Enantiomeric selectivity of ruthenium (II) chiral complexes with antitumor activity, in vitro and in vivo

Article abstract of DOI:10.1016/j.jinorgbio.2020.111339

Different enantiomers of chiral drugs show distinctive activities. Here, a pair of chiral ruthenium Λ-[Ru(phen)₂(TPEPIP)]²⁺ (Λ-Ru), and Δ-[Ru(phen)₂(TPEPIP)]²⁺ (Δ-Ru) (phen = 1,10-phenanthroline; TPEPIP = 2-(4′-(1,2,2-triphenylvinyl)-[1,1′-biphenyl]-4-yl)-1H-imidazo[4,5-f][1,10]phenanthroline) compounds have been prepared and characterized. Both have aggregation-induced emission characteristics, although Λ-Ru exhibits much higher activity, towards duplex DNA extracted from SGC-7901 cancer cells. In vitro experiments demonstrate that both Λ-Ru and Δ-Ru can induce the apoptosis of tumor cells with Λ-Ru showing greater activity than Δ-Ru. Λ-Ru aggregates in the cell nucleus of SGC-7901 within 5 h which shows that nucleic acids may be the effective target of Λ-Ru. In vivo experiments with nude mice showed that Λ-Ru can inhibit the growth and proliferation of a tumor, in tumor-bearing mice as well as targeting the tumor site, as demonstrated by fluorescence. These results demonstrate the dual-function of Λ-Ru, which could be used for real-time visualization of a chemotherapeutic agent.

Full text of DOI:10.1016/j.jinorgbio.2020.111339

Journal of Inorganic Biochemistry 216 (2021) 111339
Contents lists available at ScienceDirect
Journal of Inorganic Biochemistry
journal homepage: www.elsevier.com/locate/jinorgbio
Enantiomeric selectivity of ruthenium (II) chiral complexes with antitumor
activity, in vitro and in vivo
a
,
c
a
a
a
a
Weiwei Zhang , Yu Sun , Jingyuan Wang , Xiaoyuan Ding , Endong Yang ,
b
,
*
a,*
Lisandra L. Martin , Dongdong Sun
a
School of Life Sciences, Anhui Agricultural University, Hefei 230036, China
b
c
School of Chemistry, Monash University, Clayton 3800, Victoria, Australia
School of Biochemical Engineering, Anhui Polytechnic University, Beijing Middle Road, 241000 Wuhu, Anhui, China
A R T I C L E I N F O
A B S T R A C T
Keywords:
Different enantiomers of chiral drugs show distinctive activities. Here, a pair of chiral ruthenium Λ-[Ru
Chiral complexes
Ruthenium
Fluorescent probes
AIE
2+
2+
(
phen)
2
(TPEPIP)] (Λ-Ru), and Δ-[Ru(phen)
2
(TPEPIP)] (Δ-Ru) (phen = 1,10-phenanthroline; TPEPIP = 2-
′
′
(
4 -(1,2,2-triphenylvinyl)-[1,1 -biphenyl]-4-yl)-1H-imidazo[4,5-f][1,10]phenanthroline) compounds have been
prepared and characterized. Both have aggregation-induced emission characteristics, although Λ-Ru exhibits
much higher activity, towards duplex DNA extracted from SGC-7901 cancer cells. In vitro experiments
demonstrate that both Λ-Ru and Δ-Ru can induce the apoptosis of tumor cells with Λ-Ru showing greater activity
than Δ-Ru. Λ-Ru aggregates in the cell nucleus of SGC-7901 within 5 h which shows that nucleic acids may be the
effective target of Λ-Ru. In vivo experiments with nude mice showed that Λ-Ru can inhibit the growth and
proliferation of a tumor, in tumor-bearing mice as well as targeting the tumor site, as demonstrated by fluo-
rescence. These results demonstrate the dual-function of Λ-Ru, which could be used for real-time visualization of
a chemotherapeutic agent.
Anticancer
1
. Introduction
have one major drawback which is quenching of the fluorescence due to
aggregation [7–9]. There are numerous examples of fluorescent organic
Cancer is a leading cause of mortality, with predictions of more than
1 million cases by 2030. According to the World Health Organization,
polymers that are highly emissive in dilute solution but become weakly
luminescent as they concentrate and form aggregates [10]. Activatable
fluorophores with aggregation-induced emission (AIE) activity have
been successful is overcoming this limitation and are used to detect
binding events and have been especially successful in biosensing and
bioimaging applications [11,12].
2
in 2015, the number of deaths caused by cancer was 8.8 million, with
nearly one in six deaths globally being attributed to cancer [1,2]. There
is also an economic loss due to deaths from cancer, which is increasing
concomitantly. Some of the recent approaches to combat this prolifer-
ation of cancers, have been reviewed by Chen et al. [3], who highlighted
the use of intelligent molecules as theranostics. These intelligent mole-
cules combine cancer diagnostics, especially those developed for imag-
ing, with therapeutic activity required for cancer treatment [4–6]. Novel
fluorescent molecules, which can target cancer cells at the molecular
level, have also attracted significant interest. However, these molecules
The asymmetric synthesis of chiral secondary alcohols, which play
an important role as intermediates in organic chemistry, is a stimulating
subject. In previous studies, ruthenium complexes exhibited good anti-
tumor activities and prominent DNA-binding properties [13–15]. Pre-
viously, Sun et al. have reported that Ru-SeNPs (ruthenium-modified
selenium nanoparticles) inhibit angiogenesis suggesting possible
′
′
Abbreviations: AIE, aggregation-induced emission; ATCC, American Type Culture Collection; BDTPE, 4 -(1,2,2-triphenylvinyl)-[1,1 -biphenyl]-4-carbaldehyde
′
′
′
phenanthroline; bpy, 2,2 -bipyridine; CD, circular dichroism; CLSM, confocal laser scanning microscopy; DAPI, 4 ,6-diamidino-2-phenylindole; dppf, 1,1 -bis
diphenylphosphino)ferrocene; H&E, hematoxylin and eosin (stain); MRI, magnetic resonance imaging; MTT, thiazolyl blue tetrazolium bromide; phen, 1,10-phe-
(
nanthroline; PI, propidium iodide; RTCA, real-time cellular analysis; Ru-SeNPs, ruthenium-modified selenium nanoparticle; SEM, scanning electron microscopy;
′
TEM, transmission electron microscopy; THF, Tetrahydrofuran; TPE, tetraphenylethene; TPE-Br, (2-(4-Bromophenyl)ethene-1,1,2-triyl)tribenzene; TPEPIP, 2-(4 -
′
(
1,2,2-triphenylvinyl)-[1,1 -biphenyl]-4-yl)-1H-imidazo[4,5-f][1,10]; TUNEL, (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling).
*
Corresponding authors.
E-mail addresses: Lisa.Martin@monash.edu (L.L. Martin), sunddwj@126.com (D. Sun).
https://doi.org/10.1016/j.jinorgbio.2020.111339
Received 19 July 2020; Received in revised form 12 December 2020; Accepted 13 December 2020
Available online 28 December 2020
0
162-0134/© 2020 Published by Elsevier Inc.

W. Zhang et al.
Journal of Inorganic Biochemistry 216 (2021) 111339
Scheme 1. The sequence of reactions used to prepare the Λ-Ru and Δ-Ru complexes, as described in ref. [29].
applications as viable drug candidates for anti-angiogenesis and anti-
cancer therapies [16]. Here we employ Ru(II) complexes with poly-
pyridyl ligands, which provide a combination of modular construction
with a rigid chiral, and 3D spatial structures. The rich photophysical
properties and DNA-binding capabilities provide significant improve-
ments compared with traditional drugs. Independently, there have been
reports that Ru(II) dipyridophenazine complexes (Puckett et al. [17])
and examples of arene Ru(II) complexes (Wu et al. [15]) that have low
toxicity and were readily absorbed and rapidly excreted in vivo. Thus,
these ruthenium complexes offer enormous potential with applications
for dual imaging in antitumor treatment [18–20].
were synthesized in our previous study [29]. The synthetic strategy for
the preparation of Λ-Ru and Δ-Ru is presented in Scheme 1.
′
′
2
.3. Preparation of 4 -(1,2,2-triphenylvinyl)-[1,1 -biphenyl]-4-
carbaldehyde (BDTPE)
′
[
Pd(dppf)]Cl
2
(0.06 mmol) (dppf = 1,1 -bis(diphenylphosphino)
ferrocene) was added to a well-degassed solution of 4-(4,4,5,5-tetra-
methyl-1,3,2-dioxaborolan-2-yl)benzaldehyde (1.8 mmol), (2-(4-bro-
2 3
mophenyl)ethene-1,1,2-triyl)tribenzene (1.2 mmol), and K CO (6.1
mmol) in a mixture of methanol (12.5 mL) and toluene (12.5 mL). The
In the current study, we report on the properties of two, dual-
function ruthenium complexes that achieve real-time visualization
◦
mixture was stirred at 75 C for 18 h under argon atmosphere. The
mixture was evaporated to dryness and taken up with CH
organic layer was washed with H O, dried over NaSO , filtered, and
evaporated to dryness, producing a light-yellow powder in 88% yield.
2 2
Cl . The
(
imaging) and therapeutic activity, towards tumor cells. We modified
2
4
one of the three phen (1,10-phenanthroline) ligands in a ruthenium
complex with a tetraphenylethene (TPE) moiety, because AIE is usually
retained after chemical modification [21–24]. Importantly, previous
studies have shown that ruthenium complexes can combine with the
tumor cell nuclei of target (tumor) cells [25–27]. Thus, the target of the
tumor cell is also thought to be the nucleus, with the chiral Λ-Ru and
Δ-Ru complexes binding to duplex DNA in the tumor cells. Thus, we
expected AIE behavior from the ruthenium complexes to provide fluo-
rescence imaging of the tumor cells. This study provides a new insight
into the application of dual-function, AIE theranostics for targeted bio-
imaging and therapy using chiral ruthenium complexes.
Elemental analysis: calculated for C33
H24O: C 90.83, H 5.47; found: C
9
0.64, H 5.47%.
′
′
2
1
.4. Preparation of 2-(4 -(1,2,2-triphenylvinyl)-[1,1 -biphenyl]-4-yl)-
H-imidazo[4,5-f][1,10] phenanthroline (TPEPIP)
′
′
4
-(1,2,2-Triphenylvinyl)-[1,1 -biphenyl]-4-carbaldehyde
(0.4
CO
mmol), 1,10-phenanthroline-5,6-dione (0.4 mmol), and NH
4
(CH
3
2
)
◦
(
8 mmol) were stirred in CH
3
CO
2
H (5 mL) at 110 C for 18 h under argon
atmosphere. The mixture was then poured into a large amount of water,
the pH adjusted 7 and left overnight. The resulting solid was filtered and
washed with ethanol and then placed under high-vacuum pressure
2
. Experimental
′
′
overnight.
dazo[4,5-f][1,10] phenanthroline was obtained as a slightly pink solid
in 67% yield. Elemental analysis: calculated for C45 : C 86.23, H
.82, N 8.94; found: C 86.11, H 4.80, N 8.90%. ( H NMR (600 MHz,
2-(4 -(1,2,2-Triphenylvinyl)-[1,1 -biphenyl]-4-yl)-1H-imi-
2
.1. Materials and methods
30 4
H N
The reagents used in the synthesis were purchased from Sigma-
1
4
Aldrich Chemical Co. (USA). All solvents for synthesis were of reagent
grade and were used as received. For spectroscopic studies spectroscopy-
grade solvents were used.
DMSO‑d
6
) δ 13.78 (s, 1H), 9.03 (s, 2H), 8.93 (d, J = 7.9 Hz, 2H), 8.33 (d,
J = 8.0 Hz, 2H), 7.86 (dd, J = 26.3, 7.8 Hz, 4H), 7.60 (d, J = 7.8 Hz, 2H),
7
.13 (ddt, J = 24.0, 16.9, 8.7 Hz, 12H), 7.03 (dd, J = 15.3, 7.7 Hz, 5H).
3
ESI-MS (in CH OH, m/z):1087.2 [M]-) (Supporting Information (SI)
Fig. S1 ESI†).
2
.2. Synthesis and general characterization of the complexes
2-(4-Bromophenyl)ethene-1,1,2-triyl)tribenzene (TPE-Br) was syn-
(
2.5. Preparation of Λ-Ru
thesized according to the literature [28]. 1,10-phenanthroline-5,6-
′
′
2 2
(py) ][O,O -dibenzoyl-L-
dione, Λ-[Ru(phen)
Δ-[Ru(phen) (py)
2
(py)
2
][O,O -dibenzoyl-D-tartrate]•12H
2
O, and
TPEPIP (0.3 mmol) and Λ-[Ru(phen)
′
2
2
][O,O -dibenzoyl-L-tartrate]•12H
2
O (py = pyridine)
tartrate]•12H
2
O (0.2 mmol) were mixed in ethylene glycol (20 mL) and
2

W. Zhang et al.
Journal of Inorganic Biochemistry 216 (2021) 111339
′
refluxed for 8 h under an argon atmosphere. (Λ-[Ru(phen)
2
(py)
2
][O,O -
culture continued for 12 h. Cell viabilities were measured using a
standard MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide) colorimetric assay. These experiments were performed strictly
in the dark.
dibenzoyl-L-tartrate]•12H
2
O
was purchased from Sigma-Aldrich
Chemical Co., USA). The cooled reaction mixture was diluted with
water (50 mL) and filtered to remove solid impurities, and then satu-
rated ammonium perchlorate aqueous solution was added to the filtrate.
A deep red solid was collected and dried in a small amount of water in a
2
.10. Real-time cell analysis
vacuum and then purified by column chromatography (Al
trile/toluene 1:1), giving a yield of 68% of a dark red powder. Elemental
Cl Ru.2H O): C 60.96, H
.70, N 8.24; found: C 60.81, H 3.68, N 8.21%. ( H NMR (600 MHz,
2 3
O , acetoni-
The cell proliferation was analyzed by real-time cellular analysis
analysis: calculated for Λ-Ru (C69
H
46
N
8
2
O
8
2
(
RTCA), using a multielectrode array-based RTCA system (ACEA Bio-
sciences, Inc., San Diego, CA, USA). Cells were plated at a density of
000/well with a fresh medium to a final volume of 200 L. After cell
culture for 12 h, the medium was removed, and a fresh medium with
1
3
DMSO‑d
6
) δ 13.81 (s, 1H), 9.05 (d, J = 8.3 Hz, 4H), 9.01 (d, J = 4.3 Hz,
5
μ
2
H), 8.92 (t, J = 7.7 Hz, 2H), 8.76 (d, J = 8.2 Hz, 8H), 8.37 (s, 8H), 8.33
(
dd, J = 15.0, 8.0 Hz, 7H), 8.11 (d, J = 5.2 Hz, 4H), 8.06 (d, J = 5.2 Hz,
different concentrations of Λ-Ru and Δ-Ru (2.5
μ
M, 5
μ
M, and 10
μ
M)
4
5
H), 7.98 (d, J = 5.3 Hz, 4H), 7.88 (t, J = 6.4 Hz, 7H), 7.75 (dd, J = 8.3,
◦
was added. Cells were incubated at 37 C and 5% CO
2
in the RTCA
.4 Hz, 9H), 7.59 (dd, J = 11.9, 8.0 Hz, 7H). ESI-MS (in CH
3
OH, m/
system cradle. The signals were recorded every 10 min for 60 h. The
experiment was performed strictly in the dark.
z):1087.2 [M]-) (SI, Fig. S2 ESI†).
2
.6. Preparation of Δ-Ru
2
.11. Fluorescence microscopic observation (live/dead)
′
A mixture of TPEPIP (0.3 mmol) and Δ-[Ru(phen)
2
(py)
2
][O,O -
For the fluorescence microscopic observations, SGC-7901 cells (2.0
dibenzoyl-D-tartrate]•12H
2
O (0.2 mmol) in ethylene glycol (20 mL) was
4
×
10 cells/well) were placed in 48-well plates overnight. The cells were
M, 5
M) for 12 h. The cells were co-stained using the LIVE/DEAD
′
refluxed for 8 h under argon atmosphere. (Δ-[Ru(phen)
2
(py)
2
][O,O -
incubated with different concentrations of Λ-Ru and Δ-Ru (2.5
μ
dibenzoyl-D-tartrate]•12H
2
O were purchased from Sigma-Aldrich
μ
M, and 10
μ
Chemical Co., USA). The cooled reaction mixture was diluted with
water (50 mL). A saturated aqueous ammonium perchlorate solution
was added, mixed vigorously, and filtered. A dark red solid was
collected, washed with small amounts of water, dried under vacuum,
Kit (Sigma-Aldrich Chemical) for 30 min under dark conditions. After
removing the culture medium, calcein-AM (10 ng/mL, 1 mL) and Pro-
pidium iodide (PI) (10 ng/mL, 1 mL) were mixed and added into the
wells of the culture dishes, which were then incubated for 30 min in the
dark. Then, all dishes were washed three times with phosphate buffered
saline solution (PBS) and observed by confocal laser scanning micro-
scopy (CLSM). The experiment was performed strictly in the dark.
and purified by column chromatography (Al
:1), producing a dark red powder in 71% yield. Elemental analysis:
calculated for Δ-Ru (C69 Cl Ru.2H O): C 60.96, H 3.70, N 8.24;
found: C 61.11, H 3.69, N 8.26%. ( H NMR (600 MHz, DMSO‑d ) δ 9.04
2 3
O , acetonitrile/toluene
1
H
46
N
8
2
O
8
2
1
6
(
s, 2H), 8.90 (s, 1H), 8.75 (d, J = 8.1 Hz, 6H), 8.37 (s, 6H), 8.32 (s, 2H),
2
.12. Apoptotic cell death analysis
8
7
2
.11 (s, 3H), 8.06 (s, 4H), 7.97 (t, J = 6.8 Hz, 4H), 7.84 (s, 3H), 7.75 (s,
H), 7.54 (s, 2H), 7.44 (t, J = 7.4 Hz, 2H), 7.14 (dt, J = 16.8, 8.6 Hz,
◦
SGC-7901 cells were grown in 6-well plates overnight at 37 C and
% CO M, 5 M,
. The cells were then treated with Λ-Ru and Δ-Ru (2.5
M) and the culture continued for 12 h. The blank group was
3
6H), 7.08 (s, 7H), 7.06–6.95 (m, 20H). ESI-MS (in CH OH, m/z):1087.2
5
2
μ
μ
[
M]-) (SI, Fig. S3 ESI†).
and 10
μ
PBS. After treatment, the cells were collected and washed twice with
PBS. The cells were collected and stained with PI and Annexin V, and
then analyzed by a CytoFLEX flow cytometer (Beckman Coulter).
Annexin V could detect early apoptotic cells due to being bound to the
exposed phosphatidylserine. PI labeling was used to stain the late
apoptotic cells. The experiment was performed strictly in the dark. (PI,
Ex = 535 nm, and Em = 615 nm. Annexin V-FITC, Ex = 488 nm, and Em
2
.7. Characterization
The UV–visible absorption spectra were registered using ultraviolet-
visible spectroscopy (UV–vis, S-3100 photodiode array, Scinco Co.,
Korea). CD spectra were collected at 37 C with a JASCO J-810 spec-
◦
tropolarimeter using a 1 mm path length quartz cell. Λ-Ru and Δ-Ru (1
μ
M) was added in 0.5 mL DMSO and then diluted to obtain a stock so-
=
525 nm).
lution (buffered with Tris-HCl, TRIS is trisaminomethane, 10 mM, pH
.4). Duplex DNA (SGC-7901 cell nucleus material) was extracted from
7
2
.13. Membrane integrity and wall destruction studies
the nucleus pellet using a cell DNA extraction Kit (Sangon Biotech,
Shanghai, China). DNA stock solutions were prepared and diluted with
the Tris-HCl buffer (pH = 8).
Germ-free silicon wafers were placed into six-well plates. The SGC-
7
3
901 cells were grown on the surface of silicon wafers overnight at
◦
7 C and 5% CO
2
. The cells were subjected to the same treatment with
M, 5 M, and 10 M) for
2 h. The cells cultured without a complex were used as the blank group.
2
.8. Cell cultures
different concentrations of Λ-Ru and Δ-Ru (2.5
1
μ
μ
μ
HeLa, HepG-2, SGC-7901, A549, and MRC-5 cell lines were pur-
The cells with the silicon wafers were dehydrated with a series of
ethanol concentrations after they were fixed with 2.5% glutaraldehyde
overnight. The cells were then dried with a critical point drier and
observed by scanning electron microscope (SEM) (S-4800, Hitachi,
Japan) for the study of membrane intensity. The experiment was per-
formed strictly in the dark.
chased from ATCC (American Type Culture Collection) and maintained
in RPMI (Roswell Park Memorial Institute) with 10% fetal bovine serum
◦
at 37 C and under an atmosphere with 5% CO
2
.
2
.9. Cytotoxicity assays
The cells were seeded in 96-well plates at 37 C for 12 h. All cultured
For the study on wall destruction, the SGC-7901 cells were grown in
◦
◦
six-well plates overnight at 37 C and 5% CO
2
. The cells cultured
cells grew against the side of the well overnight, and different com-
pounds at varying concentrations were added into the plate and then cell
without a complex were used as the blank group. After being subjected
to the same treatment with different concentrations of Λ-Ru and Δ-Ru
3

W. Zhang et al.
Journal of Inorganic Biochemistry 216 (2021) 111339
Fig. 1. (A) UV ꢀ vis absorption spectrum of Λ-Ru and Δ-Ru in THF. (B) Circular dichroism (CD) spectroscopy of Λ-Ru and Δ-Ru in THF. (C) Fluorescence spectra of
Λ-Ru (10 M), with increasing H O fraction was from 0% to 99% (or 0 to 4.9-fold). (D) Fluorescence spectra of Δ-Ru (10 M), with increasing H O fraction from 0%
to 99% (or 0 to 4.9-fold). (E) Variation of the relative fluorescence intensity (I/I
-1) of Λ-Ru and Δ-Ru with increasing duplex DNA concentration; I is the fluorescence
intensity of Λ-Ru or Δ-Ru, and I is the fluorescence intensity without duplex DNA present; λex = 300 nm. (F) Images of 1 M Λ-Ru (1 M) and Δ-Ru (1 M) with
increasing concentrations of duplex DNA. (left to right concentrations are 0, 2, 4, 6, and 8 g/mL).
μ
2
μ
2
0
0
μ
μ
μ
μ
(
2.5
μ
M, 5
μ
M, and 10
μ
M) for 12 h, the cells were treated with Trypsin-
2.15. Animal model and in vivo chemotherapy
EDTA solution 0.25%. All cells (including the floating cells in the me-
dium) were collected with 1000 r/min for 5 min. The cells were ulti-
mately treated with a series of processing steps to be mounted on copper
grids and observed by transmission electron microscopy (TEM) (HT-
BALB/c male nude mice (~25 g) were purchased from the Model
Animal Research Center of Nanjing University and bred in a specific
pathogen free environment. All animals received care in compliance
with the guidelines outlined in the Guide for the Care and Use of Lab-
oratory Animals. All animal experiments were carried out according to
the protocols approved by Anhui Agricultural University Laboratory
Animal Center (Permit Number: SYXK 2016-007). Tumor models were
established by subcutaneous injection of cell suspension (SGC-7901
7
700, Hitachi, Japan). The experiment was performed strictly in the
dark.
2
.14. Real-time cell fluorescence imaging
For real-time cell fluorescence imaging, SGC-7901 cells were seeded
cells, 100
the tumor volume reached about 100 mm , the mice were randomized
into 3 groups (n = 6 per group): the untreated control Ru group, 2.5
group, and 5 M Ru group. Dosing solutions were administered via
intravenous injection every 3 days during the experiment.
μL, 1 × 10 [6]/mL) into the shoulder of the nude mice. When
◦
3
in 48-well plates at 37 C and grown for 12 h. The cells were then treated
with Λ-Ru and Δ-Ru (5
μ
M). After incubation for different time periods,
μM
fluorescence and normal images were taken under a 60 × oil-immersion
objective by using the real-time cell fluorescence imaging system. For
Λ-Ru and Δ-Ru, the excitation filter was 300 nm, and the emission filter
was 630–760 nm. The experiment was performed strictly in the dark.
μ
To determine the tumor volume, a vernier caliper was used to find
the maximum longitudinal diameter (length) and maximum transverse
diameter (width) of each tumor, and the tumor volume was calculated
using length × width [2] × 0.5. At the end of the experimental period,
with consecutive anesthetizations using 2% isoflurane in oxygen,
4

W. Zhang et al.
Journal of Inorganic Biochemistry 216 (2021) 111339
Magnetic resonance imaging (MRI) was used to obtain images of the
whole mice and tumor tissue with SGC-7901 xenograft subcutaneous
models with a 3.0-TMR scanner (General Electric, Milwaukee, WI, USA).
The mice were sacrificed, and the tumor tissues removed, rinsed with
physiological saline, and weighed. The tumor tissues were finely sliced
using a cryotome. The frozen tumor sections were then stained with
TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end
Table 1
IC50 of different compounds in various cancer cells compared to an MRC-5
control cell line.
Complex
IC50
( M)
μ
HeLa
HepG-2
SGC-7901
A549
MRC-5
Λ-Ru
26.8 ± 3.6
39.6 ± 3.4
36.7 ± 1.6
41.5 ± 3.2
46.3 ± 5.2
15.2 ± 1.5
20.4 ± 1.7
70.2 ± 9.3
49.2 ± 5.8
43.8 ± 2.5
41.7 ± 2.9
21.4 ± 1.1
7.6 ± 1.5
25.6 ± 2.8
26.1 ± 1.3
39.9 ± 2.9
34.2 ± 3.8
38.8 ± 4.4
22.6 ± 1.8
51.1 ± 3.1
36.3 ± 2.5
42.3 ± 2.7
55.7 ± 1.9
44.7 ± 2.9
36.8 ± 3.7
∆
-Ru
15.1 ± 1.3
33.2 ± 4.5
48.1 ± 2.9
43.9 ± 3.3
18.3 ± 1.3
′
labeling) and DAPI (4 ,6-diamidino-2-phenylindole) and then observed
TPE-Br
BDTPE
TPEPIP
Cisplatin
by fluorescence microscopy.
2
.16. Toxicity assay in vivo
Data are average values of at least three replicates.
To evaluate the toxicity of Λ-Ru, the mice were sacrificed after 14
days. The PBS treated mice were used as the blank group. The mice were
was calibrated by UV–visible spectroscopy prior to the titration exper-
iments. Fluorescence intensity was correlated with the concentration by
using I/I -1, where I and I are the peaks of fluorescence intensities with
treated with different concentrations of Λ-Ru (2.5
μ
M, 5
μ
M, and 10 M).
μ
The important organs (liver, spleen, kidneys, heart, and lungs) of the
mice were collected and stained with hematoxylin and eosin (H&E). The
body weight and survival rate were also measured during treatment
time.
0
0
and without DNA, respectively. Fig. 1E and F show linear growth when
the duplex DNA concentration increases within a certain range. The
fluorescence enhancement of Λ-Ru is more intense than that of Δ-Ru,
indicating high-contrast fluorescence and a high combination rate
before and after duplex DNA addition. This observation indicates that
Λ-Ru can aggregate more on the duplex DNA, compared with Δ-Ru.
When the duplex DNA concentration exceeds the threshold, the Λ-Ru
and Δ-Ru molecules may find more binding sites on different duplex
DNA chains, thereby increasing the molecular aggregation and assembly
of Λ-Ru or Δ-Ru with DNA. Thus, the fluorescence intensity of the sys-
tem slowly decreases. To provide additional evidence for the enantio-
meric selectivity of both complexes towards DNA, we undertook
viscosity measurements for both Λ-Ru and Δ-Ru complexes using SGC-
7901 DNA. Hydrodynamic measurements, such as viscosity, are regar-
ded as the most critical test of binding between two entities in solution,
especially in the absence of X-ray crystallographic data. Fig. S4 (SI)
shows the changes in the relative viscosity of SGC-7901 DNA in the
presence of ethidium bromide (EB), Λ-Ru or Δ-Ru. EB is a well-known
intercalator of DNA and these results show the expected increase in
relative viscosity due to an increase in length of the DNA double helix
through intercalation. Thus, both the Λ-Ru and Δ-Ru complexes appear
to bind to SGC-7901 DNA, similarly to EB. Of the two complexes, clearly
Λ-Ru binds more effectively and Δ-Ru less well to the SGC-7901 DNA.
However, taken together, it appears that both Ru complexes bind DNA in
a similar manner to EB; thus, we assume that they intercalate between
the base-pairs of DNA helices.
2
.17. In vivo fluorescence imaging
Male nude SGC-7901 subcutaneous models were administered
intravenous tail injections with Λ-Ru (5
μ
M). Fluorescence signal pho-
tographs (640 ± 20 nm) were taken at different time points. The fluo-
rescence images of the whole mice were taken after injection using the
IVIS spectrum in vivo fluorescence imaging system (Calipaer Perkin
Elmer).
3
. Results and discussion
3
.1. Characterization of Λ-Ru and Δ-Ru
The spectroscopic characterization of Λ-Ru and Δ-Ru were evaluated
as shown in Fig. 1. Λ-Ru and Δ-Ru are soluble in polar solvents, such as
DMSO and Tetrahydrofuran (THF), but exhibit low solubility in water.
We first measured their Ultraviolet–visible (UV–vis) absorption spectra,
where Λ-Ru and Δ-Ru showed identical absorption characteristics
(
Fig. 1A). In the present study, to further determine the factors that
govern the optical activity of a chiral complex, we investigated circular
dichroism (CD) spectral responses of Λ-Ru and Δ-Ru. The CD spectra are
shown in Fig. 1B for the two complexes where a clear mirror image
relationship between the Λ-Ru and Δ-Ru was exhibited. These data
confirmed that Λ-Ru and Δ-Ru have the correct chirality. Mixtures of
DMSO with varying amounts of water were used to check emission ac-
tivity. Fig. 1C and D show the occurrence of AIE observed for Λ-Ru and
Δ-Ru in dilute THF solution, where both exhibit low emission intensities.
With an increase in the percentage of water, AIE gradually increases,
and where the amount of water exceeds 90%, AIE quickly rises. The
maximum emission intensity of Λ-Ru is observed at 608 nm, and that of
Δ-Ru was observed at 608 nm. These characterization data confirmed
that Λ-Ru and Δ-Ru were chiral compounds, and that both these chiral
compounds had AIE properties.
3.3. Anti-tumor activity of Λ-Ru and Δ-Ru
Numerous studies have indicated that ruthenium complexes exhibit
higher antitumor activity as well as less toxicity and side effects [31–33].
In this study, we have evaluated the anticancer activity of Λ-Ru and
Δ-Ru against different cancer cells: HeLa, HepG-2, SGC-7901 and A549
cell lines. MRC-5 cells, derived from human embryo lung fibroblast,
were used as the control group. The conventional cytostatic agent
cisplatin, a treatment for numerous malignances, was used as a positive
control. Table 1 lists the IC50 values of Λ-Ru, Δ-Ru, cisplatin and other
precursor compounds. Both Λ-Ru and Δ-Ru exhibited the broad spec-
trum of anticancer activity towards several types of tumor cells, with
Λ-Ru showing greater performance. Λ-Ru showed a broad spectrum of
inhibition on the tested cells (see Table 1) with IC50 values ranging from
3
.2. Fluorescence titration of DNA
Λ-Ru and Δ-Ru are positively charged molecules, while duplex DNA
carries a large number of negative phosphate anions. Λ-Ru and Δ-Ru
molecules can attract duplex DNA [30]. To confirm the interaction,
fluorescence assays were performed by adding the duplex DNA to a stock
solution containing 1 mM Λ-Ru or Δ-Ru. The duplex DNA concentration
7.6 ± 1.5
μ
M to 26.8 ± 3.6
μ
M, across the four types of cancer cell. Of
note, was the treatment of SGC-7901 cells, in which Λ-Ru showed
significantly lower IC50 values than those of Δ-Ru and other compounds,
even cisplatin. The cytotoxicity of Λ-Ru was comparable or better than
5

W. Zhang et al.
Journal of Inorganic Biochemistry 216 (2021) 111339
Fig. 2. Real-time visualizing of SGC-7901 cell proliferation effects of Λ-Ru (A) and Δ-Ru (B). After SGC-7901 cells were cultured for 12 h, the medium was removed,
M, 5 M, and 10 M) was added. SGC-7901 cells were incubated with the different
and a fresh medium with different concentrations of Λ-Ru and Δ-Ru (2.5
μ
μ
μ
concentration Λ-Ru and Δ-Ru for 12 h. After removing the culture medium, the SGC-7901 cells were co-stained by calcein-AM and PI for 30 min in the dark.
Fluorescence microscopic images of Λ-Ru (C) and Δ-Ru (D) group were obtained through CLSM.
cisplatin for all the cell types except HeLa cells. Importantly, our control
cell line, MRC-5 cells, showed lower toxicity of the Λ-Ru isomer, than
the Δ-Ru isomer, and notably cisplatin (Λ-Ru 51.1 ± 3.1, Δ-Ru 36.3 ±
in Fig. 2C and D. The cells in the blank solution grew with a large cell
body and clear cell membrane structures. However, the treated cells
clearly exhibited morphological changes during apoptosis. With an in-
crease in concentration, cells became increasingly round, the percentage
of dead cells increased proportionally, and cell density decreased. These
effects were particularly apparent in the Λ-Ru group.
To further evaluate the obtained anticancer activity, flow cytometric
analysis was completed in which SGC-7901 cells were stained with
Annexin V and propidium iodide (PI) after treated with Λ-Ru and Δ-Ru
at different concentrations. Cells originally induced with Λ-Ru resulting
in a high percentage of late-stage apoptosis, even at lower concentra-
tions. With an increasing of concentration, this observation becomes
more apparent. Fig. 3 shows the weaker apoptotic effect of Δ-Ru,
compared with Λ-Ru, showing a slight increase in the number of cells in
early and late apoptosis but no significant effect on cell cycle
progression.
2
.5 compared with cisplatin 36.8 ± 3.7 μM). The effect of Λ-Ru on
cancer cell growth, showed a distinct preference for the SGC-7901 cells,
indicating the higher cytotoxic effects towards these SGC-7901 cells as
well as a good potency and low toxicity towards MRC-5 control cells.
To further investigate the obtained antitumor activity of Λ-Ru and
Δ-Ru, the SGC-7901 cells were cultured with different concentrations of
Λ-Ru and Δ-Ru. Adhesion and spreading were monitored by iCE-
LLigence (Fig. 2A and B) [34–36]. The results are broadly similar to MTT
analysis. Both could inhibit the proliferation of SGC-7901 cells with
increase of concentration. Compared with Δ-Ru, Λ-Ru could better
reduce the proliferation activity of SGC-7901 cells and result in cell
death, even at low concentrations.
A fluorescence experiment was also conducted to investigate and
verify the antitumor efficiency of Λ-Ru and Δ-Ru [37]. After treatment
with Λ-Ru and Δ-Ru at different concentrations, the SGC-7901 cells were
briefly stained using the LIVE/DEAD kit. The measurements are shown
6

W. Zhang et al.
Journal of Inorganic Biochemistry 216 (2021) 111339
Fig. 3. SGC-7901 cells treated with Λ-Ru and Δ-Ru (2.5
blank group was PBS.
μ
M, 5
μ
M, and 10
μ
M) for 12 h and analyzed using flow cytometry following Annexin V-FITC/PI staining. The
3
.4. Cell integrity study
potential as fluorescent agents for real-time cell imaging [39]. We
stained the live SGC-7901 cells with Λ-Ru and Δ-Ru. The typical results
The result of fluorescence assay showed the survival rate of SGC-
901 cells after treatment. The cell morphology changes were
of real-time fluorescence imaging of cells are shown in Fig. 5 and Fig. S5
(SI). The images revealed that the bright-red emission could be recorded
when the cells were treated with Λ-Ru over time. The cells were not
fluorescigenic initially; however, the fluorescence signal could be
detected over time when the cells showed characteristics of apoptosis.
The result of the aforementioned experiments indicated that Λ-Ru
showed antiproliferative and real-time visualization activities (Fig. 5).
Δ-Ru exhibited extremely low fluorescence intensity, which shows that
it was difficult for it to target tumor cell nuclei (SI, Fig. S5). The reason
for this needs to be explored in future experiments. The practical
application of Λ-Ru as an effective probe for cell imaging should
improve diagnostic analysis.
7
observed by SEM and TEM. SEM could directly observe and study the
surface morphology and other physical characteristics of cells without
altering the cell shape in samples [37]. In the blank group (Fig. 4A), the
cells had a plump cell morphology and long intercellular filaments. The
cell surface was relatively uniform and smooth, and almost no cell debris
were visible around the cells. In the treated group, the intercellular fil-
aments were almost invisible, and the cells appeared shriveled. Specif-
ically, the cell surface became broken in the 5
μM and 10
μM Λ-Ru
treatment groups. The cell morphology changed considerably, and cell
debris increased significantly. Simultaneously, regular cubes appeared
on the cell surface, which might be Λ-Ru accumulated on the cell sur-
face. Moreover, no remarkable changes were observed in the Δ-Ru
group as the concentration increased. TEM was conducted to observe the
internal structure of the cells and provide a reference for anticancer
mechanisms [38]. The blank images of the SGC-7901 cells without
exposure to Λ-Ru or Δ-Ru showed no significant changes in cell
morphology and appeared to have clear cell walls. However, significant
changes in the cell wall and internal structure could be observed after
exposure to Λ-Ru and Δ-Ru. The cell walls particularly in the Λ-Ru group
were disintegrated as concentration increased. The cytoplasm leaked,
and the nuclear structure became unclear. Many cell fragments were
observed around the cells and there were some hollow cells (Fig. 4B).
These aforementioned results suggested that antitumor activity was
related to compromised cell integrity and nuclear structure. Λ-Ru and
Δ-Ru might have combined with DNA; hence these results.
3
.6. Cytotoxicity evaluation of Λ-Ru in vivo
The potential in vivo toxicity is often a significant concern for the
clinical application of chemotherapy drugs [40]. To verify the applica-
bility of Λ-Ru in vivo, the mice treated with different concentrations of
Λ-Ru were evaluated. The mice showed slight and constant body weight
gain 14 days after treatment under lower concentrations of 2.5 and 5
Fig. 6A). Only the 10 M group slightly decreased in weight. All mice
were alive during the experiment under decreased concentration, and
even in the 10 M group, the survival rate remained at 83% (Fig. 6B). HE
staining of organ sections (Fig. 6C) showed no sign of organ damage nor
inflammation at the higher dose of 5
M of Λ-Ru compared with the
μM
(
μ
μ
μ
control group, indicating Λ-Ru has negligible side effects in vivo. The
results indicate that Λ-Ru has negligible side effects in vivo. These ob-
servations have significant implications for the efficiency and safety of
Λ-Ru in future clinical applications. A similar experiment using mice
3
.5. Cell imaging
with tumors but treated with ∆-Ru (10 M) was performed and also
μ
The obvious AIE-activity indicated that Λ-Ru and Δ-Ru exhibit
showed minimal side effects (SI, Fig. S6).
7

W. Zhang et al.
Journal of Inorganic Biochemistry 216 (2021) 111339
Fig. 4. (A) SEM images of SGC-7901 cells treated with different concentration of Λ-Ru and Δ-Ru (2.5
μ
M, 5
μ
M, and 10
μ
M) for 12 h. The blank group was PBS. Scale
M) for 12 h. The blank group was PBS.
bar = 10
μ
m. (B) TEM images of SGC-7901 cells treated with different concentration of Λ-Ru and Δ-Ru (2.5
μ
M, 5 M, and 10
μ
μ
Scale bar = 5
μ
m.
3
.7. Λ-Ru inhibits tumor growth in subcutaneous SGC-7901 tumor
xenograft mice models.
-weighted magnetic resonance imaging (MRI) is increasingly being
bearing nude BALB/c mice
T
2
used by researchers to examine tissue lesion. The emergence of tissue
edema could enhance the signal during treatment [41,42]. Thus, MRI
was performed in the present study to examine the changes in tumor
The in vivo antitumor activities of Λ-Ru were evaluated using an
SGC-7901 tumor xenograft model. When the tumor mass reached a
3
volume of approximately 100 mm , mice bearing the tumors were
2
status. In Fig. 7B, an increase in the T -weighted signal indicates more
intravenously injected with either PBS, 2.5
μ
M Λ-Ru or 5
μ
M Λ-Ru.
apoptotic cell deaths with Λ-Ru treatment, and an increase in Λ-Ru
concentration resulting in lower cancer cell densities within the tumor
Comparison of the images of tumors (Fig. 7A) with those of the control
group showed that mice treated with Λ-Ru markedly reduced the weight
and a higher degree of tumor cell death. The 5
smaller tumor volume.
μ
M group also showed a
and size of the tumor. The tumor weight and size of the 2.5 M group
μ
were less than those of the control group. However, the tumor weight of
the 5 M group was a half that of the control group. The tumor volume of
the 2.5 M group was only about a fifth of the control group. The rela-
tively stronger antitumor effect in the 5
In addition, the efficacy of treatment by Λ-Ru was assessed by his-
tological analysis from different treatment groups 21 days after the
μ
μ
treatments (SI, Fig. S7). Tumor growth curves of blank, 2.5
groups were collected. The results showed Λ-Ru treatment inhibited the
growth of the tumor (Fig. 8A). Tumor weight of mice in blank, 2.5
and 5 M groups after being treated for 21 days also showed a similar
μM and 5
μM
μ
M group suggests that Λ-Ru
inhibits tumor growth in a dose-dependent manner. These results
μM
demonstrated that the administration of Λ-Ru inhibited tumor growth in
μ
8

W. Zhang et al.
Journal of Inorganic Biochemistry 216 (2021) 111339
Fig. 5. Real time fluorescence imaging of SGC-7901 cells was induced by 5
μ
M Λ-Ru with a time from 0.5 to 5 h. All images share the same scale bar (10
μ
m).
Fig. 6. (A) Body weight change line graph after treatments with different concentration Λ-Ru (2.5
μ
M, 5
μ
M, and 10 M). (B) Survival rate histogram after treatments
μ
with different concentration Λ-Ru (2.5
00 m.
μ
M, 5
μM, and 10
μ
M). (C) H&E-stained images of major organs. Each value represents the mean ± SD (n = 6). Scale bar is
2
μ
9

W. Zhang et al.
Journal of Inorganic Biochemistry 216 (2021) 111339
Fig. 7. In vivo applications of Λ-Ru on mice tumor xenograft. (A) Photographs of the mice taken in 7, 14 and 21 days of 2.5
images of SGC-7901 tumor-bearing mice after treated with 2.5 M Λ-Ru for 21 days.
M Λ-Ru and 5
μM and 5
2
μM Λ-Ru. (B)T -weighted MR
μ
μ
result (Fig. 8B, and C). The tumor tissue is clearly damaged after the
treatment. TUNEL–DAPI staining of the tumor section was used to
evaluate cell apoptosis (Fig. 8D). DAPI could stain the cells with blue,
and TUNEL could show the apoptosis cells as green. After the treatment
with Λ-Ru, prominent necrosis was observed, indicating apoptotic cells.
mice were anesthetized and injected with Λ-Ru (5
μ
M) via the caudal
vein. The resulting fluorescence signal, shown in Fig. 9, does not appear
prominent because Λ-Ru is not aggregated after injection. The fluores-
cence signal of the mouse was enhanced gradually over time and
reached a maximum after 4 h. The fluorescence signal of the tumor in
the mouse was significantly stronger than that from normal tissue. The
result confirms that Λ-Ru can selectively detect and image SGC-7901
cells in vivo, and hence could visualize tumors.
The mice treated with 5
μM Λ-Ru showed significantly lower tumor
cellularity and more vacuolization, compared with the lower Λ-Ru
concentration group, which is consistent with the observed dose-
dependence. These results clearly suggest that Λ-Ru exhibits great po-
tential for future cancer treatment.
4. Conclusion
In summary, we have prepared two new chiral ruthenium complexes
3
.8. Fluorescence image in vivo
(
Λ-Ru and Δ-Ru) that exhibit AIE activity. These new diagnostic tools
were prepared using a synthetic pathway that exploited ligands known
to have AIE properties and ruthenium complexes with defined geometry
and coordination chemistry. Despite the low solubility in water, both
showed typical AIE characteristics and bound with duplex DNA from
Given the optical property of Λ-Ru and the aforementioned intra-
cellular fluorescence imaging, Λ-Ru was further used to visualize cell
apoptosis in living mice. An SGC-7901 tumor xenograft model was used
in this experiment. The male nude SGC-7901 tumor-bearing BALB/c
1
0

W. Zhang et al.
Journal of Inorganic Biochemistry 216 (2021) 111339
Fig. 8. Tumor tissue from SGC-7901 tumor-bearing BALB/c treated with PBS, 2.5
μ
M, and 5
μ
M Λ-Ru after 21 days. (A) Tumor growth curves. (B) Tumor weight. (C)
Images of tumors stripped from the mice after 21 days. (D) Images of apoptotic cells of the tumor tissues following use of the TUNEL-DAPI technique. The scale bar is
1
00 m.
μ
Fig. 9. Fluorescence images of SGC-7901 tumor-bearing mice after intravenous injection 5
μM Λ-Ru at different time points.
1
1

W. Zhang et al.
Journal of Inorganic Biochemistry 216 (2021) 111339
SGC-7901 cells, which was assessed by fluorescence intensity and vis-
cosity data. In relation to antiproliferative potential, Λ-Ru was better
than Δ-Ru in binding DNA from SGC-7901 cells. By using an SGC-7901
tumor xenograft mouse model, Λ-Ru was shown to inhibit tumor growth
and real-time visualization. Thus, we conclude, that Λ-Ru provides an
important advance in its capacity as a dual-function molecule for real-
time visualization as well as a therapeutic agent towards SGC-7901
cells. These results not only provide important information about a
design strategy for ruthenium complexes with AIE activity but also
about a research tool for biological studies.
[10] G. Wang, R. Zhang, C. Xu, R. Zhou, J. Dong, H. Bai, X. Zhan, ACS Appl. Mater.
Interfaces 6 (2014) 11136–11141.
[
11] Y. Hong, L. Meng, S. Chen, C.W. Leung, L.T. Da, M. Faisal, D.A. Silva, J. Liu, J.
W. Lam, X. Huang, B.Z. Tang, J. Am. Chem. Soc. 134 (2012) 1680–1689.
[12] N. Zhao, S. Chen, Y. Hong, B.Z. Tang, Chem. Commun. 51 (2015) 13599–13602.
[
[
[
13] V. Brabec, O. Nov a´ kov a´ , Drug Resist. Updat. 9 (2006) 111–122.
14] A. Bergamo, G. Sava, Dalton Trans. 13 (2007) 1267–1272.
15] Q. Wu, K. Zheng, S. Liao, Y. Ding, Y. Li, W. Mei, Mini-Rev. Med. Chem. 35 (2016)
317.
[
[
16] D. Sun, Y Liu, Q Yu, Y Zhou, R Zhang, X Chen, A Hong, J Liu, Biomaterials 34
(
2013) 171–180.
17] C.P. Popolin, M.R. Cominetti, Mini-Rev. Med. Chem. 17 (2017) 1435–1441.
[18] S. Masaoka, K. Sakai, Chem. Lett. 38 (2016) 182–183.
[
[
19] A. Mitrovi ´c , J. Kljun, I. Sosi ˇc , S. Gobec, I. Turel, J. Kos, Dalton Trans. 45 (2016)
1
6913–16921.
Declaration of Competing Interest
There are no conflicts to declare.
Acknowledgements
20] J. Diao, F. Bai, Y. Wang, Q. Han, X. Xu, H. Zhang, Q. Luo, Y. Wang, J. Inorg.
Biochem. 191 (2019) 135–142.
[
[
[
21] R. Hu, N.L. Leung, B.Z. Tang, Chem. Soc. Rev. 43 (2014) 4494–4562.
22] D. Ding, K. Li, B. Liu, B.Z. Tang, Acc. Chem. Res. 46 (2013) 2441–2453.
23] J. Mei, Y. Hong, J.W. Lam, A. Qin, Y. Tang, B.Z. Tang, Adv. Mater. 26 (2015)
5429–5479.
[
24] W.Z. Yuan, Y. Gong, S. Chen, X.Y. Shen, J.W.Y. Lam, P. Lu, Y. Lu, Z. Wang, R. Hu,
N. Xie, Chem. Mater. 24 (2012) 1518–1528.
We would like to thank our colleagues from the biotechnology center
of Anhui Agriculture University for their technical supports. This work
was supported by the National Natural Science Foundation of China
[
25] D. Sun, R. Zhang, F. Yuan, D. Liu, Y. Zhou, J. Liu, Dalton Trans. 41 (2012)
1
734–1741.
[26] X. Chen, J.H. Wu, Y.W. Lai, R. Zhao, H. Chao, L.N. Ji, Dalton Trans. 42 (2013)
386–4397.
27] L. He, X. Chen, Z. Meng, J. Wang, K. Tian, T. Li, F. Shao, Chem. Commun. 52
2016) 8095–8098.
[28] M. Banerjee, S.J. Emond, S.V. Lindeman, R. Rathore, J. Org. Chem. 72 (2007)
054–8061.
29] D. Sun, Y. Liu, D. Liu, R. Zhang, X. Yang, J. Liu, Chem. Eur. J. 18 (2012)
4
(
(
21401002), the Natural Science Foundation of Anhui Province, China
1508085QB37), and as a Major Science and Technology Project of
[
(
Anhui Province (17030701023). Financial support from the Australian
Research Council (DP170103477) to L.L.M. is gratefully acknowledged.
8
[
4
285–4295.
Appendix A. Supplementary data
[30] H.H. Gerets, K. Tilmant, B. Gerin, H. Chanteux, B.O. Depelchin, S. Dhalluin, F.
A. Atienzar, Cell Biol. Toxicol. 28 (2012) 69–87.
[
31] A.A. Almaqwashi, T. Paramanathan, P. Lincoln, I. Rouzina, F. Westerlund, M.
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.jinorgbio.2020.111339.
C. Williams, Nucleic Acids Res. 42 (2014) 11634–11641.
[32] G. Süss-Fink, J. Organomet. Chem. 751 (2014) 2–19.
[
[
33] H.J. Yu, L. Yu, Z.F. Hao, Y. Zhao, Spectrochim. Acta 124 (2014) 187–193.
34] M. Roshan Moniri, A. Young, K. Reinheimer, J. Rayat, L.J. Dai, G.L. Warnock,
Cytotechnology 67 (2015) 379–386.
References
[
35] J. Sun, Y. Chen, Y. Wu, X. Zhang, L. Jiang, Y. Zhang, Chromatographia 78 (2015)
[
[
[
1] W. Chen, R. Zheng, P.D. Baade, S. Zhang, H. Zeng, F. Bray, A. Jemal, X.Q. Yu, J. He,
CA Cancer J. Clin. 66 (2016) 115–132.
4
95–506.
[36] S. Ye, L. Jiang, J. Wu, C. Su, C. Huang, X. Liu, W. Shao, ACS Appl. Mater. Interfaces
10 (2018) 5862–5870.
2] R.L. Siegel, S.A. Fedewa, K.D. Miller, A. Goding-Sauer, P.S. Pinheiro, D. Martinez-
Tyson, A. Jemal, CA Cancer J. Clin. 65 (2015) 457–482.
[37] D. Sun, W. Zhang, Z. Mou, Y. Chen, F. Guo, E. Yang, W. Wang, ACS Appl. Mater.
Interfaces 9 (2017) 10047–10060.
3] H. Chen, Z. Gu, H. An, C. Chen, J. Chen, R. Cui, Sci. China Chem. 61 (2018)
1
503–1552.
[
38] L. Pan, J. Liu, Q. He, J. Shi, Adv. Mater. 26 (2014) 6742–6748.
[
[
4] L. Chen, J. Wu, C. Schmuck, H. Tian, Chem. Commun. 50 (2014) 6443–6446.
5] K. Horiguchi, S. Yoshikawa, A. Saito, S. Haddad, T. Ohta, K. Miyake, Y. Yamanishi,
H. Karasuyama, Biochem. Bioph. Res. Co. 479 (2016) 517–522.
6] K.N. Wang, X.J. Chao, B. Liu, D.J. Zhou, L. He, X.H. Zheng, Q. Cao, C.P. Tan,
C. Zhang, Z.W. Mao, Chem. Commun. 54 (2018) 2635–2638.
[39] Z. Wang, Y. Gu, J. Liu, X. Cheng, J.Z. Sun, A. Qin, B.Z. Tang, J. Mater. Chem. B 6
(
2018) 1279–1285.
[40] F. Hu, Y. Huang, G. Zhang, R. Zhao, H. Yang, D. Zhang, Anal. Chem. 86 (2014)
7987–7995.
[
[
[
[
[41] Y. Chang, L. He, Z. Li, L. Zeng, Z. Song, P. Li, L. Chan, Y. You, X.F. Yu, P.K. Chu,
ACS Nano 11 (2017) 4848–4858.
7] Y. Zhang, L. Pang, C. Ma, Q. Tu, R. Zhang, E. Saeed, A.E. Mahmoud, J. Wang, Anal.
Chem. 86 (2014) 3092–3099.
[
42] N.L. Leung, N. Xie, W. Yuan, Y. Liu, Q. Wu, Q. Peng, Q. Miao, J.W. Lam, B.Z. Tang,
8] R. Zhang, Y. Yuan, J. Liang, R.T.K. Kwok, Q. Zhu, G. Feng, J. Geng, B.Z. Tang,
B. Liu, ACS Appl. Mater. Interfaces 6 (2014) 14302–14310.
Chem. Eur. J. 20 (2014) 15349–15353.
9] G. Zhang, F. Hu, D. Zhang, Langmuir 31 (2015) 4593–4604.
1
2