Squaraine-Peptide Conjugates as Efficient Reporters of Neutrophil Extracellular Traps-Mediated Chronic Inflammation

The excessive and uncontrolled release of neutrophil extracellular traps (NETs) is increasingly linked to the pathogenesis of various inflammatory diseases, cardiovascular disorders, and cancers. Real-time, non-invasive detection of NETs is crucial for understanding their role in disease progression and developing targeted therapies. Current NETs detection methods often lack the necessary specificity and resolution, particularly in vivo and ex vivo settings. To address this, we have developed novel near-infrared squaraine-peptide conjugates by rational molecular design as reporters of NETosis by targeting the protease activity of neutrophil elastase (NE). These self-quenching, cell-impermeable probes enable the precise real-time detection and imaging of NETs. The Förster resonance energy transfer (FRET)-based probe, Hetero-APA, demonstrated high specificity in detecting NETs in vitro and in vivo, generating strong fluorescence in NETs-rich environments. To overcome the limitations of FRET-based probes for ex vivo imaging, we designed SQ-215-NETP, a non-FRET-based probe that covalently binds to the NE. SQ-215-NETP achieved an unprecedented imaging resolution of 90 nm/pixel in human coronary thrombi, marking the first report of such high resolution with a low molecular weight probe. Additionally, SQ-215-NETP effectively detected NETs by flow cytometry. These results highlight the potential of these probes in NETosis detection, offering promising tools for enhanced diagnostics and therapeutic strategies in managing NET-mediated inflammatory diseases and cancers.