Preparation of drug-loaded iridium oxidenanocomposites and its application in synergetic sonodynamic/chemotherapeutic therapy against cancer

Objective To prepare iridium oxide (IrOX) as a nano-sonosensitizer to load chemotherapy drug doxorubicin (DOX) in order to obtain nanocomposites IrOX@DOX with dual response of pH/ultrasound (US) irradiation, and to verify its efficacy in sonodynamic therapy (SDT)-chemotherapy for tumor treatment. Methods IrOX nanoparticles were prepared by thermal hydrolysis and then loaded with DOX. The morphological, chemical and physical properties of the nanoparticles were characterized by transmission electron microscopy (TEM), X-ray photoelectron spectroscopy (XPS), ultraviolet-visible spectrophotometer (UV-Vis) and Zeta potential analyzer. The drug loading efficiency and drug release behavior were determined with UV-vis spectrometry and ELISA. The generation of reactive oxygen species (ROS) by IrOX@DOX nanoparticles was evaluated usingelectron spin-resonance spectroscopy (ESR). The uptake of IrOX@DOX by 4T1 cells under ultrasonic irradiation was evaluated with laser confocal scanning microscopy (LCSM), and the level of intracellular ROS was investigated using fluorescence microscopy and flow cytometry. The synergistic efficacy of IrOX@DOX in SDT-chemotherapy against 4T1 cells was evaluated by CCK-8 assay and flow cytometry. In addition, the Hepa1-6 xenograft tumor model in nude mice was built and divided into 5 groups (n=3 in each group) as follows: saline, IrOX, IrOX+US, IrOX@DOX, and IrOX @DOX+US. The tumor volume, contrast-enhanced US imaging and pathological changes of each group were observed, and the inhibitory rate of tumor volume was calculated to evaluate the effect of synergistic therapy in vivo. Results IrOX nanoparticles were successfully prepared, at a diameter of about 5.0 nm, and when the ratio of IrOX to DOX was 4.0, the drug loading reached 18.7%. Prepared IrOX@DOX released less than 10% of DOX at pH 7.4, which confirmed its good stability, while the cumulative release of DOX reached 39.9% at pH 5.5, and further increased to 69.9% under US irradiation. Both ESR and UV-vis experiments demonstrated that ROS production was positively correlated with irradiation power and time. Meanwhile, LCSM showed that US promoted the uptake of IrOX@DOX by tumor cells. In vitro cytotoxicity experiments indicated the survival rate of tumor cells in the synergistic treatment group (IrOX @DOX+US) was reduced to 19.4%, significantly lower than that of the chemotherapy group alone (P < 0.05), which proved that IrOX@DOX nanocomposites achieved an excellent synergistic effect of SDT-chemotherapy under ultrasound irradiation. Furthermore, in vivo experiments showed that as compared with saline group, the tumor-inhibiting rate of IrOX+US, IrOX@DOX and IrOX@DOX+US groups were 27%, 57% and 76%, respectively, indicating significant difference (P < 0.05), and this was consistent with contrast US imaging and H＆E staining results. Conclusion IrOX@DOX nanocomposites with dual stimulation response of pH and US irradiation are successfully developed, and the synergistic treatment of SDT-chemotherapy can be achieved under US irradiation.