← Back
Dinuclear Organoruthenium Complex for Mitochondria-Targeted Near-Infrared Imaging and Anticancer Therapy to Overcome Platinum Resistance.
pubs.acs.org/IC
Article
Dinuclear Organoruthenium Complex for Mitochondria-Targeted
Near-Infrared Imaging and Anticancer Therapy to Overcome
Platinum Resistance
Jiaoyang Wang, Yufei Zhang, Yifan Li, Enbo Li, Wenjing Ye,* and Jie Pan*
Downloaded via TSINGHUA UNIV on November 10, 2022 at 02:52:05 (UTC).
See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.
Cite This: Inorg. Chem. 2022, 61, 8267−8282
ACCESS
Metrics & More
Read Online
Article Recommendations
sı Supporting Information
*
ABSTRACT: New mononuclear and dinuclear Ru(II) coordination compounds with
the 2,7-bisbenzoimidazolyl-naphthyridine ligand have been synthesized and characterized by UV−vis, NMR, and MALDI-TOF. The molecular structures for Ru(II)
compounds were determined by single-crystal X-ray diffraction. With the expansion of
ligand π-conjugation and the increase in the complexed Ru number, the maximum
emission wavelength red-shifted from 696 to 786 nm. The binding mode between
complexes and DNA was predicted by molecular docking, which is intercalations and
π−π stacking interactions with the surrounding bases. The intercalation mode of DNA
binding was then determined by DNA titration and ethidium bromide (EB)
displacement experiments. The antigrowth effects of complexes RuY, RuY1, and RuY2 were tested in HaCat (normal cells),
HeLa (cervical cancer), A549 (lung cancer), and A549/DDP (cisplatin-resistant lung cancer) through the MTT assay. The dinuclear
complex RuY2 was superior to mononuclear complexes and cisplatin in the cisplatin-resistant cell line. Confocal imaging proved that
the subcellular localization of Ru(II) complexes was mitochondria; moreover, apoptosis was detected by flow cytometry. All three
complexes showed a dose-dependent manner in all four cell lines. All Ru(II) complexes were found to have reactive oxygen species
(ROS). The finding indicated that these Ru(II) complexes caused cell death by both DNA disruption and ROS. This study helps to
explore the potential of the polynuclear Ru(II) complexes for the combination of NIR imaging and Pt-resistant cancer therapy.
■
INTRODUCTION
Platinum-based anticancer drugs have been used for chemotherapy for almost 40 years due to its effective toxicity toward
cancer cells.1 Some classic Pt drugs have been developed in
clinics and used worldwide such as cisplatin, carboplatin, and
oxaliplatin.2−4 However, some side effects have significantly
influenced their worldwide use due to drug resistance and
toxicity without selectivity.5 To eliminate such side effects and
explore the novel mechanisms of cell death, new anticancer
drug candidates are in demand urgently.
Under this circumstance, transition metals such as
ruthenium, iridium, and osmium are of interest due to their
comparative lower toxicity, better selectivity, and novel
mechanism for causing cell death. For example, these new
mechanisms include DNA binding, inhibition of the activity of
the proteins, or catalytic hydride transfer reactions in cells.6−8
Moreover, after the metal is complexed with π-conjugated
auxiliary ligands such as arene,9 polypyridine,10 1,10-phenanthroline,11 and their derivatives, with the modification of
ligands, novel transition metal complexes can lead to a
significant change in luminescence properties and anticancer
efficacy. Among these transition metal complexes, Ru(II)
complexes were attractive due to their special photophysical
properties and DNA binding mechanism for anticancer
therapy.12 Some Ru(II) complexes have been used in clinical
trials such as NAMI-A,13 KP1019,14 and (N)KP133915
© 2022 American Chemical Society
(Figure 1). However, these above-mentioned probes did not
reach the near-infrared (NIR) region, which was not suitable
for NIR imaging. NIR imaging has several advantages such as
minimum photodamage to biological tissue, deep penetration
depth, less light scattering, and fast and cheap technique.16−21
We believe that combining imaging techniques with the
anticancer properties of Ru complexes will lead to better tumor
therapeutics. Therefore, the design of ligands is very important.
With the design of π-conjugated ligands, the charge transfer of
metals to ligands can extend the emission range to the NIR
region with relatively longer wavelengths.
Polynuclear complexes provide a novel design as anticancer
agents, which may display additional advantages over
mononuclear counterparts such as longer emission wavelength,
improved anticancer efficacy, and lower toxicities. Subtle
examples are [(arene)2Ru2L]2+,5 BBR3464,22 and Ru(η6-pcymene) complexes23,24 (Figure 1). However, there are only a
few examples of polynuclear ruthenium complexes in the
reported results, and even fewer of them reached the nearReceived: March 4, 2022
Published: May 18, 2022
8267
https://doi.org/10.1021/acs.inorgchem.2c00714
Inorg. Chem. 2022, 61, 8267−8282
Inorganic Chemistry
pubs.acs.org/IC
Article
Figure 1. Representative anticancer platinum(IV) and ruthenium(II) complexes.
Figure 2. Design of a novel ligand for a polynuclear anticancer drug.
In the following synthetic work, we first complexed a Ru at the
M1 position to synthesize a mononuclear RuY1 complex.
Then, tBuOK was used as a strong base for the removal of
hydrogen chloride. The second Ru was then complexed at the
M4 position instead of M2, which may be due to the steric
hindrance effect. For other vacant coordination sites of Ru, we
used bipyridine as the ligand for complexation, which resulted
in RuY2 (Figure 2). The molecular structures for Ru(II)
compounds were determined by single-crystal X-ray diffraction. Photophysical studies, docking analysis, CT-DNA binding
mode, antigrowth effect, ROS generation, cell cycle capture,
confocal imaging, and apoptosis studies have been done.
Through these tests, we hoped that our designed dinuclear
infrared region (650−900 nm).25−27 Therefore, well-designed
polynuclear ruthenium needs to be developed, and more
extensive studies are required.
In our and other groups’ previous studies,28−32 the pyridiylbenzimidazole ligand (YL-2) had sufficient coordination sites
that provided possibilities for complexation to metal and DNA
binding and a large π-conjugated system for red-shifted
emission wavelengths. Based on the pyridiyl-benzimidazole
moiety, herein, a newly designed ligand, 2,7-bisbenzoimidazolyl-naphthyridine (YL-1), is constructed by two benzimidazoles conjugated with a naphthyridine moiety. Compared to
the pyridiyl-benzimidazole ligand (YL-2), YL-1 had an
expanded π-conjugated system and more binding sites for
polynuclear complexation (M1 to M4), as shown in Figure 2.
8268
https://doi.org/10.1021/acs.inorgchem.2c00714
Inorg. Chem. 2022, 61, 8267−8282
Inorganic Chemistry
pubs.acs.org/IC
ethanol (15 mL), and the mixture was refluxed for 24 h. The resulting
dark-purple solution was concentrated under reduced pressure to give
a dark-purple solid. The resulting solid was purified by flash column
chromatography on neutral Al2O3 (200−300 mesh) (eluents:
CH2Cl2/MeOH = 20:1) and then recrystallized by layering CH2Cl2
and MeOH with hexane and Et2O (VDCM/VMeOH/Vhexane/Vether =
3:1:4:4) to give black-purple crystals (0.031 g, 85% yield), which was
suitable for single-crystal X-ray diffraction analysis. Mp: >260 °C. 1H
NMR (400 MHz, CD3OD) δ 8.82−8.72 (m, 3H), 8.68 (dd, J = 8.3,
2.8 Hz, 1H), 8.62 (d, J = 8.2 Hz, 1H), 8.58−8.46 (m, 3H), 8.43 (d, J
= 8.1 Hz, 1H), 8.23 (t, J = 8.5 Hz, 2H), 8.11 (dt, J = 14.4, 7.9 Hz,
2H), 8.02 (d, J = 5.4 Hz, 1H), 7.99−7.76 (m, 6H), 7.73−7.60 (m,
5H), 7.52 (q, J = 4.8, 4.2 Hz, 2H), 7.40−7.09 (m, 9H), 7.01 (dd, J =
12.2, 6.3 Hz, 2H), 6.92 (d, J = 2.1 Hz, 1H), 6.82 (td, J = 7.8, 3.3 Hz,
1H), 6.63 (t, J = 7.6 Hz, 1H), 5.86 (d, J = 8.2 Hz, 1H), 5.49 (d, J = 8.2
Hz, 1H). 13C NMR (101 MHz, CD3OD) δ 160.52, 159.34, 158.21,
158.00, 157.82, 157.41, 156.95, 156.27, 156.09, 155.08, 154.62,
153.26, 152.64, 152.00, 150.53, 150.27, 149.32, 146.85, 145.28,
143.36, 141.72, 136.30, 136.06, 135.29, 134.92, 134.68, 134.49,
134.03, 133.94, 126.62, 126.41, 126.22, 126.11, 125.82, 123.22,
123.18, 123.07, 122.99, 122.90, 122.41, 122.16, 121.98, 121.42,
121.21, 118.55, 118.35, 114.77, 114.39, 113.19. HRMS: Calcd for
C62H44N14Ru22+ [M − Cl + H]2+: 1188.1949. Found: 594.09821 (z =
2).
Log Pow Determination. The log Pow determination of complexes
RuY, RuY1, and RuY2 was conducted using the shake-flask method.
RuY, RuY1, and RuY2 (40 μM) were dissolved in double-distilled
water, and the solution was filtered to remove undissolved ruthenium
complexes. Subsequently, the solution was added to an equal volume
of n-octanol (presaturated with water), shaken vigorously at 37 °C for
24 h, and centrifuged for 15 min to achieve phase separation. The
initial and final concentrations of compounds in the aqueous phase
were determined by the UV−vis spectrum method, and the water−
octanol partition coefficients (log Pow) were calculated.
Fluorescence Quantum Yield. Quantum yields (QY) of
complexes RuY, RuY1, and RuY2 were measured using Ru(bpy)32+
as a standard (λex = 450 nm in MeCN, Φ = 0.018). The QY of
complexes RuY, RuY1, and RuY2 can be calculated from eq 1
Ru(II) complexes could exert their activities through different
anticancer mechanisms and enabled near-infrared imaging.
■
Article
EXPERIMENTAL SECTION
Materials and Measurements. All chemical reagents were
purchased commercially and used directly without further purification
unless specified. The solvents MeOH and EtOH were dried with
CaH2 and then distilled before use. cis-Ru(bpy)2Cl2·2H2O was
synthesized through the reported method.33 1H and 13C NMR spectra
were recorded on a Bruker DRX-400 spectrometer. High-resolution
electrospray ionization mass spectra (HRMS) were recorded using a
6224-TOF-LC/MS (Agilent). HaCat, HeLa, A549, and A549/DDP
were purchased from Fenghui Biology Company. Cell uptake, cell
cycle distribution, cell apoptosis, and ROS experiments were
measured by flow cytometry (BD Accuri C6 Plus) and analyzed
using FlowJo software. A UH4150 UV−vis recording spectrophotometer and an RF6000 spectrophotometer (SHIMADAZU) were
used with 1 cm-path length quartz cuvettes (3 mL). Spectra were
processed using Originlab software. Experiments were carried out at
298 K unless otherwise stated.
Synthesis of the Complex RuY. Under N2, a Schlenk bottle
containing cis-Ru(bpy)2Cl2·2H2O (0.104 g, 0.2 mmol) and YL-2
(0.039 g, 0.2 mmol) was treated with methanol (20 mL), and the
mixture was refluxed for 6 h. The resulting red-brown solution was
concentrated under reduced pressure to give a red-brown solid. The
resulting solid was purified by flash column chromatography on
neutral Al2O3 (200−300 mesh) (eluents: CH2Cl2/MeOH = 20:1)
and then recrystallized by layering CH2Cl2 and MeOH with toluene
(VDCM/VMeOH/Vtol = 6:2:15) to give brown crystals (0.085 g, 66%
yield). Mp: >260 °C. 1H NMR (400 MHz, CDCl3−CD3OD) δ 8.77−
8.63 (m, 2H), 8.55−8.48 (m, 1H), 8.42 (ddd, J = 8.3, 3.5, 2.3 Hz,
2H), 8.05 (tdd, J = 7.9, 5.0, 1.5 Hz, 2H), 7.94 (td, J = 7.9, 1.6 Hz,
1H), 7.87−7.74 (m, 4H), 7.70−7.60 (m, 3H), 7.43−7.32 (m, 3H),
7.25−7.21 (m, 1H), 7.18 (ddd, J = 7.2, 5.7, 1.3 Hz, 1H), 7.10 (ddd, J
= 7.3, 5.7, 1.5 Hz, 1H), 7.02 (ddd, J = 8.2, 7.0, 1.1 Hz, 1H), 6.69
(ddd, J = 8.3, 7.0, 1.2 Hz, 1H), 5.52 (d, J = 8.0 Hz, 1H). 13C NMR
(101 MHz, CDCl3−CD3OD) δ 158.55, 158.29, 158.04, 157.27,
157.22, 155.47, 151.75, 151.53, 150.95, 150.88, 149.84, 146.92,
144.67, 137.38, 137.34, 136.89, 136.80, 136.19, 127.69, 127.15,
127.12, 126.65, 124.78, 124.52, 124.28, 123.77, 123.67, 122.48,
121.85, 121.44, 119.22, 113.08. HRMS: Calcd for C32H24N7Ru+ [M −
Cl−]+: 608.1131. Found: 608.11316.
Synthesis of the Complex RuY1. Under N2, a Schlenk bottle
containing cis-Ru(bpy)2Cl2·2H2O (0.370 g, 0.55 mmol) and YL-1
(0.181 g, 0.5 mmol) was treated with methanol (80 mL), and the
mixture was refluxed for 24 h. The resulting red-brown solution was
concentrated under reduced pressure to give a red-brown solid. The
resulting solid was purified by flash column chromatography on
neutral Al2O3 (200−300 mesh) (eluents: CH2Cl2/MeOH = 20:1)
and then recrystallized by layering CH2Cl2 and MeOH with hexane
and Et2O (VDCM/VMeOH/Vhexane/Vether = 3:1:4:4) to give dark-brown
crystals (0.34 g, 84% yield), which was suitable for single-crystal X-ray
diffraction analysis. Mp: >260 °C. 1H NMR (400 MHz, CD3OD) δ
8.76−8.61 (m, 3H), 8.54 (td, J = 8.9, 1.9 Hz, 2H), 8.46 (dt, J = 5.6,
1.3 Hz, 1H), 8.26 (dt, J = 8.2, 1.1 Hz, 1H), 8.20 (dd, J = 4.7, 2.4 Hz,
1H), 8.12 (td, J = 7.9, 1.4 Hz, 1H), 8.08−7.95 (m, 3H), 7.85−7.76
(m, 2H), 7.74−7.64 (m, 2H), 7.38 (tdd, J = 7.2, 3.5, 2.0 Hz, 5H),
7.32 (ddt, J = 5.7, 1.7, 0.9 Hz, 1H), 7.18 (ddd, J = 7.2, 5.6, 1.4 Hz,
1H), 7.08 (ddd, J = 8.3, 7.0, 1.2 Hz, 1H), 6.98 (dd, J = 6.1, 2.6 Hz,
2H), 6.70 (ddd, J = 8.3, 7.0, 1.2 Hz, 1H), 5.54−5.40 (m, 1H). 13C
NMR (101 MHz, CDCl3−CD3OD) δ 160.00, 159.30, 158.11, 157.51,
156.64, 156.36, 153.81, 152.58, 152.10, 151.44, 150.95, 148.98,
147.41, 145.71, 138.84, 138.30, 136.50, 136.06, 135.93, 134.30,
127.41, 126.65, 126.27, 124.50, 123.52, 123.41, 122.79, 122.55,
122.40, 122.16, 121.01, 119.70, 113.72. HRMS: Calcd for
C42H29N10Ru+ [M − Cl]+: 775.1620. Found: 775.1606.
Synthesis of the Complex RuY2. Under N2, a Schlenk bottle
containing cis-Ru(bpy)2Cl2·2H2O (0.016 g, 0.03 mmol), RuY1 (0.023
g, 0.03 mmol), and tBuOK (0.014 g, 0.12 mmol) was treated with
Φif =
F ifs ni2
F sfi ns2
Φsf
(1)
where Φif and Φsf are the photoluminescence QY of the sample and
that of the standard, respectively, Fi and Fs are the integrated
intensities (areas) of the sample and standard spectra, respectively, f x
is the absorption factor (also known under “absorptance”), the
fraction indices impinging on the sample that is absorbed (f x = 1 −
10−Ax, where A = absorbance), and ni and ns are the refractive indices
of the sample and reference solution, respectively.34
Molecular Docking. The structures of the three complexes were
sketched using SYBYL-X 2.1 software (Tripos Inc., St. Louis, MO),
and the energy-minimized conformations were obtained with the
Tripos force field. The docking studies of the molecules with calf
thymus DNA (CT-DNA) were accomplished by docking these
complexes into B-DNA. The crystal structure of B-DNA (PDB ID:
3IXN) was downloaded from the Protein Data Bank. Then, the
docking studies were implemented with the Surflex-Dock Geom
module in SYBYL-X 2.1. The visualization of the docking results was
performed using PyMOL software (DeLano Scientific LLC, San
Carlos, CA).
Electronic Absorption Titration Studies. DNA binding experiments were carried out in Tris-HCl buffer (5 mM Tris-HCl, 10 mM
NaCl buffer solution, pH = 7.4) using DMSO solutions of complexes
RuY, RuY1, and RuY2 and then diluting them suitably with buffer to
the required concentrations for all the complexes (RuY: 40 μM,
RuY1: 25 μM, and RuY2: 20 μM). During the experiment, the
concentration of the complexes was constant, and the same volume of
CT-DNA solution was added to the reference solution and the test
solution (ε260nm, CT-DNA = 6600 M−1 cm−1 per nucleotide) to
reduce the influence of the absorption of CT-DNA at 260 nm on the
8269
https://doi.org/10.1021/acs.inorgchem.2c00714
Inorg. Chem. 2022, 61, 8267−8282
Inorganic Chemistry
pubs.acs.org/IC
(PBS) three times and trypsinized. The fluorescence intensity of ROS
in cells containing RuY, RuY1, and RuY2 complexes was determined
by flow cytometry (BD Accuri C6 Plus).
In Vitro Cytotoxicity. The cytotoxic study was examined by 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
assay. The MTT proliferation assay is based on the reduction of the
yellow MTT tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) by mitochondrial dehydrogenases to form a
blue MTT formazan in viable cells. A total of 104 cells/wells were
inoculated in 96-well plates and cultured in 100 μL of DMEM or
RPMI 1640 or F12K medium containing 10% FBS and 1% double
antibody at 37 °C with a 5% CO2 atmosphere. The cells were grown
for 24 h, and the growth medium was replaced with fresh medium
containing different concentrations of the compounds to be assayed
and maintained at 37 °C in a 5% CO2 atmosphere for 72 h. Cisplatin
was also tested as a positive control. After the treatment, cells were
washed with phosphate-buffered saline (PBS) and incubated with
fresh DMEM or RPMI 1640 or F12K medium containing MTT (5
mg/mL). The plates were incubated for 4 h, and 150 μL of DMSO
was added to each well. The absorbance values of each well were
measured at 490 nm with a Thermo Scientific microplate reader. The
relative cell viability was calculated by the equation: cell viability (%)
= (OD treated/OD control) × 100%. The IC50 values were calculated
using SPSS software.
Cell Uptake. A549 cells were plated onto a 6-wells plate and
incubated at 37 °C with a 5% CO2 atmosphere for 24 h. The cells
were treated with RuY, RuY1, and RuY2 (20 μM) complexes for 24 h
to evaluate the intracellular incorporation. After being washed with
phosphate-buffered saline (PBS) three times, the A549 cells were
trypsinized and collected. A flow cytometer (BD Accuri C6 Plus) was
used for measuring the fluorescence intensity of cells containing
complexes RuY, RuY1, and RuY2.
Mitochondrial Transmembrane Potential. A549 cells were
planted in 35 mm glass-bottom culture dishes at a density of 1 × 104
and cultured overnight at 37 °C with a 5% CO2 atmosphere
overnight. After treating with 20 μM RuY, RuY1, and RuY2
complexes for 6 h, cells were further stained using 1 μL of the
mitochondrial membrane potential probe JC-1 for 30 min, followed
by washing three times with PBS (pH = 7.4). Confocal fluorescence
images were acquired on a Zeiss LSM 880 multiphoton laser scanning
confocal microscope (Carl Zeiss, Germany) with a 20× water
immersion objective (NA 1.0) using excitation wavelengths of 488 nm
for JC-1. The signals of JC-1 aggregates were collected at 570−620
nm, and the signals of JC-1 monomers were collected at 493−620 nm.
A549 cells were transplanted into 6-well plates with a density of
105/well and cultured at 37 °C in a 5% CO2 atmosphere for 24 h. The
complexes RuY, RuY1, and RuY2 were dissolved in DMSO and
diluted to 20 μM in the medium. Then, the mitochondrial membrane
potential probe JC-1 (0.5 μL) was added and incubated at 37 °C for
30 min in darkness. A549 cells were washed with phosphate-buffered
saline (PBS) three times and trypsinized. The fluorescence intensity
of JC-1 monomers in cells containing RuY, RuY1, and RuY2
complexes was determined by flow cytometry (BD Accuri C6 Plus).
Mitochondrial DNA Damage Assay. A549 cells were transplanted into 6-well plates with a density of 105/well and cultured at 37
°C in a 5% CO2 atmosphere for 24 h. The cells were treated with
RuY, RuY1, and RuY2 (20 μM) complexes for 6 h. After being
washed with PBS three times, the A549 cells were trypsinized and
collected. The PicoGreen concentrated solution is diluted with TE
buffer in a ratio of 1:200 to prepare the PicoGreen dyeing working
solution. The cells were resuspended with 150 μL of buffer, and then,
an equal volume of PicoGreen staining working solution was added to
mix them evenly. The reaction mixture was kept away from light and
at room temperature for about 5−10 min. The fluorescence intensity
of PicoGreen in cells containing RuY, RuY1, and RuY2 complexes
was determined by flow cytometry (BD Accuri C6 Plus). The
excitation wavelength of the PicoGreen dye is 488 nm.
Cell Cycle Distribution. Flow cytometry analysis of the cell cycle
of A549 cells caused by exposure to metal complexes was carried out
using the cell cycle analysis kit (Yeasen Biotechnology, Shanghai, Co.,
experiment. After each addition, samples were incubated under
physiological conditions (5 mM Tris-HCl, 10 mM NaCl buffer
solution, pH = 7.4) at room temperature for 5 min equilibration time,
and then, the UV−visible spectra were recorded. Spectra were
collected from 250 to 600 nm after successive addition of CT-DNA
(0−93.3 μM) into a 6 mL solution of each complex. The absorbance
(A) of the most red-shifted band of each investigated complex was
recorded after successive additions of CT-DNA. Interactions of the
complexes RuY, RuY1, and RuY2 with DNA were fitted to the
Benesi−Hildebrand equation (eq 2) to calculate the binding
constants Kb.
[DNA]
[DNA]
1
=
+
εa − εf
εb − εf
Kb(εb − εf )
Article
(2)
where [DNA] is the concentration of CT-DNA and the apparent
absorption coefficient, εa, corresponds to A/[Ru]. εb and εf refer to
the extinction coefficient of the complexes in their bound and free
forms, respectively. The plot of [DNA]/(εa − εf) versus [DNA] gives
a straight line, and the binding constant Kb was calculated as the
slope/intercept ratio.
Ethidium Bromide Displacement Experiments. The experiments were carried out by the addition of the complexes RuY, RuY1,
and RuY2 to the samples containing 100 μM CT-DNA (nucleotide)
and 20 μM EB (ethidium bromide) in a Tris buffer solution (5 mM
Tris-HCl, 10 mM NaCl buffer solution, pH = 7.4). For each sample,
the concentration of CT-DNA and EB was constant, and a solution of
different complexes RuY, RuY1, and RuY2 (dissolved with DMSO)
was added. After each addition, samples were incubated under
physiological conditions (5 mM Tris-HCl, 10 mM NaCl buffer
solution, pH = 7.4) at room temperature for 5 min equilibration time,
and then, the fluorescence spectra were recorded. The influence of the
addition of RuY, RuY1, and RuY2 (0−50 μM) to the EB-DNA
mixture was measured by recording the variations in the fluorescence
emission spectra between 550 and 800 nm with an excitation at 537
nm.
Cell Culture. For cytotoxicity determination, four cell lines were
used, HaCat (human immortal keratinocyte cell line), HeLa (human
cervical carcinoma cell line), A549 (human non-small-cell lung cancer
cell line), and A549/DDP (cisplatin-resistant non-small-cell lung
cancer cell line), purchased from Fenghui Biology Company. Frozen
HeLa cells were thawed out in a 25 cm2 cell culture flask in DMEM
(GIBCO/Invitrogen, Camarillo, CA) supplemented with 10% fetal
bovine serum (FBS, Biological Industry, Kibbutz Beit Haemek, Israel)
and 1% penicillin−streptomycin (100 U/mL penicillin and 10 μg/mL
streptomycin, Solarbio Life Science, Beijing, China) and maintained
at 37 °C in a 5% CO2 atmosphere, replacing the medium twice a
week. Frozen HaCat and A549 cells were thawed out in a 25 cm2 cell
culture flask in RPMI 1640 (GIBCO/Invitrogen, Camarillo, CA)
supplemented with 10% fetal bovine serum (FBS, Biological Industry,
Kibbutz Beit Haemek, Israel) and 1% penicillin−streptomycin (100
U/mL penicillin and 10 μg/mL streptomycin, Solarbio Life Science,
Beijing, China) and maintained at 37 °C in a 5% CO2 atmosphere,
replacing the medium twice a week. Frozen A549/DDP cells were
thawed out in a 25 cm2 cell culture flask in F12K (Nanjing SenBeiJia
Biological Technology Co., Ltd.) supplemented with 10% fetal bovine
serum (FBS, Biological Industry, Kibbutz Beit Haemek, Israel) and
1% penicillin−streptomycin (100 U/mL penicillin and 10 μg/mL
streptomycin, Solarbio Life Science, Beijing, China) and maintained
at 37 °C in a 5% CO2 atmosphere, replacing the medium twice a
week.
ROS Generation. A549 cells were transplanted into 6-well plates
with a density of 105/well and cultured at 37 °C in a 5% CO2
atmosphere for 24 h. The complexes RuY, RuY1, and RuY2 were
dissolved in DMSO and diluted to 30 μM in medium (DMSO final
concentration was 5%). Since the ROS level induced by aromaticruthenium complexes increased first and then decreased, the culture
time was selected as 6 h for study. Then, reactive oxygen probe
DCFH-DA (1 μL) was added and incubated at 37 °C for 20 min in
darkness. A549 cells were washed with phosphate-buffered saline
8270
https://doi.org/10.1021/acs.inorgchem.2c00714
Inorg. Chem. 2022, 61, 8267−8282
Inorganic Chemistry
pubs.acs.org/IC
Article
Scheme 1. (a, d) Schematic Presentation of the Synthesis of Ru(II) Complexes and (b, c) Molecular Structures of RuY1 and
RuY2a
Conditions: (i) a: MeLi, Et2O, −78 °C, 4 h; b: KMnO4, acetone, r.t., overnight, 80% total yield. (ii) SeO2, dioxane, reflux 2 h, 73% yield. (iii)
HNO3, reflux 5 h, 78% yield. (iv) 1,2-Diaminobenzene, PPA, 220 °C, 5 h, 67% yield. (v) cis-Ru(bpy)2Cl2·2H2O 1.1 equiv, MeOH, reflux 24 h, 84%
yield. (vi) cis-Ru(bpy)2Cl2·2H2O 1.0 equiv, tBuOK 4.0 equiv, EtOH, reflux 24 h, 85% yield. (vii) 1,2-Diaminobenzene, DMF/H2O, 80 °C, 16 h,
81% yield. (viii) cis-Ru(bpy)2Cl2·2H2O 1.0 equiv, MeOH, reflux 6 h, 66% yield.
a
at 37 °C in a 5% CO2 atmosphere overnight. After treating with 20
μM RuY, RuY1, and RuY2 complexes for 4 and 8 h, cells were further
stained using 1 μL of Hoechst 33342 for 15 min, 0.1 μL of
MitoTracker Green, and 1 μL of LysoTracker Green for 30 min,
followed by washing three times with PBS (pH = 7.4). Confocal
fluorescence images were acquired on a Zeiss LSM 880 multiphoton
laser scanning confocal microscope (Carl Zeiss, Germany) with a 63×
water immersion objective (NA 2.0) using excitation wavelengths of
405 nm for Hoechst 33342, 488 nm for LysoTracker Green and
MitoTracker Green, and 514 nm for complexes RuY, RuY1, and
RuY2. The signals of Hoechst 33342 were collected at 410−500 nm,
the signals of MitoTracker Green and LysoTracker Green were
collected at 493−620 nm, and the signals of RuY, RuY1, and RuY2
were collected at 600−758 nm.
Apoptosis Study. Flow cytometry analysis of apoptotic
populations of A549 and A549/DDP cells caused by exposure to
metal complexes was carried out using the annexin V-FITC apoptosis
detection kit (Yeasen Biotechnology, Shanghai, Co., Ltd.) according
to the supplier’s instructions. A549 and A549/DDP cells were
Ltd.) according to the supplier’s instructions. A549 cells were
transferred into 6-well plates, with a density of 105/well, and cultured
overnight at 37 °C in a 5% CO2 atmosphere. Then, complexes RuY,
RuY1, and RuY2 were dissolved in DMSO (1 mM) and diluted with
medium to 20 μM (the final concentration of DMSO was 1%) and
cultured at 37 °C with a 5% CO2 atmosphere for 24 h. As for the
negative control, the same volume of DMSO was added. All adherent
and floating cells were collected and washed twice with phosphatebuffered saline. After being centrifuged, 500 μL of staining solution, 5
μL of PI solution, and 10 μL of RNase A solution were added
successively and dyed at 37 °C for 0.5 h under dark conditions. Cell
pellets were washed and resuspended in PBS before being analyzed in
a flow cytometer (BD Accuri C6 Plus) using excitation of DNAbound PI at 488 nm, with emission at 585 nm. Data were processed
using FlowJo software. The cell cycle distribution is shown as the
percentage of cells containing G0/G1, S, G2/M-phase DNA as
identified by propidium iodide staining.
Confocal Imaging. A549 cells were planted in 35 mm glassbottom culture dishes at a density of 1 × 104 and cultured overnight
8271
https://doi.org/10.1021/acs.inorgchem.2c00714
Inorg. Chem. 2022, 61, 8267−8282
Inorganic Chemistry
pubs.acs.org/IC
Article
Figure 3. (a) UV−visible absorbance spectra of RuY, RuY1, and RuY2 (10 μM) in DCM. (b) Normalized emission spectra of RuY (10 μM, λex =
502 nm), RuY1 (10 μM, λex = 495 nm), and RuY2 (10 μM, λex = 509 nm) in DCM.
transferred into 6-well plates, with a density of 105/well, and cultured
overnight at 37 °C in a 5% CO2 atmosphere. Then, complexes RuY,
RuY1, and RuY2 were dissolved in DMSO (1 mM) and diluted with
medium to 100 μM (the final concentration of DMSO was 5%) and
cultured at 37 °C in a 5% CO2 atmosphere for 24 h. As for the
negative control, the same volume of DMSO was added. After
digestion with trypsin without EDTA, all adherent and floating cells
were collected and washed twice with phosphate-buffered saline. After
being centrifuged, 500 μL of binding buffer, 5 μL of annexin V-FITC,
and 10 μL PI staining solution were added successively and dyed at
room temperature for 10−15 min under dark conditions. The samples
were analyzed using a flow cytometer (BD Accuri C6 Plus).
treated with 4.0 equiv of tBuOK, dinuclear RuY2 was
successfully prepared in 85% isolated yield as a dark-purple
crystal, which was analyzed through single-crystal X-ray
diffraction, as shown in Scheme 1c. It was found from the
molecular structure of the complex RuY2 that the second Ru
center was coordinated to not the N4 atom of naphthyridine
but the C14 atom and similar to the RuY1 center with a
regular octahedral geometry. The dinuclear RuY2 was also an
ionic complex with a Cl− as the anion. In addition, as a control
trial, RuY bearing pyridyl benzimidazole YL-2 was synthesized
through the reaction of cis-Ru(bpy)2Cl2·2H2O with the ligand
pyridyl benzimidazole YL-2, which was obtained by a
condensation reaction of pyridine-2-aldehyde (5) with ophenylenediamine, as shown in Scheme 1d.
Photophysical Properties. The ground-state absorption
spectra of Ru(II) polypyridyl complexes were characterized by
strong absorption bands at ∼294 nm (RuY was ε = 77 440
M−1 cm−1, RuY1 was ε = 56 680 M−1 cm−1, and RuY2 was ε =
147 820 M−1 cm−1), which was attributed to π−π* electronic
transitions centered on aromatic rings, such as the 1,10phenanthroline moiety, bipyrimidine, and the benzimidazole
unit. A broad and intense band between 330 and 420 nm was
due to an intra-ligand charge transfer (ILCT) transition.35 This
result could be confirmed by YL-1 since YL-1 also displayed an
intense absorption at 394 nm (Figure S1). A broad band that
was found in the 450−600 nm range corresponded to dp (Ru
II)-π*-metal-to-ligand charge-transfer (MLCT) transitions.
Compared to the mononuclear Ru(II) complex RuY1, the
absorption band of the dinuclear RuY2 complex was shifted to
a longer wavelength and became broader. In the meanwhile,
the solvatochromism effect was observed when Ru(II)
complexes were dissolved in the following solvents with
increasing polarity: dichloromethane (DCM), chloroform
(CHCl3), acetonitrile (ACN), and methanol (MeOH). The
absorption spectra (displayed in Figure S2 and Table S1) of
RuY2 consist of broad bands centered at 509, 508, 506, and
495 nm in DCM, CHCl3, ACN, and MeOH, respectively. The
other two complexes, RuY and RuY1, showed the same
hypsochromic shift.
Luminescence was observed for both the ligand and its
Ru(II) complexes in solution and at room temperature. Strong
red emission bands were found at 696 nm for RuY, 740 nm for
RuY1, and 786 nm for RuY2. These results fully proved that
with the increase of ligand π-conjugation and the amount of
■
RESULTS AND DISCUSSION
Synthesis and Characterization. The synthetic procedure is shown in Scheme 1. Starting from the commercial
compound 2-methylnaphthyridine (1), the methylation gave
2,7-dimethyl-naphthyridine (2) in 80% isolated yield. Intermediate 2 underwent two-step oxidation with SeO2 and nitric
acid as the oxidants, respectively, giving the diacid product 4.
The condensation of diacid 4 with o-phenylenediamine in
polyphosphoric acid (PPA) produced the desired tetradentate
ligand 2,7-bis(1H-benzo[d]imidazol-2-yl)-1,8-naphthyridine
YL-1 in 67% isolated yield. The reaction of YL-1 with 1.1
equiv of the Ru precursor cis-Ru(bpy)2Cl2·2H2O in methanol
under refluxing conditions afforded mononuclear RuY1 in 84%
isolated yield as a dark-brown crystal, which was obtained
through recrystallization in the mixture of CH2Cl2, MeOH,
hexane, and Et2O. The molecular structure of RuY1 was
confirmed through single-crystal X-ray diffraction, as shown in
Scheme 1b. It was found that the Ru center had been
coordinated to two bipyridines and a pyridyl and a
benzimidazolyl group to yield a regular octahedral geometry.
Moreover, the formation of a covalent bond between the Ru
center and N atom of the benzimidazolyl group made the Ru
center a cation with a positive charge, and the corresponding
anion was Cl−. This kind of ionic structure led to water
solubility, which is beneficial for applying as a bioactive
molecule. The reaction of YL-1 with 2.0 equiv of the Ru
precursor cis-Ru(bpy)2Cl2·2H2O was also investigated, but
equally, mononuclear RuY1 was obtained, and we failed to
obtain a dinuclear Ru complex owing to the considerable steric
hindrance between two benzimidazolyl groups and several
bipyridines. Unexpectedly, when the reaction mixture of the
mononuclear RuY1 with cis-Ru(bpy)2Cl2·2H2O had been
8272
https://doi.org/10.1021/acs.inorgchem.2c00714
Inorg. Chem. 2022, 61, 8267−8282
Inorganic Chemistry
pubs.acs.org/IC
Article
Table 1. Fluorescence Characterization of RuY, RuY1, and RuY2
RuY
RuY1
RuY2
λabs (nm)/ε (M−1 cm−1)
λem (nm)
QYa
log Powb
pKac
294 (77 440), 351 (29 670), 502 (11 640)
294 (56 680), 429 (32 350), 495 (10 830)
294 (147 840), 396 (65 670), 509 (34 950)
696
740
786
0.015
0.0037
0.0021
0.16 ± 0.20
−0.19 ± 0.01
−0.85 ± 0.04
6.4
a
Ru(bpy)32+ was used as a reference for quantum yield measurements (λex = 450 nm). bLog Pow of RuY, RuY1, and RuY2 was determined by
measuring the octanol/H2O partition coefficients. cThe pKa value of RuY, RuY1, and RuY2 obtained from the variation of the ratio of emission
intensity at maximum versus pH (295 K, 5% DMSO in DI-water, RuY, λex = 457 nm, RuY1, λex = 477 nm, RuY2, λex = 473 nm) (Figure S3).
Interaction with DNA. To confirm whether the simulation
results of our docking analysis were correct, the UV−vis
absorption spectrum and fluorescence spectrum were used to
study the interaction of Ru(II) complexes with DNA. It has
been reported that the Ru(II) complex can bind to DNA either
by binding to the DNA groove or by partially intercalating into
DNA. After binding to DNA, they exhibited a distinct
bathochromic shift and hypochromisms in their visible
absorption and UV bands. π−π* stacking occurs when the
ligand from the complex is inserted between the DNA base
pairs; then, the π* empty orbital of the ligand is coupled with
the π electron orbital of the base, resulting in a decrease in its
energy level. Since the energy gap of the π−π* transition
decreases, the UV absorption spectrum shows a bathochromic
shift. At the same time, the coupled π orbitals are partially filled
with electrons, reducing the probability of π−π* transitions,
resulting in a hypochromatic effect.36 The titration of our
synthesized Ru(II) complexes with the calf thymus DNA (CTDNA) was monitored by UV−vis spectra; the spectral
behaviors of RuY1 and RuY2 were very similar, and they
displayed obvious hypochromism, H% (as defined by H% ≅
100 (Afree − Abound)/Afree), and bathochromism, as indicated by
Δλ (Δλ = λbound − λfree). Upon increasing concentrations of
CT-DNA to the constant spectra (saturation), the H% (Δλ)
values at ∼480 nm were found to be 45% (13 nm) and 37.5%
(16 nm) for RuY1 and RuY2, respectively. Thus, the evident
spectral changes observed from RuY1 and RuY2 indicated a
strong interaction between the complexes and the DNA,
suggesting that these three molecules may bind to DNA in the
form of partial insertion (Figure 5).
The values of the intrinsic DNA binding constant Kb were
determined by monitoring the changes in absorbance at 286
nm with increasing DNA concentrations. The intrinsic DNA
binding constants Kb obtained for RuY, RuY1, and RuY2 were
calculated to be 3.35 × 104, 2.20 × 104, and 4.73 × 104 M−1,
respectively. Among them, complex RuY2 with DNA showed
enhanced binding efficacy compared with that of complexes
RuY and RuY1; this was probably because the two Ru(II)
centers may not bind to the same double strands of the DNA
strands (Table S3).
Changes in fluorescence spectra can be used to study the
interaction between ruthenium complexes and DNA. The
emission intensity of the Ru(II) complexes was affected by the
hydrophobic environment of the base pair of the DNA. The
vibrational mode of the complexes was restricted to some
degree after binding with DNA. In the meanwhile, without the
quenching of its fluorescence by water molecules, the
fluorescence intensity of the complexes would be enhanced.33
The responsive emission spectra of RuY, RuY1, and RuY2
binding with DNA are shown in Figure 6. As can be seen from
the figure, the fluorescence intensities of the solutions of the
complexes RuY, RuY1, and RuY2 increased significantly after
the addition of CT-DNA, indicating that these three complexes
Ru complexation, the spectrum was clearly red-shifted (Figure
3 and Table 1). Notably, RuY1 and RuY2 were completely
unaffected by pH changes ranging from 2 to 13, while RuY had
a pKa of 6.4 (Figure S3). The stability of the three Ru(II)
complexes in the biological medium (PBS buffer, 10% DMSO,
pH = 7.4, incubation time = 24 h) has been checked by the
emission intensity; the results showed good chemical stability
(remained > 86%). Among them, RuY2 was the most stable,
and the stability order was RuY2 (97.6%) > RuY1 (96.4%) >
RuY (86.0%) (Figure S4).
Docking Analysis. DNA is a key therapeutic target for
metal anticancer drugs, and they may interact with DNA
through covalent bonds, intercalation, groove binding, or
electrostatic interactions. We first simulated the binding of our
synthesized complexes to DNA by docking. The molecular
docking method could offer a visual representation of the
binding pattern of small ligands to DNA, which helps
implement and verify the experimental results. As shown in
Figure 4, the binding conformations of the dinuclear
Figure 4. Docking results of complexes RuY1 (A), RuY (B), and
RuY2 (C) into B-DNA (PDB ID: 3IXN).
ruthenium(II) complexes and DNA indicated that the
complexes interacted with DNA mainly through the intercalations into the adjacent base pairs of the DNA. The
backbones of the three complexes were inserted into the same
space in varying degrees between the two bases: DT5 and
DA6. In addition to the conventional interactions including
van der Waals and hydrophobic interactions of the complexes
with DNA, the pyridine rings of complexes RuY1 and RuY and
the phenyl ring of RuY2 also formed π−π stacking interactions
with the surrounding bases. Also, this kind of intercalation
could obviously increase the distance between the two adjacent
bases. The calculated binding energies of RuY, RuY1, and
RuY2 were −17.12, −22.88, and −26.99 kJ/mol, respectively
(Table S2). It suggested that complex RuY2 is bound to DNA
with the lowest binding energy.
8273
https://doi.org/10.1021/acs.inorgchem.2c00714
Inorg. Chem. 2022, 61, 8267−8282
Inorganic Chemistry
pubs.acs.org/IC
Article
Figure 5. Absorption spectra of RuY, RuY1, and RuY2 in Tris-HCl buffer upon addition of calf thymus DNA. (a) RuY: [Ru] = 40 μM, [DNA] =
0−93.3 μM; (b) RuY1: [Ru] = 25 μM, [DNA] = 0−51.3 μM, (c) RuY2: [Ru] = 20 μM, [DNA] = 0−42 μM. The arrow shows a decrease in
absorption upon increasing DNA concentration.
Figure 6. Emission spectra of RuY (a), RuY1 (b), and RuY2 (c) in the absence and the presence of increasing concentrations of DNA. ((d) RuY:
[Ru] = 40 μM, [DNA] = 0−98.07 μM; RuY1: [Ru] = 25 μM, [DNA] = 0−46.67 μM, RuY2: [Ru] = 20 μM, [DNA] = 0−84.06 μM).
8274
https://doi.org/10.1021/acs.inorgchem.2c00714
Inorg. Chem. 2022, 61, 8267−8282
Inorganic Chemistry
pubs.acs.org/IC
Article
Figure 7. Fluorescence quenching curves of EB bound to DNA in the presence of complexes RuY (a), RuY1 (b), and RuY2 (c). [DNA] = 100 μM,
[EB] = 20 μM, and [Ru] = 0−50 μM. The arrow shows a decrease in absorption upon increasing DNA concentration.
might interact with DNA. The fluorescence intensities of the
complexes RuY, RuY1, and RuY2 increased by 64.5, 63.9, and
66.0%, respectively, at their emission maximum.
Ethidium Bromide Displacement Experiments. Ethidium bromide (EB) can be a competitive binding agent for
CT-DNA and complexes. The fluorescence of EB will be
greatly enhanced after binding with CT-DNA. Thus, if the
complexes can bind to CT-DNA in the insertion mode, they
will compete with EB for the binding site, which leads to the
fluorescence quenching of EB. The EB displacement experiment was conducted by adding complexes RuY, RuY1, and
RuY2 to the EB-DNA system (Figure 7). With the increase of
the complexes’ concentration, the fluorescence of the EB-DNA
system displayed a gradual downward trend, with the
decreasing amplitude reaching 89.9% (RuY), 98.6% (RuY1),
and 99.6% (RuY2). Notably, the fluorescence intensity of the
DNA-EB complex was reduced more remarkably by complexes
RuY1 and RuY2, probably attributed to the intercalation effect
of the 2,7-bisbenzoimidazolyl-naphthyridine ligand. Although
all three complexes have the same benzoimidazole and
pyridine moieties, due to the complexation with ruthenium,
RuY1 and RuY2 possess larger aromatic rings, which facilitated
π−π* stacking; moreover, dinuclear Ru includes four bipyridyl
ligands, which possess high potential to insert into the DNA
ligand.5 This result showed that the three complexes, RuY,
RuY1, and RuY2, could replace EB from the EB-DNA system,
indicating that these three Ru(II) complexes were likely to be
bonded to CT-DNA through the insertion mode. Although
groove binding reagents can also lower the fluorescence of the
EB-DNA system, the general groove reagents decrease the
fluorescence intensity of the system to a small extent.37 The
three complexes, RuY, RuY1, and RuY2, reduced the
fluorescence intensity of the EB-DNA system by about 90%,
indicating that the three Ru(II) complexes were bonded to
CT-DNA in the intercalation mode.
ROS Generation. Ruthenium(II) arene complexes were
reported to induce ROS production in cancer cells, which
could be responsible for the cytotoxicity observed. Therefore,
ROS levels in A549 cells induced by RuY, RuY1, and RuY2
complexes were determined by flow cytometry. As shown in
Figure 8, all three complexes significantly increased ROS levels
in A549 cells, especially the dinuclear ruthenium(II) complex
RuY2. Then, we quantified the results of flow cytometry, as
Figure 8. Analysis of ROS levels by flow cytometry after A549 cells
were treated with complexes RuY, RuY1, and RuY2 (30 μM) for 6 h
and stained with DCFH-DA.
8275
https://doi.org/10.1021/acs.inorgchem.2c00714
Inorg. Chem. 2022, 61, 8267−8282
Inorganic Chemistry
pubs.acs.org/IC
Article
Figure 9. Cell survival rate of cisplatin, carboplatin, RuY, RuY1, and RuY2 in HaCat, HeLa, A549, and A549/DDP cells incubated with the MTT
method for 72 h.
Table 2. In Vitro Cytotoxicity (IC50 μM) of RuY, RuY1,
RuY2, Cisplatin, and Carboplatin against Human Cancer
Cell Lines
shown in Figure S5. The relative order of ROS levels induced
by these complexes is RuY2 > RuY > RuY1.
In Vitro Cytotoxicity. We evaluated the effects of
cytotoxicity for Ru(II) polypyridyl complexes in vitro against
the viability of HaCat (human keratinocyte cell line), HeLa
(human cervical carcinoma cell line), A549 (human non-smallcell lung cancer cell line), and A549/DDP (cisplatin-resistant
non-small-cell lung cancer cell line) by MTT assay after 72 h,
and the concentrations were up to 50 μM (Figure 9). For
comparison purposes, the cytotoxicity of cisplatin and
carboplatin was also evaluated. The half-maximal inhibitory
concentration (IC50) values are listed in Table 2. Decreased
cell viability is depicted in a dose-dependent manner in all four
cell lines as a result of the MTT assay. Notably, RuY2
exhibited better anticancer effects than those of cisplatin and
carboplatin. The cytotoxicity of RuY, RuY1, and RuY2 was
lower than that of cisplatin and carboplatin in HaCat cells
(normal cell) but was comparative in the HeLa cells. In terms
of strong antiproliferative effects on the tested cancer cell lines,
these ruthenium(II) complexes were further investigated to
IC50 (μM)
RuY
RuY1
RuY2
cisplatin
carboplatin
HaCat
HeLa
A549
A549/DDP
44.8 ± 1.2
41.8 ± 0.3
40.8 ± 0.6
34.0 ± 0.8
39.8 ± 0.2
15.3 ± 1.0
10.0 ± 0.5
24.5 ± 1.6
7.57 ± 0.3
22.1 ± 0.6
21.3 ± 0.7
22.7 ± 0.4
14.6 ± 0.5
22.3 ± 1.7
20.0 ± 0.8
26.0 ± 1.1
29.1 ± 1.8
22.8 ± 0.7
43.0 ± 1.1
37.2 ± 0.8
determine whether cisplatin resistance could be overcome. As
shown in Table 2, the IC50 value of cisplatin against A549/
DDP was increased to 43.0 μM, while the cytotoxicity of the
complex against cisplatin-sensitive A549 and cisplatin-resistant
A549/DDP cells was almost better. The cytotoxicity test of
carboplatin followed the same trend. Especially, the IC50 of
RuY2 in the A549 cell line was 14.6 μM and in the A549/DDP
8276
https://doi.org/10.1021/acs.inorgchem.2c00714
Inorg. Chem. 2022, 61, 8267−8282
Inorganic Chemistry
pubs.acs.org/IC
Article
fluorescence of the JC-1 monomer in the cells treated with the
complex increased significantly and verified the damage of
mitochondria. The figure on the right in Figure S7 is the
quantization of the flow cytometry results, in which the
decrease of the mitochondrial membrane potential caused by
complex RuY2 was the largest.
Mitochondrial DNA Damage Assay. PicoGreen is a
fluorescent dye that detects double-stranded DNA. PicoGreen
fluoresces only when bound to double-stranded DNA. It will
not emit fluorescence when there is no DNA. The fluorescence
intensity will decrease or disappear when double-stranded
DNA is damaged or broken into single-stranded DNA. The
results are shown in Figure 12a. Compared with the control,
the fluorescence intensity of the complexes RuY, RuY1, and
RuY2 bound to PicoGreen all decreased, especially the
complex RuY2. This result indicated that the complexes
RuY, RuY1, and RuY2 could insert into double-stranded
DNA, disrupted DNA replication and transcription, and caused
irreversible damage to mitochondrial DNA.
In addition, we further validated the experimental results by
q-PCR, as shown in Figure 12b. The experimental results
showed that the proportion of damaged mitochondrial DNA
(mtDNA) to total mtDNA was significantly increased in the
complexes RuY, RuY1, and RuY2 compared to that in the
control group, which indicated that the complexes RuY, RuY1,
and RuY2 were all capable of causing damage to mtDNA, with
the complex RuY2 causing the most severe damage. This was
consistent with the results of the PicoGreen experiment and
validates our experimental results.
Cell Cycle Distribution. The perturbation effects of
cisplatin and Ru(II) polypyridyl complexes on the cell cycle
progression of A549 cells were analyzed by flow cytometry
(Figure 13). Most of the antineoplastic drugs in current use
block the cell cycle in the S or G2/M phases.40 Compared to
the control, the cancer cells that were arrested at the S phase
by our Ru(II) complexes were increased to 45.2% (RuY),
42.5% (RuY2), and 41.5% (RuY1) from 31.3% (control),
which indicated that Ru(II) complexes affected the S phase
more than other phases in A549 cells, which were similar to
cisplatin.
Confocal Imaging. To this end, monitoring the microenvironment within defined subcellular organelles is important
to help understand organelle-related pathophysiology. To
investigate the cellular distribution, the subcellular localization
of Ru(II) complexes was easily determined by confocal
microscopy in A549 cells (Figure 14) and HeLa cells (Figures
S8−S12) on account of their luminescence. To confirm their
subcellular localization, costaining experiments using commercially available organelle-specific markers were performed.
After 4 h of incubation with these complexes, the
mitochondria-specific marker (Beyotime Biotechnology, MitoTracker Green, 5.0 μg/mL) was then added. The image in the
right panel represented the merged image (yellow) of the Ru
complex (left panel, red) with the MitoTracker (middle panel,
green), clearly showing the colocalization of the Mitotracker
with the complex in mitochondria. The results showed that all
three Ru complexes targeted the mitochondria with high
Pearson’s colocalization coefficients (PCC) of 0.9502 (RuY2)
in A549 cells and 0.9807 (RuY2) in HeLa cells after 4 h of
incubation. The control experiment has been done with a
commercially available lysosome tracker and nucleus tracker;
minimal overlap was observed.
cell line was 22.8 μM, which indicated that these ruthenium(II) complexes could overcome cisplatin resistance.
Cell Uptake. Under the same experimental conditions, the
cellular uptake of RuY, RuY1, and RuY2 to A549 cells was
evaluated by flow cytometry (Figure 10), and the flow
Figure 10. Flow cytometry of RuY, RuY1, and RuY2 in the A549 cells
(a), (b), (c), and control (d), dosed concentration = 20 μM and 24 h
of incubation.
cytometric uptake was quantified, results of which are shown
in Figure S6. The results show that RuY2 has better uptake in
A549 cells than that of RuY and RuY1. This is probably due to
amphiphilic and cationic properties presenting better cell
permeability.38,39
Mitochondrial Transmembrane Potential. JC-1 is a
cationic lipid fluorescent dye, which has two states: monomer
and polymer; their emission spectra are different. The
membrane potential (Δy) of normal healthy mitochondria
has polarity. JC-1 is rapidly absorbed into mitochondria
depending on the polarity of Δy and forms a polymer in
mitochondria due to the increase in concentration. The light
emitted by the polymer is red fluorescence; when apoptosis
occurs, the mitochondrial transmembrane potential is depolarized, and JC-1 is released from the mitochondria (the red
light intensity decreases) and exists in the cytoplasm in the
form of a monomer and emits green fluorescence. The results
of confocal imaging (Figure 11) indicated that the complexes
RuY, RuY1, and RuY2 could reduce the mitochondrial
membrane potential of A549 cells. The cells displayed red
fluorescence when A549 cells were not treated with complexes
RuY, RuY1, and RuY2, indicating that JC-1 existed in the form
of aggregates, and the mitochondrial cell membrane was not
damaged. All cells exhibited green fluorescence when the cells
were treated with complexes RuY, RuY1, and RuY2, indicating
that the mitochondrial cell membrane of A549 cells was
damaged to varying degrees. JC-1 existed in the form of a
monomer and showed green fluorescence, indicating that ROS
produced by the complexes caused mitochondrial damage.
Similarly, we detected it by flow cytometry (Figure S7). The
results displayed that compared with the control group, the
8277
https://doi.org/10.1021/acs.inorgchem.2c00714
Inorg. Chem. 2022, 61, 8267−8282
Inorganic Chemistry
pubs.acs.org/IC
Article
Figure 11. Confocal imaging of A549 cell membrane potential changes induced by complexes RuY, RuY1, and RuY2 (20 μM, 6 h).
Figure 12. (a) PicoGreen staining experiment verified mitochondrial DNA damage caused by complexes RuY, RuY1, and RuY2 (20 μM, 6 h). (b)
q-PCR assay experiment verified mitochondrial DNA damage caused by complexes RuY, RuY1, and RuY2 (20 μM, 6 h).
Apoptosis Study. The determination of the cellular death
mechanism (necrosis or apoptosis) was performed using an
annexin V-FITC/propidium iodide (PI) assay (Figure 15).
Ru(II) complexes and cisplatin were incubated with A549 and
A549/DDP cells for 72 h at a concentration of 50 μM. A549
and A549/DDP cells were treated with Ru(II) complexes,
cisplatin, and negative control. The relative order of apoptosis
induced against A549 cells was RuY2 (33.9%) > RuY (30.1%)
> cisplatin (28.7%) > RuY1 (28.6%). The relative order of
apoptosis induced against A549/DDP cells was RuY2 (24.2%)
> RuY (18.3%) > RuY1 (12.5%) > cisplatin (5.52%), which
was consistent with the result of the cytotoxicity study (Figure
9). According to the results, cell death was caused by apoptosis
rather than necrosis, which proved that these Ru(II)
polypyridyl complexes induce cell death through the apoptotic
pathway.
■
CONCLUSIONS
In conclusion, three novel ruthenium(II) complexes, including
mononuclear ruthenium complexes and dinuclear ruthenium
8278
https://doi.org/10.1021/acs.inorgchem.2c00714
Inorg. Chem. 2022, 61, 8267−8282
Inorganic Chemistry
pubs.acs.org/IC
Article
Figure 13. Cell cycle analysis of cell cycle distribution upon treatment with RuY, RuY1, and RuY2 (20 μM) in A549 cells after 24 h of treatment. A
selection of histograms show the strong effects of RuY, RuY1, and RuY2 on cell cycle distribution. The DNA content of cells was analyzed by flow
cytometry upon staining with propidium iodide and evaluated with FlowJo. Cisplatin was used as a positive control.
Figure 14. Subcellular localization of RuY2 (20 μM, 4 h) in A549 cells was determined by confocal laser scanning microscopy. Colocalization
images of A549 cells costained with RuY2 and MitoTracker Green (a, 1 μL, 1 mM, 30 min), LysoTracker Green (b, 1 μL, 1 mM, 30 min), and
Hoechst 33342 (c, 1 μL, 1 mM, 15 min). Scale bar: 20 μm.
complexes, were designed and synthesized. The photoluminescent tail of the dinuclear complex RuY2 reached 900
nm, which is suitable for NIR imaging. In vitro experiments
showed that the resulting ruthenium(II) complexes exhibited
good antiproliferative activity against the tested cancer cells,
while the cytotoxicity of the complex RuY2 was superior to
that of cisplatin. Further experimental and computational
studies revealed that RuY2 had a binding constant of about
4.73 × 104 M−1 for DNA, mainly through intercalation
interactions. Furthermore, the three complexes could generate
significant levels of ROS and further induce apotheosis.
Therefore, we propose that these ruthenium(II) complexes
8279
https://doi.org/10.1021/acs.inorgchem.2c00714
Inorg. Chem. 2022, 61, 8267−8282
Inorganic Chemistry
pubs.acs.org/IC
Article
Figure 15. Flow cytometry analysis for apoptosis of A549 and A549/DDP cells induced by cisplatin and RuY, RuY1, and RuY2 at the same
concentration of 50 μM for 24 h. Lower left, living cells; lower right, early apoptotic cells; upper right, late apoptotic cells; and upper left, necrotic
cells. The inserted numbers in the profiles indicate the percentage of the cells present in this area.
Authors
induced cell death through DNA binding and ROS-mediated
apoptosis pathways, and the results of this study helped to
reveal the mechanism of action of ruthenium(II) complexes.
All three Ru(II) complexes could cross the cell membrane and
were located in the mitochondria. Notably, the complex RuY2
does not have cross-resistance with cisplatin due to different
mechanisms of action. Overall, this dinuclear ruthenium(II)
complex had unique biological properties and was a promising
model for novel anticancer drug development.
■
Jiaoyang Wang − Key Laboratory for the Synthesis and
Application of Organic Functional Molecules, College of
Chemistry and Chemical Engineering, Hubei University,
Wuhan 430062, P. R. China
Yufei Zhang − Key Laboratory for the Synthesis and
Application of Organic Functional Molecules, College of
Chemistry and Chemical Engineering, Hubei University,
Wuhan 430062, P. R. China
Yifan Li − Key Laboratory for the Synthesis and Application of
Organic Functional Molecules, College of Chemistry and
Chemical Engineering, Hubei University, Wuhan 430062, P.
R. China
Enbo Li − Key Laboratory for the Synthesis and Application of
Organic Functional Molecules, College of Chemistry and
Chemical Engineering, Hubei University, Wuhan 430062, P.
R. China
ASSOCIATED CONTENT
sı Supporting Information
*
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.inorgchem.2c00714.
Experimental details, additional spectroscopic information, and biological supplementary data (PDF)
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.inorgchem.2c00714
Accession Codes
CCDC 2130307 and 2130966 contain the supplementary
crystallographic data for this paper. These data can be obtained
free of charge via www.ccdc.cam.ac.uk/data_request/cif, or by
emailing data_request@ccdc.cam.ac.uk, or by contacting The
Cambridge Crystallographic Data Centre, 12 Union Road,
Cambridge CB2 1EZ, UK; fax: +44 1223 336033.
■
Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS
This work was funded by grants from the National Natural
Science Foundation of China [Nos. 22007029, 21502120].
■
AUTHOR INFORMATION
Corresponding Authors
Wenjing Ye − Key Laboratory for the Synthesis and
Application of Organic Functional Molecules, College of
Chemistry and Chemical Engineering, Hubei University,
Wuhan 430062, P. R. China; National & Local Joint
Engineering Research Center of High-Throughput Drug
Screening Technology, Hubei University, Wuhan 430062, P.
R. China; Email: wye@hubu.edu.cn
Jie Pan − Key Laboratory for the Synthesis and Application of
Organic Functional Molecules, College of Chemistry and
Chemical Engineering, Hubei University, Wuhan 430062, P.
R. China; orcid.org/0000-0001-9245-0700;
Email: j.pan@hubu.edu.cn
REFERENCES
(1) Johnstone, T. C.; Suntharalingam, K.; Lippard, S. J. The next
generation of platinum drugs: targeted Pt (II) agents, nanoparticle
delivery, and Pt (IV) prodrugs. Chem. Rev. 2016, 116, 3436−3486.
(2) Rosenberg, B.; Vancamp, L.; Krigas, T. Inhibition of cell division
in escherichia coli by electrolysis products from a platinum electrode.
Nature 1965, 205, 698−699.
(3) Harrap, K. R. Preclinical studies identifying carboplatin as a
viable cisplatin alternative. Cancer Treat. Rev. 1985, 12, 21−33.
(4) Cvitkovic, E.; Bekradda, M. Oxaliplatin: a new therapeutic
option in colorectal cancer. Semin. Oncol. 1999, 26, 647−662.
(5) Zhao, J.; Li, S.; Wang, X.; Xu, G.; Gou, S. Dinuclear
organoruthenium complexes exhibiting antiproliferative activity
through DNA damage and a reactive-oxygen-species-mediated
8280
https://doi.org/10.1021/acs.inorgchem.2c00714
Inorg. Chem. 2022, 61, 8267−8282
Inorganic Chemistry
pubs.acs.org/IC
endoplasmic reticulum stress pathway. Inorg. Chem. 2019, 58, 2208−
2217.
(6) Liu, P.; Wu, B.; Liu, J.; Dai, Y.; Wang, Y.; Wang, K. DNA binding
and photocleavage properties, cellular uptake and localization, and invitro cytotoxicity of dinuclear ruthenium (II) complexes with varying
lengths in bridging alkyl linkers. Inorg. Chem. 2016, 55, 1412−1422.
(7) Adhireksan, Z.; Davey, G. E.; Campomanes, P.; Groessl, M.;
Clavel, C. M.; Yu, H.; Nazarov, A. A.; Yeo, C. H. F.; Ang, W. H.;
Droge, P.; Rothlisberger, U.; Dyson, P. J.; Davey, C. A. Ligand
substitutions between ruthenium-cymene compounds can control
protein versus DNA targeting and anticancer activity. Nat. Commun.
2014, 5, No. 3462.
(8) Soldevila-Barreda, J. J.; Romero-Canelon, I.; Habtemariam, A.;
Sadler, P. J. Transfer hydrogenation catalysis in cells as a new
approach to anticancer drug design. Nat. Commun. 2015, 6, No. 6582.
(9) Zhao, J.; Zhang, D.; Hua, W.; Li, W.; Xu, G.; Gou, S. Anticancer
activity of bifunctional organometallic Ru (II) arene complexes
containing a 7-hydroxycoumarin group. Organometallics 2018, 37,
441−447.
(10) Milutinović, M. M.; Č anović, P. P.; Stevanović, D.; Masnikosa,
R.; Vraneš, M.; Tot, A.; Zarić, M. M.; Marković, B. S.; Marjanović, M.
M.; Vučićević, L.; Savić, M.; Jakovljević, V.; Trajković, V.; Volarević,
V.; Kanjevac, T.; Simović, A. R. Newly synthesized heteronuclear
ruthenium (II)/ferrocene complexes suppress the growth of
mammary carcinoma in 4T1-treated BALB/c mice by promoting
activation of antitumor immunity. Organometallics 2018, 37, 4250−
4266.
(11) Pan, J.; Jiang, L.; Chan, C.; Tsoi, T.; Shiu, K.; Kwong, D. W. J.;
Wong, W.; Wong, W.; Wong, K. Excitation energy transfer in
ruthenium (II)-porphyrin conjugates led to enhanced emission
quantum yield and 1O2 generation. J. Lumin. 2017, 184, 89−95.
(12) Brabec, V.; Kasparkova, J. Ruthenium coordination compounds
of biological and biomedical significance. DNA binding agents. Coord.
Chem. Rev. 2018, 376, 75−94.
(13) Bergamo, A.; Pelillo, C.; Chambery, A.; Sava, G. Influence of
components of tumour microenvironment on the response of HCT116 colorectal cancer to the ruthenium-based drug NAMI-A. J. Inorg.
Biochem. 2017, 168, 90−97.
(14) Gransbury, G. K.; Kappen, P.; Glover, C. J.; Hughes, J. N.;
Levina, A.; Lay, P. A.; Musgrave, I. F.; Harris, H. H. Comparison of
KP1019 and NAMI-A in tumour-mimetic environments. Metallomics
2016, 8, 762−773.
(15) Bytzek, A. K.; Koellensperger, G.; Keppler, B. K.; Hartinger, C.
G. Biodistribution of the novel anticancer drug sodium trans[tetrachloridobis (1H-indazole) ruthenate (III)] KP-1339/IT139 in
nude BALB/c mice and implications on its mode of action. J. Inorg.
Biochem. 2016, 160, 250−255.
(16) Jose, D. A.; Sakla, R.; Sharma, N.; Gadiyaram, S.; Kaushik, R.;
Ghosh, A. Sensing and bioimaging of the gaseous signaling molecule
hydrogen sulfide by near-infrared fluorescent probes. ACS Sens. 2020,
5, 3365−3391.
(17) Zhang, J.; Ye, H.; Jin, Y.; Han, D. Recent Progress in NearInfrared Organic Electroluminescent Materials. Top. Curr. Chem.
2022, 380, No. 6.
(18) Wu, C.; Mao, Y.; Wang, X.; Li, P.; Tang, B. Deep-tissue
fluorescence imaging study of reactive oxygen species in a tumor
microenvironment. Anal. Chem. 2022, 94, 165−176.
(19) Zhang, Y.; Wang, Y.; Song, J.; Qu, J.; Li, B.; Zhu, W.; Wong, W.
Y. Near-infrared emitting materials via harvesting triplet excitons:
molecular design, properties, and application in organic light emitting
diodes. Adv. Opt. Mater. 2018, 6, No. 1800466.
(20) Wu, Y.; Wu, J.; Wong, W. Y. A new near-infrared
phosphorescent iridium (III) complex conjugated to a xanthene dye
for mitochondria-targeted photodynamic therapy. Biomater. Sci. 2021,
9, 4843−4853.
(21) Wu, J.; Li, Y.; Tan, C.; Wang, X.; Zhang, Y.; Song, J.; Qu, J.;
Wong, W. Y. Aggregation-induced near-infrared emitting platinum
(ii) terpyridyl complex: cellular characterisation and lysosome-specific
localisation. Chem. Commun. 2018, 54, 11144−11147.
Article
(22) Kloster, M. B. G.; Hannis, J. C.; Muddiman, D. C.; Farrell, N.
Consequences of nucleic acid conformation on the binding of a
trinuclear platinum drug. Biochemistry 1999, 38, 14731−14737.
(23) Haribabu, J.; Sabapathi, G.; Tamizh, M. M.; Balachandran, C.;
Bhuvanesh, N. S. P.; Venuvanalingam, P.; Karvembu, R. Watersoluble mono- and binuclear Ru(η6-p-cymene) complexes containing
indole thiosemicarbazones: synthesis, DFT modeling, biomolecular
interactions, and in vitro anticancer activity through apoptosis.
Organometallics 2018, 37, 1242−1257.
(24) Zeng, L.; Gupta, P.; Chen, Y.; Wang, E.; Ji, L.; Chao, H.; Chen,
Z. The development of anticancer ruthenium (II) complexes: from
single molecule compounds to nanomaterials. Chem. Soc. Rev. 2017,
46, 5771−5804.
(25) Baggaley, E.; Gill, M. R.; Green, N. H.; Turton, D.; Sazanovich,
I. V.; Botchway, S. W.; Smythe, C.; Haycock, J. W.; Weinstein, J. A.;
Thomas, J. A. Dinuclear ruthenium (II) complexes as two-photon,
time-resolved emission microscopy probes for cellular DNA. Angew.
Chem., Int. Ed. 2014, 53, 3367−3371.
(26) Liu, H.-Y.; Zhang, S.; Cui, M.; Gao, L.; Zhao, H.; Wang, K. pHsensitive near-IR emitting dinuclear ruthenium complex for
recognition, two-photon luminescent imaging, and subcellular localization of cancer cells. ACS Appl. Bio Mater. 2020, 3, 5420−5427.
(27) Raza, A.; Archer, S. A.; Fairbanks, S. D.; Smitten, K. L.;
Botchway, S. W.; Thomas, J. A.; MacNeil, S.; Haycock, J. W. A
dinuclear ruthenium (II) complex excited by near-infrared light
through two-photon absorption induces phototoxicity deep within
hypoxic regions of melanoma cancer spheroids. J. Am. Chem. Soc.
2020, 142, 4639−4647.
(28) Havrylyuk, D.; Heidary, D. K.; Nease, L.; Parkin, S.; Glazer, E.
C. Photochemical properties and structure-activity relationships of
RuII complexes with pyridylbenzazole ligands as promising anticancer
agents. Eur. J. Inorg. Chem. 2017, 2017, 1687−1694.
(29) Yılmaz, I.; Akar, O. R.; Erkisa, M.; Selvi, S.; Ş engül, A.; Ulukaya,
E. Highly promising antitumor agent of a novel platinum (II) complex
bearing a tetradentate chelating ligand. ACS Med. Chem. Lett. 2020,
11, 940−948.
(30) Yellol, G. S.; Donaire, A.; Yellol, J. G.; Vasylyeva, V.; Janiak, C.;
Ruiz, J. On the antitumor properties of novel cyclometalated
benzimidazole Ru (II), Ir (III) and Rh (III) complexes. Chem.
Commun. 2013, 49, 11533−11535.
(31) Ye, W.; Zhao, M.; Du, W.; Jiang, Q.; Wu, K.; Wu, P.; Yu, Z.
Highly Active Ruthenium (II) Complex Catalysts Bearing an
Unsymmetrical NNN Ligand in the (Asymmetric) Transfer Hydrogenation of Ketones. Chem. - Eur. J. 2011, 17, 4737−4741.
(32) Four, M.; Riehl, D.; Mongin, O.; Blanchard-Desce, M.;
Lawson-Daku, L. M.; Moreau, J.; Chauvin, J.; Delaire, J. A.;
Lemercier, G. A novel ruthenium (II) complex for two-photon
absorption-based optical power limiting in the near-IR range. Phys.
Chem. Chem. Phys. 2011, 13, 17304−17312.
(33) Sullivan, B. P.; Salmon, D. J.; Meyer, T. J. Mixed phosphine 2,
2′-bipyridine complexes of ruthenium. Inorg. Chem. 1978, 17, 3334−
3341.
(34) Brouwer, A. M. Standards for photoluminescence quantum
yield measurements in solution. Pure Appl. Chem. 2011, 83, 2213−
2228.
(35) Barton, J. K.; Danishefsky, A.; Goldberg, J. Tris(phenanthroline)ruthenium (II): stereoselectivity in binding to
DNA. J. Am. Chem. Soc. 1984, 106, 2172−2176.
(36) Hiort, C.; Lincoln, P.; Norden, B. DNA binding of.DELTA.and.LAMBDA.-[Ru(phen)2DPPZ]2+. J. Am. Chem. Soc. 1993, 115,
3448−3454.
(37) Mardanya, S.; Karmakar, S.; Mondal, D.; Baitalik, S. Homo- and
heterobimetallic rutheniumn (II) and osmium (II) complexes based
on a pyrene-biimidazolate spacer as efficient DNA-binding probes in
the near-infrared domain. Inorg. Chem. 2016, 55, 3475−3489.
(38) Singer, S. J.; Nicolson, G. L. The fluid nosaic model of the
structure of cell membranes: cell membranes are viewed as twodimensional solutions of oriented globular proteins and lipids. Science
1972, 175, 720−731.
8281
https://doi.org/10.1021/acs.inorgchem.2c00714
Inorg. Chem. 2022, 61, 8267−8282
Inorganic Chemistry
pubs.acs.org/IC
Article
(39) Poon, C.-T.; Chan, P.; Man, C.; Jiang, F.; Wong, R. N. S.; Mak,
N.; Kwong, D. W.; Tsao, S.; Wong, W. An amphiphilic ruthenium
(II)-polypyridyl appended porphyrin as potential bifunctional twophoton tumor-imaging and photodynamic therapeutic agent. J. Inorg.
Biochem. 2010, 104, 62−70.
(40) Gümüs,̧ F.; Eren, G.; Acik, L.; Celebi, A.; Ozturk, F.; Yilmaz, S.;
Sagkan, R. I.; Gur, S.; Ozkul, A.; Elmali, A.; Elerman, Y. Synthesis,
cytotoxicity, and DNA interactions of new cisplatin analogues
containing substituted benzimidazole ligands. J. Med. Chem. 2009,
52, 1345−1357.
Recommended by ACS
Photochemistry of Heteroleptic 1,4,5,8Tetraazaphenanthrene- and Bi-1,2,3-triazolyl-Containing
Ruthenium(II) Complexes
Rayhaan Z. Boota, Paul I. P. Elliott, et al.
OCTOBER 06, 2021
INORGANIC CHEMISTRY
READ
In Vitro Photodynamic Therapy of Mononuclear and
Dinuclear Iridium(III) Bis(terpyridine) Complexes
Cuifen Lu, Wenfang Sun, et al.
AUGUST 28, 2020
ACS APPLIED BIO MATERIALS
READ
Dual-Action Ru(II) Complexes with Bulky π-Expansive
Ligands: Phototoxicity without DNA Intercalation
Nicholas P. Toupin, Jeremy J. Kodanko, et al.
FEBRUARY 25, 2020
INORGANIC CHEMISTRY
READ
Making the Right Link to Theranostics: The Photophysical
and Biological Properties of Dinuclear RuII–ReI dppz
Complexes Depend on Their Tether
Hiwa K. Saeed, Jim A. Thomas, et al.
DECEMBER 17, 2019
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
READ
Get More Suggestions >
8282
https://doi.org/10.1021/acs.inorgchem.2c00714
Inorg. Chem. 2022, 61, 8267−8282