Highly Efficient Photosensitizers with Molecular Vibrational Torsion for Cancer Photodynamic Therapy

The development of highly effective photosensitizers (PSs) for photodynamic therapy remains a great challenge at present. Most PSs rely on the heavy-atom effect or the spin–orbit charge-transfer intersystem crossing (SOCT-ISC) effect to promote ISC, which brings about additional cytotoxicity, and the latter is susceptible to the interference of solvent environment. Herein, an immanent universal property named photoinduced molecular vibrational torsion (PVT)-enhanced spin–orbit coupling (PVT-SOC) in PSs has been first revealed. PVT is verified to be a widespread intrinsic property of quinoid cyanine (QCy) dyes that occurs on an extremely short time scale (10–10 s) and can be captured by transient spectra. The PVT property can provide reinforced SOC as the occurrence of ISC predicted by the El Sayed rules (1ππ*–3nπ*), which ensures efficient photosensitization ability for QCy dyes. Hence, QTCy7-Ac exhibited the highest singlet oxygen yield (13-fold higher than that of TCy7) and lossless fluorescence quantum yield (ΦF) under near-infrared (NIR) irradiation. The preeminent photochemical properties accompanied by high biosecurity enable it to effectively perform photoablation in solid tumors. The revelation of this property supplies a new route for constructing high-performance PSs for achieving enhanced cancer phototherapy.


S2
respectively.Unless otherwise specified, all spectroscopic tests were performed in a quartz cell (10×10 mm).Fluorescence quantum yield of all the compounds were measured on the HAMAMATSU absolute fluorescence quantum yield spectrometer (Serial No. C11347).Triplet lifetime of compounds were acquired on LP980 laser flash photolysis spectrometer (Edinburgh Instruments, U.K.).The cell confocal laser scanning microscope (CLSM) images were all obtained by using Olympus FV3000 confocal laser scanning microscope.And the mice fluorescence imaging experiments were performed on NightOWL II LB983 small animal in vivo imaging system (German).

Synthesis of QTCy7-R
The synthesis route and method and structural characterization of QTCy7-R were shown in Scheme S1 and Figure S24-Figure S43.

Computational methods
Density functional theory (DFT) and time-dependent DFT (TD-DFT) were employed to rationalize the excited state properties of QTCy7-R.The ground state and excited state geometric configurations of the compounds were optimized by using B3LYP functional in combination with the 6-31G(d, p) basis set [1][2] .The solvent (dichloromethane) effect was included in all calculations based on the polarizable continuum model (PCM).Frequency analysis was performed to confirm that we have obtained stable structures on the potential energy surfaces.All calculations were performed on Guassian 16A unless otherwise noted.The S1 coordinate were obtained by optimizing molecule at S1, and S1V coordinate were obtained by optimizing molecule at S1 state with T4 initial coordinate (The intramolecular dihedral angles were given in Figure 4, Figure S6 and Figure S7).The spin-orbital coupling (SOC) values between the S1/S1V and T2 were calculated with ORCA 4.1 3 .Hole-Electron analysis was carried out using Multiwfn 3.8 4 .

ISC rate constants calculation
The ISC rate constants from singlet to triplet states were calculated according to the Marcus theory: Where   , ℏ,   , ,   ,  and Δ  represent the ISC rate constants, reduced Planck constant, spin-orbit coupling (SOC) constant (  = 〈 1 |ℋ ̂ |  〉) , reorganization energy, Boltzmann constant, temperature and energy gap from singlet to triplet states (Δ  =  1 −   ), respectively.Boltzmann distribution function for the population of the initial vibronic manifold was considered to calculate the contribution of all initial electronic states (Ei) to the total ISC rate, each transition was weighted using the Boltzmann thermal factor:

𝑖
Where (  ) and   represent Boltzmann thermal factor and the energy of each initial electronic states.

Singlet oxygen detection
Photoexcited singlet oxygen production was detected by employed 1,3diphenylisobenzofuran (DPBF) as the indicator.The absorbance of DPBF at 415 nm was regulated to about 1.0 in DCM.And the samples were irradiated with 660 nm light for various times, then the absorption spectra of different time nodes were recorded.
The singlet oxygen quantum yield (ΦΔ) was calculated by the following equation: Where "Φ" represent the singlet oxygen quantum yield, "sam" and "std" represent QTCy7-R and MB, respectively."m" is the slope of the absorbance of DPBF at 415 nm with time, F=1-10 -O.D. (O.D. is the absorbance of samples at 660 nm).

Triplet lifetime measurements
The triplet life of QTCy7-R was measured by LP980 laser flash photolysis spectrometer (Edinburgh Instruments Ltd.) in combination with a Nd:YAG laser (Surelite I-10, Continuum Electro-Optics, Inc.).The oxygen in the sample was removed by aerating nitrogen for 30 min.The samples (5 μM) were excited by a 610 nm laser pulse (1 Hz, 100 mJ per pulse, fwhm ≈ 7 ns) at 300 K.The triplet state decay kinetics were measured at the maximum absorption wavelengths of each compound.

Fluorescence lifetime and femtosecond time-resolved transient absorption spectra measurements
The fluorescence lifetimes and femtosecond time-resolved transient absorption spectra of dyes were recorded on a freshly prepared samples using the time-correlated single photon counting (TCSPC) method (PicoQuant PicoHarp 300) at 300 K.The concentration is 10 μM, solvent is DCM.

Cell incubation
Hepatoma carcinoma cells (HepG-2 cells), human mammary carcinoma (MCF-7) and mouse breast cancer cells (4T1) were maintained in Dulbecco's modified Eagle's medium (DMEM, Invitrogen) with 10 % fetal bovine serum (Invitrogen) and 1% penicillinstreptomycin.All cells were treated at 37 ºC with the humidified atmosphere containing 5% CO2 and 95% air.Before conducting cell imaging experiments, all types of cells were maintained on 35 mm glass-bottom cell dishes for 24 h.

Confocal fluorescence imaging
After culturing the cells in 35 mm glass-bottom cell dishes for 24 h, the DMEM medium was replaced with saline, then QTCy7-R was added

Mitochondrial membrane potential detection
After culturing the cells in 35 mm glass-bottom cell dishes for 24 h, the DMEM medium was replaced with saline, the cells were divided into four groups: 1) cells incubated with 3 μM QTCy7-Me/QTCy7-Ph for 90 min under condition; 2) cells incubated with 3 μM QTCy7-Me/QTCy7-Ph for 90 min and irradiated with 660 nm light (10 mW/cm 2 , 10 min).After the experiment, each group of cells was incubated with JC-1 for 20 minutes (according to the JC-1 kit instructions).Normal cells with high mitochondrial membrane potential showed red fluorescence signal (JC-1 aggregates); yet injured cells with low mitochondrial membrane potential showed green fluorescence signal (JC-1 monomer).

Intracellular ROS detection
2,7-dichlorofluorescein diacetate (DCFH-DA) Detection Kit was chosen to validate the intracellular ROS generation.After culturing the cells in 35 mm glassbottom cell dishes for 24 h, the cells were divided into ten groups: 1) cells incubated with PBS; 2) cells incubated with PBS and irradiated with 660 nm light (10 mW/cm 2 , 10 min); 3) cells incubated with QTCy7-R; 4) cells incubated with QTCy7-R and irradiated with 660 nm light (10 mW/cm 2 , 10 min).After incubated with QTCy7-R or PBS, the cells were treated with DCFH-DA for 20 min.Then the cells were exposed under NIR irradiation, after which the confocal fluorescence imaging was performed.

Confocal imaging of cell viability
HepG2 cells were incubated in 35 mm glass-bottom cell dishes for 24 h, the

S5
DMEM medium was replaced with saline to prevent false positive interference.Then the cells were exposed to different following treatments: 1) cells incubated with PBS; 2) cells incubated with PBS under 660 nm irradiation (20 mW/cm 2 , 10 min); 3) cells incubated with QTCy7-R; 4) cells incubated with QTCy7-R under 660 nm irradiation (20 mW/cm 2 , 10 min).After being different treated, the cells were stained with Calcein-AM and Propidium Iodide (PI) according to the manufacture instruction.Then the confocal fluorescence imaging was carried out and recorded with a 10 × objective lens.

Cytotoxicity assays
Cytotoxicity was tested by the reduction of methyl thiazolyl tetrazolium to formazan by succinic acid dehydrogenase in the mitochondria of living cells (MTT assay).HepG2 cells, MCF7 cells and 4T1 cells were seeded to 96-well microplates (Nunc, Denmark) with a density of 1 × 10 5 cells/mL cells in 100 μL DMEM medium and incubated at 37 ºC for 24 h.When the cell density reached about 70%, the QTCy7-R with different concentrations (4, 3, 2, 1.5, 1, 0.75, 0.5, 0.25, 0 μM) in PBS were added to the wells of cells in dark condition, respectively.For light groups, the cell incubation conditions were the same as those of the dark group and the cells were further incubated for 2 h, subsequently, the cells were subjected to 660 nm irradiation (20 mW/cm 2 ) for different time (10 min, 5 min and 2.5 min) corresponding to different light doses (12 J/cm 2 , 6 J/cm 2 and 3 J/cm 2 ).Then all the cells were incubated for 12 h, after which the MTT solution in DMEM (0.5 mg/ml, 100 μL) was added to each well and the cells were incubated at 37 ºC for another 4 h.The MTT solution was carefully removed and 100 μL DMSO was added to dissolve formazan, and the absorbance of each well was measured by a multifunctional microplate reader at 570 nm and 630 nm.The cell viability was obtained by the following equation: Where "OD" represent the absorbance at 570 nm and 630 nm, "ps" and "control" represent experimental groups and control groups, respectively.

4T1-subcutaneous tumor model and in vivo imaging experiments
Female BALB/c mice, 4-5 weeks of age, were selected to establish 4T1subcutaneous tumor model.5 × 10 6 4T1 cells were inoculated subcutaneously to their armpits.when the volume of subcutaneous tumors reached about 100 mm 3 , QTCy7-Ac (200 μM, 100 μL) was injected into 4T1 tumor-bearing BALB/c mice through tail vein.
And the fluorescence signals were monitored at different post-injection time by a NightOWL II LB983 small animal in vivo imaging system (German).For the images of major organs, the mice L) was intravenously injected with QTCy7-Ac (200 μM, 100 μL) 2 hours in advance were killed and the main organs and tumors were removed for imaging experiment.

In Vivo Biosafety Assay
The in vivo biocompatibility was evaluated by using measurement mice body weight and H&E slice histological analysis.After the PDT treatment, the mice were euthanized, and main organs including heart, liver, spleen, lung, kidneys and tumors were harvested for histological analysis by means of hematoxylin-eosin (H&E) staining.

Synthesis of compound 1
2-methylbenzothiazole (1.5 g, 10 mmol) and iodoethane (3.12 g, 20 mmol) were stirred in a quartz seal tube and heated to 110 ℃ for 16 hours.Subsequently, the reaction mixure was cool to room temperature.The precipitate was filtered and dried in vacuum to obtain a compound 1 as a white solid.(Yield: 86%)

Synthesis of compound 2a
4-methylphenol (1 g, 9.25 mmol) and hexamethylenetetramine (4.2 g, 30 mmol) were dissolved in 10 mL trifluoroacetic acid.The reaction mixture was stirred at 100 ℃ for 24 h under N2 atmosphere.Subsequently, the mixture was cooled down to 70 ℃ and 50 mL HCl (4 M) was added carefully.After 3 h reaction, the product was cooled to room temperature, the precipitate was filtered and dried in vacuum to obtain a pale yellow solid.If no precipitation formed, the product was extracted with dichloromethane and the solvent was removed under vacuum.The residue was purified by column chromatography with petroleum-ether/EtOAc (5:1) as eluent to afford a pale yellow solid.(Yield: 74%) 1

Synthesis of compound 2c
Ethyl 4-hydroxybenzoate (1 g, 6.02 mmol) and hexamethylenetetramine (2.8 g, 20 mmol) were dissolved in 10 mL trifluoroacetic acid.The reaction mixture was stirred at 100 ℃ for 48 h under N2 atmosphere.Subsequently, the mixture was cooled down to 70 ℃ and 50 mL HCl (4 M) was added carefully.After 3 h reaction, the product was cooled to room temperature, the precipitate was filtered and dried in vacuum to obtain a pale yellow solid.If no precipitation formed, the product was extracted with dichloromethane and the solvent was removed under vacuum.The residue was purified by column chromatography with petroleum-ether/EtOAc (3:1) as eluent to afford a pale yellow solid.(Yield: 60%) 1

Figure S6 .
Figure S6.(a) The views from different locations (up, front and side) of QTCy7-CHO and their torsion angles in S1 and S1V coordinates.(b) The frontier molecular orbital (FMO) and the corresponding energy of QTCy7-CHO in S1 and S1V coordinates.(c) The views from different locations (up, front and side) of QTCy7-Me and their torsion angles in S1 and S1V coordinates.(d) The frontier molecular orbital (FMO) and the corresponding energy of QTCy7-Me in S1 and S1V coordinates.

Figure S7 .
Figure S7.(a) The views from different locations (up, front and side) of QTCy7-Ph and their torsion angles in S1 and S1V coordinates.(b) The frontier molecular orbital (FMO) and the corresponding energy of QTCy7-Ph in S1 and S1V coordinates.(c) The views from different locations (up, front and side) of TCy7 and their torsion angles in S1 and S1V coordinates.(d) The frontier molecular orbital (FMO) and the corresponding energy of TCy7 in S1 and S1V coordinates.

Figure S9 .
Figure S9.(a) The hole-electron distribution of QTCy7-CHO at S1 and T2 in S1 coordinate.(b) (c) The heat map of hole-electron distribution of QTCy7-CHO at S1 and T2 in S1 coordinate.(d) The hole-electron distribution of QTCy7-CHO at S1 and T2 in S1V coordinate.(e) (f) The heat map of hole-electron distribution of QTCy7-CHO at S1 and T2 in S1V coordinate.

Figure S10 .
Figure S10.(a) The hole-electron distribution of QTCy7-Me at S1 and T2 in S1 coordinate.(b) (c) The heat map of hole-electron distribution of QTCy7-Me at S1 and T2 in S1 coordinate.(d) The hole-electron distribution of QTCy7-Me at S1 and T2 in S1V coordinate.(e) (f) The heat map of holeelectron distribution of QTCy7-Me at S1 and T2 in S1V coordinate.

Figure S11 .
Figure S11.(a) The hole-electron distribution of QTCy7-Ph at S1 and T2 in S1 coordinate.(b) (c) The heat map of hole-electron distribution of QTCy7-Ph at S1 and T2 in S1 coordinate.(d) The holeelectron distribution of QTCy7-Ph at S1 and T2 in S1V coordinate.(e) (f) The heat map of holeelectron distribution of QTCy7-Ph at S1 and T2 in S1V coordinate.

Figure S12 .S18Figure S14 .
Figure S12.(a) The hole-electron distribution of TCy7 at S1 and T2 in S1 coordinate.(b) (c) The heat map of hole-electron distribution of TCy7 at S1 and T2 in S1 coordinate.

Figure S22 .
Figure S22.(a) The picture of isolated tumors at different groups.(b) The tumor inhibition rate in different groups.

Figure S23 .
Figure S23.H&E staining assays of main organs and tumors at Dark group (first line), Irradiation group (second line), QTCy7-Ac group (third line) and QTCy7-Ac+irradiation group (final line) after 16 days of treatment.(Scale bar = 100 μm)