An RGD modified water-soluble fluorophore probe for in vivo NIR-II imaging of thrombosis

Venous thrombosis leads to severe symptoms and death through pulmonary embolism. There is a great need for high sensitivity imaging methods to identify acute patients who would benefit from thrombolysis. We designed a novel, organic near-infrared second-window (NIR-II) probe, which targets the glycoprotein IIb/IIIa receptor (GPIIb/IIIa) on activated platelets. The probe's structure was characterized by MALDI-TOF-MS, TEM, UV-visible absorption and NIR-II fluorescent spectroscopy. The probe's specificity for activated platelets was investigated in vitro and in vivo. Thrombosis in mice was induced by administration of FeCl₃ in the external jugular vein and imaged by using a NIR-II imager. The donor-acceptor-donor fluorescent core TTQ was prepared from donor and acceptor units. TTQ-PEG-NH₂ was synthesized by sequential modification of PEGylated TTQ, followed by c(RGD) condensation. Signal strength was continuously monitored for 24 h following TTQ-PEG-c(RGD) or non-specific fluorescent dye injection. The contralateral external jugular vein, sham surgery and a competitive inhibition experiment served as controls. TTQ-PEG-c(RGD) presented high NIR-II intensity, good stability and excellent affinity for activated platelets. The NIR-II fluorescence signal of TTQ-PEG-c(RGD) injected mice significantly increased at the thrombus site and peaked at 4 h, whereas there was no significant change in the control mice, and the competitive inhibition of the RGD antagonist suppressed the enhancement of the NIR-II fluorescence signal. Comparison between fresh and old thrombi confirmed that TTQ-PEG-c(RGD) could be used to distinguish a fresh thrombus from an old thrombus. TTQ-PEG-c(RGD) can specifically target thrombosis in vitro and in vivo, providing a potential tool for noninvasive diagnosis of early thrombi.