Effect of folic acid-targeted triptolide-loaded microbubbles combined with ultrasound on apoptosis of human MCF-7 breast cancer xenografts in nude mice.

BACKGROUND: Breast cancer is a leading cause of mortality among women, with its incidence and mortality rates steadily rising year after year. Despite the common use of chemotherapy and radiotherapy in clinical treatment, the prognosis often remains unsatisfactory. The majority of patients face the imminent risk of tumor recurrence and metastasis, resulting in a persistently low 5-year survival rate. This study aims to develop and evaluate a novel folic acid-targeted triptolide-loaded ultrasound microbubble (FA-TLUM) system for the targeted treatment of breast cancer. METHODS: FA-TLUM were prepared using the thin film hydration method, and ultrasound irradiation was applied to disrupt the microbubbles and facilitate drug release, resulting in effective eradication of breast cancer cells. The particle size and Zeta potential of the hydrated microbubbles were determined using dynamic light scattering, while the drug loading and encapsulation efficiency were measured using ultraviolet spectrophotometry. Inverted fluorescence microscopy was employed to observe the coupling of folate onto the microbubbles and their targeting ability in vitro. Additionally, color Doppler ultrasound imaging was performed both in vitro and in vivo using a diagnostic system, while hematoxylin and eosin (HE) staining was used to examine morphological changes in mice. Furthermore, the Cell Counting Kit-8 (CCK-8) assay was conducted to evaluate the toxic effect of microbubbles combined with ultrasound on MCF-7 cells, followed by flow cytometry analysis for cell cycle and apoptosis assessment. Finally, real-time quantitative polymerase chain reaction (RT-qPCR) and Western blotting techniques were utilized to determine messenger RNA (mRNA) and protein expression levels of apoptosis-related genes such as Caspase-3, Bax, Bcl-2, and Survivin. RESULTS: The FA-TLUM exhibited regular morphology and uniform dispersion, demonstrating favorable drug loading and encapsulation efficiency, while maintaining stability at 4 ℃. Moreover, the FA-TLUM exhibited fluorescent properties and demonstrated firm attachment to MCF-7 cells, thereby showcasing excellent targeting ability. Furthermore, the FA-TLUM microbubbles exhibited negligible cytotoxicity towards MCF-7 cells with a high level of safety in both cellular and animal models. In ultrasound imaging applications, the FA-TLUM demonstrated a remarkable enhancement effect on echo signals. Additionally, in contrast-enhanced ultrasound imaging of breast cancer xenografts in nude mice, the FA-TLUM generated a stronger contrast signal than the non-targeted TLUM, demonstrating its potential for improved tumor visualization. Notably, when combined with ultrasound treatment on MCF-7 cells, the FA-TLUM enhanced cytotoxicity leading to an increased proportion of cells in the S phase, along with significantly elevated levels of apoptosis. CONCLUSIONS: The present study successfully prepared FA-TLUM, and the combination of FA-TLUM with ultrasonic targeted fragmentation technology significantly enhances the inhibitory effect on MCF-7 cell proliferation and promotes cellular apoptosis. Therefore, this technology is anticipated to offer novel approaches and concepts for clinical breast cancer treatment.