Self‐Assembled Triple‐Targeted Radiosensitizer Enhances Hypoxic Tumor Targeting and Radio‐Immunotherapy Efficacy

Targeted delivery of radiosensitizers and real-time monitoring of hypoxia are crucial for overcoming radiotherapy resistance in hypoxic tumors. Here, we report A-Cy-Ni-RGD, a triple-targeted nitroimidazole (Ni)-linked radiosensitizer that self-assembles into nanoparticles (A-Cy-Ni-RGD NPs) for bimodal near-infrared fluorescence (NIR FL) and photoacoustic (PA) imaging-guided radio-immunotherapy. A-Cy-Ni-RGD NPs specifically accumulate in α_vβ₃-positive tumors, where they are hydrolyzed by carboxylesterase to form Cy-Ni-RGD NPs, with enhanced FL at 710 nm and dual PA signals at 680 and 730 nm. Under hypoxic conditions, nitroreductase (NTR) further reduces these NPs, covalently labeling endogenous proteins and increasing NP size. This process partially alleviates aggregation-caused quenching effect, increasing the FL₇₁₀ signal and decreasing the PA₇₃₀ signal, enabling real-time tracking of tumor-specific delivery and hypoxia. Following low-dose X-ray irradiation (2 Gy), elevated NTR expression promotes further Cy-Ni-RGD NPs reduction, enhancing proteins labeling and causing DNA damage. Moreover, radiosensitization with A-Cy-Ni-RGD NPs triggers robust immunogenic cell death, stimulating antitumor immunity that inhibits tumor growth and metastasis, significantly prolonging survival in mice with orthotopic 4T1 tumors. This work underscores the potential of self-assembling, triple-targeted radiotheranostic agents for improving tumor targeting, imaging, and radiotherapy efficacy in hypoxic tumors.