Chitosan Nanoparticles Inhibit the Growth of Human Hepatocellular Carcinoma Xenografts through an Antiangiogenic Mechanism

Materials and Methods

Reagents. Chitosan nanoparticles were prepared and characterized as described previously (29). The vehicle, acetic acid (HAC)/sodium tripolyphosphate solution, was used as the control in the animal studies. CNP with a mean particle size ranging from 52 nm to 73 nm and a positive surface charge of about 50 mV, regularly formed and well distributed in HAC/sodium tripolyphosphate solution (pH 5) were used in this study (Figure 1). The monoclonal VEGF antibody and factor VIII-related antigen (F8/86) antibody were purchased from Gene Tex (Southern California, USA). Polyclonal VEGFR2 antibody was purchased from Thermo Scientific (Shanghai, China). Dako EnVision+™ Peroxidase (Dako Corp., Carpinteria, California, USA), M-MLV reverse transcriptase (Promega Corp., Madison, Wisconsin, USA) UNIQ-10 Column total RNA isolation kit (Shanghai Sangon Biotec Inc., Shanghai, China), and SYBR Premix Ex-Taq Kit (TaKaRa Biotec Inc., Dalian, China) were used as directed.

Cells and cell culture. The human HCC cell line BEL-7402 was obtained from the Cell Bank of the Chinese Academy of Science (Shanghai, China). The cells were maintained in culture as previously described (28).

In vivo tumor model. Six-week-old female athymic nude mice were obtained from the Slac Laboratory Animal Limited Company (Shanghai, China) and housed in the animal facility under specific pathogen-free conditions. All the animal experiments were conducted in accordance with a protocol approved by Zhejiang University. Two million BEL-7402 cells were suspended in 250 μl of saline solution and injected subcutaneously into the fatty layer of the anterior forelimb using sterile technique. The animals were given free access to food and water. Tumor diameters were measured every five days using a caliper, and tumor volumes were calculated by a standard formula (width²×length/2). Given the limitation of our previous study (a single dose and one end-time point), to further understand the pharmacokinetics and safety parameters of CNP, we investigated the effects of CNP treatment at daily oral dose levels of 0, 60 and 90 mg/kg/day on HCC tumor growth for 21 days were investigated. Once tumor growth reached 100 mm³ in size, CNP or vehicle was given by oral administration once daily to groups of six animals. After 21 days, the mice were euthanized by CO₂ followed by cervical dislocation, and the tumors were excised from sacrificed animals. One-half of each tumor was immediately frozen in liquid nitrogen and then stored at −80°C until analysis. The remaining tumor halves were fixed in 10% neutral buffered formalin overnight at room temperature and then stored in 70% ethyl alcohol for subsequent histological analysis. Growth curves for the tumors in each group were plotted as a function of tumor volume (mm³; mean±S.D.) vs. time.

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Figure 1.

Size, zeta potential, and morphology of CNP. A: The size distribution by intensity of CNP. B: Zeta potential distribution of CNP. C: Atomic force micrograph (AFM) of CNP.

Histology. The fixed tumor tissues were embedded in paraffin, sectioned at ~4 μm thicknesses and mounted onto slides. After deparaffinization and hydration, the sections were either stained with hemotoxylin and eosin (H&E) or used for subsequent immunohistochemistry. The necrotic area of each tumor was quantified by morphometric determination of the proportion of the total tumor area that was necrotic in the H&E stained section.

Immunohistochemistry. The labeled polymer method was used in immunohistological staining. In brief, the sections were deparaffinized in xylene and re-hydrated in graded solutions of ethanol. Antigen retrieval was performed at 95°C for 40 min in 0.01M sodium citrate buffer (pH 6.0). All the sections were then immersed in 3% hydrogen peroxide for 30 min. After being incubated overnight with the primary antibody at 4°C, the sections were incubated with Dako EnVision+™ peroxidase for 60 min. The peroxidase reaction was visualized with “liquid 3″, 3-diaminobenzidine tetrahydrochloride (DAB) and counterstained in hematoxylin. The slides were then viewed under a brightfield microscope. Positive staining was counted in a ×400 field and five randomly chosen fields per section were selected to determine the average count of microvessel density (MVD) (30).Any single highlighted endothelial cell, an endothelial cell cluster clearly separated from adjacent microvessels, or distinct clusters of brown-staining endothelial cells was counted as a separate count. The evaluation of the expression levels of VEGF and VEGFR2 was performed in accordance with the immunoreactive score (IRS) (31): IRS=SI (staining intensity) ×PP (percentage of positive cells). SI was determined as 0, negative; 1, weak; 2, moderate and 3, strong. PP was defined as 0, negative; 1, 10% positive cells; 2, 11-50% positive cells; 3, 51-80% positive cells and 4, more than 80% positive cells.

RNA isolation and reverse transcription. The total RNA was extracted from each tumor sample using an UNIQ-10 Column total RNA isolation kit according to the manufacturer's directions. The integrity and quality of each total RNA sample was analyzed by measurement of the optical density at 260/280 nm and verified by inspection of the 18S and 28S rRNA bands after agarose gel electrophoresis. The total RNA (2 μg) was reversely transcribed using M-MLV reverse transcriptase according to the manufacturer's protocol.

Real-time quantitative RT-PCR. Real-time quantitative PCR was used to quantify the mRNA expression of total glyceraldehyde-3-phosphate dehydrogenase (GAPDH), VEGF and VEGFR2. Primers (Table I) were designed for GAPDH, VEGF and VEGFR2 using Primer Express® software v2.0 from Applied Biosystems. Real-time quantitative RT-PCR was performed in a 25 μl reaction volume containing 1 μl cDNA template, 12.5 μl SYBR Premix Ex Taq™ (2×) and 1 μl forward and reverse primers (20 nM) using an iQ™5 real-time multicolor PCR detection system (Bio-Rad, California, USA). The real-time PCR parameters used for examinating the mRNA levels were for VEGF, initial denatureation at 95°C for 10 sec, followed by 45 cycles of 5 sec denaturation at 94°C, 30 sec annealing and extension at 63°C; for VEGFR2, initial denaturation at 95°C for 10 sec, followed by 45 cycles of denaturation at 95°C for 5 sec, annealing at 58°C for 10 sec and elongation at 72°C for 8 sec, and for GAPDH, initial denatureation at 95°C for 10 sec, followed by 45 cycles of denaturation at 95°C for 5 sec, annealing and extension at 60°C for 20 sec. The fluorescence signals for VEGF, VEGFR2 and GAPDH were collected at 63°C, 72°C and 60°C respectively. Melting curves were all made from 55°C − 95°C to determine the specificity of the amplification reactions. The relative amount of target mRNA was calculated by the 2^−ΔΔCt method (32), while all the gene expression values were normalized to that of GAPDH in the same sample.

Statistical analysis. The results are presented as the means±SD. ANOVA was used to determine the differences between control and treated groups. P<0.05 was considered significant. Analysis was performed using the SAS system for windows v6.12 (SAS Institute Inc., North Carolina, USA).