Triphenylamine-appended cyclometallated iridium(III) complexes: Preparation, photophysical properties and application in biology/luminescence imaging

Article abstract of DOI:10.1016/j.jinorgbio.2019.110757

Four triphenylamine (TPA)-appended cyclometallated iridium(III) complexes were designed and synthesized. Photophysical properties of these complexes were studied, and density functional theory (DFT) was utilized to analyze the influence of the ancillary l

Full text of DOI:10.1016/j.jinorgbio.2019.110757

JournalofInorganicBiochemistry199(2019)110757
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Journal of Inorganic Biochemistry
journal homepage: www.elsevier.com/locate/jinorgbio
Triphenylamine-appended cyclometallated iridium(III) complexes:
Preparation, photophysical properties and application in biology/
luminescence imaging
⁎
Xicheng Liua, , Hailong Haoa, Xingxing Gea, Xiangdong Hea, Yifei Liub, Yan Wanga, Haojie Wanga,
⁎
Mingxiao Shaoa, Zhihong Jinga, Laijin Tiana, Zhe Liua,
a Institute of Anticancer Agents Development and Theranostic Application, The Key Laboratory of Life-Organic Analysis and Key Laboratory of Pharmaceutical
Intermediates and Analysis of Natural Medicine, School of Chemistry and Chemical Engineering, Qufu Normal University, Qufu 273165, China
b Qufu Normal University, School of Life Science, Qufu 273165, China
A R T I C L E I N F O
A B S T R A C T
Keywords:
Four triphenylamine (TPA)-appended cyclometallated iridium(III) complexes were designed and synthesized.
Photophysical properties of these complexes were studied, and density functional theory (DFT) was utilized to
analyze the influence of the ancillary ligands (TPA-modified bipyridine) to these complexes. The introduction of
TPA units could effectively adjust the lipid solubility of complexes (logP), and endowed complexes with potential
bioactivity (anticancer, antibacterial and bactericidal activity), especially in the field of anticancer (the best
Triphenylamine
Cyclometallated iridium complex
Photophysical property
Bioactivity
Luminescence imaging
value of IC50 is 4.34
0.01 μM). Interestingly, complexe 4 show some selectivity for cancer cells versus normal
cells. Meanwhile, complexes could effectively prevent the metastasis of cancer cells. Complexes can be trans-
ported by serum albumin and followed by the static quenching mechanism (Kq: 1013 M−1 s−1), disturb cell cycle
at G0/G1 phase, and induce apoptosis. The favorable fluorescence property confirmed these complexes followed
by an energy-dependent cellular uptake mechanism, effectively accumulated in lysosomes (PCC: > 0.95) and
induced lysosomal damage, and eventually leaded to cell death. Our study demonstrates that these complexes
are potential anticancer agents with dual functions, including metastasis inhibition and lysosomal damage.
1. Introduction
for the construction of novel theranostic platforms.
Lysosomes are often described as the stomachs of the cells (pH:
Iridium(III) (IrIII) complexes have recently emerged as promising
alternatives to platinum-based organometallic anticancer drugs because
of their unique anticancer mechanisms, e.g., involvement in cellular
redox reactions, inhibition of multiple protein activities and protein-
protein interactions. [1–4] Among these, cyclometalated complexes
provide excellent luminescence properties, e.g., large Stokes shifts, high
quantum yields and variable absorption/emission wavelength, thus
they have been widely used as biological imaging and fluorescence
probes. [5–8] The general formula of cyclometallated IrIII complexes
can be expressed as [Ir(ppy)2(biPy)]PF6, wherein ppy is 2-phenylpyr-
idine or its derivatives (C^N chelating ligands) and biPy is bipyridine or
its derivatives (C^N chelating ligands). Due to the easy modification of
these ligands, the luminescence and biological properties of cyclome-
tallated IrIII complexes can be readily adjusted. [9–11] More im-
portantly, the combination of potential anticancer activity and lumi-
nescence properties of cyclometallated IrIII complex makes it possible
4.5–5.5), which contain a variety of hydrolytic enzymes that are cap-
able of degrading almost all kinds of biomacromolecules delivered by
ways of phagocytosis, autophagy, and endocytosis. [12,13] In addition,
lysosome plays a significant role in intracellular transport, cell migra-
tion, metabolism, plasma membrane repair, and even apoptosis.
[14,15] Lysosomal dysfunction, a process known as lysosomal mem-
brane permeabilization, can lead to the release of cathepsins and other
hydrolases from the lysosomal lumen to the cytosol, thus induce
apoptosis. Therefore, lysosomes are emerging as attractive targets for
selective killing of cancer cells. Due to the significant acidic environ-
ment in the lysosomes of cancer cells, a large number of groups (ben-
zimidazole [16], morpholine [17], rhodamine [18] etc.) or ligands (β-
carboline [19], imine-N-heterocyclic carbene [20], 2-naphthalen-1-yl-
1H-benzoimidazole [21], etc.) containing free electron pairs have been
introduced into cyclometallated IrIII complex as lysosomal targeted and
real-time tracking drugs, and achieved the ideal results.
⁎ Corresponding authors.
E-mail addresses: chemlxc@163.com (X. Liu), liuzheqd@163.com (Z. Liu).
https://doi.org/10.1016/j.jinorgbio.2019.110757
Received 30 March 2019; Received in revised form 18 June 2019; Accepted 25 June 2019
Availableonline27June2019
0162-0134/©2019ElsevierInc.Allrightsreserved.

X. Liu, et al.
JournalofInorganicBiochemistry199(2019)110757
Fig. 1. Structures of designed cyclometalated IrIII complexes.
Triphenylamine (TPA) and its derivatives, hole-transporting and
steric hindrance of target IrIII complexes. Instead, they were char-
acterized by infrared spectroscopy (IR), nuclear magnetic resonance
(1H and 13C NMR) spectroscopy, mass spectrometry (MS) and elemental
analysis (Figs. S1–S4). These complexes were soluble in common or-
ganic solvents such as dichloromethane, methanol and dimethyl sulf-
oxide, insoluble in the ether and hexane. Aqueous stability of com-
plexes 1–4 was also determined in 50% CH3OH/50% H2O (v/v) by
ultraviolet-visible (UV–vis) absorption spectrum at 298 K for 8 h, and
no obvious changes were observed (Fig. S5). The studies confirm that
TPA-appended cyclometallated IrIII complexes possess sufficient stabi-
lity for the application of biological assays.
emitting materials, are widely used in organic photoelectric field, e.g.,
organic light-emitting diodes (OLEDs) [22], organic field effect tran-
sistors (OFETs) [23] and perovskite solar cells (PSCs) [24–26]. Recent
studies showed that possessed a promising application in biological
probes and drug targeting fields due to the advantages of low excitation
energy, small light damage and strong penetrability etc. [27–30] TPA-
appended phenanthroline chelating ligand (N^N) applied to cyclome-
tallated IrIII complex and acted as an effective phototherapy drug to-
wards HeLa cells, which can effectively induce the production of singlet
oxygen (1O2) after 730 nm laser irradiation and lead to apoptosis. [31]
Our previous studies also confirmed that half-sandwich iridium(III)
complexes with TPA-substituted dipyridine frameworks showed po-
tential anticancer activity and unique anticancer mechanism different
from cis‑platinum [32]. Inspired by above studies, four cyclometallated
IrIII complexes ([Ir(ppy)2(N^N)]PF6) with TPA-appended bipyridine
chelating ligands (N^N) and phenylpyridine (ppy) ligands were syn-
thesized and characterized (Fig. 1). The anticancer and antibacterial
activity of target complexes towards A549 (human lung cancer) cells
and E. coli (Escherichia coli) were determined by the MTT (3-(4,5-di-
methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and micro-
calorimetry assay, respectively. Because of the potential applications in
the field of anticancer, the selectivity between normal human cells and
tumor cells, the transport and anticancer mechanism of target com-
plexes were also studied. Due to the favorable fluorescent properties of
these complexes (the comparison and interpretation were also carried
out by means of quantum chemical simulation), laser confocal tech-
nology was used to investigate the cellular uptake and target sites in
cells. Above all, TPA-modified cyclometallated IrIII complexes are dis-
tinguished significative for further evaluation as anticancer drugs.
2.1. Photophysical properties
Due to the advantage of short excited state lifetime, high thermo-
stability and easily adjustable emission color, cyclometallated IrIII
complexes, the most popular luminous materials, have been extensively
used in the field of organic photoelectric field. [36] Photophysical
properties of complexes 1–4 were determined by UV–vis spectrum and
photoluminescence (PL) spectra. As shown in Fig. 2 and Table 1,
complexes 1–4 show almost the same photophysical properties, which
have
a strong intraligand absorption band (π-π*) at around
250–280 nm (ε > 18,000 M−1 cm−1) and a relatively weaker metal-
to-ligand charge-transfer (MLCT) transition band at around
350–500 nm. The room-temperature PL spectra showed that complexes
1–4 exhibited an intense yellow emission located at ~600 nm. [37,38]
To further understand how the ancillary ligands (TPA-modified bi-
pyridine) influence the photophysical properties of target complexes,
the optimal configurations, the frontier molecular orbitals and their
electron cloud distribution were analyzed by density functional theory
(DFT) calculation at the B3LYP/6-31G(d) (C, H, N, Cl)/lanl2dz (Ir)
level. [39] As shown in Fig. 3, the highest occupied molecular orbitals
(HOMOs) of complexes 1–4 are mainly localized on the bipyridine li-
gand and the terminal TPA groups, while the lowest unoccupied mo-
lecular orbitals (LUMOs) are mainly localized on the central iridium
atom, bipyridine ligand and phenyl of TPA molecule connected with
bipyridine, which indicate that the introduction of TPA molecule im-
proves the conjugate area of bipyridine chelating ligand [40], thus in-
creasing conjugate power of the whole target complexes. The dihedral
angles (θ) between P2 and P3 (benzene ring in TPA units, Table 1) are
almost the same numerically, which confirmed that the introduction of
terminal methoxyl has little effect on the TPA group. However, the
dihedral angles between P1 (pyridine ring) and P2 (benzene ring of
TPA molecule) are 24.80°, 28.92°, 25.80° and 3.55° for complexes 1–4,
respectively. Due to the introduction of trans-double bond, the minor
dihedral angle of complex 4 increase the conjugate power of the whole
2. Results and discussion
Triphenylamine-appended cyclometalated IrIII complexes were
synthesized by the reaction of [(ppy)2IrCl]2 (Dimer) and TPA-modified
bipyridine (TPA-biPy) chelating ligands (L1-L4), Scheme 1. Ad-
ditionally, TPA-biPy chelating ligands were synthesized by the classical
Suzuki reaction (L1-L3) [33] and Wittig reaction (L4) [34]. The ulti-
mate trans-configuration of L4 was obtained by the reflux reaction
using a catalytic amount of iodine, which was proved by the hydrogen
nuclear magnetic resonance (1H NMR) spectrum (hydrogen atoms of
the double bonds are shown in 6.97 ppm with the coupling constant of
16.2 Hz [35]). Target complexes were isolated as PF6 salts, obtained
with favorable yield (61–74%). Multiple attempts to grow single crys-
tals failed due to the “propeller” structure and the introduction of
terminal flexible substituents (-OCH3) for TPA units increasing the
2

X. Liu, et al.
JournalofInorganicBiochemistry199(2019)110757
Scheme 1. Synthesis process of cyclometalated IrIII complexes.
complexes, and endowed with the bigger UV–vis and PL wavelength
compared with other complexes, and which further confirmed why
complexes 1–3 show almost the same photophysical properties.
2.2. Cytotoxicity test
The cytotoxicity of target IrIII complexes was evaluated by the
standard MTT (3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium
bromide) assay towards A549 (lung cancer cell, the leading cause of
death in both developed and developing countries [41]) cells as a model
cancer cell line in vitro. The IC50 values (50% inhibitory concentration)
of target IrIII complexes, TPA-unattached structural IrIII complexes [Ir
(ppy)2(biPy)]PF6 (complex 5, Scheme S2), cis-platin and dimer of ir-
idium are listed in Table 2. As shown, except for complex 2, the other
three complexes show better antineoplastic activity towards A549 cells
than the dimer, cis-platin and complex 5, the best of which (complex 1)
was nearly five times of cis-platin and ten times of TPA-unattached
structural complex 5. Due to the minor difference in structure, com-
plexes 1, 2 and 4 show almost the similar antineoplastic activity.
However, for complex 2, the more TPA substituents, the less active it is.
Our previous studies have verified that the antineoplastic activity of IrIII
complexes can be effectively improved by the adjustment of lipid and
water solubility of complexes [42,43]. The partition coefficients (logP)
of complexes 1–4 in oil/water was determined by inductively coupled
plasma mass spectrometry (ICP-MS), and the values were −0.13, 0.98,
−0.18 and −0.23, respectively. These results indicate that the in-
troduction of too many TPA groups enhances the lipid solubility of
complex 2, but at the same time weakens its water solubility, thus af-
fecting the antineoplastic activity of these complexes.
Fig. 2. Normalized UV–vis and PL of complexes 1–4 in methanol solution
(1.0 × 10−5 mol L−1).
Table 1
Optical and structural optimization parameters of target complexes.
a
Complex
λab/nm (ε, 105 M−1 cm−1
)
λem/nmb
θ/°c
P1-P2
P2-P3
1
2
3
4
263 (2.42), 415 (0.94)
255 (2.39), 388 (0.65)
259 (1.88), 418 (0.68)
265 (2.31), 465 (1.01)
591
590
599
606
24.80
28.92
25.80
3.55
66.82
66.29
67.41
68.50
In addition, the cytotoxicities of complexes 1–4 to human bronchial
epithelial cells (16HBE) and lung epithelial cells (BEAS-2B) were fur-
ther evaluated. As shown in Table 2, complexes 1–3 show almost no
selectivity for cancer cells versus normal cells, however, complex 4
show some selectivity. This conclusion indicates the introduction of
double bond is beneficial to enhance the selectivity of this kind of
complex, which provides a structural basis for the later optimization of
structure in future work.
a
The maximum absorption peak of UV–vis was recorded in the
1.0 × 10−5 mol L−1 methanol solution.
b
PL spectra were recorded with excitation wavelength at maximum ab-
sorption peak.
c
The dihedral angles (θ) of optimized structure.
3

X. Liu, et al.
JournalofInorganicBiochemistry199(2019)110757
Fig. 3. Calculated optimal configurations, HOMO and LUMO diagrams of complexes 1–4.
Table 2
(no drugs) after 24 h. However, for complexes 1 and 4, extensive
IC50 values of IrIII complexes, cis-platin and dimer towards A549, BEAS-2B and
wound remained uncovered with the concentration of 0.25 × IC50
.
16HBE cells after 24 h.
Complex
Compared with complex 4, the effect of 2 is more distinct. The wound
closure ratio was 7.5% and 18.8% for complexes 1 and 4, respectively,
which was consistent with the result of the MTT assay (complex 1 show
better anticancer activity than 4). The results confirm that target IrIII
complexes possess the potential effect on the metastasis of cancer cells,
which are helpful for cancer therapy.
IC50 (μM)
A549
BEAS-2B
5.48
16HBE
[Ir(ppy)2(L1)]PF6 (1)
[Ir(ppy)2(L2)]PF6 (2)
[Ir(ppy)2(L3)]PF6 (3)
[Ir(ppy)2(L4)]PF6 (4)
[Ir(ppy)2(biPy)]PF6 (5)
cis-platin
4.34
80.91
5.80
0.01
1.21
5.99
83.87
2.56
0.81
4.23
0.36
8.65
0.21
0.11
0.82
1.71
0.81
> 100
5.80
0.61
10.80
40.33
21.30
57.70
> 100
> 100
2.4. Reaction with BSA
/
/
/
/
/
/
Dimer
As a transport protein in vivo, serum albumin (SA) provides a
“carrier” for drugs to reach the lesion. Studying the binding properties
between antitumor drugs and SA is crucial to understand the me-
chanism of action. [46] Due to the favorable binding features and si-
milar to human serum albumin (HSA) structure, bovine serum albumin
(BSA) could be an inexpensive model for studying the transport me-
chanism of drugs in organisms. [47] Therefore, in this study, BSA was
used to identify the reaction progress of target complexes with transport
protein.
2.3. Metastasis inhibition
Metastasis is really troublesome problem in the later period of
cancer evolution. Malignant tumor cells possess the capacity, migrate to
non-malignant tissue of distant and survive there. [44,45] Therefore,
metastasis inhibition plays a crucial role in advanced cancer treatment.
The effects of complex 1 and 4 on antimetastasis for A549 cells were
evaluated by wound healing migration assays. As shown in Fig. 4, ob-
vious migration of A549 cells and wound closure were found in control
As shown in Figs. 5a and S6, with the increase of cyclometallated
IrIII TPA complexes, the maximum absorption of 218 nm (the absorp-
tion of BSA) decreased, which indicated that these complexes could
affect BSA and lead to alpha helical interference. Additionally, a
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X. Liu, et al.
JournalofInorganicBiochemistry199(2019)110757
Fig. 4. Wound healing assay of A549 cells treated with or without complexes (1 and 4) for 24 h. (a) Images were obtained after 0 and 24 h. (b) Histograms of wound
healing assay after 24 h. Data are quoted as mean SD of three replicates.
significant red shift was found at 218 nm due to the influence of polar
solvent (water) [48]. Meanwhile, the absorption intensity at 278 nm
increase along with the addition of complexes, which is related to the
presence of aromatic acid residues (tryptophan, tyrosine and phenyla-
lanine) in BSA [49]. Simultaneous fluorescence spectra were used to
determine the detailed changes of microenvironment around BSA under
Fig. 5. (a) Absorbance of BSA (10 μM) was determined by UV–vis spectra after incubated with complex 1 (0–10 μM) in Tris-HCl/NaCl buffer solution (pH = 7.2) at
298 K. Inset: Changes of wavelength from 250 to 300 nm. (b) The fluorescence intensity of BSA (10 μM) obtained after reacted with complex 1 (0–10 μM) in Tris-HCl/
NaCl buffer solution (pH = 7.2) at 298 K. The arrows showed the changes with the increase of complex.
5

X. Liu, et al.
JournalofInorganicBiochemistry199(2019)110757
Table 3
2.7. Mitochondrial membrane potential (MMP)
The values of Ksv, Kb, Kq and n for complexes 1–4 at 298 K.
Mitochondria, the main energy producing sites of cells, play the
vital role in apoptosis. Once the integrity of mitochondrial membrane is
destroyed, which might release signal and lead to mitochondrial dys-
function, and then induce apoptosis. The degree of mitochondrial
dysfunction could be evaluated by measuring the changes of mi-
tochondrial membrane potential (MMP). [56] A549 cells were in-
cubated with complexes 1 and 4 for 24 h at the concentrations of
0.25 × IC50, 0.5 × IC50, 1.0 × IC50 and 2.0 × IC50, and stained by JC-
Complexes
Ksv (105 M−1
)
Kq (1013 M−1 S−1
)
Kb (104 M−1
)
n
1
2
3
4
1.23
2.29
1.64
0.94
0.14
1.23
2.87
1.64
0.94
14.57
10.19
12.13
10.67
1.02
0.96
0.96
0.91
0.13
0.10
0.17
the similar conditions, particularly near the fluorophore [50]. In this
study, tyrosine and tryptophan, aromatic acid residues of BSA, were
detected through the wavelength interval of Δλ = 15 nm and 60 nm by
a synchronous fluorescence. The weakened of fluorescence intensity at
291 nm and 285 nm (Δλ = 15 nm and 60 nm, respectively) for com-
plexes 1–4 were shown in Figs. S8 and S9. Interestingly, there is a minor
shift (2–3 nm) at Δλ = 15 nm, however, almost no change occurs at the
wavelength of Δλ = 60 nm, which indicate that tyrosine residues of
BSA is more involved in the interaction with target complexes, other
than tryptophan [51].
1 (a commonly used fluorescent probe to evaluate MMP). JC-1
monomer (green)/JC-1 aggregate (red) as shown in Fig. 8 and Tables
S5-S6. Compared with negative control, a significant concentration-
dependent increase was demonstrated at the indicated concentrations.
The population of mitochondrial membrane depolarized cells increased
by 24.4% and 28.1% for complexes 1 and 4 with the concentration
changed from 0.25 × IC50 to 2.0 × IC50, respectively. This observation
suggests that cyclometallated IrIII TPA complexes can cause cancer cell
death through the dysfunction of MMP.
Fluorescence emission spectra were further employed to detect the
binding characteristics between cyclometallated IrIII TPA complexes
and BSA. As shown in Figs. 5b and S7, with the increase of complexes,
the fluorescence intensity of BSA rapidly quenched. The Stern-Volmer
quenching constant Ksv, the quenching rate constant Kq, the binding
constant Kb and the number of binding sites n were determined by
classical Stern-Volmer equation [52] (Fig. S10) and the Scatchard
equation [53] (Fig. S11). As shown in Table 3, the Kq values of cyclo-
metallated IrIII TPA complexes are nearly three orders of magnitude
(1013 M−1 s−1) higher than that of the pure dynamic quenching me-
chanism, which is sufficient to demonstrate that cyclometallated IrIII
TPA complexes binding to BSA primarily follows the static quenching
mechanism [54]. In spite of almost the same number of binding sites
(n), complex 1 show the larger binding constant (Kb) than the other
three complexes, which is consistent with the result of cytotoxicity test.
2.8. Cell uptake and cellular localization
Cell imaging is a key crucial criteria to evaluate targeting sites of
complex, especially for real-time. Due to favorable luminescence
property of cyclometallated IrIII TPA complexes, laser confocal micro-
scopy was used to determine the intercellular colocalization of A549
cells. [8] Additionally, Lyso Tracker Red DND-99 (LTRD) and Mito
Tracker Deep Red (MTDR) were utilized as lysosomes and mitochondria
fluorescence probes. As shown in Fig. 9, complexes 1 and 4 could ef-
fectively accumulate in lysosomes with the Pearson's co-localization
coefficient (PCC) of 0.97 and 0.98 after incubated for 6 h. However, the
PCC of mitochondrion-targeted is 0.12 and 0.13 for complexes 1 and 4,
respectively. The results indicate that the introduction of TPA group
containing nitrogen atoms with free electron pairs facilitates the tar-
geting of acidic lysosomes in vivo for these IrIII TPA complexes. [16–21]
Interestingly, target complexes did not cause abnormal cell death im-
mediately, which made it easy to track changes in lysosomal mor-
phology timely.
2.5. Apoptosis assay
Due to the accumulation in lysosome for cyclometallated IrIII TPA
complexes, lysosomal damage assays were performed towards complex-
treated A549 cells to gain insight into the effects on lysosomes and their
surroundings. In this study, acridine orange (AO), an effective probe for
red fluorescence in lysosomes and green fluorescence in cytoplasm and
nucleus, was used to evaluate the lysosomal integrity. As shown in
Fig. 10, compared with control, there was a significant decrease in red
fluorescence and increase in green fluorescence when hatched in
complexes 1 and 4 (10 μM) for 1 h, and obvious lysosomal damage was
found after 6 h. The results confirmed that cyclometallated IrIII TPA
complexes could target lysosome, lead to lysosomal damage and the
release of cathepsins and other hydrolases from the lysosomal lumen to
the cytosol, and eventually induce apoptosis. [13] Meanwhile, this
conclusion also explains why cyclometallated IrIII TPA complexes do
not target mitochondria, but MMP changes significantly, which is
mainly due to the lysosomal damage.
Cell function decline was often associated with apoptosis. In this
study, flow cytometry was used to determine that by means of A549
cells treated with target complexes after stained with Annexin V-FITC/
PI. [55] As shown in Fig. 6 and Tables S1-S2, after incubated with
complexes 1 and 4 (0.5 × IC50, 1.0 × IC50, 2.0 × IC50 and 3.0 × IC50
)
for 24 h, the values of the late apoptotic phase indicate a significant
increase in a dose-dependent manner. With the concentration of in-
cubation changed from 0.5 × IC50 to 3.0 × IC50, the proportion of late
apoptosis increase by 67.3% and 63.8% for complexes 1 and 4, re-
spectively. However, above 86% cells survive for the control under the
same conditions. This result further confirmed that cyclometallated IrIII
TPA complexes could induce cell function decline and lead to apoptosis.
2.6. Cell cycle analysis
Cellular uptake mechanisms of drugs fall into two broad categories:
energy demand mechanism (divided into active transport and en-
docytosis) and non-energy demand mechanism (divided into passive
transport and free diffusion) [57]. In this study, chloroquine (en-
docytosis inhibitors) and carbonyl cyanide m-chlorophenylhydrazine
(CCCP, energy inhibitors) were used to determine the cellular uptake
mechanisms of cyclometallated IrIII TPA complexes. A549 cells were
incubated with complexes 1 and 4 at 277 K and 310 K for 2 h, and then
treated with chloroquine and CCCP. As shown in Figs. S12 and S13,
there were almost no changes compared with control, which confirmed
that the way of target complexes enter A549 cells is independent of
endocytosis and energy, therefore, corresponding to the mechanisms of
To investigate whether apoptosis is the result of cell cycle arrest
induced by cyclometallated IrIII complexes, flow cytometry was used to
measure the changes of cell cycle of A549 cells after treated by com-
plexes 1 and 4 with the concentration of 0.25 × IC50, 0.5 × IC50
,
1.0 × IC50 and 2.0 × IC50 for 24 h. As shown in Fig. 7 and Tables
S3–S4, 1 and 4 mainly lead to cell arrest in G0/G1 phase with the po-
pulation of 66.0% and 56.6%, respectively. Compared with control,
which nearly increased by 8.5% and 8.6%. This conclusion indicates
that cyclometallated IrIII TPA complexes might restrain the proliferation
of A549 cells to some extent by cell cycle arrest, and eventually leading
to apoptosis.
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X. Liu, et al.
JournalofInorganicBiochemistry199(2019)110757
Fig. 6. (a) Apoptosis of A549 cells was analyzed by flow cytometry after incubated with selected complexes 1 and 4 (concentrations: 0.5, 1.0, 2.0, 3.0 × IC50) and
stained by Annexin V-FITC/PI for 24 h at 310 K. (b) Histograms of four stages induced by complexes 1 and 4. Data was averaged for three replicate
experiments SD.
non-energy dependent cell uptake.
when μ = 0. Antibacterial data were presented in Table 4, cyclome-
tallated IrIII TPA complexes show certain antibacterial activity against
E. coli. However, compared with Penicillin sodium (common anti-
bacterial on the market), there is still a big gap (almost ten times) for
target complexes. Which indicate that the potential applications of
cyclometallated IrIII TPA complexes in the antibacterial field are lim-
ited.
2.9. Antibacterial and bactericidal activity
In this study, the minimum inhibitory concentration (MIC) of cy-
clometallated IrIII TPA complexes against Escherichia coli (E. coli, ATCC
8739) at 37 °C was determined by microcalorimetry. [58] As shown in
Figs. 11a and S14, the thermal power-time curves, which indicate the
growth rate of E. coli, were processed at different concentrations by
classical Logistic equation (Eq. (1)).
3. Conclusions
Four TPA-appended cyclometallated IrIII complexes were synthe-
sized and characterized. Target complexes had a certain inhibitory ef-
fect on E. coli. More importantly, complexes showed potential antic-
ancer activity against A549 cells, the best of which (complex 1) was
nearly five times of cis-platin and ten times of TPA-unattached struc-
tural IrIII complexes, meanwhile, which could prevent the metastasis of
cancer cells. Interestingly, complexe 4 show some selectivity for cancer
cells versus normal cells. Complexes could transport trough serum
protein and followed by a binding mechanism of static quenching
µt
P = Pm/(1 +
e
)
(1)
t
where μ is the growth rate constant, α is the integral constant, Pm and Pt
are the maximum heat production rate and the heat production rate at
time t, respectively. The measured experimental data Pm, Pt and t were
analyzed in the measured power-time curve. The growth rate constant μ
was obtained by linear regression analysis and further established the
relationship with the inhibitory concentration (C). As shown in
Figs. 11b and S15, the minimum inhibitory concentration was obtained
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X. Liu, et al.
JournalofInorganicBiochemistry199(2019)110757
Fig. 7. (a) The altered A549 cell cycle was analyzed by flow cytometry after induced by complexes 1 and 4 (concentrations = 0.25, 0.5,1.0 and 2.0 × IC50) for 24 h
at 310 K. (b) Histograms of cell cycle distributions for complexes 1 and 4. Data are quoted as mean SD of three replicates.
mode. Because of the favorable fluorescent properties of target IrIII
complexes, laser confocal microscopy confirmed the way enter cells is
corresponding to the mechanism of non-energy dependent, accumulate
in lysosome and lead to lysosomal damage, which induce the release of
lysozyme, thereby affecting mitochondrial membrane potential, disrupt
the cell cycle and induce apoptosis. Above all, TPA-appended cyclo-
metallated IrIII bipyridine complexes are potential anticancer agents
with dual functions, including metastasis inhibition and lysosomal da-
mage.
fetal bovine serum, penicillin/streptomycin mixture, trypsin/EDTA,
and phosphate-buffered saline (PBS) were purchased from Sangon
Biotech. A549 lung cancer cells and human bronchial epithelial normal
cells (16HBE and BEAS-2B) were obtained from Shanghai Institute of
Biochemistry and Cell Biology (SIBCB). Dimer of iridium ([Ir(ppy)2Cl]2)
[59] and chelating ligands (L1-L4) were synthesized according to the
methods of literatures [33,34,42] (Scheme S1), the data were shown in
supporting information.
NMR spectra were obtained on Bruker DPX 500 spectrometers in-
strument, with the chemical shifts reported in ppm using tetra-
methylsilane (TMS) as an internal standard. Mass spectrum (ESI-MS)
was measured on a LCQ Advantage MAX mass spectrometer. Elemental
analysis was performed on a VarioMICRO CHNOS elemental analyzer.
IR spectra were recorded on a Nicolet Nexus 470 FT-IR spectro-
photometer using KBr discs in the range 4000–400 cm−1. UV–vis
spectroscopy was performed on a PERSEE TU-1901 UV spectrometer.
Fluorescence spectra were collected by a Hitachi F-4600 fluorescence
spectrophotometer, with a 400 V voltage and 5 nm slit width for both
excitation and emission. Induction of apoptosis, cell cycle and mi-
tochondrial membrane potential (MMP) determination were carried out
by an ACEA Novocyte2040R flow cytometry. Viability assay (MTT) was
4. Experimental section
4.1. General information
Iridium trichloride, iodine, 2-phenylpyridine, 4,4′-dimethyl-2,2′-bi-
pyridyl, selenium dioxide, anhydrous potassium carbonate, n-bu-
tyllithium, 4,4′-dibromo-2,2′-bipyridine, 4-bromo-2,2′-bipyridine, 4-
bromotriphenylamine, 4-formyltriphenylamine, sodium borohydride,
triphenyl phosphine hydrobromide, triisopropyl borate, tetra-
triphenylphosphine palladium, potassium tert-butanolate and all kinds
of organic solvents (methanol, tetrahydrofuran, methylbenzene etc.)
were purchased from Rhea biotechnology co. LTD. DMEM medium,
measured using
a Perlong DNM-9606 microplate reader at an
8

X. Liu, et al.
JournalofInorganicBiochemistry199(2019)110757
Fig. 8. (a) Changes of JC-1 monomer (green)/JC-1 aggregate (red) induced by complexes 1 and 4 (concentrations = 0.25, 0.5, 1.0 and 2.0 × IC50). (b) Histograms
for the JC-1 monomers/JC-1 aggregates treated with complexes 1 and 4. Data are quoted as mean SD of three replicates.
absorbance of 570 nm. Cell uptake and cellular localization were car-
ried out on a Carl Zeiss AG */LSM/880NLO two photon laser Scanning
microscope. Antibacterial activity was measured by Thermometric AB
3114/3236 Thermal Activity Monitor (TAM) Air Isothermal
Calorimeter.
δ 167.37, 167.33, 156.24, 156.08, 151.41, 151.08, 150.26, 150.18,
150.07, 149.87, 149.36, 146.66, 144.34, 144.30, 140.00, 139.22,
131.58, 130.72, 130.33, 129.31, 127.66, 125.82, 125.57, 124.99,
124.44, 122.71, 121.65, 121.29, 120.55. ESI-MS (m/z): [M-Cl]+ Calcd
for C50H37IrN5, 900.1; Found 900.5. Elemental analysis: Found: C,
57.81; H, 3.61; N, 6.61%, calcd for C50H37N5PF6Ir: C, 57.56; H, 3.64; N,
6.63%%.
4.2. Synthesis of target complexes (1–4)
[Ir(ppy)2(L2)]PF6 (2). Yield: 73.3%. Selected IR (KBr): 1607.3,
1586.4, 1498.6, 1334.1, 1279.9, 757.7, 699.7 cm−1
;
1H NMR
General procedure: [Ir(ppy)2Cl]2 (Dimer, 53.6 mg, 0.05 mmol) and
chelating ligands (L1-L4, 0.10 mmol) were mixed in methanol (80 mL)
under nitrogen atmosphere. After stirred 12 h at room temperature,
65.2 mg (0.40 mmol) ammonium hexafluorophosphate were added to
above solution and reacted for another 6 h. The solvent was removed by
a rotary evaporator and the crude product recrystallized from methanol
and ether. Purification product was rinsed with n-hexane and dried in
vacuo. The IR (KBr), 1H NMR (500 MHz), 13C NMR (126 MHz), ESI-MS
and element analysis of complexes 1–4 are shown in Figs. S1–S4. The
data were listed as follows:
(500 MHz, DMSO) δ 9.23 (s, 2H), 8.29 (d, J = 8.2 Hz, 2H), 7.96 (dd,
J = 14.0, 8.0 Hz, 10H), 7.82 (d, J = 5.9 Hz, 2H), 7.74 (d, J = 5.7 Hz,
2H), 7.39 (t, J = 7.9 Hz, 8H), 7.23–7.15 (m, 6H), 7.14–7.10 (m, 8H),
7.03 (t, J = 7.5 Hz, 6H), 6.92 (t, J = 7.4 Hz, 2H), 6.24 (d, J = 7.4 Hz,
2H). 13C NMR (126 MHz, DMSO) δ 167.40, 156.37, 151.56, 150.07,
150.00, 149.37, 146.67, 144.36, 139.21, 131.59, 130.72, 130.32,
129.47, 127.92, 125.75, 125.59, 125.29, 125.10, 124.95, 124.46,
122.67, 121.88, 121.37, 120.54. ESI-MS (m/z): [M-Cl]+ Calcd for
C
68H50IrN6, 1143.4; Found 1143.8. Elemental analysis: Found: C,
63.69; H, 3.92; N, 6.47%, calcd for C68H50N6PF6Ir: C, 63.45; H, 3.96; N,
6.44%.
[Ir(ppy)2(L1)]PF6 (1). Yield: 67.0%. Selected IR (KBr): 1604.0,
1588.3, 1501.4, 1329.9, 762.2 cm−1
;
1H NMR (500 MHz, DMSO) δ
[Ir(ppy)2(L3)]PF6 (3). Yield: 68.2%. Selected IR (KBr): 1607.0,
9.13 (d, J = 8.2 Hz, 1H), 9.06 (s, 1H), 8.28 (dd, J = 13.9, 7.2 Hz, 3H),
7.98–7.91 (m, 6H), 7.89 (d, J = 5.5 Hz, 1H), 7.77 (d, J = 5.9 Hz, 1H),
7.73–7.68 (m, 2H), 7.65 (d, J = 5.7 Hz, 1H), 7.39 (t, J = 7.9 Hz, 4H),
7.27–7.22 (m, 1H), 7.22–7.10 (m, 8H), 7.06–6.99 (m, 4H), 6.91 (t,
J = 7.4 Hz, 2H), 6.21 (d, J = 7.6 Hz, 2H). 13C NMR (126 MHz, DMSO)
1561.8, 1503.7, 1329.1, 1251.2, 845.1, 762.2 cm−1
;
1H NMR
(500 MHz, DMSO) δ 9.11 (d, J = 8.4 Hz, 1H), 9.00 (s, 1H), 8.31–8.24
(m, 3H), 7.97–7.90 (m, 5H), 7.87 (dd, J = 11.7, 7.1 Hz, 3H), 7.73 (d,
J = 6.0 Hz, 1H), 7.69 (t, J = 6.0 Hz, 2H), 7.64 (d, J = 5.1 Hz, 1H),
9

X. Liu, et al.
JournalofInorganicBiochemistry199(2019)110757
Fig. 9. Intercellular colocalization of complexes 1 (a, b) and 4 (c, d) (15 μM, 1 h) with LTRD (100 nM, 30 min) and MTDR (50 nM, 30 min) in A549 cells. Scale bars:
20 μm.
7.17 (t, J = 6.6 Hz, 2H), 7.13 (d, J = 8.9 Hz, 4H), 7.02 (t, J = 7.6 Hz,
2H), 6.98 (d, J = 9.0 Hz, 4H), 6.91 (t, J = 7.4 Hz, 2H), 6.81 (d,
J = 9.0 Hz, 2H), 6.21 (dd, J = 7.2, 4.1 Hz, 2H), 3.76 (s, 6H). 13C NMR
(126 MHz, DMSO) δ 167.39, 167.33, 157.13, 156.11, 151.49, 151.23,
151.14, 150.26, 150.03, 149.95149.35, 144.36, 144.29, 140.59,
139.96, 139.28, 131.57, 130.71, 129.10, 128.20, 125.72, 125.55,
125.32, 124.42, 122.69, 121.17, 120.52, 117.89, 115.65, 55.78. ESI-MS
(m/z): [M-Cl]+ Calcd for C52H41IrN5O2, 960.1; Found 960.9. Elemental
analysis: Found: C, 56.95; H, 3.75; N, 6.33%, calcd for
(m/z): [M-Cl]+ Calcd for C55H45IrN5O2, 1000.2; Found 999.9.
Elemental analysis: Found: C, 56.73; H, 3.85; N, 6.16%, calcd for
C54H43O2N5PF6Ir: C, 56.40; H, 3.90; N, 6.12%.
Abbreviations
ppy
phenylpyridine
biPy
TPA
bipyridine
Triphenylamine
C
52H41O2N5PF6Ir: C, 56.61; H, 3.80; N, 6.30%.
TPA-biPy Triphenylamine-modified bipyridine
[Ir(ppy)2(L4)]PF6 (4). Yield: 61.3%. Selected IR (KBr): 1608.7,
SA
Serum Albumin
1584.7, 1504.1, 1255.0, 845.6, 757.7 cm−1; 1H NMR (500 MHz, CDCl3)
HSA
BSA
NADH
Human Serum Albumin
δ
8.65 (d, J = 6.8 Hz, 2H), 7.90 (d, J = 8.5 Hz, 2H), 7.76 (d,
Bovine Serum Albumin
J = 7.1 Hz, 2H), 7.72 (d, J = 6.0 Hz, 2H), 7.69 (dd, J = 9.3, 6.8 Hz,
3H), 7.59 (d, J = 5.6 Hz, 1H), 7.54 (d, J = 6.0 Hz, 1H), 7.50 (s, 1H),
7.47 (d, J = 2.4 Hz, 1H), 7.45 (s, 1H), 7.31 (d, J = 7.4 Hz, 1H), 7.16 (d,
J = 5.7 Hz, 1H), 7.08 (d, J = 8.9 Hz, 3H), 7.06–7.00 (m, 5H),
6.93–6.89 (m, 3H), 6.86 (t, J = 9.0 Hz, 5H), 6.31 (d, J = 7.8 Hz, 2H),
3.81 (s, 6H), 2.64 (s, 3H). 13C NMR (126 MHz, DMSO) δ 167.36,
156.92, 155.93, 155.72, 151.90, 151.56, 151.32, 150.29, 149.86,
149.34, 149.22, 148.70, 144.34, 139.59, 139.13, 137.42, 131.59,
130.66, 129.62, 129.27, 128.05, 127.03, 126.11, 125.52, 125.35,
124.39, 122.61, 121.04, 120.48, 118.22, 115.60, 55.76, 21.44. ESI-MS
Reduced Form of Nicotinamide Adenine Dinucleotide
ICP-MS Inductively Coupled Plasma Mass Spectrometry
log P
MTT
ROS
Partition Coefficient in Oil/Water
(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
Reactive Oxygen Species
MMP
PCC
Mitochondrial Membrane Potential
Pearson's Colocalization Coefficient
Minimum Inhibitory Concentration
Escherichia coli
MIC
E. coli
10

X. Liu, et al.
JournalofInorganicBiochemistry199(2019)110757
Fig. 10. Laser confocal images of A549 cells hatched by complexes 1 and 4. (a) Control group; A549 cells were incubated in complex 1 (10 μM) for 1 h (b) and 6 h (c);
A549 cells were in complex 4 (10 μM) for 1 h (d) and 6 h (e); All groups were stained with AO (5 μM) after incubation. λex = 488 nm, λem = 510 30 nm (green)
and 625 30 nm (red). Scale bar: 20 μm.
Fig. 11. (a) Pt-T curve of E. coli under the action of complex 1 was measured by microcalorimeter. (b) The μ-C curve of E. coli metabolism after incubation of complex
1.
Table 4
Declaration of Competing Interest
The authors declare no competing financial interest.
Acknowledgments
The MIC (mg mL−1) of target complexes and Penicillin sodium against E. coli.
Complex
μ-C relational
MIC(mg mL−1
)
1
y = −19.299x + 1.002
y = −18.089x + 1.076
y = −12.259x + 0.777
y = −15.238x + 0.840
y = −28.176x + 0.154
0.05192
0.05948
0.06338
0.05513
0.00547
2
3
We thank the University Research Development Program of
Shandong Province (J18KA082), the National College Students
Innovation and Entrepreneurship Training Program (2018A043), the
4
Penicillin sodium
11

X. Liu, et al.
JournalofInorganicBiochemistry199(2019)110757
National Natural Science Foundation of China (Grant No. 21671118)
and the Taishan Scholar Project of Shandong Province for support.
469–475.
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Appendix A. Supplementary data
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