Magnetic resonance molecular imaging contrast agents and their application in atherosclerosis

Heart disease is the most prevalent cause of mortality in the Western world and is most frequently caused by rupture of lesions in the arteries, which are formed by atherosclerosis. Atherosclerosis is a progressive disease, and therefore, there is a strong motivation to be able to image the stages of this disease in vivo. The pathogenesis of this disease is now well established, and a number of markers such as macrophages, vascular adhesion molecules, fibrin, and the alphanubeta3-integrin have been identified that are of particular interest for imaging. Furthermore, the differentiation between the stable and unstable plaque with imaging is a central goal of the field. Contrast can be generated in magnetic resonance imaging through the application of several types of agents such as T1, T2, chemical exchange saturation transfer or 19F-based imaging agents. Subsequent to the discussion of the above topics, we will describe some examples of molecular imaging agents that successfully detect specific markers in atherosclerotic plaques that are of interest in several stages of this diseas

Magnetic resonance molecular imaging of thrombosis in an arachidonic acid mouse model using an activated platelet targeted probe

Atherosclerotic plaque rupture leads to acute thrombus formation and may trigger serious clinical events such as myocardial infarction or stroke. Therefore, it would be valuable to identify atherothrombosis and vulnerable plaques before the onset of such clinical events. We sought to determine whether the noninvasive in vivo visualization of activated platelets was effective when using a target-specific MRI contrast agent to identify thrombi, hallmarks of vulnerable or high-risk atherosclerotic plaques. Inflammatory thrombi were induced in mice via topical application of arachidonic acid on the carotid. Thrombus formation was imaged with intravital fluorescence microscopy and molecular MRI. To accomplish the latter, a paramagnetic contrast agent (P975) that targets the glycoprotein alpha(IIb)beta(3), expressed on activated platelets, was investigated. The specificity of P975 for activated platelets was studied in vitro. In vivo, high spatial-resolution MRI was performed at baseline and longitudinally over 2 hours after injecting P975 or a nonspecific agent. The contralateral carotid, a sham surgery group, and a competitive inhibition experiment served as controls. P975 showed a good affinity for activated platelets, with an IC(50) (concentration of dose that produces 50% inhibition) value of 2.6 micromol/L. In thrombosed animals, P975 produced an immediate and sustained increase in MRI signal, whereas none of the control groups revealed any significant increase in MRI signal 2 hours after injection. More important, the competitive inhibition experiment with an alpha(IIb)beta(3) antagonist suppressed the MRI signal enhancement, which is indicative for the specificity of P975 for the activated platelets. P975 allowed in vivo target-specific noninvasive MRI of activated platelet

Tyrosine polyethylene glycol (PEG)-micelle magnetic resonance contrast agent for the detection of lipid rich areas in atherosclerotic plaque

Vulnerable or high-risk atherosclerotic plaques often exhibit large lipid cores and thin fibrous caps that can lead to deadly vascular events when they rupture. In this study, polyethylene glycol (PEG)-micelles that incorporate a gadolinium diethylenetriamine pentaacetic acid (Gd-DTPA) amphiphile were used as an MR contrast agent. In an approach inspired by lipoproteins, the micelles were functionalized with tyrosine residues, an aromatic, lipophilic amino acid, to reach the lipid-rich areas of atherosclerotic plaque in a highly efficient manner. These micelles were applied to apolipoprotein E(-/-) (ApoE(-/-)) mice as a model of atherosclerosis. The abdominal aortas of the animals were imaged using T(1)-weighted (T(1)W) high-resolution MRI at 9.4T before and up to 48 h after the administration of the micelles. PEG-micelles modified with 15% tyrosine residues yielded a significant enhancement of the abdominal aortic wall at 6 and 24 h postinjection (pi) as compared to unmodified micelles. Fluorescence microscopy on histological sections of the abdominal aorta showed a correlation between lipid-rich areas and the distribution of the functionalized contrast agent in plaque. Using a simple approach, we demonstrated that lipid-rich areas in atherosclerotic plaque of ApoE(-/-) mice can be detected by MRI using Gd-DTPA micelle

Incorporation of an apoE-derived lipopeptide in high-density lipoprotein MRI contrast agents for enhanced imaging of macrophages in atherosclerosis

Magnetic resonance (MR) imaging is becoming a pivotal diagnostic method to identify and characterize vulnerable atherosclerotic plaques. We previously reported a reconstituted high-density lipoprotein (rHDL) nanoparticle platform enriched with Gd-based amphiphiles as a plaque-specific MR imaging contrast agent. Further modification can be accomplished by inserting targeting moieties into this platform to potentially allow for improved intraplaque macrophage uptake. Since studies have indicated that intraplaque macrophage density is directly correlated to plaque vulnerability, modification of the rHDL platform may allow for better detection of vulnerable plaques. In the current study we incorporated a carboxyfluoresceine-labeled apolipoprotein E-derived lipopeptide, P2fA2, into rHDL. The in vitro macrophage uptake and in vivo MR efficacy were demonstrated using murine J774A.1 macrophages and the apolipoprotein E knock-out (apoE(-/-)) mouse model of atherosclerosis. The in vitro studies indicated enhanced association of murine macrophages to P2fA2 enriched rHDL (rHDL-P2A2) nanoparticles, relative to rHDL, using optical techniques and MR imaging. The in vivo studies showed a more pronounced and significantly higher signal enhancement of the atherosclerotic wall 24 h after the 50 micromol Gd/kg injection of rHDL-P2A2 relative to administration of rHDL. The normalized enhancement ratio for atherosclerotic wall of rHDL-P2A2 contrast agent injection was 90%, while that of rHDL was 53% 24 h post-injection. Confocal laser scanning microscopy revealed that rHDL-P2A2 nanoparticles co-localized primarily with intraplaque macrophages. The results of the current study confirm the hypothesis that intraplaque macrophage uptake of rHDL may be enhanced by the incorporation of the P2fA2 peptide into the modified HDL particl

Molecular imaging of macrophages in atherosclerotic plaques using bimodal PEG-micelles

Pegylated, fluorescent, and paramagnetic micelles were developed. The micelles were conjugated with macrophage scavenger receptor (MSR)-specific antibodies. The abdominal aortas of atherosclerotic apoE-KO mice were imaged with T1-weighted high-resolution MRI before and 24 h after intravenous administration of the contrast agent (CA). Pronounced signal enhancement (SE) (up to 200%) was observed for apolipoprotein E knockout (apoE-KO) mice that were injected with MSR-targeted micelles, while the aortic vessel wall of mice injected with nontargeted micelles showed little SE. To allow fluorescence microscopy and optical imaging of the excised aorta, the micelles were made fluorescent by incorporating either a quantum dot (QD) in the micelle corona or rhodamine lipids in the micelle. Ultraviolet (UV) illumination of the aorta allowed the identification of regions with high macrophage content, while MSR-targeted rhodamine micelles could be detected with fluorescence microscopy and were found to be associated with macrophages. In conclusion, this study demonstrates that macrophages in apoE-KO mice can be effectively and specifically detected by molecular MRI and optical methods upon administration of a pegylated micellar CA

Targeted molecular probes for imaging atherosclerotic lesions with magnetic resonance using antibodies that recognize oxidation-specific epitopes

Oxidized low-density lipoprotein plays a key role in the initiation, progression, and destabilization of atherosclerotic plaques and is present in macrophages and the lipid pool. The aim of this study was to assess the feasibility of magnetic resonance imaging of atherosclerotic lesions in mice using micelles containing gadolinium and murine (MDA2 and E06) or human (IK17) antibodies that bind unique oxidation-specific epitopes. MDA2 micelles, E06 micelles, IK17 micelles, nonspecific IgG micelles, and untargeted micelles (no antibody) were prepared and characterized with respect to pharmacokinetics and biodistribution in wild-type and atherosclerotic apolipoprotein E-deficient (apoE(-/-)) mice. Magnetic resonance imaging was performed at 9.4 T over a 96-hour time interval after the administration of 0.075-mmol Gd/kg micelles. MDA2, E06, and IK17 micelles exhibited a longer plasma half-life than IgG or untargeted micelles in apoE(-/-) but not wild-type mice. In apoE(-/-) mice, MDA2 and IK17 micelles showed maximal arterial wall uptake at 72 hours and E06 micelles at 96 hours, manifested by 125% to 231% enhancement in magnetic resonance signal compared with adjacent muscle. Confocal microscopy revealed that MDA2, IK17, and E06 micelles accumulated within atherosclerotic lesions and specifically within macrophages. Intravenous injection of free MDA2 before imaging with MDA2 micelles resulted in significantly diminished magnetic resonance signal enhancement. IgG micelles and untargeted micelles showed minimal enhancement in apoE(-/-) mice. There was no significant signal enhancement with all micelles in wild-type mice. Magnetic resonance imaging with micelles containing gadolinium and oxidation-specific antibodies demonstrates specific targeting and excellent image quality of oxidation-rich atherosclerotic lesion

Annexin A5-Functionalized Bimodal Nanoparticles for MRI and Fluorescence Imaging of Atherosclerotic Plaques

Apoptosis and macrophage burden are believed to correlate with atherosclerotic plaque vulnerability and are therefore considered important diagnostic and therapeutic targets for atherosclerosis. These cell types are characterized by the exposure of phosphatidylserine (PS) at their surface. In the present study, we developed and applied a small micellar fluorescent annexin A5-functionalized nanoparticle for noninvasive magnetic resonance imaging (MRI) of PS exposing cells in atherosclerotic lesions. Annexin A5-mediated target-specificity was confirmed with ellipsometry and in vitro binding to apoptotic Jurkat cells. In vivo T(1)-weighted MRI of the abdominal aorta in atherosclerotic ApoE(-/-) mice revealed enhanced uptake of the annexin A5-micelles as compared to control-micelles, which was corroborated with ex vivo near-infrared fluorescence images of excised whole aortas. Confocal laser scanning microscopy (CLSM) demonstrated that the targeted agent was associated with macrophages and apoptotic cells, whereas the nonspecific control agent showed no clear uptake by such cells. In conclusion, the annexin A5-conjugated bimodal micelles displayed potential for noninvasive assessment of cell types that are considered to significantly contribute to plaque instability and therefore may be of great value in the assessment of atherosclerotic lesion phenotyp

Well-defined, multifunctional nanostructures of a paramagnetic lipid and a lipopeptide for macrophage imaging

In the field of nanomedicine there is a great demand for technologies that allow the creation of self-assembled structures of which the size and morphology can be accurately controlled. In the current study, we report a nanoparticle platform that is composed of a paramagnetic lipid and a fluorescently labeled lipopeptide. By judiciously controlling the ratio of the aforementioned amphiphilic molecules, a variety of well-defined nanosized supramolecular structures with different sizes and morphologies could be created. The hydrodynamic radii of the different structures were determined by dynamic light scattering. Cryo-TEM revealed the aggregate morphology to vary from small micellar structures to plate-like and even full grown ribbons of which the aspect ratios varied from a diameter of 5-8 nm to structures with a width of up to 25 nm and infinite length. Interestingly, nuclear magnetic resonance dispersion profiling revealed excellent properties for MRI and also showed that the relaxivity of the structures was tunable and morphology dependent. Finally, macrophage cells were treated with two selected nanoparticles and were shown to be avidly taken up. In conclusion we demonstrate a methodology to create structures that (1) are paramagnetic to enable their detection with MRI, (2) exhibit fluorescent properties, (3) can be tuned to defined sizes and shapes, and (4) are efficiently taken up by macrophage cells in vitr

Detecting and assessing macrophages in vivo to evaluate atherosclerosis noninvasively using molecular MRI

We investigated the ability of targeted immunomicelles to detect and assess macrophages in atherosclerotic plaque using MRI in vivo. There is a large clinical need for a noninvasive tool to assess atherosclerosis from a molecular and cellular standpoint. Macrophages play a central role in atherosclerosis and are associated with plaques vulnerable to rupture. Therefore, macrophage scavenger receptor (MSR) was chosen as a target for molecular MRI. MSR-targeted immunomicelles, micelles, and gadolinium–diethyltriaminepentaacetic acid (DTPA) were tested in ApoE−/− and WT mice by using in vivo MRI. Confocal laser-scanning microscopy colocalization, macrophage immunostaining and MRI correlation, competitive inhibition, and various other analyses were performed. In vivo MRI revealed that at 24 h postinjection, immunomicelles provided a 79% increase in signal intensity of atherosclerotic aortas in ApoE−/− mice compared with only 34% using untargeted micelles and no enhancement using gadolinium–DTPA. Confocal laser-scanning microscopy revealed colocalization between fluorescent immunomicelles and macrophages in plaques. There was a strong correlation between macrophage content in atherosclerotic plaques and the matched in vivo MRI results as measured by the percent normalized enhancement ratio. Monoclonal antibodies to MSR were able to significantly hinder immunomicelles from providing contrast enhancement of atherosclerotic vessels in vivo. Immunomicelles provided excellent validated in vivo enhancement of atherosclerotic plaques. The enhancement seen is related to the macrophage content of the atherosclerotic vessel areas imaged. Immunomicelles may aid in the detection of high macrophage content associated with plaques vulnerable to rupture