ROS‐Responsive Nanobubbles for Dual‐Enhanced Ultrasound and Magnetic Resonance Imaging of Tumor Oxidative Stress

ABSTRACT Reactive oxygen species (ROS) are key biomarkers of oxidative stress in the tumor microenvironment (TME), yet their non‐invasive, real‐time visualization remains challenging. Here, we present biotinylated PEGylated bilirubin nanobubbles encapsulating perfluoropentane gas (bt‐PEG‐BR@PFP) as ROS‐responsive contrast agents for dual‐modality ultrasound (US) and magnetic resonance imaging (MRI). Upon ROS exposure, the bilirubin shell undergoes oxidative degradation, leading to nanobubble fusion and signal amplification in both US and T 2 *‐weighted MRI. In vitro, biotin‐mediated cellular uptake and ROS‐induced fusion were validated in A549 cancer cells. In vivo, intratumoral injection of bt‐PEG‐BR@PFP into dual‐tumor xenografts led to a >3.7‐fold increase in US signal intensity in ROS‐high A549 tumors compared to ROS‐low DU145 tumors, which was abolished by the ROS scavenger N‐acetylcysteine. Following systemic administration, the nanobubbles accumulated selectively in A549 tumors through biotin‐mediated targeting and produced ∼50‐fold higher US signal than in DU145 tumors. In contrast, the clinical agent SonoVue showed no such tumor selectivity and ROS‐responsive signal enhancement. MRI studies revealed a time‐dependent signal drop only in A549 tumors treated with bt‐PEG‐BR@PFP, consistent with ROS‐mediated nanobubble fusion. These results highlight bt‐PEG‐BR@PFP as a promising and clinically translatable platform for non‐invasive, dual‐modality imaging of tumor oxidative stress, with potential utility in various ROS‐associated pathologies.