Dual-parameter sequential ultrasound irradiation with oxygen-loaded microbubbles induces vascular normalization to potentiate sonodynamic therapy in breast cancer

The efficacy of sonodynamic ther cancer is limited by tumor hypoxia and the constrained bio-effects of single-parameter ultrasound. To address this, an optimized SDT strategy was established with microbubbles loaded with Chlorin e6 (Ce6) and oxygen (CMO) and a dual-parameter sequential ultrasound irradiation mode, which begins with a cavitation-inducing ultrasound (US_ca, 0.6 W/cm²) to normalize tumor vasculature and alleviates hypoxia, then followed by a sonochemical-activating ultrasound (US_sc, 1.4 W/cm²) to elicit potent tumor cell killing effect. It was demonstrated by cellular experiments that US_ca facilitated the release of oxygen and Ce6, allowing US_sc to exert maximal cytotoxicity effect. In vivo, US_ca pretreatment was indispensable for normalizing tumor vasculature, while US_sc alone was found to be vasculature-damaging. With the synergistic effect of sequential US_ca and US_sc ultrasound irradiation, superior anti-tumor effect was achieved, manifested as a 75 % reduction in tumor volume and significant outperformance over classical SDT mode. Overall, This work pioneers a dual-stage sequential ultrasound irradiation mode that tactically induces vascular nonmalization as a sensitizing step for subsequent SDT, culminating in a novel SDT strategy with high translational potential.