Vascular thrombus imaging in vivo via near-infrared fluorescent nanodiamond particles bioengineered with the disintegrin bitistatin (Part II)
Abstract: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-NDPNV) 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-NDPNV with a mean diameter of 700 nm. Intravital imaging (using in vivo imaging system [IVIS]) in vitro revealed that F-NDPNV-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 in the carotid artery bifurcation. 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-NDPNV in the vascular clots was further confirmed by direct counting of fluorescent particles extracted from clots following tissue solubilization. Our data suggest that F-NDPNV-Bit associate with vascular blood clots, presumably by binding of F-NDPNV-Bit to activated platelets within the blood clot. We posit that F-NDPNV-Bit could serve as a noninvasive platform for identification of vascular thrombi using NIR energy monitored by an extracorporeal device.
Keywords: fluorescent nanodiamond particles, NIR fluorescence imaging, thrombosis, biomarkers
Thromboembolic events (TEE) in cerebral and coronary vessels are the major causes of death from strokes and heart attacks in developed countries.1,2 Advanced technologies such as computed tomography (CT) scans, magnetic resonance imaging (MRI), and angiography are routinely used to identify the location of tissue ischemia or infarct and guide treatment options in conditions such as focal occlusive stroke and heart attacks.3 However, these diagnostic technologies have drawbacks that preclude their routine utilization for primary disease risk assessment in an ambulatory setting. For example, MRI requires specialized facilities and specialty-trained medical teams, and is restrictive in terms of costs and availability in non-tertiary (rural, community) medical facilities. CT scans, especially angiography, require hospital-based facilities and highly trained technical and medical personnel to acquire and interpret the complex imaging infor- mation. Furthermore, most current imaging technologies used for assessing TEE are indirect; that is, they monitor morphological changes, such as the narrowing of blood flow, rather than pinpointing vascular lesion content (eg, presence of a blood clot vs atherosclerotic lesion). Ultrasound imaging of vascular elements provides mostly dimensional information precluding assessment of specific pathological elements such as blood clots associated with a vulnerable plaque. Altogether, the ability to evaluate the risk of TEE as part of the general health management for pri- mary prevention of vascular thrombosis remains a significant unmet medical need. These considerations suggest the need for advanced technology to provide specific knowledge of “vascular pathology content”, which could be acquired nonin- vasively in real time and in an affordable ambulatory setting.
Materials and methods
Antibodies and other reagents
Bitistatin was purified from the venom of Bitis arietans (Latoxan Serpentarium, Valence, France) using two steps of reverse-phase high-performance liquid chromatography, as described previously.4 F-NDP, chemically surface- functionalized with carboxyl groups (-COOH), were purchased from Adamas Nanotechnologies (Raleigh, NC, purchased from Adamas Nanotechnologies (Raleigh, NC, USA). Two strains of F-NDP were used: green fluorescent F-NDP at 700 nm (2×108 particles/mg) and red fluores- NVN cent F-NDP with N-V color centers (F-NDPNV) at 100 nm (5×1011 particles/mg), 700 nm (2×108 particles/mg), and 10,000 nm (5×105 particles/mg). Isoflurane (IF; B34C16A) was purchased from Henry Schein (Melville, NY, USA). Ethyl alcohol (70% denatured) and PE-10 tubing were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Roboz SUT-15-1 5-0 silk suture was purchased from Roboz Surgical Instrument Co. (Gaithersburg, MD, USA). Parafilm and FeCl3 were purchased from Sigma-Aldrich (St Louis, MO, USA).
Coupling of bitistatin to F-NDP
Bitistatin was coupled to the F-NDP of all types using 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochlo- ride (EDC) as a hetero-bifunctional cross-linker,11 according to the methodology described previously.4 Coupling effi- ciency and preservation of bitistatin activity on the various functionalized nanodiamond particles (F-NDP-Bit) were verified using a semiquantitative enzyme-linked immuno- sorbent assay, as described previously.4
Caracterization of NIr emission of F-NDPNV and F-NDP
Bit was purified from B. arietans venom according to a previously described procedure21 with some minor modifi- cations. Briefly, lyophilized snake venom was dissolved in 0.1% TFA (30 mg/mL). Insoluble material was pelleted by centrifugation at 14,000 rpm for 5 minutes at room tempera- ture (RT). The supernatant was fractionated by reverse-phase high-performance liquid chromatography (HPLC) on a C18 column (250×10 mm; Vydac, Hesperia, CA, USA). The col- umn was eluted with a linear acetonitrile gradient 0%–80% over 45 minutes at a flow rate of 2 mL/min (Figure S2). Separation was monitored at 230 nm; fractions containing Bit (~22 minutes retention time) were collected manually and lyophilized in a Speed-Vac system. The lyophilized fractions were dissolved in water, and protein concentra- tions determined using the BCA assay (Pierce, Rockford, IL, USA). The crude Bit preparation (5 mg protein) was re-chromatographed using the same HPLC system but eluting the column with a shallower acetonitrile gradient (20%–80% over 120 minutes). The main peak containing purified Bit (Figure S3) was lyophilized and dissolved in deionized water to prepare the stock solution (8–10 mg/mL) used for coupling to F-NDPs. The purity of Bit, as tested by sodium dodecyl sulfate – polyacrylamide gel electrophoresis, was found to be 98%, based on the digitalized intensity (HP ScanJet G3110 and software UN-scan-It gel version 6.1 by Silk Scientific Corp., Orem, UT, USA) of the major bands at maximal concentration (Figure S4).
Characterization of NIr emission of F-NDPNV and F-NDPNVN
NNIR fluorescence profiles of F-NDP were characterized using a Tecan Infinite 200 PRO (Tecan AG, Männedorf, Switzerland). One hundred microliters of 3 mg/mL of 700 nm F-NDP or F-NDP suspended in de-ionized NV NVN (DI) water was loaded into 96-well polystyrene Fluores- 8472 submit your manuscript | www.dovepress.com Dovepress cence was scanned for all wells with excitations from 230 to 850 nm and emissions from 290 to 850 nm (Figure 1A) at 20 nm intervals. Data were processed in MATLAB 2015b (Mathworks, Natick, MA, USA). Background fluorescence was subtracted from empty wells without F-NDP, and the resulting net fluorescence value was log10-transformed for visualization.
Glass capillaries (40 mm length, 1 mm internal diameter; Thermo Fisher Scientific) were filled with equal volumes (30 μL) of suspensions of F-NDP at concentrations from 0.06 mg/mL and up to 4 mg/mL (1.8–120 μg total par- ticle mass) and sealed at each end by plasticine (Hasbro, Pawtucket, RI, USA). The NIR fluorescence intensity of the various suspensions in the capillaries was analyzed using an in vivo imaging system (IVIS; IVIS 50 Imaging System;