Molecular imaging of fibrin deposition in deep vein thrombosis using fibrin-targeted near-infrared fluorescence

Abstract

Objectives: The goal of this study was to develop and validate a new fibrin-targeted imaging agent that enables high-resolution near-infrared fluorescence (NIRF) imaging of deep vein thrombosis (DVT).

Background: NIRF imaging of fibrin could enable highly sensitive and noninvasive molecular imaging of thrombosis syndromes in vivo.

Methods: A fibrin-targeted peptide was conjugated to a near-infrared fluorophore Cy7, termed FTP11-Cy7. The NIRF peptide is based on a fibrin-specific imaging agent that has completed Phase II clinical magnetic resonance imaging trials. In vitro binding of FTP11-Cy7 to human plasma clots was assessed by using fluorescence reflectance imaging. Next, FTP11-Cy7 was intravenously injected in mice with femoral DVT induced by topical 7.5% ferric chloride treatment. Intravital fluorescence microscopy and noninvasive fluorescence molecular tomography-computed tomography were performed in 32 mice with DVT, followed by histological analyses.

Results: In vitro human clot-binding analyses showed a 6-fold higher NIRF clot target-to-background ratio (TBR) of FTP11-Cy7 than free Cy7 (6.3 ± 0.34 vs. 1.2 ± 0.03; p < 0.0001). The thrombus TBR of acute and subacute femoral DVT with FTP11-Cy7 obtained by using intravital fluorescence microscopy was >400% higher than control free Cy7. Binding of FTP11-Cy7 to thrombi was blocked by a 100-fold excess of unlabeled competitor peptide both in vitro and in vivo (p < 0.001 for each). Histological analyses confirmed that FTP11-Cy7 specifically accumulated in thrombi. Noninvasive fluorescence molecular tomography-computed tomography imaging of fibrin in jugular DVT demonstrated strong NIRF signal in thrombi compared with sham-operated jugular veins (mean TBR 3.5 ± 0.7 vs. 1.5 ± 0.3; p < 0.05).

Conclusions: The fibrin-targeted NIRF agent FTP11-Cy7 was shown to avidly and specifically bind human and murine thrombi, and enable sensitive, multimodal intravital and noninvasive NIRF molecular imaging detection of acute and subacute murine DVT in vivo.

Figure 1. Synthesis and structure of FTP11-Cy7

(A) FTP11-Cy7 is comprised of 11 amino acids conjugated to the near-infrared fluorochrome, Cy7. (B) The HPLC trace demonstrates > 98% purity. (C) Matrix-assisted laser desorption/ionization mass spectrometry shows a molecular mass of 2062 g/mol. (D) The NIRF signal of the FTP11-Cy7 related linearly to its Cy7 concentration (R² = 0.99).

Figure 2. NIRF imaging of fibrin in human plasma clots

(A) Representative light and NIRF images of in vitro binding of FTP11-Cy7 to human plasma clots obtained by fluorescence reflectance imaging. (B) Clots incubated with FTP11-Cy7 showed a 6-fold increase (p < 0.0001) in NIRF signal enhancement compared to free Cy7. The NIRF signal was significantly blocked with an excess of competitor peptide in a dose-dependent manner. *p < 0.05, ***p < 0.0001, Bars represents mean ± standard error.

Figure 3. Clearance of FTP11-Cy7in blood circulation

After injecting FTP11-Cy7 via tail vein in normal C57BL/6 mice, serial blood sampling was performed through jugular vein, and (A) Cy7 levels were measured by fluorescence reflectance imaging. (B) The blood half-life was 2.8 minutes (95% CI, 1.9–5.9).

Figure 4. High-resolution IVFM detection of fibrin deposition in femoral DVT

Femoral veins were treated with topical FeCl₃ to induce DVT. Following FTP11-Cy7 imaging agent injection, IVFM of (A) acute DVT or (B) subacute DVT was performed. Representative images from multiple mice in each group are shown. Both acute and subacute thrombi were enhanced by FTP11-Cy7. (C) In contrast, control free Cy7 did not enhance venous thrombi. (D) The thrombus TBR was significantly higher in the FTP11-Cy7 group compared to the control free Cy7 group. (E) FTP11-Cy7 based NIRF signal deposition in acute thrombi was blocked by pre-injection of a 100-fold excess of unlabeled FTP11. *p < 0.05, **p < 0.001, Scale Bars, 100 μm. V indicates vein.

Figure 5. Thrombus-specific binding of FTP11-Cy7 in murine venous thrombi

Fluorescence microscopy and hematoxylin and eosin photomicrographs. FITC (green), Cy7 (red), merged image (FITC + Cy7), and H&E were shown in each panel. FTP11-Cy7 accumulated into both (A) acute and (B) sub-acute thrombus. (C) In contrast, free Cy7 did not bind thrombus. Lower left panel shows enlarged thrombosed vein. Scale bars, 100 μm.

Figure 6. Noninvasive molecular imaging of fibrin in jugular DVT by integrated FMT-CT

In C57BL/6 mice, the left jugular vein was treated with FeCl₃ to induce DVT, and then FMT-CT was performed at day 3. (A, B) CT and FMT-CT co-registered images of 2D fusion and (C) 3D fusion are shown. Arrows indicate a thrombosed left jugular vein. (D) The TBR of thrombosed jugular veins was significantly higher than sham-operated jugular veins (p < 0.05, n = 9 mice).

Figure 7. Ex vivo targeting of FTP11-Cy7 to thrombosed jugular veins

(A) Fluorescence reflectance image of Cy7 channel and (B) ex vivo image of color photograph. High NIRF signal in jugular vein is matched to the thrombosed lesion. (C) The thrombus TBR of the left thrombosed jugular vein was 50% greater than the sham-operated right jugular vein (p < 0.05, n = 9 mice). Arrows indicates a thrombosed left jugular vein.