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 traditional CAM test was used to study the effects of the drugs on angiogenesis in vivo. The anti-angiogenic ability of the drug is expressed as a percentage of neovascularization. 28, 29 Under the stereoscopic microscope, the CAM vascular network of the control group showed a clear vein-like distribution after the addition of physiological saline and bFGF ( Figure 4C ). The number of small vessels generated around the main vessel increased, the small blood vessels grew exuberantly, and then, produced more branches, thereby increasing the density of the whole blood vessels in the field. Within the same drug group, the density of the blood vessels decreased significantly when the concentration of the drug increased, which showed that the activity of the CAM was positively correlated with the concentration. At the same concentrations, the activity of the CS&ES2-AF mixture group was slightly less than the other two groups. The chemically modified product CS-ES2-AF had the smallest number of small blood vessels around the main blood vessels, and almost no visible small blood vessels existed in the field of view under the stereoscope. To corroborate the above results, the newly generated vessels in each group were statistically analyzed ( Figure 4F ). After drug treatment for 24 hours, the neovascularization rate of the control group was 48.75%±2.27%. When the concentrations of drug were 10 μg/mL, 25 μg/mL, and 50 μg/mL, the neovascularization rates in the CS-ES2-AF group were 34.40%±1.54%, 27.14%±2.08%, and 18.25%±2.62%, respectively. At these same concentrations, the proportions of new vessels in the ES2-AF and CS&ES2-AF groups were 32.29%±2.54%, 28.02%±1.23%, and 26.02%±2.28%; and 38.11%±1.81%, 30.51%±3.13%, and 23.79%±2.29%, respectively. Comparing the groups, it was found that when the concentration was 10 μg/mL-25 μg/mL, there was no significant difference in the activity of ES2-AF and the modified product CS-ES2-AF. When the concentration was 10 μg/mL, the activity of CS-ES2-AF was lower than that of the unmodified polypeptide ES2-AF. This might be due to the water-soluble CS forming a gel-like barrier near ES2-AF at low concentrations, affecting the contact between ES2-AF and the chicken embryos, thereby reducing the effect of ES2-AF, and making the conjugates less active at low concentration. When the concentration was 50 μg/mL, the CS-ES2-AF activity was higher than the unmodified polypeptide ES2-AF. This might be due to the increase in stability of CS-ES2-AF in vivo, thereby improving the activity.