Novel cationic lipid that delivers siRNA and enhances therapeutic effect in lung cancer cells

Abstract

We have developed lipid-polycation-DNA (LPD) nanoparticles containing DOTAP and targeted with polyethylene glycol (PEG) tethered with anisamide (AA) to specifically deliver siRNA to H460 human lung carcinoma cells which express the sigma receptor. A novel non-glycerol based cationic lipid which contains both a guanidinium and a lysine residue as the cationic headgroup, i.e. DSGLA, downregulated pERK more efficiently in H460 cells than DOTAP. As demonstrated by using fluorescently labeled siRNA, LPD-PEG-AA prepared with DSGLA efficiently delivered siRNA to the cytoplasm of the H460 cells. Although the siRNA delivered by LPD-PEG-AA containing either DOTAP or DSGLA could effectively silence EGFR expression, a synergistic cell killing effect in promoting cellular apoptosis was only observed with DSGLA. The fluorescently labeled siRNA was efficiently delivered into the cytoplasm of H460 xenograft tumor by the LPD-PEG-AA containing either DOTAP or DSGLA 4 h after intravenous injection. Three daily injections (0.6 mg/kg) of siRNA formulated in the LPD-PEG-AA containing either DOTAP or DSGLA could effectively silence the epidermal growth factor receptor (EGFR) in the tumor, but the formulation containing DSGLA could induce more cellular apoptosis. A significant improvement in tumor growth inhibition was observed after dosing with LPD-PEG-AA containing DSGLA. Thus, DSGLA served as both a formulation component as well as a therapeutic agent which synergistically enhanced the activity of siRNA.

Figure 1

The structure of DSGLA and pERK inhibition induced by lipids. (A) The structure of DSGLA. (B) pERK and ERK expression in H460 cells after incubation with 10 μM DSGLA and DOTAP for various times.

Figure 2

Intracellular uptake of siRNA and EGFR expression inhibited by siRNA formulation in H460 cells in vitro. Fluorescence photographs of cultured H460 cells after treatment with 5′-cy3 labeled siRNA against an irrelevant target in LPD-PEG or LPD-PEG-AA with DSGLA or DOTAP as the cationic lipid for 4 h (A). Quantitative measurement of mean fluorescence intensity (MFI) of cy3 siRNA uptake by flow cytometry. Data = mean ± SD, n = 3 (B). (C) Western blot analysis of EGFR and β-Actin in H460 cells treated with LPD-PEG with formulations containing anisamide ligand (AA+) or without (AA−). Formulations were prepared with either DOTAP or DSGLA.

Figure 3

Apoptosis induced by siRNA formulation in vitro. Cells treated with different formulations for 72 h and analyzed for TUNEL staining (A), or AIF distribution (B). Cells with nuclear AIF are shown by arrows. Formulations contained anisamide ligand (AA+) or without (AA−). Formulations were prepared with either DOTAP or DSGLA.

Figure 4

Tissue distribution and intracellular uptake of siRNA in different formulations. Fluorescence signal of cy3 labeled siRNA in different tissues observed by confocal microscopy.

Figure 5

EGFR and p-ERK expression in H460 xenograft tumor. Western blot analysis of EGFR (A) and p-ERK (B) in the H460 xenograft tumor after treatment with different formulations.

Figure 6

Synergistic apoptosis induction in H460 xenograft tumor. TUNEL staining (positive cells indicated by dark arrows) (A) and AIF expression and localization (cells with nuclear AIF indicated by red arrows) (B) in H460 tumor cells after treatment with EGFR siRNA with different formulations in vivo. (C) Quantitative analysis of TUNEL positive staining and nuclear translocation of AIF in the tumors treated with different formulations. a, EGFR siRNA in DSGLA AA+; b, EGFR siRNA in DSGLA AA−; c, EGFR siRNA in DOTAP AA+; d, EGFR siRNA in DOTAP AA−; e, control siRNA in DSGLA AA+; f, free siRNA. Data = mean ± SD, n = 3–4. * indicates P < 0.05.

Figure 7

H460 xenograft tumor growth inhibition by siRNA in different formulations. Solid arrows indicate the intravenous administrations of siRNA (0.6 mg/kg). Data = mean, n = 5–7. SD of the data points is not shown for clarity.