br Human umbilical vein endothelial cells HUVECs
Human umbilical vein endothelial A769662 (HUVECs) and human gastric cancer cell line SGC-7901 were provided by the Institute of Pharmacol-ogy, Ocean University of China. The cells were maintained in RPMI 1640 medium containing 10% FBS, 100 U/mL penicillin and 100 μg/mL strep-tomycin at 37 °C with 5% CO2 in a humidified incubator. The BALB/c nude mice (female, 6 weeks old) with number of animal license SCXK (Xiang) 2016–0002 were obtained from Hunan SJA Laboratory Animal Co., Ltd. (Changsha, China) and were kept under standard conditions. All animal experimental procedures were conducted in accordance
with the ethical guidelines of Shandong Province Experimental Animal Management Committee and were approved by Laboratory Animal Care and Use Committee of Ocean University of China.
2.2. Synthesis of CNC and FITC-CNC
CNC was synthesized by amidation reaction as previously described [18,35]. In brief, CMCS (0.2 g) was dissolved in deionized water (20 mL), and the pH of the solution was regulated to 9.0 with pyridine. Then, cal-culated amounts of NCTD (1.0 equiv.) was dissolved in acetone and then added into the CMCS solution dropwise. The reaction was performed under magnetic stirring (500 rpm) at 25 °C for 24 h. The resultant prod-uct was precipitated by adding excess ethanol. Impurities were re-moved by rinsing with excess acetone, then the precipitate was purified by absolute ethanol for 3 times. Finally, the white powdery product was vacuum-dried at 25 °C.
The chemical structure of CNC conjugates was characterized by fourier-transformed infrared (FTIR) and 1H nuclear magnetic resonance (NMR), which was performed using a NEXUE 470 instrument (Nicolet Co. USA) and a Bruker DPX300 (Bruker, Germany) spectrometer, re-spectively. For FTIR spectrum, samples were scanned against a blank KBr pellet background at wavelength range of 4000–500 cm−1. More-over, the 1H NMR spectra was assessed using D2O as solvent with ace-tone as the internal standard at 25 °C. Elemental analysis (C, H and
N) of CMCS and CNC were conducted using a CHN-O-Rapid elemental analyzer (Heraeus, Germany). The grafting ratio of NCTD to CNC conju-gates was determined by the results of the elemental analysis. FITC-labeled CNC (FITC-CNC) was synthesized according to the pre-vious method with minor modifications . Briefly, CNC (450 mg) and FITC (15 mg) were dissolved in phosphate buffer saline (pH 8.3), re-spectively. Then, FITC was added into CNC solution and the mixture was reacted under magnetic stirring in the darkness at 4 °C for 24 h. The product was subsequently dialyzed in distilled water until no fluo-rescence was detected. Finally, FITC-CNC was obtained by freeze-drying and stored in the dark at 4 °C until the experiment. The labeling effi-ciency of FITC was determined by a UV spectrophotometer and standard solutions.
2.3. In vitro cytotoxicity assay
The in vitro cytotoxicity of CNC and free NCTD were evaluated by MTT assay using SGC-7901 cells. Cells in the logarithmic phase were separately seeded in 96-well plates at a density of 2 × 104 cells/well in RMPI 1640 medium for 24 h. Subsequently, the medium was removed and replaced with fresh medium containing different concentrations of CNC (12.5, 50, 200 μg/mL) and free NCTD (40 μg/mL). After incuba-tion for 24, 48 and 72 h, 20 μL MTT at a concentration of 5 mg/mL was added to each well and incubated with cells for another 4 h at 37 °C. Fi-nally, supernatant fluid was removed, and the formazan crystals formed in each well were solubilized in 150 μL dimethyl sulfoxide. The cell via-bility was recorded according to the absorbance using a microplate reader (Thermo Fisher Scientific, INC, USA) at wavelength of 492 nm and determined by comparing with control wells containing only cell culture medium.
2.4. Cellular uptake
SGC-7901 cells were seeded in 24-well plates at a density of 8 × 104 cells/well in RPMI 1640 medium. The cells were incubated at 37 °C for 24 h to forming a cell monolayer. Then, the original medium was re-placed by fresh medium containing FITC-CNC (100 μg/mL). After incu-bation for 12, 24 and 36 h, respectively, the medium in each well was removed and the cells were washed with PBS for three times. Cellular uptake about the conjugates at different time points was observed and photographed by confocal laser scanning microscopy (CLSM).
Fig. 1. (A) Synthesis of norcantharidin-conjugated carboxymethyl chitosan (CNC) through amidation reaction. (B) FTIR spectra of CMCS and CNC. (C) 1H NMR spectra of CMCS and CNC.
2.5. Transwell assay
Migration of HUVECs were measured using a transwell chamber with polycarbonate membranes. In total, cells were suspended and then plated into the upper chamber at a density of 2 × 105 cells/well in RPMI 1640 medium with 1% FBS, followed by filling 500 μL complete medium into the lower chamber. Subsequently, the upper chamber was filled with different concentrations of CNC (12.5, 50, 200 μg/mL) and NCTD (40 μg/mL), in which cells were allowed to migrate for 24 h. The compete medium supplemented with 10% FBS was used as a chemoattractant. After incubation, the non-migrated cells on the upper surface of the polycarbonate membranes were scraped off using cotton swabs. The cells migrated to the lower surface were fixed with methanol and stained with 0.1% crystal violet for 15 min. Cell migratory ability was assessed and quantified by counting stained cells in five ran-dom fields at 100× magnification with a light microscope.