Enzymatic In Situ Self‐Assembly of a Fluorogenic Photosensitizer Induces PANoptosis for Photodynamic‐Immunotherapy

ABSTRACT PANoptosis, an inflammatory programmed cell death that integrates pyroptosis, apoptosis, and necroptosis, holds great promise for cancer immunotherapy. However, conventional strategies relying on PANoptosome assembly are hindered by complex upstream signaling requirements. Here, we report P–CHD–TPP, an enzyme‐instructed in situ self‐assembling probe that triggers PANoptosome‐independent PANoptosis via persistent mitochondrial disruption. Upon alkaline phosphatase (ALP)‐triggered dephosphorylation, P–CHD–TPP self‐assembles into fluorescent nanoparticles decorated with triphenylphosphonium (TPP) and 1,3‐cyclohexanedione (CHD) groups, which are rapidly internalized by tumor cells and preferentially accumulate in mitochondria. The TPP groups in the assembled nanoparticles moderately disrupt mitochondrial homeostasis, leading to protein sulfenylation. Then, the CHD moiety covalently labels and inactivates these sulfenylated proteins, effectively disabling the endogenous reactive oxygen species (ROS) scavenging system. Upon near‐infrared irradiation, a massive ROS burst is generated, inflicting severe and sustained mitochondrial damage. This cascade induces rapid PANoptosis‐associated pathways and triggers profound mitochondrial DNA release to activate the cGAS–STING pathway. The combined activation elicits robust antitumor immunity, effectively suppressing orthotopic 4T1 tumor growth and prolonging survival. This work establishes a mitochondrial sulfenylated protein‐labeling strategy that amplifies oxidative stress, providing a paradigm for PANoptosis‐based photodynamic‐immunotherapy.