2-Nitroimidazole-ruthenium polypyridyl complex as a new conjugate for cancer treatment and visualization

Article abstract of DOI:10.1016/j.jinorgbio.2014.02.001

A novel long-lifetime highly luminescent ruthenium polypyridyl complex containing 2-nitroimidazole moiety [Ru(dip)₂(bpy-2-nitroIm)]Cl ₂ (dip = 4,7-diphenyl-1,10-phenanthroline, bpy-2-nitroIm = 4-[3-(2-nitro-1H-imidazol-1-yl)propyl]-2,2′-bipyridine) has been designed cancer treatment and imaging. The luminescence properties of the synthesized compound strongly depend on the oxygen concentration. Under oxygen-free conditions quantum yield of luminescence and the average lifetime of emission were found to be 0.034 and 1.9 μs, respectively, which is ca. three times higher in comparison to values obtained in air-equilibrated solution. The binding properties of the investigated ruthenium complex to human serum albumin have been studied and the apparent binding constant for the formation of the protein-ruthenium adduct was determined to be 1.1 × 10⁵ M ^{- 1}. The quantum yield and the average lifetime of emission are greatly enhanced upon binding of ruthenium compound to the protein. The DNA binding studies revealed two distinguished binding modes which lead to a decrease in luminescence intensity of ruthenium complex up to 60% for [DNA]/[Ru] < 2, and enhancement of emission for [DNA]/[Ru] > 80. Preliminary biological studies confirmed fast and efficient accumulation of the ruthenium complex inside cells. Furthermore, the ruthenium complex was found to be relatively cytotoxic with LD₅₀ of 12 and 13 μM for A549 and CT26 cell lines, respectively, under normoxic conditions. The retention and cellular uptake of ruthenium complex is enhanced under hypoxic conditions and its LD ₅₀ decreases to 8 μM for A549 cell line.

Full text of DOI:10.1016/j.jinorgbio.2014.02.001

2-Nitroimidazole-ruthenium polypyridyl complex as a new conjugate for
cancer treatment and visualization
Olga Mazuryk, Monika Maciuszek, Graz˙yna Stochel, Franck Suzenet,
Małgorzata Brindell
PII:
DOI:
Reference:
S0162-0134(14)00040-3
doi: 10.1016/j.jinorgbio.2014.02.001
JIB 9467
To appear in:
Journal of Inorganic Biochemistry
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Revised date:
Accepted date:
15 July 2013
2 February 2014
2 February 2014
Please cite this article as: Olga Mazuryk, Monika Maciuszek, Graz˙yna Stochel, Franck
Suzenet, Malꢀgorzata Brindell, 2-Nitroimidazole-ruthenium polypyridyl complex as a new
conjugate for cancer treatment and visualization, Journal of Inorganic Biochemistry (2014),
doi: 10.1016/j.jinorgbio.2014.02.001
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ACCEPTED MANUSCRIPT
2-Nitroimidazole-ruthenium polypyridyl complex as a new
conjugate for cancer treatment and visualization
Olga Mazuryka,#, Monika Maciuszeka,b,#, Grażyna Stochela, Franck Suzenetb,*, Małgorzata
Brindella,*
a
Department of Inorganic Chemistry, Faculty of Chemistry, Jagiellonian University,
Ingardena 3, 30-060 Krakow, Poland
b Institut of Organic and Analytical Chemistry, University of Orléans, UMR-CNRS 7311, rue
de Chartres, BP 6759, 45067 Orléans Cedex 2, France
*Corresponding authors:
Dr. Małgorzata Brindell
Faculty of Chemistry
Jagiellonian University
Ingardena 3, 30-060 Krakow, Poland
brindell@chemia.uj.edu.pl
phone: +48126632221
fax: +48126340515
Dr. Franck Suzenet
Institut de Chimie Organique et Analytique
UMR-CNRS 7311, Université d’Orléans
rue de Chartres, 45067 Orléans, France
franck.suzenet@univ-orleans.fr
phone: +33238494580
fax: +33238417281
#Both authors contributed equally as the first co-authors.
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Abstract
A novel long-lifetime highly luminescent ruthenium polypyridyl complex containing 2-
nitroimidazole moiety [Ru(dip)2(bpy-2-nitroIm)]Cl2 (dip = 4,7-diphenyl-1,10-phenanthroline,
bpy-2-nitroIm = 4-[3-(2-nitro-1H-imidazol-1-yl)propyl]-2,2'-bipyridine) has been designed
cancer treatment and imaging. The luminescence properties of the synthesized compound
strongly depend on the oxygen concentration. Under oxygen-free conditions quantum yield of
luminescence and the average lifetime of emission were found to be 0.034 and 1.9 µs,
respectively, which is ca. three times higher in comparison to values obtained in air-
equilibrated solution. The binding properties of the investigated ruthenium complex to human
serum albumin have been studied and the apparent binding constant for the formation of the
protein-ruthenium adduct was determined to be 1.1 × 105 M–1. The quantum yield and the
average lifetime of emission are greatly enhanced upon binding of ruthenium compound to
the protein. The DNA binding studies revealed two distinguished binding modes which lead
to a decrease in luminescence intensity of ruthenium complex up to 60% for [DNA]/[Ru] < 2,
and enhancement of emission for [DNA]/[Ru] > 80. Preliminary biological studies confirmed
fast and efficient accumulation of the ruthenium complex inside cells. Furthermore, the
ruthenium complex was found to be relatively cytotoxic with LD50 of 12 and 13 µM for A549
and CT26 cell lines, respectively, under normoxic conditions. The retention and cellular
uptake of ruthenium complex is enhanced under hypoxic conditions and its LD50 decreases to
8 µM for A549 cell line.
Keywords: ruthenium polypyridyl complex, theranostic, cytotoxicity, optical imaging,
luminescence, DNA
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1. Introduction
Recently a new approach based on combining of diagnostic and therapeutic properties
in one compound or platform (in case of nanoparticles) so called theranostics has attracted
interest of researchers [1, 2]. Such strategy offers many advantages over classical one, among
others the real-time monitoring of the therapeutic effect which can help to optimize the
treatment schedule for each patient individually. Moreover, the visualization of drug
biodistribution and its accumulation allow for better protection of healthy tissues by the
controlling drug administration as well as using protective agents.
Polypyridyl ruthenium complexes are very interesting class of compounds which by a
proper designing can combine properties needed for the treatment of the cancer as well its
visualization. The unique optical features (intense and long lifetime luminescence strongly
depending on the molecular oxygen concentration, emission close to near-infrared) together
with favorable biological properties (ability to pass cellular membrane, reasonable solubility
in aqueous media) give rise to their application in an optical imaging. They can be applied as
probes not only for the fluorescence molecular tomography (FMT) but also for the
fluorescence lifetime imaging microscopy (FLIM), where the signal-to-background ratios can
be significantly improved by suppressing short-lived autofluorescence. Recently, a series of
phosphorescent ruthenium-complexes of the type [Ru(bpy)2L]2+, where L is phenanthroline
ligand modified by hydrophobic substituents (an alkyl side chain or an aromatic hydrocarbon)
have been employed for non-invasive imaging of physiological hypoxia in living organism
[5]. It is well-known, that state of hypoxia is a consequence of chaotic and poor vascular
organization in the tumor leading to lower efficiency in delivering of oxygen and nutrition.
Moreover, it is suggested that hypoxia causes resistance to standard therapies and promotes a
more malignant phenotype [6]. Therefore, the targeting of cancer cells by using the hypoxia
state as a unique feature of solid tumors seems to be very attractive [7].
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Lately several research groups have taken an advantage of photophysical properties of
polypyridyl ruthenium complexes and functionalized them with different moieties such as
porphyrins (photodynamic therapy) [8], coumarins (esterase sensing) [9], estradiols (targeting
estrogen receptor-α ER- α) [10], squalene (cellular imaging tool) [11] and short peptides
(targeting the nucleus) [12, 13]. The aim of our study was to modify of polypyridyl ruthenium
complexes to increase cellular accumulation, to obtain better selectivity toward hypoxic
tissues as well as to combine cytotoxic activity with imaging properties. Several recent studies
have shown that the ruthenium complexes comprising two 4,7-diphenyl-1,10-phenantroline
(dip) ligands exhibits better uptake by cells over complexes with simple bpy or phen ligands
[14, 15]. This feature encourages us to use [Ru(dip)2L]2+ platform for designing of new
complexes.
2-Nitroimidazoles are the group of bioreductive prodrugs which are developed toward
their application in diagnosis and therapy of hypoxic tissues characteristic for tumors [16].
Pimonidazole (-((2-nitroimidazol-1-yl)methyl)-1-piperidineethanol) has been commonly
applied for the detection and quantification of hypoxia [16, 17] and currently is studied as an
invasive hypoxia diagnostic tool for pancreatic tumor in a pre-operation treatment [18]. This
approach is limited by the requirements for biopsy of the tissue followed by the complex
immunohistochemical test. To overcome this problem the hypoxic tracer can be labeled with
radionuclides, the most often 18F for PET imaging [7]. Alternatively, 2-nitroimidazole moiety
can be attached to the another unit responsible for imaging. The first attempts to obtain the
near-infrared fluorescent probes for imaging of tumor hypoxia by combining of 2-
nitroimidazole moiety with NIR fluorophore (indocyanine green, tricarbocyanine) have been
recently reported and tested in vivo [23, 24].
In view of the recent findings we report the synthesis of a new agent for cancer
treatment and its visualization based on ruthenium polypyridyl complex and 2-nitroimidazole
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as depicted in Scheme 1. The synthesis, photophysical, protein/DNA-binding and biological
properties of [Ru(dip)2(bpy-2-nitroIm)]2+ are discussed in the context of its potential use in
anticancer therapy combine with visualization of cancer tissue.
Scheme 1
2. Materials and methods
2.1. Synthetic procedures
Materials and general procedures are described in supplementary information. 2,2’-
Bipyridyl-1-oxide (2), 4-nitro-2,2’-bipyridyl-1-oxide (3) and 4-bromo-2,2’-bipyridine (4) were
prepared according to published procedures [25].
2.1.1. 3-(2,2'-bipyridin-4-yl)prop-2-yn-1-ol (5)
In a sealed tube under argon atmosphere 4-bromo-2,2’-bipyridine (4, 0.05 g, 0.23
mmol) and propargyl alcohol (0.019 g, 0.35 mmol, 0.02 mL, 1.5 eq) were added to 2.5 mL of
anhydrous DME. To the reaction mixture 1.5 mL of triethylamine was added and the solution
was degassed. Finally Pd(PPh3)4 (0.0129 g, 0.011 mmol, 0.05eq) was added. The mixture was
heated to 120 °C for 3 h under microwave irradiations. After cooling down, solution was
filtrated on the celite. Filtrate was evaporated and crude product was dissolved in the small
amount of acetone. Then, under vigorous stirring diethyl ether and next hexane were added to
allow triphenylphosphine derivative to precipitate. Mixture was filtrated on a Büchner funnel
over a large plug of silica and washed with diethyl ether and hexane. The filtrate was
evaporated under reduced pressure to afford the final product as a yellowish solid in 56%
yield. IR (cm-1): 664, 787, 853, 905, 973, 993, 1034, 1088, 1213, 1391, 1461, 1538, 1566,
1581, 1601, 2850, 2914, 3031, 3217. 1H-NMR (400 MHz, CDCl3): δ/ppm = 2.19-2.12 (1H, t),
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4.53 (2H, d, J = 6.2 Hz), 7.28 (1H, dd, J = 5.0, 1.4Hz), 7.36 (1H, m) 7.87-7.79 (1H, m), 8.38
(1H, dd, J = 8.0, 1.0 Hz), 8.44 (1H, s), 8.63 (1H, d), 8.68(1H, dd, J = 3.5, 1.3 Hz); 13C NMR
(101 MHz, CDCl3) δ/ppm = 156.25, 155.41, 149.15, 149.23, 137.02, 131.78, 125.35, 124.03,
123.30, 121.17, 92.01, 83.38, 51.44. HRMS [M+H]+ m/z calculated for C13H11N2O: 211.2319;
found: 211.0868.
2.1.2. 4-(3-hydroxypropyl)-2,2’-bipyridine (6)
3-(2,2'-Bipyridin-4-yl)prop-2-yn-1-ol (5, 150 mg) was dissolved in 25 mL of ethanol
and the solution was degassed with argon. Pd/C (0.030 g) was added and reaction mixture was
bubbled with hydrogen. Reaction was stirred at room temperature for 3 h under H2
atmosphere. The solution was filtrated on celite and concentrated under reduced pressure to
obtain an oily residue. Purification through a short column chromatography on silica gel
afforded the desired product as a creamy oil in 98% yield. 1H NMR (400 MHz, CDCl3) δ 8.67
(d, 1H), 8.56 (dd, J = 4.8, 2.7 Hz, 1H), 8.39 (d, J = 8.0 Hz, 1H), 8.24 (s, 1H), 7.82 (t, J = 6.8
Hz, 1H), 7.34 – 7.27 (m, 1H), 7.16 (s, 1H), 3.69 (s, 2H), 2.81 (t, J = 8.9 Hz, 2H), 2.46 (s, 1H),
13
2.02 – 1.91 (p, J = 9.3 Hz, 2H). C-NMR (400 MHz, CDCl3): δ/ppm = 156.22, 155.99,
152.06, 149.03, 137.04, 127.81, 123.71, 121.36, 121.31, 61.58, 33.03, 31.59. HRMS [M+H]+
m/z calculated for C13H15N2O: 215.2716; found: 215.1180.
2.1.3. 4-(3-bromopropyl)-2,2'-bipyridine (7)
130 mg of 4-(3-hydroxypropyl)-2,2’-bipyridine (6) was dissolved in 2.4 mL of a 1:1
mixture of HBr (33% in acetic acid) and water. The reaction mixture was then heated to reflux
overnight. After cooling down to room temperature, a saturated aqueous solution of sodium
bicarbonate was added until the pH reached 5-6. The aqueous phase was extracted three times
with dichloromethane (3 × 10 mL). Organic phases were dried over MgSO4, filtered and
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evaporated under reduce pressure. Purification on a short aluminia column with
DCM/Petroleum ether (8/2) as eluent afforded compound 7 as a pink-orange oil in 50% yield.
1H-NMR (400 MHz, CDCl3): δ/ppm = 8.67 (1H, dd, J 4.8, 8 Hz), 8.56 (1H, dd, J 10.9, 5.0
Hz), 8.41-8.35 (1H, m), 8.26 (1H, s), 7.85-7.75 (1H, m), 7.30 (1H, dt, J =1 0.9, 4.8Hz), 7.16
13
(1H, d, J = 4.9 Hz), 3.42 (2H, t, J = 6.7 Hz), 2.87 (2H, t, J = 7.5 Hz), 2.31-2.20 (2H, m). C
NMR (101 MHz, CDCl3) δ 156.28, 156.09, 150.57, 149.32, 149.11, 136.96, 124.05, 123.75,
121.24, 121.07, 33.61, 33.00, 32.62. HRMS [M+H]+ m/z calculated for C13H1479BrN2: 277.08;
found: 277.0338.
2.1.4. 4-[3-(2-nitro-1H-imidazol-1-yl)propyl]-2,2'-bipyridine (8, bpy-2-nitroIm)
2-Nitroimidazole (0.033 g, 1 eq), potassium carbonate (1 eq) and 4-[3-(bromopropyl]-
2,2'-bipyridine (7, 0.08g) were dissolved in 2 mL of dry DMF. The mixture was heated at
110°C for 3 hours under argon atmosphere. After cooling down to room temperature, most of
the DMF was evaporated under the reduced pressure and the residue was dissolved in AcOEt
(10 mL) and washed 3 times with water (3 x 10mL). Organic layer was dried over magnesium
sulfate, filtrated and evaporated under reduced pressure. Crude product was washed with ice-
cold n-pentane and filtrated on Millipore apparatus. The resulting pinkish solid was isolated in
35% yield and used without any further purification. The melting point was found in the range
104-106 °C. IR (cm-1): 3123, 1599, 1583, 1557, 1456, 1284, 1244, 1179, 1122, 1092, 991,
1
856, 759, 636. H NMR (400 MHz, CDCl3) δ 8.67 (dd, J = 3.9, 0.8 Hz, 1H), 8.60 (t, J = 4.2
Hz, 1H), 8.41 (d, J = 8.0 Hz, 1H), 8.25 (s, 1H), 7.82 (td, J = 7.8, 1.8 Hz, 1H), 7.32 (ddd, J =
7.4, 4.7, 1.1 Hz, 1H), 7.15 (s, 1H), 7.14 (s, 1H), 7.06 (d, J = 3.1 Hz, 1H), 4.46 (dd, J = 12.9,
5.6 Hz, 2H), 2.84 – 2.74 (t, 2H), 2.36 – 2.24 (p, 2H). 13C NMR (101 MHz, CDCl3) δ 156.47,
155.86, 149.72, 149.53, 149.12, 137.03, 128.57, 125.77, 123.90, 123.60, 121.26, 120.72,
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49.60, 32.07, 30.78. HRMS [M+H]+ m/z calculated for C16H16N5O2: 310.3313; found:
310.1297.
2.1.5. [Ru(dip)2(bpy-2-nitroIm)]Cl2
The synthetic procedure was adapted from the literature [26]. In the round-bottomed
flask Ru(dip)2Cl2DMFH2O (52 mg, 0.06 mmol, 0.92 eq, synthesized according to literature
procedure[26]), NaOAc (13.5 mg, 0.1625 mmol, 2.5 eq) and bpy-2-nitroIm (20 mg, 0.065
mmol, 1 eq) were dissolved in a methanol/water mixture (25 mL, 4/1 v/v). The solution was
heated to reflux for 31 h. The reaction mixture was then cooled to rt, and concentrated to one-
third by evaporation of the solvent. Brine was added to the mixture, and the resulting
precipitate was filtered. The solid was dissolved in methanol and the solution was filtrated.
Filtrate was evaporated and residue was dissolved in a small volume of dichloromethane.
Toluene was then added slowly to the filtrate and a new precipitation occurred. The product
was filtrated and isolated as an orange solid. The resulting filtrate was treated as the previous
one and more product was obtained after the solution was left standing at 4 °C for a few days.
Solids were combined and dried under vacuum to give the final product (50 mg, 72%) as a
microcrystalline dark orange powder. IR (cm-1) = 3360, 3055, 1619, 1556, 1478, 1440, 1415,
1357, 1080, 834, 766, 737, 702 HRMS [M]2+ m/z calculated for [C64H47N9O2Ru]2+ 537.6451
found: 537.6450 (see Fig. S1) The purity of the obtained compound was further checked by
TLC (all types mentioned in the general part) as well as HPLC analysis (see Fig. S2).
2.2. Spectroscopic measurements
UV–visible absorption spectra were recorded on a Perkin Elmer Lambda 35
spectrophotometer using quartz cells with a 1-cm optical pathlength. Optical properties were
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examined in aqueous solutions containing DMSO (<0.04% v/v). Luminescence measurements
were performed on a spectrofluorimeter Perkin Elmer LS55 in a quartz cell with a 1-cm
pathlength. The spectra were recorded at the room temperature in aqueous solution containing
small amount of DMSO (<0.008% v/v). The emission spectra were recorded between 470 and
860 nm upon excitation at 463 nm. The average of three scans was subjected to smoothing.
For determination of the quantum yield of luminescence (Φ), aqueous solutions of
[Ru(bpy)3]2+ with a small amount of DMSO (<0.008% v/v) were used as standards (Φ = 0.028
[27] and 0.042 [28] for air-equilibrium and deoxygenated conditions, respectively). The
spectra were recorded at the concentration less than 0.05 absorbance unit at the excitation
wavelength. Values were calculated according to the following equation [4]:
2
Φ = Φref × [Aref/A] × [I/Iref] × [n2/nref ]
where I is the integrated intensity of luminescence, A is the optical density, and n is the
refractive index, ref refers to the values for reference. The mean value from minimum three
independent experiments was calculated.
The luminescence lifetime measurements were performed with a single photon
counting technique using Fluorolog-3, Horiba Jobin Yvon. The excitation wavelength was set
at 464 nm (NanoLed Diodes) and the average lifetime of luminescence was monitored at 621
nm. Luminescence decays were collected with 1000 counts in the peak. The instrument
response functions were measured using a light scattering solution of Ludox (colloidal silica,
Sigma-Aldrich). Experiments were conducted at room temperature. The DAS6 software
(HORIBA Scientific) was used for deconvolution of the obtained decays and for calculation
of the lifetime values. The quality of the fit was judged by the χ2 parameter (the goodness of
fit evaluation). One-exponential fit was determined to be an optimal description of the
obtained results for the free compound; but after binding with protein and DNA single-
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exponential decay was not efficient and two-exponential fit was modeled (similar as in
reference [29]).
2.3. Protein and DNA-binding studies
The protein solution was prepared by dissolving of human serum albumin (HSA) in
water and its concentration was determined spectrophotometrically from the molar
absorptivity of 4.4 × 104 cm1 M1 at 280 nm [30]. The quantum yield of luminescence for
ruthenium complex in the present of HSA (1 μM) was measured using the same procedure as
described for ruthenium complex alone.
Calf thymus deoxyribonucleic acid was purchased from Sigma-Aldrich and its stock
solution was prepared by dissolving of solid DNA in water. DNA concentrations per
nucleotide were determined by absorption spectroscopy using the molar absorption coefficient
of 6600 M–1cm–1 at the wavelength of 260 nm. DNA binding experiments were performed in
0.05 M Tris/HCl buffer (pH 7.4) at 37 °C. The emission titration studies were performed by
using fixed concentration of ruthenium compound (3 µM). The DNA aliquots were added and
after 5 min of incubation fluorescence spectra upon excitation of 463 nm were measured. The
average of three scans was subjected to smoothing and the fluorescence intensities were
corrected due to dilution effects.
2.4. Biological studies
For biological evaluation murine colon carcinoma cell line (CT26) as well as human
lung adenocarcinoma epithelial cell line (A549) were used. Cells were cultured in DMEM
medium supplemented with ferine serum (10%) and antibiotics (1%) – penicillin (100
units/mL) and streptomycin (100 μg/mL) at atmosphere of 5% CO2 at 37 °C. For hypoxia
treatments, cells were placed in a humidified atmosphere containing 94% N2, 5% CO2 and 1%
O2 gas mixture in hypoxic station Whitley H35. Cells and medium intended for hypoxic
experiments were preincubated at hypoxic chamber for 24 h. The evaluation of accumulation
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of [Ru(dip)2(bpy-2-nitroIm)]Cl2 in CT26 cells under normoxic conditions was conducted
using 96 wells plates with the seeding density of cell at 30 000 cells per well. Cells were
cultivated for 2 days, then the medium was washed out and dissolved ruthenium compound
was added into the wells. [Ru(dip)2(bpy-2-nitroIm)]Cl2 was dissolved in DMSO and diluted
in PBS with Ca2+/Mg2+ to the required concentration (0-50 × 10-6 M) immediately prior to
addition into the wells. The PBS buffer was used to avoid interaction of the ruthenium
complex with medium ingredients due to a high lipophilicity of the compound. The final
DMSO concentration was kept at 0.5% v/v level, since this amount of DMSO does not
influence cells viability (LD50 for aqueous solution of DMSO was found to be of 5 μM,
results not shown). Compound was incubated with the cells for 4 and 24 hours and then was
washed out. Cells were washed with PBS buffer (with Ca2+/Mg2+) and were kept in PBS for
further studies. The luminescence measurements were recorded using Infinite 200 microplate
reader (Tecan) with 463 nm as an excitation wavelength and 621 nm as an emission
wavelength. Each well was measured at 25 points and the mean value was calculated.
Cellular uptake for A549 cells under both normoxic and hypoxic conditions was
monitored by flow cytometry. A549 cells were seeded on a 24 well plates with a density
35 000 cells per well. 24 h after the seeding [Ru(dip)2(bpy-NitroIm)]2+ and [Ru(dip)2(bpy)]2+
were added at 2, 4 and 6 µM concentrations. Cells were incubated in medium without and
with serum (2% FBS) under hypoxic and normoxic conditions for 24 h. After incubation, cells
were washed twice with PBS, trypsinisized and analyzed by LSRII cytometer (Becton
Dickinson) with λext at 488 nm and λem at 610 ± 10 nm. Experiment was analyzed using BD
FACSDiva Software. 10 000 singular gated events were collected. Moreover, the ability of
ruthenium compounds to retain in cells under hypoxic conditions was assessed also after 24 h
of cell cultivation in medium not supplemented with these complexes. After 24 h of
incubation with [Ru(dip)2(bpy-NitroIm)]2+ and [Ru(dip)2(bpy)]2+ (2, 4 and 6 µM, diluted in
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DMEM with 2 % FBS), cells were washed twice with PBS, trypsinisized and the first half of
cells was analyzed by flow cytometry. Another half was seeded on the new well, fresh
medium was applied, and cells remained under hypoxic conditions for another 24 h, followed
by trypsinisization and analyzing by flow cytometry.
The cytotoxicity studies on CT26 cells were performed using MTT test [31]. Cells
were cultivated in 96 wells plates for a day prior to addition of the investigated compounds.
After 4 or 24 h incubation of the CT26 cells either with ligand alone (bpy-2-nitroIm, 8) or the
investigated ruthenium complex in the PBS solution were washed out and subsequently 200
μl of fresh medium was applied to allow cells repair existed damages. After 24 h, the medium
was replaced with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide salt (MTT)
dissolved in DMEM (0.5 mg/ml) to determine the cell viability. After 3 h of incubation the
MTT solution was washed out and crystals were dissolved in methanol:DMSO at volume
ratio of 1:1. Then the absorbance value at 565 nm was recorded using Infinite 200 microplate
reader (Tecan). Three series of minimum two plates were performed and SD of a mean value
was calculated.
The evaluation of the cytotoxicity of [Ru(dip)2(bpy-2-nitroIm)]+ on A549 cells was
conducted using Alamar Blue assay. The ruthenium complex dissolved in medium (DMSO
0.1 %) was incubated with the cells for 24 h under hypoxia or normoxia conditions and then
was washed out. Cells were washed with PBS buffer (with Ca2+/Mg2+) and resazurin sodium
salt (25 10-6 M) diluted in PBS was added to the wells. The fluorescence measurements were
recorded using Infinite 200 microplate reader (Tecan) with λext at 565 nm and λem at 605 nm.
Each well was measured at 25 points and the mean value was calculated. Experiments were
repeated three times in triplicates. The viability was calculated with regard to the untreated
cells control. The LC50 values were determined using Hill equation (Origin 9.0) [31].
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For cell imaging Olympus fluorescence microscope IX51 equipped with XC10 camera
was used. Cells were seeded into round dishes with density 300 000 cells/dish. After
cultivation for 24 h cells were incubated with different concentration of [Ru(dip)2(bpy-2-
nitroIm)]Cl2 (0, 0.5, 1, 5 and 10 μM). The ruthenium complex was dissolved in PBS buffer
with Ca2+/Mg2+ with small amount of DMSO (0.5% v/v). After incubation (1 h, 37 °C)
ruthenium compound was washed out and cells were washed with PBS three times. Next,
keeping cells in PBS buffer, images of [Ru(dip)2(bpy-2-nitroIm)]Cl2 embedded in CT26 cells
were taken using 470-495 nm excitation filter.
3. Results and discussion
3.1. Synthesis
The synthesized complex is composed of ruthenium polypyridyl moiety as the
luminescent dye with the 2-nitroimidazole moiety attached with alkyl spacer to the bipyridyl
ligand as a potential hypoxia sensitive unit. The overview of the synthetic pathway is
presented in Scheme 2. 4-Bromo-2,2’-bipyridine (4) was obtained from commercially
available 2,2’-bipyridine in a three steps sequence [25, 32, 33]. A Sonogashira cross-coupling
reaction with propargylic alcohol under microwaves activation afforded the alkyne
intermediate (5). After hydrogenation and reaction with hydrobromic acid, the
bromopropylbipyridine (7) was reacted with 2-nitroimidazole to afford the desired bpy-2-
nitroIm (8). The Ru(dip)2Cl2 synthesized according to published procedure [26] was reacted
with (8) in methanol/water mixture (4/1 v/v) following literature method developed for the
substitution with monocarboxylic bipyridyl ligand [26]. Purity and identity of the final
compound [Ru(dip)2(bpy-2-nitroIm)]Cl2 was confirmed by the presence of single peak in
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HPLC chromatogram and in high resolution mass spectrum with m/z = 537.6450 for
[C6H47N9O2Ru]2+ (see Supplementary material Fig. S1 and S2).
Scheme 2
3.2. Photophysical characterization
Due to quite satisfactory solubility of the compound in water, all photophysical
properties were measured in aqueous solution with a small addition of DMSO (<0.04%). The
absorption spectrum of [Ru(dip)2(bpy-2-nitroIm)]2+ is shown in Fig. 1. The narrow and
intense band at 278 nm (ε = 88 400 ± 100 M-1cm-1) have been assigned to a spin allowed 1LC
(1π→π* bpy-centered) transition [34]. The shoulder at 314 nm (ε = 25 900 ± 100 M-1cm-1)
1
originates from a LC transition of phenanthroline moiety. At visible region of spectrum two
bands at 433 nm (ε = 19 100 ± 100 M-1cm-1) and 463 nm (ε = 19 700 ± 100 M-1cm-1) are
1
attributed to the spin allowed MLCT d→π* transitions (assignment based on [34]). New
synthesized ruthenium complex was found to be luminescent in aqueous solution with the
emission maximum occurring at 621 nm (excitation at 463 nm). A very large Stoke’s shift of
150 nm is observed, which is not encounter for NIR organic dyes proposed as selective
fluorescent probe for hypoxia [23, 24].
Fig. 1.
The quantum yield of luminescence of the studied complex was determined in aqueous
solution at room temperature both in air equilibrated (Φair = 0.010 ± 0.0004) and
deoxygenated solution (ΦAr = 0.034 ± 0.0004). The increased quantum yield in oxygen-free
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solution is an important feature since the synthesized complex is designed for imaging of
cancer tissues which very often exhibit lower oxygen concentration. Average emission
lifetime for [Ru(dip)2(bpy-2-nitroIm)]2+ in air-equilibrated water solution was found to be 700
± 10 ns (660 ± 10 in TRIS buffer, pH 7.4) suggesting that the emission takes place from
triplet sate, typically found for these type of ruthenium complexes. In the oxygen-free solution
emission lifetime dramatically increases up to 1.9 ± 0.1 µs, that makes [Ru(dip)2(bpy-2-
nitroIm)]2+ an interesting candidate for luminescence reporter for monitoring the oxygen
concentration using fluorescence lifetime imaging microscopy. Moreover, the compound is
very stable in aqueous solution as confirmed by the absence of changes in the absorption and
emission spectra during at least 24 h. Sufficient solubility in water, high molar absorptivity
together with the increased yield of luminescence and average lifetime in oxygen-free
solution create a strong opportunity to use of [Ru(dip)2(bpy-2-nitroIm)]2+ as luminescent
probe for imaging cancer cells.
3.3. Reactivity towards biomacromolecules (human serum albumin and calf thymus DNA)
3.3.1. Enhancement of ruthenium complex luminescence in the presence of human serum
albumin
The interaction of [Ru(dip)2(bpy-2-nitroIm)]2+ with human serum albumin (HSA) was
found to be a moderate with an association constant for the formation of HSA-Ru-complex
adducts ca. 105 M-1 (more details concerning this experiment are available in Supplementary
Information). This correlates very well with association constants calculated for other
ruthenium polypyridyl complexes [35]. The binding of [Ru(dip)2(bpy-2-nitroIm)]2+ to HSA
also effect its luminescence spectra leading to gradual increase of the emission intensity and
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shift of the maximum toward shorter wavelengths (Fig. S4, experimental details are described
in Supplementary Information). The observed increase for both the luminescence quantum
yield as well as the average lifetime of emission is dependent on protein concentration as
shown in Fig. 2. It is very likely that the interaction of [Ru(dip)2(bpy-2-nitroIm)]2+ with HSA
leads to spatial separation of nitroimidazole moiety from Ru center by protein scaffold. In this
way protein can prevent from quenching its luminescence by this residue. It can be assumed
that if such interaction occurs inside the cells also with another proteins, the enhancement of
the luminescence should be manifested. Taking into account increased amount of the proteins
inside cells (50 – 400 mg/ml) [36] this feature is very interesting in the context of application
of [Ru(dip)2(bpy-2-nitroIm)]2+ as potential cellular dye. Previously, reported increased
photoluminescence lifetimes for ruthenium compound [Ru(bpy)2(5-iodoacetamido-1,10-
phenantroline)](PF6)2 from 490 ns for free complex to 809 and 895 ns after covalent binding
with human serum albumin and human immunoglobulin G, respectively, have been also
reported [29].
Fig. 2.
3.3.2. Unusual effect of calf thymus DNA on luminescence of ruthenium complex
DNA-binding ability of [Ru(dip)2(bpy-2-nitroIm)]2+ is rather moderate (ca. 5.0 × 105
M–1, for details see Supplementary Information) in comparison to classical intercalators
[Ru(bpy)2(dppz)]2+ (3.1 × 106 M–1) or [Ru(phen)2(dppz)]2+ (5.1 × 106 M–1) [37]. These and the
other typical intercalators after addition of the DNA show a substantial increase of
luminescence [38] which can arise either from the protection of the ruthenium complex from
quenching by water molecules by the hydrophobic environment inside the DNA or by the
reduction of the complex mobility leading to the decrease of the vibration mode of relaxation
[43]. In contrast for the studied ruthenium complex the small excess of the DNA ([DNA]/[Ru]
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≤ 10) results in decreases of luminescence intensity down to 65 % (Fig. S7A). Slight decrease
of the average lifetime of luminescence is also observed from τunbound = 660 ± 10 ns to a τbound
= 540 ± 10 ns for [DNA]/[Ru] ratio = 10. The Stern-Volmer analysis shows that Ksv equals
1.96 × 104 M-1, and taking into account the lifetime of luminescence for ruthenium complex
(τ0 = 660 ± 10 ns in TRIS/HCl buffer), the bimolecular quenching constant kq is 3.0 × 1010 M–
1
s-1. Such value suggests that the observed quenching comes probably from diffusion
interaction between the DNA and ruthenium complex and/or weak binding interaction
between molecules [4]. This weak binding is accompanying by electrostatic interaction as
confirmed by the observed quicker quenching of luminescence in water than in the buffer
solution for [DNA]/[Ru] < 10. The presence of surface interaction is typically found for such
systems since ruthenium complexes possess positive charge (2+) [44].
Addition of higher excess of the DNA up to [DNA]/[Ru] ratio of 80 does not cause
any changes in luminescence spectra of the ruthenium complex. However, further increased in
the DNA concentration leads to recovery of the luminescent properties by ruthenium complex
and even the enhancement of the emission at large DNA excess (Fig. S7B). A small
hypsochromic shift of emission maximum ca. 2 nm is observed. Increased the average
lifetime of luminescence is also detected from τunbound = 660 ± 10 ns to a τbound = 1.24 ± 0.02
µs for [DNA]/[Ru] ratio = 200. The increased in ruthenium complex luminescence and
average lifetime of luminescence is observed only at very high concentration of the DNA,
unlike as for other intercalative and major groove binding ruthenium complexes for which
[DNA]/[Ru] ratio of 1 is usually enough for the substantial luminescence intensification [38,
41, 45, 46]. Interestingly, ruthenium compound in the presence of high concentration of the
DNA promotes the DNA condensation possibly after its structure distortion. This type of
interaction is often observed for the polyamines interacting with DNA [42]. The packing of
the DNA around the ruthenium complex changes its environment into more rigid and less
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polar one. Entrapment of ruthenium complex in such matrix results in the hypsochromic shift
of emission peak as well as the increase of luminescence intensity. Similar photophysical
behavior is observed for the [Ru(bpy)3]2+ assembled into sol-gel materials [47]. The
aggregation of the complex on the DNA template has been reported for mixed ligand
ruthenium(II) complexes with 5,6-dimethyl-1,10-phenantroline [48].
Ruthenium(II) complexes with polypyridyl ligands can interact with the DNA in
several noncovalent modes such as electrostatic binding, groove binding or intercalation [49].
[Ru(dip)2(bpy-2-nitroIm)]2+ interacts with DNA at least in two different ways. On small
excess of the DNA the ruthenium complex exhibits tight interaction with helix, probably by
partial intercalation or groove binding, as it is confirmed by UV-visible and luminescence
titration and ethidium bromide competitive binding experiment (more details concerning these
experiments are available in Supplementary Information). At higher excess of the DNA the
additional interaction takes place (probably strong hydrophobic interaction with the DNA
surface) as it is confirmed by the increased intensity of luminescence of ruthenium compound.
The classical intercalation binding mode can be excluded as shown by the thermal
denaturation studies (compare the Supplementary Information).
3.5. Preliminary cellular studies – normoxic conditions
Evaluation of biological properties of [Ru(dip)2(bpy-2-nitroIm)]2+ was conducted on
CT26 mouse carcinoma cells. The uptake of this complex was investigated by taking an
advantage of its luminescence properties using microplates reader for the quantification of its
internalization as well as microscopy imaging for the assessment of its staining properties. It
was found that [Ru(dip)2(bpy-2-nitroIm)]2+ quickly and easily accumulates in cancer cells.
Even 1 h of incubation is sufficient to observe an efficient cell luminescence. The lengthening
of incubation time from 4 to 24 h does not intensify the emission properties of the cells as
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shown in Fig. 3. A linear correlation between the amount of incubated compound and the
level of luminescence expressed by the cells observed in the concentration range 1-50 M
suggests that [Ru(dip)2(bpy-2-nitroIm)]2+ can easily and efficiently accumulate in the cells. At
low concentration of dye (1 M ) the luminescence intensification relative to the blank has a
value of ca. 20 for both incubation times, which is quite promising for its application in
optical imaging. This value is at least two times higher compare to the compounds of type
[Ru(dip)2(L)]2+ (where L is CH3bpyCOOH, bpy(COOH)2, dppz) [14, 50]. The presence of dip
ligands facilitate the uptake of the complex, probably due to their lipophilicity, which helps in
the passive crossing of the cell membrane.[14, 50] The detailed mechanism of uptake for
these type of complexes is poorly understand and both the passive and active transports are
suggested [50-52].
Fig. 3.
The excellent luminescent properties were further confirmed by the microscopy
fluorescence imaging performed on the CT26 cells incubated with [Ru(dip)2(bpy-2-
nitroIm)]2+ for 1 h (Fig. 4A). At concentration of 10 µM the compound is transported inside
the cells and stains quite homogenously the cytoplasm as well as different organelles, which is
seen by the local increase of luminescence intensity. These results are in good correlation with
the observed increased in luminescence intensity of ruthenium compound after protein
binding. Weak labeling in the nucleus compartment is observed, which can arise either from
the weak interaction between ruthenium complex and nucleus DNA or the quenching of the
ruthenium complex luminescence by intercalating. Lowering the complex concentration (5
µM) leads to staining only selected cells while further decrease of [Ru(dip)2(bpy-2-nitroIm)]2+
down to 1 µM causes differentiation in its accumulation in the cell. However the detailed
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evaluation of its distribution within the cell is not possible due to too low magnification. The
recent data for similar type of complexes ([Ru(dip)2(L)]2+, where L is CH3bpyCOOH,
bpy(COOH)2, dppz) [14, 50] have also shown that nucleus compartment is rather weakly
labelled.
Fig. 4.
The cytotoxic effect of the synthesized compound was tested using the standard MTT
test. The [Ru(dip)2(bpy-2-nitroIm)]2+ was found to be quite cytotoxic with LD50 = 13 ± 3 µM
measured after 4 h of incubation with cells (Fig. 5). The increase of exposure time up to 24 h
does not influence the cytotoxicity, and this correlates very well with the observed lack of
changes in the luminescence level. The nitroimidazole moiety alone exhibit rather weak
toxicity (Fig. 5), therefore it can be assumed that the antiproliferative effect arise from
ruthenium residue. The application of the studied compound as a dye in optical imaging is
therefore limited to the concentration of 1 M and lower, for which the growth inhibition is
marginal. However at higher doses [Ru(dip)2(bpy-2-nitroIm)]2+ could be utilized as a
cytotoxic agent enabling visualization of the field of action.
Fig. 5.
3.6. Preliminary in vitro studies under hypoxic conditions
Behavior of [Ru(dip)2(bpy-NitroIm)]2+ under hypoxic in relation to normoxic
conditions was studied using human lung adenocarcinoma epithelial (A549) cell line. This
cell line was selected because it is known to express nitroreductase[53], enzyme which can
reduced the nitroimidazole moiety. The studied ruthenium complex exhibits dose-dependent
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growth inhibitory effect on tested A549 cell line. The cytotoxicity of [Ru(dip)2(bpy-
NitroIm)]2+ increased clearly under hypoxic conditions: LC50 decreased from 11.8 ± 1.2 µM
under normoxic conditions to 7.7 ± 0.4 µM under 1% of oxygen. These results can be
explained in terms of higher accumulation of ruthenium compound under hypoxic conditions
or the involvement of additional mechanism of toxicity triggered by hypoxia.
The cellular uptake of [Ru(dip)2(bpy-NitroIm)]2+ was monitored directly by following
the luminescent signal of treated cells using flow cytometry. It is known, that the stress
triggered by hypoxic conditions results in worse growth as well as slower metabolism of cells
compare to those under normoxia. Therefore, in order to appropriate evaluate the change in
the uptake under both conditions, structurally similar compound [Ru(dip)2(bpy)]2+, was used
as a reference. It is presume, that the uptake of [Ru(dip)2(bpy)]2+ is not directly dependent on
hypoxic/normoxic conditions, so its accumulation (under normoxia and hypoxia) was applied
as a base level of the uptake under corresponding conditions. The relative intensity of
luminescence of cells treated with [Ru(dip)2(bpy-NitroIm)]2+ was calculated as a percentage
of the luminescence intensity of cells incubated with [Ru(dip)2(bpy)]2+ at 2 µM concentration
under the same conditions. As shown in Fig. 6, the relative luminescence intensity of cells
incubated with [Ru(dip)2(bpy-NitroIm)]2+ under hypoxic conditions is always higher than for
cells incubated under normoxic conditions. The difference is more pronounce with increased
concentration of the studied compound. When studies are carried out in medium containing
serum still the uptake of [Ru(dip)2(bpy-NitroIm)]2+ is higher under hypoxia, however a
distinct decrease in accumulation of ruthenium complex is observed regardless of applied
conditions. This points to formation of adducts between ruthenium complex and serum
components (most probably albumin) which are not accessible for cells and therefore the
accumulation of [Ru(dip)2(bpy-NitroIm)]2+ becomes lower.
Fig. 6.
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To confirm the ability of new synthesized ruthenium complex to be “trapped” inside
the cells under hypoxic conditions, the intensity of luminescence of treated cells was
measured after one day of growth in ruthenium free medium. Retention factor was presented
as a percentage of cells luminescence intensity compared to an original luminescence
intensity of the cells measured directly after 24 h of the incubation with ruthenium complex.
Results were compared with retention ability of [Ru(dip)2(bpy)]2+ for which the uptake and
the loss is not expected to be related to hypoxic conditions. As shown in Table 1
[Ru(dip)2(bpy-NitroIm)]2+ preserves its luminescence in the cells up to 83–89% and this level
is ca. 30% higher than found for [Ru(dip)2(bpy)]2+. The main difference between these two
complexes arises from the presence of nitroimidazole moiety. Therefore, one can speculate
that [Ru(dip)2(bpy-NitroIm)]2+ can be trapped inside the cells due to reduction of nitro group
follow by its binding to biomolecules under hypoxic conditions.
Table 1.
4. Conclusions
The photophysical and biological properties of [Ru(dip)2(bpy-2-nitroIm)]2+ indicate
that this compound has potential to be applied as optical imaging probe and cytotoxic agent.
Preliminary results have shown that this compound is more toxic towards hypoxic tissues,
exhibits higher accumulation inside hypoxic cells, which combined with higher luminescence
parameters (quantum yield, luminescence lifetime) in deoxygenated atmosphere, makes
reasonable assumption that it can be applied for detection of hypoxic tissues inside body. Our
future work will focus on in vitro/in vivo studies toward verification its potential as a hypoxia
marker. Such achievement would be of particular benefit since the assessment of hypoxia
state is one of the most important parameter needed for estimation of cancer progress and
choosing the appropriate treatment.
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This dye can be also regarded as an alternative for pimonidazole, a currently exploring
hypoxia marker, which administrated prior to surgery can help in identification of lower
oxygen content [18]. As already mentioned, the visualization procedure involves application
of a complicated immunochemical test, while for the studied compound irradiation with an
appropriate light beam (around 450 nm) would most probably be sufficient. The intra-
operative multispectral system has been already successfully tested for clinical use for folate
and fluoresceine isothiocyanate conjugate [54]. Combination of two methods (fluorescence
imaging during cytoreductive procedure) allowed 5-fold increase of tumor deposits detected
by surgeons as well as making possible the excision of tumors of size < 1 mm. Using other
types of optical probes, for example based on ruthenium complexes, could allow for
application of these techniques in a wider range of surgery procedures.
5. Abbreviations
bpy
2,2’-bipyridine
bpy-2-nitroIm
DMEM
dip
4-[3-(2-nitro-1H-imidazol-1-yl)propyl]-2,2'-bipyridine
Dulbecco's modified Eagle's medium
4,7-diphenyl-1,10-phenanthroline
dimethoxyethane
DME
DMF
dppz
N, N-dimethylformamide
dipyrido[3,2-a:2’,3’-c]phenazine
ethidium bromide
EB
HSA
human serum albumin
HRMS
LC
high resolution mass spectrometry
liquid chromatography
LD50
lethal dose, 50%
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MLCT
MTT
NIR
metal to ligand charge transfer
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide salt
near infrared
PBS
Phosphate buffered saline
PET
positron emission tomography
1,10-phenanthroline
phen
THF
tetrahydrofuran
Acknowledgments
Financial support from the National Science Center (grant no. N N204 247340) is
acknowledged. The research was carried out with equipment purchased with financial support
from the European Regional Development Fund within the framework of the Polish
Innovation Economy Operational Program (contract no. POIG.0 2.01.00-12-0 23/08). O.M.
acknowledges the financial support from the project Interdisciplinary PhD Studies "Molecular
sciences for medicine" (co-financed by the European Social Fund within the Human Capital
Operational Programme). M.M thanks for support from Bourses du Gouvernement Français.
We thank Prof. A. Józkowicz for the access to hypoxia station.
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Schemes and Figures Captions
Scheme 1. [Ru(dip)2(bpy-2-nitroIm)]2+
Scheme 2. The synthetic pathway for the bpy-2-nitroIm (8) ligand.
Fig. 1. Absorption (A) and emission spectra (E) (excitation at 463 nm) of [Ru(dip)2(bpy-2-
nitroIm)]2+ in H2O.
Fig. 2. Luminescence quantum yield (■) and average lifetime (●) of ruthenium complex for
different [HSA]/[Ru] ratios. Experimental conditions: [HSA] = 1 μM, [Ru]= 1-3 μM; PBS pH
7.4.
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Fig. 3. Luminescence intensity of seeded CT26 cells measured after incubation with
[Ru(dip)2(bpy-2-nitroIm)]2+ during 4 () and 24 h () monitored at 621 nm upon excitation
at 463 nm, [CT26] = 30 × 103 cell/well.
Fig. 4. Fluorescent (A) and the corresponding bright-field (B) microscopic images of CT26
cells treated with [Ru(dip)2(bpy-2-nitroIm)]2+. Experimental conditions: [CT26] = 300 × 103
cell/well; [Ru] = 10 × 10–6 M.
Fig. 5. Cytotoxic effect of [Ru(dip)2(bpy-2-nitroIm)]2+ () and bpy-2-nitroIm () on CT26
cell after incubation during 4 h. Cell viability was assessed by MTT assay.
Fig. 6. Relative luminescence intensity of A549 cells incubated with [Ru(dip)2(bpy-
NitroIm)]2+ in medium without (S-) or with (S+, 2%) serum under normoxic and hypoxic
conditions.
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Table 1. Retention of ruthenium complexes in A549 cells under hypoxic conditions estimated
by the monitoring of luminescence intensity conserved inside the cells cultivated for 24 h in
medium (2% FBS) without addition of these complexes.
Concentration [µM] [Ru(dip)2(bpy-NitroIm)]2+ [Ru(dip)2(bpy)]2+
2
4
6
83 %
85 %
89 %
57 %
53 %
63 %
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