Non Anticoagulant Heparin Modified Anti-angiogenic Peptide ES2 And Its Anti-tumor Activity

Protein and peptide drugs have shown great value in the treatment of cancer and other major diseases,with high efficacy,strong specificity,low toxicity and side effects,and clear biological functions,which have irreplaceable therapeutic effects on certain diseases.Endostatin(ES)is a polypeptide with a molecular weight of 20kDa,which is the most studied endogenous angiogenesis inhibitor.It can play an anti-tumor role by inhibiting the formation of neovascularization near the tumor and its tissues.ES2(60-70,IVRRADRAAVP)is a segment of amino acid in ES that can effectively inhibit the proliferation and migration of endothelial cells,and its activity of inhibiting neovascularization in vivo is about three times than ES.However,compared with small-molecule chemical drugs,there are common shortcomings such as poor stability,short half-life,and immunogenicity.Pegylation can solve these shortcomings,but because Pegylation is not degradable in vivo,there is a certain risk in its long-term use;in addition,because Pegylation does not have any biological function,its role is relatively single.Therefore,Pegylation is not the best choiceIn recent years,polysaccharide has made great progress in anti-tumor research,and it has become one of the important sources of anti-tumor drug screening because of its high safety,difficult drug resistance and relatively small side effects.Studies have shown that heparin(HP)can combine with over-expressed neovascularization-related growth factors(VEGF,bFGF)at the tumor site to inhibit its activity,thereby blocking angiogenesis at the tumor site to achieve the purpose of tumor treatment.Because of its anticoagulant activity,it has a greater risk as an anti-tumor drug in vivo.Some studies have found that the antitumor activity of heparin is largely independent of its anticoagulant activity.Therefore,the chemical modification of heparin can not only reduce its anticoagulant activity,but also maintain or improve its anti-tumor proliferation and metastasis activity.During the chemical modification of heparin,the non-sulfated uronic acid C2-C3 ortho hydroxyl group is cleaved to form ethylene glycol cleaved heparin(GSHP).Due to the destruction of the pentose structure,its anticoagulant activity is significantly reduced.GSHP-ES2 conjugates were prepared by connecting GSHP and ES2 via amide bond.The structure of intermediates and end products was confirmed by HPLC,1H NMR,multi angle laser light scattering and MS.The biological activity of GSHP-ES2 in vitro and in vivo was investigated,APTT detected that the conjugate has low anticoagulant activity,and the stability of the conjugate was higher than ES2 under different temperature and pH conditions;The anti-neovascularization study of the conjugate in vivo and in vitro revealed that the activity of GSHP-ES2 was higher than ES2;Pharmacokinetic studies have found that the half-life of GSHP-ES2 was 3.67 times longer than ES2,and compared with ES2,the conjugate has a higher concentration in the liver,and the retention time was longer than other tissues;Targeting studies found that GSHP-ES2 has significantly higher affinity for endothelial cells and P-selectin than ES2;Examining the anti-tumor activity of GSHP-ES2 in vivo,it was found that the anti-tumor activity of GSHP-ES2(38.36%)was significantly higher than that of ES2(24.77%)On this basis,in order to increase the antitumor activity,cysteamine(Cys)containing disulfide bond was connected with GSHP by amide bond,natural antitumor drug paclitaxel(PTX)was connected with GSHP by esterification reaction,and then ES2 and Cys were connected by amide bond to prepare PTX-GSHP-CYS-ES2 conjugate.Using glutathione(GSH)to investigate the environmental responsiveness of PTX-GSHP-CYSES2,it was found that PTX-GSHP-CYS-ES2 exhibited redox-sensitive drug release behavior;Using endothelial cells EAhy926 and high-metastatic melanoma cells B16F10 as models,the anti-angiogenic ability of PTX-GSHP-CYS-ES2 was examined by CCK-8 assay,wound-healing assay,tube formation assay,transwell assay,etc.The results showed that it all showed dose-dependent inhibition effect;The mechanism of PTX-GSHP-CYS-ES2 inhibition of neovascularization was investigated through apoptosis and cycle,and it was found that the conjugate can induce apoptosis of endothelial cells and block the cell cycle in S phase.Thus,the synergistic antitumor drugs with tumor microenvironment sensitivity and the ability of "one drug and two targets"(one dose,polypeptide for neovascularization and chemotherapy for tumor cells)were obtained,which laid the foundation for the development of targeted antitumor polypeptide drugs.