Arginine-based cationic liposomes for efficient in vitro plasmid DNA delivery with low cytotoxicity

Abstract

Background: Currently available gene delivery vehicles have many limitations such as low gene delivery efficiency and high cytotoxicity. To overcome these drawbacks, we designed and synthesized two cationic lipids comprised of n-tetradecyl alcohol as the hydrophobic moiety, 3-hydrocarbon chain as the spacer, and different counterions (eg, hydrogen chloride [HCl] salt or trifluoroacetic acid [TFA] salt) in the arginine head group.

Methods: Cationic lipids were hydrated in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer to prepare cationic liposomes and characterized in terms of their size, zeta potential, phase transition temperature, and morphology. Lipoplexes were then prepared and characterized in terms of their size and zeta potential in the absence or presence of serum. The morphology of the lipoplexes was determined using transmission electron microscopy and atomic force microscopy. The gene delivery efficiency was evaluated in neuronal cells and HeLa cells and compared with that of lysine-based cationic assemblies and Lipofectamine™ 2000. The cytotoxicity level of the cationic lipids was investigated and compared with that of Lipofectamine™ 2000.

Results: We synthesized arginine-based cationic lipids having different counterions (ie, HCl-salt or TFA-salt) that formed cationic liposomes of around 100 nm in size. In the absence of serum, lipoplexes prepared from the arginine-based cationic liposomes and plasmid (p) DNA formed large aggregates and attained a positive zeta potential. However, in the presence of serum, the lipoplexes were smaller in size and negative in zeta potential. The morphology of the lipoplexes was vesicular. Arginine-based cationic liposomes with HCl-salt showed the highest transfection efficiency in PC-12 cells. However, arginine-based cationic liposomes with TFA salt showed the highest transfection efficiency in HeLa cells, regardless of the presence of serum, with very low associated cytotoxicity.

Conclusion: The gene delivery efficiency of amino acid-based cationic assemblies is influenced by the amino acids (ie, arginine or lysine) present as the hydrophilic head group and their associated counterions.

Keywords: cationic liposome; counterions; cytotoxicity; pDNA; transfection efficiency.

Figure 1

Syntheses of arginine-based cationic lipids. Abbreviations: BOP, benzotriazol-1-yloxytris (dimethylamino) phosphonium hexafluorophosphate; EtOAc, ethyl acetate; DCM, dichloromethane; HCl, hydrogen chloride; TEA, triethylamine; TFA, trifluoroacetic acid.

Figure 2

Transmission electron microscope images of the arginine-based cationic liposomes constructed with (A) a and (B) b. The cationic liposomes were stained with 1% phosphotungstic acid. Atomic force microscope images of the liposomes constructed with (C) a and (D) b.

Figure 3

Morphology of the lipoplexes prepared at 10/1 (w/w) lipid-to-plasmid (p) DNA ratio. Transmission electron microscope images of a/pDNA (A) and b/pDNA (B); atomic force microscope images of a/pDNA (C) and b/pDNA (D).

Figure 4

Size and zeta potential of the lipoplexes prepared from the arginine-based cationic liposomes and plasmid (p) DNA at a varying lipid-to-pDNA ratios (w/w). Lipoplexes were prepared and diluted with Dulbecco’s modified Eagle’s medium in the absence (A) a and (C) b and in the presence of serum (B) a and (D) b. Notes: The differences in size and zeta potential of the lipoplexes observed in the presence and in the absence of serum were statistically significant (P < 0.01). Data are presented as the mean ± standard deviation (n = 5).

Figure 5

Gel retardation assay of the lipoplexes prepared at different lipid (0.6–6.0 μg) to plasmid (p) DNA (0.2 μg) ratios (ie, 3/1 to 30/1 w/w or 1.28 to 12.8 N/P ratios) in the absence and presence of serum, (A) a and (B) b. Notes: “Neg. control” (the right-most well of A) denotes that Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS) was loaded. “Pos. control” (the right-most well of B) denotes that 0.2 μg plasmid DNA without DMEM and FBS was loaded.

Figure 6

The transfection efficiency of each of the amino acid-based cationic assemblies, (a–d), was compared with that of Lipofectamine™ 2000 (LA2000) in PC-12 and HeLa cells. Transfection experiments were performed in the absence of serum in PC-12 cells (A) and HeLa cells (B) and in the presence of serum in PC-12 cells (C) and HeLa cells (D). Notes: Units of luciferase activity are plotted against the various lipid-to-plasmid (p) DNA ratios (w/w). Data are presented as the mean ± standard deviation (n = 5); *P < 0.05; **P < 0.01. Abbreviation: RLU, relative light unit.

Figure 7

Cytotoxicity of the arginine-based cationic liposomes was compared with that of Lipofectamine™ 2000 (LA2000) in PC-12 cells (A) and HeLa cells (B). Notes: Cytotoxicity experiment was performed in the presence of serum (10% fetal bovine serum) and estimated through water-soluble tetrazolium-1 assay. Data were analyzed and are presented plotted in a semi-logarithmic plot.