Hybrid PET- and MR-driven attenuation correction for enhanced 18F-NaF and 18F-FDG quantification in cardiovascular PET/MR imaging.

BACKGROUND: The standard MR Dixon-based attenuation correction (AC) method in positron emission tomography/magnetic resonance (PET/MR) imaging segments only the air, lung, fat and soft-tissues (4-class), thus neglecting the highly attenuating bone tissues and affecting quantification in bones and adjacent vessels. We sought to address this limitation by utilizing the distinctively high bone uptake rate constant Ki expected from 18F-Sodium Fluoride (18F-NaF) to segment bones from PET data and support 5-class hybrid PET/MR-driven AC for 18F-NaF and 18F-Fluorodeoxyglucose (18F-FDG) PET/MR cardiovascular imaging. METHODS: We introduce 5-class Ki/MR-AC for (i) 18F-NaF studies where the bones are segmented from Patlak Ki images and added as the 5th tissue class to the MR Dixon 4-class AC map. Furthermore, we propose two alternative dual-tracer protocols to permit 5-class Ki/MR-AC for (ii) 18F-FDG-only data, with a streamlined simultaneous administration of 18F-FDG and 18F-NaF at 4:1 ratio (R4:1), or (iii) for 18F-FDG-only or both 18F-FDG and 18F-NaF dual-tracer data, by administering 18F-NaF 90 minutes after an equal 18F-FDG dosage (R1:1). The Ki-driven bone segmentation was validated against computed tomography (CT)-based segmentation in rabbits, followed by PET/MR validation on 108 vertebral bone and carotid wall regions in 16 human volunteers with and without prior indication of carotid atherosclerosis disease (CAD). RESULTS: In rabbits, we observed similar (< 1.2% mean difference) vertebral bone 18F-NaF SUVmean scores when applying 5-class AC with Ki-segmented bone (5-class Ki/CT-AC) vs CT-segmented bone (5-class CT-AC) tissue. Considering the PET data corrected with continuous CT-AC maps as gold-standard, the percentage SUVmean bias was reduced by 17.6% (18F-NaF) and 15.4% (R4:1) with 5-class Ki/CT-AC vs 4-class CT-AC. In humans without prior CAD indication, we reported 17.7% and 20% higher 18F-NaF target-to-background ratio (TBR) at carotid bifurcations wall and vertebral bones, respectively, with 5- vs 4-class AC. In the R4:1 human cohort, the mean 18F-FDG:18F-NaF TBR increased by 12.2% at carotid bifurcations wall and 19.9% at vertebral bones. For the R1:1 cohort of subjects without CAD indication, mean TBR increased by 15.3% (18F-FDG) and 15.5% (18F-NaF) at carotid bifurcations and 21.6% (18F-FDG) and 22.5% (18F-NaF) at vertebral bones. Similar TBR enhancements were observed when applying the proposed AC method to human subjects with prior CAD indication. CONCLUSIONS: Ki-driven bone segmentation and 5-class hybrid PET/MR-driven AC is feasible and can significantly enhance 18F-NaF and 18F-FDG contrast and quantification in bone tissues and carotid walls.

Probing nanoparticle translocation across the permeable endothelium in experimental atherosclerosis.

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.

Three-dimensional dynamic contrast-enhanced MRI for the accurate, extensive quantification of microvascular permeability in atherosclerotic plaques.

Atherosclerotic plaques that cause stroke and myocardial infarction are characterized by increased microvascular permeability and inflammation. Dynamic contrast-enhanced MRI (DCE-MRI) has been proposed as a method to quantify vessel wall microvascular permeability in vivo. Until now, most DCE-MRI studies of atherosclerosis have been limited to two-dimensional (2D) multi-slice imaging. Although providing the high spatial resolution required to image the arterial vessel wall, these approaches do not allow the quantification of plaque permeability with extensive anatomical coverage, an essential feature when imaging heterogeneous diseases, such as atherosclerosis. To our knowledge, we present the first systematic evaluation of three-dimensional (3D), high-resolution, DCE-MRI for the extensive quantification of plaque permeability along an entire vascular bed, with validation in atherosclerotic rabbits. We compare two acquisitions: 3D turbo field echo (TFE) with motion-sensitized-driven equilibrium (MSDE) preparation and 3D turbo spin echo (TSE). We find 3D TFE DCE-MRI to be superior to 3D TSE DCE-MRI in terms of temporal stability metrics. Both sequences show good intra- and inter-observer reliability, and significant correlation with ex vivo permeability measurements by Evans Blue near-infrared fluorescence (NIRF). In addition, we explore the feasibility of using compressed sensing to accelerate 3D DCE-MRI of atherosclerosis, to improve its temporal resolution and therefore the accuracy of permeability quantification. Using retrospective under-sampling and reconstructions, we show that compressed sensing alone may allow the acceleration of 3D DCE-MRI by up to four-fold. We anticipate that the development of high-spatial-resolution 3D DCE-MRI with prospective compressed sensing acceleration may allow for the more accurate and extensive quantification of atherosclerotic plaque permeability along an entire vascular bed. We foresee that this approach may allow for the comprehensive and accurate evaluation of plaque permeability in patients, and may be a useful tool to assess the therapeutic response to approved and novel drugs for cardiovascular disease.

PET/MR imaging of inflammation in atherosclerosis.

Myocardial infarction, stroke, mental disorders, neurodegenerative processes, autoimmune diseases, cancer and the human immunodeficiency virus impact the haematopoietic system, which through immunity and inflammation may aggravate pre-existing atherosclerosis. The interplay between the haematopoietic system and its modulation of atherosclerosis has been studied by imaging the cardiovascular system and the activation of haematopoietic organs via scanners integrating positron emission tomography and resonance imaging (PET/MRI). In this Perspective, we review the applicability of integrated whole-body PET/MRI for the study of immune-mediated phenomena associated with haematopoietic activity and cardiovascular disease, and discuss the translational opportunities and challenges of the technology.

Systematically evaluating DOTATATE and FDG as PET immuno-imaging tracers of cardiovascular inflammation.

In recent years, cardiovascular immuno-imaging by positron emission tomography (PET) has undergone tremendous progress in preclinical settings. Clinically, two approved PET tracers hold great potential for inflammation imaging in cardiovascular patients, namely FDG and DOTATATE. While the former is a widely applied metabolic tracer, DOTATATE is a relatively new PET tracer targeting the somatostatin receptor 2 (SST2). In the current study, we performed a detailed, head-to-head comparison of DOTATATE-based radiotracers and [18F]F-FDG in mouse and rabbit models of cardiovascular inflammation. For mouse experiments, we labeled DOTATATE with the long-lived isotope [64Cu]Cu to enable studying the tracer's mode of action by complementing in vivo PET/CT experiments with thorough ex vivo immunological analyses. For translational PET/MRI rabbit studies, we employed the more widely clinically used [68Ga]Ga-labeled DOTATATE, which was approved by the FDA in 2016. DOTATATE's pharmacokinetics and timed biodistribution were determined in control and atherosclerotic mice and rabbits by ex vivo gamma counting of blood and organs. Additionally, we performed in vivo PET/CT experiments in mice with atherosclerosis, mice subjected to myocardial infarction and control animals, using both [64Cu]Cu-DOTATATE and [18F]F-FDG. To evaluate differences in the tracers' cellular specificity, we performed ensuing ex vivo flow cytometry and gamma counting. In mice subjected to myocardial infarction, in vivo [64Cu]Cu-DOTATATE PET showed higher differential uptake between infarcted (SUVmax 1.3, IQR, 1.2-1.4, N = 4) and remote myocardium (SUVmax 0.7, IQR, 0.5-0.8, N = 4, p = 0.0286), and with respect to controls (SUVmax 0.6, IQR, 0.5-0.7, N = 4, p = 0.0286), than [18F]F-FDG PET. In atherosclerotic mice, [64Cu]Cu-DOTATATE PET aortic signal, but not [18F]F-FDG PET, was higher compared to controls (SUVmax 1.1, IQR, 0.9-1.3 and 0.5, IQR, 0.5-0.6, respectively, N = 4, p = 0.0286). In both models, [64Cu]Cu-DOTATATE demonstrated preferential accumulation in macrophages with respect to other myeloid cells, while [18F]F-FDG was taken up by macrophages and other leukocytes. In a translational PET/MRI study in atherosclerotic rabbits, we then compared [68Ga]Ga-DOTATATE and [18F]F-FDG for the assessment of aortic inflammation, combined with ex vivo radiometric assays and near-infrared imaging of macrophage burden. Rabbit experiments showed significantly higher aortic accumulation of both [68Ga]Ga-DOTATATE and [18F]F-FDG in atherosclerotic (SUVmax 0.415, IQR, 0.338-0.499, N = 32 and 0.446, IQR, 0.387-0.536, N = 27, respectively) compared to control animals (SUVmax 0.253, IQR, 0.197-0.285, p = 0.0002, N = 10 and 0.349, IQR, 0.299-0.423, p = 0.0159, N = 11, respectively). In conclusion, we present a detailed, head-to-head comparison of the novel SST2-specific tracer DOTATATE and the validated metabolic tracer [18F]F-FDG for the evaluation of inflammation in small animal models of cardiovascular disease. Our results support further investigations on the use of DOTATATE to assess cardiovascular inflammation as a complementary readout to the widely used [18F]F-FDG.

Employing nanobodies for immune landscape profiling by PET imaging in mice.

Noninvasive immunoimaging holds great potential for studying and stratifying disease as well as therapeutic efficacy. Radiolabeled single-domain antibody fragments (i.e., nanobodies) are appealing probes for immune landscape profiling, as they display high stability, rapid targeting, and excellent specificity, while allowing extremely sensitive nuclear readouts. Here, we present a protocol for radiolabeling an anti-CD11b nanobody and studying its uptake in mice by a combination of positron emission tomography imaging, ex vivo gamma counting, and autoradiography. Our protocol is applicable to nanobodies against other antigens. For complete details on the use and execution of this protocol, please see Priem et al. (2020), Senders et al. (2019), or Rashidian et al. (2017).

Imaging Cardiovascular and Lung Macrophages With the Positron Emission Tomography Sensor 64Cu-Macrin in Mice, Rabbits, and Pigs.

BACKGROUND: Macrophages, innate immune cells that reside in all organs, defend the host against infection and injury. In the heart and vasculature, inflammatory macrophages also enhance tissue damage and propel cardiovascular diseases. METHODS: We here use in vivo positron emission tomography (PET) imaging, flow cytometry, and confocal microscopy to evaluate quantitative noninvasive assessment of cardiac, arterial, and pulmonary macrophages using the nanotracer 64Cu-Macrin-a 20-nm spherical dextran nanoparticle assembled from nontoxic polyglucose. RESULTS: PET imaging using 64Cu-Macrin faithfully reported accumulation of macrophages in the heart and lung of mice with myocardial infarction, sepsis, or pneumonia. Flow cytometry and confocal microscopy detected the near-infrared fluorescent version of the nanoparticle (VT680Macrin) primarily in tissue macrophages. In 5-day-old mice, 64Cu-Macrin PET imaging quantified physiologically more numerous cardiac macrophages. Upon intravenous administration of 64Cu-Macrin in rabbits and pigs, we detected heightened macrophage numbers in the infarcted myocardium, inflamed lung regions, and atherosclerotic plaques using a clinical PET/magnetic resonance imaging scanner. Toxicity studies in rats and human dosimetry estimates suggest that 64Cu-Macrin is safe for use in humans. CONCLUSIONS: Taken together, these results indicate 64Cu-Macrin could serve as a facile PET nanotracer to survey spatiotemporal macrophage dynamics during various physiological and pathological conditions. 64Cu-Macrin PET imaging could stage inflammatory cardiovascular disease activity, assist disease management, and serve as an imaging biomarker for emerging macrophage-targeted therapeutics.

Probing myeloid cell dynamics in ischaemic heart disease by nanotracer hot-spot imaging.

Ischaemic heart disease evokes a complex immune response. However, tools to track the systemic behaviour and dynamics of leukocytes non-invasively in vivo are lacking. Here, we present a multimodal hot-spot imaging approach using an innovative high-density lipoprotein-derived nanotracer with a perfluoro-crown ether payload (19F-HDL) to allow myeloid cell tracking by 19F magnetic resonance imaging. The 19F-HDL nanotracer can additionally be labelled with zirconium-89 and fluorophores to detect myeloid cells by in vivo positron emission tomography imaging and optical modalities, respectively. Using our nanotracer in atherosclerotic mice with myocardial infarction, we observed rapid myeloid cell egress from the spleen and bone marrow by in vivo 19F-HDL magnetic resonance imaging. Concurrently, using ex vivo techniques, we showed that circulating pro-inflammatory myeloid cells accumulated in atherosclerotic plaques and at the myocardial infarct site. Our multimodality imaging approach is a valuable addition to the immunology toolbox, enabling the study of complex myeloid cell behaviour dynamically.

Nanoimmunotherapy to treat ischaemic heart disease.

Atherosclerosis is a chronic disease of the large arteries and the underlying cause of myocardial infarction and stroke. Atherosclerosis is driven by cholesterol accumulation and subsequent inflammation in the vessel wall. Despite the clinical successes of lipid-lowering treatments, atherosclerosis remains one of the major threats to human health worldwide. Over the past 20 years, insights into cardiovascular immunopathology have provided a plethora of new potential therapeutic targets to reduce the risk of atherosclerosis and have shifted the therapeutic focus from lipids to inflammation. In 2017, the CANTOS trial demonstrated for the first time the beneficial effects of targeting inflammation to treat cardiovascular disease by showing that IL-1ß inhibition can reduce the recurrence rate of cardiovascular events in a large cohort of patients. At the same time, preclinical studies have highlighted nanotechnology approaches that facilitate the specific targeting of innate immune cells, which could potentially generate more effective immunomodulatory treatments to induce disease regression and prevent the recurrence of cardiovascular events. The clinical translation of such nanoimmunotherapies and their application to treat patients with ischaemic heart disease are challenges that lie ahead.

Hybrid PET/MR Kernelised Expectation Maximisation Reconstruction for Improved Image-Derived Estimation of the Input Function from the Aorta of Rabbits.

Positron emission tomography (PET) provides simple noninvasive imaging biomarkers for multiple human diseases which can be used to produce quantitative information from single static images or to monitor dynamic processes. Such kinetic studies often require the tracer input function (IF) to be measured but, in contrast to direct blood sampling, the image-derived input function (IDIF) provides a noninvasive alternative technique to estimate the IF. Accurate estimation can, in general, be challenging due to the partial volume effect (PVE), which is particularly important in preclinical work on small animals. The recently proposed hybrid kernelised ordered subsets expectation maximisation (HKEM) method has been shown to improve accuracy and contrast across a range of different datasets and count levels and can be used on PET/MR or PET/CT data. In this work, we apply the method with the purpose of providing accurate estimates of the aorta IDIF for rabbit PET studies. In addition, we proposed a method for the extraction of the aorta region of interest (ROI) using the MR and the HKEM image, to minimise the PVE within the rabbit aortic region-a method which can be directly transferred to the clinical setting. A realistic simulation study was performed with ten independent noise realisations while two, real data, rabbit datasets, acquired with the Biograph Siemens mMR PET/MR scanner, were also considered. For reference and comparison, the data were reconstructed using OSEM, OSEM with Gaussian postfilter and KEM, as well as HKEM. The results across the simulated datasets and different time frames show reduced PVE and accurate IDIF values for the proposed method, with 5% average bias (0.8% minimum and 16% maximum bias). Consistent results were obtained with the real datasets. The results of this study demonstrate that HKEM can be used to accurately estimate the IDIF in preclinical PET/MR studies, such as rabbit mMR data, as well as in clinical human studies. The proposed algorithm is made available as part of an open software library, and it can be used equally successfully on human or animal data acquired from a variety of PET/MR or PET/CT scanners.

Nanobody-Facilitated Multiparametric PET/MRI Phenotyping of 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 (64Cu), 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 (68Ga), a short-lived radioisotope with high clinical relevance, and used in an ensuing atherosclerosis progression PET/MRI study. Macrophage burden was longitudinally studied by 68Ga-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 (18F)-labeled fluorodeoxyglucose (18F-FDG) and 18F-sodium fluoride (18F-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.

Imaging-assisted nanoimmunotherapy for atherosclerosis in multiple species.

Nanomedicine research produces hundreds of studies every year, yet very few formulations have been approved for clinical use. This is due in part to a reliance on murine studies, which have limited value in accurately predicting translational efficacy in larger animal models and humans. Here, we report the scale-up of a nanoimmunotherapy from mouse to large rabbit and porcine atherosclerosis models, with an emphasis on the solutions we implemented to overcome production and evaluation challenges. Specifically, we integrated translational imaging readouts within our workflow to both analyze the nanoimmunotherapeutic's in vivo behavior and assess treatment response in larger animals. We observed our nanoimmunotherapeutic's anti-inflammatory efficacy in mice, as well as rabbits and pigs. Nanoimmunotherapy-mediated reduction of inflammation in the large animal models halted plaque progression, supporting the approach's translatability and potential to acutely treat atherosclerosis.

Development and Multiparametric Evaluation of Experimental Atherosclerosis in Rabbits.

Several animal models have been developed to study atherosclerosis. Here we present a rabbit atherosclerosis model generated by surgical denudation of the aortic endothelium in combination with a high-fat and cholesterol-enriched diet. This model is characterized by the formation of vascular lesions that exhibit several hallmarks of human atherosclerosis. Due to the rabbit's relative large size, as compared to rodents, this model is suited for the imaging-guided evaluation of novel therapeutic strategies using clinical scanners. In this chapter, we present an extensive outline of the procedures to induce aortic atherosclerotic lesions in rabbits as well as methods to evaluate the disease, including noninvasive in vivo multiparametric imaging and histopathology.

PET/MR Imaging of Malondialdehyde-Acetaldehyde Epitopes With a Human Antibody Detects Clinically Relevant Atherothrombosis.

BACKGROUND: Oxidation-specific epitopes (OSEs) are proinflammatory, and elevated levels in plasma predict cardiovascular events. OBJECTIVES: The purpose of this study was to develop novel positron emission tomography (PET) probes to noninvasively image OSE-rich lesions. METHODS: An antigen-binding fragment (Fab) antibody library was constructed from human fetal cord blood. After multiple rounds of screening against malondialdehyde-acetaldehyde (MAA) epitopes, the Fab LA25 containing minimal nontemplated insertions in the CDR3 region was identified and characterized. In mice, pharmacokinetics, biodistribution, and plaque specificity studies were performed with Zirconium-89 (89Zr)-labeled LA25. In rabbits, 89Zr-LA25 was used in combination with an integrated clinical PET/magnetic resonance (MR) system. 18F-fluorodeoxyglucose PET and dynamic contrast-enhanced MR imaging were used to evaluate vessel wall inflammation and plaque neovascularization, respectively. Extensive ex vivo validation was carried out through a combination of gamma counting, near infrared fluorescence, autoradiography, immunohistochemistry, and immunofluorescence. RESULTS: LA25 bound specifically to MAA epitopes in advanced and ruptured human atherosclerotic plaques with accompanying thrombi and in debris from distal protection devices. PET/MR imaging 24 h after injection of 89Zr-LA25 showed increased uptake in the abdominal aorta of atherosclerotic rabbits compared with nonatherosclerotic control rabbits, confirmed by ex vivo gamma counting and autoradiography. 18F-fluorodeoxyglucose PET, dynamic contrast-enhanced MR imaging, and near-infrared fluorescence signals were also significantly higher in atherosclerotic rabbit aortas compared with control aortas. Enhanced liver uptake was also noted in atherosclerotic animals, confirmed by the presence of MAA epitopes by immunostaining. CONCLUSIONS: 89Zr-LA25 is a novel PET radiotracer that may allow noninvasive phenotyping of high-risk OSE-rich lesions.

Efficacy and safety assessment of a TRAF6-targeted nanoimmunotherapy in atherosclerotic mice and non-human primates.

Macrophage accumulation in atherosclerosis is directly linked to the destabilization and rupture of plaque, causing acute atherothrombotic events. Circulating monocytes enter the plaque and differentiate into macrophages, where they are activated by CD4+ T lymphocytes through CD40-CD40 ligand signalling. Here, we report the development and multiparametric evaluation of a nanoimmunotherapy that moderates CD40-CD40 ligand signalling in monocytes and macrophages by blocking the interaction between CD40 and tumour necrosis factor receptor-associated factor 6 (TRAF6). We evaluated the biodistribution characteristics of the nanoimmunotherapy in apolipoprotein E-deficient (Apoe-/-) mice and in non-human primates by in vivo positron-emission tomography imaging. In Apoe-/- mice, a 1-week nanoimmunotherapy treatment regimen achieved significant anti-inflammatory effects, which was due to the impaired migration capacity of monocytes, as established by a transcriptome analysis. The rapid reduction of plaque inflammation by the TRAF6-targeted nanoimmunotherapy and its favourable toxicity profiles in both mice and non-human primates highlights the translational potential of this strategy for the treatment of atherosclerosis.

Author Correction: Efficacy and safety assessment of a TRAF6-targeted nanoimmunotherapy in atherosclerotic mice and non-human primates.

In the version of this Article originally published, the surname of the author Edward A. Fisher was spelt incorrectly as 'Fischer'. This has now been corrected.

A systematic comparison of clinically viable nanomedicines targeting HMG-CoA reductase in inflammatory atherosclerosis.

Atherosclerosis is a leading cause of worldwide morbidity and mortality whose management could benefit from novel targeted therapeutics. Nanoparticles are emerging as targeted drug delivery systems in chronic inflammatory disorders. To optimally exploit nanomedicines, understanding their biological behavior is crucial for further development of clinically relevant and efficacious nanotherapeutics intended to reduce plaque inflammation. Here, three clinically relevant nanomedicines, i.e., high-density lipoprotein ([S]-HDL), polymeric micelles ([S]-PM), and liposomes ([S]-LIP), that are loaded with the HMG-CoA reductase inhibitor simvastatin [S], were evaluated in the apolipoprotein E-deficient (Apoe-/-) mouse model of atherosclerosis. We systematically employed quantitative techniques, including in vivo positron emission tomography imaging, gamma counting, and flow cytometry to evaluate the biodistribution, nanomedicines' uptake by plaque-associated macrophages/monocytes, and their efficacy to reduce macrophage burden in atherosclerotic plaques. The three formulations demonstrated distinct biological behavior in Apoe-/- mice. While [S]-PM and [S]-LIP possessed longer circulation half-lives, the three platforms accumulated to similar levels in atherosclerotic plaques. Moreover, [S]-HDL and [S]-PM showed higher uptake by plaque macrophages in comparison to [S]-LIP, while [S]-PM demonstrated the highest uptake by Ly6Chigh monocytes. Among the three formulations, [S]-PM displayed the highest efficacy in reducing macrophage burden in advanced atherosclerotic plaques. In conclusion, our data demonstrate that [S]-PM is a promising targeted drug delivery system, which can be advanced for the treatment of atherosclerosis and other inflammatory disorders in the clinical settings. Our results also emphasize the importance of a thorough understanding of nanomedicines' biological performance, ranging from the whole body to the target cells, as well drug retention in the nanoparticles. Such systematic investigations would allow rational applications of nanomaterials', beyond cancer, facilitating the expansion of the nanomedicine horizon.

Hyaluronan Nanoparticles Selectively Target Plaque-Associated Macrophages and Improve Plaque Stability in Atherosclerosis.

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.

Polyglucose nanoparticles with renal elimination and macrophage avidity facilitate PET imaging in ischaemic heart disease.

Tissue macrophage numbers vary during health versus disease. Abundant inflammatory macrophages destruct tissues, leading to atherosclerosis, myocardial infarction and heart failure. Emerging therapeutic options create interest in monitoring macrophages in patients. Here we describe positron emission tomography (PET) imaging with 18F-Macroflor, a modified polyglucose nanoparticle with high avidity for macrophages. Due to its small size, Macroflor is excreted renally, a prerequisite for imaging with the isotope flourine-18. The particle's short blood half-life, measured in three species, including a primate, enables macrophage imaging in inflamed cardiovascular tissues. Macroflor enriches in cardiac and plaque macrophages, thereby increasing PET signal in murine infarcts and both mouse and rabbit atherosclerotic plaques. In PET/magnetic resonance imaging (MRI) experiments, Macroflor PET imaging detects changes in macrophage population size while molecular MRI reports on increasing or resolving inflammation. These data suggest that Macroflor PET/MRI could be a clinical tool to non-invasively monitor macrophage biology.