Improved In Vivo Detection Of Atherosclerotic Plaques With A Tissue Factor-targeting Magnetic Nano Probe

Sudden rupture of atherosclerotic plaques causes acute cardiovascular and cerebrovascular pathology.Patients may have no clinical symptomsand until the plaque ruptures,leading to myocardial infarction or stroke,which is the main cause of mortality and morbidity in such patients.Accurate detection of plaque can effectively avoid such situations.Molecular imaging can present disease states because of high specificity.By selecting appropriate molecular targets,we can monitor the plaque progression and direct therapy.Tissue factor(TF)is a key factor affecting the progression of atherosclerosis and thrombosis,and thus can be used as a potential target for the detection of atherosclerotic plaque.In this study,a TF-targeted magnetic nanoprobe was constructed to study its imaging effect on atherosclerotic plaque as a contrast agent,thus achieving molecular imaging diagnosis of atherosclerotic plaques.In addition,types of activated TF transform from"encryption"to"decryption",promoting the formation of vurerable atherosclerotic plaques.Activated TF acts as a common factor in the coagulation pathway and inflammatory response.It is an ideal target for early detection of vulnerable plaques.By detecting“decrypted”TF,early warning of vulnerable plaque and accurate molecular diagnosis can be achieved.The whole study is divided into five chapters.In the first chapter,EGFP-EGF1-SPIONs were successfully constructed.CCK-8 test suggests EGFP-EGF1-SPIONs have good stability and suitable biocompatibility.They can be used as contrast agents to travel through the human body because of the size.In addition,EGFP-EGF1-SPIONs had higher relaxation rate(r2)of 357.0 m M-1s-1 and better negativeness than superparamagnetic iron oxide nanoparticles(SPIONs).The contrast enhancement effect allowed EGFP-EGF1-SPIONs in further study of molecular imaging as magnetic resonance contrast agents.In the second chapter,the biosafety of EGFP-EGF1-SPIONs and the ability to specifically target TF were verified.EGFP-EGF1-SPIONs did not affect cell viability at safe doses.In vitro,mouse aortic endothelial cells(MAECs)were treated with LPS to overexpress TF.Immunofluorescence showed that EGFP-EGF1-SPIONs specifically binded to TF-overexpressing cells.Additionally,after knocking down of TF expression by transfecting TF-si RNA,the uptake of EGFP-EGF1-SPIONs was also significantly reduced,further indicating that EGFP-EGF1-SPIONs can specifically bind to TF,targeting cells,tissues or organs with high expression of TF.In the third chapter,in vivo imaging of atherosclerotic mouse plaques with EGFP-EGF1-SPIONs was studied.Firstly,a mouse model of atherosclerosis was established.Immunohistochemistry showed that TF was highly expressed in plaque,and the expression of TF was positively correlated with plaque instability.Meanwhile,as plaque progressed,more TF was expressed.Subsequently,magnetic resonance imaging of mice indicated the signal intensity of the plaque site was significantly reduced after administration of EGFP-EGF1-SPIONs.Prussian blue staining showed EGFP-EGF1-SPIONs deposited in the plaque,consistent with imaging results.EGFP-EGF1-SPIONs could specifically target atherosclerotic plaques because of specific aggregation in the plaque.Therefore,EGEP-EGF1-SPIONs can effectively target atherosclerotic plaques with significant MRI T2 enhancement and can be used as molecular probes for the detection of atherosclerotic plaques.In the forth chapter,we designed multiple EGF1 derivative polypeptides,E1 and E2by serving aa248-259 of TF as a ligand by the GLIDE program,which targeted"decrypted"TF with optimal conformational sensitivity.Then the affinity of TF and EGF1 derivative polypeptides was detected with surface plasmon resonance(SPR)technique,suggesting that the affinity of E1 and TF was significantly higher than that of EGF1 and TF,which laid the foundation of building probes more sensitive to vulnerable plaques.In the fifth chapter,EGF1,E1,E2 were conjugated with SPIONs,respectively.E1-SPIONs and E2-SPIONs have higher relaxation rates than EGF1-SPIONs with better negative contrast enhancement effects.Therefore,E1-SPIONs and E2-SPIONs can be used as sensitive magnetic resonance contrast agents,and further in vitro and in vivo studies are to be carried out.In summary,EGFP-EGF1-SPIONs showed improved accuracy,superior contrast effects,and better cytocompatibility compared with common contrast agents in the detection of atherosclerotic plaques of apolipoprotein E knockout(Apo E^-/-)mice using magnetic resonance imaging.In conclusion,EGFP-EGF1-SPION is a promising TF-targeting nanoprobe to precisely and specifically detect atherosclerotic plaques,which may improve molecular imaging diagnosis of cardiovascular and cerebrovascular events for the comprehensive evaluation of atherosclerosis.Meanwhile,the probe is further optimized by screening EGF1 derivative polypeptides with optimal conformational sensitivity to prepare for study of accomplishing more accurate molecular diagnosis.