High nuclearity carbonyl clusters as near-IR contrast agents for photoacoustic in vivo imaging

High nuclearity carbonyl clusters of ruthenium and osmium are found to exhibit good photoacoustic (PA) activity in the near-IR (NIR) region. Their potential as PA contrast agents for full body imaging has been demonstrated for the first time with mice; intravenous administration of the osmium carbonyl cluster Na2[Os10(μ6-C)(CO)24] afforded up to a four-fold enhancement of the PA signal in various tissues. The cluster exhibits low toxicity, high stability and superior PA stability compared to the clinically approved NIR dye, indocyanine green.

28. Table S1.ICP-MS analysis of Os content in various organs 24 h after injection of 9. S2.ICP-MS analysis of Os content in various organs organs 96 h after injection of 9. S3.ICP-MS analysis of background Os content in various organs in control animals (without the introduction of 9).S4.Calculation of the percentage retention of injected dose (% ID) of 9 in various organ tissues 24 h after injection of 9.

Experimental section a. General methods
All reactions and manipulations were carried out under an argon atmosphere using standard Schlenk techniques.Solvents that were used for reaction were distilled over the appropriate drying agents under argon before use.Purification of compounds was generally carried out by column chromatography on silica gel, or by preparative thin-layer chromatography (TLC) using 20 cm x 20 cm plates pre-coated with silica gel 60 F 254 Infrared (IR) spectra were recorded on a Bruker Alpha FT-IR spectrometer.Solution spectra were recorded in DCM solution, unless otherwise stated, in a solution IR cell with NaCl windows and a path length of 0.1 mm, at a resolution of 2 cm -1 .Alternatively, IR spectra of aqueous samples were recorded by drop casting the mixture (10 μL) onto a CaF 2 disc, followed by drying under a stream of nitrogen before recording on the spectrometer.Optical absorption spectra were recorded using a Hitachi U-2900 double-beam spectrophotometer using quartz cuvette with path length of 10 mm.For samples of smaller volume (100 μL), the optical absorption spectra were recorded using a Beckman Coulter DU 730 spectrometer.HRMS were recorded in ESI mode on a Waters UPLC-Q-Tof MS mass spectrometer.

b. Synthesis of cluster 9
The synthesis was adapted from the microwave-assisted synthesis of cluster 8. 7 A Discover-SP microwave reactor (2450 MHz, CEM Corp., Matthews, NC) and thick-walled 35 mL glass vessel with Teflon-lined caps (CEM Corp., Matthews, NC) were used.In a typical reaction, a 35 mL microwave vessel was charged with Os 3 (CO) 12 (150 mg, 0.1654 mmol) and diglyme (7 mL).The mixture was irradiated with 300 W with high stirring rate, till the temperature reached 230 o C.
The power was subsequently reduced to 100 W for additional heating for 10 min.The reaction mixture was cooled to 50 o C and vented, before irradiated at 300 W for 4 min.The cooling, venting and irradiation at 300 W for 4 min was repeated once.The solvent was removed by evaporation over a hot water bath and the residue was extracted with acetone (20 mL) and filtered over a thin layer of celite.The solvent was removed and the residue dissolved in MeOH (10 mL).NaCl (20 mg, 0.34 mmol) was added to the solution and stirred at 60 o C for 1 h.Solvent was removed and the residue was purified by TLC using acetone-hexane (3:2, v/v) as eluant to give cluster 9 as a dark red band.

c. Cell culture and cell viability assay
Experimental cultures of oral squamous cell carcinoma (OSCC) cell line were obtained from the American Type Culture Collection (ATCC) and cultured in tissue culture dishes (Nunc Inc., IL).The cells were maintained in Dulbecco's modified Eagle's medium (DMEM; Biowest, France) supplemented with 10 % fetal bovine serum (FBS; Gibco ® , NY), 1 % L-glutamine (PAA Laboratories, Austria), and 1 % penicillin/streptomycin (HyClone, UT) at 37 °C in 5 % CO 2 atmosphere.Phosphate-buffered saline (PBS) was obtained from PAA Laboratories.Stock solutions of cluster 9 and [(Ph 3 P) 2 N]Cl in sterile-filtered dimethyl sulfoxide (DMSO) were prepared and serial diluted to lower concentrations.For treatment with the compounds, cells were seeded in wells within 96-well plate in growth medium at the same initial density of 10 000 cells per well, and allowed to adhere and grow for 24 h.They were serum-starved in serum-free DMEM for 24 h, followed by treatment with the indicated concentrations of compounds in serum-free DMEM (1 % DMSO) for 24 h.Control cells were treated with vehicle (1 % DMSO).
To each well, 10 μL of MTS reagent (MTS Cell Proliferation Assay Kit, BioVision, CA) was added and then left to incubate in a 37 °C incubator with 5% CO 2 for 2 h.The absorbance intensities at 490 nm were then measured and the cell proliferation relative to the control sample was calculated.Each sample was analyzed in triplicates and was corrected with background intensities from same incubation conditions without the cells.IC 50 was determined from three separate experiments and the mean value calculated.

d. Gel electrophoresis
Agarose gel (1%, UltraPure TM Agarose, Invitrogen) was cast with and immersed in 0.5 x TAE (Tris-acetate-EDTA buffer, prepared from 50 x stock solution, pH 8.0, 1 st BASE).Cluster 9 incubated in 10% DMSO in modified DMEM (30 L) for different duration (10 min, 1, 3, 5 and 7 days) was loaded into the respective wells of the gel and electrophoresed in a horizontal electrophoresis system (Wide Mini-sub® cell GT Basic System, Biorad, electrode spacing of 15 cm) at 150 V for 30 min.

e. Multispectral optoacoustic tomography (MSOT) experimental parameters and protocol
All phantom and in vivo mouse imaging experiments were performed using a real-time MSOT imaging system; inVision 128 (ithera Medical GmbH, Germany). 8Optical excitation was provided by an optical parametric oscillator with a tunable NIR wavelength range from 680 to 900 nm, which was in turn pumped by a Q-switched Nd:YAG laser with a pulse duration of 10 ns and repetition rate of 10 Hz.Light was delivered by a fiber bundle divided into 10 output arms to illuminate the sample from multiple angles around the imaging plane.PA signals were acquired using a 128-element concave transducer array spanning a circular arc of 270°.This transducer array had a central frequency of 5 MHz, which provided a transverse spatial resolution in the range of 150-200 μm.One transverse image slice was acquired from each laser pulse, resulting in a frame rate of 10 Hz.During image acquisition, the sample was translated through the transducer array along its axis across the volume region of interest (ROI), in order to capture the corresponding transverse image slices.

f. Evaluation of PA activity of clusters 4-9 in phantom model
The phantom is made of polyurethane and is cylindrical in shape with a diameter of 2 cm, which is specially designed to mimic the shape, size, and optical properties of the mouse.In addition, it has two inner cylindrical channels, each with an inner diameter of 3 mm, one for holding the control medium and the other for holding the dissolved contrast agent in the same medium.The control medium and contrast agent suspension (170 μL each) were pipetted into the respective channels of the phantom, and placed in the MSOT imaging chamber using a phantom holder.For data acquisition, we set up an ROI of multiple transverse slices with a step size of 1 mm across the channel portion which contained the probe and control, applied excitation wavelengths from 680 to 900 nm with an interval of 10 nm for each transverse slice, and recorded the averaged PA signals from 10 frames for each wavelength and position.The acquired images were reconstructed using model based approach and PA signals were quantified using ViewMSOT at the respective  max of each probe from the reconstructed images. 9The PA amplitude of the probe was determined by subtracting the values of the channel containing the control from that of the probe, and the mean was calculated from 10 different transverse slices.(200 μL, 500 μM) in 10% DMSO in heparinized saline (v/v) was injected intravenously through the tail vein of each mouse anaesthetized under isoflurane.Ultrasound gel was applied on the mouse skin surface for in vivo imaging, and measurements were recorded in temperaturecontrolled water medium for good acoustic coupling.An animal holder with a thin polyethylene membrane was used to prevent direct contact between the mouse and the water.Seven laser excitation wavelengths (680, 700, 730, 750, 810, 850, and 900 nm) were chosen for multispectral imaging on the basis of the absorption maxima and minima of the contrast agent, and oxy-and deoxyhemoglobin.PA signals were acquired across multiple transverse slices from the liver to the lower abdomen region inclusive of the tumor at a step size of 0.5 mm and averaged over 10 frames for each wavelength.

h. Image processing and analysis for in vivo experiments
Images acquired from the in vivo experiments were reconstructed using direct back projection approach for offline analysis.After image reconstruction, spectral unmixing was performed to resolve individual components from different chromophores in the system.For each pixel, the method fits the total measured optoacoustic spectrum to the known absorption spectra of the individual chromophores, based on least-squares linear regression.Images were exported out from ViewMSOT and further analyzed using Fiji (version 2.0.0), an open source imageprocessing package. 10Regions of Interest (ROIs) were chosen from liver, spleen, kidney and tumor and quantified for multispectrally unmixed PA signals before and after probe injection.PA signals at all time-points were normalized against that of the pre-injection scan and expressed as fold change over the same.

i. Microwave acid digestion and ICP-MS analysis of osmium content of tissue samples
The mice were euthanized after 24 and 96 h respectively, the organs extracted, washed with deionized water and snap-freezed in liquid nitrogen for future use.The selected organs were freeze-dried over a period of five days and the dried masses measured, prior to microwave acid digestion.A programmable Milestone microwave system (ETHOS EZ, Italy) was used to digest and prepare the samples for ICP analysis.Samples were placed inside the Teflon microwave vessels and 12 mL HCl (37%, ultrapure grade, J. T. Baker, Canada) were added.Ultra-pure water with 18.2 MΩ•cm resistivity was used (ELGA, PURLAB Option -Q, UK) for sample and standard preparations.Using the microwave radiation (Power 1100W) the temperature was ramped to 185 °C in 20 min and then held at that temperature for 15 min.The vessels were opened after cooling down to room temperature for several hours, to prevent any sample loss.The extracts were diluted with ultrapure water, filtered through 0.2 µm syringe filters, and carefully transferred to ICP sample vials for elemental analysis.Samples were analyzed using an Agilent 7700 series ICP-MS (Japan) equipped with a third generation He reaction / collision cell (ORS3) to minimize interference.An external calibration curve for osmium was constructed, before each analytical run, from serial dilutions of an osmium standard for ICP-MS (1000 mgL -1 , Inorganic Venture, Christiansburg, VA, USA) with high purity deionized water.

j. Histopathology of organ tissues of mice
Six mice were intravenously injected with cluster 9 (200 L, 500 M, 10% DMSO in saline) and euthanized after 24 h and 96 h (three mice at each time point) respectively.For the control sample, four mice were injected with 10% DMSO in saline instead.The mice were anaesthetized using ketamine (150 mg/mL) and xylazine (10 mg/kg) and euthanized by cervical dislocation to collect various organs for histopathology analysis.All organs were immediately fixed in 10% neutral buffered formalin (Sigma) and submitted to the Advanced Molecular Pathology Laboratory (AMPL), IMCB for histopathology evaluation.The pathology report was prepared by an experienced pathologist.All bright field images were captured using the Nikon Ni-E upright microscope, with Plan Apo 20x DIC M N2 objective and Nikon DS-Ri2 camera.
The images were viewed using NIS-Elements software.The blood volume of mouse was estimated to be 79 (77-80) μL g -1 . 12,13 ence, this value was used for the calculation of blood volume:

10 .
Figure S9.Irradiation of a solution of 9 (200 μM) in 10% DMSO in modified DMEM at 760 nm for various time intervals: a) FTIR spectra ( CO ) obtained from the dried samples, and b) integrals of the  CO absorbance.11. Figure S10.a) Time course studies of 9 and ICG over 120 min with irradiation at the respective  max : PA amplitude as a function of time, b) optical absorbance as a function of time, c) optical absorption spectra of 9 and d) ICG.12. Figure S11.a) Optical absorption spectra of ICG of various concentrations, b) plot of absorbance of ICG at λ max= 790 nm as a function of concentration, c) PA (red) and optical absorption (black) spectra of ICG, d) plot of PA amplitude at λ max= 790 nm as a function of concentration of ICG in 10% DMSO in modified DMEM (v/v).13. Figure S12.PA signal from 9 across a single slice of the mouse abdomen showing liver before and after injection up to 24 h.14. Figure S13.a) Enlarged images of the tumors at various time point with the PA intensities normalized (top).Fold change in PA intensity in the tumor over that at t=0 in the tumor (bottom).b) Tumor-to-muscle ratio of PA amplitude at various time points up till 24 h.15. Figure S14.ICP-MS analysis of the percentage retention of injected dose (% ID) of 9 per organs, 24 h after injection.Error bars represent standard error of the mean, n = 3. 16. Figure S15.Histopathology (H & E staining) of kidneys, heart and lungs tissues of mice, 24 h and 96 h after intravenous injection of 9, with that of control samples.17. Figure S16.Percentage weight change of control mice and mice injected with 9 up till 24 h and 96 h with respect to initial weight.Error bars represent standard error of the mean, n = 3 for samples with 9 and n = 2 for control samples.18. Figure S17.IR spectrum ( CO ) of 1 in DCM.19. Figure S18.IR spectrum ( CO ) of cluster 2 in hexane.20. Figure S19.IR spectrum ( CO ) of cluster 3 in DCM. 21. Figure S20.IR spectrum ( CO ) of cluster 4 in DCM.22. Figure S21.IR spectrum ( CO ) of cluster 5 in DCM.23. Figure S22.IR spectrum ( CO ) of cluster 6 in DCM.24. Figure S23.IR spectrum ( CO ) of cluster 7 in DCM.25. Figure S24.IR spectrum ( CO ) of cluster 8 in DCM. 26. Figure S25.IR spectrum ( CO ) of cluster 9 in acetonitrile.27. Figure S26.HRMS-ESI spectrum of cluster 9 with the calculated isotopic pattern.

g.
In vivo longitudinal monitoring of probe biodistribution in mouse xenograft model All animal experiments were performed in compliance with the protocol #120774 and #151085 approved by the Institutional Animal Care and Use Committee, Agency for Science, Technology and Research (A*STAR).Xenograft models were established on three Balb/C nude mice by injecting subcutaneously into the right flank of each mouse, a cell suspension (0.1 mL) containing 5×10 6 oral squamous carcinoma cells (OSCC) and matrigel (BD Biosciences, San Jose, CA) in a 1:1 volume ratio.When the tumor volume reached a palpable size, a solution of 9

Figure
Figure S1.i) Optical absorption spectra of various concentrations (in DMSO), ii) plot of optical absorbance (at respective λ max ) as a function of concentrations of a) cluster 4, b) cluster 5 and cluster 6.

Figure S9 .Figure S10 .FigureFigure S12 .
Figure S9.Irradiation of a solution of 9 (200 μM) in 10% DMSO in modified DMEM at 760 nm for various time intervals: a) FTIR spectra ( CO ) obtained from the dried samples, and b) integrals of the  CO absorbance.

Figure S13 .Figure S14 .
Figure S13.a) Enlarged images of the tumors at various time point with the PA intensities normalized (top).Fold change in PA intensity in the tumor over that at t=0 in the tumor (bottom).Error bars represent standard error of the mean, n = 3. b) Tumor-to-muscle ratio of PA amplitude at various time points up till 24 h.Error bars represent standard error of the mean, n = 3.

Figure S15 .
Figure S15.Histopathology (H & E staining) of kidneys, heart and lungs tissues of mice, 24 h and 96 h after intravenous injection of 9, with that of control samples.Scale bar = 100 m.

Figure S16 .
Figure S16.Percentage weight change of control mice and mice injected with 9 up till 24 h and 96 h with respect to initial weight.Error bars represent standard error of the mean, n = 3 for samples with 9 and n = 2 for control samples.