To cage or to be caged? The cytotoxic species in ruthenium-based photoactivated chemotherapy is not always the metal† †Electronic supplementary information (ESI) available: Synthetic procedures, singlet oxygen quantum yield, partition coefficient and cellular uptake measurements, cell culture and EC

In metal-based photoactivated chemotherapy (PACT), two photoproducts are generated by light-triggered photosubstitution of a metal-bound ligand: the free ligand itself and an aquated metal complex.


Irradiation experiments followed by MS, UV-Vis
UV-Vis spectroscopy was performed using a Cary Varian spectrometer equipped with temperature control set to 298 K and a magnetic stirrer. For the irradiation, a LED light source (λ ex = 445 nm, with a Full Width at Half Maximum of 22 nm) with a light intensity of 15.50 or 13.65 mW·cm -2 (for [2]Cl 2 and [3]Cl 2 respectively) was used. Experiments were performed in a quartz cuvette containing 3 mL of solution. A stock solution of the desired complex was prepared using demineralized water, which was then diluted in the cuvette to the desired working concentration. When the experiment was carried under N 2 the sample was deoxygenated for 15 min by gentle bubbling of N 2 and the atmosphere was kept inert during the experiment by a gentle flow of nitrogen on top of the cuvette. A UV-Vis spectrum was measured every 30 s for the first 10 min, every 1 min for the next 10 min, and eventually every 10 min until the end of the experiment. Data was analysed with Microsoft Excel. The quantum yields of the photoreactions (Φ PR ) were calculated by modelling the time evolution of the absorbance spectrum of the solution using the Glotaran software (see Figure S2-S2). Experimental conditions are detailed in Table S1.   Table S1.

Blue light irradiation in the cell irradiation setup: which dose is necessary?
In order to assess which light dose should be used for photocytotoxicity assay, the photochemical reactivity of [1]Cl 2 and [2]Cl 2 was measured in 96-well plates, i.e. in the conditions of the cell experiments, but without cells and using UV-vis spectroscopy to measure to which extent the compounds are activated at different light doses. Two solution of each compound were prepared in Opti-MEM complete (40 µM and 200 µM) and distributed in a 96-well plate. The plate was irradiated with blue light (454 nm) at different irradiation times (0, 2, 5, 8, 10 min) using the blue LED source described in details in Hopkins et al. 6 At 40 µM and below both complexes received enough light at 10 min irradiation (dose 6.5 J.cm -2 ) to be fully activated. At 200 µM complex [2]Cl 2 was only partly activated ( Figure S4). Higher light doses would be necessary to fully activate the highest concentrations used for [2]Cl 2 , but they would also be inherently cytotoxic to A549 cells, as described in Hopkins et al. 6 Thus, 10 minutes irradiation, for a dose of 6.5 J.cm -2 , was chosen for all photocytotoxicity experiments.

Singlet Oxygen quantum yield measurement
The quantum yield of singlet oxygen generation was determined in a custom-built setup ( Figure S5), in which both UV-Vis absorption and infrared emission spectroscopy could be performed. All optical parts were connected with optical fibers from Avantes (Apeldoorn, The Netherlands), with a diameter of 200-600 µm. For each measurement, 500 µL of sample, consisting of the compound in deuterated methanol (A 450 ≤ 0.1 for 4.0 mm pathlength), was placed in a stirred 104F-OS semi-micro fluorescence cuvette (Hellma Analytics, Müllheim, Germany) in a CUV-UV/VIS-TC temperature-controlled cuvette holder from Avantes. The sample was allowed to equilibrate at 293 K for 5 minutes. Emission spectroscopy was performed with a 450 nm fiber-coupled laser (Laser system LRD-0450; Laserglow, Toronto, Canada), at 50 mW optical power (4 mm beam diameter; 0.4 W.cm -2 ) at a 90° angle with respect to the spectrometer. The excitation power was measured using a S310C thermal sensor connected to a PM100USB power meter (Thorlabs, Dachau, Germany). Infrared emission spectra were measured from 1000 nm to 1400 nm using an Avantes NIR256-1.7TEC spectrometer, The infrared emission spectrum was acquired within 9 seconds, after which the laser was turned off directly. UV-Vis absorption spectra before and after emission spectroscopy were measured using an Avalight-DHc halogen-deuterium lamp (Avantes) as light source (turned off during emission spectroscopy) and an Avantes 2048L StarLine UV-Vis spectrometer as detector, both connected to the cuvette holder at a 180° angle. No difference in UV-Vis absorption spectrum was found due to exposure to the blue laser, showing that the singlet oxygen emission is that of the starting compound. All spectra were recorded with Avasoft 8.5 software from Avantes and further processed with Microsoft Office Excel 2010 and Origin Pro 9.1 software. where Φ Δ is the quantum yield of singlet oxygen generation, A 450 is the absorbance at 450 nm, E is the integrated emission peak of singlet oxygen at 1274 nm, and sam and std denote the sample and standard, respectively.

Partition coefficient (logP)
The partition coefficient determination was adapted from Wang et al. 5 Stock solutions of [1]Cl 2 , [2]Cl 2 , and [3]Cl 2 were prepared in octanol-saturated water (1 mM). Aliquots of the stock solutions (0.2 mL) were transferred per triplicate to 15 mL centrifuge tubes and diluted up to 1 mL with octanol-saturated water to give 0.2 mM solutions. Then, 1 mL of water-saturated octanol was added and the mixtures were shaken in a IKA Vibrax shaker for 1 h at 2200 rpm. Then, the mixtures were centrifuged (4300 rpm, 10 min, room temperature). Aliquots of the water layer (0.2 mL) were diluted with MilliQ water (2.4 mL) and 65% HNO 3 (0.4 mL) per duplicate, to give a final solution at 5% HNO 3 . The ruthenium content of these samples was determined by ICP-OES using a Vista-MPX CCD Simultaneous ICP-OES. The partition coefficient values can be found in Table 2  where [Ru] total is the concentration of Ru in the control sample (where no water-saturated octanol was added) and [Ru] aq is the concentration of Ru in the aqueous layer as a mean of the six replicates. Penicillin and streptomycin were purchased from Duchefa and were diluted to a 100 mg/mL penicillin/streptomycin solution (P/S). Trypsin and Opti-MEM (without phenol red) were purchased from Gibco Life Technologies. Trypan blue (0.4 % in 0.81% sodium chloride and 0.06 % potassium phosphate dibasic solution) was purchased from BioRad. Plastic disposable flasks and 96-well plates were purchased from Sarstedt. Cells were counted by using a BioRad TC10 automated cell counter with Biorad cellcounting slides. UV/Vis measurements for analysis of 96-well plates were performed with a M1000 Tecan Reader. Cells were inspected with an Olympus IX81 microscope.

Cell culture
Cells were cultured in Dulbecco's Modified Eagle Medium containing phenol red, supplemented with 8.0% v/v fetal calf serum (FCS), 0.2% v/v penicillin/streptomycin and 0.9% v/v glutamax. Cell were incubated at 37 ºC at 7.0% CO 2 in 75 cm 2 T-flask and splitted once a week at 80-90% confluency. Cell were cultured for a maxium of 8 weeks for all biological experiment, and passaged at least twice after being thawed.

Cell-irradiation setup
The cell-irradiation system consisted of a Ditabis thermostat (980923001) fitted with two flat-bottomed micro-plate thermoblocks (800010600) and a 96-LED array fitted to a standard 96-well plate. Plates were incubated in the dark for an additional 6 h. After this period, half of the plates were irradiated for 10 min with blue light (λ = 454 ± 11 nm, power density = 10.5 ± 0.7 mW cm -2 , irradiation time = 10 min, light dose = 6.5 Jcm -2 ) and the other half were kept in the dark. After irradiation all the plates were incubated for an additional 66 h (making a total assay of 96 h) The cells were fixated by adding cold TCA (10 % w/v; 100 µL) in each well and the plates were stored at 4 ºC for at least 4 h as part of the sulforhodamine B (SRB) assay that was adapted from Vichai et al. 7 In short, after fixation TCA medium mixture was removed from the wells, rinsed with demineralized water three times and air dried. Then, each well was stained with 100 µL SRB (0.6 % w/v in 1% v/v acetic acid) for 30 min, the SRB was removed by washing with acetic acid (1 % v/v), and air dried. The SRB dye was solubilized with Tris base (10 mM; 200 µL), and the absorbance in each well was read at λ = 510 nm by using a M1000Tecan Reader.
The SRB absorbance data per compound per concentration were averaged over three identical wells (technical replicates, n t = 3) in Excel and made suitable for use in GraphPad Prism. Relative cell populations were calculated by dividing the aver-age absorbance of the treated wells by the average absorbance of the untreated wells. In any case it was checked that the cell viability of the untreated cells of the samples irradiated were similar (maximum difference of 10%) to the unirradiated samples to make sure no harm was done by the light. The data from three independent biological replications was plotted versus log(concentration) [µm]. By using the dose-response curve for each compound under dark-and irradiated conditions, the effective concentration (EC 50 ) was calculated by fitting the curves to a non-linear regression function with fixed y maximum (100 %) and minimum (0 %) (relative cell viability) and a variable Hill slope, which resulted in the simplified two-parameter Hill-slope equation. Photo indices (PI) reported in Table 1 were calculated, for each compound by dividing the EC 50 value obtained in the dark by the EC 50 value determined under light irradiation.

Cell uptake
Cell uptake studies for complexes [1]Cl 2 and [2]Cl 2 were conducted on A549 lung cancer cells. 8×10 5 cells were seeded at t = 0 h in Opti-MEM complete (3 mL) in 6 cm diameter dishes. At t = 24 h cells were treated with solutions of [1]Cl 2 and [2]Cl 2 to give a final concentration of 20 and 80 µM respectively in a total volume of 6 mL. After 6 h of drug incubation at 37 ºC, the medium was aspirated and the cells were washed twice with 4 mL PBS. Then, the cells were trypsinized (1 mL), suspended with Opti-MEM (3 mL), and centrifuged (1200 rpm, 4 min). After aspiration of the supernatant, the cells were re suspended in PBS (1mL) and counted. After a second centrifugation, the supernatant was discarded and the pellets were resuspended in MilliQ water (154 µL) and 65% HNO 3 (up to 2 mL) for overnight digestion. Then, 1 mL of the solution was diluted with MilliQ water to obtain a final concentration of 5% HNO 3 . For ICP-MS measurements, the system was optimized with a ruthenium-platinum solution. The calibration range was from 0 to 25 µg/L, and obtained detection limit for all isotopes was 0.01 µg/L. Silver and Indium were used for internal standard, to correct for sample dependent matrix effects. No reference sample was available; therefore several samples were spiked with a known concentration. The recoveries of the spiked concentrations were all within a 10% deviation. The data from two independent biological replications were used to obtain the uptake values shown in Table 1.