Study On The Constraction Of Tumor Microenvironment Sensitive Chondroitin Sulfate-ES2 And Its Anti-tumor Effect

In recent years,the morbidity and mortality of malignant tumors have shown an increasing trend around the world,which has seriously endangered people’s lives and health.Currently,related anti-tumor research has also become the focus of scientists.Based on the study of malignant tumors,it is found that tumor growth and metastasis require new blood vessels to provide nutrients.If the nutrient supply of tumor tissue is blocked,the growth and migration of tumor can be inhibited.This discovery has promoted the development of anti-angiogenesis and anti-tumor drugs.ES2 is a sequence of endostatin,composed of 11 amino acids.It can exert anti-tumor effects by inhibiting the formation of new blood vessels near the tumor and its tissues.Its biological activity is three times that of endostatin.Compared with small-molecular chemical drug,ES2 has some shortcomings such as short half-life and poor stability.Presently,a variety of methods have been developed to improve the shortcomings of polypeptide drugs,such as polyethylene glycol(PEG),glycosylation,and fatty acid modification.Chondroitin sulfate(CS)is a biological endogenous glycosaminoglycan.Its own physical and chemical properties,including biodegradability,cellular compatibility,low immunogenicity,and high solubility,provide the possibility for CS as a drug carrier.There are overexpressed CD44 receptors on the surface of a large number of tumor cells.Chondroitin sulfate is one of the effective ligands of CD44 receptors.Therefore,using chondroitin sulfate as a drug carrier can actively target tumor tissues.At the same time,using chondroitin sulfate to modify peptide drugs can enhance the stability of polypeptide drug and prolong its half-life.Paclitaxel(PTX)is a natural anti-tumor drug.Paclitaxel can selectively bind to tubulin,thereby hindering the normal division of cells,and ultimately leading to the death of tumor cells.As a chemotherapeutic drug widely used in clinical practice,paclitaxel has some shortcomings that cannot be ignored,such as extremely poor water solubility,low targeting,and side effects.Paclitaxel cannot obtain the ideal therapeutic effect in clinical practice because of these shortcomings.Studies have found that the content of glutathione(GSH)in the tumor site is significantly higher than that in normal tissues.According to this feature,the antiangiogenic peptide ES2 was modified with chondroitin sulfate to obtain the glycopeptide conjugate CS-CYS-ES2(CCE)by using cysteamine(CYS)containing disulfide bonds as the intermediate ligator.On this basis,in order to increase the antitumor activity of the drug,the natural anti-tumor drug paclitaxel was linked to CCE through esterification reaction,and another binding compound PTX-CS-CYS-ES2(PTX-CCE)was obtained,which could target both neovascularization and tumor cells.These two conjugates can be targeted to tumor tissue under the active targeting effect of CS.Under the stimulation of high GSH in tumor tissue,the disulfide bond on cystamine breaks rapidly,and ES2 frees into endothelial cells to play a role.In addition,the PTX in PTX-CCE can be quickly targeted to tumor cells under the action of CS targeting and play an anti-tumor effect.After the CCE and PTX-CCE conjugates were prepared,different concentrations of GSH were used to investigate the responsive release behavior of the two conjugates.It was found that the two drugs have the ability to quickly release the anti-angiogenic peptide ES2 in a 10 mM environment.The ES2 cumulative release rate of CCE and PTX-CCE is(84.26%±4.81%)and(93.99%±3.89%),respectively.The stability of the CCE and PTX-CCE conjugates under the different pH,temperatures and long-term storage conditions was explored.It is found that the two drugs are relatively stable and can still maintain high stability under different conditions.Using EAhy 926 endothelial cells as a research model,the anti-angiogenic activity of the two conjugates was explored by CCK-8 experiment,scratch experiment,lumen formation experiment and other methods.It was found that the combination of CCE and PTX-CCE can inhibit the proliferation,migration and formation of endothelial cells,and with the increase of the concentration,its inhibitory ability becomes stronger.Among them,the inhibitory effect of PTX-CCE on endothelial cells is better than that of CCE.In addition,through apoptosis and cycle experiments,We found that CCE and PTX-CCE conjugates can promote endothelial cell apoptosis in a concentration-dependent manner,and CCE and PTX-CCE can block endothelial cells in G2/M phase and S phase,respectively.Using B16F10 highly metastatic melanoma cells as a research model,we found that two conjugates can inhibit the proliferation,migration and invasion of B16F10 cells in a concentrationdependent manner by CCK-8 experiment,scratch experiment,lumen formation experiment and other methods.A mouse melanoma model was constructed,and CCE and PTX-CCE conjugates were injected into mice through tail vein injection.It was found that the two drugs can inhibit the growth of melanoma.Among them,the tumor inhibition rates of CCE and PTX-CCE were 58.82%and 66.75%,respectively.PTXCCE showed better anti-tumor activity in vivo.In summary,CCE and PTX-CCE conjugates exhibit good anti-angiogenic and anti-tumor activities,laying a foundation for the development of anti-tumor target drugs.