Materials:
Bi(NO3)3·5H2O (99.0%, Aladdin), NH4VO3 (99.9%, Aladdin), Fe(acac)3 (99.9%, Simga-Aldrich), oleyamine (OLA, 70%, Simga-Aldrich), 1,2-hexadecanediol (90%, Aladdin), oleic acid (OA, 90%, Simga-Aldrich), SDS (99%, Simga-Aldrich), 1-octadecene (ODE, 90%, Simga-Aldrich), tris(hydroxymethyl) aminomethans (Tris, >99%, Aladdin), DA (99.0%). g-H2AX (phospho S139) antibody [EP854(2)Y] (Alexa Fluor 568) (ab206901) was purchased from Abcam. Crystal violet staining solution, CCK-8, hoechst 33342 and ROS assay kit were purchased from Beyotime.
Preparation of BiVO4 NPs:
1 mmol Bi(NO3)2·5H2O, 2 mL OLA, 2 mL OA and 10 mL ODE were added into a 100 mL three-necked flask. The temperature was raised to 175 °C in nitrogen atmosphere under vigorous stirring. When the solution was completely clear, the solution was dropped to 130 °C, followed by the addition of 10 mL water containing 2 mmol NH4VO3. The resulting solution was heated at 100 °C for 5 min and cooled down to room temperature naturally. After that, the solution was uniformly mixed with ethanol, and the bottom aqueous layer of the mixture was discarded. The upper organic layer was mixed with water and ethanol for twice, and the bottom aqueous layer of the mixture was discarded. The final product was washed by ethanol for another three times and dispersed in toluene.
Preparation of Fe3O4 NPs:
OA-capped Fe3O4 NPs were prepared by the thermal decomposition route. 2 mmol Fe(acac)3, 2 mL OA, 2 mL OLA, 5 mmol 1,2-hexadecanediol and 20 mL benzyl ether were added into a 100 mL three-necked flask. The mixture was heated at 200 °C for 30 min under nitrogen atmosphere, followed by reflux at 265 °C for 30 min. After that, the solution was dropped to room temperature naturally, and the product was washed for three times by ethanol and finally dispersed in toluene.
Preparation of SDS-capped BiVO4/Fe3O4 SPs:
2 mL toluene containing 50 mg BiVO4 NPs and 20 mg Fe3O4 NPs was added into 5 mL aqueous solution containing 10 mg SDS. After ultrasonic stirring for 10 min, the resulting emulsions were heated at 60 °C for another 30 min to evaporate toluene. After that, solution was centrifuge at 3000 r/min for 5 min, and the SDS-capped BiVO4/Fe3O4 SPs were obtained.
Preparation of BiVO4/Fe3O4@PDA SPs:
DA monomer was added into 10 mL Tris-buffer solution (10 mM, pH 8.5) containing 5 mg SDS-capped BiVO4/Fe3O4 SPs. After stirring for 3 h, the reaction solution was centrifuged at 5000 r/min for 30 min. Then, BiVO4/Fe3O4@PDA SPs were obtained.
Characterization:
TEM was taken on a Hitachi H-800 electron microscope (200 kV) coupled with a CCD camera. UV-vis absorption spectra were obtained using a Lambda 800 UV-vis spectrophotometer. HRTEM and EDS were performed on a JEM-2100F electron microscope at an acceleration voltage of 200 kV with an EDS detector. XRD was implemented on an Empyrean X-ray diffractometer with Cu K radiation (λ= 1.5418 Å).
Cell experiment:
Cytotoxicity assay:
We used CCK-8 and KB cells to test the cytotoxicity of SPs. In the 96-well plate, we cultured 5000 cells for each well. The cells were incubated at 37 °C with 5% CO2 for 24 h. Then the cells were treated with different concentrations of SPs and further incubated for 24 h. After that, the medium was removed and the cells were washed twice with PBS. Solarbio 1640 medium is re-added with 10 μL of CCK-8 and the cells were continued to culture for 1 h. Finally, the microplate reader was used to measure the absorbance at 450 nm.
Clonogenic assay:
In the 6-well plate, we cultured 1000 KB cells for each well. After co-cultivation with SPs (100 μg/mL) overnight, the cells were first treated with NIR (0.33 W/cm2, 10 min), then X-rays (0, 2, 4, 6, 8 Gy) sequentially. After that, the cells were washed with PBS and continued to incubate for 10 days. Finally, the cells were stained with crystal violet staining solution. Surviving fraction (SF) was calculated by (surviving colonies)/(cells seeded × plating efficiency). The mean surviving fraction was obtained from three parallel samples. The SF and the radiation dose can be fitted using the following formula: SF = exp[-(αD+βD2)]
ROS in cells:
We use ROS assay kit to detect ROS in cells. In the 6-well plate, we cultured 50000 KB cells in each well. We used SPs (100 μg/mL) to co-cultivate with cells overnight, followed by washing the cells with PBS for three times. We added the 1000-fold diluted ROS assay kit to each well and washed the cells with PBS for three times after incubation for 20 min. Then we treated the cells with X-ray (6 Gy) or NIR (0.33 W/cm2, 10 min). The cells were first irradiated by NIR, then by X-ray. Finally, the cells were observed using the FV1000 laser scanning confocal microscopy (excitation wavelength: 488 nm; emission wavelength: 525 nm).
DNA double-strand breaks:
In the 6-well plate, we cultured 50000 KB cells in each well. We used SPs (100 μg/mL) to co-cultivate with cells overnight, followed by treatment of cells with X-ray (6 Gy) or NIR (0.33 W/cm2, 10 min). The cells were first irradiated by NIR, then by X-ray. We fixed the cells with paraformaldehyde (4 %) for 10 min and washed with PBS for three times. It was then permeabilized with methanol and washed with PBS for three times. The cells were exposed in blocking buffer for 1 h and further incubated with 100-fold γ-H2AX (phospho S139) antibody [EP854(2)Y] (Alexa Fluor 568) (ab206901) at 4 °C overnight and then washed with PBS for three times (excitation wavelength: 578 nm; emission wavelength: 603 nm). The cells were stained with Hoechst 33342 (excitation wavelength: 350 nm; emission wavelength: 461 nm) and washed with PBS for three times. Finally, the cells were observed using FV1000 laser scanning confocal microscopy.
Animal experiment:
In vivo RT/PTT synergistic treatment:
KB cells were subcutaneously injected into the right leg of BALB/c nude mice. When the average tumor volume reached 75 mm2, mice were randomly divided into 7 groups with 5 mice in each group according to different treatment conditions: (1) PBS, (2) SPs, (3) NIR, (4) X-rays, (5) SPs+NIR, (6) SPs+X-rays, (7) SPs+NIR+X-rays. We injected 20 μL of SPs (5 mg/mL) into the tumor via intratumoral injection. Mice in the corresponding groups were then treated with NIR (0.33 W/cm2, 10 min) or X-ray (6 Gy). Tumor volumes and body weights of mice in each group were recorded after this treatment. Finally, all the mice were sacrificed and the blood, heart, liver, spleen, lung, kidney and tumor were taken. The blood was centrifuged and the serum was taken for the blood analysis. The tumors and other organs were weighed and used for H&E staining.
CT imaging:
In vivo and in vitro CT imaging were used U-SPECT+/CT (MILABS). For in vitro imaging, we prepared aqueous solutions of different concentrations of SPs and iopromide and measured their HU values. For in vivo imaging, BALB/c nude mice harboring KB tumors were intratumoral injected with 50 μL, 10 mg/mL SPs solution.
MR imaging:
We used SIEMENS Avanto 1.5T clinical MRI unit to assess the MR imaging property of SPs. For in vitro imaging, we prepared aqueous solutions of different concentrations of SPs for measuring. For in vivo imaging, BALB/c nude mice with KB tumors were intratumoral injected with 20 μL, 10 mg/mL SPs solution.
PA imaging:
In vivo and in vitro PA imaging was used MSOT INVISIO-256 (iThera Medical). For in vitro imaging, we prepared aqueous solutions of different concentrations of SPs for measuring. For in vivo imaging, BALB/c nude mice with KB tumors were intratumoral injected with 20 μL, 10 mg/mL SPs solution.