Spatially Confined Reactive Oxygen Species Generation Enabled by a Bacteria-Driven Intracellular Delivery Platform Augments Sonodynamic Therapy for Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) confers resistance to chemotherapy and is prone to metastasis. Sonodynamic therapy (SDT) presents a potential dual-mechanism strategy, wherein both ROS-mediated cytotoxicity target tumor cells and immune activation counteract metastasis. However, the short lifespan and limited diffusion of reactive oxygen species (ROS) hinder their effective interaction with intracellular targets, fundamentally limiting the sonochemical efficiency and therapeutic outcome of SDT. Enhancing proximity between ROS generation and intracellular targets is critical. Here, we developed a biohybrid system based on attenuated Salmonella (Sal@ICG) for intracellular sonosensitizer delivery, thereby confining the ROS generation in close spatial proximity to intracellular targets. Following biosafety and invasion validation, attenuated Salmonella was covalently functionalized with the sonosensitizer indocyanine green (ICG). By harnessing the tumor-targeting and invasive properties of attenuated Salmonella, the Sal@ICG facilitated efficient intracellular accumulation of ICG, minimized its degradation, and prolonged tumor retention. At 72 h postinjection, Sal@ICG demonstrated approximately 20-fold higher tumor accumulation compared to free ICG, ensuring a high payload of sonosensitizer at the target site. Under ultrasound irradiation conditions that induce cavitation, it enabled spatially confined and enhanced ROS generation near intracellular targets, which significantly augmented the sonochemical efficacy, as evidenced by boosted cytotoxicity, induced immunogenic cell death, and inhibited growth of both primary tumors and metastasis. In summary, this work developed a bacteria-based biohybrid system for intracellular sonosensitizer delivery, which fundamentally enhances the local sonochemical process, amplifying ROS-mediated effects and offering a promising therapeutic platform for TNBC and a novel strategy for modulating the local sonochemical environment using microbial vectors.