Macrophage PET imaging in mouse models of cardiovascular disease and cancer with an apolipoprotein-inspired radiotracer.

Macrophages are key inflammatory mediators in many pathological conditions, including cardiovascular disease (CVD) and cancer, the leading causes of morbidity and mortality worldwide. This makes macrophage burden a valuable diagnostic marker and several strategies to monitor these cells have been reported. However, such strategies are often high-priced, non-specific, invasive, and/or not quantitative. Here, we developed a positron emission tomography (PET) radiotracer based on apolipoprotein A1 (ApoA1), the main protein component of high-density lipoprotein (HDL), which has an inherent affinity for macrophages. We radiolabeled an ApoA1-mimetic peptide (mA1) with zirconium-89 (89Zr) to generate a lipoprotein-avid PET probe (89Zr-mA1). We first characterized 89Zr-mA1's affinity for lipoproteins in vitro by size exclusion chromatography. To study 89Zr-mA1's in vivo behavior and interaction with endogenous lipoproteins, we performed extensive studies in wildtype C57BL/6 and Apoe-/- hypercholesterolemic mice. Subsequently, we used in vivo PET imaging to study macrophages in melanoma and myocardial infarction using mouse models. The tracer's cell specificity was assessed by histology and mass cytometry (CyTOF). Our data show that 89Zr-mA1 associates with lipoproteins in vitro. This is in line with our in vivo experiments, in which we observed longer 89Zr-mA1 circulation times in hypercholesterolemic mice compared to C57BL/6 controls. 89Zr-mA1 displayed a tissue distribution profile similar to ApoA1 and HDL, with high kidney and liver uptake as well as substantial signal in the bone marrow and spleen. The tracer also accumulated in tumors of melanoma-bearing mice and in the ischemic myocardium of infarcted animals. In these sites, CyTOF analyses revealed that natZr-mA1 was predominantly taken up by macrophages. Our results demonstrate that 89Zr-mA1 associates with lipoproteins and hence accumulates in macrophages in vivo. 89Zr-mA1's high uptake in these cells makes it a promising radiotracer for non-invasively and quantitatively studying conditions characterized by marked changes in macrophage burden.

Protocol for the development of mRNA lipid nanoparticle vaccines and analysis of immunization efficiency in mice.

Here, we present a protocol for the development of mRNA-loaded lipid nanoparticle (LNP) vaccines for target antigen sequences of interest. We describe key steps required to design and synthesize mRNA constructs, their LNP encapsulation, and mouse immunization. We then detail quality control assays to determine RNA purity, guidelines to measure RNA immunogenicity using in vitro reporter systems, and a technique to evaluate antigen-specific T cell responses following immunization.

Multiparametric Immunoimaging Maps Inflammatory Signatures in Murine Myocardial Infarction Models.

In the past 2 decades, research on atherosclerotic cardiovascular disease has uncovered inflammation to be a key driver of the pathophysiological process. A pressing need therefore exists to quantitatively and longitudinally probe inflammation, in preclinical models and in cardiovascular disease patients, ideally using non-invasive methods and at multiple levels. Here, we developed and employed in vivo multiparametric imaging approaches to investigate the immune response following myocardial infarction. The myocardial infarction models encompassed either transient or permanent left anterior descending coronary artery occlusion in C57BL/6 and Apoe-/-mice. We performed nanotracer-based fluorine magnetic resonance imaging and positron emission tomography (PET) imaging using a CD11b-specific nanobody and a C-C motif chemokine receptor 2-binding probe. We found that immune cell influx in the infarct was more pronounced in the permanent occlusion model. Further, using 18F-fluorothymidine and 18F-fluorodeoxyglucose PET, we detected increased hematopoietic activity after myocardial infarction, with no difference between the models. Finally, we observed persistent systemic inflammation and exacerbated atherosclerosis in Apoe-/- mice, regardless of which infarction model was used. Taken together, we showed the strengths and capabilities of multiparametric imaging in detecting inflammatory activity in cardiovascular disease, which augments the development of clinical readouts.

Embracing nanomaterials' interactions with the innate immune system.

Immunotherapy has firmly established itself as a compelling avenue for treating disease. Although many clinically approved immunotherapeutics engage the adaptive immune system, therapeutically targeting the innate immune system remains much less explored. Nanomedicine offers a compelling opportunity for innate immune system engagement, as many nanomaterials inherently interact with myeloid cells (e.g., monocytes, macrophages, neutrophils, and dendritic cells) or can be functionalized to target their cell-surface receptors. Here, we provide a perspective on exploiting nanomaterials for innate immune system regulation. We focus on specific nanomaterial design parameters, including size, form, rigidity, charge, and surface decoration. Furthermore, we examine the potential of high-throughput screening and machine learning, while also providing recommendations for advancing the field. This article is categorized under: Nanotechnology Approaches to Biology > Nanoscale Systems in Biology Diagnostic Tools > In Vivo Nanodiagnostics and Imaging Therapeutic Approaches and Drug Discovery > Nanomedicine for Oncologic Disease.

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.

Trained Immunity-Promoting Nanobiologic Therapy Suppresses Tumor Growth and Potentiates Checkpoint Inhibition.

Trained immunity, a functional state of myeloid cells, has been proposed as a compelling immune-oncological target. Its efficient induction requires direct engagement of myeloid progenitors in the bone marrow. For this purpose, we developed a bone marrow-avid nanobiologic platform designed specifically to induce trained immunity. We established the potent anti-tumor capabilities of our lead candidate MTP10-HDL in a B16F10 mouse melanoma model. These anti-tumor effects result from trained immunity-induced myelopoiesis caused by epigenetic rewiring of multipotent progenitors in the bone marrow, which overcomes the immunosuppressive tumor microenvironment. Furthermore, MTP10-HDL nanotherapy potentiates checkpoint inhibition in this melanoma model refractory to anti-PD-1 and anti-CTLA-4 therapy. Finally, we determined MTP10-HDL's favorable biodistribution and safety profile in non-human primates. In conclusion, we show that rationally designed nanobiologics can promote trained immunity and elicit a durable anti-tumor response either as a monotherapy or in combination with checkpoint inhibitor drugs.

Tumor Targeting by αvß3-Integrin-Specific Lipid Nanoparticles Occurs via Phagocyte Hitchhiking.

Although the first nanomedicine was clinically approved more than two decades ago, nanoparticles' (NP) in vivo behavior is complex and the immune system's role in their application remains elusive. At present, only passive-targeting nanoformulations have been clinically approved, while more complicated active-targeting strategies typically fail to advance from the early clinical phase stage. This absence of clinical translation is, among others, due to the very limited understanding for in vivo targeting mechanisms. Dynamic in vivo phenomena such as NPs' real-time targeting kinetics and phagocytes' contribution to active NP targeting remain largely unexplored. To better understand in vivo targeting, monitoring NP accumulation and distribution at complementary levels of spatial and temporal resolution is imperative. Here, we integrate in vivo positron emission tomography/computed tomography imaging with intravital microscopy and flow cytometric analyses to study αvß3-integrin-targeted cyclic arginine-glycine-aspartate decorated liposomes and oil-in-water nanoemulsions in tumor mouse models. We observed that ligand-mediated accumulation in cancerous lesions is multifaceted and identified "NP hitchhiking" with phagocytes to contribute considerably to this intricate process. We anticipate that this understanding can facilitate rational improvement of nanomedicine applications and that immune cell-NP interactions can be harnessed to develop clinically viable nanomedicine-based immunotherapies.

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.