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Dual-energy computed tomography imaging of atherosclerotic plaques in a mouse model using a liposomal-iodine nanoparticle contrast agent - PubMed

  • ️Tue Jan 01 2013

Dual-energy computed tomography imaging of atherosclerotic plaques in a mouse model using a liposomal-iodine nanoparticle contrast agent

Rohan Bhavane et al. Circ Cardiovasc Imaging. 2013.

Abstract

Background: The accumulation of macrophages in inflamed atherosclerotic plaques has long been recognized. In an attempt to develop an imaging agent for detection of vulnerable plaques, we evaluated the feasibility of a liposomal-iodine nanoparticle contrast agent for computed tomography imaging of macrophage-rich atherosclerotic plaques in a mouse model.

Methods and results: Liposomal-iodine formulations varying in particle size and polyethylene glycol coating were fabricated and shown to stably encapsulate the iodine compound. In vitro uptake studies using optical and computed tomography imaging in the RAW 264.7 macrophage cell line identified the formulation that promoted maximal uptake. Dual-energy computed tomography imaging using this formulation in apolipoprotein E-deficient (ApoE(-/-)) mice (n=8) and control C57BL/6 mice (n=6) followed by spectral decomposition of the dual-energy images enabled imaging of the liposomes localized in the plaque. Imaging cytometry confirmed the presence of liposomes in the plaque and their colocalization with a small fraction (≈2%) of the macrophages in the plaque.

Conclusions: The results demonstrate the feasibility of imaging macrophage-rich atherosclerotic plaques using a liposomal-iodine nanoparticle contrast agent and dual-energy computed tomography.

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Figures

Figure 1
Figure 1

Effect of liposome size and incubation time on the uptake of rhodamine-labelled non-PEGylated liposomes (A) and PEGylated liposomes (B) by RAW 264.7 macrophages. Cell nucleus was stained with DAPI (blue color). Images were acquired at 40X magnification.

Figure 2
Figure 2

Quantitative analysis of uptake of rhodamine-labeled non-PEGylated liposomes (A) and PEGylated liposomes (B) by RAW 264.7 macrophages. A total of 100 cells were used for the analysis. CT number of iodine-containing macrophages (C, D). Macrophages were incubated with non-PEGylated liposomal-iodine (C) and PEGylated liposomal-iodine (D). Error bars represent standard deviations.

Figure 3
Figure 3

Effect of liposomal-iodine uptake on cell viability of RAW 264.7 macrophages. The macrophages were incubated with non-PEG liposomal-iodine (A) and PEGylated liposomal-iodine (B) for different incubation times and the cell viability assessed using the MTS assay. Error bars represent standard deviation. Untreated cells were used as controls and their viability numbers were averaged and normalized to 100%, and all other test cases normalized against this standard.

Figure 4
Figure 4

3D volume-rendered image and orthogonal slices demonstrating co-visualization of blood-pool (red) and calcified plaques (yellow regions) in an ApoE−/− mouse. Images were acquired within 2 hours post-administration of the liposomal contrast agent.

Figure 5
Figure 5

Representative grayscale (rows 1 and 3) and corresponding decomposed (rows 2 and 4) coronal images of an ApoE−/− and control C57BL/6 mouse before and after administration of liposomal-iodine contrast agent. Red denotes iodine and green denotes calcium. The white arrows indicate region of signal enhancement due to accumulation of liposomal-iodine in plaques.

Figure 6
Figure 6

Histological examination of plaque section showing localization of rhodamine in regions of macrophage infiltration. A: H&E Section showing plaque and vessel wall (tunica media and portions of the adventitia) (10x). B: Inset from A, enlarged. C: DAPI –FITC-rhodamine merged image showing blue: cell nuclei, red: rhodamine in liposomes, green: macrophages labeled by Mac-2 FITC. D: cropped image of C used in imaging cytometric analysis, with computer generated tesselation to approximate cell boundaries.

Figure 7
Figure 7

Imaging cytometric analysis of aorta sections from mice that received rhodamine-labeled liposomes (red dots). ApoE−/− mice with no liposomes (black dots), and background C57BL/6 animals with liposome treatment (blue dots) were used as controls. Note that the C57BL/6 animals show practically no rhodamine or FITC signal, indicating no macrophages and no liposomes in the aortic wall. Untreated ApoE−/− mice show significant FITC signal indicating a significant macrophage level. The corresponding rhodamine signal is attributed to tissue-background and establishes the baseline criterion. The treated ApoE−/− mice show a significant elevation in rhodamine levels for a fraction of the cells (upper right quadrant, ~2% of cells), suggesting that avid uptake of the rhodamine is limited to about 2% of the macrophages in the plaques that are imaged.

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