Characterization and bioactivity of self-assembled anti-angiogenic chondroitin sulfate-ES2-AF nanoparticle conjugate

Xing, Liang; Sun, Feng; Wang, Zhendong; Li, Yan; Yang, Zhifang; Wang, Fengshan; Zhai, Guangxi; Tan, Haining

The inhibitory effect of ES2-AF and CS-ES2-AF on the proliferation of the endothelial cells was measured by MTT assay. 9, 21, 22, 42 As shown in Figure 3A , when the concentrations of ES2-AF were 5, 25, 50, 100, and 200 μg/mL, the IRs of EA.hy926 endothelial cells were 8.38%±1.29%, 25 .72%±3.55%, and 33.01%±3.63%, respectively. From these results, it can be seen that the IRs of ES2-AF and CS-ES2-AF on EA.hy926 cells showed concentrationdependent effect ranging from 5 μg/mL to 200 μg/mL. The inhibitory rates of ES2-AF and CS-ES2-AF were similar (5 μg/mL-100 μg/mL), which indicated that the activity of ES2-AF was similar to CS-ES2-AF at relatively low concentrations. When the concentration increased, the IR of CS-ES2-AF was higher than that of ES2-AF at the same concentration, which indicated that the activity of CS-ES2-AF was significantly better than that of ES2-AF at high concentrations. The activity of polypeptide drugs is often related to their structure. Compared to small molecule drugs, polypeptide drugs often have poor aqueous stability due to their spatial structure. When the anti-angiogenic peptide ES2-AF was modified with CS, the stability of the conjugate in aqueous solution improved. This is due to the fact that the CS group in the aqueous solution forces the water molecules around the ES2-AF polypeptide to enhance the stability of ES2-AF. Therefore, it can be inferred that the higher the concentration of CS, the better the stability of ES2-AF. In addition, the results of the MTT assay are also related to the state of endothelial cells and the degree of differentiation. Therefore, when the MTT assay is used to evaluate the inhibitory effect of drugs on endothelial cell proliferation, cells with similar passage numbers should be selected for experiments. cell migration assay EA.hy926 cells were co-cultured in cell culture medium containing different concentrations of ES2-AF, CS&ES2-AF, and CS-ES2-AF for 48 hours and photographed under a fluorescence microscope to obtain a graph of the cell migration. The initial position of the scratch was marked with a black solid line ( Figure 4A ). As shown in Figure 4D For different samples at the same concentration, the activity of CS-ES2-AF was higher than the activity of ES2-AF, and when the concentrations were 50 μg/mL and 100 μg/mL, the effect of CS-ES2-AF on the endothelial cell migration was significantly better than that of ES2-AF. Like the MTT assay results, even when the concentration was relatively low, CS could improve the solubility of ES2-AF. CS could increase the number of water molecules near ES2-AF and increase the stability of ES2-AF. Thus, CS-ES2-AF could inhibit the endothelial cell metastasis more efficiently than ES2-AF. When the concentration was increased to 200 μg/mL, the inhibitory effect of CS-ES2-AF on the endothelial cells was similar to that of ES2-AF. Thus, CS could promote the adhesion and growth of endothelial cells, and, to a certain extent, counteract the inhibitory effect of ES2-AF.