A br B br solubility
solubility and potential enhanced antitumor effects. Therefore, the anti-tumor efficacy of CNC was studied systematically in the following sections.
3.2. In vitro cytotoxicity of CNC
The in vitro cytotoxicity assessment was conducted by MTT assay in SGC-7901 GSK J4 at gradient concentrations of CNC. As shown in Fig. 2A, the results clearly disclosed that the inhibition rates of CNC on the pro-liferation of SGC-7901 cells increased in a dose- and time-dependent manner. In comparison, the optimal formulation CNC (200 μg/mL) de-creased the cell viability to 52.84% after incubation for 24 h, which was much lower than that of free NCTD (40 μg/mL) at equivalent dose. The improved efficacy of CNC conjugates might be due to the ac-tive cellular uptake of NCTD and the higher intracellular concentration
Effect of CNC on the growth of SGC-7901 gastric tumor tissue.
Group Dosage (mg/kg) Tumor weight (g) Inhibition rate (%)
. Moreover, CNC exhibited similar cytotoxicity compared with free NCTD at equivalent dose after incubation for 48 and 72 h. These results indicated that CNC exerted excellent inhibitory effects on the prolifera-tion of tumor cells, which was superior to that of free NCTD.
3.3. Cellular uptake of CNC
The FITC content in the FITC-CNC was 2.91% (w/w). The cellular up-take of the CNC in SGC-7901 cells at different time points was observed and photographed by CLSM. As shown in Fig. 2B, the FITC-CNC was slowly endocytosed into cells with the extension of incubation time. After incubation for 12 h, weak fluorescence was observed in the SGC-7901 cells. The fluorescence became more extensive and intense after incubation for 24 and 36 h, indicating that the uptake of the conjugates in SGC-7901 cells was time-dependent. In the study of Xu et al., it was revealed that the cellular uptake may be attributed to the electrostatic ionic interaction between tumor cells and the conjugates . The above results suggested that CNC conjugates exerted the antitumor ef-fects by internalizing into tumor cells.
3.4. Anti-angiogenesis effect of CNC in vitro
The migration of endothelial cells is considered a key step involved in the course of angiogenesis . To evaluate the anti-angiogenesis ef-fects of CNC in vitro, transwell assay and tube formation assay were car-ried out in HUVECs. As shown in Fig. 2C and D, the migratory cells were dramatically decreased after incubation with CNC in a dose-dependent manner. In particular, the inhibition rate of migration was increased to 69.33% relative to the control group when the concentration of CNC was 200 μg/mL (Fig. 2D). As for tube formation assay, HUVECs in the control group were observed to connect closely, differentiate and form tube-like networks (Fig. 2E). When HUVECs were incubated with CNC and free NCTD, they were unable to maintain complete capillary-like structures. Particularly, the high dose of CNC conjugates thoroughly broke up the enveloped vessel-like networks formed by HUVECs and showed the maximum inhibition to tube formation (Fig. 2F). Based on the above results, CNC could significantly reduce the migratory ability and inhibit the tube formation of HUVECs, thus it might be employed as a potential anti-angiogenesis agent in clinical application.
3.5. In vitro apoptosis analysis
Hoechst 33258 can stain concentrated nuclei, and apoptotic cells can be distinguished from normal and necrotic cells . Cell apoptosis in-duced by the conjugates was investigated via assessment of nuclear morphology after staining the cells with Hoechst 33258. As shown in Fig. 3A, cells treated with CNC exhibited apparent nucleus fragmenta-tion, chromatin condensation and apoptotic body formation, which were typical characteristics of apoptotic cells. However, no significant apoptosis was observed in control group. The nucleuses in control group were stained homogeneously under confocal microscope. More-over, the apoptosis rates of SGC-7901 cells treated with different formu-lations were determined by flow cytometry. As shown in Fig. 3B, the apoptosis rate of untreated SGC-7901 cells was 5.93%. In contrast, CNC significantly induced apoptosis of SGC-7901 cells in vitro, and mainly in-creased the early apoptosis rates. The percentages of early and late apo-ptosis of CNC treated cells for 24 h were 25.88% and 12.26%, while those in NCTD treated group were 9.40% and 14.93%, respectively. Previous studies revealed that free NCTD could induce apoptosis in human colon carcinoma, hepatoma and glioblastoma cells . It was noted
that NCTD induced apoptosis via both extrinsic and intrinsic pathways by activation of the CD95 receptor system and many Caspases as well as the regulation of Bcl-2 protein family . In our study, CNC as a mac-romolecular derivative could change nuclear morphology of tumor cells and induce a higher apoptosis rate compared with free NCTD at equiv-alent dose.