Gemini Lipopeptide Bearing an Ultrashort Peptide for Enhanced Transfection Efficiency and Cancer-Cell-Specific Cytotoxicity

Abstract

Cationic gemini lipopeptides are a relatively new class of amphiphilic compounds to be used for gene delivery. Through the possibility of incorporating short peptides with cell-penetrating functionalities, these lipopeptides may be advantageous over traditional cationic lipids. Herein, we report the design, synthesis, and application of a novel cationic gemini lipopeptide for gene delivery. An ultrashort peptide, containing four amino acids, arginine-cysteine-cysteine-arginine, serves as a cationic head group, and two α-tocopherol moieties act as hydrophobic anchoring groups. The new lipopeptide (ATTA) is incorporated into the conventional liposomes, containing 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine (DOPE), at different molar ratios. The formulated liposomes are characterized and screened for better transfection efficiency. Transfection activity in multiple human cell lines from cancerous and noncancerous origins indicates that the inclusion of an optimal ratio of ATTA in the liposomes substantially enhances the transfection efficiency, superior to that of a traditional liposome, DOTAP-DOPE. Cytotoxicity of ATTA-containing formulations against multiple cell lines indicates potentially distinct activity between cancer and noncancer cell lines. Furthermore, lipoplexes of the ATTA-containing formulations with anticancer therapeutic gene, plasmid encoding tumor necrosis factor-related apoptosis-inducing ligand (pTRAIL), induce obviously more cytotoxicity than conventional formulations. The results indicate that arginine-rich cationic lipopeptide appears to be a promising ingredient in gene delivery vector formulations to enhance transfection efficiency and cell-selective cytotoxicity.

Scheme 1. Synthesis of Cationic Gemini Lipopeptide (ATTA)

Figure 1

Chemical structures of cationic lipids (ATTA and DOTAP) and the neutral lipid (DOPE).

Figure 2

Physicochemical characterization and the reduction-responsive stability test of liposomal formulations. (A) Hydrodynamic diameters of liposomes at different lipid molar ratios (n = 3). (B) Stability of hydrodynamic diameters of liposomes over a period of 7 days (n = 3). (C) Zeta potentials of liposomes at different lipid molar ratios (n = 3). (D) TEM images of DTD 11 and DTDA 111. (E) Time-dependent relative size variations of DTDA 110.5, DTDA 111, DTDA 112, and DTD 11 with 10 mM dithiothreitol (DTT) treatment (n = 3).

Figure 3

(A) Gel retardation images of lipoplexes at different N/P ratios ranging from 1 to 15. (B) Reduction-responsive DNA release from DTDA 111 lipoplexes with DTT (10 or 50 mM) treatment.

Figure 4

In vitro pEGFP gene transfection efficiency mediated by DTDA 111 compared with that by DTD 11 and lipofectamine in HEK-293 cells. (A) Representative fluorescence microscopy images after pEGFP gene transfection mediated by DTD 11 and DTDA 111 at various N/P ratios ranging from 3 to 9. The scale bar is 200 μm. (B) Relative transfection efficiencies of various lipid formulations quantified from the fluorescence intensities of images (A) using ImageJ software. The ratio of the fluorescence intensity of DTDA 111 to the fluorescence intensity of DTD 11 at a given N/P ratio was calculated. The ratio of the fluorescence intensity of lipofectamine to the fluorescence intensity of DTD 11 at N/P ratio 3 was considered for the lipofectamine value. (C) Fluorescence intensities of pEGFP-transfected cells by various lipid formulations as quantified using ImageJ software (n = 3, *P < 0.05, **P < 0.01).

Figure 5

In vitro pEGFP gene transfection efficiency mediated by DTDA 111 compared with that by DTD 11 and lipofectamine in HEL-299 cells. (A) Representative fluorescence microscopy images after pEGFP gene transfection mediated by DTD 11 and DTDA 111 at various N/P ratios ranging from 3 to 9. The scale bar is 200 μm. (B) Relative transfection efficiencies of various lipid formulations quantified from the fluorescence intensities of images (A) using ImageJ software. The ratio of the fluorescence intensity of DTDA 111 to the fluorescence intensity of DTD 11 at a given N/P ratio was calculated. The ratio of fluorescence intensity of lipofectamine to the fluorescence intensity of DTD 11 at N/P ratio 3 was considered for the lipofectamine value. (C) Fluorescence intensities of pEGFP-transfected cells by various lipid formulations as quantified using ImageJ software (n = 3, **P < 0.01).

Figure 6

In vitro pEGFP gene transfection efficiency mediated by DTDA 111 compared with DTD 11 and lipofectamine in three cancer cell lines (PC3, U87, and A549). (A) Representative fluorescence microscopy images after pEGFP gene transfection mediated by DTD 11 (at N/P ratio 3), DTDA 111 (at N/P ratio 5), and lipofectamine. The scale bar is 200 μm. (B) Relative transfection efficiencies of DTD 11, DTDA 111, and lipofectamine, quantified from the fluorescence intensities of images (A) using ImageJ software.

Figure 7

Relative cell viabilities of liposomes. DTD 11- and DTDA 111-based liposomes at various concentrations of 1–100 μg/mL against (A) HEK-293, (B) HEL-299, (C) PC3, (D) U87, (E) A549, and (F) MDA-MB cells (n = 6, *P < 0.05, **P < 0.01, ***P < 0.001).

Figure 8

Comparison of relative cell viabilities of lipoplexes derived from the control pEGFP plasmid and therapeutic pTRAIL plasmid using DTD 11, DTDA 111, and lipofectamine formulations against (A) PC3, (B) U87, and (C) A549 cells (n = 3, *P < 0.05, **P < 0.01, ***P < 0.001). (D) Microscopy observation of variations in U87 cell morphologies upon transfection with various lipoplexes based on the control pEGFP plasmid and therapeutic pTRAIL plasmid. The scale bar is 200 μm.