Construction Of Tumor Vessels Targeting Magnetic Nano-procoagulant Protein And Identification Of Its Activity

Tumor vascular embolization is one of the important options of interventional oncology.However,conventional embolic agents still face some shortcomings.It is an urgent problem to develop new embolic agents.The purpose of this study was to develop a tumor vascular targeting magnetic nano-procoagulant protein tTF-EG3287/CMC/Fe3O4 for tumor vascular embolization,and to identify its selective vascular thromboembolic activity and its anti-tumor activity.The magnetic nano-procoagulant protein tTF,EG3287/CMC/Fe3O4 consists of the fusion protein tTF-EG3287 and the magnetic nanocarrier CMC/Fe3O4.The truncated tissue factor(tTF)is a recombinant form of the tissue factor(TF),which only contains the extracellular region of TF.tTF is not procoagulant while free in blood circulation system,but restore the procoagulant activity of TF after binding to the cell membrane of vascular endothelial cell by a ligand.Neuropilin-1(NRP-1)a membrane protein which specificity high express on a variety of tumor tissues and tumor-associated blood vessels.It is a potential tumor vascular molecular target for tumor vascular targeted therapy.EG3287 is a targeting polypeptide that targets NRP-1.The fusion protein tTF-EG3287 consisting of tTF and EG3287 can bind to NRP-1,which is highly expressed in tumor blood vessels,by targeting polypeptide EG3287.After binding,the membrane-bound tTF restores procoagulant activity and leading coagulation reaction in the tumor blood vessels,and then causing thromboembolism and blocking the vascular supply of the tumor.However,the targeting peptide EG3287 is low affinity,so the fusion protein tTF-EG3287 is not satisfactory for targeting thrombus.In order to make the fusion protein tTF-EG3287 reach the tumor area efficiently and quickly.In this study,Fe3O4 with a particle size of about 10 nm was prepared by chemical coprecipitation.And then combined with carboxymethyl chitin(CMC)by chemical crosslinking to form a magnetic nanocarrier of CMC coated Fe3O4(CMC/Fe3O4).The magnetic nanocarrier is then crosslinked with the fusion protein to form a magnetic nano-procoagulant protein tTF-EG3287/CMC/Fe304.In vitro,we assessed NRP-1 targeting ability of the magnetic nano-procoagulant protein by using confocal microscopy and flow cytometry,and evaluated potential procoagulant activity of the magnetic nano-procoagulant protein by Spectozyme FXa assay,and evaluated the cytotoxicity of the magnetic nano-procoagulant protein by CCK-8.The in vitro results indicated that the magnetic nano-procoagulant protein contains strong NRP-1 targeting ability and potential procoagulant activity.Moreover,the magnetic nano-procoagulant protein is low cytotoxicity and have good biocompatibility.In vivo,magnetic targeting ability of the magnetic nano-procoagulant protein was detected by in vivo imaging system.HepG2 tumor bearing mice models revealed that intravenous administration of the magnetic nano-procoagulant protein induced intravascular thrombosis specifically on tumor-associated blood vessels,resulting in tumor growth retardation or regression.No apparent side effects,such as thrombosis in other organs or other treatment-related toxicity were observed.Our data showed that the magnetic nano-procoagulant protein may be a promising embolic agent for the embolic therapy of solid tumors.In summary,this study successfully constructed a novel magnetic nano-procoagulant protein tTF-EG3287/CMC/Fe3O4,which may be a promising embolic agent for the embolic therapy of solid tumors.