siRNA-containing liposomes modified with polyarginine effectively silence the targeted gene

Abstract

Development of RNA interference (RNAi) technology utilizing the short interfering RNA sequences (siRNA) based 'targeted' therapeutics has focused on creating methods for delivering siRNAs to cells and for enhancing siRNA stability in vitro and in vivo. Here, we describe a novel approach for siRNA cellular delivery using siRNA encapsulated into liposomes additionally bearing arginine octamer (R8) molecules attached to their outer surface (R8-liposomes). The R8-liposomal human double minute gene 2 (HDM2)-siRNA demonstrated a significant stability against degradation in the blood serum (siRNA-loaded R8-liposomes remained intact even after 24-h incubation), and higher transfection efficiency into all three tested lung tumor cell lines. siRNA delivery successfully proceeds in the presence of plasma proteins, and R8-liposomes demonstrate low non-specific toxicity. The mechanism of action of R8-liposome-encapsulated siRNA is associated with the RNAi-mediated degradation of the target mRNA. siRNA in R8-liposomes effectively inhibited the targeted gene and significantly reduced the proliferation of cancer cells. The approach offers the potential for siRNA delivery for various in vitro and in vivo applications.

Fig. 1

Formation, characterization and assembly mechanism of R8-lipo-siRNA. (a) Synthetic scheme for intracellular chemical conjugate between an octamer of l-arginine and pNP-PEG-DPPE (R8-PEG-PE) by coupling pNP-PEG-PE to amino group of R8 molecule with the formation of a stable carbamate. (b) Freeze-fracture EM of R8-lipo-siRNAs. (c) Freeze-fracture EM of cross-fracture through R8-lipo-siRNAs. (d) Size distribution pattern of R8-lipo-siRNAs measured by dynamic light scattering. (e) Gel-electrophoresis results of mock (lane 1), free-siRNAs (lane 2), Comp-R8-lipo-siRNA (lane 3), R8-lipo-siRNA (lane 4) and 0.5% SDS treated R8-lipo-siRNAs (lane 5). (f) Schematic of the siRNA encapsulation in a small particle and assembly a large particle.

Fig. 2

Serum stability of siRNA in different lipid preparations and FITC labeled siRNA. (a) Free-siRNAs, (b) FITC labeled siRNA, (c) R8-lipo-siRNAs and (d) the samples from (c) treated by 0.5% SDS at 37 °C for 10 min, (e) Lipofectamine2000-siRNA complex prepared according to manufacturer's procedure with positive charge excess (+ 15 ± 2.3), (f) DOTAP-siRNA complex prepared by hydration of DOTAP film with a siRNA solution at charge ratios (DOTAP/siRNA) of 5-to-1, (g) free-siRNAs (lane 1), FITC labeled siRNA (lane 2), Lipofectamine2000-siRNA complex (lane 3), DOTAP-siRNA complex (lane 4), R8-lipo-siRNA (lane 5) were untreated with 50% mouse serum.

Fig. 3

Translocation of R8-liposomes. Translocation of R8-liposomes was monitored by Leica confocal microscope. Overlay images of both liposomal labels and nucleus (Hoechst 33342) were show here. The cells were treated with the same lipid concentrations (20 μg/ml) of R8-liposomes labeled with 0.1% molar ratio of Rh-PE (Red) in the medium containing 10% FBS at various temperatures for various times and subjected to Hoechst 33342 (Blue) staining. (a) All three lung cancer cells (NCI-H446, A549 and SK-MES-1) were treated with R8-liposomes at 37 °C for 3 h. (b) SK-MES-1 cells were treated with R8-liposomes at 37 °C for 1 h, 3 h and 6 h. (c) SK-MES-1 cells were treated with R8-liposomes at 4 °C for 1 h, 3 h and 6 h.

Fig. 4

Translocation of a FITC labeled HDM2-siRNA into lung cancer cells using R8-liposome. The cells were incubation with either R8-liposome (20 μg/ml) or liposome without R8 (20 μg/ml) and fluorescent HDM2-siRN (200 nM) at 37 °C for 4 h without FBS. Overlay images of both siRNA labels (Green) and nucleus (Hoechst 33342, Blue) were monitored by Leica confocal microscope. (a) All three lung cancer cells (NCI-H446, A549 and SK-MES-1) were treated with R8-lipo-siRNA. (b) All three lung cancer cells were treated with lipo-siRNA without R8.

Fig. 5

Serum effects on R8-mediated translocation of R8-lipo-siRNA into A549 cells. The cells were incubated with R8-liposome (20 μg/ml) containing fluorescent HDM2-siRN (200 nM) at 37 °C for 4 h with 2%, 5%, 10% and 15% FBS. Overlay images of both siRNA labels (Green) and nucleus (Hoechst 33342, Blue) were monitored by Leica confocal microscope.

Fig. 6

HDM2-siRNA reduces expression of HDM2 gene in SK-MES-1 cells. The cells were treated with the various concentrations of HDM2-siRNA in R8-liposomes, and the expression levels of HDM2 were examined by the RT-PCR and Western bolt. (a) HDM2-mRNA levels in the cells treated with 200 nM mock siRNA (lane 3) and HDM2-siRNA different concentrations (lanes 4–7, corresponding 50–200 nM) for 24 h, untreated cells as a control (lane 2). β-Actin was used as a loading control. (b) HDM2 protein levels in the cells treated with 200 nM mock siRNA (lane 2) and HDM2-siRNA deferent concentrations (lanes 3–6, corresponding 50–200 nM) for 24 h, untreated cells as a control (lane 1). β-Actin was used as a loading control.

Fig. 7

Toxicity of R8-liposome. Comparative cytotoxicity of R8-liposome and Lipofectamine2000 toward SK-MES-1 cells at different lipid concentrations (μg/ml) for either 24-h (a) or 48-h (b) incubation was tested. Cell viability in R8-liposomes-free or Lipofectamine2000-free medium was taken as control (100%).

Fig. 8

Effect of HDM2-siRNA in R8-liposomes on the growth inhibition of SK-MES-1 cells. Cells were treated using R8-lipo-HDM2-siRNA and R8-lipo-mock siRNA, where the siRNA concentrations varied from 50 to 100 and 200 nM, the corresponding lipid concentrations were from 7.5 to 15 and 30 μg/ml. Cell viability in R8-liposomes-free medium was taken as 100%.