Activity-Based Fluorescent Molecular Logic Gate Probe for Dynamic Tracking of Mitophagy Induced by Oxidative Stress

Abstract

Visualizing and modulating the mitophagy process is essential for understanding the role of mitophagy in cellular homeostasis, physiology, and pathology. To overcome the sensing limitation of available mitophagy probes to only lysosome fusion or degradation, a molecular logic gate probe showing multiple fluorescence responses to different mitophagy stages was proposed in this study to sense the oxidative stress-induced mitophagy via a dual-channel mode. This new fluorescent molecular logic gate probe, Mito-PN, was composed by integrating a peroxynitrite-responsive 1,8-naphthalimide with an acidity-activatable rhodamine spirolactam and possesses the mitochondria-targeting capability due to its triphenylphosphonium group. This probe is able to sense both the mitophagy initiation triggered by peroxynitrite and lysosome fusion at different fluorescence wavelengths. It can be rapidly activated by mitochondrial peroxynitrite to turn on the green fluorescence of naphthalimide, and subsequent lysosome/mitophagosome fusion activates the probe with protons to generate red fluorescence. Moreover, our preliminary results demonstrate that the fluorescence response of Mito-PN to peroxynitrite-induced mitophagy can be discriminated from the mitophagy stimulated by carbonyl cyanide m-chlorophenyl hydrazone, which further proves the specific mitophagy tracking ability of Mito-PN. Overall, this research offers a potentially powerful tool for studying the role played by peroxynitrite in mitophagy and provides a versatile strategy for monitoring oxidative stress-related pathological processes.

Heavy Atom‐Free, Mitochondria‐Targeted, and Activatable Photosensitizers for Photodynamic Therapy with Real‐Time In‐Situ Therapeutic Monitoring

“…As shown in Figure S11, at pH>7.0, BDP1 / BDP2 were nearly nonfluorescent due to the PeT‐induced fluorescence quenching; by comparison, at pH<7.0, their fluorescence was gradually enhanced and reached maximum at pH≈4.2, indicating that in weakly acidic lysosomes, the fluorescence of BDP1 / BDP2 could be lighted up by protonating their amino group, as the case of a recently reported lysosome probe bearing 2‐aminophenol unit as electron donor [58] . Further, we evaluated the fluorescence response of BDP1 (as a representative) to the extensive mitophagy induced by CCCP (carbonyl cyanide m ‐chlorophenylhydrazone), a protonophore known to cause extensive mitophagy and cell apoptosis by collapsing mitochondrial membrane potential [62, 63] . The process could be monitored under CLSM by a commercial mitophagy probe Mtphagy Dye (MD), essentially a mitochondria‐immobilized fluorescent pH probe based on PeT mechanism [64] .…”

“…[58] Further, we evaluated the fluorescence response of BDP1 (as a representative) to the extensive mitophagy induced by CCCP (carbonyl cyanide m-chlorophenylhydrazone), a protonophore known to cause extensive mitophagy and cell apoptosis by collapsing mitochondrial membrane potential. [62,63] The process could be monitored under CLSM by a commercial mitophagy probe Mtphagy Dye (MD), essentially a mitochondriaimmobilized fluorescent pH probe based on PeT mechanism. [64] As shown in Figure 7, when A549 cells were co-incubated with BDP1 and Mtphagy Dye (MD) for 30 min, only faint fluorescence was observed in both green (BDP1 channel: 493-550 nm) and red channel (MD channel: 650-747 nm), indicating that no extensive mitophagy occurred and both dyes were still located at mitochodria; in contrast, when A549 cells were co-incubated with BDP1 and MD for 30 min and then treated with CCCP for 6 h, the fluorescence in green and red channels was lighted up simultaneously, which overlaped well with a high Pearson's correlation coefficient (R = 0.89), indicating that the extensive mitophagy has occurred after CCCP stimulation and that BDP1 experienced the same fate as MD.…”

Advances in Applications of Molecular Logic Gates

“…How Mito-PN works in the mitophagy process. Reproduced from ref . Copyright 2021 American Chemical Society.…”

“…The logic gate can be used in the field of disease diagnosis. 66 presented a novel method for monitoring oxidative stress and subsequent mitophagy with a single fluorescent molecular logic gate probe by caging two fluorophores, 1,8-naphthalimide and rhodamine, with AP and spirolactam, respectively. Changing the recognition unit allows the molecular logic system to be extended to a general method for monitoring mitochondrial autophagy induced by oxidative stress (Figure 24).…”

“…Changing the recognition unit allows the molecular logic system to be extended to a general method for monitoring mitochondrial autophagy induced by oxidative stress (Figure 24). 66 3.4. Organic Molecule Detection.…”