Molecular Thrombosis Imaging In The Second Near-infrared Window Using A NIR-Ⅱ Fluorophore-peptide Probe

Objective:Venous thromboembolism（VTE）mainly includes deep vein thrombosis（DVT）and pulmonary embolism（PE）,and its early diagnosis is essential for clinical treatment.The purpose of this study is to construct a novel organic second near-infrared window（NIR-II）fluorescent probe,which can target glycoprotein IIb/IIIa receptor（GP IIb/IIIa）on activated platelets,to use NIR-II imaging technology to image venous thrombus and identify fresh venous thrombus from old venous thrombus according to its imaging characteristics.Methods:Construction of a novel organic NIR-II fluorescent nanoparticles di（thiophen）-thiadiazolo-quinoxaline TTQ-PEG-c（RGD）targeting activated platelets GP IIb/IIIa.The probe’s structure was characterized by MALDI-TOF-MS,TEM,UV-visible absorption and NIR-II fluorescence emission spectrum,and the cytotoxicity of the probe was detected by the MTT assay.In vivo and in vitro experiments were carried out to verify the targeting of the probe for thrombus.The in vivo experiment is divided into experimental group（injection of targeted probe TTQ-PEG-c（RGD））and control group（contralateral external jugular vein,non-targeted probe group and competitive inhibition experimental group）.Venous thrombus was induced by local application of Fe Cl₃in the external jugular vein of mice.The signal was continuously monitored for 24 hours following tail vein injection of TTQ-PEG-c（RGD）or non-specific fluorescent dye.Results:The fluorescent core TTQ-TF was prepared by using TTQ as acceptor and TF as donor.TTQ-PEG-NH₂was synthesized by modifying polyethylene glycol（PEG）on TTQ,and then modifying c（RGD）to form TTQ-PEG-c（RGD）.A novel organic NIR-II nano-fluorescent probe TTQ-PEG-c（RGD）was successfully constructed.In vitro characterization studies have shown that TTQ-PEG-c（RGD）presented high NIR-II intensity,good stability.Cytotoxicity experiments show that the probe has no obvious cytotoxicity and has good safety and biocompatibility.In vitro thrombus imaging experiments showed that compared with the control probe TTQ-PEG,the NIR-II fluorescence signal of thrombus immersed in combination with the targeting probe TTQ-PEG-c（RGD）was significantly increased（P<0.05）,and TTQ-PEG-c（RGD）could target thrombus in vitro.In vivo thrombus imaging experiments showed that in mice injected with the targeted probe TTQ-PEG-c（RGD）,the NIR-II fluorescence signal was generated in the thrombotic sites of the external jugular vein and reached a peak at 4 h after injection.Competitive antagonists of RGD significantly inhibited NIR-II fluorescence signals.Then,fresh venous thrombus and old venous thrombus were constructed respectively.After 4 hours of injection of targeted probe into tail vein,the NIR-II fluorescence signal in the thrombotic side of fresh thrombosis group was significantly stronger than that of old thrombosis group（P<0.05）,indicating that TTQ-PEG-c（RGD）can distinguish fresh venous thrombus from old venous thrombus in vivo.Conclusion:TTQ-PEG-c（RGD）,as a novel organic NIR-II nano-fluorescent probe,can specifically target thrombus in vitro and in vivo,and can distinguish fresh venous thrombus from old venous thrombus in vivo,providing potential clinical practical value for non-invasive diagnosis of venous thrombosis.