Background

[PubMed]

Macrophages are key cellular mediators of inflammation in atheroma and participate in all stages in evolving atherosclerotic plaques (1, 2). The atheroma lesion is normally initialized by recruiting monocytes in inflamed intima. Monocytes mature into macrophages under the in situ stimulation of overexpressed macrophage colony-stimulating factor. As cholesteryl esters gradually accumulate in cytoplasm, macrophages are converted into foam cells at the early stage of atheroma. The accumulation of foam cells leads to the formation of fatty streaks and the deposition of fibrous tissues, which indicates the progression of atheroma into an intermediate stage. Fibrous caps are formed on the surface of a lipid-rich core and result in vulnerable atherosclerotic plaque as the smooth muscle cells synthesize bulk extracellular matrix. Finally, the rupture of the atherosclerotic plaques and the calcification of vessel walls progressively occlude the lumen. The vulnerable plaques are considered as a “high-risk” stage, which contain much higher levels of macrophages than that in any other stage (3). Thus, measuring macrophage density in plaques becomes an alternative approach to evaluate the vulnerable plaques (4). Atherosclerotic plaques can be generated in rabbits by balloon injury in the aortas followed by hypercholesterolemic diet (4). This animal model provides high levels of macrophages with sizes similar to those found in the human coronary atherosclerotic plaques, suitable for examining the effects of antiatherosclerotic drugs on atherosclerotic plaque size and composition.

Multi-detector row computed tomography (MDCT) has been used as a robust imaging modality for non-invasive assessment of coronary arteries (5, 6). In particular, the development of 64-slice MDCT (64-MDCT) provides fast gantry rotation time (0.33 s) and small imaging voxel size (0.4 mm³) (7), which allows for reliable assessment of atherosclerotic plaques (8). Because their CT attenuation differences are much larger than 30 HU (a criteria in delineation of tissues), calcified plaques (391–419 HU), fibrous plaques (70–104 HU), and lipid-rich plaques (soft plaques, 14–49 HU) can be directly differentiated with the use of MDCT (8). To measure the macrophage density in the plaques requires a macrophage-specified CT contrast agent (4). Iodinated compounds have been used as CT contrast agents for many years. Small molecules such as iopamidol can attenuate the X-ray density up to 30 HU at a dosage of 1 mg iodine/g tissue (9), but they are not specific imaging agents for macrophages. Contrast agents can be incorporated into macrophages through phagocytosis, a special macrophage uptake process. Sparingly soluble crystals, supramolecular aggregates, large micelles, or emulsions of iodinated lipids are suitable for this purpose. All of these contrast agents should have a sufficiently large size to be marked (“opsonized”) by circulating proteins (“opsonins”), then followed by phagocytosis (9). Ethyl-3,5-bis(acetylamino)-2,4,6-triiodobenzoate nanoparticles (N1177) is an emulsified suspension that is composed of crystalline iodinated particles dispersed with surfactant (4). The iodinated particles, ethyl-3,5-bis(acetylamino)-2,4,6-triiodobenzoate, are an esterified derivative of the X-ray contrast agent diatrizoic acid with a very low aqueous solubility (~2 μg/ml). Two biocompatible surfactants, a polyoxyethylene-polyoxypropylene block co-polymer (poloxamer 338) and a polyethylene glycol, are added to stabilize the particles and prevent aggregation.

Synthesis

[PubMed]

Hyafil et al. reported the detailed synthesis of N1177 (4). Initially, 6-ethoxy-6-oxohexy-3,4-bis(acetylamino)-2,4-triiodobenzoate (C₁₉H₂₃I₃N₂O₆), also called iodinated aroyloxy ester, was obtained by condensation of 6-bromohexanoate with sodium diatrizoate in dimethyl formamide followed by precipitation in dimethyl sulfoxide. The produced iodinated aroyloxy ester was milled with poloxamer 338 in the presence of inert beads to generate a nanoparticulate suspension, which was separated from the beads by membrane filtration with membrane pore diameter of 2–5 μm. Then, polyethylene glycol 1450 and tromethamine were added to the obtained suspension to stabilize the particles and to maintain a neutral pH. The final product (N1177) was composed of 150 mg/ml of iodinated aroyloxy ester, 30 mg/ml of poloxamer 338, 150 mg/ml of polyethylene glycol 1450, and 0.36 mg of tromethamine, and remained stable for ~8 months. The size of N1177 particle ranged from 153 nm to 408 nm with a mean value of 259 nm. Transmission electron microscopy of N1177 demonstrated that cores formed by electron-dense granules were covered by molecular polymers, and iodine was present in the electron-dense granules.

In Vitro Studies: Testing in Cells and Tissues

[PubMed]

Hyafil et al. examined the in vitro uptake of N1177 in mouse macrophages (4). After incubation with N1177 (n = 6 mice) or iopamidol (n = 6 mice) as control for 1 h, numerous dark granules were observed in the cytoplasm of macrophages that were incubated with N1177. The cell-internalized iodine was quantified with inductively coupled plasma mass spectrometry, which revealed a substantially higher uptake of N11777 by macrophages as compared to uptake by iopamidol (4,920 ± 1,019 versus 56 ± 11 μg iodine/g cell pellets).

Animal Studies

Human Studies

[PubMed]

No publication is currently available.

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