Plasmid DNA

The plasmid pSGR-JFH1 (DDBJ/EMBL/GenBank accession number AB114136) containing a HCV 2a subgenomic replicon (JFH1) (provided by Takaji Wakita, Tokyo Metropolitan Institute for Neuroscience, Tokyo, Japan) was used to construct a HCV-replicable cell line, Huh7.5.1-HCV. The plasmid pCDNA3.1(+)-3FLAG-Core (pCore-3FLAG) expressing FLAG-tagged core protein was constructed by PCR amplification of the core from plasmid HFL (provided by Dr. Rice, Rockefeller University, USA) containing a full length of HCV 1a and subcloned into the eukaryotic expression vector pCDNA3.1(+)-3FLAG (p-3FLAG). The plasmid containing the full sequence of the HCV genome 5′UTR (pGL3-5′UTR-luc) was constructed by PCR amplification from plasmid HFL and subcloned into the Luciferase Reporter Vector, pGL3-luciferase (pGL3-luc). The HCV genome 5′UTR was used as the promoter for reporter gene luciferase. All these plasmids were constructed previously in our laboratory and are shown in Figure. S1.

Reagents and antibodies

The primary antibodies used for this study were as follows: the mouse anti-HCV NS5A monoclonal antibody [H26] (Abcam, USA), mouse anti-HCV NS3 monoclonal antibody (Abcam, USA), goat anti-HCV core polyclonal antibody (Santa Cruz, USA) and mouse anti-β-actin monoclonal antibody (Boster, China). DAPI (Santa Cruz, USA) and Lipofectamine 2000 (Invitrogen, USA) was used per the manufacturer’s instructions.

Cell culture

The human hepatocellular carcinoma cell line Huh7.5.1 was maintained in the Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS; Gibco, USA), 100 U/ml penicillin and 100 μg/ml streptomycin. The replication ability of the JFH-1 replicon in Huh7.5.1 cells was established by transfecting RNA transcribed from linearized pSGR-JFH1 into Huh7.5.1 cells. Transfected cells were cultured for 3 weeks with G418 (Sigma, USA), resulting in an HCV-replicable cell line (Huh7.5.1-HCV). All other procedures were performed as previously described52. The Huh7.5.1-HCV cells were maintained in DMEM supplemented with 10% FBS, 100 U/ml penicillin, 100 μg/ml streptomycin and 400 μg/ml G418. The cell culture was maintained at 37 °C in a humid atmosphere containing 5% CO 2 .

Synthesis of siRNAs

The siRNAs against the HCV 2a replicon (JFH1), HCV 1a 5′UTR and the core gene were designed to the most conserved target region of these genes using the Ambion’s siRNA desigion tool http://www.ambion.com/techlib/misc/siRNA_finder.html. Negative control siRNAs (scrambled siRNAs) were designed using scrambled sequences. All siRNAs were chemically synthesized by invitrogen (USA) and are shown in Table S1.

Preparation and characterization of nanoscale VE-DC/siRNA

Cholesterol-based cationic liposomes were prepared by the thin-film hydration method53. Briefly, 1,2-dioleoyl-3-trimethylammoniumpropane (DOTAP) (Avanti, USA) and cholesterol (GENVIEW, USA) (molar ratio = 1:1) were dissolved in 10 ml trichloromethane. Removal of the organic solvent by rotary evaporation under vacuum at 40 °C, resulted in a thin film, which was then combined with 10 ml trichloromethane and 3 ml phosphate buffered saline (PBS) and sonicated with a probe sonicator at 100 W for 2 min at 37 °C to form a mixture. The mixture was rotated under a partial vacuum at 40 °C to prepare a gel state and then 7 ml PBS was added. The gel state was rotated for 30 min at ordinary pressure at 40 °C, resulting in the formation of a cationic liposome suspension, referred to as DC. Vitamin E coupled DCs (VE-DCs) were prepared by mixing vitamin E (SIGMA, USA) with the cationic liposome suspensions at a 1:2 (mol/mol) ratio overnight at 4 °C. The VE-coupled DCs carrying siRNA (VE-DC-siRNA) were prepared by mixing a solution of siRNA with VE-coupled liposomes at a 1:1 (mol ∕mol) ratio for 10 min at 37 °C. The size and surface charge (zeta potential) of the nanoscale VE-DC/siRNA were determined with a laser particle size analyzer (Rise-2008, China) and the experiment was repeated three times. The homogeneity and dispersivity of the samples were analyzed by transmission electron microscopy (HITACHI7500, Japan). The optimal molar ratio of VE-DC and siRNA for transfection of siRNA into Huh7.5.1 cells was determined by treating the cells with VE-DC carrying Cy3-labeled siRNA (Cy3 bound to the sense strand of siRNA) (VE-DC/siRNA-Cy3) in fractionated molar ratios ranging from 4:1 to 1:4 for 24 h. The transfection efficiency of Huh7.5.1 cells by VE-DC/siRNA with fractionated molar ratios was then determined by flow cytometry (Becton Dickinson, USA).

In vitro stability assays

Naked siRNA, DC/siRNA and VE-DC/siRNA were incubated in 50% human serum at 37 °C for 0, 3, 6, 12, 24, 48, 72 and 96 h. Aliquots taken at different time points were treated with 0.1% SDS and immediately stored at 72 °C. All samples were separated in 2% agarose gels and stained with Good ViewTM (SBS Genetech, China). The results were recorded with a gel imaging system (Gel Doc 1000, Bio-Rad, USA).

In vitro cytotoxicity of VE-DC/siRNA

The CCK-8 assay was used to assess the cytotoxicity of VE-DC/siRNA using a Cell Counting Kit (Dojindo, Japan) following the manufacturer’s protocol. The data were reported as the fold change over the untreated control.

Cellular uptake of VE-DC/siRNA

Huh7.5.1 and Huh7.5.1-HCV cells were plated onto cleaned-up cover slips. DC/siRNAs-Cy3, VE-DC/siRNAs-Cy3 and liposome/siRNAs-Cy3 were added to the cells at a final siRNA concentration of 40 nM. At 24 h post-treatment, the cells were washed with PBS, fixed with 4% paraformaldehyde and exposed to DAPI for 1 min to stain nuclei. The subcellular localization of Cy3-labeled siRNAs was assessed by fluorescence microscopy (Leica DM4000, Germany).

Immunofluorescent staining

Cells or frozen sections were washed with PBS and fixed in 4% paraformaldehyde, then permeabilized with 0.2% Triton X-100. Cover slips were rinsed and incubated with blocking serum and then incubated overnight at 4 °C with primary anti-HCV Core or anti-HCV NS3 antibody. The cells were then washed with PBS and stained with the corresponding FITC-conjugated secondary antibody. The nuclei were visualized by staining the cells with DAPI. The fluorescent images were then observed and analyzed by fluorescence microscopy.

Real time quantitative PCR analysis

Cells were transfected with DC/siRNAs, VE-DC/siRNAs, or liposome/siRNAs for 24 h and then lysed with Trizol (Invitrogen, Carlsbad, CA, USA). Complementary single-stranded DNA was synthesized from total RNA by reverse transcription (TaKaRa, Japan). Quantification of cDNA targets was performed on CFX96TM Real-Time-PCR Detection System (Bio-Rad, USA). Primers were synthesized by Invitrogen and are listed in Table S2.

Western blotting

The levels of HCV NS3, NS5A and core protein in cells or liver tissues were evaluated by western blotting. Briefly, samples containing equal amount of protein were separated by SDS-PAGE and blotted onto PVDF membranes. The membranes were blocked with 5% bovine serum albumin and incubated with anti-HCV Core, anti-HCV NS3, anti-NS5A, or anti-β-actin antibodies, following by incubation with secondary antibodies conjugated with horseradish peroxidase. The proteins of interest were detected using the SuperSignal West Pico Chemiluminescent Substrate kit (ThermoFisher Scientific, USA). The results were recorded by the Bio-Rad Electrophoresis Documentation (Gel Doc 1000, Bio-Rad, USA) and Quantity One Version 4.5.0.

In vitro luciferase assay

The luciferase assay was performed as previously described54. Briefly, the Huh7.5.1 cells were seeded and grown in each well of 24-well culture plates, transfected with plasmid transfection mix (pRL-PK Renal luciferase, 100 ng; pGL3-5′UTR-luc or pGL3-luc, 900 ng) for 24 h and then treated with DC/siRNA or VE-DC/siRNA for another 24 h. The cells were then lysed in 1× luciferase lysis buffer (Promega, USA) and the lysates were assayed for luciferase activity on a multiple function enzyme analyzer. The firefly luciferase activity was normalized for the transfection efficiency based on renilla luciferase activity.

Animals

Female BALB/c mice, 6 weeks of age, were purchased from the animal center at Chongqing Medical University. All the in vivo experiments were approved and conducted in accordance with the guidelines established by the University Animal Care and Use Committee for Laboratory Animal Research in Chongqing Medical University.

In vivo distribution of siRNA delivered by VE-DC

VE-DC/siRNA-Cy3 was intravenously injected into BALB/c mice. At 1 h after injection, the livers and other organs (heart, lungs, spleen and kidneys) were harvested from each mouse. Tissue specimens were immediately embedded in OCT medium and cryogenically sectioned. Sections of these tissues were then stained with DAPI and slides were finally examined by fluorescence microscopy.

In vivo HCV siRNA treatment

We evaluated the effect of siRNA treatment on HCV by conducting two sets of experiments. The first set of experiments involved the constructions of an HCV mouse model by hydrodynamic injection of 5 μg pCore-3FLAG or control p-3FLAG. Twenty-four hours later, PBS, naked siRNA, DC/siRNA (siRNA-core, 0.8 mg/kg), or VE-DC/siRNA (siRNA-core, 0.8 mg/kg) was intravenously administered (n = 4, each group). On day 2 after siRNA treatment, the mice were sacrificed and liver tissues were homogenized. The HCV core protein level was determined by immunofluorescent staining and western blotting. The second set of experiments involved the construction of the other HCV mouse model by hydrodynamic injection of 5 μg pGL3-5′UTR-luc. 24 h later, PBS, naked siRNA (siRNA-5′UTR, 0.8 mg/kg), DC/siRNA (siRNA-5′UTR, 0.8 mg/kg) or VE-DC/siRNA (siRNA-5′UTR, 0.8 mg/kg) were intravenously administered. On day 2 after siRNA treatment, whole body images showing RNAi activity were analyzed using a Xenogen IVIS Spectrum instrument (PERKIN ELMER, USA).

Hematological and serum biochemical and histological analysis

Blood samples were drawn from the orbits of mice 24 h after injection of VE-DC/siRNA. Hematological parameters (white blood cells, red blood cells and platelets) were determined by a full automatic blood cell analysis (SYSMEX XS-500i, Japan). The total protein, alanine aminotransferase (ALT), blood urine nitrogen (BUN) and creatinine levels in serum were assayed using an automated chemical analyzer Hitachi7600-110 (Japan). Serum IFN-a level was detected using an ELISA Kit (DGE, China) following the manufacturer’s protocol. Heart, liver, spleen, lung and kidney tissues were collected from mice 24 h after injection of VE-DC/siRNA, fixed and embedded in paraffin wax and then sectioned into 5 μm thick slices. Then sections were stained with H-E and examined by light microscopy for alteration of histological structures.

Statistical analysis

All values in the text and figures are presented as the means ± standard deviation (SD). The differences were analyzed using t test and one-way ANOVA followed by the Student-Newman-Keuls test and all statistical analyses were performed using GraphPad Prism software (GraphPad Software, La Jolla, California, USA). Statistical differences are presented at probability levels of *p < 0.05, **p < 0.01 and ***p < 0.001.