NIR-II Imaging for Tracking the Spatiotemporal Immune Microenvironment in Atherosclerotic Plaques

The inflammatory immune microenvironment is responsible for atherosclerotic plaque erosion and rupture. Near-infrared-II (NIR-II) fluorescence imaging has the potential to continuously monitor the spatiotemporal changes in the plaque immune microenvironment. Herein, we constructed three different NIR-II probes based on benzo[1,2-<i>c</i>;4,5-<i>c</i>']bis[1,2,5]thiadiazole-4,7-bis(9,9-dioctyl-9<i>H</i>-fluoren-2-yl)thiophene (denoted as BBT-2FT): VHPK/BBT-2FT NPs, where VHPK is a specific peptide targeting vascular cell adhesion molecule-1; iNOS/BBT-2FT NPs for modulating the polarization of M1 macrophages by inducible NO synthase (iNOS) antibodies; and Arg-1/BBT-2FT for counterbalancing the inflammatory responses of M1 macrophages. These tracers enable precise tracking of atherosclerotic plaques and M1 and M2 macrophages through NIR-II imaging. VHPK/BBT-2FT NPs can accurately trace atherosclerotic plaques at various stages. Arg-1/BBT-2FT NPs precisely located M2 macrophages in the early plaque microenvironment with upregulation of peroxisome proliferator-activated receptor γ (PPAR-γ), signal transducer and activator of transcription (STAT) 6, and ATP-binding cassette transporter A1 (ABCA1), indicating that M2 macrophage polarization is crucial for early plaque lipid clearance. Meanwhile, iNOS/BBT-2FT NPs accurately tracked M1 macrophages in the advanced plaque microenvironment. The results showed that M1 macrophage polarization induces the formation of an inflammatory microenvironment through anaerobic glycolytic metabolism and pyroptosis in the advanced hypoxic plaque microenvironment, as indicated by the upregulation of hypoxia-inducible factor 1 alpha (HIF-1α), STAT1, NOD-, LRR-, and pyrin domain-containing protein 3 (NLRP3), pyruvate dehydrogenase kinase 1 (PDK1), and glucose transporter 1 (GLUT-1). Combining immunological approaches with NIR-II imaging has revealed that hypoxia-induced metabolic reprogramming of macrophages is a key factor in dynamic changes in the immune microenvironment of atherosclerotic plaques. Furthermore, our strategy shows the potential for real-time diagnosis and clinical prevention of unstable plaque rupture in atherosclerosis.