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

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 T₂*-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.