Analytica chimica acta · 2025
Fluorescent light-up probes for legumain imaging in macrophages: Minimal disturbance to macrophage cytoskeleton.
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Zhang Jiajing, Chen Xiaotong, Ming Ruiqi, Huang Lili, Zhang Ruoyu
Аннотация
BACKGROUND: Legumain is a ubiquitously expressed protease playing vital parts in multiple pathological conditions, such as cancerous and inflammatory diseases including pancreatitis, fibrosis and atherosclerosis. The multiple links between legumain and various disease may derive from its role in immune cell infiltration and antigen presenting process. Despite its established clinical associations, the mechanistic interplay between legumain mediated immune cross-talk and disease pathogenesis remains poorly understood. However, there are very limited real-time legumain probes reported. The fact emphasizes the importance of developing legumain-specific fluorescent probe, particularly in leukocytes such as macrophages.
RESULTS: First, a series legumain-specific AIE-peptide probes have been developed and applied for the real-time fluorescent imaging of legumain in subphenotypes of macrophages. Human macrophages were derived from THP-1 cells and induced into M0, M1 and M2 macrophage, which was validated by flow cytometry by recognizing the fluorescent labeled surface markers. The probes showed significant real-time fluorescent turn-on upon legumain, which is useful for further investigation of inflammatory diseases. The good selectivity of the probes was proved by legumain inhibitor and immunofluorescence staining. In addition, by proper design, targeted legumain imaging in M2 macrophages can also be achieved. It is also noteworthy that the probes exert minimal disturbance to the cytoskeleton of macrophages, which indicates their good compatibility in sustaining the normal biological functions of macrophages.
SIGNIFICANCE: The probes have successfully realized the specific legumain sensing in human macrophages, both for any subphenotype or for targeted imaging in M2 macrophages. It is also noteworthy that the probes exerted minimal side effects on the cytoskeleton morphology of macrophages, which will benefit the fluorescent sensing of targets which levels are sensitive to the probe disturbance.
RESULTS: First, a series legumain-specific AIE-peptide probes have been developed and applied for the real-time fluorescent imaging of legumain in subphenotypes of macrophages. Human macrophages were derived from THP-1 cells and induced into M0, M1 and M2 macrophage, which was validated by flow cytometry by recognizing the fluorescent labeled surface markers. The probes showed significant real-time fluorescent turn-on upon legumain, which is useful for further investigation of inflammatory diseases. The good selectivity of the probes was proved by legumain inhibitor and immunofluorescence staining. In addition, by proper design, targeted legumain imaging in M2 macrophages can also be achieved. It is also noteworthy that the probes exert minimal disturbance to the cytoskeleton of macrophages, which indicates their good compatibility in sustaining the normal biological functions of macrophages.
SIGNIFICANCE: The probes have successfully realized the specific legumain sensing in human macrophages, both for any subphenotype or for targeted imaging in M2 macrophages. It is also noteworthy that the probes exerted minimal side effects on the cytoskeleton morphology of macrophages, which will benefit the fluorescent sensing of targets which levels are sensitive to the probe disturbance.