A planar electronic acceptor motif contributing to NIR-II AIEgen with combined imaging and therapeutic applications

Designing molecules with donor–acceptor–donor (D–A–D) architecture plays an important role in obtaining second near-infrared region (NIR-II, 1000–1700 nm) fluorescent dyes for biomedical applications; however, this always comes with a challenge due to very limited electronic acceptors. On the other hand, to endow NIR-II fluorescent dyes with combined therapeutic applications, trivial molecular design is indispensable. Herein, we propose a pyrazine-based planar electronic acceptor with a strong electron affinity, which can be used to develop NIR-II fluorescent dyes. By structurally attaching two classical triphenylamine electronic donors to it, a basic D–A–D module, namely Py-NIR, can be generated. The planarity of the electronic acceptor is crucial to induce a distinct NIR-II emission peaking at ∼1100 nm. The unique construction of the electronic acceptor can cause a twisted and flexible molecular conformation by the repulsive effect between the donors, which is essential to the aggregation-induced emission (AIE) property. The tuned intramolecular motions and twisted D–A pair brought by the electronic acceptor can lead to a remarkable photothermal conversion with an efficiency of 56.1% and induce a type I photosensitization with a favorable hydroxyl radical (OH˙) formation. Note that no additional measures are adopted in the molecular design, providing an ideal platform to realize NIR-II fluorescent probes with synergetic functions based on such an acceptor. Besides, the nanoparticles of Py-NIR can exhibit excellent NIR-II fluorescence imaging towards orthotopic 4T1 breast tumors in living mice with a high sensitivity and contrast. Combined with photothermal imaging and photoacoustic imaging caused by the thermal effect, the imaging-guided photoablation of tumors can be well performed. Our work has created a new opportunity to develop NIR-II fluorescent probes for accelerating biomedical applications.

UV-Vis spectra were measured on a SHIMADZU UV-2600i UV-Vis spectrophotometer.
Fluorescence spectra were performed on EDINBURGH FS5/FLS 1000 spectrofluorometers.TEM image was taken with a JEM-1400Flash JEOL transmission electron microscope.DLS was performed with a NANO ZS (Malvern Panalytical Co., UK) at a fixed angle of 90°.The cellular fluorescence images were taken with a confocal laser scanning microscope (CLSM, ZEISS-LSM900).The cell viability was investigated by CCK-8 kit, and the absorbance of the samples were recorded at 450 nm with a microplate reader (synergyH1, BioTek).The cell apoptosis was investigated with a BD FACSAria SORP fluorescence activated cell sorting (FACS).Temperature change and photothermal images were studied by an E6 camera (FLIR System).The in vivo fluorescence imaging was performed with a commercial NIR-II in vivo imaging system (MARS-HS, Artemis Intelligent Imaging).Photoacoustic imaging was investigated based on a commercial optoacoustic imaging system (Vevo LAZR, Fuji Film Visual Sonics).Phototherapeutic experiments were conducted by an 808 nm infrared semiconductor laser (Changchun radium photoelectric technology).

Synthesis of Py-NIR
Into a 100 mL round bottom flask was added 3 (500 mg, 0.72 mmol), 5,6-diaminopyrazine-2,3dicarbonitrile (4, 150 mg, 0.94 mmol), 20 mL of 1,2,4-trimethylbenzene and trace ptoluenesulfonic acid.The mixture was allowed for stir at 150 °C for 4 days.After that, the mixture was condensed by reduce pressure and the crude product was purified on a silica geo column with petroleum ether/dichloromethane (v/v = 1:1) as eluent.A black powder of 70 mg was obtained in a yield of 11.9 %. 1

Theoretical calculation
The optimization of S1 conformation and calculation of electrostatic potential map were carried out with a Gaussian 09 package 1 at TD-PBE0/6-31G(d,p).Empirical dispersion was taken to obtain a better description of the weak interaction.The SMD model was adopted to simulate the THF environment.[4]

Fabrication of NPs
Py-NIR (1 mg) and DSPE-mPEG2000 (10 mg) were first dissolved in 1 mL of THF, which was then added into 10 mL of water dropwise in 1 min under ultrasound.Followed by another 2-minute ultrasound, the organic solvent was removed by dialysis (3500 kDa molecular weight cutoff) and the NPs were concentrated by centrifuge ultrafiltration.The concentration of NPs based on Py-NIR was calculated based on a standard curve by the UV-Vis spectra analysis.

Photothermal conversion evaluation
To evaluate the effect of Py-NIR NPs concentration on the photothermal conversion properties, the NPs with different concentration of 25, 50, 75, 100 and 150 μM (based on dye) were irradiated by 808 nm laser (1.0 W cm -2 ).Similarly, to evaluate the powder dependence, the 808 nm laser with different laser power densities (0.6, 0.8, 1.0, 1.2, 1.4 W cm -2 ) were applied to the photothermal experiments.The photothermal stability was studied by a heating-cooling experiment with 6 cycles.
The variation in temperature was recorded by a FLIR E6 camera.The photothermal conversion efficiency of NPs was evaluated by the reported method. 5

Cell culture
Mouse breast cancer 4T1 cell line was provided by Chinese Academy of Science Cell Bank for Type Culture Collection, and grown in 1640 culture medium having 10% FBS and 1% antibiotics (penicillin-streptomycin) at 37 °C under 5% CO2.

Intracellular ROSs generation
4T1 cells were seeded in glass bottom dish and cultured for 24 h, After that, the cells were incubated with or PBS or Py-NIR NPs (50 μM, based on dye) in fresh medium for 12 h, followed by incubated with DCFH-DA (10 μM) in 1 mL fresh serum-free medium at 37 °C for another 20 min.The cells were washed and irradiated by 808 nm laser (1.0 W cm -2 ) for 5 min if necessary and imaged by CLSM.Excitation wavelength: 488 nm; emission filter: 500550 nm.

Live-dead cell staining
4T1 cells were seeded in glass bottom dish and cultured for 24 h.Then, the cells were divided into four groups with treatments of PBS, PBS + laser, Py-NIR NPs, and Py-NIR NPs + laser, respectively.For all groups, the cells were incubated for 12 h before further treatments.For groups involving light irradiation, the 808 nm laser (1.0 W cm -2 ) was used to irradiate the cells for 5 min.
The concentration of Py-NIR NPs was 50 μM based on dye.After these treatments, the cells were incubated at 37 °C for another 30 min, and then stained by PI (60 μg mL -1 ) and FDA (100 μg mL - 1 ) for 10 min.After that, the cells were gently washed and then imaged by CLSM.Excitation wavelength: 488 nm (FDA) and 561 nm (PI); emission filter: 500550 nm (FDA) and 600700 nm (PI).

Cell apoptosis evaluation
4T1 cells (10 5 cells per well) were seeded and cultured in 6-well plates for 24 h.After that, the cells were incubated with the Py-NIR NPs (50 μM, based on dye) in the culture media for 12 h and irradiated by 808 nm laser (1.0 W cm -2 ) for 5 min.The cells treated with PBS, Py-NIR NPs and PBS plus laser were set as the controls.Then, the cells were incubated at 4 °C for another 0.5 h, washed with PBS, and collected by centrifugation at 1000 rpm for 5 min at 4 °C.Afterwards, the cells were stained with an Annexin V-FITC/PI Apoptosis Detection Kit based on manufacturer's instructions and analyzed by FACS.

Phototoxicity test
4T1 cells were seeded on a 96-well plates at a density of 5 × 10 3 cells per well and cultured for 24 h.After removing the culture medium, the new mediums containing Py-NIR NPs with different concentrations (0, 10, 20, 30, 40 and 50 μM, based on dye) were adopted.After 12 h incubation, the cells were irradiated by 808 nm laser (1.0 W cm -2 ) for 5 min.At the same time, the cells incubated with the NPs in the absence of light irradiation were taken for the dark cytotoxicity study.
After further 12 h incubation, the culture media were removed, and the cells were washed with PBS for three times and incubated with fresh FBS-free medium containing 10% CCK-8 for 2 h in the dark.Then, the absorption wavelength at 450 nm of the products was recorded with a microplate reader.The results were embodied as the viable percentage of cells after light irradiation versus the cells without light irradiation.The relative cell viability was calculated based on the following formula: Cell viability (%) = (ODsample -ODbackground)/(ODcontrol -ODbackground) × 100%.

In vivo NIR-II fluorescence, photoacoustic and photothermal imaging
For NIR-II fluorescence or photoacoustic imaging, the orthotopic 4T1 breast tumor bearing mice were administered with 200 μL of Py-NIR NPs (2 mM, based on the dye) via tail vein.Then, the in vivo NIR-II fluorescence images were captured at predetermined time intervals (3, 6, 12 and 24 h) of post-injectionon by a NIR-II in vivo imaging system with the long pass (LP) filter of 1000 nm under an excitation wavelength of 808 nm.Besides, the in vivo photoacoustic imaging was collected on a commercial optacoustic imaging system at the same time intervals after intravenous injection of the NPs.The NIR-II fluorescence and photoacoustic images at 0 h were obtained before the NPs injection.In order to evaluate the distributions of Py-NIR NPs in the tissues, the mice were sacrificed at 24 h post-injection.The major organs including heart, liver, spleen, lung, and kidney, and tumor were excised.Then, they are washed with saline several times followed by NIR-II fluorescence imaging and quantitative analyses.For in vivo photothermal imaging, the infrared thermal images of mice were taken with an IR camera under the irradiation of 808 nm laser (1.0 W cm -2 ) while they are intravenously injected with 200 μL of Py-NIR NPs (2 mM, based on dye) for 12 h.Meanwhile, the mice treated with saline under the same irradiation condition was employed as a control.

In vivo antitumor efficacy
To evaluate the in vivo antitumor efficacy of Py-NIR NPs, the orthotopic 4T1 breast tumor bearing mice with the tumor volume reached ~ 100 mm 3 were randomly divided into 4 groups (n = 5), which were Saline, Saline + L, Py-NIR NPs and Py-NIR NPs + L, respectively.For these groups, 200 µL of saline or Py-NIR NPs (2 mM, based on dye) were intravenously injected into the mice before further treatments.In groups needing light irradiation, the 808 nm laser (1.0 W cm -2 ) was employed to irradiate the tumor site in each group for 10 min after intravenous injection of saline or Py-NIR NPs (2 mM, based on dye) for 12 h.After different treatments, the mouse body weight and tumor volume were recorded every 3 days during the 15-day study duration.The tumor size was measured by a vernier caliper, and the tumor volume (V) was estimated using the formula: V = (length × width 2 )/2.Relative tumor volume (RTV) was evaluated as RTV = V/V0, while V0 was the initial tumor volume.Relative body weight (RBW) was evaluated as RBW = (W-W0)/W0, while W0 was the initial mouse body weight.

Histological and hematological analyses
At the 15th day of various treatments, the blood samples of the mice were extracted for serum biochemistry and hematology analyses.After that, the mice were sacrificed.The tumors and major organs including heart, liver, spleen, lung and kidney were excised, fixed in 4% (v/v) formalin saline overnight, embedded in paraffin and then sectioned at the thickness of 5 μm.For the group treated with Py-NIR NPs plus laser, tumor samples were collected at 12 h after light irradiation.
Then, the obtained tumor sections from four groups were subjected to H&E, CD31, Ki67 and TUNEL staining and investigated by an inverted optical microscopy to evaluate the histopathological behavior.Besides, the slices of major organs were conducted by H&E staining to evaluate the biosafety.

Statistical Analysis
Data are donated as the mean ± standard deviation (SD).The significance between experimental and control groups was determined by unpaired 2-tailed Student's t-test with the GraphPad Prism All the animal experiments conducted in this work were approved by the Administrative Committee on Animal Research in Shenzhen Graduate School, Peking University (SYXK(YUE)2017-0172). Female BALB/c mice aged 4~5 weeks were provided by Beijing Vital River Laboratory Animal Technology.The mice were housed under a pathogen-free condition and fed with standard laboratory water and chow.Orthotopic 4T1 breast tumor model was established by orthotopical injection of 4T1 cells (5 × 10 5 ) suspended in PBS into the upper right mammary fat pads.The 4T1 tumor bearing mice were subsequently employed when the tumor volumes reached ~ 100 mm 3 .

Figure S9 .
Figure S9.UV-vis spectra of Py-NIR NPs in (A) water and (B) PBS during two weeks.

Figure S13 .Figure S14 .
Figure S13.PL spectra of DHR123 in the presence of Py-NIR NPs under continuous 30-minute

Figure 15 .
Figure 15.Cell viabilities of HUVEC (human umbilical vein endothelial cell), LO2 (human normal liver cell) and 3T3 (mouse fibroblast cell) after incubation of Py-NIR NPs with different concentrations for 24 h.

Figure S17 .Figure S18 .
Figure S17.NIR-II fluorescence imaging of heart, liver, spleen, lung, kidney and tumor excised from the mice after 24 h post-injection of the Py-NIR NPs.

Table S1 .
Routine blood indices of mice after different treatments.