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New cyclometalated Ir(iii) complexes with NCN pincer and meso-phenylcyanamide BODIPY ligands as efficient photodynamic therapy agents
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Cite this: RSC Adv., 2017, 7, 34160
New cyclometalated Ir(III) complexes with NCN
pincer and meso-phenylcyanamide BODIPY ligands
as efficient photodynamic therapy agents†
Leila Tabrizi
*ab and Hossein Chiniforoshan*b
A new class of cyclometalated iridium(III) with NCN pincer and meso-phenylcyanamide BODIPY ligands of
the formula [Ir(L)(PPY)(Pcyd-BODIPY)], 1, [Ir(L)(PIQ)(Pcyd-BODIPY)], 2, in which HL: 5-methoxy-1,3-bis (1methyl-1H-benzo[d]imidazol-2-yl)benzene, Pcyd-BODIPY: 8-(4-phenylcyanamide) BODIPY, PPY: 2phenylpyridine, PIQ: 1-phenylisoquinoline have been synthesized and studied for photodynamic therapy
(PDT). The photophysical properties, DNA binding, DNA photocleavage, cellular uptake, thioredoxin
Received 17th May 2017
Accepted 2nd July 2017
reductase activity, reactive oxygen species (ROS) generation, and cellular apoptosis of the complexes
DOI: 10.1039/c7ra05579j
have also been studied. Therefore, we present a conceivable biologically applicable PDT modality that
rsc.li/rsc-advances
develops persuasively designed photoactivatable Ir(III) complexes.
1. Introduction
Photodynamic therapy (PDT) has been successfully used for
cancer treatment and involves the interaction of the photosensitizer, light, and molecular oxygen.1–5 Recently, a rutheniumbased photosensitizer (TLD-1433) has been approved for
phase Ib clinical trials in Canada for non-muscle invasive
bladder cancer.6–8
Some cyclometalated Ru(II), Ir(III), and Pt(II) complexes, Ru(II)
arene complexes, and Re(I) tricarbonyl complexes have proven
themselves for photocytotoxicity studies.9–15 Cyclometalated IrIII
complexes have recently received great interest for PDT agents
due to their exceptional properties such as simple color tuning,
energy-level control, high quantum yields, large Stokes shis,
long-lived phosphorescence, and ROS generation under hypoxia
conditions via electron (type I) or energy transfer (type II).16–27
BODIPY (boron dipyrromethene) derivatives have found to
be applicable as potential applications for biological labeling,
cellular imaging, and uorescence sensing, unique narrow
emission with high quantum yield in biological and medical
elds. Furthermore, it has been found that the heavy atom
substituted BODIPY dyes containing an electron-withdrawing
acetoxymethyl moiety in the meso-position could be enhanced
photostability for the PDT agents.28–32 Recently, our groups
focused attention in the development Ni(II), Zn(II), Cd(II), Co(II),
Ru(II), Ir(III), Pt(II), Pd(II), and triorganotin(IV) complexes
a
School of Chemistry, National University of Ireland, Galway, University Road,
Galway, Ireland. E-mail: LEILA.TABRIZI@nuigalway.ie
b
Department of Chemistry, Isfahan University of Technology, Isfahan, Iran. E-mail:
Chinif@cc.iut.ac.ir
† Electronic supplementary
10.1039/c7ra05579j
information
34160 | RSC Adv., 2017, 7, 34160–34169
(ESI)
available.
See
DOI:
containing phenylcyanamide derivatives as monodentate
ligands in regard to traditional chemotherapy and PDT applications.33–39 With the aim of enhancing the photophysical and
photobiological properties of BODIPY ligands in cyclometalated
Ir(III) complexes, we have designed new monodentate ligand by
linking meso-BODIPY ligands to phenylcyanamide ligands.
Here we present the synthesis, characterization, photophysical properties, and photocytotoxicity explorations of two
new cyclometalated Ir(III) complexes of the type [Ir(L)(PPY)(PcydBODIPY)], 1, [Ir(L)(PIQ)(Pcyd-BODIPY)], 2, in which HL: 5methoxy-1,3-bis (1-methyl-1H-benzo[d]imidazol-2-yl)benzene,
Pcyd-BODIPY: 8-(4-phenylcyanamide) BODIPY, PPY: 2-phenylpyridine, PIQ: 1-phenylisoquinoline.
2.
Results and discussion
2.1. Synthesis and characterization
The ligand 8-(4-phenylcyanamide) BODIPY (Pcyd-BODIPY) was
synthesized from 8-(4-anilino) BODIPY as previously reported.40
The ligand HL was synthesized as previously reported by us.41
Reaction of IrCl3$xH2O with pincer ligand HL in MeOH/CH2Cl2
(1 : 1, v/v) and then PPY (2-phenylpyridine) or PIQ (1-phenylisoquinoline) and Pcyd-BODIPY in MeOH/CH2Cl2 (1 : 2, v/v) in
pH 7 gave the complexes 1 and 2 (Fig. 1).
The ligand Pcyd-BODIPY and complexes were prepared in
excellent yield and in high purity as described in the experimental section. Complexes were characterized by elemental
analysis, IR, 1H NMR, 13C NMR spectroscopy (Fig. S1–S6†) and
ESI-TOF mass spectrometry (Fig. S7–S9†).
The formation of Pcyd-BODIPY, 1, and 2 is clearly evidenced
by the parent peaks in the mass spectra. The mass spectra of
Pcyd-BODIPY, 1, and 2 show peaks of (M + H)+ centered at m/z ¼
365, 1078, and 1128, respectively (Fig. S7–S9†).
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Fig. 1
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Structure of complexes 1 and 2.
The disappearance of the H1 signal (7.54 ppm in ligand HL)
and the upeld shi of the signal of H3/H5 of the central phenyl
ring (7.29–7.35 ppm in ligand HL and 6.75 and 6.79 ppm in 1
and 2, respectively) in the 1H NMR spectra of 1 and 2 are
consistent with the coordination of Ir to the deprotonated
pincer ligand. The H40 resonance is shied downeld due to the
ring current effect of the second ligand in complexes 1 and 2
(7.84–7.79 ppm in ligand HL and 8.31 and 8.80 ppm in 1 and 2,
respectively).41
The proton chemical shis of Pcyd-BODIPY decrease with
respect to the free ligand due to coordination of ligand PcydBODIPY to Ir. Also, the NH proton of ligand Pcyd-BODIPY disappeared due to coordination of the cyanamide group to Ir in
complexes 1 and 2.
The n (NCN) band of ligand Pcyd-BODIPY is observed at 2225
cm1, when the Pcyd-BODIPY ligand coordinates to Ir, n (NCN)
of complexes 1 and 2 is shied to lower energies (2092 and 2088
cm1, respectively) representing the coordination of cyanamide
through the cyano nitrogen.
2.2. Photophysical properties
The photophysical properties of Ir(III) complexes in acetonitrile
and phosphate buffer solution (PBS) were considered at 298 K
(Fig. S10, S11, and Table S1†). The complexes 1 and 2 display the
absorption bands of lower intensity in the 300–480 nm region due
to intraligand (IL) p / p* and n / p* transitions and singlet
and triplex metal-to-ligand charge transfer (1MLCT and 3MLCT).42
In addition, the absorption spectra of complexes 1 and 2 are
dominated by the intense bands at 541 and 526 nm in acetonitrile, respectively, due to the S0 / S1 (p–p*) transition localized
on the BODIPY unit.43 The complexes 1 and 2 exhibited red
emissions in acetonitrile and PBS upon excitation at 500 nm.
The luminescence quantum yields (Fem) and luminescence
lifetimes were evaluated in acetonitrile at room temperature
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(Table S1†). The luminescence lifetimes of complexes 1 and 2
are around 700 ns. These complexes have higher quantum
yields and longer lifetimes than [Ru(bpy)3]2+ and Ru(II) pincer
complexes.37,42
2.3. Singlet oxygen sensitization
The major cytotoxic species in PDT is the singlet oxygen
to induce cancer cell death.44 Therefore, the determination of
1
O2 quantum yields of complexes 1 and 2 was made upon
irradiation at 500 nm (Table S2†). The 1O2 photosensitizing
properties of 1 and 2 were investigated by direct and indirect
measurement.37
The complexes 1 and 2 have F (1O2) values of 79% and 92%
in acetonitrile (500 nm, direct method), respectively. The obtained values for 1 and 2 in acetonitrile are comparable with 1O2
quantum yields reported for related compounds.37,45–47 These
data display that the complexes 1 and 2 have a great efficacy in
the photosensitization of molecular oxygen. Moreover, the
quantum yields of 1O2 production in PBS are low (Table S2†).
Thus, the effect of solvent on the efficiency of 1O2 production is
obvious. In acetonitrile, the photosensitization of molecular
oxygen is effective due to the absence of quenching mechanism
on the triplet excited state.48
As a result, these complexes can produce 1O2 when they
accumulate in hydrophobic compartments and complex 2 has
the highest quantum yield of singlet oxygen production, emission and uorescence lifetime.
2.4. Interaction of 1–3 with DNA
2.4.1. DNA binding studies. DNA is generally the primary
intracellular target of anticancer drugs, so the interaction
between small molecules and DNA can block the division of
cancer cells and cause in cell death.49,50 The interaction of 1 and
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Absorption spectra of complexes 1 and 2 in Tris–HCl, pH 7.2, buffer upon addition of CT DNA. [Complex] ¼ 5.0 mM, [DNA] ¼ 0–100 mM.
The arrows show the direction of increasing DNA concentration.
Fig. 2
2 with CT DNA was studied by UV-vis absorption and uorescence spectroscopy.
Electronic absorption spectroscopy is an efficient method to
study the binding mode of metal complexes to DNA. The
binding ability of complexes 1 and 2 with CT DNA was considered by considering their effects on UV absorption. The Kb
values were measured in Tris–HCl buffer (pH 7.4) by changing
the concentration of DNA from 0 to 100 mM while keeping the
complex concentration constant (5 mM). The absorption spectra
of 1 and 2 in the absence and presence of increasing amounts of
CT DNA are shown in Fig. 2.
The complexes 1 and 2 exhibited hypochromicity upon
adding CT DNA. Such marked changes in the intensity of
spectral bands conrm an intimate association of the complex
with CT DNA. The Kb values of complexes 1 and 2 are (4.39
0.2) 105 M1 and 1.31 106 M1, respectively (Fig. 2, inset).
Moreover, the s values of complexes 1 and 2 are (0.51 0.03)
and (0.91 0.03), respectively. A low value of s (<1) could be due
to an aggregation of hydrophobic molecules on the DNA surface
by p stacking.51
This result suggests that the two complexes maybe bind to
DNA by partial intercalation, involving a strong stacking interaction between the aromatic chromophore and the base pairs of
the DNA. In addition, complex 2 binds with DNA more strongly
than the complex 1 due to the stronger electrostatic interaction
between DNA and complex 2.
The binding of the complexes 1 and 2 to CT DNA was further
considered by a competitive binding uorescence assay using
ethidium bromide (EB). The EB–DNA system usually displays
a major increase in the uorescence emission when EB binds to
DNA, and a decrease is observed when EB is displaced by
another DNA intercalate molecule.52,53
Fig. 3 shows the uorescence spectra of the EB–DNA system
on addition of increasing amounts of complexes 1 and 2. The
intensity of the emission band at 601 nm of the EB–DNA system
decreases by increasing concentrations of 1 and 2.
The Ksv values that were obtained from the experiments are
(1.02 0.01) 105 M1 and (2.23 0.03) 105 M1 for
complexes 1 and 2, respectively (Fig. 3, inset). Kapp was found to
be (1.15 0.02) 105 M1 and (2.62 0.02) 105 M1 for
complexes 1 and 2, respectively.
These results show the same magnitude of the intrinsic
binding constants determined from the absorption spectra
titration, further demonstrating the absorption spectral observations that the complex 2 with the ligand PIQ can intercalate to
DNA more strongly than the complex 1.
2.4.2. DNA photocleavage studies. The DNA photocleavage
activity of 1 and 2 was considered using SC pUC19 DNA in Tris–
HCl/NaCl buffer by irradiating the samples with visible light of
500 nm. The complexes remain cleavage inactive in dark
condition thereby prohibit possibility of any hydrolytic
cleavage. All the complexes display photoinduced DNA cleavage
activity in micromolar complex concentration. The complexes 1
display 73% and complex 2 show 84% cleavage of SC DNA to its
nicked circular (NC) form on photoirradiation at 500 nm with
a complex concentration of 100 mM for 1 h (Fig. 4).
Fig. 3 Fluorescence quenching curves of EtBr bound to DNA: 1 and 2. [DNA] ¼ 10 mM, [EB] ¼ 10 mM, [complex] ¼ 0–100 mM (inset: plot of I0/I
vs. [Q]).
34162 | RSC Adv., 2017, 7, 34160–34169
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IC50 values for all the complexes incubated with MRC-5 and
HeLa cells in the dark and upon light irradiation
Table 1
IC50 mM
Cleavage of SC pUC19 DNA (0.2 mg, 30 mM) by the complexes 1
and 2 (100 mM) in 50 mM Tris–HCl/NaCl buffer (pH, 7.2) on photoirradiation at 500 nm for 1 h: (lane 1) DNA control; (lane 2) DNA + HL;
(lane 3) DNA + PPY; (lane 4) DNA + PIQ; (lane 5) DNA + Pcyd-BODIPY;
(lane 6) DNA + 1 (in dark); (lane 7), DNA + 2 (in dark); (lane 8) DNA + 1;
(lane 9) DNA + 2.
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Fig. 4
HeLa (dark)a
HeLa (light)b
PI (HeLa)
MRC-5 (dark)a
MRC-5 (light)b
PI (MRC-5)
a
Addition of hydroxyl radical scavengers like DMSO, KI,
catalase and superoxide radical scavengers such as superoxide
dismutase, SOD did not inhibit the DNA photocleavage reaction
whereas singlet oxygen quenchers such as 2,2,6,6-tetramethyl-4piperidone (TEMP), 1,4-diazabicyclo-[2.2.2]octane (DABCO),
and NaN3 inhibited the DNA photocleavage activity signicantly
(Fig. 5). These results suggested 1O2 are likely to be the reactive
species that contribute to the cleavage process.
2.5. Biological activity
2.5.1. Photostability and stability in human plasma. To
assess the photostability, complexes 1 and 2 were exposed to
continuous light irradiation (500 nm) and found to reveal
excellent photostability (Fig. S12†). To evaluate the stability of
the complexes 1 and 2 in biological media, the stability studies
was performed in human plasma by HPLC. As clearly evident
from the HPLC-UV traces (Fig. S13, Table S3†), 1 and 2 displayed
no obvious decomposition in plasma aer three days and
should be stable under physiological conditions.
2.5.2. Dark cytotoxicity and phototoxicity. The dark- and
photo-cytotoxicity of the complexes 1 and 2 toward cervical
cancer (HeLa) and non-cancerous (MRC-5) cell lines was
evaluated using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide) assay. The untreated control
cells remained unaffected upon exposure to the same irradiation procedure and the light alone was found to be nontoxic.
The complexes 1 and 2 are signicantly less toxic in the dark
with IC50 values of 60 mM (Table 1). Complexes 1 and 2,
however, were signicantly photocytotoxic in HeLa cells when
irradiated with visible light of 500 nm giving IC50 values of 0.78
0.1 mM and 0.53 0.1 mM, respectively. Upon light irradiation, complex 2 with the ligand PIQ showed a higher cytotoxic
activity in HeLa cells than 1. These IC50 values of complexes 1
Fig. 5 Photo-induced DNA cleavage activity of complex 2 (100 mM) in
the presence of different additives at 500 nm for an exposure time of
1 h: (lane 1) DNA control; (lane 2) DNA + DMSO (2 mL) + 2; (lane 3) DNA
+ KI (0.2 mM) + 2; (lane 4) DNA + catalase (4 U) + 2; (lane 5) DNA +
SOD (4 U) + 2; (lane 6) DNA + TEMP (0.2 mM) + 2; (lane 7) DNA +
DABCO (0.2 mM) + 2; (lane 8) DNA + NaN3 (0.2 mM) + 2.
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Pcyd-BODIPY
1
2
Cisplatin
78.52 0.2
11.34 0.4
6.92
80.15 0.2
53.41 0.1
1.50
59.31 0.3
0.78 0.1
76.04
60.58 0.1
23.82 0.1
2.54
60.11 0.2
0.53 0.1
113.41
61.03 0.1
31.41 0.2
1.94
9.2 1.7
27.5 1.1
—
17.2 1.3
16.4 1.1
—
24 h incubation. b Irradiation with light at 500 nm (10 J cm2).
and 2 are well comparable with the activity of photofrin (IC50 in
HeLa cells ¼ 4.3 0.2 mM (visible light) and >41 mM (dark)),
which is approved PDT agent.54
The phototoxicity index (PI ¼ IC50 (dark)/IC50 (light)) values
of complexes 1 and 2 in HeLa cells are 76.04 and 113.41,
respectively. These values are auspicious considering that the
commonly used photosensitizer available on the market (photofrin) with PI > 10.55
The phototoxicity of complexes 1 and 2 on the MRC-5 cell
line was also considered (Table 1), which followed the same
trend observed for HeLa cells. Notably, 1 and 2 are only
moderately photocytotoxic against the nontumorigenic MRC-5
cell line (IC50 ¼ 23.82 and 31.41 mM).
The ligand Pcyd-BODIPY is less toxic in the dark with IC50
values of 78 mM, but was signicantly photocytotoxic in HeLa
cells when irradiated with visible light of 500 nm giving IC50
value of 11.34 0.4 mM (Table 1). Also, Pcyd-BODIPY is
moderately photocytotoxic against the nontumorigenic MRC-5
cell line with IC50 ¼ 53.41 mM.
These complexes are comparable with cyclometalated Ir(III)
complexes of styryl-BODIPY ligands with IC50 values 2.58–6.18
mM and 8.16–9.81 mM on photoirradiation and in the dark for
LLC cells (lung cancer cells) cell lines, respectively.56
2.5.3. Cellular uptake. The amounts of iridium associated
with HeLa cells incubated with the complexes 1 and 2 were
determined using ICP-MS. An average cell contained 7.28 0.2
and 13.06 0.1 fmol of iridium for complexes 1 and 2,
respectively, which is comparable to those reported in the
cellular uptake studies of other iridium complexes.57,58 The
complex 1 exhibited lower cellular uptake efficiency than the
complex 2. The cellular uptake efficiency of complexes 1 and 2 is
closely related to their cytotoxicity that is in accordance with
other reported studies.57,58
2.5.4. ROS generation and cellular apoptosis. To evaluate
the potential of complexes 1 and 2 to generate intracellular
reactive oxygen species (ROS), HeLa cells were treated with of
complexes 1 and 2 for 12 h and stained with 20 ,70 -dichlorouorescein diacetate (DCFH-DA) (Fig. 6). DCFH-DA can be
oxidized by cellular ROS to produce 2,7-dichlorouorescein
(DCF), thereby providing an emission band centered at 525 nm
(lex ¼ 490 nm).27 To determine the formation of ROS inside the
cells, HeLa cells were incubated with complexes 1 and 2 for 4 h
in the dark, and then were exposed to light irradiation (500 nm)
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Cellular ROS generation by complexes 1 and 2 in HeLa cells in dark or light conditions as estimated by DCFDA assay (pre-incubation: 4 h in
dark, light exposure: 500 nm, 1 h).
Fig. 6
for 1 h. The results exhibit that the complexes generate ROS in
HeLa cells only upon photo-irradiation but not in the dark
(Fig. 6). A good shi in the uorescence bands was observed for
the cells treated with the photo-irradiated complex 2 as
compared to complex 1 that could be related to the higher
cellular uptake of complex 2 than 1 (Fig. 6 and 7).
To evaluate the number of cells undergoing apoptosis, the
Annexin-V-FITC/PI assay was performed by complexes 1 and 2 in
HeLa cells. Cells were incubated with the complexes for 4 h in
dark followed by light irradiation (500 nm). Complex 2 induced
features of early apoptosis in 86% of the cells upon light (500
nm) exposure in comparison with 64% for complex 1 under
identical experimental conditions. The observed early apoptotic
cell population in the dark was 16.46% and 16.17% for 1 and 2,
respectively, demonstrating the importance of light exposure to
observe apoptotic cell death (Fig. 8).
2.5.5. TrxR inhibition. As thioredoxin reductase is signicant in the intracellular redox metabolism and also plays
important roles as target for new anticancer compounds, TrxR
inhibition of complexes 1 and 2 were evaluated at increasing
concentrations, and the dose–effect curves are displayed in
Fig. 9. Furthermore, the TrxR-inhibitory activities of the
complexes were also compared with auranon, a gold phosphine complex widely used as positive TrxR inhibitor.59 As
presented in Fig. 9, under the same concentrations, the
DCFH-DA assay showing generation of ROS in HeLa cells
treated with complexes 1 and 2 for 4 h in the dark and exposed to light
(500 nm).
Fig. 7
34164 | RSC Adv., 2017, 7, 34160–34169
Fig. 8 Dot plots of annexinV-FITC/PI assay on HeLa cells treated with
complexes 1 and 2 either kept in dark or exposed to photoreactor (500
nm) as labeled showing % cell populations in respective quadrants:
FITC/PI lower left (Q3), viable cells; FITC+/PI lower right (Q4), early
apoptotic cells; FITC+/PI+ upper right (Q2), late apoptotic cells; FITC/
PI+ upper left (Q1), necrotic cells. Representative data from three
independent experiments are shown (pre-incubation: 4 h in dark, light
exposure: 500 nm, 1 h).
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Inhibitory effects of complexes 1 and 2 on TrxR. Error bars
indicate the standard deviation.
Fig. 9
complexes 1 and 2 were more effective than the positive control
auranon.
3.
Conclusions
Two new cyclometalated Ir(III) complexes bearing NCN pincer,
meso-phenylcyanamide BODIPY, and 2-phenylpyridine (1) or 1phenylisoquinoline (2) ligands have been demonstrated as
photodynamic therapy agents (PDT). The DNA-binding interactions of the complexes with CT DNA display that the binding
mode is mainly non-covalent via intercalation. The complexes 1
and 2 show cleavage SC-DNA efficiently on photoactivation at
500 nm with the formation of singlet oxygen (1O2) as reactive
species to contribute in the cleavage process. The complexes 1
and 2 are signicantly less toxic in the dark with IC50 values of
60 mM, while, upon light irradiation (500 nm), 1 and 2 were
signicantly photocytotoxic in HeLa cells with IC50 values of
0.78 0.1 mM and 0.53 0.1 mM, respectively. These complexes
showed very good uptake and efficient ROS generation in HeLa
cell lines upon photoexposure to light of 500 nm. Moreover, the
complexes 1 and 2 revealed exceptional TrxR-inhibitory activities in comparison with auranon. Therefore, the selenoenzyme
TrxR may be as a protein target for cyclometalated Ir(III)
complexes 1 and 2. Overall, these designing of cyclometalated
iridium complexes can open new opportunities for metal-based
PDT agents that may be used for a wide variety of applications
as potential anticancer drugs.
4. Experimental
4.1. General procedures, materials and physical
measurements
All manipulations were carried out under nitrogen atmosphere
using standard Schlenk techniques and vacuum-line systems.
Work-up procedures were performed in air. All materials used
were received from commercial sources unless stated otherwise.
The pincer ligand HL was synthesized as previously reported by
us.41 8-(4-Anilino) BODIPY (boron dipyrromethene diuoro) was
prepared according to literature method.60 All solvents were
dried by standard procedures and freshly distilled prior to use.
Human cervical cancer (HeLa), human broblast (MRC-5)
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nontumorigenic cells, and human plasma were supplied by
the National Cell Bank of Pasteur Institute, Tehran, Iran. All
reagents and cell culture media were purchased from Gibco
Company (Germany). CT DNA was obtained from SigmaAldrich.
Fourier transform infrared spectra were recorded on a PerkinElmer Spectrum 400 (FT-IR/FT-NIR spectrometer). 1H, 13C {1H}
nuclear magnetic resonance (NMR) spectra were recorded on
a Bruker-400 MHz spectrometer at ambient temperature in DMSOd6. Elemental analyses were performed with an EA 3000 CHNS.
Electronic absorption spectra were recorded on a JASCO 7580 UVvis-NIR double-beam spectrophotometer. Emission intensity
measurements were carried out using a Hitachi F 4500 spectrouorometer. Luminescence quantum yields were measured in
degassed acetonitrile using [Ru(bpy)3]Cl2 as the reference.
4.2. Synthesis of ligands and the complexes 1 and 2
4.2.1. Synthesis of 8-(4-phenylcyanamide) BODIPY, (PcydBODIPY). The ligand Pcyd-BODIPY was synthesized from 8-(4anilino) BODIPY as previously reported.40 Recrystallization from
ethyl acetate/hexane (1 : 1) produced analytically pure orange
solid (304 mg, 83.4%, and 1 mmol). Anal. calc. (%) for
C20H19BF2N4 (364.19): C, 65.96; H, 5.26; N, 15.38; found (%): C,
65.92; H, 5.22; N, 15.33. IR (KBr): 2225 (vs., NCN), 1548, 1512 (m,
C]C, C]N), 1192 (m, B–F) cm1. TOF-MS: 365 [M + H]+. 1H
NMR (DMSO-d6): d 8.132 (s, 1H, H–NH), 6.95 (d, 2H, H-11a or
12a, 3J 7.6), 6.77 (d, 2H, H-11a or 12a, 3J 7.6), 6.09 (s, 2H, H-2a
and H-6a), 2.28 (s, 6H, H-9a), 1.60 (s, 6H, H-10a). 13C NMR
(DMSO-d6): d 150.0 (Ar), 140.1 (Ar), 138.0 (Ar), 136.0 (Ar), 133.1
(Ar), 131.6 (Ar), 129.9 (Ar), 127.1 (Ar), 116.8 (Ar), 112.0 (NCN),
12.5 (C–CH3), 10.2 (C–CH3).
4.2.2. Synthesis of [Ir(L)(PPY)(Pcyd-BODIPY)], 1. IrCl3$xH2O (0.29 g, 1 mmol) and pincer ligand HL (390 mg, 1 mmol)
in MeOH/CH2Cl2 (20 mL, 1 : 1, v/v) was reuxed under an inert
atmosphere of nitrogen for 12 h. To this solution, PPY (2-phenylpyridine) (155 mg, 1 mmol) and Pcyd-BODIPY (8-(4-phenylcyanamide) BODIPY) (364 mg, 1 mmol) in MeOH/CH2Cl2
(30 mL, 1 : 2, v/v) was added. The pH of the solution was
adjusted to 7 using triethylamine. The solution was reuxed
again under an inert atmosphere for 24 h. The reaction mixture
was then cooled to room temperature and then evaporated
under vacuum. The residual orange solid was dissolved in
CH2Cl2 and puried by column chromatography on silica gel.
The desired product was eluted with CH2Cl2/MeOH (10 : 1, v/v)
and subsequent recrystallized from CH2Cl2/diethyl ether to
afford an orange solid (954 mg, 88.6% yield, and 1 mmol). Anal.
calc. (%) for C54H45BF2IrN9O (1077.02): C, 60.22; H, 4.21; N,
11.70; found (%): C, 60.18; H, 4.18; N, 11.65. IR (KBr): 2092 (vs.,
NCN), 1542, 1507 (m, C]C, C]N), 1187 (m, B–F) cm1. TOFMS: 1078 [M + H]+. 1H NMR (DMSO-d6): d 8.31 (d, 2H, H-40 , 3J
8.0), 8.24 (d, 1H, H-5b, 3J 7.6), 7.96 (t, 1H, H-4b, 3J 7.6), 7.86 (d,
1H, H-12b, 3J 7.6), 7.68 (d, 1H, H-2b, 3J 7.6), 7.25 (t, 2H, H-60 , 3J
7.6), 6.94–7.01 (m, 9H, H-phenyl), 6.75 (s, 2H, H-3,5), 6.61 (d,
2H, H-11a or 12a, 3J 7.6), 6.35 (d, 1H, H-9b, 3J 7.6), 6.00 (s, 2H, H2a,6a), 3.80 (s, 3H, H-7), 3.49 (s, 6H, H-100 ), 2.09 (s, 6H, H-9a),
1.60 (s, 6H, H-10a). 13C NMR (DMSO-d6): d 162.9 (Ar), 159.9
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(Ar), 153.1 (Ar), 149.9 (Ar), 147.9 (Ar), 144.1 (Ar), 141.0 (Ar), 138.5
(Ar), 136.6 (Ar), 134.5 (Ar), 131.1 (Ar), 129.9 (Ar), 127.5 (Ar), 125.8
(Ar), 123.0 (Ar), 121.1 (Ar), 119.2 (Ar), 117.9 (Ar), 114.9 (Ar), 110.0
(NCN), 51.4 (C-7), 29.9 (C-100 ), 11.0 (C–CH3), 9.0 (C–CH3).
4.2.3. Synthesis of [Ir(L)(PIQ)(Pcyd-BODIPY)], 2. The
synthesis was carried out as described for 1, with PIQ (1-phenylisoquinoline) (205 mg, 1 mmol) instead of PPY. The residual
orange solid was dissolved in CH2Cl2 and puried by column
chromatography on silica gel. The desired product was eluted
with CH2Cl2/MeOH (10 : 1, v/v) and subsequent recrystallized
from CH2Cl2/diethyl ether to afford an orange solid (927 mg,
82.3% yield, and 1 mmol). Anal. calc. (%) for C58H47BF2IrN9O
(1127.08): C, 61.81; H, 4.20; N, 11.18; found (%): C, 61.76; H,
4.16; N, 11.12. IR (KBr): 2088 (vs., NCN), 1538, 1503 (m, C]C,
C]N), 1185 (m, B–F) cm1. TOF-MS: 1128 [M + H]+. 1H NMR
(DMSO-d6): d 9.04 (d, 1H, H-8b, 3J 7.6), 8.80 (d, 2H, H-40 , 3J 8.0),
8.39 (d, 1H, H-16b, 3J 7.6), 8.05 (d, 1H, H-5b, 3J 7.6), 7.92 (t, 1H,
H-7b, 3J 7.6), 7.80 (t, 1H, H-6b, 3J 7.6), 7.60 (d, 2H, H-3b,4b, 3J
7.6), 7.29 (t, 1H, H-15, 3J 7.6), 6.99–7.06 (m, 10H, H-phenyl), 6.79
(s, 2H, H-3,5), 6.61 (d, 2H, H-11a or 12a, 3J 7.6), 6.00 (s, 2H, H2a,6a), 3.70 (s, 3H, H-7), 3.40 (s, 6H, H-100 ), 2.18 (s, 6H, H-9a),
1.49 (s, 6H, H-10a). 13C NMR (DMSO-d6): d 166.1 (Ar), 162.3
(Ar), 159.9 (Ar), 156.9 (Ar), 154.0 (Ar), 151.2 (Ar), 150.0 (Ar), 148.0
(Ar), 144.9 (Ar), 142.0 (Ar), 139.1 (Ar), 137.1 (Ar), 135.0 (Ar), 132.6
(Ar), 130.9 (Ar), 128.0 (Ar), 125.9 (Ar), 122.6 (Ar), 121.1 (Ar), 118.5
(Ar), 117.1 (Ar), 113.0 (Ar), 109.9 (NCN), 52.5 (C-7), 28.5 (C-100 ),
12.1 (C–CH3), 8.0 (C–CH3).
4.3. Singlet oxygen quantum yields
The singlet oxygen quantum yields were measured by two
different methods, direct and indirect evaluation, as previously
reported.37
4.4. DNA binding studies
I0/I ¼ 1 + Ksv[Q]
where, I0 and I are the uorescence intensities exhibited in the
absence and presence of the compounds, respectively; [Q]
corresponds to the concentration ratio of the compound to
DNA. The slope of the plot of I0/I versus [Q] gives Ksv.
The apparent binding constant (Kapp) of the complexes could
be calculated using the following equation:
Kapp ¼ KEB[EB]50%/[complex]50%
where KEB is the DNA-binding constant of EB (KEB ¼ 1.0
107 M 1), and [EB]50% and [complex]50% are the EB and complex
concentrations that reduce the uorescence by 50%.
The photocleavage of SC pUC19 DNA (0.2 mg, 30 mM) by the
complexes 1 and 2 (30 mM, 0.2 mg) in presence of complexes (100
mM) was performed in Tris–HCl/NaCl buffer (50 mM, pH 7.2) by
photo-irradiation of the samples with light of 500 nm (10 J cm2)
by agarose gel electrophoresis. Mechanistic studies were performed using different additives as ROS scavengers/quenchers
(TEMP, 0.2 mM; DABCO, 0.2 mM; NaN3, 0.2 mM; KI, 0.2 mM;
DMSO, 2 mL; catalase, 4 units; SOD, 4 units) prior to the addition
of the complexes. Aer incubation of the sample for 1 h in dark
and quenched by gel loading dye, solution was loaded on 1%
agarose gel having 1 mg mL1 ethidium bromide. Electrophoresis
was run for 2.0 h at 60 V in Tris–acetate EDTA (TAE) buffer in dark
room. The quantication of cleavage products was performed
using UVITEC Fire Reader V4 gel documentation system and UVI
band soware. The error observed in measuring the band
intensities was in the range of 4–7%.
4.5. Stability studies
The complexes were dissolved in 10 mM Tris–HCl buffer (pH
7.4). The concentration of CT DNA was determined from the UV
absorption intensity at 260 nm with 3 ¼ 6600 M1 cm1.61 The
CT DNA solution in Tris–HCl buffer gave an absorbance ratio at
260 and 280 nm (A260/A280) of about 1.8–1.9 indicating that the
CT DNA was sufficiently free of protein. The DNA concentration
was varied between 0 and 100 mM, while the complex concentration was kept constant at 5.0 mM at room temperature.
The intrinsic equilibrium binding constant (Kb) and the
binding site size (s) values were obtained from a nonlinear
tting according to the equation:62
(3a 3f)/(3b 3f) ¼ [b (b2 2Kb2Ct[DNA]/s)1/2]/2KbCt
(1)
b ¼ 1 + KbCt + Kb[DNA]/2s
(2)
where 3a is the extinction coefficient observed for the charge
transfer absorption band at a given DNA concentration, 3f is the
extinction coefficient of the complex free in solution, 3b is the
extinction coefficient of the complex when is fully bound to
DNA, Ct is the total complex concentration, and [DNA] is the
DNA concentration in nucleotides.
34166 | RSC Adv., 2017, 7, 34160–34169
The observed linearity in the plot of I0/I vs. the concentration
ratio of the complexes to DNA is in good agreement with the
linear Stern–Volmer equation:63
The stability of the complexes in plasma was assessed using
a procedure analogous to that recently reported.37 An aliquot (25
mL) of a solution containing the respective complexes 1 and 2
(nal concentration 20 mM) and diazepam (nal concentration
10 mM) in DMSO was added to human plasma solution (975 mL)
to give a total volume of 1000 mL. The resulting mixture was
incubated for 72 h at 37 C with continuous and gentle shaking
(ca. 300 rpm). The reaction was stopped by adding acetonitrile
(2 mL), and the mixture was centrifuged for 45 min at 1000g at
4 C. The acetonitrile was evaporated, and the residue was
suspended in acetonitrile/H2O (1 : 1, v/v; 200 mL). The
suspension was ltered and the ltrate was analyzed by HPLCUV. A 0.1 mL aliquot of the solution was injected into an HPLC
system (Thermo, USA) connected to a UV/Vis spectrophotometer. A Hypersil Gold Dim (100 2.1 mm, Thermo, USA)
reversed-phase column was used at a ow rate of 0.5 mL min 1.
The runs were performed with a linear gradient of A (acetonitrile (Sigma-Aldrich HPLC-grade)) and B (distilled water containing 0.1% HCOOH): t ¼ 0–3 min, 20% A; t ¼ 7 min, 50% A;
t ¼ 20 min, 90% A; t ¼ 23 min, 100% A; t ¼ 25 min, 100% A; t ¼
28 min, 20% A.
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Recoveries of diazepam and complexes 1 and 2 were determined by comparing the peak area ratios of the spiked drug-free
plasma to the peak area ratios obtained by direct injection of the
solutions of the same concentration of complexes 1 and 2 or
diazepam (20 mM for complexes 1 and 2 and 10 mM for
diazepam).
RSC Advances
37 C under a 5% CO2 atmosphere for 5 min. The medium was
removed, and the cell layer was washed gently with 10 mL PBS.
Then, the cells were digested with 65% HNO3 (2 mL) at 50 C for
2 h and trypsinized and harvested with 20 mL PBS. The solution
was analyzed using NexION 2000 B ICP Mass.
4.9. DCFDA assays
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4.6. Cell culture
Human cervical cancer (HeLa) and human broblast (MRC-5)
nontumorigenic cells were maintained in Dulbecco's Modied
Eagle Medium (DMEM) supplemented with 10% FBS, 100 IU
mL1 of penicillin, 100 mg mL1 of streptomycin and 2 mM
Glutamax at 37 C in a humidied incubator at 5% CO2. The
adherent cultures were grown as monolayer and were passaged
once in 4–5 days by trypsinizing with 0.25% trypsin–EDTA.
DCFDA assay was done to quantify ROS on the oxidative
production of uorescent DCF (lem ¼ 525 nm). HeLa cells (2
105 cells plated in duplicate) were incubated in the presence or
absence of complexes 1 and 2 (concentration ¼ IC50 values in
light) for 4 h in the dark with or without photoirradiation in
DPBS (500 nm, 1 h). Cells were trypsinized and treated with 1
mM DCFDA for 15 min, and the stained cells were determined by
ow cytometry.
4.7. Cytotoxicity
4.10.
The photocytotoxicity of the complexes was studied using
a standard MTT assay.64 Approximately 8000 HeLa cancer cells
and MRC-5 nontumorigenic cells were plated separately in two
different 96 wells culture plates in DMEM containing 10% FBS.
Stock solutions of the complexes were prepared by dissolving
the complexes in aqueous solutions with DMSO as the cosolvent and diluting with cell culture medium to the desired
concentrations. The nal DMSO concentration never exceeded
1% v/v. Cells were then treated with increasing concentrations
of compounds for 24 h. Dark controls, or cytotoxicity (CT)
assays, refer to assays that include metal complex but were not
exposed to light, and light controls refer to light exposed assays
that did not contain metal complex. Photocytotoxicity (PCT)
assays contained metal complex and were exposed to light. For
phototoxicity studies, cells were treated for 4 h only with
increasing concentrations of the compounds. Aer that, the
medium was removed and replaced by fresh complete medium
prior to 20 min irradiation at 50 nm (10 J cm 2). Aer 44 h in
the incubator, the medium was replaced by 100 mL complete
medium containing resazurin (0.2 mg mL1 nal concentration). Aer 4 h incubation at 37 C, uorescence of the highly
red uorescent resorun product was quantied at 590 nm
emission with 540 nm excitation wavelength in a SpectraMax
M5 microplate reader. Light doses were evaluated with a Gigahertz Optic X1-1 optometer. Cell counts were carried out
immediately aer light exposure. All experiments were carried
out in triplicate, and the graphed data is the average of three
trials.
The LED light sources were monitored using an International Light Technologies Powermeter (ILT2400) equipped with
SED 623 detector and spectral range 200–2100 nm. Light doses
were evaluated with a Gigahertz Optic X1-1 optometer. The light
doses employed in this work are comparable to those employed
for activation of other metal-based phototoxic compounds (2–
11.5 J cm2 at 400–600 nm).65–69
HeLa cells (2 105) were incubated (concentration ¼ IC50
values in light) for 24 h followed by light treatment in DPBS (500
nm). Cells were further kept for 12 h post-treatment and then
trypsinized. Annexin VFITC (2 mL) and propidium iodide (PI; 1
mL) were then added to clear suspensions of cells in 500 mL of
a 1 binding buffer and kept in the dark for 10 min. The
percent cell population in each quadrant was estimated by FACS
analysis. Experiments were performed in triplicate along with
untreated controls for reference.
4.8. ICP-MS
Cells grown in a 60 mm tissue culture dish were incubated with
the iridium(III) complexes (100 mM) in glucose-free medium at
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4.11.
Annexin V-FITC/PI assay
TrxR inhibition
Thioredoxin reductases (Trx-R) sourced from rat liver was obtained from Sigma-Aldrich. It is a buffered aqueous glycerol
solution, $100 units per mg protein, in 50 mM Tris–HCl, pH
7.5, 300 mM NaCl, 1 mM EDTA, and 10% glycerol. The
complexes 1, 2, and auranon were preincubated for 5 min at
room temperature; the reaction started with 2 mM 5,50 dithiobis(2-nitrobenzoic acid) (DTNB), and the increase of the
absorbance was monitored at 412 nm over 5 min at 25 C. The
reaction velocity in the presence of inhibitor was normalized
relative to the control to generate percent activity and plots of
percent activity versus concentration were constructed to obtain
IC50 values (that is, the concentration that inhibited 50% of
enzyme activity).
Acknowledgements
We are grateful for general support from the Department of
Chemistry, Isfahan University of Technology (IUT).
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