A molecular rotor-based NIR probe with large stokes shift for highly sensitive tracking of mitochondrial viscosity in live tumors

Abnormal mitochondrial viscosity is closely associated with tumor initiation and progression. Therefore, the development of fluorescent probes capable of real-time, precise monitoring of mitochondrial viscosity changes in tumor cells holds significant potential for the early diagnosis and therapeutic evaluation of tumors. This study designed and synthesized a series of near-infrared (NIR) fluorescent probes ( W1–W3 ) with D-π-A structural framework based on a phenothiazine-bithiophene-pyridinium moiety. All three probes ( W1 – W3 ) featured emission at approximately 650 nm, accompanied by a substantial Stokes shift of about 150 nm. Among them, W2 and W3 exhibited excellent responsiveness to viscosity. Their molecular rotor character results in quenched fluorescence in low-viscosity environments and intensified emission in high-viscosity media, enabling highly sensitive detection of viscosity changes. Cellular experiments revealed that W2 possesses outstanding biocompatibility and precise mitochondria-targeting ability. Remarkably, it achieved effective bioimaging at a concentration as low as 500 nM. The probe also succeeded in dynamically monitoring viscosity changes during drug-induced (e.g., DXM, rotenone) mitochondrial dysfunction. Notably, W2 detected increased mitochondrial viscosity in tumor cells triggered by apoptosis inducers (e.g., DOX, cisplatin), thereby confirming its capability to track dynamic changes in mitochondrial viscosity. In vivo and in vitro imaging results established the potential of probe W2 for tumor imaging. With its capability for dynamic viscosity monitoring from cellular to in vivo levels, probe W2 positions itself as a promising tool to advance applications in early tumor diagnosis, drug efficacy assessment, and precision therapy.