Elucidating the Molecular Interactions of Engineered Protein Nanoparticles in Imaging Atherosclerotic Plaques

Conventional atherosclerotic plaques diagnosis using magnetic resonance imaging (MRI) modality is challenged due to the requirements for an ultra-sensitive contrast agent that allows early detection for improved prognosis. The FDA approved MRI contrast agent, superparamagnetic iron oxide nanoparticle (SPION), requires a long time (i.e., 2 days) to concentrate at the plaque site with limited and untargeted accumulation. The consequence is extended waiting time for patients and possibly administrative burden on the healthcare system. Furthermore, these contrast agents are typically monofunctional and do not allow for longitudinal tracking of the plaque’s progression. In previous work, we have shown that protein nanoparticles (PNPs) based on archaeal ferritin, a thermostable natural iron storage protein, can serve as a dual functional platform for both contrast agent in magnetic resonance (MR) imaging and molecular carrier [1, 2]. PNPs with magnetic iron nanocores of ~8 nm show high contrast enhancement in MR imaging [3], while loading of cyclodextrin shows reduction of lipid content in foam cells [4]. The reduction is comparable to standard statin treatment. These in vitro studies demonstrate the potential of the PNPs both as diagnostic agent and carriers for therapeutic molecules. We have also demonstrated cellular targeting via conjugation with reporter moieties, such as antibody, antibody fragments, and peptides, on the surface of the PNPs [5, 6]. Recently, we have started exploring PNPs for atherosclerotic plaque imaging. In a preliminary study, we confirm that fluorescently-labeled PNPs accumulate at the plaque sites using the ApoE-/- mouse model and they enhance the contrast in MR imaging. PNPs do not elicit acute immunological response as determined by immunodiffusion method and preliminary in vivo studies. Despite these promising material properties, the potential of PNPs with iron nanocore as a multi-functional carrier for staging of atherosclerotic plaque is relatively unexplored. No studies have been reported on their pharmacokinetics, improved accumulation at plaque sites, and molecular/cellular understanding on the fate of the PNPs in the plaque and their interactions with the resident macrophages. Understanding these interactions are important for future designs of better MR imaging contrast agents and improved diagnosis on atherosclerotic progression. In this proposal, our goal is to elucidate PNP molecular interactions with atherosclerotic plaque to better inform the design and engineering of the PNPs for faster targeted accumulation and better MR imaging contrast enhancement at the plaque sites. Hence, paving ways for the design and developments of future contrast agents. Particularly, in identifying vulnerable atherosclerotic plaques for the prevention of myocardial infarction or stroke. We hypothesize that engineered PNPs with ligands specific to different stages of the plaques will accumulate within 1 hour, enter the plaque sites, and interact with the macrophages. The plaque progression at different stages of atherosclerotic disease at the aortic root of an Apolipoprotein E-deficient (ApoE-/-) mouse model will be tracked longitudinally by in vivo MRI using the engineered PNPs. The engineered PNPs are expected to shorten the imaging interval, reduce the waiting time for the patients and improve accuracy in staging of the plaques. Co-delivery of active compounds to reduce the chance of thrombus formation and suppress inflammation will be possible in the future. To test the hypothesis, we will perform experiments with three specific aims: 1. Design, engineer, and evaluate PNPs for enhancing targeting at the plaque sites. Ligands that can recognize surface markers at inflamed endothelial cell lining (e.g., VCAM-1 and P-Selectin) for plaque location will be displayed on the external surface of the PNPs. 2. Evaluate the contrast enhancement and quantitatively track the plaque progression at different stages of atherosclerotic disease by in vivo MR imaging using the engineered PNPs. 3. Examine the fate of the PNPs and their interactions with macrophages within the plaques.