Self-Reporting Photodynamic Nanobody Conjugate for Precise and Sustainable Large-Volume Tumor Treatment

Abstract Nanobodies (Nbs), the smallest antigen-binding fragments with high stability and affinity, bring a new opportunity to improve the specificity of photosensitizers for tumor tissue in photodynamic therapy (PDT) through an active targeting strategy. Nonetheless, the rapid metabolism of Nbs in vivo restricts the accumulation and retention of the photosensitizer at the tumor site, especially in large-volume tumors. Herein, we developed photodynamic conjugates, MNB-Pyra Nbs, through site-specific modification of 7D12 Nbs with the type I photosensitizer MNB-Pyra in a 1:2 ratio. The π − π stacking interactions of MNB-Pyra molecules within the conjugate causes fluorescence quenching, while the photosensitizer with long-term retention could be released by reactive oxygen species (ROS) cleavage, which is accompanied by fluorescence recovery for self-reporting. Moreover, the conjugation of MNB-Pyra and Nbs greatly improved the metabolism of the benzophenothiazine photosensitizer in vivo, leading to the clearance of MNB-Pyra Nbs in 24 h without illumination. Ultimately, a single dose of MNB-Pyra Nbs demonstrated highly effective tumor suppression (tumor inhibition rate > 95%) with high biosafety in the large-volume tumor model after three rounds of PDT. This self-reporting photodynamic nanobody conjugate is expected to promote the development of personalized precision PDT.