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

In SPR study, the pH value of the suitable CD44 protein solution was screened to ensure the coupling of more CD44 protein to the CM5 chip, which facilitated the screening of the drug in the next step. When the ligand protein CD44 is coupled to the chip surface, it is necessary to dissolve the protein in a buffer solution with pH lower than its isoelectric point and higher than pH 3.5, so that the protein surface is positively charged, and the carboxyl groups with negative charges on the chip surface are combined by electrostatic adsorption. The isoelectric point of CD44 was pH 5.15, so the optimum pH value of CD44 protein solution was 3.8 after screening. The samples were dissolved in PBS buffer and diluted with running buffer (50 mM phosphate, 100 mM sodium chloride, and 0.01% vol/vol Tween 20, pH 7.4). Then, different concentrations of the sample were flowed through the surface of the chip immobilized with the CD44 receptor protein and the K d values were obtained by Biacore T2000. In general, when the value of KD was in the range of 10 -5 -10 -12 , it meant that the sample had a strong affinity with the ligand protein. 33 Because CS is the ligand of CD44 receptor and can specifically bind to CD44 receptor, it had a stronger affinity toward CD44 receptor than the unmodified ES2-AF. Therefore, CS was used to modify ES2-AF to improve the targeting of drugs to tumor tissues.