Activation of apoptosis by rationally constructing NIR amphiphilic AIEgens: surmounting the shackle of mitochondrial membrane potential for amplified tumor ablation

In recent years, the use of aggregation-induced emission luminogens (AIEgens) for biological imaging and phototherapy has become an area of intense research. However, most traditional AIEgens suffer from undesired aggregation in aqueous media with “always on” fluorescence, or their targeting effects cannot be maintained accurately in live cells with the microenvironment changes. These drawbacks seriously impede their application in the fields of bio-imaging and antitumor therapy, which require a high signal-to-noise ratio. Herein, we propose a molecular design strategy to tune the dispersity of AIEgens in both lipophilic and hydrophilic systems to obtain the novel near-infrared (NIR, ∼737 nm) amphiphilic AIE photosensitizer (named TPA-S-TPP) with two positive charges as well as a triplet lifetime of 11.43 μs. The synergistic effects of lipophilicity, electrostatic interaction, and structure-anchoring enable the wider dynamic range of AIEgen TPA-S-TPP for mitochondrial targeting with tolerance to the changes of mitochondrial membrane potential (ΔΨm). Intriguingly, TPA-S-TPP was difficult for normal cells to be taken up, indicative of low inherent toxicity for normal cells and tissues. Deeper insight into the changes of mitochondrial membrane potential and cleaved caspase 3 levels further revealed the mechanism of tumor cell apoptosis activated by AIEgen TPA-S-TPP under light irradiation. With its advantages of low dark toxicity and good biocompatibility, acting as an efficient theranostic agent, TPA-S-TPP was successfully applied to kill cancer cells and to efficiently inhibit tumor growth in mice. This study will provide a new avenue for researchers to design more ideal amphiphilic AIE photosensitizers with NIR fluorescence.

Human cervical carcinoma cells (HeLa cells) were obtained from Korean Cell Line Bank (Seoul, Korea). 4T1 breast cancer cells were purchased from Institute of Basic Medical Sciences (IBMS) of the Chinese Academy of Medical Sciences (China). Cells were cultured in medium supplemented with 10% fetal bovine serum (Invitrogen) according to the guideline of ATCC. The cells were seeded in confocal culture dishes and then incubated for 24 h at 37 ºC under a humidified atmosphere containing 5% CO 2 .

Cell imaging
HeLa cells were seeded in the culture dishes at approximately concentration of 1 × 10 5 cells/mL and allowed to culture for 24 h at 37 ºC in a 5% CO 2 humidified incubator. Then, the culture medium was remove and added Hank's Balanced Salt Solution (HBSS) containing the TPA-S-D and TPA-S-TPP (5 µM) for 0, 0.5 h, 1.0 h, 1.5 h, 2.0 h, 3.0 h, and 4.0 h at 37 ºC under a humidified atmosphere containing 5% CO 2 , followed by washing thrice with DPBS. Under the confocal fluorescence microscope with a 60 × objective lens, AIEgens were excited at 559 nm (FV 1200, Olympus) and the emission was collected at 655-755 nm.

Co-localization experiments
HeLa cells were seeded in the culture dishes at approximately concentration of 1 × 10 5 cells/mL and allowed to culture for 24 h at 37 ºC in a 5% CO 2 humidified incubator. The culture medium was remove and added Hank's Balanced Salt Solution (HBSS) containing the TPA-S-TPP (5 µM) for 1.5 h at 37 ºC under a humidified atmosphere containing 5% CO 2 . Then, commercial Hoechst 33342, Lyso-tracker green, and Mito-tracker green were added into the cells under the direction of the instructions, respectively. After the incubation of another 30 min, HeLa cells were washed twice with DPBS and imaged by FV 1200 confocal microscopy.

Mitochondria-locating of AIEgen affected by CCCP
HeLa cells were seeded in the culture dishes at approximately concentration of 1 × 10 5 cells/mL and allowed to culture for 24 h at 37 ºC in a 5% CO 2 humidified incubator. The culture medium was remove and added HBSS containing the TPA-S-TPP for 2 h. and then added CCCP (10 μM) for another 20 min incubation. Cells were washed twice with DPBS and imaged by FV 1200 confocal microscopy. Similar to the above method, the order of addition should be reversed (CCCP then probes) and then the cells imaged by confocal fluorescence microscopy.
HeLa cells were treated with Mito-tracker Red CMXRos (200 nM) for 20 min, and then added CCCP (10 μM) for another 20 min incubation. Cells were washed twice with PBS and imaged by FV 1200 confocal microscopy. Similar to the above method, the order of addition should be reversed (CCCP then probes) and then the cells imaged by confocal fluorescence microscopy.
Similarly, HeLa cells were treated with Rhodamine 123 (2 µM) for 20 min, and then added CCCP (10 μM) for another 20 min incubation. Cells were washed twice with PBS and imaged by FV 1200 confocal microscopy. Similar to the above method, the order of addition should be reversed (CCCP then probes) and then the cells imaged by confocal fluorescence microscopy

Intracellular singlet oxygen detection
Singlet oxygen ( 1 O 2 ) generation inside cells under white light irradiation was measured using DCF-DA (2, 7dichlorofluorescein diacetate) Detection Kit. HeLa cells were cultured in the confocal culture dishes at 37 ºC. The culture medium was remove and added Hank's Balanced Salt Solution (HBSS) containing the TPA-S-TPP (5 µM) for 1 h at 37 ºC under a humidified atmosphere containing 5% CO 2 . Then, DCF-DA (10 µM) was added into the cells. After the incubation of 20 min, HeLa cells were washed twice with DPBS and then exposed to white light irradiation (60 mW/cm 2 ) for various time (0, 5 min, and 10 min). Additionally, the NaN 3 (50 µM) was also added into cells before photo irradiation for the consumption of ROS generation. In control group, cells only were treated with DCF-DA (10 µM). The cell images were obtained using the FV 1200 confocal microscopy with the excitation of 473 nm and the emission of 490-540 nm.

MTT assays
Measurement of cell viability was tested by reducing of 3-(4, 5)-dimethylthiahiazo (-2-yl)-3, 5diphenytetrazoliumromide (MTT) to formazan crystals using mitochondrial dehydrogenases. HeLa cells were seeded in 96-well microplates (Nunc, Denmark) at a density of 1×10 5 cells/mL in 100 μL medium containing 10% FBS. After 24 h of cell attachment, the cells were then cultured in free medium (without FBS) containing various concentrations of probes for 2 h incubation at 37 o C. Then, the free medium was changed to medium supplemented with 10% FBS and irradiated with or without white light (100 mW/cm 2 ) for 30 min. After 24 h incubation, MTT (5 mg/mL) was added to each well and the plates were incubated at 37 ºC for another 4 h in a 5% CO 2 humidified incubator. The medium was then carefully removed, and the purple crystals were lysed in 100 μL DMSO for 15~20 min. Optical density of solutions was determined on a microplate reader at 650 nm. Cell viability was expressed as a percent of the control culture value, and it was calculated using the following equation, Cells viability (%) = (OD dye -OD blank )/ (OD control -OD blank ) × 100

Annexin V-FITC/propidium iodide (PI) apoptosis detection
HeLa cells were cultured in the confocal culture dishes at 37 ºC under a humidified atmosphere containing 5% CO 2 .

Mitochondria membrane potential (MMP) analysis
HeLa cells were cultured in the confocal culture dishes at 37 ºC under a humidified atmosphere containing 5% CO 2 .
HeLa cells were then washed by DPBS and incubated with HBSS containing TPA-S-TPP (5 µM) for 2 h. The cells were washed again with DPBS, added fresh cell culture medium, and then irradiated with white light for 20 min. After 30 min incubation under cell incubator, HeLa cells were treated with fresh cell culture medium containing fluorescent probe JC-1 (2 µg/mL) at 37 °C according to the manufacture instruction. Finally, the cells were washed with DPBS three times and imaged by the FV 1200 confocal microscopy.

Cleaved caspase 3 immunofluorescence
HeLa cells were cultured on slide for 24 h and treated with the TPA-S-TPP for 2 h and irradiated with light (0, 5 min, 10 min, and 20 min) and washed with PBS and then fixed with 4% paraformaldehyde for 15 min, followed by permeabilization with PBS containing 0.1% Triton X-100 for 15 min at room temperature. The cells were incubated with blocking buffer (10% normal goat serum in PBS) for 1 h and then incubated with primary antibodies (Cell Signaling Tech.) in 1.5% normal goat serum for overnight at 4 ºC. To remove excess primary antibodies, cells were washed with PBS three times at 5 min intervals. Secondary antibodies conjugated with Alexa Fluor 488 (Cell Signaling Tech.) were added to the cells, which were incubated in the dark for 1 h at room temperature. The HeLa cells were then washed with PBS three times at 5 min intervals and mounted using UltraCruz Mounting Medium with DAPI (Santa Cruz Biotech.) Fluorescence images were detected using a confocal microscopy (FV1200, Olympus) with DAPI (ex. 405 nm/em. 430-455 nm) and Alexa Fluor 488 (ex. 473 nm/em. 490-590 nm).

Dead/Live cell co-staining
HeLa cells were cultured in the confocal culture dishes at 37 ºC under a humidified atmosphere containing 5% CO 2 .
Then, cells were treated by different treatments, as following: group 1, irradiated with white light for 10 min (Control

In vivo imaging and phototherapy evaluation
After some day's inoculation, tumors grew to about 150 mm 3 in volume before used for in vivo imaging and phototherapy. The xenograft tumor mice were given with AIEgen TPA-S-TPP (200 µM, 100 µL) through in situ injection within the period of mice anaesthesia. After that, in vivo images of 4T1 tumor-bearing Balb/c mice at various times (1 h, 2 h, 4 h, 6 h, 12 h, 24 h, and 48 h) were gathered through small-animal imaging equipment (NightOWL II LB983) with a 550 nm excitation laser and a 700 nm emission filter. In the part of PDT therapy, 4T1 tumor-bearing mice were randomly divided into four groups (n = 5 per group): 1, "PBS + dark"; 2, "PBS + light"; 3, "TPA-S-TPP + dark"; and "TPA-S-TPP + light". After intratumor injection of TPA-S-TPP (200 µM, 100 µL), the tumor region was exposed to 550 nm Xe lamp (200 mW/cm 2 ) for 30 min. Then, the tumors volume of all mice was measured every two days using a caliper for 14 days after different treatments. The tumor volumes were measured with a caliper using the following formula, V (mm 3 ) = a (mm) × a (mm) ×b (mm)/2 V represents the tumor volume; a represents the width of tumor on the mice; b represents the length of tumor on the mice. Meanwhile, the body weight of mice was also recorded using analytical balance.

H&E staining of tissue slices
After the treatment of 14 days, the mice were euthanized, and main organs (heart, liver, spleen, lung, and kidneys) were obtained for histological analysis through the standard H&E staining.