Tuning Lipophilicity to Develop an MMP-Independent Fluorescent Probe for Visualizing Mitochondrial Peroxynitrite in...

Area of Science:

• Biomedical Engineering
• Molecular Biology
• Biochemistry

Background:

• Mitochondrial peroxynitrite (ONOO-) is a key mediator in oxidative stress and disease pathogenesis.
• Existing ONOO- detection probes rely on mitochondrial membrane potential (MMP), limiting their use in disease states.
• Real-time detection of mitochondrial ONOO- is crucial for understanding cellular redox homeostasis.

Purpose of the Study:

• To develop and evaluate an MMP-independent, near-infrared fluorescent probe for mitochondrial ONOO- detection.
• To create a tool for real-time monitoring of mitochondrial ONOO- dynamics in biological systems.
• To investigate the probe's utility in cellular and animal models of disease.

Main Methods:

• Rational design and synthesis of a series of fluorophores (Flu-(1-5)) with varying alkyl chain lengths to optimize lipophilicity and mitochondrial retention.
• Screening of fluorophores for mitochondria-targeting capability under ONOO- stress.
• Characterization of the selected probe (Mito-ONOO-) for Stokes shift, response time, mitochondrial localization, selectivity, and sensitivity in HepG2 cells and a DILI mice model.

Main Results:

• Flu-5 was selected for its optimal lipophilicity and mitochondrial retention, leading to the development of Mito-ONOO-.
• Mito-ONOO- exhibited a significant Stokes shift (88 nm), rapid response (~1 min), and high mitochondrial localization (PC = 0.96).
• The probe successfully visualized mitochondrial ONOO- in living HepG2 cells and monitored ONOO- dynamics in a drug-induced liver injury mouse model.

Conclusions:

• Mito-ONOO- is an effective MMP-independent probe for sensitive and selective detection of mitochondrial peroxynitrite.
• The probe allows for real-time imaging of mitochondrial ONOO- in live cells and disease models.
• Mito-ONOO- holds significant potential for studying mitochondrial redox homeostasis and its role in pathological conditions.