Two-wavelength near-infrared fluorescence for the quantitation of drug antiplatelet effects in large animal model systems

ObjectiveIntraoperative imaging of intravascular thrombi is limited by the inability of visible light to penetrate thick-walled vessels. Near-infrared (NIR) light has relatively high tissue penetration and low autofluorescence and scatter, offering significant advantages. We hypothesized that the development of 700-nm NIR fluorophores for platelet labeling, in conjunction with existing 800-nm NIR fluorophores, would permit simultaneous and separable quantitation of intravascular thrombi and measurement of the antiplatelet effect of drugs.MethodsWe synthesized a series of lipophilic, cationic, polymethine indocyanine dyes (MHI-86, 94, 106, and 114) that emit at approximately 700 nm. Platelet uptake was optimized in vitro and the bioactivity and blood half-life of labeled platelets was characterized in vitro and in vivo. FeCl3-induced injury of the femoral arteries and intravascular thrombus formation was performed in 35-kg Yorkshire pigs. A combination of 700-nm and 800-nm NIR fluorophore-labeled platelets was used in conjunction with the fluorescence-assisted resection and exploration imaging system to image and quantify the antiplatelet effect of cilostazol and acetylsalicylic acid.ResultsMHI-114 was incorporated at nearly 4.1 × 106 molecules per platelet without affecting platelet function. When infused into pigs, the signal-to-background ratio of MHI-114-labeled platelets exhibited a blood half-life of 16.4 ± 2.2 (mean ± SEM; n = 3) minute and generated a signal-to-background ratio of 2.5 ± 0.5 (mean ± SEM; n = 3) at the site of thrombi. Using dual-NIR-labeled platelet populations, cilostazol and acetylsalicylic acid were found to cause a reduction in platelet incorporation into thrombi of 51 ± 2% and 10 ± 1% (mean ± SEM; n = 3), respectively, relative to vehicle-only treated control thrombi.ConclusionsNew platelet-avid 700-nm NIR fluorophores permit simultaneous two-wavelength NIR fluorescence imaging and quantitation of intravascular thrombi in intact vessels approaching the size of humans and can be used to study the antiplatelet effect of drugs.

Clinical RelevanceOccult thrombus formation during surgery leads to significant morbidity and mortality. We describe the synthesis of a novel family of 700-nm near-infrared fluorophores that concentrate into platelets and render them highly fluorescent while maintaining bioactivity. Using these platelets and a near-infrared imaging system, we can detect thrombi in thick-walled vessels intraoperatively with high signal-to-background ratio ≈ 2.5. The optical properties and blood half-life of these 700-nm fluorophores complement a previously described 800-nm platelet-avid fluorophore and enable the assessment of antiplatelet interventions intraoperatively.