An 111In-labelled bis-ruthenium(ii) dipyridophenazine theranostic complex: mismatch DNA binding and selective radiotoxicity towards MMR-deficient cancer cells† †Electronic supplementary information (ESI) available. See DOI: 10.1039/d0sc02825h

Auger electron emitter indium-111 demonstrates cancer-selective radiotoxicity and SPECT imaging compatibility when conjugated to a ruthenium(ii) polypyridyl complex.

H 3 [2] 4+ was prepared by reacting [1] 2+ with cyclic DTPA in a 2:1 molar ratio in dry DMF overnight at 60 C. The crude product was precipitated by the addition of diethyl ether and collected by centrifugation. The reaction mixture was then analyzed by reverse-phase HPLC. The major peak at 12 min 03 s (Method A) was characterised as H 3 [2] 4+ by high resolution mass spectrometry. The major peak at 12 min 03 s (HPLC Method A) was collected by reversed-phase HPLC. The solution was pH adjusted to neutral and the acetonitrile removed by rotary evaporation. Addition of NH 4 PF 6 gave a red-orange precipitate which was collected by centrifugation and washed with diethyl ether. The solid was dried using a Virtis Advantage Plus freeze dryer. NMR: 400 MHz, ((CD 3  Due to the hygroscopicity of the dinuclear Ru(II) compounds, we were unable to obtain reliable elemental analyses, even after vacuum drying. All Ru(II) compounds were used as their chloride salts in biological studies.

Radiolabelling
A typical radiolabelling reaction involved adding 2 l of a H 3 [2]

High resolution mass spectrometry
Samples were diluted to a final concentration of 1 M in 0.1 % formic acid. 50 µl was injected on to a 1290 UPLC coupled to a 6530 QTOF mass spectrometer fitted with a 2.1 mm x 12.5 mm Zorbax 5 µm 300SB-C3 guard column (Agilent Technologies, Santa Clara, USA). Solvent A was 0.1 % formic acid in HPLC grade water; solvent B was 0.1 % formic acid in LC-MS grade methanol (Fisher Scientific, Loughborough, UK). Initial conditions were 10 % B at 1 ml/min. A methanol gradient was developed from 10 % to 80 % over 34 seconds, followed by isocratic at 95 % B for 41 seconds, followed by 15 seconds re-equilibration at 10 % B. The mass spectrometer was operated in positive ion, 2 GHz detector mode. Source parameters were drying gas 350 C, flow 12 L/min, nebulizer 60 psi, capillary 4000 V. Fragmentor was 250 V, collision energy 0 V and data acquired from 100-3200 m/z. Data analysis was performed using Masshunter Qualitative Analysis B0.7 proprietary software and deconvolution performed using the resolved isotope method.

Oligonucleotides synthesis and purification
Oligonucleotides were synthesized on an Applied Biosystems 394 automated DNA/RNA synthesizer using a standard 1.0 μmole phosphoramidite cycle of acid-catalyzed detritylation, coupling, capping, and iodine oxidation. Stepwise coupling efficiencies and overall yields were determined by the automated trityl cation conductivity monitoring facility and was >98.0 %. Standard DNA phosphoramidites and additional reagents were purchased from Link Technologies Ltd, Sigma-Aldrich, Glen research and Applied Biosystems Ltd. All -cyanoethyl phosphoramidite monomers were dissolved in anhydrous acetonitrile to a concentration of 0.1 M immediately prior to use with coupling time of 50 s. Cleavage and deprotection were achieved by exposure to concentrated aqueous ammonia solution for 60 min at room temperature followed by heating in a sealed tube for 5 h at 55 °C. Purification was carried out by reversed-phase HPLC on a Gilson system using a Brownlee Aquapore column (C8, 8 mm x 250 mm, 300 Å pore) with a gradient of acetonitrile in triethylammonium bicarbonate (TEAB) increasing from 0 % to 50 % buffer B over 30 min with a flow rate of 4 mL/min (buffer A: 0.1 M triethylammonium bicarbonate, pH 7.0, buffer B: 0.1 M triethylammonium bicarbonate, pH 7.0 with 50 % acetonitrile). Elution was monitored by ultraviolet absorption at 298 nm. After HPLC purification, oligonucleotides were freeze dried then dissolved in water without the need for desalting. All purified oligonucleotides were characterised by electrospray mass spectrometry. Mass spectra of oligonucleotides were recorded using a XEVO G2-QTOF MS instrument in ES-mode. Data were processed using MaxEnt and in all cases confirmed the integrity of the sequences.

DNA binding
Calf thymus DNA was dissolved in aqueous Tris-buffer (25 mM NaCl, 5 mM Tris, pH = 7) and broken into an average of 150-200 base pair fragments by sonication (2 × 15 mins). Purity was determined by UV-vis spectroscopy, with A 260 /A 280 > 1.8 indicating a protein-free sample. Luminescent titrations were carried out with the addition of an aqueous solution of concentrated DNA to 30 M of each Ru(II) complex. After each addition of DNA, the solution was mixed by pipette and allowed to equilibrate for 5 min. Luminescence spectra were recorded on a Tecan Infinite 200 PRO Microplate Reader ( ex = 405 nm,  em = 500-800 nm). DNA was added until the MLCT emission reached a maximum (typical DNA concentration at saturation = 80-90 M). The AUC (area under curve) for each luminescence spectrum were used to construct nonlinear Scatchard plots (r/Cf versus r, an example within Fig S7b) and fit to the McGhee-von Hippel model, 2 in which neither the site size nor binding constant were defined, to determine K b and n. Hairpin DNA and each 10-mer DNA duplex were dissolved in 10 mM phosphate buffer, 200 mM NaCl at pH 7.0. A 3 M concentration of each was then added to 30 M of each Ru(II) complex in 10 mM phosphate buffer, 200 mM NaCl at pH 7.0 and the fluorescence spectrum recorded on a Tecan Infinite 200 PRO Microplate Reader ( ex = 405 nm,  em = 500-800 nm). The emission intensity was determined as the AUC.

Ultraviolet DNA melting studies
UV DNA melting was monitored on Cary 4000 Scan UV-Visible Spectrophotometer (Varian) using an adapted method of a recent publication. 3 A 3 M concentration of each oligonucleotide +/-30 M of each Ru(II) complex in 10 mM phosphate buffer, 200 mM NaCl at pH 7.0 was employed. The samples were initially denatured by heating to 85 C at 10 C min -1 , cooled to 20 C at 1 C min -1 and then heated to 85 C at 1 C min -1 . Spectra were recorded at 260 nm. Eight successive melting curves were measured and T m values were calculated from their derivatives using Cary Win UV Thermal application Software. T m = T m (with compound) -T m (without compound).

Cell culture
HCT-116, HT-29, WI-38 and HeLa cells were cultured in DMEM supplemented with 10% FBS and penicillin/streptomycin. DLD-1 and DLD-1+Chr2 cells were cultured in RPMI supplemented with 10% FBS and penicillin/streptomycin. DLD1+Chr2 cells were maintained under selective pressure of 400 µg/mL geneticin (G418 sulfate, Roche). Cell lines were maintained at 37 °C in an atmosphere of 5% CO 2 and sub-cultured by Trypsin. Cell lines were used at passage numbers 40 or lower and routinely confirmed to be mycoplasma-free.

Subcellular distribution
Non-radioactive compounds: HCT-116 cells were grown in 6 well plates, treated with Ru(II) compound for 24 h, washed with cold PBS (2 × 2 mL) before washing with acidified PBS (pH 2.5) to remove the surface-bound fraction. 0.4 mL EZ lysis buffer was added, cells were detached by scraping, collected into eppendorf tubes, vortexed briefly and left for 5 minutes on ice. Samples were centrifuged (500 g, 5 min) and the supernatant (cytosol fraction) aspirated. The pellet (nuclear fraction) was re-suspended in 200 μl RIPA buffer. Successful fractionation of the two subcellular compartments was verified by immunoblotting using anti-α-tubulin (Sigma) and anti-histone H2AZ (Abcam) for cytosol and nuclear fractions, respectively. Protein content of each fraction was determined by BCA assay and ruthenium content determined by ICP-MS analysis, as described in detail in a recent publication. 4 Radioactive compounds: The adherence of [ 111 In]In 3+ to the negatively-charged tissue culture plastic well plates gave a false signal by the method above. To counter this, 1x10 6 cells in eppendorfs were treated with 1 MBq/ml [ 111 In][In-2] 4+ for 2 h with gentle shaking. Cells were collected by centrifugation (2000 rpm, 5 min) before processing as described above. Protein content of each fraction was determined by BCA assay and radioactivity was measured using a WIZARD-2 Automatic Gamma Counter.

MTT assay
Cancer cell lines were seeded in 96 well plates at 10,000 cells/well and allowed to adhere for 24 h before treatment. Cell lines were treated with a concentration gradient of each compound in triplicate. After incubation, 0.5 mg/ml MTT (thiazolyl blue tetrazolium bromide) dissolved in serumfree medium was added for 60 minutes and the formazan product eluted using acidified isopropanol. The absorbance at 540 nm was quantified by plate reader (reference wavelength 650 nm). The metabolic activity of the cell population was determined as a percentage of a negative (solvent) control. WI-38 cells were seeded in 24 well plates at 10,000 cells/well and allowed to adhere for 24 h.  4+ for 24 h before exposure to 0-6 Gy 137 Cs--rays. Cells were washed with PBS and collected using Trypsin. Samples were counted and cells re-seeded in 6 well plates at 1000-5000 cells/well (exact number optimised for each cell line and condition to ensure adequate colonies for counting).
External beam: DLD-1 or DLD-1+Chr2 cells were seeded in 6 well plates at 1500-48000 cells/well (depending on dose to be received) and allowed to adhere for 4 h. Cells were then exposed to 0-8 Gy 137 Cs--rays.
In all cases, colonies were grown for >7 days, washed in PBS, and stained with 1 % methylene blue (Alfa Aesar) in 50% methanol (Thermo Fisher Scientific). Colonies containing 50 cells or greater were counted using a Gelcount instrument and accompanying software (Oxford Optronix). Plating efficiencies were determined for each treatment condition and normalised to an untreated control to provide the surviving fraction (S. F.).

Animal procedures and SPECT imaging
All animal procedures were performed in accordance with the UK Animals (Scientific Procedures) Act 1986 and with local ethical committee approval. DLD-1 tumours were established by subcutaneous injection of 2 × 10 6 cells suspended in 200 μL 1:1 RPMI:Matrigel into the right dorsal flank of female BALB/c Nu/Nu mice (n=3). SPECT imaging was performed using a VECTor 4 imaging system (MILabs) using a rat collimator with 1.8 mm pinholes. Continuous SPECT data were acquired for 10-70 minutes after intravenous administration of [ 111 In][In-2] 4+ (7-9 MBq) followed by a CT image on the same system. Image acquisition and processing was performed as described in detail elsewhere using the PMod software package. 6 Mice were kept under anaesthesia by inhalation of 2% isofluorane in air and maintained at 37 °C during the imaging session. Mice were revived and 24 h after the SPECT imaging session they were euthanised by cervical dislocation. Selected organs, tissues and blood were removed, and the percentage of the injected dose per gram (% ID/g) of each sample was calculated. No adverse effects of the compound were observed.