Target delivery of small interfering RNAs with vitamin E-coupled nanoparticles for treating hepatitis C

RNA interference (RNAi) represents a promising strategy for the treatment of HCV infection. However, the development of an effective system for in vivo delivery of small interfering RNA (siRNA) to target organ remains a formidable challenge. Here, we develop a unique nanoparticle platform (VE-DC) composed of α-tocopherol (vitamin E) and cholesterol-based cationic liposomes (DOTAP-Chol) for systemic delivery of siRNAs to the liver. A HCV-replicable cell line, Huh7.5.1-HCV, and a transient HCV core expressing cell line, Huh7.5.1-Core, were constructed and used to assess the in vitro anti-HCV activity of VE-DC/siRNAs. A transient in vivo HCV model was also constructed by hydrodynamic injection of pCDNA3.1(+)-3FLAG-Core (pCore-3FLAG) plasmid expressing core protein or pGL3-5′UTR-luciferase (pGL3-5′UTR-luc) plasmid expressing luciferase driven by HCV 5′UTR. Nanoscale VE-DC/siRNA was intravenously injected to assess the liver-targeting property as well as antiviral activity. The nanoscale VE-DC effectively exerted an anti-HCV activity in the in vitro cell models. Post-administration of VE-DC/siRNAs also effectively delivered siRNAs to the liver, suppressing core protein production and firefly luciferase activity, without inducing an innate immunity response or off-target and toxicity effects. The VE-DC platform has high potential as a vehicle for delivery of siRNAs to the liver for gene therapy for targeting hepatitis C.


Results
Inhibition of HCV gene expression by efficient siRNA in vitro. Following transfection with RNA transcribed from linearized pSGR-JFH1 and selection by cultivation with G418, the HCV-replicable cell line Huh7.5.1-HCV was constructed and the expression of HCV nonstructural protein 3 (NS3) and 5A (NS5A) were examined by western blotting and immunofluorescent staining assays. HCV NS3 and 5A were detected in Huh7.5.1-HCV, but not in Huh7.5.1 (Fig. 1A,B). Three siRNAs (siNS5B, siNS4A, siNS5A) were designed to target the replicon of HCV virus gene for destruction of its replication, their efficacy was examined by real-time PCR and western blot analysis for the expression of HCV NS3 or NS5A gene. All three siRNAs efficiently suppressed gene and protein expression of NS3 or NS5A (Fig. 1C-E), but the greatest efficacy was observed using siNS5A to target HCV replicon, so it was selected for subsequent experiments.
The plasmid pCore-3FLAG expressing the HCV core protein was transfected into Huh7.5.1 cells and core protein expression was examined at 48 h post-transfection by western blotting and immunofluorescent staining analysis (Fig. 1F,G). The core protein was detected using its specific antibody. Two core siRNA (siRNA-core 1 and siRNA-core 2) were designed to target the HCV core protein, and their efficacy was examined by real-time PCR and western blotting. Both core-specific siRNAs suppressed the core protein expression at the gene and protein levels (Fig. 1H,I), but the greatest efficacy was observed using siRNA-core 1, so it was selected for subsequent experiments.
Characterization of nanoscale VE-DC/siRNA. We determined the optimal VE-DC/siRNA ratio for transfection of siRNA (siNS5A) into Huh7.5.1 cells by incubating VE-DC carrying Cy3-labeled siRNA (VE-DC/ siRNA-Cy3) in varied molar ratios ranging from 4:1 to 1:4. After transfection of Huh7.5.1 cells with fractionated molar ratios of VE-DC/siRNA-Cy3 ( Fig. 2A), the transfection efficiency was determined using flow cytometry. The highest mean fluorescence intensity (MFI) was observed at a molar ratio of 1:1 in the incubation solution ( Fig. 2B,C). We used complexes at this incubation molar ratio in subsequent experiments. The particle size distribution and zeta potential of the VE-DC/siRNA were determined at 37 °C with a laser particle size analyzer as 118.0 ± 7.7 nm and 40.6 ± 3.4 mV for DC/siRNA and 125.5 ± 9.0 nm and 39.1 ± 4.8 mV for VE-DC/siRNA (Fig. 2D,E). The VE-DC/siRNA had a spherical morphology and homogeneous distribution, as determined from TEM images (Fig. 2F), and its diameter was consistent with the results from the laser particle size analyzer.
Naked siRNA, DC/siRNA and VE-DC/siRNA in 50% human serum were compared for their in vitro stability. We observed intact siRNA even after 24 h in 50% serum in the DC/siRNA or VE-DC/siRNA groups. By contrast, naked siRNA was completely degraded within 6 h under the same conditions (Fig. 2G). These data suggested that the delivery vehicle may significantly improve the stability of siRNA in serum.
The cytotoxicity of VE-DC/siRNA was addressed by measuring cell viability by the CCK8 assay (Fig. 2H). The cell viabilities were much higher for the VE-DC/siRNA-treated Huh7.5.1 cells than for the cells treated with commercial liposomes. Treatment with a concentration of commercial liposomes of 100 nM resulted in only 45% Scientific RepoRts | 6:24867 | DOI: 10.1038/srep24867 viable cells. By contrast, DC/siRNA or VE-DC/siRNA treatments showed low cytotoxicity, even at concentrations up to 200 nM, which was much higher than the actual amount used for gene transfection.
Silencing of target genes in liver of mice by VE-DC/siRNA. The liver-specific expression of the HCV core gene was established by hydrodynamic tail vein injection of plasmid pCore-3FLAG in Balb/c mice (Fig. 4A). The core protein expression was evaluated by western blotting (Fig. 4B) and immunofluorescent staining (Fig. 4C) assays. We assessed the in vivo silencing ability of VE-DC/siRNA (siRNA-core) by administering VE-DC/siRNA to this mouse model, which harbors liver-specific expression of the core protein. We first investigated whether delivery of VE-DC/siRNA-Cy3 to the liver had been successful. The mice were injected with VE-DC/siRNA-Cy3 via the tail vein and the livers were extirpated and observed by fluorescence microscopy at 1 h after injection. We observed a marked accumulation of the Cy3 signal in the liver, while a weak signal was found in the control liver sections from the mice injected with naked siRNA-Cy3 or DC/siRNA-Cy3 (Fig. 4D). These results demonstrated that VE-DC was more effective at delivering siRNA into the liver cells.
We also investigated the in vivo distribution of siRNA delivered by VE-DC. At 1 h after injection, the fluorescence intensity was higher in the liver than in other internal organs, such as heart, spleen, lung, and kidney, which suggested that VE-DC facilitated the liver-specific cellular uptake of siRNA (Fig. 4E). Mice were also sacrificed on the second day after injection of VE-DC/siRNA for measurement of expression of core protein in the liver by western blotting (Fig. 4F,G) and immunofluorescent staining (Fig. 4H) assays. The DC/siRNA inhibited core protein expression by up to 36.17% in the livers of some mice (probability, one in five), but this non-targeted delivery was not statistically significant. By contrast, VE-DC/siRNA reduced the target core protein by an average of 83.01%. Taken together, these results suggest that VE-DC facilitated liver-specific cellular uptake of siRNA, which subsequently suppressed HCV core protein production.
We validated the repressive effect of VE-DC/siRNA on HCV replication using DC/siRNA (siRNA-5′ UTR) or VE-DC/siRNA (siRNA-5′ UTR) to treat the Huh7.5.1 cells. The cells were co-transfected with pRL-PK Renal luciferase and pGL3-5′ UTR-luc, and the relative luciferase activity was detected by a multiple function enzyme analyzer. The relative luciferase activity was significantly lower in VE-DC/siRNA-treated cells than in naked siRNA-treated or DC/siRNA-treated cells (Fig. 5A). We evaluated the destructive effect of VE-DC/siRNA on HCV replication in a model of liver-specific expression of luciferase regulated by HCV 5′ UTR, which we established by the hydrodynamic tail vein injection of plasmid pGL3-5′ UTR-luc in Balb/c mice (Fig. 5B). These mice transiently expressed luciferase in the liver, and we determined the changes in liver-specific expression of luciferase by whole body image analysis 24 h after injection of VE-DC/siRNA (siRNA-5′ UTR) (Fig. 5C). The relative luciferase activity was significantly lower in the VE-DC/siRNA group than in the naked siRNA-treated or DC/ siRNA-treated cells, suggesting that VE-DC facilitated liver-specific cellular uptake of siRNA and subsequent efficient silencing of HCV replication.
No side effects are produced by VE-DC/siRNA. In addition to therapeutic efficacy, we also evaluated the in vivo side effects of the VE-DC/siRNA. Hematoxylin-eosin (H-E) staining results indicated no noticeable histological changes in the tissues from heart, liver, spleen, lung, and kidney, suggesting no organ toxicity (Fig. 6A). The complete blood count (CBC) results showed that white blood cells, red blood cells, and platelets did not differ between the control group and VE-DC/siRNA group ( Fig. 6B-D). In addition, no abnormalities were evident in biochemical parameters, including total protein, alanine aminotransferase (ALT), blood urine nitrogen (BUN), and creatinine ( Fig. 6E-H). These results suggest that VE-DC/siRNA had no effect on the blood system, on liver and kidney functions, and on the histopathology of organs. We examined whether VE-DC/siRNA caused a type I IFN response by measuring the induction of interferons (IFNs) in serum 3 h after the injection of VE-DC/siRNA. The serum level of IFN-α was barely detectable by ELISA (Fig. 6I), and no increases in IFN-β gene expression were observed in the liver by real-time PCR (Fig. 6J). These results suggest that VE-DC/siRNA does not trigger an immune reaction.

Discussion
Safe and efficient gene delivery is a major task that requires the development of new gene vectors with low cytotoxicity and high gene transfection performance. Previous studies have used hydrodynamic injection 27 , viral vectors 28 , and cationic liposomes 29 as the main methods of in vivo gene delivery. However, hydrodynamic injection is unsuitable for human therapy, and application of viral vectors may result in severe side effects. By contrast, since the physical properties of cationic liposomes are well understood, liposomes are good prospects for clinical applications. In this paper, we designed and synthesized a novel siRNA-packing nanoscale cholesterol-based cationic liposomes aimed at destroying HCV RNA and coupled this with VE promoted delivery of siRNA to the liver. The biological behaviors of the VE-DC/siRNA platform were evaluated and confirmed high delivery efficiency and low toxicity, indicating it as potential gene delivery vector.
Several delivery systems for siRNA based on liposomes show great promise due to their favorable characteristics, such as the ease of large-scale production and biocompatibility. Their use in gene therapy is currently under investigation in several clinical trials for the treatment of diseases 30 . However, the use of liposomes as siRNA carriers has some limitations in terms of gene transfection efficiency, the need for high dose injections, and poor targeted tissue distribution in vivo 31 . An in vivo study in mice showed that intravenous injection of DOTAP/siRNA complexes had an effective effect than injection of naked siRNA, but it also produced potent cytotoxicity and immune responses 32 . Conjugation of cholesterol to siRNA molecules could improve the delivery efficiency of siRNA targeting apolipoprotein B in the liver, but it required high doses of cholesterol-conjugated siRNA (50 mg/kg), which limits their therapeutic applications in humans due to cost 33 . Cholesterol-conjugated siRNA has also been reported to silence the herpes simplex virus-related genes after intravaginal administration without inflammation or interferon response at the administrative site, but it still need a high dose of cholesterol/siRNA 34 . The gene transfection efficiency and cytotoxicity strongly depend on the molecular backbone of the delivery vectors. Thus, the selection of appropriate molecular building blocks and construction of low cytotoxicity and highly efficient gene vectors are important in gene therapy.
Vitamins are organic compounds and essential for liver tissue cells, but they cannot be synthesized within the cells. Recent studies have demonstrated that modification of delivery systems with vitamins is a very promising strategy for liver targeting [35][36][37] . Vitamin E is an antioxidant, and its uptake strengthens the immune system response against viruses and bacteria, and its uptake is not toxic, even at high doses [38][39][40] . The natural isomer of VE, α -tocopherol, is abundant in human diets and easily absorbed 41 . For these reasons, we designed (VE)-coupled lipid nanoparticles carrying siRNAs against the HCV replicon, the 5′ UTR, and the core gene.
Hydrodynamic gene delivery using a rapid injection of a relatively large volume of DNA solution has opened up a new avenue for gene studies in vivo. This method is simple and effective. Wide success in applying hydrodynamic principles to delivery of DNA, RNA, proteins, and synthetic compounds, into the cells in various tissues of small animals, has inspired the recent attempts at establishing a hydrodynamic procedure for clinical use 42,43 . In our present study, the liver-specific expression of the HCV core or liver-specific expression of luciferase regulated by HCV 5′ UTR was established by hydrodynamic tail vein injection of plasmid pCore-3FLAG or pGL3-5′ UTR-luc in Balb/c mice. Nanoscale VE-DC/siRNA (siRNA-core or siRNA-5′ UTR) was intravenously injected to assess the liver-targeting property as well as the antiviral activity. The importance of VE in the delivery of siRNAs in vivo was examined by comparing the in vivo silencing activity of VE-coupled lipid nanoparticles (VE-DC) with equal amounts of uncoupled DC. The VE-coupled DC enabled the efficient in vivo delivery of siRNA to the liver, and resulted in a more efficient silencing effect. The relatively low dose of siRNA (0.8 mg/kg per single injection) used in our in vivo study was less than doses previously shown to have in vivo therapeutic effects 44,45 . Therefore, coupling of DC with VE not only increases the liver targeting property but also facilitates the liver-specific cellular uptake of siRNA, which efficiently suppressed HCV replication and HCV core protein production.
Previous studies have suggested that LDL particles can mediate the delivery of cholesterol-siRNAs into liver tissues and that the particles then enter hepatocytes through a lipoprotein receptor-mediated pathway 46 . Our data showed an enhanced liver-targeting property and liver-specific cellular uptake after coupling of lipid nanoparticles with VE. In contrast to cholesterol, VE is an exogenous lipid that cannot be synthesized in vivo, suggesting that its transport mechanism and cellular uptake in the liver might differ from those of cholesterol. One possible mechanism might involve a high binding affinity of VE to the novel binding glycoprotein, afamin, which shows multiple VE-binding sites and is highly expressed in the liver 47,48 . The possible role of afamin in facilitating liver-specific cellular uptake of VE-DC/siRNAs will be elucidated in future studies. The intracellular fate of VE-DC/siRNAs might involve degradation of the VE-DC/siRNAs in endolysosomes, release of siRNAs into the cytosol, and binding to target mRNA.
Notably, no prominent in vivo side effects were noted after intravenous injection of VE-DC/siRNAs in our blood system, biochemical, and histopathology analyses. The dosage of α -tocopherol, when used as a nutritional supplement is 15 mg/day 49 . When 0.8 mg/kg of VE-DC/siRNA was injected, the amount of α -tocopherol delivered was only 6.5 μ g for each mouse, which is a very small dose. The formulation in lipid nanoparticles can activate the innate immune responses through activation of Toll-like receptors, which has direct effects in modulating viral replication 50,51 . We excluded the possibility that the anti-HCV effect of VE-DC/siRNAs might be confounded by the immune stimulation of an innate immune response by investigating the IFN-α /β response, which involves the expression of the IFN-β and IFN-α genes. Of specific interest is the finding that VE-DC/ siRNA did not induce an increase of IFN-α in the serum or of IFN-β mRNA in the liver. One possible mechanism of escape from an immunostimulatory effect in VE-DC/siRNA-injected mice could reflect the use of low doses of cholesterol-based cationic liposome DC in our VE-DC, which might not have been sufficient to induce an immunostimulatory effect. The other possible mechanism is that composition of nanoscale VE-DC with VE may formulate a different molecular backbone which is different from before, VE itself might exert an unclear neutralize effect on in vivo side effects. So these possibilities should be verified in our further research. The results presented here, taken together, indicate that VE-DC/siRNAs are a noninvasive delivery vector for siRNA.
In summary, we confirmed the effectiveness and safety of VE-coupled siRNA-packing nanoscale cholesterol-based cationic liposomes for in vivo delivery of siRNA. The actual delivery pathway of VE-DC-siRNA remains to be established for better optimization of its use, but this study represents an important step in advancing the use of gene delivery vectors potentially very useful systems for gene therapy.

Methods
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 (pGL3luc). 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.  All other procedures were performed as previously described 52 . The Huh7.5.1-HCV cells were maintained in DMEM supplemented with 10% FBS, 100 U/ml penicillin, 100 μ g/ml streptomycin and 400 μ g/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 method 53 . 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 View TM (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 described 54 . 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 μ g 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 μ g 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 μ m thick slices. Then sections were stained with H-E and examined by light microscopy for alteration of histological structures.