Low‐Power Ultrasound with Microbubbles Improve Oxygenation and Doxorubicin Uptake in Hypoxic Triple‐Negative Breast Cancer

Objectives Triple‐negative breast cancer (TNBC) exhibits poor chemotherapy response and prognosis due to the lack of targeted therapies and a hypoxic, poorly vascularized tumor microenvironment. This study demonstrated the efficacy of low‐power ultrasound‐stimulated microbubble (USMB) therapy in enhancing drug delivery and improving the TNBC microenvironment, with a focus on its relationship with nitric oxide (NO). Methods A 4T1 murine TNBC model was established. Multi‐parametric imaging techniques, including contrast‐enhanced ultrasound (CEUS), ultra‐resolution microscopy imaging (URM), and photoacoustic imaging (PAI) were innovatively combined to assess tumor perfusion, oxygenation, and drug delivery following USMB treatment. Epirubicin penetration and targeted intratumoral distribution were quantitatively analyzed using high‐performance liquid chromatography (HPLC) and CD31 immunofluorescence under confocal microscopy. NO inhibition experiments were conducted to evaluate the involvement of the endothelial NO synthase (eNOS)/NO pathway. Results Group USMB significantly increased tumor perfusion within 4 hours post‐treatment ( p < .001), reduced perfusion‐deficient regions, and raised oxygenation to 1.4‐fold of baseline. Doxorubicin (Dox) concentration was 3.15‐fold higher than the chemotherapy group, with wider distribution. Blocking eNOS/NO signaling markedly attenuated the perfusion‐enhancing effects of USMB, implicating NO as a key mediator. Conclusion USMB improves the TNBC microenvironment and enhances drug delivery by promoting perfusion and oxygenation, likely via NO signaling. These findings support USMB as a promising strategy for TNBC combination therapy and highlight NO as a potential therapeutic target.