Targeted iron oxide particles for in vivo magnetic resonance detection of atherosclerotic lesions with antibodies directed to oxidation-specific epitopes

Abstract

Objectives: The aim of this study was to determine whether iron oxide particles targeted to oxidation-specific epitopes image atherosclerotic lesions.

Background: Oxidized low-density lipoprotein plays a major role in atherosclerotic plaque progression and destabilization. Prior studies indicate that gadolinium micelles labeled with oxidation-specific antibodies allow for in vivo detection of vulnerable plaques with magnetic resonance imaging (MRI). However, issues related to biotransformation/retention of gadolinium might limit clinical translation. Iron oxides are recognized as safe and effective contrast agents for MRI. Because the efficacy of passively targeted iron particles remains variable, it was hypothesized that iron particles targeted to oxidation-specific epitopes might increase the utility of this platform.

Methods: Lipid-coated ultra-small superparamagnetic iron particles (LUSPIOs) (<20 nm) and superparamagnetic iron particles (<40 nm) were conjugated with antibodies targeted to either malondialdehyde-lysine or oxidized phospholipid epitopes. All formulations were characterized, and their in vivo efficacy evaluated in apolipoprotein E deficient mice 24 h after bolus administration of a 3.9-mg Fe/kg dose with MRI. In vivo imaging data were correlated with the presence of oxidation-specific epitopes with immunohistochemistry.

Results: MRI of atherosclerotic lesions, as manifested by signal loss, was observed after administration of targeted LUSPIOs. Immunohistochemistry confirmed the presence of malondialdehyde-epitopes and iron particles. Limited signal attenuation was observed for untargeted LUSPIOs. Additionally, no significant arterial wall uptake was observed for targeted or untargeted lipid-coated superparamagnetic iron oxide particles, due to their limited ability to penetrate the vessel wall.

Conclusions: This study demonstrates that LUSPIOs targeted to oxidation-specific epitopes image atherosclerotic lesions and suggests a clinically translatable platform for the detection of atherosclerotic plaque.

Copyright © 2011 American College of Cardiology Foundation. Published by Elsevier Inc. All rights reserved.

Figures

Figure 1. Diagram

Schematic diagram of the lipid-coated iron oxide particles formed.

Figure 2. TEM

Transmission electron microscopy (TEM) and negative TEM of untargeted lipid-coated ultrasmall superparamagnetic iron oxide particle (LUSPIO) and lipid-coated superparamagnetic iron oxide particle (LSPIO) formations. The red asterisks indicate the iron core. The red arrow points to the lipid layer associated with the particles.

Figure 3. Nuclear Magnetic Relaxation Dispersion Profiles

The longitudinal (r1) and transverse (r2) nuclear magnetic relaxation dispersion profiles of untargeted LSPIOs and LUSPIOs. Abbreviations as in Figure 2.

Figure 4. Macrophage Uptake of Iron Oxide Particles

In vitro J774A.1 macrophage uptake of untargeted lipid-coated iron particles (negative control), targeted lipid-coated iron particles, and Feridex (Berlex, Inc.) (positive control). All cells were incubated with 1 mmol/l Fe for 24 h. Both Perl’s staining for iron oxide uptake and confocal microscopy (rhodamine-labeled LUSPIOs/LSPIOs) were performed. In addition, iron oxide particle uptake was determined with inductively coupled plasma mass spectrometry and reported in pg Fe/cell. Abbreviations as in Figure 2.

Figure 5. LUSPIO MRI of ApoE−/− Mice

(A) Representative in vivo LUSPIO magnetic resonance images obtained before (PRE) and 24 h after (POST) administration of a 3.9-mg Fe/kg dose in apolipoprotein E deficient (apoE−/−) mice. The yellow arrows indicate the position of the lumen associated with the abdominal aorta. All gradient echo (GRE) images shown were obtained with an echo time of 7 ms. Corresponding white marker gradient echo acquisition for superparamagnetic particles with positive contrast (GRASP) images are also shown. The apoE−/− mice were administered free excess MDA2 antibody at the time of MDA2-labeled LUSPIO injection, for the competitive inhibition results. Matched histology sections show iron deposition (blue) within foam cells associated with the arterial wall. Relative percentage change in the R2* values associated with the arterial wall after administration of the LUSPIO formulations are also shown (B). The error bars reflect the SD associated with mean value obtained for mice receiving untargeted LUSPIOs and antibody-labeled LUSPIOs. MRI = magnetic resonance imaging; other abbreviations as in Figures 2 and 4.

Figure 6. Ultrasmall Iron Oxide Particle MRI of ApoE−/− Mice

Representative in vivo LSPIO magnetic resonance images obtained before and 24 h after administration of a 3.9-mg Fe/kg dose in apoE−/− mice. Only the untargeted and E06 formulations are shown in A. The yellow arrows indicate the position of the lumen associated with the abdominal aorta. The GRE images shown were obtained with an echo time of 7 ms. Corresponding white marker GRASP images are also shown. Matched histology sections show limited iron deposition (blue) within foam cells associated with the arterial wall. Relative percentage change in the R2* values associated with the arterial wall after administration of the LUSPIO formulations are also shown (B). The error bars reflect the SD associated with mean value obtained for mice receiving untargeted and antibody-labeled LSPIOs. Abbreviations as in Figures 2, 4, and 5.

Figure 7. Immunohistochemistry

Immunohistochemistry (A) and confocal microscopy (B) of MDA2-labeled LUSPIOs in the arterial wall of apoE−/− and wild-type mice. The arterial wall was stained for MDA-lysine (MDA3 staining), macrophages (Mac3), and iron oxide (Perl’s) deposition. Confocal microscopy with a multi-channel laser so that colocalization between the cell nuclei (blue), macrophages (green), and MDA2-labeled LUSPIOs (red) could be performed. Abbreviations as in Figures 2, 4, and 5.