FeCo/Graphite Nanocrystals for Multi-Modality Imaging of Experimental Vascular Inflammation Hisanori Kosuge1, Sarah P. Sherlock2, Toshiro Kitagawa 1 , Masahiro Terashima1, Joe¨ lle K. Barral3, Dwight G. Nishimura3, Hongjie Dai2, Michael V. McConnell1,3* 1 Division of Cardiovascular Medicine, Stanford University, Stanford, California, United States of America, 2 Department of Chemistry, Stanford University, Stanford, California, United States of America, 3 Electrical Engineering, Stanford University, Stanford, California, United States of America Abstract Background: FeCo/graphitic-carbon nanocrystals (FeCo/GC) are biocompatible, high-relaxivity, multi-functional nanopar- ticles. Macrophages represent important cellular imaging targets for assessing vascular inflammation. We evaluated FeCo/ GC for vascular macrophage uptake and imaging in vivo using fluorescence and MRI. Methods and Results: Hyperlipidemic and diabetic mice underwent carotid ligation to produce a macrophage-rich vascular lesion. In situ and ex vivo fluorescence imaging were performed at 48 hours after intravenous injection of FeCo/GC conjugated to Cy5.5 (n = 8, 8 nmol of Cy5.5/mouse). Significant fluorescence signal from FeCo/GC-Cy5.5 was present in the ligated left carotid arteries, but not in the control (non-ligated) right carotid arteries or sham-operated carotid arteries (p = 0.03 for ligated vs. non-ligated). Serial in vivo 3T MRI was performed at 48 and 72 hours after intravenous FeCo/GC (n = 6, 270 mg Fe/mouse). Significant T2* signal loss from FeCo/GC was seen in ligated left carotid arteries, not in non-ligated controls (p = 0.03). Immunofluorescence staining showed colocalization of FeCo/GC and macrophages in ligated carotid arteries. Conclusions: FeCo/GC accumulates in vascular macrophages in vivo, allowing fluorescence and MR imaging. This multi- functional high-relaxivity nanoparticle platform provides a promising approach for cellular imaging of vascular inflammation. Citation: Kosuge H, Sherlock SP, Kitagawa T, Terashima M, Barral JK, et al. (2011) FeCo/Graphite Nanocrystals for Multi-Modality Imaging of Experimental Vascular Inflammation. PLoS ONE 6(1): e14523. doi:10.1371/journal.pone.0014523 Editor: Stefan Kiechl, Innsbruck Medical University, Austria Received July 2, 2010; Accepted December 16, 2010; Published January 14, 2011 Copyright: 2011 Kosuge et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This study was supported by grants from the National Institutes of Health (R01-HL078678, nih.gov), the American Heart Association (0825272-F, heart.org), and Stanford Bio-X Interdisciplinary Initiatives Program (biox.stanford.edu), as well as MRI research support from GE Healthcare (gehealthcare.com). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: Drs. McConnell and Nishimura receive research support from GE Healthcare. Dr. McConnell is on a Scientific Advisory Board for Kowa, Inc. This does not alter the authors’ adherence to all the PLoS ONE policies on sharing data and materials. The other authors have declared that no competing interests exist. * E-mail: mcconnell@stanford.edu Introduction Inflammation is a major contributor to atherosclerosis, a leading cause of death worldwide [1–3]. Macrophages in the vessel wall have been associated with atherosclerotic plaque rupture and acute myocardial infarction [4–7]. Thus, imaging of vascular macrophages may be useful for characterizing plaque biological activity. Cellular and molecular imaging of atherosclerosis and vascular inflammation has made significant progress, but remains chal- lenging [8–22]. FeCo/graphitic-carbon nanocrystals (FeCo/GC) are composed of an iron-cobalt (FeCo) core with a graphitic- carbon shell [23]. We have shown that the FeCo core provides higher relaxivity for MRI compared to other commercially available contrast agents, while the graphite shell enables biocompatibility and high optical absorbance in the near-infrared region for potential thermal therapy [23]. We have also shown the potential of FeCo/GC for stem cell imaging in vitro as well as high- resolution vascular angiography in vivo [23,24], but in vivo cellular imaging of FeCo/GC has not been studied. The aim of the current study was to investigate FeCo/GC for in vivo macrophage uptake and imaging in a murine model of vascular inflammation. We show carotid artery macrophages take up FeCo/GC and can be imaged by in situ fluorescence and in vivo high-field MRI. Results Fluorescence imaging of carotid arteries after administration of FeCo/GC Both in situ and ex vivo fluorescence imaging at 48 hours showed high signal from the ligated left carotid arteries, confirming FeCo/ GC uptake in the vascular lesions (Figure 1A; yellow arrows). In contrast, there was no significant FeCo/GC accumulation seen in the non-ligated right carotid arteries (Figure 1A, red arrows). Furthermore, sham operated mice did not show significant signal in either left or right carotid arteries (Figure 1B). Quantitative analysis of both in situ and ex vivo signal intensities showed that the ligated left carotid arteries had significantly higher fluorescence than the control (non-ligated) right carotid arteries PLoS ONE | www.plosone.org 1 January 2011 | Volume 6 | Issue 1 | e14523