments): (a) pCMV-Luc plasmid, and (b) pEGFP-C3 plasmid. In both experiments: lane 1, pDNA control; lane 2, pDNA–DNase I; lanes 3–6, C₃(C₁₆His)₂/DOPE-pDNA lipoplexes at two molar fractions of the C₃(C₁₆His)₂ cationic lipid in the mixed lipid (lanes 3–4, α = 0.2; lanes 5–6, α = 0.5) and two effective charge ratios of the lipoplex lanes 3 and 5, ρ_eff = 4; lanes 4 and 6, ρ_eff= 10).

Figure 5

Transfection efficiency levels of C₃(C₁₆His)₂/DOPE-pDNA lipoplexes in HeLa (solid bars) and COS-7 cells (dashed bars) at two molar fractions of the C₃(C₁₆His)₂ cationic lipid in the C₃(C₁₆His)₂/DOPE mixed lipid (α = 0.2 and 0.5): (a) in terms of ng of luciferase/mg of protein for plasmid pCMV-Luc, and (b) in terms of mean fluorescence intensity (MFI) for plasmid pEGFP-C3. The experiments were performed in the presence of 10% of fetal bovine serum (FBS). The green and blue bars correspond to effective charge ratios ρ_eff = 4 and 10 of the lipoplex, respectively. Gray bar: Lipo2000* as the positive control. The data represent the mean ± SD of three wells and are representative of three independent experiments.

Figure 6

Cell viability of COS-7 cells in the presence of C₃(C₁₆His)₂/DOPE-pDNA lipoplexes at two molar fractions of the C₃(C₁₆His)₂ cationic lipid in the C₃(C₁₆His)₂/DOPE mixed lipid (α = 0.2 and 0.5): (a) with pCMV-Luc plasmid and (b) with pEGFP-C3 plasmid. The green and blue bars correspond to effective charge ratios ρ_eff = 4 and 10 of the lipoplex, respectively. Gray bar: Lipo2000* as the positive control. The data represent the mean ± SD of three wells and are representative of three independent experiments.

Figure 7

Transfection activity of C₃(C₁₆His)₂/DOPE-pDNA lipoplexes in COS-7 cells at two molar fractions of the C₃(C₁₆His)₂ cationic lipid in the C₃(C₁₆His)₂/DOPE mixed lipid (α = 0.2 and 0.5) carrying plasmid pCMV-IL12. The green and blue bars correspond to effective charge ratios ρ_eff = 4 and 10 of the lipoplex, respectively. Gray bar: Lipo2000* as the positive control. The data represent the mean ± SD of three wells and are representative of three independent experiments.