Charge transfer modulation in anionic Cy5-Protein afterglow nanoprobe enables precise pancreatic cancer surgery and...

Reactive oxygen species-mediated afterglow (ROSAF) probes offer great potential in cancer theranostics for their high signal-to-background ratios (SBRs) and intrinsic photodynamic activity. However, previous ROSAF probes often require nanocarriers for tumor delivery and suffer from leakage-related signal distortion and oxygen-dependent efficacy loss in hypoxic tumor. Herein, we engineered the negative charge transfer in anionic pentamethine cyanine (ACy5) by rationally introducing strongly electron-withdrawing meso-substituents based on SOCT-ISC. The optimized ROSAF, ACy5-NPy, exhibits a 110 nm red shift in absorption/emission compared to classical Cy5 and stably binds serum albumin (SA). Upon complexation with SA, ACy5-NPy transforms into a powerful type-I ROSAF nanoprobe (ACy5-NPy@BSA), showing a 27.7-fold afterglow enhancement over its monomeric form even under hypoxia. Thanks to oxygen independence and innate tumor-targeting, ACy5-NPy@BSA enables 30-min high-contrast afterglow imaging of pancreatic tumors, with a SBR up to 33.3 and precise lesion delineation. This allows precise afterglow surgical navigation and thorough resection, preventing recurrence for 24 days even in multifocal lesions. Moreover, it mediates efficient PDT, significantly suppressing pancreatic tumor growth and metastasis by activating pyroptosis, supported by the reduced serum levels of cancer markers. Systematic modulation of negative charge transfer yields the first protein afterglow nanoprobe, providing a new strategy for afterglow probe design in cancer management.