Journal of the American College of Cardiology
Volume 59, Issue 6, February 2012 DOI: 10.1016/j.jacc.2011.10.881
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Pre-Clinical Research

In Vivo Detection of Oxidation-Specific Epitopes in Atherosclerotic Lesions Using Biocompatible Manganese Molecular Magnetic Imaging Probes

Karen C. Briley-Saebo, Tuyen Hoang Nguyen, Alexander M. Saeboe, Young-Seok Cho, Sung Kee Ryu, Eugenia Volkava, Stephen Dickson, Gregor Leibundgut, Philipp Weisner, Simone Green, Florence Casanada, Yury I. Miller, Walter Shaw, Joseph L. Witztum, Zahi A. Fayad and Sotirios Tsimikas

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vol. 59 no. 6 616-626
DOI: 
https://doi.org/10.1016/j.jacc.2011.10.881
Published By: 
Journal of the American College of Cardiology
Print ISSN: 
0735-1097
Online ISSN: 
1558-3597
History: 
  • Received June 30, 2011
  • Revision received October 31, 2011
  • Accepted October 31, 2011
  • Published online February 7, 2012.
Copyright & Usage: 
American College of Cardiology Foundation

Author Information

  1. Karen C. Briley-Saebo, PhD⁎,†,⁎ (saebo{at}mountsinai.org),
  2. Tuyen Hoang Nguyen⁎,
  3. Alexander M. Saeboe⁎,
  4. Young-Seok Cho, MD§,,
  5. Sung Kee Ryu, MD, PhD§,#,
  6. Eugenia Volkava⁎,
  7. Stephen Dickson, MS⁎,
  8. Gregor Leibundgut, MD§∥,
  9. Philipp Weisner, MD∥,
  10. Simone Green, BS§,
  11. Florence Casanada, BS§,
  12. Yury I. Miller, MD, PhD∥,
  13. Walter Shaw, PhD⁎⁎,
  14. Joseph L. Witztum, MD∥,
  15. Zahi A. Fayad, PhD⁎,‡ and
  16. Sotirios Tsimikas, MD§,⁎ (stsimikas{at}ucsd.edu)
  1. ⁎
  2. †
  3. ‡
  4. §
  5. ∥
  7. #
  8. ⁎⁎
  1. ↵⁎Reprint requests and correspondence
    : Dr. Sotirios Tsimikas, University of California San Diego, Cardiology, 9500 Gilman Drive, BSB 1080, La Jolla, California 92093-0682
  2. ↵⁎Dr. Karen C. Briley-Saebo, Imaging Science Laboratory, Department of Radiology, Mount Sinai School of Medicine, One Gustave Levy Place, New York, New York, 10029

Abstract

Objectives This study sought to evaluate the in vivo magnetic resonance imaging (MRI) efficacy of manganese [Mn(II)] molecular imaging probes targeted to oxidation-specific epitopes (OSE).

Background OSE are critical in the initiation, progression, and destabilization of atherosclerotic plaques. Gadolinium [Gd(III)]-based MRI agents can be associated with systemic toxicity. Mn is an endogenous, biocompatible, paramagnetic metal ion that has poor MR efficacy when chelated, but strong efficacy when released within cells.

Methods Multimodal Mn micelles were generated to contain rhodamine for confocal microscopy and conjugated with either the murine monoclonal IgG antibody MDA2 targeted to malondialdehyde (MDA)-lysine epitopes or the human single-chain Fv antibody fragment IK17 targeted to MDA-like epitopes (“targeted micelles”). Micelle formulations were characterized in vitro and in vivo, and their MR efficacy (9.4-T) evaluated in apolipoprotein-deficient (apoE−/−) and low-density lipoprotein receptor negative (LDLR−/−) mice (0.05 mmol Mn/kg dose) (total of 120 mice for all experiments). In vivo competitive inhibition studies were performed to evaluate target specificity. Untargeted, MDA2-Gd, and IK17-Gd micelles (0.075 mmol Gd/kg) were included as controls.

Results In vitro studies demonstrated that targeted Mn micelles accumulate in macrophages when pre-exposed to MDA-LDL with ∼10× increase in longitudinal relativity. Following intravenous injection, strong MR signal enhancement was observed 48 to 72 h after administration of targeted Mn micelles, with colocalization within intraplaque macrophages. Co-injection of free MDA2 with the MDA2-Mn micelles resulted in full suppression of MR signal in the arterial wall, confirming target specificity. Similar MR efficacy was noted in apoE−/− and LDLR−/− mice with aortic atherosclerosis. No significant differences in MR efficacy were noted between targeted Mn and Gd micelles.

Conclusions This study demonstrates that biocompatible multimodal Mn-based molecular imaging probes detect OSE within atherosclerotic plaques and may facilitate clinical translation of noninvasive imaging of human atherosclerosis.

Key Words

Oxidized low-density lipoproteins (OxLDL) have been identified as one of the main factors in the initiation, progression, and destabilization of atherosclerotic plaques (1–4). The presence of oxidation-specific epitopes (OSE) within the arterial wall initiates the recruitment and activation of monocytes, thereby promoting macrophage accumulation. Once activated, intraplaque macrophages take up OxLDL in an unregulated fashion, forming foam cells that are retained in the arterial wall. Apoptosis and necrosis of foam cells amplifies chronic inflammatory responses and eventually leads to plaque rupture.

Since OxLDL is immunogenic, murine and human monoclonal antibodies that bind unique OSE can be generated and have been identified, characterized, and purified and shown to bind to murine and human atherosclerotic plaques. Recently, we have reported the use of oxidation-specific antibody (MDA2, E06, and IK17)-labeled gadolinium (Gd) micelles and iron oxide nanoparticles for the in vivo detection of OSE in atherosclerotic lesions of apoE−/− mice using magnetic resonance imaging (MRI) (5,6) and showed that these nanoparticles accumulate within intraplaque macrophages. In vitro cell studies strongly suggested that uptake is specific and receptor mediated, and relies upon interaction of the targeted nanoparticle with endogenous OxLDL. Once taken up by macrophages, the entire lipid nanoparticle is metabolized within intracellular vesicles (lysosomes/endosomes). Although chelated Gd is renally excreted (>99.9%) bioretention and in vivo biotransformation of Gd(III) micelles was observed due to their prolonged half-life. This may lead to systemic toxicity in renally impaired patients (7–9).

Manganese (Mn(II)) is an endogenous paramagnetic metal that has been used in other Food and Drug Administration-approved contrast agents for hepatocyte targeting and MR liver indications (10,11). Although biocompatible, Mn is typically not used in MR contrast agent development due to its low magnetic moment that limits MR signal enhancement relative to Gd. However, studies have indicated that if Mn is delivered into a cell, interaction with intracellular components and metalloproteinase results in significant (>20-fold) increases in MR efficacy (Fig. 1) (12–14). On the basis of prior studies with targeted Gd micelles, we hypothesized that it may be possible to produce Mn probes that exhibit limited MR efficacy in the vascular phase in which the Mn remains completely chelated but high MR efficacy once Mn is released within intraplaque macrophages and foam cells. The intracellular release of Mn would enhance the MR signal by increasing the number of water exchange sites (q ≫ 0) and by decreasing the molecular tumbling rates due to interaction with macromolecules and cell membranes. Chelation in the vascular phase has 2 significant effects: 1) it reduces potential cardiotoxicity associated with bolus injection of free Mn (MnCl2); and 2) background signal from blood is nominal since there are no inner sphere water exchange sites available (q = 0) if diethylenetriamine pentaacetic acid (DTPA) is used as the chelating agent. The antibody associated with the surface of the particle allows for binding of the micelle to endogenous OSE found in circulation and/or within the arterial wall (5,6). The Mn micelle-Ab-OxLDL complex is then taken up by intraplaque foam cells/macrophages, as shown with Gd micelles. This degradation would result in the intracellular release of the Mn metal ions that are then either taken up by metalloproteinase or interact with the cell membrane.

Figure 1
Figure 1

Diagram of the Efficacy of OSETargeted Mn Micelles in the Vascular Phase

Mn is chelated to DTPA. Mn is released with the endosomes/lysosomes after uptake into intraplaque macrophage/foam cells (black circle). Due to intracellular release of Mn and subsequent interaction with intracellular components, the magnetic resonance (MR) signal is greatly enhanced. The r1 values are derived from Table 2. OSE = oxidation-specific epitope.

The aim of the current study was, therefore, to synthesize, characterize, and evaluate the MR efficacy of targeted Mn molecular imaging probes to OSE in murine models of atherosclerosis and to compare their imaging properties with previously described Gd targeted probes (5).

Materials and Methods

Antibodies and micelle synthesis

MDA2 is a murine immunoglobulin G (IgG) monoclonal antibody that binds malondialdehyde (MDA)-lysine epitopes present on OxLDL (15,16). IK17 is a human single-chain Fv fragment that recognizes an MDA-related epitope expressed on both MDA-LDL and copper-oxidized LDL. The following micelle formulations were prepared: untargeted Mn and Gd micelles, MDA2-Mn and Gd micelles, and single-chain Fv fragment IK17-Mn micelles.

Characterization

All micelle formulations were characterized with respect to particle size, zeta potential, Mn or Gd content, in vitro MR efficacy, vascular stability, and uptake by J447A.1 murine macrophages. Relaxometry was used to evaluate transmetallation of the Mn and Gd from untargeted formulations to other proteins in human plasma.

Animal models

All mice used in the current study are summarized in Table 1.

View this table:
Table 1

Overview of All In Vivo Studies

Neurotoxicity

Since studies have shown that high repeat intravenous administration of MnCl2 may lead to neurotoxicity, the uptake of the polyethylene glycol (PEG) micelles was evaluated in murine models of Alzheimer's disease (17). This model was chosen because studies have shown significant OSE deposition in the lesions of these mice (18). Due to the high endogenous concentration of Mn, dysprosium (Dy) micelles were prepared as described in the previous text for Mn and Gd micelles. The DyDTPA-bis(stearyl-amid) was provided by Avanti Polar Lipids, Alabaster, Alabama). It should be noted that due to the high electronic relaxation rate and susceptibility of Dy, uptake of Dy into tissue results in MR signal loss (19). Mice were injected with 0.075 mmol Dy/kg of either untargeted (n = 3) or MDA2-labeled (n = 3) Dy micelles. MRI was performed prior to injection and over a 72-h time interval post-injection. Immediately following the last scan, mice were sacrificed, saline perfused, and the brain excised, cleaned, and weighed. The concentration of Dy (per gram wet weight) was then determined using inductively coupled plasma mass spectroscopy (ICP-MS).

In vivo MRI

All in vivo MRI was performed at 9.4-T (400 MHz, Bruker Instruments, Billerica, Massachusetts), using previously reported methodology (5,6). In short, all animals underwent a pre-injection MR scan within 24 h prior to the administration of the various micelle formulations. MRI was performed over a 1-week time interval after tail vein injection of either a 0.050 mmol Mn/kg dose or a 0.075 mmol Gd/kg dose of micelles. MRI of the abdominal aorta was performed using a T1-weighted black blood spin-echo sequence (TR/TE/flip = 800 ms/8.6 ms/30°, number of excitations = 16, field of view = 2.6 cm × 2.6 cm) with a microscale in-plane resolution of 0.098 mm2. In order to quantitatively evaluate the MR data, signal intensity (SI) measurements were obtained using regions of interest within the aortic wall on slices (n > 3) exhibiting signal modulation post-contrast using ImageJ software (National Institutes of Health, Bethesda, Maryland). SI measurements of adjacent muscle and the standard deviation associated with noise were also obtained for each slice. The percent normalized enhancement (%NENH), relative to muscle, was then determined for the aortic vessel wall and liver according to established methods (5). The %NENH values reflect the percent relative change in the contrast-to-noise ratios as a function of time post-injection. For neuroimaging, T2-weighted spin-echo sequences were used (TR/TE = 125 ms/18 ms, number of excitations = 8, field of view = 2.6 cm × 2.6 cm, 12 slices). The anatomy of the brain was used to match the images pre- and post-injection. The SI measurements were obtained using regions of interest within the cerebellum, cerebrum, and locations exhibiting lesion deposition (slices: n > 2). The %NENH, relative to muscle, was then determined.

Confocal microscopy

To evaluate J774A.1 macrophage uptake or colocalization of the rhodamine-labeled micelles within the arterial wall, confocal microscopy was performed as reported previously (5).

In vivo competitive inhibition studies

The specificity of the MDA2-Mn micelles for MDA-lysine epitopes within the vessel wall of apoE−/− mice was evaluated using in vivo competitive inhibition, as previously described (5,6).

Immunohistochemistry

MDA epitopes detected using purified guinea pig antiserum MDA3 (1:500 dilution), as described previously (6,15).

The study materials and methods are described in greater detail in the Online Appendix.

Results

Physical properties, relaxivities, and half-life of the various micelle formulations

The micelle formulations were 10 to 22 nm in size (Table 2), similar to other PEG-DSPE–based micelles (5,20). The smaller hydrated particle size observed for Mn micelles relative to Gd was likely due to limited water coordination associated with the q = 0 Mn formulations. The addition of the antibodies resulted in an increase in the mean hydrated particle size of all formulations tested. Due to the higher magnetic moment, as well as the ability to coordinate water protons within the inner sphere (q = 1 for GdDTPA), all Gd formulations exhibited significantly greater r1 values in HEPES buffer relative to equivalent Mn micelles (21). The r1 values observed for the Mn micelles were, however, higher than those expected for q = 0 complexes (21). The elevated r1 values are likely caused by an added second sphere contribution due to the presence of uncoordinated carboxylic groups. All antibody-labeled micelles exhibited increased r1 values when analyzed in apoE−/− mouse blood, relative to values measured in wild-type mouse blood (Table 2). The intracellular r1 values obtained for MDA2-Mn micelles (45.5 ± 5 s−1nM−1), which were pre-incubated with MDA-LDL in J744A.1 macrophages, were significantly greater than the values obtained in either HEPES buffer (4.1 ± 0.2 s−1nM−1) or apoE−/− mouse blood (8.1 ± 0.4 s−1nM−1). No significant intracellular r1 values were observed for untargeted Mn micelles, thereby suggesting limited intracellular uptake of the untargeted formulation. The surface charge, as defined by the zeta potential, was significantly greater for Gd versus micelles (−6 ± 1 mV vs. −4 ± 1 mV, p < 0.01).

View this table:
Table 2

Physical Properties, Relaxivities and Half-Life of the Various Micelles

Vascular stability

Untargeted Gd micelles show no significant change in the r1 values after 24-h incubation but did show an initial increase in the r1 values in plasma (time <5 min) relative to buffer (r1 = 14 vs. 10.4 s−1mM−1, p < 0.01). This may be indicative of an initial and potentially rapid transmetallation that occurs within 5 min post-incubation. For the untargeted Mn micelles, the r1 values decreased by 6% over the 24-h time period studied (3.75 vs. 3.50 s−1mM−1). The initial r1 values in plasma were not significantly different than those observed in buffer, indicating slower kinetics of the transmetallation relative to Gd.

The IK17-Gd and IK17-Mn micelles exhibited 3.5% and 1.5% transmetallation after incubation with 20 μmol/l zinc and copper, respectively, suggesting that both the Gd and Mn formulations are susceptible to in vivo transmetallation with endogenous metal ions (Fig. 2).

Figure 2
Figure 2

Percent Transmetallation of IK17-Labeled Mn(II) and Gd(III) Micelles After Exposure to Copper and Zinc

All samples were prepared in 2% bovine serum albumin, and the percent transmetallation determined using relaxometry methods. *p < 0.05, #p < 0.01 represents Mann-Whitney test for differences between groups at each condition.

Macrophage uptake of antibody-micelle formulations

Significant cell association (binding and/or uptake) of IK17-Mn by J774A.1 macrophages pre-exposed to MDA-LDL was noted (Fig. 3). Only limited unspecific uptake was observed for the untargeted micelles. These results are consistent with previously reported results that show specific uptake of OxLDL-targeted Gd micelles by in vitro macrophages (6).

Figure 3
Figure 3

Confocal Imaging of Activated J774A.1 Macrophages

Images of the macrophages were made 24 h after incubation with untargeted and IK17-labeled Mn(II) and Gd(III) micelles. All cell nuclei were DAPI labeled (blue), and all micelles were rhodamine labeled (red).

Pharmacokinetics and biodistribution

Targeted (with both MDA2 and IK17) Mn and Gd micelles had significantly longer (5 to 10×) circulating half-lives than untargeted micelles in apoE−/− mice (Table 2). The endogenous background concentration of Mn in apoE−/− mouse blood was 0.32 ± 0.05 μg/ml. Multiexponential decay was observed for all Mn formulations, thereby suggesting recirculation of metabolized Mn. Additionally, the variation observed in the pKa between the Mn and Gd micelles is likely due to differences in dose or number of particles injected (0.05 mmol Mn/kg and 0.075 mmol Gd/kg), as well as differences in surface charge (zeta potential) associated with the particles (−4 mV for Mn micelles and −6 mV for Gd micelles).

The biodistribution of untargeted and MDA2-Mn micelles in the liver, kidney, and aorta are shown in Figure 4. Compared with MDA2-Gd micelles, significantly higher uptake (per gram wet weight) of MDA2-Mn micelles was observed within the aorta, and significantly lower uptake in the liver 24 h post-injection. Untargeted micelles exhibited limited uptake in the arterial wall. Whereas the excretion kinetics of MDA2-Gd out of the arterial wall was complex, the elimination of MDA2-Mn micelles was mono-exponential with an elimination half-life of 58.7 h, and all Mn was eliminated from the kidneys within 168 h post-injection. Residual kidney uptake (0.97% injected dose [%ID]) was observed for the MDA2-Gd micelles at 1 week post-injection. No significant uptake (<0.05%) of untargeted or MDA2-Mn micelles was observed in the following tissue at any of the time points tested: lung, bone, and heart. At 24 h post-injection, approximately 0.1% of the MDA2-labeled Mn(II) formulation was present within the spleen. MDA2-Gd micelles, however, exhibited significant uptake in the lung (0.47% ID), heart (0.35% ID), bone (0.17% ID), and spleen (6.27% ID) at 48 h post-injection.

Figure 4
Figure 4

Biodistribution of Untargeted Mn(II) Micelles, MDA2-Labeled Mn(II) Micelles, and Gd(III) Micelles

Biodistribution is shown for the liver (A), kidney (B), and aorta (C) of apoE−/− mice. Dosages of 0.05 mmol/kg and 0.075 mmol/kg Mn(II) and Gd(III) were administered, respectively. The percent injected doses (%ID) were determined based upon the wet weight of the tissue. Kruskal-Wallis test was used to assess differences between the 3 types of micelles. *p < 0.05 comparing MDA2-Mn and MDA2-Gd micelles. #p < 0.05 comparing untargeted and MDA2-labeled Mn(II) formulations. p.i. = post-injection.

The initial lower uptake of Mn micelles within the liver, spleen, and kidney is likely a function of dose (lower concentration of Mn micelles injected relative to Gd micelles). However, the higher uptake of targeted Mn micelles within the arterial wall may be related to charges that may influence the binding of the targeted particles to endogenous OSE. Variations in the excretion kinetics, however, are likely related to the rate of intracellular metabolism of Mn and Gd.

Neurotoxicity

No significant (p > 0.1) change in the MR SI was observed in the brain of Alzheimer's mice after administration of either untargeted or targeted micelles. Additionally, all ex vivo Dy tissue concentrations determined by ICP-MS were below method detection limits. As a result, the Dy concentration within the brain could not be accurately determined. On the basis of these findings, it is evident that the PEG micelles are unable to cross the intact blood-brain barrier.

In vivo MRI

Serial imaging was performed at baseline and at 24, 48, 72, and 96 h and 1 week post-injection of targeted micelles. Significant and robust MR signal enhancement was observed in the arterial wall of apoE−/− mice following administration of a 0.05 mmol Mn/kg dose of MDA2-Mn and IK17-Mn micelles (Fig. 5), relative to untargeted formulations. Maximum signal enhancement was observed 48 to 72 h after administration of the OSE-targeted Mn micelles. Quantitatively, MDA2 and IK17-Mn micelles exhibited 141 ± 20% and 125 ± 6%, respectively (p < 0.001 for each compared with pre-injection values) higher signal than adjacent muscle at 48 h post-injection. Additionally, MR signal enhancement of 124 ± 7% was observed in LDLR−/− mice 48 h after administration of MDA2-Mn micelles (Fig. 6). Although the mean values were lower in LDLR−/− mice, no statistically significant difference was observed between the apoE−/− and LDLR−/− mice. In vivo competitive inhibition studies in apoE−/− mice showed a 92% (p < 0.001) reduction in MR signal for mice administered free MDA2 at the time of MDA2-Mn micelle injection.

Figure 5
Figure 5

Representative MR Images of the Abdominal Aorta of apoE−/− Mice Following Injection of Mn Micelles

Images of the abdominal aorta (white arrows) were made after a dose of 0.05 mmol Mn/kg Mn of micelles were injected. The percent normalized enhancement (%NENH) values represent the relative change in the contrast-to-noise ratios pre- and post-imaging. For the competitive inhibition (Compt. Inh.) studies, 3 mg of free MDA2 antibody was co-injected with the MDA2-labeled Mn micelles to compare the 4 types of micelles. *p < 0.05, #p < 0.01 represents Kruskal-Wallis tests for differences between groups compared with the untargeted (Untar.) Mn micelles. MR = magnetic resonance; p.i. = post-injection; Pre = pre-injection.

Figure 6
Figure 6

Representative MRI of apoE−/− and LDLR−/− Mice 48 and 72 h After Administration of MDA2-Labeled Mn Micelles

No significant differences were noted between groups using the Mann-Whitney test. MR = magnetic resonance. Abbreviations as in Figure 5.

MRI of the MDA2-Gd micelles showed maximum signal occurring (%NENH = 156 ± 21%) at 1 week after the administration of a 0.075 mmol Gd/kg dose (Fig. 7). The results obtained in the current study were similar to those reported for 12- to 13-month-old apoE−/− mice following administration of MDA2-Gd micelles (5). No significant difference in the MR efficacy was observed between the MDA2-Mn and MDA2-Gd micelles at the optimal imaging time points post-injection (48 h vs. 1 week, respectively).

Figure 7
Figure 7

Comparison of MDA2-Labeled Gd and Mn Micelles in apoE−/− Mice

Mice were administered 0.075 mmol/kg and 0.05 mmol/kg Gd and Mn, respectively. *p < 0.05, #p < 0.01 represents Mann-Whitney test for differences between groups at each time point. Abbreviations as in Figure 5.

Plaque macrophage uptake of targeted micelles

Confocal microscopy confirmed the uptake of MDA2-Mn micelles within atherosclerotic lesions and specifically within intraplaque foam cells 48 h post-injection (Fig. 8). Rhodamine-labeled targeted micelles were not observed in the extracellular matrix. Additionally, rhodamine was not present within the arterial wall after 1 week post-injection. These results are consistent with the findings reported for MDA2-Gd micelles (5). For the untargeted Mn micelles, no significant rhodamine signal was observed within the arterial wall at any of the time points tested. MDA epitopes were present in regions exhibiting plaque deposition and signal enhancement by MRI (Fig. 9), consistent with histological findings from prior radionuclide imaging studies using the same targeting antibodies (15,16).

Figure 8
Figure 8

Confocal Microscopy of the Abdominal Arterial Wall of an apoE−/− Mouse 48 h After Administration of MDA2-Labeled Mn Micelles

Cell nuclei were stained with DAPI (blue, upper left corner), micelles were labeled with rhodamine (red, upper left corner), and macrophages were RPE-labeled anti-CD68 (green, lower left corner). The composite image of all channels is shown in the bottom right corner.

Figure 9
Figure 9

Representative Correlation Between In Vivo MRI and the Presence of MDA Epitopes

The aorta was imaged in vivo prior to and 48 h after the administration of 0.05 mmol Mn/kg MDA2-labeled micelles. The mouse was sacrificed immediately after imaging, and the immunohistology was performed. The aorta was opened longitudinally and stained with Sudan (red). The yellow line in the Sudan-stained aorta reflects the area of magnetic resonance imaging (MRI) and histological assessment for MDA epitopes. Strong MR signal was observed in regions exhibiting plaque deposition and the presence of MDA-lysine epitopes (red).

Discussion

This study demonstrates the novel development of Mn-based MR molecular imaging probes and their successful application of serial, noninvasive imaging of atherosclerotic plaques in 2 murine models of atherosclerosis. These multimodal micelles are composed of an optical tag, the Mn for MRI, and oxidation-specific antibodies to enhance specific targeting of atherosclerotic lesions. Importantly, although MnDTPA-based probes theoretically exhibit less MR efficacy than equivalent GdDTPA probes, these specifically designed Mn probes accumulated within intraplaque macrophages, presumably due to binding of extracellular OxLDL in the vessel wall or plasma, and subsequent uptake in macrophages through scavenger receptors. The intracellular accumulation of targeted Mn micelles and demetallation resulting in free Mn, noted both in vitro and in vivo, resulted in significant increases in intracellular r1 values, enhanced MR efficacy, and effective imaging of atherosclerotic lesions. Furthermore, specific targeting of OSE was demonstrated by the fact that untargeted control micelles provided limited lesion enhancement and that cotreatment of animals with free MDA2 and MDA2-Mn micelles showing markedly reduced MR signal enhancement due to blocking of available antibody binding sites. Since Mn is a relatively nontoxic cation, and noncardiovascular formulations are FDA approved, these data provide a framework for the clinical translation of this approach to imaging human atherosclerosis and vulnerable plaques.

Mn is a paramagnetic endogenous metal ion that is considered safe when chelated during the bolus injection (22,23). Chelation of Mn during the bolus is critical in order to eliminate cardiotoxicity caused by the competition of Mn ions with calcium in normal cardiomyocytes. However, studies have shown that even soluble Mn (as MnCl2) is safe if it is administered as a slow infusion (24). In the current study, DTPA was used to chelate Mn in the vascular phase. Although limited vascular transmetallation was observed, the kinetics associated with the vascular release of Mn was relatively slow (>5 min). These findings are consistent with reported MnDTPA-BMA data that show relatively slow kinetics with transmetallation half-lives in human plasma of >2 min (non–first-order kinetics) (25). Reported studies using similar GdDTPA lipid micelle constructs have shown 1.6% transmetallation of Gd 3 h after injection in mice (26). Although minor vascular transmetallation is observed with the Mn micelle formulations, toxicity issues caused by the biotransformation and bioretention of Mn chelates are minimal compared with that of equivalent Gd formulations (7,27). Additionally, since the PEG micelles are unable to cross the intact blood-brain barrier, issues related to neurotoxicity are expected to be nominal. The small concentration of Mn that may transmetallate in the vascular phase is also not expected to induce neurotoxicity. Reported toxicity studies using FDA-approved MnDPDP (Teslascan [mangafodipir trisodium], General Electric, Princeton, New Jersey) that undergoes 20% transmetallation in the vascular phase does not induce clinical signs indicative of neurotoxicity (28).

Studies have shown that most cells quickly metabolize or utilize soluble Mn either via integration into Mn-specific metalloproteins or interaction with other cellular components such as the cell membrane (12,13). The in vivo biodistribution data showed 97% clearance of the MDA2-Mn micelles within 1 week post-injection. The MDA2-Gd micelles, however, exhibited only 72.9% clearance within 1 week post-injection. The variation in the retention is due to differences in the rate of metabolism of Mn and Gd micelles. For example, confocal studies show that the targeted PEG micelles were completely degraded within intraplaque macrophages 72 h after injection (rhodamine signal is no longer present). However, as confirmed by ICP-MS as well as MR imaging, Gd is still present within the arterial wall. These data illustrate that the targeted micelles and Gd or Mn label track together at early time points post-injection (<72 h). However, as the intraplaque macrophages internalize and degrade the particles, the lipids forming the micelles and MR label are no longer colocalized within the arterial wall. During intracellular metabolism, the cells degrade the particles, and the MR label is released from the chelate. For targeted Mn micelles, the Mn interacts with cellular components and eventually is either exocytosed or integrated into the endogenous Mn pool. However, demetallation of Gd results in the formation of insoluble Gd salts that remains within the intracellular vesicles (8). So, whereas the half-life of Mn within the arterial wall was approximately 60 h (or 2.5 days), MDA2-Gd micelles exhibit prolonged retention of Gd (>1 week).

The results of the current study strongly indicate that the novel Mn probes allow for specific delivery of Mn to the target cells such as macrophages. This was demonstrated by several lines of evidence. 1) The strong MR signal enhancement observed within the arterial wall 48 to 72 h post-injection of MDA2- or IK17-Mn micelles. Strong signal enhancement may only be observed after intracellular demetallation of Mn. 2) Consistent with specific targeting mechanisms, pre-injection with free MDA2 resulted in complete MR signal inhibition of MDA2-Mn micelles. If passive unspecific uptake mechanisms were present, then the addition of excess free MDA2 would not limit uptake into intraplaque macrophages, and the MR signal would not be affected. 3) Confocal microscopy confirmed uptake of the micelles within intraplaque macrophages and the presence of MDA epitopes. 4) The fact that the MR signal and rhodamine signal returned to baseline within 1 week post-injection is indicative that the intraplaque macrophages/foam cells are able to effectively degrade and clear the demetallated Mn and lipid fragments within a relatively short time period post-injection. The molecular mechanisms through which targeted micelles accumulate in macrophages after being exposed to OxLDL are actively being investigated and may be mediated through macrophage scavenger receptors, macropinocytosis, or other mechanisms. Future studies are underway to fully define the mechanism of intracellular uptake.

This study significantly advances the approach of targeting OSE for molecular imaging of atherosclerotic lesions noninvasively by providing both a more easily translatable approach and higher specificity for targeting OxLDL-enriched macrophages. Prior studies using radiolabeled oxidation-specific antibodies have shown that their uptake in mouse and rabbit atherosclerotic lesions is strongly correlated with plaque burden, measured both as percent atherosclerosis surface area and aortic weight (15,16,29). Furthermore, in dietary regression studies, it was demonstrated that reduced plaque uptake of radiolabeled antibodies was highly sensitive to regression of atherosclerosis and removal of OSE (29). In fact, areas within the aorta where the loss of OSE was greatest were characterized by phenotypes of plaque stabilization, such as absence of OxLDL and macrophages, and accumulation of smooth muscle cells and collagen. Furthermore, in rabbits with very advanced plaques containing fibrous lesions, there was also evidence of reduced OxLDL content and antibody accumulation. If these results can be confirmed in patients, one would be able to quantitate the burden of OSE and monitor their regression.

We have recently reported that targeted Gd micelles may be used to accurately identify OSE within the arterial wall of apoE−/− mice using high-resolution MR imaging (5). We have validated and significantly extended these observations by performing comparative studies of transmetallation, serial pharmacokinetics, biodistribution, and imaging to 1 week with both Gd and Mn IK17 micelles and MDA2 micelles in age-matched LDLR−/− and apoE−/− mice. Although MDA2-Mn micelles were administered at a dose that was 33% lower than that of Gd, equivalent MR signal enhancement was observed at the optimal imaging time points post-injection. For targeted Mn micelles, maximum signal enhancement was observed 48 to 72 h post-injection, with no significant signal observed after 1 week. MDA2-Gd micelles, however, exhibited maximum enhancement after 1 week post-injection. However, like all lipid-based nanoparticles, a fraction of the injection dose was sequestered by liver, causing bioretention of Mn and Gd (5). Unlike with targeted Mn micelles, Gd remains in the plaque for at least a week. Reported studies indicate that intracellular uptake of Gd may promote cell apoptosis, which may result in proinflammatory effects (8,30). However, the intracellular toxicity is strongly dependent upon intracellular uptake into vesicles (endosomes/lysosomes) and the formation of toxic insoluble Gd salts. Liver uptake of Mn, however, is not toxic, and Mn-based targeted liver agents have been approved by the FDA (11).

To avoid potential toxic effects of free Gd, we have also successfully imaged atherosclerotic lesion in apoE−/− mice with MDA2-, IK17-, and E06-targeted lipid-coated ultra-small superparamagnetic iron oxide particles (31). Once taken up by macrophages, iron oxide particles generate MR signal loss due to the generation of strong T2/T2* effects. This approach is qualitatively different than using untargeted iron oxide–based nanoparticles that nonspecifically accumulate in intraplaque macrophages (32,33). A recent therapeutic study in patients showed a significant change in MR signal after 6 to 12 weeks of high-dose atorvastatin treatment (relative to baseline T2* values), and it is anticipated that targeted iron oxide nanoparticles will enable specific detection and monitoring of high-risk atherosclerotic plaque (31,32).

The clinical relevance of OSE-targeted imaging shown in the current study was recently demonstrated in coronary sections from sudden death victims where the presence of oxidized phospholipids and IK17 epitopes was strongest in late lesions with macrophage-rich areas, lipid pools, and the necrotic core, and were most specifically associated with unstable and ruptured plaque. This was further demonstrated in living patients with material derived from carotid, coronary, and renal distal protection devices, demonstrating the presence of oxidized lipids in clinically relevant lesions (34).

Study limitations

Although similar imaging efficacy was observed in 2 atherosclerotic mouse strains, the current study did not specifically image vulnerable plaques. Dedicated toxicology studies will be needed to formally test the safety of the current targeted manganese nanoparticles. Due to limitations in the current study design, it was not possible to evaluate the mechanism of uptake of the targeted micelles in intraplaque macrophages. Future studies are planned in macrophages scavenger receptor knockout mice to evaluate the effect of macrophage phenotype on intracellular uptake. Additionally, the current study used extreme in vitro conditions to evaluate transmetallation. Since other cations, as well as endogenous proteins and macromolecules, may influence transmetallation, these studies will be repeated using more physiological in vivo conditions. Additionally, direct methods such as ICP–high performance liquid chromatography will be used to evaluate the in vivo concentration of metabolites that are indicative of transmetallation (ZnDTPA, CuDTPA, CaDTPA).

Conclusions

The current study demonstrates the use of novel biocompatible Mn molecular imaging probes for the in vivo detection of OSE within atherosclerotic lesions. These observations support the concept that active targeting of Mn to OxLDL-rich intraplaque macrophages/foam cells will allow for sensitive and robust in vivo detection and monitoring of high-risk atherosclerotic lesions.

Appendix

For an expanded Methods section, please see the online version of this paper.

Appendix

Online Appendix

In Vivo Detection of Oxidation-Specific Epitopes in Atherosclerotic Lesions Using Biocompatible Mn(II) Molecular Magnetic Imaging Probes

[S0735109711049862_mmc1.doc]

Footnotes

  • This investigation was supported by the Fondation Leducq (Drs. Witztum and Tsimikas) and by the National Institutes of Health (grant R21 HL091399, to Dr. Briley-Saebo, the principal investigator). Drs. Tsimikas and Witztum are co-inventors of patents, owned by the University of California, on the potential clinical use of antibodies MDA2 and IK17, and have equity interest in Atherotope, Inc. Drs. Fayad and Briley-Saebo are consultants to Atherotope, Inc. Dr. Witztum is a consultant for Isi and Regulus; and has equity interest in Atherotope Inc. Dr. Tsimikas has equity in Atherotope Inc.; and is a consultant for Isis, Aterovax, Genzyme, Quest, and Amarin. All other authors have reported that they have no relationships relevant to the contents of this paper to disclose. Stanley L. Hazen, MD, PhD, served as Guest Editor of this paper.

Abbreviations and Acronyms
%ID
percent injected dose(s)
%NENH
percent normalized enhancement
ICP-MS
inductively coupled plasma mass spectroscopy
MR
magnetic resonance
MRI
magnetic resonance imaging
OSE
oxidation-specific epitope(s)
OxLDL
oxidized low-density lipoprotein(s)
SI
signal intensity
  • Received June 30, 2011.
  • Revision received October 31, 2011.
  • Accepted October 31, 2011.

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  • Online Appendix

    In Vivo Detection of Oxidation-Specific Epitopes in Atherosclerotic Lesions Using Biocompatible Mn(II) Molecular Magnetic Imaging Probes

    [S0735109711049862_mmc1.doc]

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