Abundant, active inflammatory cells are a hallmark of high-risk atherosclerotic plaques¹. High-density lipoprotein (HDL) is a natural nanoparticle composed of phospholipids, cholesterol and apolipoprotein A-I (APOA1), which has been shown to have atheroprotective properties. It is believed that these properties are mainly mediated by cholesterol efflux from macrophages in plaques and its transport to the liver for excretion². The recent development of combined PET/MRI scanners and new advances in radiolabeling technology gives the opportunity to investigate the behavior of HDL nanoparticles in vivo. Using a unique set-up combining PET/CT and PET/MRI, we non-invasively assessed the pharmacokinetics, distribution, metabolism and turnover of ⁸⁹Zr-HDL^3-4 in a rabbit model of atherosclerosis. Nanoparticles were prepared by conjugation of DFO-p-NCS to APOA1, and then radiolabeled with ⁸⁹Zr^3-4. Atherosclerosis was induced in male New Zealand White rabbits using a combination of high-fat diet and double balloon injury of the abdominal aorta (n=13)⁵. Non-atherosclerotic rabbits were used as controls (n=8). Rabbits were injected with ⁸⁹Zr-HDL and Cy5.5-HDL. For pharmacokinetics, blood was sampled via the ear vein at 30 min, 24, 48, 72 and 120 h post injection and its radioactivity content measured on an automatic gamma counter. Rabbits were imaged on a Siemens mMR 3T PET/MRI scanner and on a Siemens mCT Biograph PET/CT scanner. PET/MRI and PET/CT scans were acquired within the first hour and then at 24, 48, 72 and 120 h after ⁸⁹Zr-HDL administration. For PET/MRI, after scout scans, the PET scan was initiated and co-acquired with a radial VIBE MR sequence, used to analyze organ nanoparticle uptake. Time-of-flight (TOF) non-contrast enhanced angiography was also performed for localization of arterial anatomical landmarks. Animals were sacrificed after the last imaging session. For ex vivo validation, tissue samples were harvested for radioactivity counting and imaged using near infrared fluorescence (NIRF) to quantify Cy5.5-HDL uptake. To determine the distribution of ⁸⁹Zr-HDL, digital autoradiography was also performed. Statistical analyses were conducted using non-parametric unpaired Mann-Whitney tests (p<0.05 considered significant).

Pharmacokinetics. Blood time-activity curves are shown in figure 1A. Radioactivity clearance was 1.13 and 1.05 days in control and atherosclerotic animals, respectively. Radioactivity distribution in selected tissues was measured at 5 days post injection (p.i.) (figure 1B). Kidneys showed the highest uptake values, higher for atherosclerotic animals compared to controls, although these differences were not statistically significant. Spleen and liver uptakes were around 0.1 %ID/g.

Imaging. Representative PET/CT fusion images of the abdominal region at three different time points can be seen in figure 1D. Figure 1E shows representative PET/MRI fusion images of the same animals shown in figure 1D. PET images obtained with both scanners were almost identical and a strong correlation was found between the SUVs measured from both systems (figure 1C). Time-activity curves based on SUVs obtained from PET/CT and PET/MRI images are shown in figure 2. Initially, within the first hour after intravenous administration, images were dominated by a strong blood pool signal (figures 1D and 1E), as well as high signal from liver, spleen and kidneys. At later time points, images showed very high radioactivity accumulation in kidneys, which was significantly higher in atherosclerotic animals at all time points investigated (figure 2). Bone radioactivity was also detected over the five-day period (SUVs around 5 g/mL). Ex vivo analysis proved that this was mostly due to bone marrow uptake, as opposed to mineral bone. Bone marrow uptake was higher in animals with atherosclerosis (0.17 ± 0.03 vs. 0.13 ± 0.01 %ID/g for controls). PET-measured SUVs were in good agreement with SUVs determined by ex vivo gamma counting (R²=0.95). Aortic radioactivity was also measured at 1, 2, 3 and 5 days. Increased accumulation of activity was found at day 5 p.i. in atherosclerotic aortas compared to controls by PET/CT imaging (figure 3A).

Plaque targeting. Radioactivity concentration was significantly higher in atherosclerotic aortas (P = 0.03) as determined by gamma counting at 5 days p.i. (figure 3A). Autoradiography of explanted aortas revealed a patchy distribution of radioactivity in atherosclerotic aortas, showing preferential accumulation in lesions (figure 3B). Analysis by NIRF imaging showed increased accumulation of Cy5.5-HDL in atherosclerotic aortas (figure 3B). A good correlation was found between total radiant efficiency and radioactivity concentration (figures 3C).

Here we demonstrated that it is possible to investigate the in vivo behavior of ⁸⁹Zr-HDL's by PET/CT and PET/MRI. This multimodal imaging approach allows the non-invasive evaluation of HDL’s trafficking, distribution, metabolism and turnover. Ultimately, this non-invasive imaging tool may be useful to identify patients amenable to HDL therapy and subsequent treatment monitoring in a theranostic fashion.