Bright red aggregation-induced emission nanoparticles for multifunctional applications in cancer therapy

Developing multifunctional photosensitizers (PSs) is needed to effectively simplify cancer treatment, but it remains a big challenge. Here, two red-emitting AIE-active, donor–acceptor (D–A) PSs with small ΔEST and their AIE nanoparticles, are rationally designed and synthesized. The PS1 NPs exhibit bright red-emission with high quantum yield, appropriate 1O2 generation ability and good biocompatibility. More importantly, PS1 NPs can strongly light up the cytoplasm by gently shaking the cells for only 5 s at room temperature, indicating ultrafast staining and mild incubation conditions. In vitro and in vivo cell tracing demonstrate that PS1 NPs can track cells over 14 days, and effectively inhibit tumor growth upon irradiation. To the best of our knowledge, this work is the first example of a PS that integrates image-guided PDT, ultrafast staining and long-term tracing functions, demonstrating the “all-in-one” concept which offers great advantages for potential clinical applications.


Materials
Materials obtained from commercial suppliers were used without further purification unless otherwise stated. All glassware, syringes, magnetic stirring bars and needles were thoroughly dried in a convection oven. All other materials for organic synthesis were purchased from Energy Chemical (China). The cell viability (live dead cell staining) assay kit was purchased from Jiangsu KeyGEN Biotechnology Co., Ltd. (China). Milli-Q water was collected from a Milli-Q system (Millipore).

Density functional theory calculations
All the calculations were performed in the gas phase using a Gaussian 09 program. 1 The groundstate structure was optimized with B3LYP method and 6-311G(d, p) basis set. Then, a vertical excitation was carried out based on the optimized structure with the same method, from which the ground-state molecular orbital energy was obtained.

Cell culture
HeLa cells (the human cervical cancer cell line) and H22 cells (the murine hepatic carcinoma cell line) were purchased from Jilin University and grown in Dulbecco's modified Eagle's medium (DMEM, GIBCO) supplemented with 10% heat-inactivated fetal bovine serum (FBS, GIBCO), 100 U mL penicillin and 100 µg mL streptomycin (Sigma). The cells were maintained in a humidified incubator at 37 o C with 5% CO 2 .

Cytotoxicity assay
HeLa cells were harvested in a logarithmic growth phase and seeded in 96-well plates at a density of 10 4 cells per well for 24 h. Then different concentrations of PS1/PS2 and PS1/PS2 NPs of various concentrations (0-20 μg mL -1 ) were added into cell culture medium separately. After incubating for 6 h, the light group received white irradiation (20 mW cm -2 ) for 60 min, then continued incubation to 24 h.
The dark control group received nothing and were incubated for the same time as the control group.
Subsequently the MTT (20 µL) was added to the medium for 4 h. The absorbance of MTT was measured by a Bio-Rad 680 microplate reader at 490 nm.

Intracellular ROS assays
HeLa cells were seeded in 6-well culture plates at a density of 5×10 4 cells per well for 24 h. Then PS2 and PS2 NPs (10 μg mL -1 ) were added into the cell culture medium separately. After incubating for 6 h, the light group received irradiation (20 mW cm -2 ) for 20 min, whereas the dark control group received nothing. Then using the DMEM solution without FBS the cells were washed. The DMEM solution containing DCFH-DA (10 -5 mol L -1 ) was added and incubated for 30 min. The cells were observed as soon as possible by CLSM with excitation at 488 nm.

Live/ dead cell staining assays
HeLa cells were harvested in a logarithmic growth phase and seeded in 96-well plates at a density of 10 4 cells per well for 24 h. The PS1/PS2 and PS1/PS2 NPs (20 μg mL -1 ) were added into cell culture medium separately. After incubating for 6 h, the light group received white irradiation (20 mW cm -2 ) for 60 min, then incubation was continued to 24 h. The dark control group received nothing and was incubated for the same time as the control. After staining with calcein-AM/PI for 40 min, phosphatebuffered saline (PBS) was used to wash the cells. Finally the cells were observed by Nikon C1 Si laser scanning confocal microscopy.

Cellular Uptake
The cellular uptake measurement was investigated by CLSM. Cells harvested in a logarithmic growth phase were seeded in 6-well plates at a density of 5×10 4 cells/well and incubated in DMEM for 24 h. The medium was then replaced by 2 mL of DMEM containing AIE NPs (0.5 μg mL -1 or 2 μg mL -1 or 2 μg mL -1 or 3 μg mL -1 ) and incubated for different times at 37 °C and further washed 3 times with PBS buffer. For the CLSM, the cells were fixed with 4% paraformaldehyde solution for 10 min. Then, the cells were washed with PBS and observed using confocal laser scanning microscopy with excitation at 488 nm (CLSM, Zeiss LSM 700).

Long-Term Cellular Tracing
Cells harvested in a logarithmic growth phase were seeded in 6-well plates at a density of 5×10 4 cells/well and incubated in DMEM for 24 h. The medium was then replaced by 2 mL of DMEM containing 20 μg mL -1 of AIE NPs and incubated for 6 h at 37 °C (day 0). Then the cells were diluted and subcultured in 6-well plates for 0 to 15 days regeneration, respectively. Upon reaching the designated day, the cells were washed with PBS buffer and then fixed with 4% of paraformaldehyde solution for 10 min. Later, the cells were washed with PBS and observed using confocal laser scanning microscopy with excitation at 488 nm (CLSM, Zeiss LSM 700).

Tumor-bearing mouse model
6 All animal studies were performed in strict accordance with the NIH guidelines for the care and use of laboratory animals (NIH Publication No. 85-23 Rev. 1985) and were approved by the guidelines of the Committee on Animal Use and Care of the Chinese Academy of Sciences. Male mice were purchased from Jilin University, China (6 weeks, 15-20 g). Inoculating the subcutaneous murine U14 cells (100 μL) into the right thigh established the tumor-bearing mouse model.

In vivo PDT
After one week, the mice were intratumoral injected with PS1 NPs (100 μg mL -1 , 100 μL), the control group received saline instead. After 1 h the tumor site of the mouse was subjected to laser irradiation (200 mW cm -2 ) for 20 min. The tumor volume and body weight of the mice were measured every 2 days for 14 days. The tumor size was measured every other day and calculated as follows: volume = (tumor length) × (tumor width) 2 /2.

Histological analysis
The mice were sacrificed at day 14, and major organs and the tumors were collected and fixed in 4% paraformaldehyde. Then they were embedded into paraffin, and sliced at a thickness of 5 μm. Slices were stained with hematoxylin and eosin (H&E) and imaged by optical microscopy.

Synthesis of S1
4-(Diphenylamino)phenylboronic acid (0.694 g, 2.4 mmol), 4-bromobenzophenone (0.522 g, 2.0 mmol), Pd(PPh 3 ) 4 (0.058g, 0.05 mmol) and sodium carbonate solution (2 M, 10 mL) was dissolved in THF (40 mL) and CH 3 OH (10 mL) was stirred at 80 °C for 24 h under a nitrogen atmosphere. After cooling down the reaction mixture to ambient temperature, it was extracted with dichloromethane and washed with water. The dichloromethane layer was separated and dried over MgSO 4 . After solvent evaporation, the crude product was purified by column chromatography on silica gel using nhexane/dichloromethane (1/1, v/v) as the eluent to afford S1 as a yellow liquid (

Synthesis of PS2
To the solution of compound S2 (0.669 g, 1.0 mmol) and malononitrile (0.198 g, 3.0 mmol) in dichloromethane (60 mL) was added titanium tetrachloride (0.4 mL, 3.5 mmol) slowly at 0 °C. After the reaction mixture was stirred for 30 min, pyridine (0.3 mL, 3.5 mmol) was injected and stirred for another 30 min. Then the mixture was heated at 40 °C for 4 h. After the mixture was cooled down to room temperature, the reaction was quenched by water (300 mL) and the mixture was extracted with dichloromethane. The collected organic layer was washed with brine, dried over Na 2 SO 4 and concentrated under reduced pressure. After solvent evaporation, the crude product was purified by column chromatography on silica gel using n-hexane/dichloromethane (1/2, v/v) as the eluent to afford PS2 as a red liquid (0.535 g, 75% yield  12, 147.46, 147.32, 144.58, 135.94, 133.34, 131.38, 130.72, 129.41, 129.37, 127.94, 127.89,