A nuclear targeted dual-photosensitizer for drug-resistant cancer therapy with NIR activated multiple ROS† †Electronic supplementary information (ESI) available. See DOI: 10.1039/c6sc00737f

A nuclear targeted dual-photosensitizer was developed for photodynamic therapy against multidrug resistant cancer. Multiple reactive oxygen species (ROS) could be generated in the nucleus to directly break DNA double strands with a single 980 nm NIR laser irradiation, regardless of drug resistance.

(Synergy 2, Biotek, USA) in the MTT assay. Confocal fluorescence imaging studies were performed with a TCS SP5 confocal laser scanning microscopy (Leica Co., Ltd. Germany) with an objective lens (×40, ×60). The sections were also observed through Nikon eclipse 80i microscope.
Synthesis of NaYF 4 :20%Yb 3+ ,0.1%Er 3+ ,0.2%Tm 3+ nanocrystals. The β-phase NaYF 4 :Yb 3+ ,Er 3+ ,Tm 3+ nanoparticles were synthesized using a solvothermal method with some modifications. Briefly, 1 mmol rare earth chlorides YCl 3 , YbCl 3 , ErCl 3 and TmCl 3 with a stoichiometric ratio of 79.7:20:0.1:0.2 were dispersed in 8 mL of oleic acid and 18 mL of 1-octadecene, and then the mixture was heated to 156 o C for half an hour under stir. After a homogeneous solution was formed, the mixture was cooled down to room temperature, then 10 mL of methanol solution containing sodium hydroxide (0.1 g, 2.5 mmol) and NH 4 F (0.148 g, 4 mmol) was added dropwise. After stirring for 30 min, the temperature was raised to 100 o C to make the methanol volatilizing. Then the mixture was heated to 295 o C in an argon atmosphere for 90 min and cooled to room temperature. The precipitates were washed with methanol and cyclohexane (1:1, v/v) for times and redispered in 10 mL of cyclohexane.
Synthesis of UCNPs@TiO 2 core-shell nanoparticles. Before the synthesis, the oleic acid ligand on the surface of upconversion nanoparticles should be removed firstly.
0.5 mL of as-synthesized oleate-conjugated UCNPs was dispersed in a 10 mL of HCl solution (pH=2) under vigorous stirring for 4 h to make the carboxylate groups of the oleate ligand protonated (to yield oleic acid). After the reaction was completed, diethyl ether was added to the solution to remove the oleic acid by extraction. Then the oleate-free UCNPs in aqueous phase were obtained by centrifugation. The UCNPs@TiO 2 core/shell nanoparticles were synthesized via a kinetics-controlled method. 45 The OA-free upconversion nanoparticles were dispersed in 25 mL absolute ethanol and mixed with concentrated ammonia solution (75 μL, 28 wt%) under ultrasound for 15 min. Subsequently, 120 μL of tetrabutyl titanate (TBOT) was added dropwise in 5 min, and the reaction was performed for 24 h at 45 o C under continuous gentle stirring. The final products were separated under centrifugation, followed by washing with deionized water and ethanol for 3 times, respectively. After dried at 100 o C, the UCNPs@TiO 2 was gained and redispersed in deionized water for further use.
Co-localization into nucleus. MCF-7 cells were first cultured in a confocal dish for 24 h. Then, the cells were treated with UCNPs@TiO 2 -Ce6-TAT and UCNPs@TiO 2 -Ce6 (0.1 mg/mL) in DMEM culture medium. After 4 h incubation, cells were washed several times with PBS buffer to remove the nanoparticles outside the cells. Then, fresh DMEM culture medium was added and the cells were incubated for another 8h.
Then, the cells were washed with PBS twice and stained with Hoechst 33342 for 15 min. Subsequently, the cells were observed using confocal laser scanning microscopy (CLSM) and confocal images were captured with 405 nm excitation for Hoechst 33342 (emission=430-480 nm) and 633 nm excitation for Ce6 (emission=650-700 nm). The liner fluorescence profile was quantified using Image-Pro Plus Imaging software. In vitro therapeutic effects.

Intracellular tracking UCNPs@TiO
(1) MCF-7 cells were cultured in 96-well microtiter plates and incubated at 37 °C in 5% CO 2 for 24 h. UCNPs@TiO 2 -Ce6-TAT (0.1 mg/mL) was incubated with the cells in DMEM culture medium for 12 h followed by washing the cells with PBS buffer to remove the nanoparticles that were not uptake into the cells. Then, MCF-7 cells were treated with irradiation of different laser powers and irradiation times (0.5, 1, 2 W•cm -2 and 1, 2, 3, 4, 5 min, respectively). The cells in the control group were without any treatment. Then, the cells were further cultured for 24 h. 150 µL MTT solution (0.5 mg/mL) was then added to each well.
After incubation for 4 h, the MTT solution was removed, and 150 µL of DMSO was added to each well under slight shake in the dark. Considering the therapeutic effects and the lower phototoxicity to normal cells, the parameter of 1 W•cm -2 and 5 min was chosen in the following therapeutic application. (2) MCF-7 cells were cultured in 96well microtiter plates and incubated at 37 °C in 5% CO 2 for 24 h. UCNPs@TiO 2 -Ce6, UCNPs@TiO 2 -TAT, and UCNPs@TiO 2 -Ce6-TAT (0.1 mg/mL) were incubated with the cells in DMEM culture medium for 12 h, respectively, followed by washing the cells with PBS buffer to remove the nanoparticles that were not uptake into the cells.
The cells in the control group were without any treatment. Then, the cells were further cultured for 24 h. Then, 150 µL of MTT solution (0.5 mg/mL) was added to each well.
After incubation for 4 h, the MTT solution was removed, and 150 µL of DMSO was added to the well under slight shake in the dark. (3) MCF-7 cells were cultured in 96well microtiter plates and incubated at 37 °C in 5% CO 2 for 24 h. UCNPs@TiO 2 -Ce6-TAT (0.1 mg/mL) without irradiation and only irradiation with 980 nm laser (1 W•cm -2 , 5 min) were performed on two groups of cells, respectively. The cells in the control group were without any treatment. Then, the cells were further cultured for 24 h. Then all groups of cells were washed with PBS buffer to remove the nanoparticles that were not uptake into the cells. Afterward, 150 µL of MTT solution (0.5 mg/mL) was added to each well. After incubation for 4 h, the MTT solution was removed, and 150 µL of DMSO was added to the well under slight shake in the dark. The absorbance was measured at 490 nm with microplate reader.
In vitro DNA damage study. DNA damage was detected using H2A.X Phosphorylation Assay Kit. Take MCF-7 cells for example. In brief, 2×10 6 MCF-7 cells were incubated with UCNPs@TiO 2 -Ce6, UCNPs@TiO 2 TAT, UCNPs@TiO 2 -Ce6-TAT (0.1mg/mL) for 12 h, respectively, and treated with 980 nm laser irradiation for 5 min. Then each group of cells was trypsinized and centrifuged (1000 rpm, 3 min). The cells were resuspend with 1X fixation solution at a cell density of 2×10 6 per mL and incubated for 20 min in ice to fix the cells, followed by washed the cells using PBS to remove the fixative. The cells pellets were then gently resuspend in 1X permeabilization solution to a density of 2×10 6 cells per mL (50 μL per 1×10 5 cells).
Subsequently, either FITC conjugated anti-phospho-histone H2A.X (Ser139) or the negative control FITC conjugated normal mouse IgG (3.5 μL/1×10 5 cells) was added and the cells were incubated on ice for 20 min. Then add 1 mL of 1X wash solution aid in removing excess FITC labeled antibody. Cell images were acquired using the ImageStream X multispectral imaging flow cytometer (Amnis Corporation) with 488 nm excitation for FITC (emission=500-560 nm). Cell images were analyzed using IDEAS® image analysis software (Amnis).
Animal tumor xenograft models. All animal experiments were carried out and accorded with the Principles of Laboratory Animal Care (People's Republic of China).
Female nude mice (4-6 week old, ~20 g) were raised on normal conditions of 12 h light and dark cycles and given access to food and water ad libitum.   Figure S6. H&E staining images of five major organs (heart, liver, spleen, lung, and kidney) in MCF-7 tumor-bearing mice (left) and MCF-7/Dox tumor-bearing mice at 7 day after different treatment groups: control, UCNPs@TiO 2 -Ce6, UCNPs@TiO 2 -TAT, and UCNPs@TiO 2 -Ce6-TAT with irradiation for 5 min at the tumor region.
The power of the irradiation was 1 W·cm -2 . And no histopathological abnormalities were observed in each group.