MRI to detect atherosclerosis with gadolinium‐containing immunomicelles targeting the macrophage scavenger receptor

Abstract

The ability to specifically image macrophages may enable improved detection and characterization of atherosclerosis. In this study we evaluated the in vitro uptake of gadolinium (Gd)-containing immunomicelles (micelles linked to macrophagespecific antibody), micelles, and standard contrast agents by murine macrophages, and sought to determine whether immunomicelles and micelles improve ex vivo imaging of apolipoprotein E knockout (ApoE KO) murine atherosclerosis. Murine RAW 264.7 macrophages were incubated with Gd-DTPA, micelles, and immunomicelles. Cell pellets were prepared and imaged using a 1.5 T MR system with an inversion recovery spin-echo sequence to determine the in vitro T 1 values. Ex vivo analysis of mouse aortas was performed using a 9.4T MR system with a high-spatial-resolution sequence (78 ؋ 39 ؋ 78 m Lesions that result in acute coronary syndromes are typically less than 50% stenosed (1) and are therefore difficult to detect using x-ray coronary angiography. Noninvasive assessment of atherosclerosis by magnetic resonance imaging (MRI) can be used to characterize carotid or aortic plaques (2), but imaging of the coronary arteries is currently limited. The specific targeting of certain plaque components with a paramagnetic contrast agent may therefore improve characterization of atherosclerosis by MRI and perhaps ultimately aid in assessing coronary lesions.In acute coronary syndromes, the lipid core of responsible lesions is rich in macrophage-derived foam cells and other inflammatory cells (3,4). The macrophage scavenger receptor A (MSR-A) is important in the progression of atherosclerosis (5,6) and is expressed at elevated levels in lesions (7). We thus selected the MSR as a target to identify atherosclerotic disease by MRI. In the current study we show that immunomicelles can be produced by linking a monoclonal antibody specific for the murine MSR types I and II (CD204) to gadolinium (Gd)-containing micelles. Our goals were to evaluate the in vitro uptake of immunomicelles, micelles, and standard (Gd-DTPA) MRI contrast agents by murine macrophages, and to determine whether immunomicelles and micelles improve ex vivo imaging of atherosclerotic plaque. MATERIALS AND METHODS Micelle and Immunomicelle FormulationMixed micelles were made from phospholipids (Palmitoyl-oleoyl phosphatidylcholine (POPC), 1,2-Dipalmitoylsn-glycero-3-phosphoethanolamine-N-7-nitro-2-1,3-benzoxadiazol-4-yl (DPPE-NBD), 1,2-Dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-biotinyl (DPPE-Biotin)), a surfactant (Tween 80), and an aliphatic Gd complex (Gd-DOTA-C 16 ) as previously described (8). The POPC and Gd complex were dissolved in a 1:1 chloroform : methanol solution (5 mL). Evaporation under nitrogen flux yielded a thin film that was then rehydrated in hot water (2 mL, 70°C). This solution was sonicated for 15 min at 70 W at a 90% cycle duty while the temperature was kept at 65°C by means of a thermostatic bath. After sonication, Tween 80 was added, followed by another 15 min of sonication. Green fluorescent micelles were ...

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

“…The results presented in this study are consistent with previously published findings that show uptake of immunomicelles in cultured macrophages and in ex vivo atherosclerotic aortas (18). In addition, a quantitative correlational relationship was revealed between the MRI enhancement seen by using immunomicelles and standard macrophage immunostaining (Fig.…”

“…As shown previously, MSR-targeted immunomicelles have been shown to be quite effective in the delivery of a high number of Gd complexes (18). The result has been excellent targeting of macrophages in vitro and in atherosclerotic plaques ex vivo (18).…”

“…Gd-carrying micelles and biotinylated Gd micelles were synthesized by using previously described techniques (18,20). Please see supporting information (SI) Text for a complete description.…”

“…Please see supporting information (SI) Text for a complete description. The biotinylated monoclonal rat anti-mouse antibody to murine MSR-A types I and II (anti-CD204, monoclonal IgG, Serotec, Raleigh, NC) was used to make immunomicelles by linking the biotinylated micelle with the biotinylated anti-CD204 by a biotin-avidin-biotin bridge using established protocols (18,32). Using established techniques (20), the r 1 of the immunomicelle formulations were determined, in aqueous solution at 60 MHz and 40°C (Bruker Medical, Ettingen, Germany), from a linear fit of Gd concentration (mM Gd) vs. 1/T 1 (1/T 1 ϭ r 1 , s Ϫ1 ).…”

Preparation and initial characterization of biodegradable particles containing gadolinium-DTPA contrast agent for enhanced MRI

“…Faranesh and colleagues (18) demonstrated the utility of the inclusion of small amounts of Gd-DTPA in PLGA microparticles for tracking a drug delivery vehicle. Additionally, several groups have created gadolinium-containing micelles, microspheres, nanoparticles, and liposomes for the purposes of targeting delivery or modifying the biodistribution to increase the site-specific concentration of gadolinium over that of aqueous gadolinium or gadolinium directly conjugated to the targeting molecule (19)(20)(21)(22)(23)(24)(25)(26). Toward the same goals, this article details a method for the entrapment of hydrophilic agents, specifically Gd-DTPA, with very high loading in a polymeric particulate system as well as work toward achieving a targetable imaging agent particle formulation for the detection of staged atherosclerosis.…”