A Non-Invasive Photoacoustic Imaging with Erythrocyte Derived Optical Nanoparticles to Detect CAD in In Vivo Mice (2019)

230 Objectives: Coronary artery disease (CAD) claims more lives each year than all forms of cancer and chronic lower respiratory diseases combined. However, not all CAD show up in a CT scan and MRI’s inability to accurately estimate plaque burden and their degree of stenosis makes predicting heart attack very difficult. Thus, catheterization angiography, an invasive method, is the clinical standard for CAD diagnosis. Here, we used a novel molecular contrast, near-infrared (NIR) erythrocyte-mimicking transducers(NETs), in combination with a photoacoustic imaging system for non-invasive detection of CAD. Methods: Ten weeks old male C57BL/6 mice (n=9) were used in this study. They underwent left anterior descending (LAD) coronary artery ligation to mimic vulnerable atherosclerotic plaques and their rupture leading to myocardial infarction. These mice were healed for one week before 150 µL of NETs suspension in phosphate buffer saline (90 nm diameter, 20 µM) were IV injected via tail vein one hour prior to in vivo imaging with VevoSonic 2100/LAZR and IVIS-200 for photoacoustic and fluorescence imaging, respectively. NETs were derived from mice hemoglobin-depleted erythrocyte ghosts doped with Indocyanine Green (ICG). The NETs were characterized for their diameter distribution and absorption before IV injection. The LAD mice models were exposed to laser light to excite the NETs at 800 nm and 650 nm for photoacoustic and fluorescence imaging, respectively. The experiments were done under anesthesia according to an approved protocol. For further verification, animals were euthanized, and chests were cut open for imaging the organs, specially the heart with IVIS-200, followed by harvesting tissues for histochemical analysis. Results: Pre-characterization showed that NETs had peak diameter of 89.92±3.35 nm (Fig. 1a) with emission peaked at 700 nm for 650 nm excitation (Fig. 1b). Absorption of NETs over 600-800 nm originates from the presence of ICG (Fig.1c). The radiant efficiency was also measured for NETs, fabricated using 20 µM and 1 mM ICG, in two separate vials using IVIS-200 prior to the in vivo experiments (Fig. 2a). We found a radiant efficiency of 5.43×107 (p/sec/cm2/sr)/(µM/cm2)for 20 µM NETs and none for 1 mM NETs, suggesting aggregation-induced quenching. The schematic diagram and photograph of mouse heart illustrated the ligation location at LAD coronary artery (Figs. 2b-c). In the area above the ligation (Fig. 2d, yellow arrow), we observed 256-fold (±13.73) higher (Fig. 2e) photoacoustic signal outlining the accumulation of NETs within the macrophages. Histological images illustrated infarct region (double dotted line, Fig. 2c) with tissue necrosis below the ligation of the left ventricle (Figs. 2f-h). Fluorescence signals were detected in area of NETs accumulation (LAD coronary, thymus, and liver) (Fig. 3a). Similar signals were observed when the chest was cut open (Fig. 3b). Statistical data showed that in vivo heart (1.76×107±1.85×106) had 1.32-fold and 1.96-fold higher fluorescence signals compared to thymus (1.33×107±7.69×105) and liver (8.99×106±8.05×105), respectively (Fig. 3c). After opening the chest (Fig. 3d), mice heart, thymus, and liver followed the same trend with 2-fold higher signal (Fig. 3c) than in vivo. The decreased signal from in vivo image is due to high light scattering skin and fat layers. For further verification, we observed scarring, granulation, and fibrosis in the area with infarct in the left ventricle (red arrow) and septum (Figs. 3e-f); however, viable tissue was observed in the right ventricle (blue arrow). Liver demonstrated normal portal tract (Fig. 3g), with no parenchymal necrosis, inflammation, fibrosis, or other pathologic changes, and hence indicative of the biocompatibility of NETs. Conclusions: Non-invasively detecting atherosclerotic plaques and stenosis using NETs may drive a paradigm shift in the diagnosis of CAD, enabling clinical detection and improving risk assessment.