Ex Vivo High-Dose Ionizing Irradiation-Conditioned Nanovesicles for Enhancing the Immune Effects of Tumor Radiotherapy.

Radiotherapy (RT) exhibits a dose-dependent duality in its impact on antitumor immunity. While high-dose irradiation induces immunogenic cell death (ICD) and enhances immune responses, conventional RT is limited by toxicity and may paradoxically promote immunosuppression via myeloid-derived suppressor cells (MDSCs) infiltration and M2 macrophage polarization. To address this challenge, we leveraged ex vivo high-dose irradiation to generate tumor-derived exosomes (cExo) enriched with immunostimulatory components. Proteomic analysis revealed that cExo were enriched with damage-associated molecular patterns (DAMPs), including high mobility group box 1 protein (HMGB1) and calreticulin (CRT), as well as major histocompatibility complex I (MHC I), thereby enhancing dendritic cells (DCs) activation and antigen cross-presentation. Building upon these findings, we developed a hybrid nanoplatform (cExo-Lip) by fusing immunogenic cExo with liposomes loaded with plasmid IL-12 (pIL-12). This design compensates for the limited immunogenicity of low-dose irradiation by delivering MHC I and DAMPs via cExo, thereby enhancing DCs maturation and cytotoxic T lymphocyte (CTLs) priming. Moreover, sustained IL-12 expression further remodels the tumor immune microenvironment by reprogramming M2 macrophages toward a tumoricidal M1 phenotype and enhancing CTLs activation, thereby synergistically amplifying RT-induced immune responses. In B16F10 melanoma mouse models, combining 6 Gy RT with pIL-12@cExo-Lip resulted in 91.07% tumor suppression. Our approach uniquely capitalizes exosomes treated by ex vivo high-dose irradiation for the first time to overcome the limitations of clinical dose constraints and synergizes RT's immune-priming potential with cytokine delivery to combat the challenges in clinical cancer radiotherapy and enhance its therapeutic efficacy.