FeCo/GC Macrophage Imaging Figure 2. Serial in vivo MRI of carotid arteries with FeCo/GC. (A) The ligated left carotid artery (yellow arrow), as expected, was smaller than the non-ligated right carotid artery (red arrow) prior to FeCo/GC injection (Pre). After FeCo/GC injection, T2* signal loss of the ligated left carotid artery was seen at 48 and 72 hours. Luminal area reduction was not seen in the control (non-ligated) right carotid artery. (B) Quantitative analysis showed significant % reduction in lumen area of ligated left vs. non-ligated right carotid arteries. {p = 0.03 vs. Pre, *p = 0.03 vs. right carotid. doi:10.1371/journal.pone.0014523.g002 Vascular inflammation has been studied by a variety of imaging modalities, including optical, nuclear, x-ray computed tomogra- phy, and MRI [8–22]. Prior optical studies have used biolumi- nescence, magneto-optical particles, or enzymatically activated near-infrared agents [8,12,14,17,21]. MRI studies have primarily used ultra-small superparamagnetic iron-oxide agents for detecting macrophage infiltration in vascular lesions in both experimental and clinical studies [15–17,22] Gadolinium-based micelles and lipoproteins have also been used for macrophage MRI [9,19]. One study used a combined carbon nanotube/iron-oxide complex for macrophage cell imaging in vitro [20]. In addition to the advantageous MRI properties of FeCo/GC discussed above, FeCo/GC can be conjugated with targeting ligands or therapeutic drugs. We have also shown that the graphite shell provides high optical absorbance for photothermal therapy [23], while the FeCo component has the potential for magnetic hyperthermia [25]. Thus, FeCo/GC nanoparticles have a wide range of imaging, targeting, and therapeutic capabilities and warrant further study. While the carotid ligation model is advantageous in providing a discrete, macrophage-rich vascular lesion, it cannot replicate the complex, chronic plaques that develop in humans. Fluorescence imaging required in situ carotid exposure due to limitations in fluorescence tissue penetration. Fully noninvasive fluorescence imaging may be enabled with alternative techniques, such as fluorescence molecular tomography [8,14]. The MRI approach is likely made more challenging by using a carotid ligation model, as the ligation decreases both lumen size and flow. Aortic imaging in a transgenic hyperlipidemic mouse model is an alternative [8]. Finally, while preliminary studies have shown no acute or chronic toxicity of FeCo/GC [24], more extensive, large-animal toxicity studies are needed prior to clinical translation. In conclusion, the present study provides evidence that FeCo/ GC nanoparticles are promising multi-modality contrast agents for vascular macrophage imaging. Further studies of this multi- functional nanomaterial may enable improved imaging, and therapy, of vascular inflammation in patients. Materials and Methods Ethics statement This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Administrative Panel on Laboratory Animal Care at Stanford University (Assurance # A3213-01). All procedures were performed under isoflurane anesthesia, and all efforts were made to minimize suffering. Animals A macrophage-rich murine carotid ligation model was used [21,22]. Eight-week-old male FVB mice (n = 17) were fed high fat diet containing 40% kcal fat, 1.25% (by weight) cholesterol and 0.5% (by weight) sodium cholate (D12109, Research Diets, Inc. New Brunswick, NJ, USA) [26]. After 1 month on the diet, diabetes was induced by 5 daily intraperitoneal injections of streptozotocin (STZ, 40 mg/kg, Sigma-Aldrich, Saint Louis, MO, USA) dissolved in a citrate buffer (pH 4.5, Sigma-Aldrich, USA) PLoS ONE | www.plosone.org 3 January 2011 | Volume 6 | Issue 1 | e14523