Ultrasound-responsive doxorubicin microbubbles engineered by QbD: enhanced in vitro anti-breast-cancer efficacy with attenuated cardiac cell toxicity

Ultrasound-responsive microbubbles offer an externally controlled strategy for localized drug delivery, but their performance is often limited by empirical formulation development and incomplete evaluation of formulation-related safety controls. In this study, a Quality by Design (QbD) approach was applied to develop doxorubicin-loaded chitosan microbubbles for ultrasound-triggered anticancer delivery. A Box–Behnken design was used to evaluate the effects of chitosan, palmitic acid, and Pluronic F68 concentrations on microbubble size, encapsulation efficiency, and ultrasound-triggered burst behavior. The optimized formulation occupied a constrained design space that balanced microbubble size of approximately 3.2 μm, encapsulation efficiency of approximately 70–75%, and burst response above 80% within 40 s of ultrasound exposure. The optimized microbubbles showed low passive doxorubicin release under non-sonicated conditions and rapid release following ultrasound activation. In MCF-7 breast cancer cells, DOX-MB + US enhanced cytotoxicity and apoptosis compared with free doxorubicin and DOX-MB without ultrasound. The apparent IC₅₀ of DOX-MB + US in MCF-7 cells was 0.0013 µg/mL, compared with 0.62 µg/mL for free doxorubicin and 5.44 µg/mL for DOX-MB without ultrasound. The newly included Blank MB + US control produced only moderate bioeffects compared with DOX-MB + US, supporting that the stronger cytotoxic response of DOX-MB + US was associated with both ultrasound-mediated microbubble activation and doxorubicin delivery. In H9c2 cardiomyoblast cells, DOX-MB without ultrasound showed reduced baseline toxicity compared with free doxorubicin, with an apparent IC₅₀ of 8.72 µg/mL versus 5.14 µg/mL, respectively. Selectivity analysis was interpreted as comparative in vitro indices, with the high SI₃ value considered a trigger-dependent therapeutic window estimate rather than a conventional matched-condition selectivity index. These findings indicate that QbD-guided formulation control, combined with ultrasound activation, can reduce non-triggered doxorubicin availability while enhancing anticancer activity under insonated conditions. The study provides an in vitro proof-of-concept for ultrasound-responsive doxorubicin microbubbles as externally controlled delivery systems. Further acoustic calibration, three-dimensional tumor models, hemocompatibility testing, pharmacokinetic evaluation, biodistribution analysis, and in vivo tumor-localized ultrasound studies are required before translational conclusions can be made.