In Vivo Selectivity and Localization of Reactive Oxygen Species (ROS) Induction by Osmium Anticancer Complexes That Circumvent Platinum Resistance

Platinum drugs are widely used for cancer treatment. Other precious metals are promising, but their clinical progress depends on achieving different mechanisms of action to overcome Pt-resistance. Here, we evaluate 13 organo-Os complexes: 16-electron sulfonyl-diamine catalysts [(η6-arene)Os(N,N′)], and 18-electron phenylazopyridine complexes [(η6-arene)Os(N,N’)Cl/I]+ (arene = p-cymene, biphenyl, or terphenyl). Their antiproliferative activity does not depend on p21 or p53 status, unlike cisplatin, and their selective potency toward cancer cells involves the generation of reactive oxygen species. Evidence of such a mechanism of action has been found both in vitro and in vivo. This work appears to provide the first study of osmium complexes in the zebrafish model, which has been shown to closely model toxicity in humans. A fluorescent osmium complex, derived from a lead compound, was employed to confirm internalization of the complex, visualize in vivo distribution, and confirm colocalization with reactive oxygen species generated in zebrafish.

treated with either complex 1 or 10 (1× IC50) for 24 h. Cells were then washed with PBS and harvested using trypsin/EDTA (2 mL). Cells were counted, and a pellet was obtained by centrifugation (1000 rpm, 5 min, 277 K). Cells were transferred to an 1.5 mL Eppendorf tube by centrifugation using PBS, and the supernatant removed. Ultra-pure nitric acid (72% v/v, freshly-distilled, 200 µL) was added directly to the Eppendorf, which was incubated overnight at 350 K. The resulting digested cell solution was diluted 1:20 with Type I milli-Q water (containing 100mg/L ascorbic acid and 10 mM thiourea) to obtain final working acid concentration (3.6% v/v). The solution was analysed for Os-189 using an Agilent 7900 series ICP-MS in both no-gas and He-gas mode, using an internal standard of Er-166.
Calibration standards (0.1-1000 ppb) for Os were freshly prepared in 3.6% nitric acid containing thiourea and ascorbic acid, as described for ICP-OES experiments. Data were acquired and processed using Agilent Mass Hunter 4.3 for Windows. All data were acquired as biological (experimental) triplicates, and standard deviations calculated. Final values were reported as ng osmium per million cells.
The experiment was repeated with the following modifications: after exposure to Os complexes, cells were recovered in either Os(II)-free culture medium, or culture medium containing verapamil (variable concentration depending on the experiment, 0-100 µM) for up to 72 h.
Reactive oxygen species (ROS) determination using flow cytometry. The generation of ROS and superoxide in A2780 ovarian cancer cells was evaluated using the ROS/Superoxide detection kit (Enzo Life Sciences) following the manufacturer's protocol. A 6-well plate was seeded with 1.5 × 106 A2780 cells per well, and incubated for 24 h (310 K). After this time, cells were exposed to equipotent concentrations of the osmium complexes (1.0 × IC50) for 24 h. Cells were washed with PBS and collected as pellets by centrifugation (1000 rpm, 5 min, 277 K). The pellets were re-suspended in 500 μL PBS and stained with green fluorescent reagent for total ROS reading in the FL1-green channel (2 μL) and orange fluorescent reagent for superoxide reading in the FL2-red channel (2 μL). Positive control samples were treated with pyocyanin (5 μL, 30 min). Single stained positive control samples were used for compensation purposes. Data were obtained using a Becton Dickson FACScan Flow Cytometer and processed using FlowJo V10 for Windows.
Reactive oxygen species (ROS) determination using confocal microscopy. Briefly, 2000 A2780 cancer cells were seeded in 8-well black plates and incubated for 24 h. After this time, cells were treated with Os(II) complex 6 (0.5× and 1× IC50; 24 h exposure). Cells were then washed with PBS, and stained using green ROS detection reagent (2 µM, dark, 20 min; Enzo Life Sciences). After removal of excess dye with PBS wash, cells were analysed using a Zeiss LSM880 confocal microscope (Argon laser; excitation: 458, 488, 561 nm; green emission for ROS: 493-550 nm; red emission for complex 6: 568-750 nm). Data were processed using Zen 2.3 for Windows.

In vivo biological studies
Zebrafish studies. Zebrafish experiments were carried out under AWERB.10/16-17 in the School of Life Science, University of Warwick, UK, under the guidance of Dr. Karuna Sampath, using Singapore wild-type zebrafish (SGWT) embryos (< 5 dpf). All animals were maintained in accordance with ASPA 1986. Warwick University is a member of the Institute of Animal Technology and the Laboratory Animal Science Association, and all animal work carried out was approved by the University Ethical Review Committee.
Singapore wild-type (SGWT) zebrafish were housed in 3.5 L tanks, and monitored at least once a day.
Fish were provided with food (live and powder) four times daily during the week and twice daily on weekends. Regular system checks were carried out daily to ensure water quality and parameters are maintained. Light cycle: 14 hours day, 10 hours night. Fish were mated once a week using 2 pairs per 1 L breeding tank. Pairs were set up the previous evening. Males and females were separated by a divider. The divider was removed around 9am (dawn). Embryos were collected and placed into petri dishes with fresh egg water. Confocal microscopy (zebrafish). This method was adapted from an existing literature procedure. 2,3 Approximately 10-20 zebrafish embryos per sample were incubated in a P100 dish containing 30 mL of Os(II) arene complex (1× or 2× LC50 concentration) prepared in fish water (DMSO not exceeding 1% v/v) for 96 h at 301.5 K. Untreated embryos were also incubated for 96 h for use as either negative or positive control samples.

Determination of LC50 concentration for Os(II) arene complexes in zebrafish
1% low-melting point agarose was prepared in Hanks Balanced Salt Solution (HBSS), and the aquatic anaesthetic Tricaine was diluted to achieve a final concentration of 0.2 mg mL -1 . After exposure to Os(II) complexes, zebrafish embryos were collected and washed with HBSS (3x 10 mL). 20 untreated embryos were exposed to the positive control, Rotenone (50 μM, 2 min) then washed with HBSS (3x S6 (20 uM) for total ROS (Enzo Life Sciences). Zebrafish were washed using HBSS, anaesthetised using tricaine (5-10 min, 298 K) the mounted in 200 µL agarose in a glass-bottom dish for confocal imaging.