Of the cardiovascular syndromes, atherosclerosis has received the most attention from nanomedicine researchers. This is likely due to the number of deleterious consequences resulting from the rupture of inflamed, vulnerable plaques. It is also resultant of the number of potential targets within the lesions, including an abundance of specific cell types, such as macrophages, and the upregulation of cell surface receptors, such as vascular cell adhesion molecule-1 (VCAM-1). Clinically, there exists a need to identify vulnerable lesions before the onset of symptoms.
A number of strategies have been investigated for the detection of lesions, such as the fluorescent or radiolabeling of antibodies or other affinity ligands targeted to specific receptors [2
]. With regard to nanoagents, most of the synthesized agents have relied upon the innate uptake of the particles by phagocytic macrophages within the lesions. This is readily exemplified in a number of recent articles by Nahrendorf et al. In their work, the authors utilize epichlorohydrin-crosslinked dextran-coated iron oxide (CLIO) nanoparticles for the targeted imaging of lesions in vivo. In fact, the authors were able to synthesize a multimodal nanoagent capable of detecting atherosclerotic plaques in apolipoprotein E–deficient (apoE−/−
) mice by fluorescence, magnetic resonance, and nuclear imaging [8•
]. To the surface coating of this agent was conjugated a near-infrared fluorophore (VivoTag 680; VisEn Medical, Woburn, MA), as well as diethylenetriaminepentaacetic acid to allow for the coordination of 64
Cu. The main advantage of this system is that the detection threshold for positron emission tomography (PET) imaging with 64
Cu is 50-fold lower than what is readily detectable by MRI, as determined in phantoms in vitro. When utilized in vivo in atherosclerotic plaque–laden mice, the nanoagent readily accumulated in the aortic arch and root, as determined by PET-CT imaging, with a target-to-background ratio (TBR) of 5.1±0.9, compared with surrounding tissues. The mice were subsequently subjected to MRI, which demonstrated a significant decrease in signal intensity in the aortic root, a phenomenon that is well known for the accumulation of iron oxide nanoparticles. After sacrifice, the aortas of the mice were excised and digested to form a single-cell suspension, which was then labeled with a cocktail of fluorescently labeled antibodies and subject to flow cytometry in order to identify the cell types in which the agent accumulated. As expected, most of the cells that accumulate the nanoagent are monocytes and macrophages (73.9%). Neutrophils (17.2%), endothelial cells (4.2%), smooth muscle cells (0.4%), lymphocytes (4.3%), and other cells (6.6%) took up the remaining agent, although to a lesser degree than macrophages. Encouragingly, iron oxide–based nanosensors have already demonstrated significant utility in the clinical arena (please see the article by Young et al. in this issue of Current Cardiovascular Imaging Reports
The role that protease-activatable nanosensors may play in the detection of atherosclerotic lesions has also been investigated [9
]. Three differently sized nanoagents were synthesized (5, 25, and 40 nm) in order to allow for the investigation of the differential localization and activation kinetics of each agent. These nanosensors are fluorogenic in the presence of cathepsin B, a protease that is abundantly expressed by macrophages and monocytes. Initially, the blood half-lives of the respective agents were assayed and demonstrated increased retention with increasing agent size. The agents were then injected into plaque-laden apoE−/−−
mice and allowed to circulate for 24 h prior to the mice being sacrificed, and the aortas were excised and imaged by fluorescence reflectance imaging (FRI). The largest agent (40 nm) exhibited the most significant activation within the diseased tissue (TBR of 7.13±1.36), whereas the 5-nm nanoagent exhibited the least (TBR of 4.76±0.56). The utility of the probes was further investigated by hybrid fluorescence-mediated tomography/x-ray CT (FMT-CT) imaging. This allows for the anatomical localization of the fluorescence signal to be determined. As was illustrated ex vivo, the largest probe demonstrated the most significant accumulation within atherosclerotic lesions, especially the aortic root, 24 h after injection, whereas the 5-nm particle had the fastest “wash in” and “wash out” kinetics. The authors further utilized the 40-nm particle to examine the effectiveness of statin therapies in decreasing inflammation, as atorvastatin is known to reduce the recruitment of monocytes, partly through the reduction of VCAM-1 expression. As compared with the control mice that did not receive the statin, the treated mice demonstrated a 2.6-fold decrease in probe signal in the FMT-CT (), which was further correlated with quantitative polymerase chain reaction for expression of cathepsin B.
Fig. 1 The protease sensor with the highest sensitivity, the 40-nm agent, was used in a therapy trial to investigate its potential as a noninvasive imaging biomarker in drug development. a and b, Fluorescence-mediated tomography (FMT)–CT datasets in (more ...)
Fayad and coworkers have also utilized the phagocytic activity of macrophages for the delivery of a novel CT contrast agent to atherosclerotic lesions [10
]. This polyiodinated nanoparticle, based upon surfactant-dispersed crystalline ethyl-3,5-bis(acetylamino)-2,4,6-triiodobenzoate (N1177), possessed a mean diameter of 259 nm and resulted in 67 mg of iodine per milliliter of agent. When injected into healthy New Zealand white rabbits, the agent displayed significantly longer retention times than a conventional, clinically used contrast agent (iopamidol), and decreased rapidly by 2 h post-injection. N1177 was next investigated in atherosclerotic lesion–laden rabbits. At 2 h after intravenous injection, where the background signal in the vasculature is expected to be low, accumulation of the agent within macrophage-rich lesions led to significant enhancement of the plaques in the CT images. The authors have recently explored the correlation between the intensity of the CT enhancement of these particles with PET imaging using 18
F-FDG, which has shown promise in the imaging of inflamed atherosclerotic lesions [11
]. As hypothesized, N1177 accumulation within atherosclerotic lesions corresponded well with the elevated metabolic activity associated with 18
F-FDG uptake. These results were further correlated with macrophage burden by immunohistology.
While significant focus has been placed on the imaging of atherosclerotic vascular disease using nanoparticulate agents, researchers are beginning to utilize the platforms already developed for diagnostic applications to co-deliver therapeutics. We have detailed the synthesis of light-activated theranostic nanoparticles for the treatment of atherosclerosis via the focal ablation of inflammatory macrophages [12
]. These agents, based upon CLIO, contain near-infrared light-activated photosensitizers, as well as near-infrared fluorophores, each at spectrally distinct wavelengths. This allows for the determination of agent localization prior to therapy. In vitro, the agent demonstrated exceptional uptake and cell killing in murine macrophages. Agent localization and therapeutic efficacy were next examined in the carotid artery of 28-week-old apoE−/−
mice on a high-fat diet. As determined by intravital fluorescence microscopy (IVFM), the nanoagent demonstrated significant accumulation within atherosclerotic lesions. After the initial imaging session, the carotid artery was irradiated with a 650-nm laser, in order to bring about the therapeutic effect, and the mice were allowed to recover. Twenty-four hours later, one cohort of mice was sacrificed for the histological determination of focal cell killing by terminal deoxynucleotidyl transferase nick end-labeling (TUNEL) assay. As compared with the control mice, those that received the agent and light exhibited extensive cell killing within the lesions. One week after the initial imaging session, the other cohort of mice was reinjected with the agent and examined by IVFM. Whereas the control mice demonstrated uptake of the agent within the plaques comparable to the initial imaging session, there was very little localization to the treated lesions, intimating the elimination of the phagocytic inflammatory cells.