FeCo/graphite nanocrystals for multi-modality imaging of experimental vascular inflammation

Abstract

Background: FeCo/graphitic-carbon nanocrystals (FeCo/GC) are biocompatible, high-relaxivity, multi-functional nanoparticles. 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 µg 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.

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.

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.

Figure 3. Immunostaining of macrophages and FeCo/GC.

(A) Immunohistochemical staining showed macrophages (by anti-Mac3 antibody) infiltrating the neointima of the ligated left carotid artery. (B) Immunohistochemical staining for smooth muscle cells (by anti-SMA antibody). (C) Quantitative analysis of proportion of neointima composed of macrophages and smooth muscle cells. (D) Immunofluorescent staining demonstrated macrophages (green), FeCo/GC-Cy5.5 (red), nuclei (blue), and combined staining with strong colocalization (yellow), particularly near the luminal border (arrow). (E) Immunofluorescent staining of the macrophage-rich liver also demonstrated strong colocalization (yellow) of FeCo/GC and macrophages. (F) By contrast, the macrophage-poor heart tissue showed scant FeCo/GC uptake, with the few spots of uptake colocalizing with macrophages (arrows).