Discrete bimodal probes for thrombus imaging

Abstract

Here we report a generalizable solid/solution-phase strategy for the synthesis of discrete bimodal fibrin-targeted imaging probes. A fibrin-specific peptide was conjugated with two distinct imaging reporters at the C- and N-termini. In vitro studies demonstrated retention of fibrin affinity and specificity. Imaging studies showed that these probes could detect fibrin over a wide range of probe concentrations by optical, magnetic resonance, and positron emission tomography imaging.

Figure 1. MR Imaging of Fibrinogen (fgn) and Fibrin Clots

1-mL centrifuge tubes were scanned on a clinical 1.5T MRI at room temperature. Top row shows axial slices corresponding to the point indicated by the line through the coronal images (bottom row). A) pure water; B) 15 μM fgn; C) 25 μM [Gd(HP-DO3A)] in 15 μM fgn; D) 25 μM [Gd(HP-DO3A)] in 15 μM fibrin gel; E) 25 μM Gd-^NPep-Fl in 15 μM fgn; F) 25 μM Gd-^NPep-Fl in 15 μM fibrin gel.

Figure 2. Fluorescence Images of Fibrinogen (fgn) and Fibrin Clots

Phantoms (A–D) were scanned on an IVIS Spectrum imager. Fluorescence images (bottom) are overlaid on photographs (top) of 1-mL glass tubes containing 1-mL of A) 50 nM Gd-^NPep-Fl in 6 μM fgn; B) 50 nM Gd-^NPep-Fl in 6 μM fibrin gel; C) 50 nM fluorescein in 6 μM fibrin gel D) 50 nM fluorescein in 6 μM fgn. E) Fluorescence confocal microscopy image of fibrinogen polymerized on a microscopy slide in the presence of Gd-^NPep-Fl.

Figure 3. PET Imaging of Fibrin Clots

Phantoms in 4 mL glass culture tubes were scanned on a clinical PET head scanner. Each phantom contained fibrinogen (fgn) at a final concentration of 6 μM, 2.3 nmol of peptide or DOTA, a total volume of 2 mL, and a total activity of 10 μCi. A) ⁶⁴CuDOTA in fgn; B) ⁶⁴CuDOTA in fibrin gel; C) Fl-^NPep-⁶⁴Cu in fgn; D) Fl-^NPep-⁶⁴Cu in fibrin gel E) ⁶⁴Cu-^NPep-Fl in fgn; F) ⁶⁴Cu-^NPep-Fl in fibrin gel.

Scheme 1. Multimodal Probe Synthesis

Cartoon depicting mixed solid/solution phase synthesis of multimodal probes. See Supporting Information for full synthetic details.