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Novel hydroxybutyl chitosan nanoparticles for siRNA delivery targeting tissue factor inhibits proliferation and induces apoptosis in human vascular smooth muscle cells

Chitosan, a polysaccharide isolated from shrimp and other crustacean shells, has been widely investigated for DNA and siRNA delivery. Despite substantial effort having been made to improve chitosan as a non-viral gene delivery vector, the application is severely limited by its poor solubility under physiological conditions. Hydroxybutyl chitosan (HBC), a modified chitosan, is soluble under neutral conditions. Tissue factor (TF) is involved in the pathogenesis of cardiovascular diseases by promoting thrombus formation and inducing the migration and proliferation of vascular smooth muscle cells. Targeting TF is an attractive therapeutic strategy for cardiovascular diseases. In the present study, the use of HBC for the transfer of TF-siRNAs into human umbilical vein smooth muscle cells (HUVSMCs) was investigated, and the effects of TF knockdown on cell proliferation and apoptosis were examined. HBC/siRNA nanoparticles were produced by mixing HBC and siRNA solutions with the assistance of tripolyphosphate buffer. The transfection efficiency with these nanoparticles was 74±2.5%, which was determined using a fluorescence-labeled siRNA under fluorescence microscopy. The delivery of HBC/TF-siRNA resulted in reductions in the production of cellular and soluble TF protein in HUVMSCs, which were measured using western blotting and enzyme-linked immunosorbent assay, respectively. TF knockdown led to inhibited cell proliferation, as assessed using a Cell Counting Kit-8 assay, and increased cell apoptosis, determined using Annexin V-fluorescein isothiocyanate staining. These findings suggested that HBC may be a promising vector for siRNA delivery, and that in vivo HBC/siRNA nanoparticle delivery targeting TF may be a potential option for the treatment of cardiovascular diseases, which warrants further investigation.


Introduction
Chitosan, a linear polysaccharide isolated from shrimp and other crustacean shells, possesses low toxicicity, is biodegradable and exhibits favorable biocompatible properties, rendering it a promising material for biomedical applications (1)(2)(3). Notably, several studies have investigated the potential of chitosan for drug delivery, bio-coating and the transferring of genetic materials (1)(2)(3)(4)(5)(6). However, one setback, which limits the use of chitosan, is its insolubility under neutral conditions (3). Previously, a modified chitosan, namely hydroxybutyl chitosan (HBC), has been developed and exhibits advantages over the parental chitosan. HBC is prepared by the conjugation of hydroxybutyl groups to the hydroxyl and amino reactive sites of chitosan (7). This change confers the polymer solubility under neutral conditions, whilst maintaining low toxicity and retaining its biodegradable and appreciable biocompatible properties (7)(8)(9).
Tissue factor (TF) is an essential cofactor in the initiation of coagulation (10). It is constitutively expressed and can be upregulated by various stimuli, including platelet-derived growth factor (PDGF) in vascular smooth muscle cells (11,12). In response to cytokines and growth factors, endothelial cells, monocytes and macrophages also produce TF (13)(14)(15)(16)(17)(18)(19). Two forms of TF, membrane-bound and soluble TF, have been identified (10,12,(20)(21)(22). TF has been implicated in the pathogenesis of cardiovascular diseases, including hypertension, atherosclerosis, acute coronary syndrome and restenosis following percutaneous coronary intervention, by promoting thrombus formation and inducing the migration and proliferation of vascular smooth muscle cells (23)(24)(25)(26)(27)(28)(29). Therefore, targeting TF has been suggested as an attractive strategy for the treatment of cardiovascular diseases (30). In the present study, the use of HBC for the transfer of siRNAs into primary human umbilical vein vascular smooth muscle cells (HUVSMCs) targeting TF was investigated, and the efficiency of HBC-mediated siRNA transfer was examined, as were the effects of TF knockdown on HUVSMC proliferation and apoptosis.

Materials and methods
siRNAs. All siRNAs were purchased from Invitrogen (Thermo Fisher Scientific, Inc., Waltham, MA, USA). A fluorescent FAM-labeled siRNA was used to examine transfer efficiency. An siRNA targeting human TF was designed, according to the published human TF cDNA sequence (gene accession number: M16553.1; GenBank; http://www.ncbi.nlm.nih. gov/nuccore/M16553.1). The sequence of the antisense strand was 5'-AUU UGU AGU GCC UGA AGC GCTT-3'. A scrambled RNA sequence was used as control.
Preparation of HBC/siRNA nanoparticles. HBC/siRNA nanoparticles were prepared as follows: The HBC solution (1 mg/ml) was made by dissolving HBC (molecular weight: 130-160 kDa; degree of deacetylation: 86%) provided by the Ocean University of China (Qingdao, China) in 0.2 M acetic acid (pH 5.5; Sigma-Aldrich, , St. Louis, MO, USA). The siRNA (final concentration, 20 µmol/l) was mixed with tripolyphosphate (TPP) solution (0.5 mg/ml; Sigma-Aldrich) under magnetic stirring. HBC/siRNA nanoparticles were then produced by slowly adding 1 ml of the siRNA/TPP solution to 3 ml of the HBC solution with stirring for 30 min. The loading efficiency of the siRNA within HBC was determined as follows: The HBC/siRNA mixture, prepared as above, was centrifuged at 13,000 x g for 15 min, following which the supernatant was collected and the siRNA content in the supernatant was determined by measuring the absorbance at 260 nm using a spectrophotometer (UV-1100; Shimadzu Corporation, Kyoto, Japan). The loading efficiency was calculated as a percentage, as follows: Total siRNA used -free siRNA remaining in the supernatant) / total siRNA used.
Cell culture and siRNA transfer. Primary HUVSMCs (Bai Li Biological Technology, Shanghai, China) were maintained at 37˚C in RPMI-1640 medium (Gibco, Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (FBS, Gibco; Thermo Fisher Scientific, Inc.), 100 U/ml penicillin and 0.1 mg/ml streptomycin (Gibco; Thermo Fisher Scientific, Inc.). Cells were used between passages four and nine. For siRNA transfer, the cells were detached with 0.25% trypsin (Thermo Fisher Scientific, Inc.), seeded on 24-well plates (1x10 5 cells/well) and incubated overnight at 37˚C in complete RPMI-1640 medium. Following replacing of the medium with 250 µl Opti-MEM serum-free medium (Gibco; Thermo Fisher Scientific, Inc.), HBC/siRNA nanoparticle solution was added (siRNA final concentration, 200 nM). The cells were incubated at 37˚C for 4 h to allow for siRNA uptake. Following incubation, the medium containing nanoparticles was replaced with fresh complete RPMI 1640 medium and the cells were maintained in culture and assayed at different time points (24,48 and 72 h), as indicated. A total of 24 h post-transfection, five high-power fields were randomly selected under a fluorescence microscope (Nikon E600; Nikon Corporation, Tokyo, Japan), and total fluorescent cells in each field were counted. The percentage of average fluorescent cells over average total cells from the five fields was used to determine the transfection efficiency TF protein measurement. As HUVSMCs express minimal basal TF, the cells examined in the present study were treated with PDGF-BB to enhance the production of TF protein. PDGF-BB (10 ng/ml, Sigma-Aldrich) was added to the cell culture 24 h following HBC/siRNA transfection. Inhibition of the PDGF-BB-induced expression of TF by TF-siRNA was then determined 48 h following siRNA transfection. The levels of soluble and cellular TF protein were measured using an enzyme-linked immunosorbent assay (ELISA) and western blotting, respectively. For the soluble TF assay, the cultured conditioned medium was collected, centrifuged at 10,000 x g for 10 min and the level of soluble TF in the medium was measured using a Human F3/Tissue Factor ELISA kit (cat. no. RAB0642-1KT; Sigma-Aldrich), according to the manufacturer's protocol. For western blotting, total protein was extracted using radioimmunoprecipitation assay lysis buffer (Sigma-Aldrich) and protein concentration was determined using a bicinchoninic acid assay (Pierce Biotechnology, Inc., Rockford, IL, USA), according to the manufacturer's protocol. The proteins (50 µg) were separated by 10% SDS-PAGE (Thermo Fisher Scientific, Inc.) and electrically transferred onto nitrocellulose membrane (Thermo Fisher Scientific, Inc.). The membrane was blocked with phosphate-buffered saline (pH 7.5) with 0.1% Tween-20 (PBST; Sigma-Aldrich) and 10% non-fat milk for 1 h at room temperature, and incubated overnight at 4˚C with mouse anti-human TF monoclonal antibody (1:1,000; cat. no. ab17375; Abcam, Cambridge, MA, USA), followed by three washes with PBST. The membrane was then incubated with a horseradish peroxidase (HRP)-conjugated goat anti-mouse secondary antibody (1:5,000; cat. no. ab6789; Abcam) at room temperature for 1 h. Following three washes with PBST, the specific TF band was visualized using an ECL detection kit (Abcam). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) on the same membrane was probed with a mouse anti-human GAPDH monoclonal antibody (1:5,000; cat. no. ab9484; Abcam) as done with TF. Densitometric analysis was performed, and relative quantitation of TF normalized to GAPDH was determined using Image J software (National Institutes of Health, Bethesda, MA, USA).
Cell proliferation assay. HUVSMC proliferation was assessed 48 h following the addition of the HBC/siRNA nanoparticles using a Cell Counting Kit-8 (CCK-8; Sigma-Aldrich), according to the manufacturer's protocol. Briefly, the culture medium was replaced with fresh complete RPMI 1640 medium (without phenol red) containing 10% CCK-8 reagent. Statistical analysis. Data are expressed as the mean ± standard deviation. Statistical analyses of the differences were performed using one way analysis of variance. Comparisons between two groups were performed using the least-significant difference test. SPSS 17.0 (SPSS, Inc., Chicago, IL, USA) and PRISM software (GraphPad Software, Inc., La Jolla, CA, USA) were used for statistical analyses and plotting. P<0.05 was considered to indicate a statistically significant difference.

Preparation of HBC siRNA nanoparticles and siRNA transfer.
HBC and siRNA solutions were mixed and stirred to form HBC/siRNA nanoparticles. TPP was used as a cross-linker to further stabilize the nanoparticle. This method resulted in an siRNA loading efficiency of 93.4±0.7% within HBC. Treatment of the HUVSMCs with nanoparticles, which were formed by mixing HBC and FAM-labeled siRNAs, showed green fluorescence in the transfected cells, as observed under fluorescence microscopy (Fig. 1). Counting of the numbers of fluorescent and total cells revealed a 74±2.5% transfection efficiency.

Knockdown of TF by HBC/TF-siRNA nanoparticles in
HUVSMCs. Inhibition of the PDGF-BB-induced expression  In the cells transfected with scrambled siRNA, the level of soluble TF was 79.0±3.0 pg/ml, which was not significantly different from that of the untreated cells ( Fig. 2A). Western blotting revealed that the relative cellular level of TF in the TF-siRNA-transfected cells was 38.5±2.6% of that in the non-transfected cells (n=12; P<0.05). By contrast, scrambled siRNA transfection caused no significant reduction in the level of cellular TF ( Fig. 2B and C).
TF knockdown inhibits HUVSMC proliferation. HUVSMC proliferation was measured 48 h following siRNA transfection using a CCK-8 kit. No significant differences were observed between the OD 450 values of the non-transfected and scrambled siRNA-treated HUVSMCs. By contrast, TF-siRNA transfection significantly inhibited HUVMSC proliferation ( Fig. 3; n=4).
TF knockdown increases HUVSMC apoptosis. HUVMSC apoptosis was assayed using Annexin V staining and flow cytometric analysis 72 h following siRNA delivery. As shown in Fig. 4, cell apoptotic rates were 5.1±0.71 and 7.73±0.87% in the non-transfected and HBC/scrambled siRNA nanoparticle-transfected cells, respectively. Statistical analysis demonstrated no significant difference between the levels of apoptosis in these two groups. However, the apoptotic rate in the TF-knockdown HUVMSCs was 42.25±1.82%, which was significantly higher than those observed in the non-transfected and scrambled siRNA-transfected cells (P<0.05; n=4).

Discussion
Despite significant efforts that have been made to improve chitosan as a non-viral gene delivery vector, the application is significantly limited by its poor solubility under   (31). HBC, a soluble derivative of chitosan under neutral conditions, has been investigated as a scaffold for tissue engineering (32,33), however its use as a gene delivery carrier remains to be fully elucidated. In the present study, HBC was investigated as a vehicle for siRNA delivery targeting TF in HUVSMCs. HBC/siRNA nanoparticles were produced by mixing HBC and siRNA solutions with TPP as a cross-linker. This resulted in >90% of the siRNAs being loaded within HBC. Furthermore, treatment of the HUVSMCs with the HBC/fluorescence-siRNA nanoparticles resulted in a transfection efficiency of ~74%. These results validated that HBC was a promising vehicle for siRNA delivery.
siRNA binds an mRNA sequence through complementarity, which leads to the cleavage of target mRNA and, eventually, the inhibition of gene expression (34). siRNA has been investigated extensively for its therapeutic potential, for example in the use of siRNAs to silence tumor genes, or genes in which overexpression has been associated with disease (35,36). TF is involved in thrombus formation and stimulates the migration and proliferation of vascular smooth muscle cells, contributing to the pathogenesis of cardiovascular diseases (23)(24)(25)(26)(27)(28)(29). As a consequence, inhibition of the action of TF has been suggested as an attractive therapeutic approach for cardiovascular diseases (30). Following confirmation of effective siRNA delivery with HBC, the present study assessed whether HBC/TF-siRNA nanoparticles were able to inhibit the expression of TF in HUVSMCs, and examined the effect of TF knockdown on cell proliferation and apoptosis. The results showed that HBC-mediated TF-siRNA transfer suppressed the production of cellular and soluble TF, which led to the significant inhibition of cell proliferation and increase of cell apoptosis.
In conclusion, the present study demonstrated that HBC can be successfully applied for siRNA loading. HBC/siRNA nanoparticles mediated high siRNA transfection efficiency in HUVMSCs, and treatment of HUVSMCs with HBC/TF-siRNA nanoparticles inhibited the protein expression of TF, which led to decreased cell proliferation and enhanced cell apoptosis. These findings suggested that HBC may be a promising vector for siRNA delivery, and that in vivo HBC/siRNA nanoparticle delivery targeting TF is a potential therapeutic option for the treatment of cardiovascular diseases, warranting further investigation.