Traditional imaging of atherosclerosis has focused on the caliber of the arterial lumen or the structure of plaque. Advances in the basic and clinical biology of atherosclerosis have identified inflammation as a key process contributing to lesion initiation, progression, and complication. This recognition has spurred considerable effort to image inflammation in atheromata. The application of nanotechnology offers new approaches to the design of diagnostic agents. Optical and magnetic resonance imaging have employed nanoparticles20, 21
, but this technology has only recently been explored for the design of new generations of PET imaging agents. Here we report on the development and validation of a novel, flexible nanoparticle platform for PET imaging. The synergy of a high inherent phagocytic avidity of dextran coated nanoparticles and derivatization of these nanomaterials with a radiotracer provides a highly sensitive tool to assess Mø burden in murine atherosclerosic lesions. Furthermore, the tri-modal character of 64
Cu-TNP allows hybrid imaging, and rigorous probe validation by fluorescence-based techniques on the cellular and molecular level.
Clinically, interest has burgeoned in developing strategies to identify inflamed, presumably rupture-prone atherosclerotic plaques. Such functional imaging might allow identification of high risk patients, and help direct therapy to prevent cardiovascular events22, 23
. Goals for a suitable technology include high sensitivity, high specificity for biological processes leading to rupture of a plaque, and practicability. Phantom imaging shown in established a high sensitivity for PET to detect low concentrations of 64
Cu-TNP. TNP employed in this study was administered at 1.5 mg Fe/kg bodyweight, well below the dose currently in clinical trials for oncology (2.6 mg Fe/kg)24
and well below the dose used experimentally for imaging of atherosclerosis in rabbits (14-56 mg Fe/kg)3, 4
. We anticipate that the detection threshold could easily be improved by several orders of magnitude with further chemical optimization aiming at higher specific activity of the nanoparticle, as well as with the higher sensitivity of next generation PET imaging systems.
In vivo PET-CT imaging after injection of 64Cu-TNP showed robust PET signal in regions of mouse atheromata. In vivo magnetic resonance and ex vivo fluorescence imaging established the distribution of the nanomaterials to atherosclerotic lesions. Phagocytic cells implicated in lesion growth and vulnerability ingested 64Cu-TNP as shown by immunofluorescence and flow cytometry. After in vivo distribution of 64Cu-TNP, cells that express Mø surface markers showed the highest nanoparticle uptake as characterized by maximal mean fluorescence units per cell in flow cytometry.
Although thorough toxicity tests remain to be performed, we expect 64
Cu-TNP to be safe at the trace amounts used. Comparable iron oxide nanoparticles have been frequently used for MRI in animal models and in patients. In vitro studies have shown that high concentrations of nanoparticles may change the cytokine profile of macrophages25
, while a dose dependent shift towards an antiflammatory phenotype was observed. It is unclear how the relatively high doses in this in vitro study reflect the in vivo situation. Nevertheless, it seems unlikely that iron oxide nanoparticle uptake would drive phagocytes towards a more inflammatory state. 64
Cu has been used as a PET tracer in patients26
. A recent study by Lewis et al. found no signs of acute toxicity of a 64
Cu derivative in hamsters27
. The metal copper can be toxic in high doses, however it is here injected in a chelated state, preventing any potential toxicity of free copper. For instance, Gadolinium is highly toxic, but is frequently used as a contrast agent in clinical MRI as Gd-DTPA.
FDG and 64
Cu-TNP PET-CT imaging established a similar but not identical macroscopic distribution for both probes, with peak vascular signal observed in the aortic root and arch. Microscopic probe distribution could be assessed for 64
Cu-TNP but not for 18
FDG since the radioactive signal was too low and decayed too fast to enable ex vivo autoradiography or gammacounting. Previously, 18
FDG distribution has been believed to correlate with plaque macrophage content in patients12, 15
and in the rabbit plaques28, 29
. However, unequivocal cellular distribution studies have not been published to date, given the experimental difficulty (rapid decay, unavailability of fluorescent analogs for correlative flow cytometry or fluorescence microscopy). Indeed, a recent study in apoE-/-
mice identified brown fat and not atherosclerotic lesions as a dominant source of 18
FDG signal in vivo16
The multimodality capabilities of the nanomaterials developed here facilitated rigorous validation of the origin of the signal and the fate of the imaging probe. The addition of a fluorochrome for optical imaging proved particularly helpful to test the novel probe and determine its fate at a cellular level in vivo. We anticipate that nanomaterials such as the one described here will advance both basic research and clinical applications for several reasons. First, the tri-modality diagnostic capability will prove synergistic for hybrid imaging systems currently entering clinical use. Secondly, PET imaging has a high inherent sensitivity, allows for quantification of the signal, and will facilitate whole body screening of the entire arterial tree. Thirdly, the described nanotechnology platform is being introduced into the clinic24
and is versatile. The carbohydrate coating can be aminated to attach linkers and affinity ligands to specifically target these nanomaterials to other molecular and cellular structures such as adhesion molecules2
, Mø subpopulations17
or cells undergoing apoptosis30