Chondroitinized Modification Of Anti Angiogenesis Peptide EA And Its Stability,Anti Angiogenesis And Anti Tumor Effects And Mechanisms

In recent years,the mortality rate of cancer has gradually increased and shown a trend of youthfulness,seriously threatening human life and health.Research has found that the growth and metastasis of tumors are closely related to the generation of new blood vessels near the tumor.Inhibiting angiogenesis and blocking the blood supply to tumors,thereby inhibiting tumor growth and metastasis,is one of the effective methods for treating solid tumors.ES2-AF(EA)is an anti angiogenic peptide obtained through solid-phase synthesis.Previous studies have shown that it can effectively inhibit endothelial cell proliferation and migration,but it has drawbacks such as short half-life and poor stability.To address these issues,it is proposed to chemically modify EA.Chondroitin sulfate(CS)is an endogenous polysaccharide with good biocompatibility and biodegradability.At the same time,it has a high affinity for the CD44 protein overexpressed on the tumor surface and can target tumor cells.Therefore,this study used CS to modify EA and obtained CS-ES2-AF(CS-EA)polymer.Previous studies have shown that CS-EA has a longer half-life and better tumor targeting ability than EA,but its stability,anti angiogenesis and anti-tumor effects and mechanisms still need to be studied.Based on the previous research,this paper focuses on the unresolved issues mentioned above.The main content and results are as follows:1.Preparation of CS-EAThe AF peptide and ES2 peptide are fused through solid phase synthesis,and EA polypeptide withamolecular weight of 2254.53 and a purity of 96.30%was successfully prepared.CS is modified with tetrabutylammonium hydroxide to obtain a CS derivative that is easily soluble in DMSO,and under the action of condensing agent BOP and catalyst DIPEA,CS is coupled with polypeptide EA in the form of amide bonds to obtain glycopeptide conjugate CS-EA.2.CCK-8 method to detect CS-EA stability and inhibit endothelial cell proliferationThrough CCK-8 reagent kit detection,the results showed that the biological activities of EA and CS-EA decreased with the prolongation of incubation time at 25℃ and 37℃ and the stability at 25℃ was better than that at 37℃;During storage in a 4℃ refrigerator,the biological activity of the two drugs showed a slow decreasing trend over time,and after 30 days,they still retained more than 70%of their activity;After incubation at different pH levels,the activity of the two drugs significantly decreased,and CS-EA was able to retain more biological activity over a wider pH range;In the stability study,the stability of CS-EA is always better than that of EA.EA,EA&CS and CS-EA all inhibited endothelial cell proliferation,and the inhibitory ability was concentration-dependent,among which CS-EA inhibition ability was the best.3.Uptake and uptake pathway of CS-EA in endothelial cellsQualitative detection of the uptake and uptake mechanism of CS-EA in endothelial cells by flow cytometry quantification and laser confocal photography.The results showed that the EA modified by CS was more easily uptaken by cells,and the uptake ability was time-dependent.Other than that.Both EA and CS-EA are entered via the clathrin and lipid raft pathways.4.CS-EA pro-endothelial cell apoptosis mechanismTaking EAhy 926 cells as the research object,the mechanism of apoptosis of CS-EA endothelial cells was studied through experiments such as mitochondrial membrane potential,reactive oxygen species,and cycle.The results showed that CS-EA can reduce the expression of anti apoptotic protein Bcl-2,leading to a decrease in mitochondrial membrane potential,which in turn leads to the disconnection of mitochondrial respiratory chain and the production of a large amount of reactive oxygen species(ROS).ROS activates Caspase 3 by oxidizing cardiolipin in the inner mitochondrial membrane to release Cyto-c from the inner membrane,which induces endothelial apoptosis through the mitochondrial apoptosis pathway.In addition,CS-EA can also block endothelial cells in G1 phase,reducing the division and proliferation of endothelial cells.By downregulating the expression of angiogenesis-related proteins VEGF and CD31,thereby inhibiting the generation of new blood vessels,further inhibiting the proliferation and migration of endothelial cells.5.Study on the antitumor effect of CS-EA in vivoA mouse model of B16 melanoma was constructed to study the inhibitory effect of EA,EA&CS,CS-EA on tumors.The results showed that all three drugs could inhibit the growth of melanoma in mice,and the tumor inhibition rate of EA after CS modification was significantly improved,and the tumor inhibition rate in the CS-EA group was 68.79%.After fourteen days of continuous administration,all groups of mice gained weight,but no obvious toxic side effects appeared.HE staining analysis was performed on the heart,liver,spleen,lung and kidney of mice,and the results showed that the morphology of organ cells in each group was normal.Immunofluorescence detection of some cytokines expression in tumor tissue showed that all three drugs could upregulate the expression levels of Caspase 3 and Cyto c,while reducing the expression levels of Bcl-2,VEGF,and CD31.In summary,the stability and anti-angiogenic activity of EA after CS modification were significantly improved,and CS-EA had the effect of inhibiting the growth of melanoma,and good safety,without significant toxic and side effects on mice.This study lays a theoretical foundation for the development of new angiogenesis related drugs.