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

Wound-healing assay EA.hy926 cells were seeded onto six-well plates and treated with different concentrations of CS-ES2-AF or ES2-AF (50 μg/mL, 100 μg/mL, and 200 μg/mL; concentrations refer to the ES2-AF portion only) for 48 hours. Three parallel cell-free gaps were created in each well with a pipette tip. Images were taken using an inverted fluorescence microscope, and the cells that migrated into the wound area were calculated. 23, 24 Tube formation assay EA.hy926 cells were propagated in DMEM supplemented with 10% FBS. The cells (4×10 4 /well) were seeded onto 48-well plates coated with Matrigel, followed by treatment with DMEM containing different concentrations of CS-ES2-AF or ES2-AF (50 μg/mL, 100 μg/mL, or 200 μg/mL; concentrations refer to the ES2-AF portion only). Then, bFGF was added to each well and made up to a final concentration of 5 ng/mL. Peak tube formation was observed after 8 hours of culture, and five random fields were observed under a microscope. The number of branches per field of each tube for each treatment was quantified. This experiment was independently repeated five times. [23] [24] [25] [26] [27] caM assay