A mitochondria-targeted nanoradiosensitizer activating reactive oxygen species burst for enhanced radiation therapy

We developed a novel strategy for enhanced radiation therapy based on a mitochondria targeted titanium dioxide-gold nanoradiosensitizer.


Preparation of TiO 2 -Au NPs.
Au NPs were anchored on the surface of TiO 2 -NH 2 by the coordination bonds. 4 mg as-prepared TiO 2 -NH 2 NPs were added to 24 mL yellowish Au NPs solution. The mixture was then stirred at room temperature for 24 h. Finally, the precipitates were centrifuged (10,000 rpm/min, 10 min), washed with water and PBS buffer (10 mM, pH = 7.4) for three times, and redispersed in PBS buffer (1 mg/mL).
Preparation of TiO 2 -Au-TPP and TiO 2 -Au-TPP-IR806. TiO 2 -Au-TPP-IR806 was prepared by coupling the carboxyl groups of the TPP bromide and infrared dye IR806 with the amino group on the surface of TiO 2 -Au to form the amido bonds. EDC, NHS, IR806 and TPP bromide was added in 10 mL of MES buffer (10 mM, pH = 6.0) and amount of EDC, NHS, IR806 and TPP bromide are shown in Table 1. The reaction was performed for 1 h at room temperature in the dark to activate carboxylate groups and then both of them were added to above TiO 2 -Au solution under gentle stirring for 24 h, which lead the amido bonds formation. Later, the precipitates were centrifuged (10,000 rpm/min, 10 min), and washed with methanol and PBS buffer (10 mM, pH = 7.4) for three times, and finally redispersed in PBS buffer (1 mg/mL).
The content of TPP bromide groups were calculated according to the standard linear calibration curve of each group using subtraction through UV-Vis absorption spectra.  with TiO 2 -Au-TPP and X-ray irradiation. After 8 h, X-ray treatment was performed with X-ray for 4 Gy and medium was replaced with serum and bicarbonate-free assay medium 1 h before oxygen consumption rate. After incubation, the sensor cartridge was loaded with oligomycin (10 μM, port A), carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP, 20 μM, port B) and rotenone (10 μM, port C) to measure the OCR. Cells were washed once with serum-free un-buffered assay medium containing 10 mM sodium pyruvate and 25 mM glucose. Before OCR measurement, cells were pre-incubated with nanoprobes in regular media for 8 h followed by washing once with assay medium, and cells were kept in 500 μL/well of assay medium.
Once the sensor cartridge was equilibrated, the calibration plate was replaced with the assay plate.
Colony formation assay. MCF-7 cells were cultured in 60 mm dishes and incubated at 37 °C under 5% CO 2 in DMEM for 24 h. The cells were then subjected to 5 different treatments: blank as the control group, TiO 2 -Au-TPP only, X-ray irradiation only, TiO 2 -Au with X-ray irradiation and TiO 2 -Au-TPP with X-ray irradiation. After 8 h, the cells were washed three times with PBS buffer (10 mM, pH = 7.4) to remove the excess NPs that were not uptaken into the cells.
Subsequently, X-ray treatment was performed with X-ray for 4 Gy and those cells were then incubated in fresh cell culture medium at 37 °C under 5% CO 2 in DMEM for another 10 days, before they were fixed with 4% paraformaldehyde and stained with 0.2% crystal violet. The resulting colonies with more than 50 cells each were counted. The surviving fraction = (surviving colonies) / (cells seeded × plating efficiency). The mean surviving fraction was obtained from three parallel tests. 6 Cell migration assay. The same setup of colony formation assay was applied with the Migration assay until washing with PBS buffer for three times and then the X-ray treatment.