Rationally designed ruthenium complexes for 1- and 2-photon photodynamic therapy

Computational details

All calculations were performed at DFT (ground state) and TD-DFT (excited state) level using the global hybrid B3LYP functional. The Los Alamos (LANL2) effective core potential and the corresponding triple-zeta basis set was applied to describe the Ruthenium atom, with all other atoms were described by a Pople double-zeta basis set with a single set of polarization functions on non-hydrogen atoms (6–31G(d)). All structures used correspond to ground state minima on the ground state potential energy surfaces. Solvent effect (acetonitrile) were taken into account using the PCM model for structural optimizations while single point calculations in the gas phase were performed to simulate 1P and 2P absorption spectra. All calculations were performed using the Gaussian suite of programs except for the 2PA spectra which were simulated using the DALTON software.

Materials

All chemicals were purchased from commercial sources and used without further purification. If necessary, solvents were dried over molecular sieves. The Ru(II) precursors Ru(DMSO)₄Cl₂⁴⁹ and Ru(2,2′-bipyridine)₂Cl₂⁵⁰ were synthesized according to previously reported procedures. The pooled human plasma was obtained from Biowest. The cell culture media and reagents were purchased from Fisher Scientific.

Instrumentation and methods

¹H and ¹³C-NMR spectra were measured on a Bruker 400 MHz or 500 MHz NMR spectrometer. Chemical shifts (δ) are reported in parts per million (ppm) referenced to tetramethylsilane (δ 0.00) ppm using the residual proton solvent peaks as internal standards and coupling constants (J) in Hertz (Hz). The multiplicity of the peaks is abbreviated as follows: s (singlet), d (doublet), dd (doublet of doublet), t (triplet), m (multiplet). ESI-MS experiments were carried out using a LTQ-Orbitrap XL from Thermo Scientific and operated in positive ionization mode with a spray voltage of 3.6 kV. No Sheath and auxiliary gas was used. Applied voltages were 40 and 100 V for the ion transfer capillary and the tube lens, respectively. The ion transfer capillary was held at 275 °C. Detection was achieved in the Orbitrap with a resolution set to 100,000 (at m z⁻¹ 400) and a m z⁻¹ range between 150 and 2000 in profile mode. Spectrum was analyzed using the acquisition software XCalibur 2.1 (Thermo Fisher Scientific). The automatic gain control allowed accumulation of up to 2 × 10⁵ ions for FTMS scans, maximum injection time was set to 300 ms and 1 µscan was acquired. Ten microliters was injected using a Thermo Finnigan Surveyor HPLC system (Thermo Fisher Scientific) with a continuous infusion of methanol at 100 µL min⁻¹. Elemental microanalyses were performed on a Thermo Flash 2000 elemental analyzer. For analytic and preparative HPLC the following system has been used: 2 × Agilent G1361 1260 Prep Pump system with Agilent G7115A 1260 DAD WR Detector equipped with an Agilent Pursuit XRs 5C18 (Analytic: 100 Å, C18 5 μm, 250 × 4.6 mm, Preparative: 100 Å, C18 5 μm 250 × 300 mm) column and an Agilent G1364B 1260-FC fraction collector. The solvents (HPLC grade) were millipore water (0.1% trifluoroacetic acid, solvent A) and acetonitrile (0.1% trifluoroacetic acid, solvent B). ICP-MS experiments were carried out on an iCAP RQ ICP-MS instrument (Thermo Fisher).

Synthesis

(E,E′)-4,4′-Bisstyryl-2,2′-bipyridine: The synthesis of (E,E′)-4,4′-Bisstyryl-2,2′-bipyridine is already published but in this study another synthetic route was employed. 4,4′-Dimethyl-2,2′-bipyridine (1000 mg, 5.43 mmol, 1.0 equiv.) was dissolved in dry N,N-dimethylformamide under nitrogen atmosphere and benzaldehyde (1.2 mL, 11.84 mmol, 2.2 equiv.) was added to the solution. Afterwards potassium tert-butoxide (2436 mg, 21.72 mmol, 4.0 equiv.) was added slowly. The color of the solution turned to green and the mixture was stirred for 24 h. After that the mixture was poured into water (400 mL) and the suspension cooled down to 5 °C. The crude product which precipitated, was filtered and washed with methanol. The product was purified by recrystallization from boiling acetic acid. The obtained solid was dissolved in dichloromethane and the mixture was washed with a 5% aqueous lithium chloride solution, brine and water. The solvent was removed and the product was isolated by recrystallization from boiling acetic acid. 1292 mg of (E,E′)-4,4′-Bisstyryl-2,2′-bipyridine (3.58 mmol, 66%) were yielded as a beige solid. The experimental data obtained is in agreement with the previous literature⁵¹.

(E,E′)-4,4′-Bis[p-(N,N-dimethylamino)styryl]-2,2′-bipyridine: The synthesis of (E,E′)-4,4′-Bis[p-(N,N-dimethylamino)styryl]-2,2′-bipyridine is already published but in this study another synthetic route was employed. 4,4′-Dimethyl-2,2′-bipyridine (1000 mg, 5.43 mmol, 1.0 equiv.) was dissolved in dry N,N-dimethylformamide (100 mL) under nitrogen atmosphere and potassium tert-butoxide (2437 mg, 21.72 mmol, 4.0 equiv.) was added slowly. After 1.5 h of stirring, 4-(dimethylamino)benzaldehyde (1701 mg, 11.40 mmol, 2.1 equiv.) was added to the reaction mixture. The color of the solution turned to yellow and the mixture was heated at 90 °C for 19 h. After that the mixture was poured into water (400 mL) and the suspension cooled down to 5 °C. The crude product which precipitated, was filtered and washed with water and diethyl ether. The product was isolated by recrystallization from dichloromethane/pentane. 1541 mg of (E,E′)-4,4′-Bis[p-(N,N-dimethylamino)styryl]-2,2′-bipyridine (3.45 mmol, 64%) were yielded as a yellow solid. The experimental data obtained is in agreement with the previous literature⁵².

(E,E′)-4,4′-Bis[p-methoxystyryl]-2,2′-bipyridine: The synthesis of (E,E′)-4,4′-Bis[p-methoxystyryl]-2,2′-bipyridine is already published but in this study another synthetic route was employed. 4,4′-Dimethyl-2,2′-bipyridine (532 mg, 2.89 mmol, 1.0 equiv.) was dissolved in dry N,N-dimethylformamide (25 mL) under nitrogen atmosphere and 4-methoxybenzaldehyde (0.88 mL, 7.22 mmol, 2.5 equiv.) was added to the solution. Afterwards potassium tert-butoxide (1360 mg, 12.13 mmol, 4.2 equiv.) was added slowly. The color of the solution turned to green and the mixture was stirred for 24 h. After that the mixture which turned bright was poured into water (200 mL) and the suspension cooled down to 5 °C. The crude product which precipitated, was filtered, and washed with methanol. The product was purified by recrystallization from boiling acetic acid. The obtained solid was dissolved in dichloromethane and the mixture was washed with a 5% aqueous lithium chloride solution, brine and water. The solvent was removed and the product was isolated by recrystallization from boiling acetic acid. 925 mg of (E,E′)-4,4′-Bis[p-methoxystyryl]-2,2′-bipyridine (2.20 mmol, 76%) were yielded as a beige solid. The experimental data obtained is in agreement with the previous literature⁵².

[Ru(E,E′)-4,4′-Bisstyryl-2,2′-bipyridine)₃][PF₆]₂ (1): (E,E′)-4,4′-Bisstyryl-2,2′-bipyridine (400 mg, 1.11 mmol, 4.0 equiv.) and Ru(DMSO)₄Cl₂ (134 mg, 0.28 mmol, 1.0 equiv.) were suspended in dry ethanol (150 mL) under nitrogen atmosphere and the mixture was refluxed for 24 h. Then the solution was cooled down and undissolved residue was removed by filtration. To the residual solution a saturated aqueous solution of ammonium hexafluorophosphate was added. The crude product, which precipitated as a hexafluorophosphate salt was collected by centrifugation and washed with ethanol, water and diethyl ether. The product was dissolved in dichloromethane and washed with a 5% aqueous lithium chloride solution, brine and water. After drying, 323 mg of 1 (0.22 mmol, 79%) were yielded as a red solid. ¹H-NMR (500 MHz, CD₃CN): δ 8.76 (d, J = 1.8 Hz, 6H), 7.78 (d, J = 16.4 Hz, 6H), 7.76 (d, J = 5.9 Hz, 6H), 7.71–7.69 (m, 12H), 7.51 (dd, J = 5.9, 1.8 Hz, 6H), 7.49–7.46 (m, 12H), 7.44–7.40 (m, 6H), 7.33 (d, J = 16.4 Hz, 6H); ¹³C-NMR (125 MHz, CD₃CN): δ 158.2, 152.4, 147.6, 137.3, 136.8, 130.6, 130.1, 128.4, 125.4, 125.1, 121.7; HRMS (m/z): [M]²⁺ calcd. for C₇₈H₆₀N₆Ru, 591.1956; found, 591.1978; analysis (calcd., found for C₇₈H₆₀F₁₂N₆P₂Ru + 4^.H₂O): C (60.66, 60.62), H (4.44, 4.43), N (5.44, 5.76). [Ru(E,E′)-4,4′-Bisstyryl-2,2′-bipyridine)₃][Cl]₂: The counter ion hexafluorophosphate was exchanged to chloride by elution with methanol from the ion exchange resin Amberlite IRA-410. Analysis (calcd., found for C₇₈H₆₀Cl₂N₆Ru): C (74.75, 74.36), H (4.83, 4.51), N (6.71, 6.37).

[Ru((E,E′)-4,4′-Bis[p-(N,N-dimethylamino)styryl]-2,2′-bipyridine)₃][PF₆]₂ (2): (E,E′)-4,4′-Bis[p-(N,N-dimethylamino)styryl]-2,2′-bipyridine (338 mg, 0.76 mmol, 4.0 equiv.), Ru(DMSO)₄Cl₂ (92 mg, 0.19 mmol, 1.0 equiv.) and lithium chloride (401 mg, 9.46 mmol, 50.0 equiv.) were dissolved in dry N,N-dimethylformamide (50 mL) under nitrogen atmosphere. The mixture was refluxed for 48 h. The solution was then cooled down and a saturated aqueous solution of ammonium hexafluorophosphate was added. The crude product, which precipitated as a hexafluorophosphate salt was collected by centrifugation and washed with ethanol, water and diethyl ether. The residue was dissolved in dichloromethane and washed with a 5% aqueous lithium chloride solution, brine and water. The solvent was removed under reduced pressure and the crude product recrystallized from dichloromethane/pentane. The product was isolated via fractionated precipitation from acetonitrile by adding dropwise diethyl ether. 86 mg of 2 (0.05 mmol, 26%) were yielded as a black solid. ¹H-NMR (500 MHz, CD₃CN): δ 8.62 (d, J = 1.9 Hz, 6H), 7.66 (d, J = 16.2 Hz, 6H), 7.65 (d, J = 6.1 Hz, 6H), 7.55–7.52 (m, 12H), 7.38 (dd, J = 6.1, 1.9 Hz, 6H), 7.03 (d, J = 16.2 Hz, 6H), 6.81–6.78 (m, 12H), 3.01 (s, 36H); ¹³C-NMR (125 MHz, CD₃CN): δ 159.4, 158.1, 152.6, 148.3, 137.7, 130.0, 124.5, 124.4, 120.6, 119.7, 113.2, 40.4; HRMS (m/z): [M]²⁺ calcd. for C₉₀H₉₀N₁₂Ru, 720.3222; found, 720.3247; analysis (calcd., found for C₉₀H₉₀F₁₂N₁₂P₂Ru): C (62.46, 62.54), H (5.24, 5.17), N (9.71, 9.79). [Ru((E,E′)-4,4′-Bis[p-(N,N-dimethylamino)styryl]-2,2′-bipyridine)₃][Cl]₂: The counter ion hexafluorophosphate was exchanged to chloride by elution with methanol from the ion exchange resin Amberlite IRA-410. Analysis (calcd., found for C₉₀H₉₀Cl₂N₁₂Ru): C (71.51, 71.19), H (6.00, 5.93), N (11.12, 10.84).

[Ru((E,E′)-4,4′-Bis[p-methoxystyryl]-2,2′-bipyridine)₃][PF₆]₂ (3): (E,E′)-4,4′-Bis[p-methoxystyryl]-2,2′-bipyridine (286 mg, 0.68 mmol, 4.0 equiv.) and Ru(DMSO)₄Cl₂ (82 mg, 0.17 mmol, 1.0 equiv.) were suspended in dry ethanol (50 mL) under nitrogen atmosphere. The mixture was refluxed for 15 h. The solution was then cooled down and undissolved solid was removed by filtration. A saturated aqueous solution of ammonium hexafluorophosphate was added and the crude product, which precipitated as a hexafluorophosphate salt was collected by filtration. The solid was washed with water and diethyl ether. The residue was purified via fractionated precipitation from acetonitrile by adding dropwise diethyl ether. The collected product was dissolved in dichloromethane and washed with a 5% aqueous lithium chloride solution, brine and water. After drying, 218 mg of 3 (0.13 mmol, 76%) were yielded as a black solid. ¹H-NMR (500 MHz, CD₃CN): δ 8.79 (d, J = 1.7 Hz, 6H), 7.79 (d, J = 16.4 Hz, 6H), 7.71 (d, J = 6.0 Hz, 6H), 7.64 (d, J = 8.9 Hz, 12H), 7.44 (d, J = 6.0, 1.7 Hz, 6H), 7.17 (d, J = 16.4 Hz, 6H), 7.00 (d, J = 8.9 Hz, 12H), 3.83 (s, 18H). ¹³C-NMR (125 MHz, CD₃CN): δ 162.0, 158.2, 152.2, 148.0, 137.0, 130.1, 129.5, 125.0, 122.7, 121.3, 115.5, 56.2; HRMS (m/z): [M]²⁺ calcd. for C₈₄H₇₂N₆O₆Ru, 681.2290; found, 681.2273; analysis (calcd., found for C₈₄H₇₂F₁₂N₆O₆P₂Ru): C (61.05, 61.17), H (4.39, 4.44), N (5.09, 5.21). [Ru((E,E′)-4,4′-Bis[p-methoxystyryl]-2,2′-bipyridine)₃][Cl]₂: The counter ion hexafluorophosphate was exchanged to chloride by elution with methanol from the ion exchange resin Amberlite IRA-410. Analysis (calcd., found for C₈₄H₇₂Cl₂N₆O₆Ru): C (70.38, 70.62), H (5.06, 5.28), N (5.86, 5.57).

[Ru(2,2′-bipyridine)((E,E′)-4,4′-Bis[p-(N,N-dimethylamino)styryl]-2,2′-bipyridine)₂][PF₆]₂ (4): (E,E′)-4,4′-Bis[p-(N,N-dimethylamino)styryl]-2,2′-bipyridine (220 mg, 0.49 mmol, 2.0 equiv.), Ru(DMSO)₄Cl₂ (119 mg, 0.25 mmol, 1.0 equiv.), and lithium chloride (1044 mg, 24.63 mmol, 100 equiv.) were suspended in dry N,N-dimethylformamide (30 mL) under nitrogen atmosphere. The mixture was refluxed for 4 h. The solution was then cooled down and water was added. The crude product, which precipitated was collected by filtration and washed with water and diethyl ether. The formation of [Ru((E,E′)-4,4′-Bis[p-(N,N-dimethylamino)styryl]-2,2′-bipyridine)₂Cl₂] was analyzed via HPLC. [Ru((E,E′)-4,4′-Bis[p-(N,N-dimethylamino)styryl]-2,2′-bipyridine)₂Cl₂] and 2,2′-bipyridine (47 mg, 0.3 mmol, 1.2 equiv.) were suspended in dry ethanol (50 mL) under nitrogen atmosphere. The mixture was refluxed for 7 h. The solution was then cooled down and undissolved solid was removed by filtration. A saturated aqueous solution of ammonium hexafluorophosphate was added and the crude product, which precipitated as a hexafluorophosphate salt was collected by filtration. The solid was washed with water and diethyl ether. The residue was purified via preparative HPLC as a trifluoroacetic acid salt. The solvents were millipore water with 0.1% trifluoroacetic acid (solvent A) and acetonitrile (solvent B). The following HPLC gradient has been used: 0–3 min: isocratic 50% A (50% B); 3–17 min: linear gradient from 50% A (50% B) to 0% A (100% B); 17–23 min: isocratic 0% A (100% B). The flow rate was 20 mL min⁻¹ and the chromatogram was detected at 250, 350, and 450 nm. The collected product was dissolved in water and a saturated aqueous solution of ammonium hexafluorophosphate was added. The product, which precipitated as a hexafluorophosphate salt was collected by filtration and washed with water, diethyl ether and pentane. 89 mg of 4 (0.06 mmol, 24%) were yielded as a dark red solid. ¹H-NMR (400 MHz, CD₃CN): δ 8.61 (s, 4H), 8.50 (d, J = 8.2 Hz, 2H), 8.04 (td, J = 8.0, 1.5 Hz, 2H), 7.86 (ddd, J = 5.7, 1.4, 0.6 Hz, 2H), 7.66 (dd, J = 16.2, 1.9 Hz, 4H), 7.64 (d, J = 6.3 Hz, 2H), 7.56–7.50 (m, 10H), 7.43–7.34 (m, 6H), 7.02 (dd, J = 16.2 Hz, 4H), 6.81–6.77 (m, 8H), 3.02 (s, 12H), 3.01 (s, 12H); ¹³C-NMR (100 MHz, CD₃CN): δ 158.1, 158.0, 152.7, 152.6, 151.9, 151.9, 148.5, 138.3, 137.9, 130.0, 128.4, 125.1, 124.4, 120.7,119.6, 113.2, 40.4; HRMS (m/z): [M]²⁺ calcd. for C₇₀H₆₈N₁₀Ru, 575.2330; found, 575.2347; analysis (calcd., found for C₇₀H₆₈F₁₂N₁₀P₂Ru): C (58.37, 58.19), H (4.76, 4.62), N (9.72, 9.72).

[Ru(2,2′-bipyridine)((E,E′)-4,4′-Bis[p-methoxystyryl]-2,2′-bipyridine)₂][PF₆]₂ (5): (E,E′)-4,4′-Bis[p-methoxystyryl]-2,2′-bipyridine (490 mg, 1.17 mmol, 2.0 equiv.), Ru(DMSO)₄Cl₂ (282 mg, 0.58 mmol, 1.0 equiv.), and lithium chloride (2470 mg, 58.26 mmol, 100 equiv.) were suspended in dry N,N-dimethylformamide (75 mL) under nitrogen atmosphere. The mixture was refluxed for 6 h. The solution was then cooled down and purged into water. The crude product, which precipitated was collected by filtration and washed with water and diethyl ether. The formation of [Ru((E,E′)-4,4′-Bis[p-methoxystyryl]-2,2′-bipyridine)₂Cl₂] was analyzed via HPLC. [Ru((E,E′)-4,4′-Bis[p-methoxystyryl]-2,2′-bipyridine)₂Cl₂] and 2,2′-bipyridine (109 mg, 0.70 mmol, 1.2 equiv.) were suspended in dry ethanol (100 mL) under nitrogen atmosphere. The mixture was refluxed for 6 h. The solution was then cooled down and undissolved solid was removed by filtration. A saturated aqueous solution of ammonium hexafluorophosphate was added and the crude product, which precipitated as a hexafluorophosphate salt was collected by centrifugation. The solid was washed with water and diethyl ether. The residue was purified via preparative HPLC as a trifluoroacetic acid salt. The solvents were millipore water with 0.1% trifluoroacetic acid (solvent A) and acetonitrile (solvent B). The following HPLC gradient has been used: 0–3 min: isocratic 50% A (50% B); 3–17 min: linear gradient from 50% A (50% B) to 0% A (100% B); 17–23 min: isocratic 0% A (100% B). The flow rate was 20 mL min⁻¹ and the chromatogram was detected at 250, 350, and 450 nm. The collected product was dissolved in water and a saturated aqueous solution of ammonium hexafluorophosphate was added. The product, which precipitated as a hexafluorophosphate salt was collected by filtration and washed with water, diethyl ether and hexane. 248 mg of 5 (0.18 mmol, 31%) were yielded as a dark red solid. ¹H-NMR (400 MHz, CD₃CN): δ 8.72 (d, J = 1.6 Hz, 4H), 8.51 (d, J = 8.2 Hz, 2H), 8.06 (td, J = 8.0, 1.3 Hz, 2H), 7.85 (dd, J = 5.6, 1.1 Hz, 2H), 7.75–7.68 (m, 6H), 7.67–7.61 (m, 8H), 7.60 (d, J = 5.9 Hz, 2H), 7.46–7.39 (m, 6H), 7.17 (dd, J = 16.4, 2.1 Hz, 4H), 7.05–6.99 (m, 8H), 3.85 (s, 6H), 3.84 (s, 6H); ¹³C-NMR (100 MHz, CD₃CN): δ 162.0, 158.1, 152.6, 152.2, 148.1, 138.6, 137.1, 132.8, 130.1, 129.5, 128.5, 125.2, 125.0, 122.7, 121.3, 115.5, 56.2; HRMS (m/z): [M]²⁺ calcd. for C₆₆H₅₆N₆O₄Ru, 549.1698; found, 549.1707; analysis (calcd., found for C₆₆H₅₆F₁₂N₆O₄P₂Ru + C₆H₁₄): C (57.96, 58.33), H (4.44, 4.08), N (5.87, 5.79).

[Ru(2,2′-bipyridine)₂((E,E′)-4,4′-Bis[p-(N,N-dimethylamino)styryl]-2,2′-bipyridine)][PF₆]₂ (6): Ru(2,2′-bipyridine)₂Cl₂ (350 mg, 0.72 mmol, 1.0 equiv.) and (E,E′)-4,4′-Bis[p-(N,N-dimethylamino)styryl]-2,2′-bipyridine (388 mg, 0.87 mmol, 1.2 equiv.) were suspended in dry ethanol (50 mL) under nitrogen atmosphere and the mixture was refluxed for 6 h. Then the solution was cooled down and a saturated aqueous solution of ammonium hexafluorophosphate was added. The crude product, which precipitated as a hexafluorophosphate salt was collected by filtration and washed with water and diethyl ether. The product was isolated via fractionated precipitation from acetonitrile by adding dropwise diethyl ether. 449 mg of 6 (0.39 mmol, 54%) were yielded as a dark red solid. ¹H-NMR (500 MHz, CD₃CN): δ 8.62 (d, J = 1.7 Hz, 2H), 8.50 (d, J = 8.2 Hz, 4H), 8.07–8.02 (m, 4H), 7.87–7.84 (m, 2H), 7.75–7.72 (m, 2H), 7.67 (d, J = 16.3 Hz, 2H), 7.57–7.52 (m, 4H), 7.52–7.49 (d, J = 6.0 Hz, 2H), 7.44–7.34 (m, 6H), 7.02 (d, J = 16.3 Hz, 2H), 6.81–6.76 (m, 4H), 3.01 (s, 12H); ¹³C-NMR (125 MHz, CD₃CN): δ 158.1, 158.0, 158.0, 152.7, 152.7, 152.6, 151.9, 148.8, 138.6, 138.0, 130.0, 128.5, 128.5, 125.2, 124.4, 124.3, 120.8, 119.5, 113.1, 40.4; HRMS (m/z): [M]²⁺ calcd. for C₅₀H₄₆N₈Ru, 430.1439; found, 430.1441; analysis (calcd., found for C₅₀H₄₆F₁₂N₈P₂Ru): C (52.22, 51.97), H (4.03, 4.04), N (9.74, 9.71).

[Ru(2,2′-bipyridine)₂((E,E′)-4,4′-Bis[p–methoxystyryl]-2,2′-bipyridine)][PF₆]₂(7): Ru(2,2′-bipyridine)₂Cl₂ (432 mg, 0.89 mmol, 1.0 equiv.) and (E,E′)-4,4′-Bis[p-methoxystyryl]-2,2′-bipyridine (450 mg, 1.07 mmol, 1.2 equiv.) were suspended in dry ethanol (100 mL) under nitrogen atmosphere and the mixture was refluxed for 6 h. Then the solution was cooled down and a saturated aqueous solution of ammonium hexafluorophosphate was added. The crude product, which precipitated as a hexafluorophosphate salt was collected by filtration and washed with water and diethyl ether. The product was isolated via fractionated precipitation from acetonitrile by adding dropwise diethyl ether. 358 mg of 7 (0.32 mmol, 36%) were yielded as a dark red solid. ¹H-NMR (500 MHz, CD₃CN): δ 8.71 (d, J = 1.4 Hz, 2H), 8.51 (dd, J = 8.2, 0.7 Hz, 4H), 8.06 (td, J = 8.0, 1.5 Hz, 4H), 7.86–7.84 (m, 2H), 7.76–7.73 (m, 2H), 7.72 (d, J = 16.4 Hz, 2H), 7.66–7.62 (m, 4H), 7.59 (d, J = 6.0 Hz, 2H), 7.45–7.38 (m, 6H), 7.16 (d, J = 16.4 Hz, 2H), 7.03–6.98 (m, 4H), 3.83 (s, 6H). ¹³C-NMR (125 MHz, CD₃CN,): δ 162.0, 158.1, 158.0, 152.7, 152.6, 152.2, 148.2, 138.7, 138.7, 137.1, 130.1, 129.5, 128.6, 128.5, 125.2, 125.0, 122.6, 121.4, 115.5, 56.2; HRMS (m/z): [M]²⁺ calcd. for C₄₈H₄₀N₆O₂Ru, 417.1123; found, 417.1126; analysis (calcd., found for C₄₈H₄₀F₁₂N₆O₂P₂Ru): C (51.30, 51.23), H (3.59, 3.48), N (7.48, 7.61). [Ru(2,2′-bipyridine)₂((E,E′)-4,4′-Bis[p-(N,N-methoxy)styryl]-2,2′-bipyridine)][Cl]₂: The counter ion hexafluorophosphate was exchanged to chloride by elution with methanol from the ion exchange resin Amberlite IRA-410. Analysis (calcd., found for C₄₈H₄₀Cl₂N₆O₂Ru): C (63.70, 63.51), H (4.46, 4.30), N (9.29, 9.11).

X-ray crystallography

Single-crystal X-ray diffraction data were collected at 160(1) K for compounds L-H, L-NMe₂, L-OMe, and 3 on a Rigaku OD XtaLAB Synergy Dualflex (Pilatus 200K detector) diffractometer associated with an Oxford liquid-nitrogen Cryostream cooler. The wavelength used for all experiments is 1.54184 Å and corresponds to the Cu K_α radiation (from a micro-focus sealed X-ray tube). The single crystals were selected and cut (if needed) in polybutene oil, mounted on a flexible loop, and transferred to the diffractometer on the goniometer head. The program suite CrysAlisPro (version 1.171.39.13a) was used for the pre-experiments, data collections, data reductions, and also for the analytical absorption corrections⁵³ based on the indexed faces. Using the Olex2 software⁵⁴, the crystal structures were solved with the SHELXT⁵⁵ small molecule structure solution program and refined with the SHELXL program package⁵⁶ by full-matrix least-squares minimization on F². Molecular graphics were generated using Mercury 4.0⁵⁷. The crystal data collections and structure refinement parameters are summarized in Tables S1 and S2. CCDC 1951466 (for L-H), CCDC 1951467 (for 3), CCDC 1951468 (for L-NMe₂), and CCDC 1951469 (for L-OMe) contain the supplementary crystallographic data for these compounds, and can be obtained free of charge from the Cambridge Crystallographic Data Center via www.ccdc.cam.ac.uk/data_request/cif.

In the crystal structure of L-H, the substituted bipyridine is located on a center of inversion, only one half of the molecule had to be refined, the second part being reproduced by a symmetry operation. The main species cocystallized with solvent molecules of acetic acid in a ratio 1:2, respectively.

In the crystal structure of L-OMe, there are two crystallographically independent bipyridine molecules in the asymmetric unit: one complete molecule and two half molecules on special positions for which the second half is reproduced by a symmetry operation. Solvent molecules of acetic acid cocrystallized with the main species in a ratio 2:1.

In the crystal structure of 3, the asymmetric unit contains two dicationic Ru species, four anionic hexafluorophosphate counterions and one solvent molecule of tetrahydropyran (disordered over three positions). The model was difficult to refine because of the large number of non-hydrogen atoms in the asymmetric unit, and of the disorders observed for the flexible terminal C=C(H)-C₆H₄OCH₃ groups. The crystal and the data collection were of a rather good quality but the final refinement parameters remained relatively high (R₁ = 12% and wR₂ = 40%) or poor (Goof = 1.5). The solvent molecules could be identified in residual density peaks but refinement was complicated and finally the real geometry of the tetrahydropyran molecule was substituted by a regular planar six-membered ring. Many restraints and constraints were used to get stable refinement cycles but the result seems to be reliable, especially there is no ambiguity about the main charged species.

Spectroscopic measurements

The absorption spectra of the sample was measured with a SpectraMax M2 Spectrometer (Molecular Devices). For the measurements of the emission, the sample was irradiated at 355 nm with a NT342B Nd-YAG pumped optical parametric oscillator (Ekspla). The emission was focused at right angle to the excitation pathway and directed to a Princeton Instruments Acton SP-2300i monochromator. The signal was detected with a XPI-Max 4 CCD camera (Princeton Instruments).

Two-photon absorption cross section

The two-photon absorption cross section spectra of the sample was determined upon direction comparison to Rhodamine B. The sample was irradiated with an OpoletteTM 355II (pulse width ≤ 100 fs, 80 MHz repetition rate, Spectra Physics Inc.) laser. The experimental excitation and detection conditions were conducted with negligible reabsorption processes, which could affect two-photon absorption measurements. The quadratic dependence of the two-photon induced luminescence intensity on the excitation power was verified at an excitation wavelength of 800 nm. The two-photon absorption cross section was calculated using the following equation:

σ₂ = two-photon cross section, Φ = luminescence quantum yield, c = concentration, I = integrated luminescence intensity, n = refractive index

Luminescence quantum yield

The sample was prepared in an acetonitrile solution with an absorption of 0.1 at 355 nm. The sample was irradiated at 355 nm with a NT342B Nd-YAG pumped optical parametric oscillator (Ekspla). The emission was focused at right angle to the excitation pathway and directed to a Princeton Instruments Acton SP-2300i monochromator. The signal was detected with a XPI-Max 4 CCD camera (Princeton Instruments). The luminescence quantum yields were determined by comparison with the reference [Ru(2,2′-bipyridine)₃]Cl₂ in acetonitrile (Φ_em = 5.9%)⁵⁸ applying the following formula:

Φ_em = luminescence quantum yield, F = fraction of light absorbed, I = integrated emission intensities, n = refractive index, A = absorbance of the sample at irradiation wavelength.

Lifetime

The sample was prepared in an air saturated as well as a degassed acetonitrile solution with an absorption of 0.2 at 355 nm. The sample was irradiated at 355 nm with a NT342B Nd-YAG pumped optical parametric oscillator (Ekspla). The emission was focused at right angle to the excitation pathway and directed to a Princeton Instruments Acton SP-2300i monochromator. The signal was detected with a R928 photomultiplier tube (Hamamatsu).

Electron spin resonance (ESR)

The sample (10 μM) was dissolved in acetonitrile or PBS containing 20 mM TEMP (2,2,6,6–tetramethylpiperidine) as a ¹O₂ scavenger or 20 mM DMPO (5,5-dimethyl-1-pyrroline N-oxide) as a ^•OOH or ^•OH radical scavenger. Capillary tubes were filled with the solution and sintered by fire. EPR spectra were recorded on a Bruker A300 spectrometer with 1 G field modulation, 100 G scan range, and 20 mW microwave power. The samples were measured in exclusion from light and after irradiation (450 ± 10 nm, 60 s, and 21.8 mW cm⁻²).

Singlet oxygen

Direct evaluation: the sample was prepared in an air-saturated acetonitrile or deuterated water solution with an absorption of 0.2 at 450 nm. The sample was irradiated at 450 nm with a mounted M450LP1 LED (Thorlabs), whose light was focused with aspheric condenser lenses. Using a T-Cube LED Driver (Thorlabs), the intensity of the irradiation was varied and monitored with an optical power and energy meter. The emission was focused at right angle to the excitation pathway and directed to a Princeton Instruments Acton SP-2300i monochromator. To cut off light at wavelengths shorter than 850 nm, a longpass glass filter was placed in front of the monochromator entrance slit. The signal was detected with an EO-817L IR-sensitive liquid-nitrogen cooled germanium diode detector (North Coast Scientific Corp.). The luminescence signal, centered at 1270 nm, was measured from 1100 to 1400 nm. The obtained data were analyzed upon plotting the integrated luminescence peaks against the percentage of the irradiation intensity. The slope of the linear regression was calculated and compared with the reference Rose Bengal (Φ = 76%)⁵⁹. The absorption of the sample was corrected with an absorption correction factor. The singlet oxygen quantum yields were calculated using the following formula:

Φ = singlet oxygen quantum yield, S = slope of the linear regression of the plot of the areas of the singlet oxygen luminescence peaks against the irradiation intensity, I = absorption correction factor, I₀ = light intensity of the irradiation source, and A = absorption of the sample at irradiation wavelength.

Indirect evaluation: measurement in acetonitrile: the sample was prepared in an air-saturated acetonitrile solution with an absorption of 0.2 at the irradiation wavelength, N,N-dimethyl-4-nitrosoaniline aniline (RNO, 24 µM) and imidazole (12 mM). Measurement in PBS buffer: the sample was prepared in an air-saturated PBS solution containing the complex with an absorption of 0.2 at the irradiation wavelength, N,N-dimethyl-4-nitrosoaniline aniline (RNO, 20 µM) and histidine (10 mM). The samples were irradiated for various time points with an Atlas Photonics LUMOS BIO irradiator. The absorption of the samples was constantly monitored with a SpectraMax M2 Microplate Reader (Molecular Devices). The difference in absorption (A₀ − A) at 420 nm for the measurement in acetonitrile or at 440 nm for the measurement in PBS was determined. The difference in absorption was then plotted against the irradiation times and the slope of the linear regression calculated. The absorption of the sample was corrected with an absorption correction factor. The singlet oxygen quantum yields were calculated using the same formulas as used for the direct evaluation.

Stability in human plasma

The stability of the sample was evaluated upon incubation in pooled human plasma with caffeine as an internal standard, which previously demonstrated to be stable under these conditions⁶⁰. The sample (20 μM) and caffeine (40 μM) were prepared in dimethyl sulfoxide stock solutions. One aliquot of both solutions was added to 975 μL of human plasma achieving a total volume of 1000 μL. The resulting solutions were incubated upon continuous gentle shaking (ca. 300 rpm) for 48 h at 37 °C. After this time, the incubation was ended by addition of 3 mL of methanol. The mixture was centrifuged for 60 min at 3000 rpm/958 gand 4 °C. The solution was filtered through a 0.2 μm membrane filter. The solvent was evaporated and the residue was redissolved in 1:1 (v/v) acetonitrile/water with 0.1% trifluoroacetic acid. The resulting solution was then filtered through a 0.2 μm membrane filter and analyzed with a HPLC System. The solvents (HPLC grade) were millipore water with 0.1% trifluoroacetic acid (solvent A) and acetonitrile (solvent B). Method M1: 0–3 min: isocratic 50% A (50% B); 3–17 min: linear gradient from 50% A (50% B) to 0% A (100% B); Method M2: 0–3 min: isocratic 95% A (5% B); 3–17 min: linear gradient from 95% A (5% B) to 0% A (100% B); 17–23 min: isocratic 0% A (100% B). The flow rate was 1 mL min⁻¹ and the chromatogram was detected at 250 nm.

Photostability

The sample was prepared in an air-saturated acetonitrile solution. Using a Atlas Photonics LUMOS BIO irradiator, the sample was constantly irradiated at 450 nm in time intervals from 0 to 10 min (13.2 J cm⁻²). The absorption spectra from 350 to 700 nm was monitored with a SpectraMax M2 Microplate Reader (Molecular Devices) at each time interval. As positive and negative controls, respectively, [Ru(2,2′-bipyridine)₃]Cl₂ and Protoporphyrin IX were used.

Photobleaching

The sample was dissolved in air saturated 0.7 mL CD₃CN and stored in a NMR tube in the dark. A ¹H-NMR spectrum was measured directly after preparation to ensure the identity of the sample. The sample was then irradiated at 450 nm with an Atlas Photonics LUMOS BIO irradiator (10 min, 13.2 J cm⁻²). Following this treatment, a ¹H-NMR spectrum was again measured.

Distribution coefficient

Using the “shake-flask” method, the distribution coefficient between the PBS and octanol phase of the sample was determined. The PBS and octanol phases were previously saturated in each other. The sample was dissolved in the phase, which is dissolving best the compound. This solution was added to an equal volume of the other phase and mixed at 190 rpm overnight with a sample mixer. Following this, the solutions were equilibrated for 4 h. The phases were carefully separated from each other. The amount of the sample in each phase was determined by ICP-MS. The evaluation was repeated three times and the ratio between the organic and aqueous phase determined.

Cell culture

Human glioblastoma astrocytoma (U373) cells were cultured in MEM medium supplemented with 10% FBS, 1% NEAA (nonessential amino acids), and 1% penicillin/streptomycin. Human cervical carcinoma (HeLa), doxorubicin-resistant human colon adenocarcinoma (SW620/AD300), and the mouse colon carcinoma (CT-26) cells were cultured in DMEM medium supplemented with 10% FBS and 1% penicillin/streptomycin. Human retinal epithelial (RPE-1) cells were cultured in DMEM-F12 medium supplemented with 10% FBS and 1% penicillin/streptomycin. All cell lines were obtained from the American Type Culture Collection and cultured at 37 °C and 5% CO_2. Before an experiment, the cells were passaged three times.

Time-dependent cellular uptake

A total of 6 × 10⁶ cells were incubated with the sample (10 μM, 2% DMSO, v%) for varying time points (0.5, 1, 2, 4, 8, 12 h) at 37 °C. After this time, the media was removed and the cells washed with cell media. The cells were trypsinised, harvested, centrifuged, and resuspended. The number of cells on the dish was counted. The sample was digested using a 60% HNO₃ solution for 3 days and, following this, diluted to a 2% HNO₃ solution in water. The Ru content was determined with an ICP-MS apparatus and the results compared with the Ru references. The Ru content was then associated with the number of cells.

Cellular uptake mechanism

The cellular uptake mechanism was investigated upon pretreatment of 1 × 10⁶ cells with pathways inhibitors and determination of the amount of Ru inside the cells with an ICP-MS apparatus.

Control: the cells were incubated with the sample (10 μM, 2% DMSO, v%) for 1 h at 37 °C and then washed with PBS. The cells were detached with trypsin, harvested, centrifuged, and resuspended. The number of cells on the dish was counted. The sample was digested using a 60% HNO₃ solution for 3 days and following this diluted to a 2% HNO₃ solution in water. The Ru content was determined with an ICP-MS apparatus and the results compared with the Ru references. The Ru content was then associated with the number of cells.

Low temperature: the cells were preincubated at 4 °C for 1 h. After this time, the cells were incubated with the sample (10 μM, 2% DMSO, v%) for 1 h at 37 °C and then washed with PBS. The cells were detached with trypsin, harvested, centrifuged, and resuspended. The number of cells on the dish was counted. The sample was digested using a 60% HNO₃ solution for 3 days and following this diluted to a 2% HNO₃ solution in water. The Ru content was determined with an ICP-MS apparatus and the results compared with the Ru references. The Ru content was then associated with the number of cells.

Metabolic inhibition: the cells were preincubated with 2-deoxy-D-glucose (50 mM) and oligomycin (5 μM) for 1 h. After this time, the cells were incubated with the sample (10 μM, 2% DMSO, v%) for 1 h at 37 °C and then washed with PBS. The cells were detached with trypsin, harvested, centrifuged, and resuspended. The number of cells on the dish was counted. The sample was digested using a 60% HNO₃ solution for 3 days and following this diluted to a 2% HNO₃ solution in water. The Ru content was determined with an ICP-MS apparatus and the results compared with the Ru references. The Ru content was then associated with the number of cells.

Endocytic inhibition: the cells were preincubated with ammonium chloride (50 mM) or chloroquine (100 μM) for 1 h. After this time, the cells were incubated with the sample (10 μM, 2% DMSO, v%) for 1 h at 37 °C and then washed with PBS. The cells were detached with trypsin, harvested, centrifuged, and resuspended. The number of cells on the dish was counted. The sample was digested using a 60% HNO₃ solution for 3 days and following this diluted to a 2% HNO₃ solution in water. The Ru content was determined with an ICP-MS apparatus and the results compared with the Ru references. The Ru content was then associated with the number of cells.

Cation transporter inhibition: the cells were preincubated with tetraethylammonium chloride (1 mM) for 1 h. After this time, the cells were incubated with the sample (10 μM, 2% DMSO, v%) for 1 h at 37 °C and then washed with PBS. The cells were detached with trypsin, harvested, centrifuged, and resuspended. The number of cells on the dish was counted. The sample was digested using a 60% HNO₃ solution for 3 days and following this diluted to a 2% HNO₃ solution in water. The Ru content was determined with an ICP-MS apparatus and the results compared with the Ru references. The Ru content was then associated with the number of cells.

Intracellular distribution by confocal luminescence imaging

A total of 1 × 10⁴ cells were seeded on confocal dishes and allowed to adhere overnight. The cells were incubated with the sample (10 μM, 2% DMSO, v%) for 8 h at 37 °C in the dark. After this time, the cells were washed with PBS. The cells were further each incubated with LysoTracker Green, MitoTracker Deep Red, Cell-light Golgi-GFP, ER tracker Green and Hoechst 33342 for 30 min at 37 °C in the dark. The cells were washed three times with PBS. Confocal images were taken with a LSM 880 (Carl Zeiss) laser scanning confocal microscope. The organelle trackers were excited and detected as recommended by the supplier. The samples were detected under usage of their 1-Photon (λ_ex = 458 nm, λ_em = 600–750 nm) or 2-Photon (λ_ex = 800 nm, λ_em = 600–750 nm) luminescence properties.

Intracellular distribution by ICP-MS

A total of 10 × 10⁶ cells were incubated with the sample (10 μM, 2% DMSO, v%) for 8 h at 37 °C in the dark. After this time, the cells were detached with trypsin and harvested. The number of cells was counted and equally divided into four portions. In the first portion, the nucleus was extracted using a nucleus extraction kit (Sangon Biotech); in the second portion, the mitochondria was extracted using a mitochondria extraction kit (Sangon Biotech), and in the third portion, the lysosome was extracted using a lysosome extraction kit (GenMed Scientific). Using the fourth portion, the cytoplasm was extracted. The cells were detached with trypsin, harvested, and centrifuged. The cell pellet was resuspended in lysis buffer and the cells lysed. The cytoplasm was separated by centrifugation(Optima MAX-XP ultracentrifuge, Beckman Coulter) for 150 min at 200,000 g at 4 °C. The supernatant solution was separated. The sample was digested using a 60% HNO₃ solution for 3 days and following this diluted to a 2% HNO₃ solution in water. The Ru content was determined with an ICP-MS apparatus and the results compared with the Ru references. The Ru content was then associated with the number of cells.

One-photon induced cellular singlet oxygen generation

A total of 1 × 10⁴ cells were seeded on confocal dishes and allowed to adhere overnight. The cells were incubated with the sample (10 μM, 2% DMSO, v%) for 4 h at 37 °C in the dark. After this time, the cells were washed with PBS. The cells were further incubated with 2′,7′-dichlorofluorescein diacetate for 30 min. The cells were washed three times with PBS. The sample was irradiated upon 1-Photon excitation (λ_ex = 488 nm). Confocal images (λ_ex = 488 nm, λ_em = 510–550 nm) were taken with a LSM 880 (Carl Zeiss) laser scanning confocal microscope.

Two-photon induced cellular singlet oxygen generation

A total of 1 × 10⁴ cells were seeded on confocal dishes and allowed to adhere overnight. The cells were incubated with the sample (10 μM, 2% DMSO, v%) for 4 h at 37 °C in the dark. After this time, the cells were washed with PBS. The cells were further incubated with 2′,7′-dichlorofluorescein diacetate for 30 min. The cells were washed three times with PBS. The sample was irradiated upon 2-Photon excitation (λ_ex = 800 nm). Confocal images (λ_ex = 488 nm, λ_em = 510–550 nm) were taken with a LSM 880 (Carl Zeiss) laser scanning confocal microscope.

(Photo-)cytotoxicity on 2D cell monolayers

A total of 4 × 10³ cells were seeded on 96-well plates and allowed to adhere overnight. The cells were treated with increasing concentrations of the sample diluted in cell media achieving a total volume of 200 μL. The cells were incubated with the sample for 8 h and, after this time, the medium refreshed. To study the phototoxic effect of the sample, the cells were exposed to 480 nm (spectral half-width: 20 nm, 10 min, 5.2 mW cm⁻², 3.1 J cm⁻²) or 540 nm (spectral half-width: 30 nm, 40 min, 4.0 mW cm⁻², 9.5 J cm⁻²) light using an Atlas Photonics LUMOS BIO irradiator. To study the dark cytotoxicity of the sample, the cells were not irradiated and the medium exchanged. The cells were grown for an additional 40 h at 37 °C. After this time, the medium was replaced with fresh medium containing resazurin with a final concentration of 0.2 mg mL⁻¹. The cells were incubated for 4 h and the amount of the fluorescent product resorufin determined (λ_ex = 540 nm, λ_em = 590 nm) with a SpectraMax M2 Microplate Reader (Molecular Devices). The obtained data was analyzed with the GraphPad Prism software.

Cell death mechanism

The cell death mechanism was investigated by measuring the cell viability upon preincubation with various cell death pathway inhibitors. 3-Methyladenine (100 μM), Z-VAD-FMK (20 μM), cycloheximide (0.1 μM), and necrostatin-1 (60 μM) were preincubated with the cells for 40 min. Following this, the sample (5 μM) was added and incubated for 8 h. The cells were washed, irradiated with a LED (500 nm, 10 min, 10.0 mW cm⁻², 6 J cm⁻²) light source, and incubated for 12 h. After this time, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide was added and the cells were incubated for additional 4 h. The liquid was disposed and 150 μL DMSO was added. The plates were shaken for 2 min and the absorbance at 595 nm was measured using a microplate reader (TecanInfiniteF200, Bio-rad).

Generation and analysis of 3D multicellular tumor spheroids (MCTS)

A suspension of 0.75% agarose in PBS was heated inside a high-pressure autoclave. The hot emulsion was transferred into a 96-well plate (50 μL per well). The plates were exposed for 3 h to UV irradiation and allowed to cool down. After this time, a cell suspension of 3 × 10³ cells was seeded on top the agarose ground layer. Within 2–3 days, MCTS were formed from the cell suspension. The MCTS were cultivated and maintained at 37 °C in a cell culture incubator at 37 °C with 5% CO₂ atmosphere. The culture medium was replaced every 2 days. The formation, integrity, diameter, and volume of the MCTS was monitored by an Axio Observer Z1 (Carl Zeiss) phase contrast microscope. The volume was calculated using the following equation: V = 4/3πr³. The luminescence images along the z-axis were captured by 1-Photon (λ_ex = 458 nm, λ_em = 600–750 nm) or 2-Photon (λ_ex = 800 nm, λ_em = 600–750 nm) excitation in the z-stack mode with a an LSM 880 (Carl Zeiss) laser scanning confocal microscope equipped with Argon or Coherent Chameleon 2-Photon laser and a GaAsP detector.

3D multicellular tumor spheroids (MCTS) growth inhibition assay

MCTS were treated with the sample (20 μM 1–7, 20 μM tetraphenylporphyrin H₂TPP, 10 μM cisplatin, 30 μM cisplatin, 2% DMSO, v%) by replacing 50% of the media with drug supplemented media in the dark for 3 days. After this time, the MCTS were exposed to a 1-Photon (500 nm, 16.7 min, 10.0 mW cm⁻², and 10 J cm⁻²) irradiation using a LED light source or 2-Photon (800 nm, 10 J cm⁻² with a section interval of 5 μm) irradiation using a LSM 880 (Carl Zeiss) laser scanning confocal microscope equipped with a Coherent Chameleon 2-Photon laser. The cell culture media was replaced every 2 days. The integrity and diameter of the MCTs was monitored with an Axio Observer Z1 (Carl Zeiss) phase contrast microscope every 24 h.

3D multicellular tumor spheroids (MCTS) viability assay

MCTS were treated with the sample (20 μM, 2% DMSO, v%) by replacing 50% of the media with drug supplemented media in the dark for 3 days. After this time, the MCTS were exposed to a 1-Photon (500 nm, 16.7 min, 10.0 mW cm⁻², and 10 J cm⁻²) irradiation using a LED light source or 2-Photon (800 nm, 10 J cm⁻² with a section interval of 5 μm) irradiation using a LSM 880 (Carl Zeiss) laser scanning confocal microscope equipped with a Coherent Chameleon 2-Photon laser. The cell culture media was replaced every 2 days. Two days after the irradiation the MCTS viability was tested using a Viability/Cytotoxicity Kit for mammalian cells (Invitrogen). Living cells can be identified from dead cells through the presence of ubiquitous intracellular esterase activity which can be monitored by the enzymatic conversion of the non-fluorescent calcein AM to the intensely fluorescent calcein (λ_ex = 495 nm, λ_em = 515 nm). As the spectroscopic properties of the dead cell probe EthD-1 overlaps with the one of the investigated compounds, this probe was not used and only calcein AM as a probe for living cells was used. The MCTS were incubated with calcein AM (2 μM) for 30 min and images of the MCTS taken with an Axio Observer Z1 (Carl Zeiss, Germany) inverted fluorescence microscope.

(Photo-)cytotoxicity on 3D multicellular tumor spheroids (MCTS)

The cytotoxicity of the compounds in 3D MCTS was assessed by measurement of the ATP concentration. MCTS were treated with increasing concentrations of the compound (2% DMSO, v%) by replacing 50% of the media with drug supplemented media and incubation for 12 h. After this time, the MCTS were divided in three identical groups. The first group was strictly kept in the dark. The second group was exposed to a 1-Photon irradiation (500 nm, 16.7 min, 10.0 mW cm⁻², and 10 J cm⁻²) using a LED light source and the third group was exposed to a 2-Photon irradiation (800 nm, 10 J cm⁻² with a section interval of 5 μm) using a LSM 880 (Carl Zeiss, Germany) laser scanning confocal microscope equipped with a Coherent Chameleon 2-Photon laser. After the irradiation, all groups were incubated additional 48 h. The ATP concertation was measured using a CellTiter-Glo 3D Cell Viability kit (Promega) by measuring the generated chemiluminescence with an infinite M200 PRO (Tecan) plate reader. The obtained data was analyzed with the GraphPad Prism software.

In vivo experiment

Six weeks old nu/nu female mice were purchased from Charles River. For the tumor xenograft, 6 × 10⁶ SW620/AD300 cells were subcutaneously (s.c.) injected with a 150 μL Matrigel and saline (1:1, v/v) suspension into the mice. After a week, the tumor volumes of the mice reached ~80 mm³. All experiments were conducted in compliance with the ethical regulations for animal testing and received approval of the experimental animal managing committee of Sun Yat Sen University.

In vivo biodistribution

Three nude mice were tail-intravenous (i.v.) injected with 2 mg kg⁻¹ of the sample. One hour later, the mice were sacrificed and the major organs (including the heart, liver, spleen, lung, kidney, brain, intestine, and blood) were separated, weighted and ground. The grinded organ as well as the blood were digested using a 60% HNO₃ solution for 3 days, and following this, diluted to a 2% HNO₃ solution in water. The Ru content was determined with an ICP-MS apparatus and the results compared with the Ru references.

In vivo time-dependent biodistribution

Six SW620/AD300 nude tumor-bearing nude mice were separated into three groups, and i.v. injected with 2 mg kg⁻¹ of the sample. 30 min, 1 h, 2 h later, the mice were euthanized and the distribution of the sample determined using the previous described method.

In vivo (Photo-)cytotoxicity

30 SW620/AD300 nude tumor-bearing nude mice were randomly separated into six groups, resulting in five mice for each group. After been anaesthetized, the mice was fixed in a warm three-axes holder. Before irradiation, the tumor site was disinfected with ethanol. The tumor was exposed to 1P or 2P light. Following the irradiation, the operative area was disinfection by iodophor.

Group 1: injected 7 (2 mg kg⁻¹, 50 μL) intravenously and irradiate under 800 nm laser (50 mW, 1 kHz, pulse width 35 fs, 5 s mm⁻¹) 1 h after injection;

Group 2: injected 7 (2 mg kg⁻¹, 50 μL) intravenously and irradiate under 500 nm light (60 min, 10.0 mW cm⁻², 36 J cm⁻²) 1 h after injection;

Group 3: injected by physiological saline (50 μL) and treated with 800 nm laser (50 mW, 1 kHz, pulse width 35 fs, 5 s mm⁻¹) 1 h after injection;

Group 4: injected by physiological saline (50 μL) and treated with 500 nm light (60 min, 10.0 mW cm⁻², 36 J cm⁻²) 1 h after injection;

Group 5: intravenous injected 7 (2 mg kg⁻¹, 50 μL);

Group 6: injected 50 μL physiological saline.

The mice were anesthetized by injection of a 4% chloral hydrate aqueous solution (0.2 mL, 20 g⁻¹) before the treatment. The tumor volume and body weight were measured and recorded every 2 days. Tumor volume was calculated by the following formula:

Histological examination

The tumor and all major organs (including the heart, liver, spleen, lung, kidney, brain, intestine, and blood) were collected and a slice of each one was fixated with 4% paraformaldehyde. The obtained slices were stained with hematoxylin and eosin. The histological images were taken with a Carl Zeiss Axio Imager Z2 microscope.

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.