br integrated optical density IOD was
integrated optical density (IOD) was scaled by Image-Pro Plus 6.0 soft-ware (NIH, USA).
2.10. Determination of TNF-α and VEGF levels in serum
The blood was centrifuged at rate of 2000 g for 15 min to separate the serum, which was refrigerated at −20 °C for further analysis. Serum samples were diluted at proper proportions and the
concentrations of TNF-α and VEGF in serum were measured by ELISA kits according to the manufacturer's instructions.
SGC-7901 cells treated with different concentrations of CNC and free NCTD for 48 h were lysed in RIPA lysis buffer containing PMSF and pro-tease inhibitors to extract the whole protein. The cellular lysates were
Fig. 3. CNC induces apoptosis of SGC-7901 cells in vitro. (A) CLSM photographs of cells stained with Hoechst 33258. (B) Apoptotic cell populations of SGC-7901 cells determined by flow cytometric analysis with Annexin V-FITC and propidium iodide (PI) staining after incubation for 24 h. (a) cells treated with complete medium; (b) cells treated with CNC at the concentration of 12.5 μg/mL; (c) cells treated with CNC at the concentration of 50 μg/mL; (d) cells treated with CNC at the concentration of 200 μg/mL; (e) cells treated with NCTD at the concentration of 40 μg/mL. Data represents mean ± SD (n = 6), *P b 0.05, **P b 0.01 significant difference compared with control group; #P b 0.05, ##P b 0.01 significant difference compared with NCTD group.
centrifuged at 10000 g for 15 min at 4 °C and supernatants were col-lected. The concentrations of total protein were assessed with the BCA protein assay kit. Equal amounts of protein in the cell extracts were sep-arated by 12.5% sodium dodecyl sulfate-polyacrylamide gel electropho-resis (SDS-PAGE) and transferred onto PVDF membranes. After blocking with 5% BSA for 1 h, the membranes were probed with specific primary FF-MAS against MMP-2 (1:1000 dilution), MMP-9 (1:1000 dilution) and β-actin (1:400 dilution) overnight at 4 °C. Subsequently, the mem-branes were washed three times and incubated with the corresponding horseradish peroxidase (HRP)-conjugated goat anti-rabbit/mouse IgG secondary antibody for 2 h at 25 °C. Finally, the protein bands were de-veloped with ECL luminescence reagent. The relative density of the im-munoreactive bands was quantified with Image J software (NIH, USA) using β-actin as an internal control.
2.12. Statistical analysis
All data were presented as mean ± standard deviation (SD) and an-alyzed using SPSS version 15.0 (SPSS, Inc., Chicago, IL, USA). Treatment comparisons were made by analysis of variance and Student's t-test. Contrasts with *P b 0.05 or **P b 0.01 were considered statistically signif-icant. The images were quantified using Image-Pro Plus 6.0 and Image J software (NIH, USA).
3. Results and discussion
3.1. Characterization of CNC conjugates
CMCS was synthesized by chemical reaction between chitosan and monochloroacetic acid, which was determined with a molecular weight of 194.6 kDa, 94.66% degree of deacetylation and 108.41% degree of sub-stitution. The CNC conjugates were synthesized by conjugating NCTD with CMCS through amide formation (Fig. 1A). Based on previous liter-atures, the FTIR spectrum of CMCS (Fig. 1B) exhibited characteristic ab-sorption bands at 1409 cm−1 and 1593 cm−1, corresponding to the – CH2COOH group and carboxy group, respectively, indicating that the carboxymethylation occurred on both the hydroxyl and amino groups of chitosan . Additionally, the absorption peak at 1066 cm−1 was at-tributed to the stretching vibration of C-O . The absorption bands at 2913 cm−1 and 1324 cm−1 were assigned to the stretching vibration and bending vibration of C-H . In the spectrum of CNC, a new ab-sorption peak emerged at 1696 cm−1, which was assigned to the ab-sorption peak of amide bond, suggesting that NCTD had been attached to CMCS backbone successfully.
The structure of CNC was further confirmed by the 1H NMR spectra shown in Fig. 1C. The proton assignment of CMCS was as follows (ppm): 2.02 (CH3, acetamido group of chitosan), 2.72 (CH, carbon 2 of glucosamine ring), 3.62 (CH, carbon 2 of glucosamine ring with the substituted amino group), 3.7–4.0 (CH, carbon 3, 4, 5 and 6 of glucos-amine ring), 4.41 (CH, carbon 1 of glucosamine ring) . According to the previous research, the characteristic proton signals of CMCS ap-peared at range of 4.0–4.1 ppm, indicating that carboxymethyl groups were linked to chitosan . In the 1H NMR spectrum of CNC, the char-acteristic peaks for methylene groups (H4’, 5′) of NCTD were identified at 1.45–1.57 ppm, suggesting that NCTD was conjugated to the CMCS backbone. Moreover, compared with that of CMCS, new proton signals observed at 3.08 ppm and 4.76 ppm were assigned to the protons in succinyl group (OCOCH2CH2COOH) and CHOCH group of NCTD, respec-tively. The FTIR and 1H NMR results together confirmed the structure of CNC. The elemental analysis method was applied to determine the con-tents of carbon, hydrogen and nitrogen in the conjugates, and the result illustrated that the content (wt%) of NCTD was 20.05 (Table 1). In the previous studies, NCTD-CSs were synthesized by formation of ester bonds between chitosan and NCTD . In our study, CNC conjugates were prepared by conjugating CMCS with NCTD via amide bonds ac-cording to our optimized method, which displayed excellent water