A specific and adaptable approach to track CD206⁺ macrophages by molecular MRI and fluorescence imaging

<b>Rationale:</b> The mannose receptor (CD206, expressed by the gene <i>Mrc1</i>) is a surface marker overexpressed by anti-inflammatory and pro-tumoral macrophages. As such, CD206⁺ macrophages play key roles in the immune response to different pathophysiological conditions and represent a promising diagnostic and therapeutic target. However, methods to specifically target these cells remain challenging. In this study, we describe a multi-mannose approach to develop CD206-targeting fluorescent and MRI agents that specifically and sensitively detect and monitor CD206⁺ macrophage immune response in different disease conditions. <b>Methods:</b> We designed and synthesized fluorescent agents MR1-cy5 and MR2-cy5, and MRI agents Mann2-DTPA-Gd and MannGdFish. Cellular assays using pro-inflammatory and anti-inflammatory macrophages differentiated from RAW 264.7 cells were performed, and signals were detected by fluorescence microscopy and inductively coupled plasma mass spectrometry (ICP-MS) to validate specificity <i>in vitro</i>. <i>In vivo</i> specificity and efficacy of the agents were evaluated by MRI in a subcutaneous wound healing model and experimental glioma with <i>Mrc1</i> ^+/+ without and with D-mannose treatment, <i>Mrc1</i> ^+/-, and <i>Mrc1</i> ^-/- mice, and in stroke. One-way ANOVA and two-way ANOVA tests were used for data analysis. P < 0.05 was considered statistically different. <b>Results:</b> Both <i>in vitro</i> fluorescence imaging with MR2-cy5, ICP-MS with Mann2-DTPA-Gd, and <i>in vivo</i> MRI in <i>Mrc1</i> ^-/- mice confirmed the specificity of our approach. Mann2-DTPA-Gd MRI can track the changes of CD206⁺ macrophages at different stages of wound healing, correlating well with flow cytometry data using another anti-inflammatory macrophage marker (arginase-1). The specificity and efficacy of Mann2-DPTA-Gd were further validated in experimental glioma, in which Mann2-DTPA-Gd imaging detected CD206⁺ tumor-associated macrophages (TAMs), demonstrated significantly decreased signals in <i>Mrc1</i> ^+/- mice and <i>Mrc1</i> ^-/- mice, and tracked treatment changes in D-mannose-treated <i>Mrc1</i> ^+/+ mice. Furthermore, Mann2-DTPA-Gd can report microglia/macrophages and correlate with histology in stroke. The more Gd-stable agent MannGdFish demonstrated similar efficacy as Mann2-DTPA-Gd <i>in vivo</i> with favorable biodistribution and pharmacokinetics. <b>Conclusion:</b> We have developed a fluorescent agent (MR2-cy5) and MRI agents (Mann2-DTPA-Gd and MannGdFish) with two mannose moieties that are highly specific to CD206 and can track CD206⁺ macrophages in disease models of wound healing, tumor, and neurological disease. Importantly, MannGdFish, with its high specificity, stability, favorable biodistribution, and pharmacokinetics, is a promising translational candidate to noninvasively monitor CD206⁺ macrophages in repair/regeneration and tumors in patients. In addition, with the specific binding motif to CD206, other imaging modalities and therapeutic agents could also be introduced for theranostic applications.