An Enzymatic in situ Self-Assembly Radiosensitizer for Hypoxic Tumor Radio-Immunotherapy via Low-Power X-ray-Induced Pyroptosis.

X-ray-induced cell pyroptosis represents a promising strategy for enhancing antitumor immunity. However, existing clinical radiosensitizers are hindered by inadequate tumor targeting and inefficiency in triggering pyroptosis, limiting the full potential of radioimmunotherapy. Herein, we introduce P-Ni-RGD, an enzymatically triggered in situ self-assembly radiosensitizer, as a pyroptosis inducer to improve cancer radioimmunotherapy. P-Ni-RGD incorporates an alkaline phosphatase (ALP)-responsive self-assembly scaffold, a cyclic RGD ligand, and a nitroimidazole (Ni) oxygen-mimetic radiosensitizer. Following systemic administration, P-Ni-RGD self-assembles into nanoparticles within the tumor microenvironment, specifically in response to ALP activation, enhancing tumor accumulation and enabling tumor-targeted delivery of the radiosensitizer and targeting ligand. In situ self-assembly results in the modulation of near-infrared fluorescence and photoacoustic bimodal imaging signals. Guided by these imaging modalities, low-dose X-ray irradiation (2 Gy) of orthotopic breast 4T1 tumors induces reactive oxygen species and promotes nitroreductase-mediated reduction of Ni to reactive intermediates within tumor cells. This cascade triggers the labeling of DNA repair proteins, upregulates key molecules involved in oxidative stress and DNA damage response, disrupts redox homeostasis, amplifies oxidative DNA damage, and impairs DNA repair. Accumulating DNA damage activates the AIM2 inflammasome, culminating in gasdermin D (GSDMD) dependent pyroptosis. This mechanism not only disrupts tumor cell viability but also provokes a potent antitumor immune response, significantly inhibiting primary and distant 4T1 tumors and prolonging survival. Our findings highlight the promise of combining enzymatic in situ self-assembly with low-dose X-rays to induce tumor-specific pyroptosis, offering an approach to enhance radiotherapeutic outcomes in hypoxic tumors.