Vascular thrombus imaging in vivo via near-infrared fluorescent nanodiamond particles bioengineered with the disintegrin bitistatin (Part II)

The aim of this feasibility study was to test the ability of fluorescent nanodiamond particles (F-NDP) covalently conjugated with bitistatin (F-NDP-Bit) to detect vascular blood clots in vivo using extracorporeal near-infrared (NIR) imaging. Specifically, we compared NIR fluorescence properties of F-NDP with N-V (F-NDP(NV)) and N-V-N color centers and sizes (100–10,000 nm). Optimal NIR fluorescence and tissue penetration across biological tissues (rat skin, porcine axillary veins, and skin) was obtained for F-NDP(NV) with a mean diameter of 700 nm. Intravital imaging (using in vivo imaging system [IVIS]) in vitro revealed that F-NDP(NV)-loaded glass capillaries could be detected across 6 mm of rat red-muscle barrier and 12 mm of porcine skin, which equals the average vertical distance of a human carotid artery bifurcation from the surface of the adjacent skin (14 mm). In vivo, feasibility was demonstrated in a rat model of occlusive blood clots generated using FeCl(3) in the carotid artery bifurcation. Following systemic infusions of F-NDP(NV)-Bit (3 or 15 mg/kg) via the external carotid artery or femoral vein (N=3), presence of the particles in the thrombi was confirmed both in situ via IVIS, and ex vivo via confocal imaging. The presence of F-NDP(NV) in the vascular clots was further confirmed by direct counting of fluorescent particles extracted from clots following tissue solubilization. Our data suggest that F-NDP(NV)-Bit associate with vascular blood clots, presumably by binding of F-NDP(NV)-Bit to activated platelets within the blood clot. We posit that F-NDP(NV)-Bit could serve as a noninvasive platform for identification of vascular thrombi using NIR energy monitored by an extracorporeal device.