Molecular Engineering of Amphiphilic Probes for Enhanced Extracellular Vesicle Imaging and Tumor Theranostics

The advancement of extracellular vesicle (EV)-based theranostics is significantly hampered by the limitations of conventional EV labeling dyes such as poor water solubility and single functionality, hindering precise tracking and therapeutic integration. To address these challenges, we present a molecular engineering strategy to design carbazole-based amphiphilic fluorescent probes for EVs, leveraging modulation via twisted intramolecular charge transfer (TICT). The optimized probe, CTTP3, exhibits a remarkable 208-fold fluorescence enhancement upon EV binding, attributed to TICT suppression. Theoretical calculations confirm that the designed molecular architecture facilitates stable membrane insertion. Notably, CTTP3-labeled EVs demonstrate dual functionality: fluorescence emission for high-contrast imaging of tumor-targeted EV trafficking and efficient generation of various reactive oxygen species (ROS) upon light irradiation. The fluorescence labeling enables real-time visualization of EV endocytosis dynamics. In addition, the labeled EVs achieve efficient tumor eradication through photodynamic therapy, achieving 76% tumor growth inhibition in vivo. This work established a molecular toolkit for precise and efficient EV-based phototheranostics.