Atherosclerosis is at the onset of the cardiovascular diseases that are among the leading causes of death worldwide. Currently, high-risk plaques, also called vulnerable atheromatous plaques, remain often undiagnosed until the occurrence of severe complications, such as stroke or myocardial infarction. Molecular imaging agents that target high-risk atheromatous lesions could greatly improve the diagnosis of atherosclerosis by identifying sites of high disease activity. Moreover, a “theranostic approach” that combines molecular imaging agents (for diagnosis) and therapeutic molecules would be of great value for the local management of atheromatous plaques. The aim of this study was to develop and characterize an innovative theranostic tool for atherosclerosis. We engineered oil-in-water nano-emulsions (NEs) loaded with superparamagnetic iron oxide (SPIO) nanoparticles for magnetic resonance imaging (MRI) purposes. Dynamic MRI showed that NE-SPIO nanoparticles decorated with a polyethylene glycol (PEG) layer reduced their liver uptake and extended their half-life. Next, the NE-SPIO-PEG formulation was functionalized with a fully human scFv-Fc antibody (P3) recognizing galectin 3, an atherosclerosis biomarker. The P3-functionalized formulation targeted atheromatous plaques, as demonstrated in an immunohistochemistry analyses of mouse aorta and human artery sections and in an Apoe^−/− mouse model of atherosclerosis. Moreover, the formulation was loaded with SPIO nanoparticles and/or alpha-tocopherol to be used as a theranostic tool for atherosclerosis imaging (SPIO) and for delivery of drugs that reduce oxidation (here, alpha-tocopherol) in atheromatous plaques. This study paves the way to non-invasive targeted imaging of atherosclerosis and synergistic therapeutic applications.

Atherosclerosis is a chronic, progressive inflammatory disease that may develop into vulnerable lesions leading to thrombosis. This pathology is characterized by the deposition of lipids within the arterial wall and infiltration of immune cells leading to amplification of inflammation. Nowadays there is a rising interest to assess directly the molecular and cellular components that underlie the clinical condition of stroke and myocardial infarction. Single chain fragment variable (scFv)-phages issuing from a human combinatorial library were selected on the lesions induced in a rabbit model of atherosclerosis after three rounds of in vivo phage display. We further implemented a high-throughput flow cytometry method on rabbit protein extracts to individually test one thousand of scFv-phages. Two hundred and nine clones were retrieved on the basis of their specificity for atherosclerotic proteins. Immunohistochemistry assays confirmed the robustness of the designed cytometry protocol. Sequencing of candidates demonstrated their high diversity in VH and VL germline usage. The large number of candidates and their diversity open the way in the discovery of new biomarkers. Here, we successfully showed the capacity of combining in vivo phage display and high-throughput cytometry strategies to give new insights in in vivo targetable up-regulated biomarkers in atherosclerosis.

Background: Atherosclerosis is a complex pathology in which dysfunctional endothelium, activated leucocytes, macrophages, and lipid‐laden foam cells are implicated, and in which plaque disruption is driven by many putative actors. This study aimed to identify accurate targetable biomarkers using new in vivo approaches to propose tools for improved diagnosis and treatment. Methods and Results: Human scFv (single‐chain fragment variable) selected by in vivo phage display in a rabbit model of atherosclerosis was reformatted as scFv fused to the scFv‐Fc (single‐chain fragment variable fused to the crystallizable fragment of immunoglobulin G format) antibodies. Their reactivity was tested using flow cytometry and immunoassays, and aorta sections from animal models and human carotid and coronary artery specimens. A pool of atherosclerotic proteins from human endarterectomies was co‐immunoprecipitated with the selected scFv‐Fc followed by mass spectrometry for target identification. Near‐infrared fluorescence imaging was performed in Apoe ^−/− mice after injection of an Alexa Fluor 647–labeled scFv‐Fc‐2c antibody produced in a baculovirus system with 2 additional cysteine residues (ie, 2c) for future coupling to nano‐objects for theranostic applications. One scFv‐Fc clone (P3) displayed the highest cross‐reactivity against atherosclerotic lesion sections (rabbit, mouse, and human) and was chosen for translational development. Mass spectrometry identified galectin‐3, a β‐galactoside‐binding lectin, as the leader target. ELISA and immunofluorescence assays with a commercial anti‐galectin‐3 antibody confirmed this specificity. P3 scFv‐Fc‐2c specifically targeted atherosclerotic plaques in the Apoe ^−/− mouse model. Conclusions: These results provide evidence that the P3 antibody holds great promise for molecular imaging of atherosclerosis and other inflammatory pathologies involving macrophages. Recently, galectin‐3 was proposed as a high‐value biomarker for the assessment of coronary and carotid atherosclerosis.

Different types of macrophages (Mφ) are involved in atherogenesis, including inflammatory Mφ and foamy Mφ (FM). Our previous study demonstrated that two-photon excited fluorescence (TPEF) imaging of NADH and FAD autofluorescence (AF) could distinguish experimental models that mimic the different atherosclerotic Mφ types. The present study assessed whether optical differences correlated with phenotypic and functional differences, potentially guiding diagnostic and therapeutic strategies. Phenotypic differences were investigated using three-dimensional principal component analysis and multi-color flow cytometry. Functional analyses focused on cytokine production, metabolic profiles, and cellular oxidative stress, in LDL dose-dependent assays, to understand the origin of AF in the FAD spectrum and assess FM ability to transition toward an immunoregulatory phenotype and function. Phenotypic studies revealed that FM models generated with acetylated LDL (Mac) were closer to immunoregulatory Mφ, while those generated with oxidized LDL (Mox) more closely resembled inflammatory Mφ. The metabolic analysis confirmed that inflammatory Mφ primarily used glycolysis, while immunoregulatory Mφ mainly depended on mitochondrial respiration. FM models employed both pathways; however, FM models generated with high doses of modified LDL showed reduced mitochondrial respiration, particularly Mox FM. Thus, the high AF in the FAD spectrum in Mox was not linked to increased mitochondrial respiration, but correlated with the dose of oxidized LDL, leading to increased production of reactive oxygen species (ROS) and lysosomal ceroid accumulation. High FAD-like AF, ROS, and ceroid accumulation were reduced by incubation with α-tocopherol. The cytokine profiles supported the phenotypic analysis, indicating that Mox FM exhibited greater inflammatory activity than Mac FM, although both could be redirected toward immunoregulatory functions, albeit to different degrees. In conclusion, in the context of immunoregulatory therapies for atherosclerosis, it is crucial to consider FM, given their prevalence in plaques and our results, as potential targets, regardless of their inflammatory status, alongside non-foamy inflammatory Mφ.