Delivery and release of curcumin by a hypoxia-activated cobalt chaperone: a XANES and FLIM study

[CoCl(H2O)(tpa)](ClO4)2, 5 [Co(cys)(tpa)]ClO4, 6 K[Co(cys)(H2O)2] and facK3[Co(cys)3] 7 were prepared according to literature procedures. All reactions involving ruthenium were carried out under nitrogen using standard Schlenk techniques. Caution! Many of the procedures involved the preparation of perchlorate salts of metal complexes. While no issues were experienced, such compounds should be handled with care, as they are potentially hazardous. Particular care was taken with the tris(2-methylpyridyl)amine perchlorate (tpa·nClO4) ligand as organic perchlorate molecules are potentially explosive.


Delivery and release of curcumin by a hypoxia-activated cobalt chaperone: a XANES and FLIM study
Anna K. Renfrew*, Nicole S. Bryce and Trevor W. Hambley

General synthesis for complexes 1a -c
Ruthenium complexes were prepared according to the following procedure.
[RuCl 2 (tpa)]ClO 4 (145 mg, 0.25 mmol) was refluxed in water (50 mL) for 30 min, during which the yellow suspension became a green or purple solution.0.25 mmol of HL (where L corresponds to acetylacetone, dibenzolymethane or curcumin, respectively) and triethylamine (0.2 mL, 0.3 mmol) were added and the solution was heated at reflux for a further 16h during which a red precipitate formed.Excess NaClO 4 was added and the resulting precipitate was collected by suction filtration and washed with water (2 x 5 mL) and diethyl ether (2 x 10 mL).

General synthesis for complexes 2b and 2c
[Co(Cl 2 )tpa]ClO 4 (130 mg, 0.25 mmol) was dissolved in water (50 mL) and the pH adjusted to 7 using 1M NaOH.After refluxing for 10 minutes, 1 equivalent of HL and 1.2 equivalents of triethylamine added and the solution was heated at reflux for a further 3h, during which time an orange precipitate formed.The solution was cooled and excess NaClO 4 added to give an orange powder, which was collected by suction filtration and washed with water (2 x 5 mL) and diethyl ether (2 x 10 mL).

Methods and Instrumentation
1 H NMR spectra were collected at 300 K on a Bruker 300 MHz spectrometer using commercially available deuterated solvents.Isotopic impurities were used as internal reference signals.Signals for 1b, 1c, 2b and 2c were assigned from 2D COSY spectra.Mass spectrometry was performed using Electro-Spray Ionisation using a Finnigan LCQ-8 spectrometer.Elemental analyses (C, H, N) were conducted by the Chemical & MicroAnalytical Services Pty Ltd, Campbell Microanalytical Laboratory, at the University of Otago.

Electrochemistry
Cyclic voltammograms were recorded on a BAS100B instrument using a glassy carbon working electrode, a silver/silver chloride reference electrode and a platinum auxiliary electrode.Scans were performed at a scan rate of 100 mV s−1 and the solutions were degassed with argon for at least ten minutes prior to scanning.All experiments were performed at room temperature and were iR compensated.Scan were run in DMF, with 0.1 M tetrapropylammonium perchlorate as the supporting electrolyte, and the ferrocene/ferrocenium couple was used as an internal reference ([Fe(η 5 -C5H5)2] 0/+ = +0.72V vs. NHE in DMF at 25 °C).
UV-vis spectra UV-visible measurements were performed on a Cary 1E UVvisible spectrophotometer using a 1 cm x 1 cm quartz cuvette.Scans were run at room temperature as 25 x10 - 5 M solutions of 1:25 MeOH/TRIS buffer (pH 7.4).

Fluorescence spectra
Fluorescence measurements were performed using a Varian Cary Eclipse fluorescence spectrophotometer, using a 1 cm x 1 cm quartz cuvette.Scans were run at room temperature as 25 x10 -5 M solutions of 1:25 MeOH/TRIS buffer (pH 7.4) at 100 nm/min with excitation and emission slit widths of 10 nm.All solutions were prepared immediately prior to analysis.Emission scans were run between 460 and 700 nm using an excitation wavelength of 420 nm.

Reductions in situ
Emission scans were collected between 460 and 700 nm with an excitation wavelength of 420 nm every 30 minutes over 20 h of 25 x10 -5 M solutions of 1:25 MeOH/TRIS (pH 7.4) following the addition of 10 molar equivalents of (L)-ascorbic acid or (L)-glutathione (reduced).Deoxygenated solutions were purged by passing a stream of nitrogen through the solutions for 15 minutes prior to addition of the reducing agent and the cuvettes tightly sealed.The extent of fluorescence return was calculated with respect to the fluorescence emission intensity of free curcumin under the same conditions.
Cell culture DLD-1 human colon carcinoma cells were purchased from ATCC and used within 2 months of resuscitation.Cells were maintained in Advanced DMEM (Invitrogen) and supplemented with 2% FBS and 2mM Glutamine in a humidified environment at 37 o C and 5% CO 2 .

Cytotoxicity assay
Cytotoxicity was determined using the MTT assay. 1 x10 5 cells per well were plated on to 96-well plates and allowed to adhere overnight.Freshly prepared media/DMSO (90:10) solutions of the complexes and free ligands were added to triplicate wells at concentrations spanning a 4-log range (final DMSO concentrations <0.5%) and incubated for 72 hrs.3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (1.0 mM) was added to each well and the cells incubated for a further 4 hrs.The culture medium was removed and the resulting purple precipitate dissolved in DMSO (100 μL).The plate was shaken for 5 seconds and the absorbance measured immediately at 600 nm using a Victor 3 V microplate reader (Perkin Elmer).IC 50 values were determined as the drug concentration required to reduce the absorbance to 50% of that of the untreated control wells.At least three independent experiments were performed for each compound with triplicate readings in each experiment.
Preparation of monolayer cell samples for imaging 5 x10 3 cells were plated on to 2 mL Matek dishes and allowed to adhere overnight.The cells were treated with Spheroids were treated with media/methanol (90:10) solutions of curcumin, 1c and 2c to give a final concentration of 20 uM (0.5% methanol).After 24 h, the media was removed, cells were rinsed 3 times with PBS and imaged on a Leica TCS SP5 multiphoton microscope.

Preparation of spheroid samples for imaging
Spheroids were formed by plating 1.5 x10 4 cells onto 0.75% agarose coated 96 well plates and incubated without movement for 72 hrs in a humidified environment at 37 o C and 5% CO 2 .Spheroids were treated with media/methanol (90:10) solutions of curcumin, 1c, 2c, [RuCl 2 (tpa)]ClO 4 , and [CoCl 2 (tpa)]ClO 4 to give a final concentration of 20 uM (1% methanol).After 24 hrs, spheroids were transferred to a Matek dish, washed 3 times with PBS and imaged on a Leica TCS SP5 multiphoton microscope using a HC PL APO 20x/0.70IMM objective.The samples were excited with 800 nm light and the emission wavelengths collected between 540 -750 nm.

Fluorescence Lifetime Imaging
Fluorescence lifetime images were collected on a Leica TCS SP5 MP FLIM system.The system had a tunable Mai Tai Deep See multiphoton laser with a repetition rate of 80 mHz (Spectra-Physics) attached to a Leica DMI6000B-CS inverted microscope.Samples were illuminated with 800 nm light and emitted light was collected in the descanned internal FLIM detectors over the 540-750 nm range using a HC PL APO 20x/0.70IMM (glycerol) objective lens.The data was collected with the aid of the B&H SPCM software.Fluorescence lifetimes were determined using time correlated single photon counting (TCSPC) and analyzed with SPCImage software (version 3.1.0.0).The instrument response function was derived from the decay curve of a 0.5 % solution of Rose Bengal and the mono-exponential decay curve of a solution of FITC confirmed.512 x 512 images were collected and 4X binning applied for analysis to ensure greater than 100 000 photons per pixel were analyzed.A minimum of 10 x 4 second accumulation scans were taken for each sample.For cell free analysis, 50 μM solutions of compounds in methanol at pH 7.4 (acidified with concentrated HCl) were analysed.Analysis of fluorescence lifetimes in spheroids required individually fitted bi-exponential curves to regions of interest defined by the outer edge of the spheroid to obtain average χ 2 values closest to 1.A total of 9 spheroids from 3 independent experiments were analyzed.
Preparation of cell samples for XANES 4 x10 6 cells were seeded onto a 6 cm dish and allowed to adhere overnight.Cells were treated with a media/DMSO (90:10) solution of 2c (prepared immediately prior to dosing) to give a final concentration of 30 μM (1% DMSO).Following incubation for 4 h, the growth medium was removed, the cells trypsinised and 5 mL of PBS solution added.The cells were centrifuged at 2000 rpm for 4 min, the supernatant removed, the washing repeated and then aqueous ammonium acetate (5 mL, 100 mM) added.After further centrifuging, the supernatant was again removed and 70% ethanol solution (0.5 mL) added, before the samples were centrifuged once more.The ethanol was removed and the resulting cell pellet freeze-dried for two days.The experiment was performed in triplicate.XANES analysis of the cell pellets was performed by packing the samples into polycarbonate sample holders (8 x 3 x 3 mm) which were secured with Kapton tape.
XANES spectra XANES spectra were collected at the X-ray Absorption Spectroscopy beamline at the Australian Synchrotron, using a Si(III) channel cut monochromator.This synchrotron light source operates the storage ring at 3 GeV with a ring current of 200 mA.Energies were calibrated with cobalt foil with the first inflection point being assigned as 7,709.5 eV.XANES spectra were recorded in fluorescence mode at the cobalt Kedge at temperature of 10 K.The scans were averaged using weights based on the signal-to-noise ratios, with each spectrum checked individually before averaging.The spectra were averaged with Average 2.0 and were normalized with Athena using appropriate pre-edge and post-edge values.

Supporting References
(ESI) for Chemical Science This journal is © The Royal Society of Chemistry 2013

Figure S1
Figure S1 Increase in fluorescence emission over time of solutions of 1c in the presence of 10 equivalents of ascorbate (top) and glutathione (centre) and 2c in the presence of 10 equivalents of glutathione (bottom), (25 uM 1:25 MeOH/TRIS buffer, λ ex = 420 nm).

Table S1 E
pc and E pa values for all complexes in DMF vs. Fc/Fc + (mV).