Chitosan Oligosaccharide Modification Of Anti - Neovascularization Peptide ES - 2 And Its Anti - Neovascularization Activity

Endostatin(ES) is an endogenous inhibitor of angiogenesis, which is isolated and purified from the supernatant of mouse endothelial cells. By inhibiting the formation of new blood vessels around the tumor tissue, it can be achieved to cut off the nutrition supply of the tumor and the metabolic pathway of waste, thereby playing an anti tumor activity.ES-2(IVRRADRAAVP) is a short fragement of ES, it has significant antiangiogenesis and antitumor activity, and it is much easier to obtained by solid phase synthesis. But it also has some shortcomings such as short half-time, poor stability and instable biological activity in vivo.These shortcomings greatly limit its further application. It was reported that chemical modification might be a good way to overcome these disadvantageChitosan oligosaccharide(COS) has a certain anti-tumor activity, with a clear structure.After quaternary ammonium, a better water-soluble and activity can be ontained.In this study, 6-O-quaternary ammonium bromide COS(O-HTCCOS)was used to modify ES-2, and 6-O- quaternary ammonium COS-ES-2( O-HTCCOS-ES-2) was successfully synthesized. As we found in the study, O-HTCCOS-ES-2 has not only retains the ES-2anti-angiogenesis and anti-tumor activity, but also integrated the good characteristics of chitosan oligosaccharide, sobetter stability, better targeting and higher cellular affinitys. The main results of this paper are as as follows:1. Synthesis and characterization of 6-O-quaternized COS(O-HTCCOS)After three steps of chemical reaction, quaternary ammonium salt was attached to the hydroxyl in C6 of the side chain of COS. Then, after further dialysis and lyophilization, better water soluble quaternary ammonium COS, the total yield was 95.26 %. Using IR spectrum and1 HNMR analysis, the structure of O-HTCCOS was studied The percentage of free amino groups of O-HTCCOS was 87.35 %.Better water solubility and higher free amino acid content of O-HTCCOS was ontained which is more favorable for the sfollowing modification.2. Synthesis and characterization of 6-O-quaternary ammonium COS-ES-2peptide(O-HTCCOS-ES-2)In the study, EDC/NHS were used as crosslinking agent after a two step reaction, by the ES-2 and O-HTCCOS were attached together, and the modification rate was more than 50 %.SephadexG-25 gel chromatography was used to separate and purify the final O-HTCCOS-ES-2pure. MALDI-TOF-MS was used to study the structure of O-HTCCOS-ES-2 pure, and the ratio of ES-2 to O-HTCCOS was 1:1, which means that one molecule of ES-2 was modified byonemolecule of O-HTCCOS.3. Stability Evaluation of O-HTCCOS-ES-2O-HTCCOS-ES-2, ES-2, and the mixture of O-HTCCOS and ES-2 were keeped at 25℃and37℃ for certain times, then the changes of their anti-endothelial cells proliferation activity was studied, then the stabilities of the three peptides were compared. The results showed that the stability of O-HTCCOS-ES-2 is better than that of ES-2 and the mixture of ES-2 and O-HTCCOS.4. Study on cell affinity of O-HTCCOS-ES-2Flow cytometry was used to study cell affinity of O-HTCCOS-ES-2, ES-2 and the mixture of O-HTCCOS and ES-2. The results showed that, compared with ES-2 or the mixture of O-HTCCOS and ES-2, The affinity of O-HTCCOS-ES-2 to endothelial cells is much stronger,and it will be much easier for O-HTCCOS-ES-2 to enter the cells.5. Study of bioactivity of O-HTCCOS-ES-25.1 Study of anti--endothelial cells proliferation activity of O-HTCCOS-ES-2 in vitro(1) CCK-8 method was used to study the inhibition of O-HTCCOS-ES-2, ES-2, the mixture of O-HTCCOS and ES-2 on endothelial cell proliferation in vitro. The results showed that different concentrations of O-HTCCOS-ES-2, ES-2 and the mixture of O-HTCCOS and ES-2could significantly inhibit the proliferation of endothelial cells, and O-HTCCOS-ES-2 displayed the best anti-proliferation activity in a dose depent manner.(2) The anti-angiogenic activity of O-HTCCOS-ES-2, ES-2, the mixture of O-HTCCOS and ES-2 were determined using tube formation assay. The results showed that at different concentrations, O-HTCCOS-ES-2, ES-2, the mixture of O-HTCCOS and ES-2 showed significant anti-angiogenesis activity in dose depent manner in this tube formation assay. And the activity of O-HTCCOS-ES-2 was much better than that of ES-2 and the mixture. Study on the anti-angiogenesis activity of O-HTCCOS-ES-2 in vivo.5.2 Study of anti--endothelial cells proliferation activity of O-HTCCOS-ES-2 in vivo(1) Chick embryo chorioallantoic membrane assay was used to study the anti-angiogenesis activity of O-HTCCOS-ES-2, ES-2 and the mixture of O-HTCCOS and ES-2. The results showed that O-HTCCOS-ES-2, ES-2 and the mixture all showed certain inhibition on chick embryo chorioallantoic membrane angiogenesis. The anti-angiogenesis effect of O-HTCCOS-ES-2 is better than that of ES-2 and mixture, and the effect of anti-angiogenesis in vivo is enhanced with the increase of the drug concentration.(2) The research of the O-HTCCOS-ES-2, ES-2 and the mixture on the inhibition of angiogenesis in zebrafish model segments showed that O-HTCCOS-ES-2, ES-2 and the mixture of O-HTCCOS and ES-2 can inhibit the formation of the new blood vessel in the body ofzebrafish, and the anti angiogenesis effect of O-HTCCOS-ES-2 is the best.5.3 Study on the anti-cell migration activity of O-HTCCOS-ES-2The inhibitory effect of O-HTCCOS-ES-2 on endothelial cell migration was studied using the scratch test and Transwell chamber method.Both two experimental results showed that the O-HTCCOS-ES-2, ES-2 and the mixture of O-HTCCOS and ES-2 can inhibit the migration of endothelial cells, and O-HTCCOS-ES-2 displayed the best ability. And inhibition was carried out in a concentration dependent manner.