Therapeutic and diagnostic nanomaterials are being intensely studied for several diseases, including cancer and atherosclerosis. However, the exact mechanism by which nanomedicines accumulate at targeted sites remains a topic of investigation, especially in the context of atherosclerotic disease. Models to accurately predict transvascular permeation of nanomedicines are needed to aid in design optimization. Here we show that an endothelialized microchip with controllable permeability can be used to probe nanoparticle translocation across an endothelial cell layer. To validate our in vitro model, we studied nanoparticle translocation in an in vivo rabbit model of atherosclerosis using a variety of preclinical and clinical imaging methods. Our results reveal that the translocation of lipid-polymer hybrid nanoparticles across the atherosclerotic endothelium is dependent on microvascular permeability. These results were mimicked with our microfluidic chip, demonstrating the potential utility of the model system. nanotechnology | cardiovascular disease | microfluidics | noninvasive imaging

Hyaluronan is a biologically active polymer, which can be formulated into nanoparticles. In our study, we aimed to probe atherosclerosis-associated inflammation by using hyaluronan nanoparticles and to determine whether they can ameliorate atherosclerosis. Hyaluronan nanoparticles (HA-NPs) were prepared by reacting amine-functionalized oligomeric hyaluronan (HA) with cholanic ester and labeled with a fluorescent or radioactive label. HA-NPs were characterized in vitro by several advanced microscopy methods. The targeting properties and biodistribution of HA-NPs were studied in apoe–/– mice, which received either fluorescent or radiolabeled HA-NPs and were examined ex vivo by flow cytometry or nuclear techniques. Furthermore, three atherosclerotic rabbits received 89Zr-HA-NPs and were imaged by PET/MRI. The therapeutic effects of HA-NPs were studied in apoe–/– mice, which received weekly doses of 50 mg/kg HA-NPs during a 12-week high-fat diet feeding period. Hydrated HA-NPs were ca. 90 nm in diameter and displayed very stable morphology under hydrolysis conditions. Flow cytometry revealed a 6- to 40-fold higher uptake of Cy7-HA-NPs by aortic macrophages compared to normal tissue macrophages. Interestingly, both local and systemic HA-NP–immune cell interactions significantly decreased over the disease progression. 89Zr-HA-NPs-induced radioactivity in atherosclerotic aortas was 30% higher than in wild-type controls. PET imaging of rabbits revealed 6-fold higher standardized uptake values compared to the muscle. The plaques of HA-NP-treated mice contained 30% fewer macrophages compared to control and free HA-treated group. In conclusion, we show favorable targeting properties of HA-NPs, which can be exploited for PET imaging of atherosclerosis-associated inflammation. Furthermore, we demonstrate the anti-inflammatory effects of HA-NPs in atherosclerosis.

Atherosclerosis is a major cause of global morbidity and mortality that could benefit from novel targeted therapeutics. Recent studies have shown efficient and local drug delivery with nanoparticles, although the nanoparticle targeting mechanism for atherosclerosis has not yet been fully elucidated. Here we used in vivo and ex vivo multimodal imaging to examine permeability of the vessel wall and atherosclerotic plaque accumulation of fluorescently labeled liposomal nanoparticles in a rabbit model. We found a strong correlation between permeability as established by in vivo dynamic contrast enhanced magnetic resonance imaging and nanoparticle plaque accumulation with subsequent nanoparticle distribution throughout the vessel wall. These key observations will enable the development of nanotherapeutic strategies for atherosclerosis.

OBJECTIVES This study sought to develop an integrative positron emission tomography (PET) with magnetic resonance imaging (MRI) procedure for accurate atherosclerotic plaque phenotyping, facilitated by clinically approved and nanobody radiotracers. BACKGROUND Noninvasive characterization of atherosclerosis remains a challenge in clinical practice. The limitations of current diagnostic methods demonstrate that, in addition to atherosclerotic plaque morphology and composition, disease activity needs to be evaluated. METHODS We screened 3 nanobody radiotracers targeted to different biomarkers of atherosclerosis progression, namely vascular cell adhesion molecule (VCAM)-1, lectin-like oxidized low-density lipoprotein receptor (LOX)-1, and macrophage mannose receptor (MMR). The nanobodies, initially radiolabeled with copper-64 ( 64 Cu), were extensively evaluated in Apoe –/– mice and atherosclerotic rabbits using a combination of in vivo PET/MRI readouts and ex vivo radioactivity counting, autoradiography, and histological analyses. RESULTS The 3 nanobody radiotracers accumulated in atherosclerotic plaques and displayed short circulation times due to fast renal clearance. The MMR nanobody was selected for labeling with gallium-68 ( 68 Ga), a short-lived radioisotope with high clinical relevance, and used in an ensuing atherosclerosis progression PET/MRI study. Macrophage burden was longitudinally studied by 68 Ga-MMR–PET, plaque burden by T2-weighted MRI, and neovascularization by dynamic contrast-enhanced (DCE) MRI. Additionally, inflammation and microcalcifications were evaluated by fluorine-18 ( 18 F)-labeled fluorodeoxyglucose ( 18 F-FDG) and 18 F-sodium fluoride ( 18 F-NaF) PET, respectively. We observed an increase in all the aforementioned measures as disease progressed, and the imaging signatures correlated with histopathological features. CONCLUSIONS We have evaluated nanobody-based radiotracers in rabbits and developed an integrative PET/MRI protocol that allows noninvasive assessment of different processes relevant to atherosclerosis progression. This approach allows the multiparametric study of atherosclerosis and can aid in early stage anti-atherosclerosis drug trials.