Inhibitory Effect and Mechanism of Paclitaxel-Loaded Magnetic Iron Oxide Nanomicrobubbles Combined with Ultrasound Therapy on Breast Cancer Xenografts in Nude Mice Running Title: the Application of Paclitaxel-Loaded Fe3O4 Nanomicrobubbles

Abstract This study aimed to investigate the therapeutic efficacy and underlying mechanisms of paclitaxel (PTX)-loaded magnetic iron oxide nanomicrobubbles (PTX@Fe3O4 NMBs) combined with ultrasound therapy in breast cancer xenograft models. The PTX@Fe3O4 NMBs were synthesised using a modified microfluidic approach and characterised for size, stability and drug loading. Female BALB/c nude mice bearing MCF-7 xenografts were randomly divided into five groups: control, free PTX (TPL), blank NMBs with ultrasound (TPL + US), targeted NMBs with ultrasound (TLUM + US) and combination therapy with ultrasound (FA-TLUM + US). Tumour growth was monitored for 21 days. Contrast-enhanced ultrasound imaging was performed to evaluate tumour perfusion. Apoptotic mechanisms were assessed through quantitative real-time polymerase chain reaction (PCR) and Western blotting. The PTX@Fe3O4 NMBs showed a mean diameter of 186 ± 15 nm, a polydispersity index value of 0.118, a zeta potential of −21.3 ± 2.1 mV, drug encapsulation of 85.4% ± 3.2% and loading capacity of 8.6% ± 0.4% (w/w), with ≤5% drug leakage over 14 days at 4°C. The FA-TLUM + US group achieved the highest tumour growth inhibition rate of 78.6%, significantly superior to other treatment groups (p < 0.001). Contrast-enhanced ultrasound imaging demonstrated enhanced signal intensity and prolonged retention time in targeted NMB groups. Quantitative PCR analysis revealed significant upregulation of pro-apoptotic genes (Bax increased 5.0-fold, caspase-3 increased 4.2-fold) and downregulation of anti-apoptotic genes (Bcl-2 decreased by 76%, survivin decreased by 72%) in the combination therapy group. The PTX@Fe3O4 NMBs combined with ultrasound therapy demonstrated potent antitumour efficacy through enhanced drug delivery and activation of mitochondrial apoptotic pathways, representing a promising theranostic platform for breast cancer treatment.