FeCo/GC Macrophage Imaging Figure 1. Fluorescence imaging of FeCo/GC in mouse carotid arteries. (A) Both in situ and ex vivo images at 48 hours after FeCo/GC-Cy5.5 injection showed enhanced fluorescence signal localized to the ligated left carotid artery (yellow arrows), but not to the contralateral non-ligated right carotid (red arrows). (B) Sham-operated mice showed no significant signal in either the left or right carotid arteries. (C) Quantitative analysis of both in situ and ex vivo fluorescence signal intensity showed significantly greater fluorescence from ligated left carotid arteries compared to non- ligated right carotid arteries. {p = 0.03 vs. right carotid, *p = 0.046 vs. sham left carotid. doi:10.1371/journal.pone.0014523.g001 (Figure 1C; in situ (left vs. right): 2.360.4 vs. 0.760.1, p = 0.03, ex vivo (left vs. right): 59.2617.2 vs. 0.360.3, p = 0.03). Also, the ligated left carotids had significantly higher fluorescence than sham left carotids (in situ (ligated vs. sham): 2.360.4 vs. 0.960.1, p = 0.046, ex vivo (ligated vs. sham): 59.2617.2 vs. 0.360.3, p = 0.046). In vivo MRI of carotid arteries after administration of FeCo/GC MRI prior to FeCo/GC administration (Figure 2A-Pre) showed that the ligated left carotid artery (yellow arrow), as expected, was smaller than the non-ligated right carotid artery (red arrow). Post FeCo/GC injection, serial MRI showed reduction in left carotid lumen area due to T2* signal loss from FeCo/GC accumulation at both 48 and 72 hours. In contrast, luminal area reduction due to T2* signal loss was not seen in the control (non-ligated) right carotid arteries (Figure 2A). The measured % reduction of carotid lumen area at 48 and 72 hours for the ligated left carotid arteries was significantly greater than for the non-ligated controls, indicating the accumulation of FeCo/GC in the carotid lesion (Figure 2B; 48 hours (left vs. right): 60.1610.7% vs. 16.969.9%, p = 0.03; 72 hours (left vs. right): 83.964.6% vs. 21.367.2%, p = 0.03). Furthermore, preliminary testing (n = 3) of an eight-fold lower FeCo/GC dose at higher field strength (7T) also showed T2*-induced lumen reduction in ligated left vs. right carotid arteries (28.065.7% vs. 25.5661.2%, p = 0.1). Histology Immunohistochemistry for macrophages demonstrated that there was substantial macrophage infiltration in the neointima of ligated left carotid arteries, accounting for 18.961.9% of the neointimal area (Figure 3A,C). There was also significant smooth muscle cell proliferation, accounting for 34.563.7% of the neointimal area (Figure 3B,C). Furthermore, carotid immunoflu- orescence staining showed that FeCo/GC colocalized with macrophages (Figure 3D). The specificity of FeCo/GC for macrophages was further confirmed by immunofluorescence staining showing abundant uptake in the macrophage-rich liver and scant uptake in the macrophage-poor heart (Figure 3E,F). Discussion In an experimental model of vascular inflammation, we have shown that a FeCo-based, graphite-coated nanoparticle can accumulate in vivo in vascular macrophages and can be imaged by fluorescence and MRI. To the best of our knowledge, this study is the first to show the use of FeCo or graphite-coated nanoparticles for in vivo macrophage imaging. We have previously reported that FeCo/GC exhibited higher T1 and T2 relaxivities than commer- cially available iron-oxide- and gadolinium-based contrast agents, with cell hyperthermia capabilities, and without evidence of in vitro or in vivo toxicity [23,24]. Furthermore, we have shown that FeCo/ GC has a long circulation time and can allow very high-resolution MR angiography (down to 100 mm) [24]. By demonstrating in vivo vascular macrophage uptake and imaging, we show that the potent MRI characteristics and potential therapeutic capabilities of FeCo/ GC can be applied more broadly to atherosclerosis diagnosis and therapy – from high-resolution angiography to detection of vascular inflammation to macrophage thermal ablation. PLoS ONE | www.plosone.org 2 January 2011 | Volume 6 | Issue 1 | e14523