2+significantly inhibits glioblastoma growth: In vitro and vivo with fewer side-effects than cisplatin

Article abstract of DOI:10.1039/d0dt01877e

To overcome the acquired resistance and the significant side-effects of the reported drugs, four new ruthenium(ii) complexes with alkynyl (Ru1, Ru2, Ru3, Ru4) were designed and synthesized. Ru1, Ru2, Ru3 and Ru4 were characterized by ESI-MS, 1H NMR, 1H-1H COSY NMR and elemental analysis. Compared with Ru2, Ru3, Ru4 and cisplatin, the anti-tumor experiments in vitro and vivo confirmed that Ru1 could most effectively inhibit tumor growth. In the experiments of safety evaluation in vivo, Ru1 could avoid any detectable side-effects compared with cisplatin. DNA binding experiments and cell cycle experiments showed that Ru1 exhibited the strongest DNA binding ability and interfered with the cell cycle by inserting DNA to inhibit tumor growth. The study demonstrated that Ru1 had the potential to be an exciting new drug candidate for glioblastoma treatment. This journal is

Full text of DOI:10.1039/d0dt01877e

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ISSN 0306-0012
COMMUNICATION
Douglas W. Stephen et al.
N-Heterocyclic carbene stabilized parent sulfenyl, selenenyl,
and tellurenyl cations (XH , X = S, Se, Te)
+
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DOI: 10.1039/D0DT01877E
ARTICLE
2
+
[
Ru(phen) podppz] significantly inhibits glioblastoma growth in
2
vitro and vivo with fewer side-effects compared with cisplatin
#a
#b
a
a
a
a
*a
Ruihao Li , Yabin Ma , Xiaochun Hu , Wenjing Wu , Xuewen Wu , Chunyan Dong , Shuo Shi , Yun
Lin
Received 00th January 20xx,
Accepted 00th January 20xx
*a
DOI: 10.1039/x0xx00000x
To overcome the acquired resistance and the significant side-effects of the reported drugs, four new ruthenium (II)
complexes with alkynyl (Ru1, Ru2, Ru3, Ru4) were designed and synthesized. Ru1, Ru2, Ru3 and Ru4 were characterized by
1
1
1
ESI-MS, H NMR, H- H COSY NMR and elemental analysis. Compared with Ru2, Ru3, Ru4 and cisplatin, the anti-tumor
experiments in vitro and vivo confirmed that Ru1 could most effectively inhibit tumor growth. In the experiments of safety
evaluation in vivo, Ru1 could avoid any detectable side-effects compared with cisplatin. DNA binding experiments and cell
cycle experiments showed that Ru1 exhibited the strongest DNA binding ability and interfered with the cell cycle by inserting
DNA to inhibit tumor growth. The study demonstrated Ru1 owned potential to be an exciting new drug candidate for
glioblastoma treatment.
apoptosis of tumor cells. It can also interfere cellular transcript-
tion processes by inserting DNA, which eventually lead to
preventing RNA polymerases from binding with DNA and
Introduction
Glioblastoma multiforme (GBM), emanated from the glial cell,
is the most prevailing malignant tumor of the central nervous
system and the most aggressive form of glioma. The median
survival is less than 2 years with the current standard treatment
of maximal safe resection, combination of chemotherapy given
with radiation therapy. Temozolomide (TMZ) was used to treat
GBM for over a decade, but its therapeutic effects are limited
by acquired resistance.
In 1969, Bamett Rosenberg, American biophysicist, accident-
ally discovered the anti-tumor ability of cisplatin (DDP), and DDP
2
4,25
inhibiting the growth of tumor cells.
Although numerous previous studies have shown the anti-
tumor effects and the ability of fluorescence imaging of Ru (II)
1
2
6, 27
complexes in vitro and vivo experiments,
Ru (II) complexes
have not been approved for clinical use. Recent study indicated
that alkynyl groups could not only improve the ability of
substances to enter cells but increase the ability of Ru (II)
2
3
2
8-31
complexes to bind to DNA.
4
, 5
gradually became the first-line drug in clinical practice. The
major issues of DDP chemotherapy are the resistance and side
effects,6 such as renal toxicity, myelosuppression, nausea,
vomiting, and toxicity, which limits its use in tumor therapy.
Ruthenium and platinum belong to 8 subgroup of transition
-9
1
0
metal elements and own similar nature, but the richer
valences (II, III, IV) of ruthenium result in more diverse physical
and chemical properties.
1
1-13
Recently, the good bioco-
mpatibility and powerful anti-tumor effect of ruthenium (II)
complexes provide a new idea and a good alternative to
platinum drugs. 1
4-21
Ru (II) complexes can intercalate into the structure of double-
2
2, 23
stranded DNA to interfere with the self-replication of DNA,
Scheme. 1 (A) structures of Ru (II) complexes. (B) The
thus affecting the progress of cell cycle and inhibiting division schematic description of the anti-tumor mechanism of Ru1.
and proliferation of tumor cells, and finally inducing the
a. Shanghai East Hospital, Shanghai Key Lab of Chemical Assessment and
Sustainability, School of Chemical Science and Engineering, Tongji University,
1
239 Siping Road, 200092 Shanghai, P.R. China.
b.
Department of Pharmacy, Shanghai East Hospital, Tongji University, Shanghai
00120, PR China
2
#.
Contribute equally to this work
E-mail: ylin@tongji.edu.cn, shishuo@tongji.edu.cn
Electronic Supplementary Information (ESI) available: [details of any supplementary
information available should be included here]. See DOI: 10.1039/x0xx00000x
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In this study, we have synthesized and characterized four new Meanwhile, the results showed a significant difference in the
View Article Online
2
+
DOI: 10.1039/D0DT01877E
ruthenium (II) complexes with alkynyl, [Ru(phen)
2
po-dppz] , ability of DDP and Ru1 to reduce cell viability. The antitumor
2
+
2+
2+
Ru(bpy)
2
podppz] , [Ru(phen)
2
ppip] , [Ru(bpy)
2
ppip] . In vitro ability of Ru1 is the most prominent candidate among the four
studies verified the long-term stability in phosphate-buffered new synthesized Ru (II) complexes. Therefore, further exper-
saline (PBS) and water. The anti-tumor mechanism of Ru1 was iments were performed to assess the antitumor capacity of Ru1.
investigated via DNA binding experiments and cell cycle assay.
In vivo safety evaluation demonstrated that Ru1 could avoid any
detectable side-effects compared with cisplatin. In vivo anti-
tumor GBM models, Ru1 could effectively inhibit tumor growth,
suggested that Ru1 could be utilized as a potential drug for GBM
treatment.
Results and discussion
Synthesis and Characterization
Initially, the compounds pip, hdppz, cis-[Ru(phen)
and cis-[Ru(bpy) Cl ]·2H O were synthesized according to the
literature references.
lexes RA, RB, RC and RD were similarly obtained according to
2 2 2
Cl ]·2H O
2
2
2
3
2-35
Subsequently, the reported comp-
3
4-38
the literatures (Fig.S1).
The synthetic route to the alkynyl complexes is summarized
in Scheme 2. RA, RB, RC and RD were individually reacted with
propargyl bromide in the presence of anhydrous potassium
carbonate in DMF resulting in the formation of alkynyl
2
+
2+
2+
(
[
[Ru(phen)
Ru(bpy)
2 2 2
podppz] , Ru(bpy) podppz] , [Ru(phen) ppip] ,
2
+
2
ppip] (Ru1, Ru2, Ru3, Ru4) respectively, in very good
yield. All the new compounds were fully characterized by H
NMR, H- H COSY, TOF-MS, and elemental analysis (Fig.S2-S5).
These complexes exhibit a weak solubility in water, but show
a high solubility in DMSO. All Ru (II) complexes displayed rem-
arkable stability in PBS or water (containing 0.5% DMSO) at
1
1
1
2
98ꢀK for at least 5ꢀdays, as verified by UV–Vis spectroscopy (Fig.
S6).
UV–Vis Absorption Titration
Fig.1 (A) In vitro cytotoxicity of Ru1, Ru2, Ru3, Ru4 and DDP
To clarify the nature of the different cytotoxicities induced by against U87 MG cells with different drug dosages for 24 h via
complexes, UV absorption spectroscopy studies were carried out to the CCK-8 assay. (B) Fluorescent inverted microscope
investigate the DNA binding ability of Ru (II) complexes. When calf- images of U87 MG cells treated with PBS, DDP and Ru1 for
thymus DNA (CT-DNA) was added into Ru1 solution, obvious
hypochromism (H) was observed for both intra-ligand charge
transfer (ILCT) (λꢀ=ꢀ250-260ꢀnm) band and metal-to-ligand charge
transfer (MLCT) (λꢀ=ꢀ400-420ꢀnm). However, slight hypochromism
was observed for the other complexes. The extent of the
2
4 h. Live and dead cells were stained by Calcein AM
AM)/Propidium Iodide (PI) for 30 min and presented in
(
green and red colors in those images, respectively. Scale bar
is 100 µm.
hypochromism commonly parallels the intercalative binding affinity. In Vitro Toxicity
These results demonstrated that Ru1 exhibits the strongest DNA
binding ability (Fig.S8).
To screen the most powerful anti-tumor capability of Ru (II)
complexes, we assess the cytotoxicity of them against GBM in
vitro and vivo. The in vitro cytotoxicity of Ru1, Ru2, Ru3, Ru4
and DDP against U87 MG cells was investigated by the CCK-8
assay. Compared with Ru2, Ru3, Ru4 and DDP, Ru1 exhibited the
strongest anti-proliferative effect due to the strongest DNA
binding ability inducing cell apoptosis and inhibiting cell cycle
Since the octahedral complex binds to DNA in three
dimensions, both its ancillary ligand and intercalative ligand can
3
9-41
tune the DNA binding affinity.
Increasing the surface area of
the ancillary ligand and intercalative ligand leads to a substa-
ntially increased intercalative binding affinity. On-going from
the ancillary ligand phen to bpy, the hydrophobicity of the
ancillary ligand phen of Ru1 is greater than that of the bpy ligand
of Ru2. Meanwhile, the surface area for intercalative ligand
podppz is bigger than ppip, which is advantageous to DNA
binding of Ru1, too. Therefore, synthetically considering these
factors, Ru1 exhibiting the strongest DNA binding ability and the
best anti-proliferative effect could be well understood.
(Fig.1A). Because of the poor solubility of Ru complexes in
water, these complexes were first dissolved in DMSO and then
diluted into the test solution appropriately. Meanwhile, the
vitro cytotoxicities of 0.25% and 0.5% DMSO in controlled
buffers against U87 MG cells were also invested. The results
confirmed
2
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inducing cellular apoptosis of U87 than DDP, which was
View Article Online
DOI: 10.1039/D0DT01877E
consistent with the results of cytotoxicity tests.
Cell Cycle Analysis
Completion of cell cycle is crucial for DNA replication. And the
dynamic balance of cell cycle activities is an important factor to
maintain the stable transmission of genetic information. To
further investigate the mechanisms of inhibiting tumor cell
proliferation of Ru1, the effect of Ru1 and DDP on the cell cycle
were assessed in U87 MG cells by flow cytometry. 33.9% of cells
were blocked in the S phase by DDP, while 48.6% of cells treated
with Ru1 in the S phase were observed, which confirmed that
Ru1 and DDP could play an anti-tumor role through inhibition of
cell cycle stranded in the S phase (Fig.2B). Moreover, Ru1
showed more excellent inhibition ability of the cell cycle than
DDP. Taken together, these results demonstrated that DNA
damage caused by Ru1 can efficiently inhibit cell cycle arrest
and induce cellular apoptosis.
In Vivo Anti-Tumor Study
To investigate the in vivo anti-tumor efficacy of Ru1, mice
bearing subcutaneous U87 MG tumor were treated with PBS,
DDP and Ru1 at a single dosage of 5 mg/kg via intraperitoneal
injection. The in vivo antitumor experiment further confirmed
excellent tumor growth inhibition achieved by Ru1 (Fig.3A).
Tumor volumes of mice were measured every 2 days for 10 days
after administration. Ru1 could inhibit tumor growth more
effectively, comparing with the PBS group and the DDP group
Fig.2 Cellular apoptosis and cell cycle analysis in U87 MG
cells in response to PBS, DDP and Ru1. (A) Flow cytometry of
(Fig.3B).
5
μL Annexin V and 5 μL PI labelled U87 MG cells exposed
to PBS, DDP or Ru1 for 24 h. (B) Flow cytometry of PI labelled
U87 MG cells incubated with PBS, DDP, or Ru1 for 24 h.
Quantitative flow cytometry analysis of cell cycle ratios.
(Single asterisks indicate p < 0.05, double asterisks indicate
p < 0.01, and triple asterisks indicate p <0.001).
that the addition of small amount of DMSO was not toxic to
U87, and the anti-tumor effect was caused by the ruthenium
complexes themselves (Fig.S7).
Live & Dead Viability Assay
Moreover, to observe the anti-proliferative ability of DDP and
Ru1, U87 MG cells were stained with AM/PI after administered
with the same concentration of DDP and Ru1. It was found that
most U87 MG cells remain alive (represented by green color)
after treatment with DDP, suggesting DDP has no significant
efficacy on damaging of U87 (Fig.1B). In contrast, the treatment
with Ru1 resulted in a large quantity of dead cells (represented
by red color). This result sufficiently demonstrates Ru1 has
stronger ability of killing U87 MG cells than DDP.
Fig.3 (A) The image of tumors isolated from the nude mice.
3 groups, (1) PBS, (2) DDP, (3) Ru1). (B) Tumor growth curves
(
Cellular Apoptosis
and (C) histogram of average tumor weight measured on
day 10 after various treatments. (D) Apoptotic tumor cells in
tumor sections were stained with TUNEL (green) observed
by fluorescence microscopy. Cell nuclei were stained with
DAPI (blue). (E) H&E stained of tumors of mice from each
group (Single asterisks indicate p < 0.05, double asterisks
indicate p < 0.01, and triple asterisks indicate p <0.001).
Scale bars: 100 µm.
Flow cytometry was used to continue our exploration on the
mechanism of cytotoxicity of Ru1 against tumor cells. Apoptotic
and necrotic cells were detected in U87 MG cells via dual
fluorescence staining of Annexin V /PI. As shown in Fig.2A, only
2
3.7% of U87 MG cells were apoptotic after administered with
DDP (75 µmol/L). While almost 48.6% of U87 MG cells were
apoptotic after treated with an equal concentration of Ru1. This
result demonstrated that Ru1 owned the stronger ability of
This journal is © The Royal Society of Chemistry 20xx
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Histopathological Examination
View Article Online
DOI: 10.1039/D0DT01877E
H&E staining of the major organs showed that liver damage
caused by DDP, including cells edema, small vacuolar degener-
ation in cytoplasm, pyknosis in cell nucleus via our histological
observations. There was no abnormality of the renal pathology
observed in the DDP group, which only caused renal dysfunction
instead of obvious tissue damage. The Ru1 group showed
negligible pathological injury in line with the PBS group
(Fig.5A−E). Biochemistry tests, blood routine examination and
histological results further confirmed the lower systemic
toxicity and better in vivo safety of Ru1.
Fig.4 In vivo toxicity and safety studies of DDP and Ru1 in
nude mice. Mice were injected with PBS, DDP and Ru1 at a
single dosage of 5 mg per kilogram of animal body weight.
Experimental section
(
A) ALT and (B) AST levels represent liver function. (C) BUN
Materials
and (D) CREA represent kidney function. (E) WBC counts.
Data were expressed as mean ± SD (n=3). (F) The relative
body weight of mice. Data were expressed as mean ± SD
Cisplatin was obtained from Dalian Meilun Biotechnology Co.
LTD. (Dalian, China). Phosphate Buffered Saline (PBS),
Dulbecco’s Modified eagle’s medium (DMEM/high glucose) and
trypsin-EDTA were purchased from HyClone (USA). Fetal bovine
serum (FBS) were obtained from Clarkbio FB25015 (Shanghai,
china). Cell counting Kit-8 (CCK-8), Annexin V-FITC Apoptosis
Detection Kit, Cell Cycle Detection Kit and Live & Dead Viability
Assay Kit were purchased from Jiangsu KeyGEN BioTECH Corp.
Ltd. (Jiangsu, China). Ethanol and dimethyl sulfoxide (DMSO)
were obtained from Sinopharm Chemical Reagent Co. Ltd.
BALB/c nude mice were purchased from Silaike Experimental
Animal Centre (Shanghai, China). This study was approved by
the Ethics Committee of Tongji University.
(n=4). (Single asterisks indicate p < 0.05, double asterisks
indicate p < 0.01, and triple asterisks indicate p < 0.001).
The tumor weight was measured to assess the therapeutic
effect, too. The results were consistent with the tumors volume
as expected: tumor weights for the Ru1 group and DDP group
on day 10 were 300 ± 17 mg and 770 ± 18 mg, respectively
(
Fig.3C). The sections from tumors in each group were stained
4
2
with TUNEL for the evaluation of apoptosis at the histological
level (Fig.3D). Compared with the DDP groups, tumors treated
with Ru1 showed the higher apoptosis due to the suppression
of DNA replication and transcription. H&E staining of tumors in
mice from each group showed the necrosis degeneration of
tumor cells for the Ru1 group was more obvious than the other
groups (Fig.3E).
Physical measurements
1
H NMR spectra was collected at 300 K on a Bruker spectr-
1
1
ometer. H- H COSY NMR spectra were recorded on a Bruker
AV-400M spectrometer. Mass spectra were measured on an
Agilent TOF-G6230B mass spectrometer. Microanalysis of
elements (C, H, and N) was carried out using a Thermo Flash
In Vivo Safety Evaluation of DDP and Ru1
To investigate the in vivo safety and biocompatibility of Ru1 and
DDP, the blood of all mice was collected after treatment on day
2
000 analyzer.
1
0 for biochemistry tests and blood routine examination. Blood
biochemistry analysis showed that blood urea nitrogen (BUN),
creatinine (CREA), aspartate aminotransferase (AST) and
alanine aminotransferase (ALT) levels of mice treated with DDP
were significantly higher than that of the other two (Fig.4A-D).
There was no significate difference in alkaline phosphatase
(ALP) between DDP group and other groups (Fig.S9). According
to the results, the function of liver and kidney was obviously
damaged in DDP group. No significant hematological toxicity
was caused by Ru1. The mice treated with DDP presented a
significant decrease in white blood cell (WBC) count (Fig.4E),
suggesting the occurrence of myelosuppression, which is one of
the main side-effects of DDP. The decrease in WBC count was
significantly attenuated with Ru1 treatment. Other major
indicators of blood routine were not statistically different
among these three groups (Fig.S10). We also measured the
changes of body weight to evaluate the systemic toxicity of the
Fig.5 (A-E) H&E stained histological sections. (A) Heart, (B)
liver, (C) spleen, (D) lung and (E) kidney of mice from each
group were observed. Scale bars: 100 µm.
Synthesis and characterization
chemotherapeutic agents. Mice treated with Ru1 presented no [Ru(phen)
2
podppz]2+ (Ru1)
significate weight loss compared with the PBS group, whereas [Ru(phen) hdppz]2+ (RA) was prepared and characterized acco-
2
the therapy of DDP reduced the weight of mice noticeably rding to the literature.36 Firstly, RA (0.4 mmol, 420 mg) and
(Fig.4F).
2 3
K CO (0.4 mmol, 55 mg) were suspended in DMF (4 mL). Then,
4
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ARTICLE
Scheme. 2 Synthetic route for Ru1, Ru2, Ru3 and Ru4
propargyl bromide (0.8 mmol, 66 μL) was added and the (dd, J = 8.2, 5.3 Hz, 1H), 7.78 (dd, J = 5.7, 0.7 Hz, 1H), 7.71 (dd, J
reaction mixture was stirred at room temperature for 36 h = 8.2, 5.3 Hz, 1H), 7.61 – 7.49 (m, 3H), 7.42 (ddd, J = 7.3, 5.8, 1.2
under argon. Upon addition of 10 mL water, the precipitated Hz, 1H), 7.34 (ddd, J = 7.3, 5.8, 1.2 Hz, 1H), 5.17 (d, J = 2.4 Hz,
2
+
28 8
complex was washed with water (4×30 mL), and purified by 2H), 3.03 (t, J = 2.4 Hz, 1H). TOF-MS for C41H N ORu :
2
+
chromatography over alumina by using MeCN as eluent, yield: Calcl.750.14 found m/z 375.07 (M /2). Anal. data for
1
about 55% (236 mg) . H NMR (600MHz, CD
3
CN) δ 9.42 (dd, J
41 28 12 8 2
C H F N OP Ru: calc (%): C, 47.36; H, 2.71; N, 10.78. found
=
1
8
5
8.2, 1.0 Hz, 1H), 9.26 (dd, J = 8.2, 0.9 Hz, 1H), 8.71 (dd, J = 8.3, (%): C, 47.45; H, 2.69; N, 10.81.
2
+
.2 Hz, 1H), 8.69 – 8.65 (m, 3H), 8.40 (dd, J = 5.3, 1.2 Hz, 1H), [Ru(phen)
.34 – 8.30 (m, 4H), 8.27 (dd, J = 5.3, 1.2 Hz, 1H), 8.17 (dd, J = [Ru(phen)
2
ppip] (Ru3)
pip] (RC) was similarly obtained according to the
8
2
+
2
3
2 3
.4, 1.2 Hz, 1H), 8.08 (dddd, J = 21.9, 16.4, 6.9, 4.5 Hz,4H), 7.98 literature. Firstly, RC (0.4 mmol, 419 mg) and K CO (0.4 mmol,
(
(
dd, J = 8.7, 0.9 Hz, 1H), 7.80 (dd, J = 8.2, 5.3 Hz, 1H), 7.75 – 7.68 55 mg) were suspended in DMF (4 mL). Propargyl bromide (0.8
m, 4H), 7.57 (dd, J = 7.8, 0.8 Hz, 1H), 7.51 (dd, J = 8.2, 5.4 Hz, mmol, 66 μL) was then added and the reaction mixture was
1
C
H), 5.18 (d, J = 2.4 Hz, 2H), 3.02 (t, J = 2.4 Hz, 1H). TOF-MS for stirred at room temperature for 48 h under argon. Upon
H N ORu : Calcl.798.14, found m/z 399.0804 (M /2). Anal. addition of 10 mL water, the precipitated complex was washed
45 28 8
2
+
2+
data for C45
found (%): C, 49.55; H, 2.57; N, 10.27.
H
28
F
12
N
8
OP
2
Ru: calc (%): C, 49.69; H, 2.59; N, 10.30. with water, and purified by chromatography over alumina by
1
using MeCN as eluent. yield 50% (0.2I7 mg). H NMR (600MHz,
DMSO) δ 9.19 (d, J = 8.6 Hz, 1H), 9.10 (d, J = 7.3 Hz, 1H), 8.82 –
2
+
Ru(bpy)
Ru(bpy)
ording to the literature. Ru2 was synthesized similarly to Ru1. (t, J = 5.0 Hz, 4H), 8.08 (d, J = 5.4 Hz, 1H), 7.97 – 7.94 (m, 2H),
RB (0.4 mmol, 400 mg) and K CO
(0.4 mmol, 55 mg) were 7.90 (dd, J = 8.6, 5.3 Hz, 1H), 7.83 – 7.71 (m, 8H), 5.62 – 5.51 (m,
suspended in DMF (4 mL). Then, propargyl bromide (0.8 mmol, 2H), 3.89 (t, J = 2.3 Hz, 1H). TOF-MS for C46
2
podppz] (Ru2)
hdppz]2+ (RB) was prepared and characterized acc- 8.76 (m, 4H), 8.41 (d, J = 5.4 Hz, 4H), 8.19 (d, J = 5.3 Hz, 1H), 8.10
[
2
3
7
2
3
2
+
H N
30 8
Ru :
6 μL) was added and the reaction mixture was stirred at room Calcl.796.16, found m/z 398.08 (M /2). Anal. data for
temperature for 36 h under argon. Upon addition of 10 mL
Ru: calcd (%): C, 50.88; H, 2.79; N, 10.32. found
water, the precipitated complex was washed with water, and (%): C, 50.95, H, 2.80; N, 10.34.
2
+
6
46 30 12 8 2
C H F N P
2
+
[
Ru(bpy)
2
ppip] (Ru4)
pip]2+ (RD) was prepared and characterized according
purified by chromatography over alumina by using MeCN as
eluent, yield: 52.5% (215 mg). H NMR (600MHz, CD
1
3
CN) δ 9.42 [Ru(bpy)
2
3
5
(
(
(
dd, J = 8.2, 1.1 Hz, 1H), 9.27 (dd, J = 8.2, 1.0 Hz, 1H), 8.69 – 8.57 to the literature. Ru4 was synthesized similarly to Ru3. RD (0.4
m, 4H), 8.24 (dd, J = 5.3, 1.2 Hz, 1H), 8.23 – 8.16 (m, 3H), 8.09 mmol, 400 mg) and K CO (0.4 mmol, 55 mg) were suspended
dddd, J = 16.4, 14.3, 8.2, 4.6 Hz, 3H), 8.00 – 7.91 (m, 4H), 7.87 in DMF (4 mL). Propargyl bromide (0.8 mmol, 66 μL) was added
2
3
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Journal Name
and the reaction mixture was stirred at room temperature for suspend the cells. Subsequently, 5 μL Annexin V-FVIieTwCAartniclde O5nlμinLe
8 h under argon. Upon addition of 10 mL water, the PI were added and mixed at room temperDaOtIu: r1e0.1a0n3d9/oDu0tDTo0f1l8ig7h7Et
4
precipitated complex was washed with water, and purified by for 15 minutes. Flow cytometry was performed within 1 h.
chromatography over alumina by using MeCN as eluent. yield Live & Dead Viability Assay
about 55.8% (223 mg). H NMR (600MHz, DMSO) δ 9.22 (d, J = U87 MG cells were seeded at a density of 1×10 cells/well in 6-
1
5
8
8
.6 Hz, 1H), 9.14 (d, J = 8.2 Hz, 1H), 8.90 (dd, J = 8.2, 3.9 Hz, 2H), well plates overnight. The cells were administered with Ru1,
.86 (dd, J = 11.0, 8.5 Hz, 2H), 8.24 (t, J = 7.9 Hz, 2H), 8.17 – 8.09 PBS and DDP for 24 h. Next, the cells were stained with AM/PI
(
(
m, 4H), 8.02 (dd, J = 8.5, 5.3 Hz, 1H), 7.97 – 7.93 (m, 3H), 7.87 solution (2 µL AM ,8 µL PI, 10 mL PBS) for 30 min and imaged by
t, J = 4.8 Hz, 2H), 7.76 – 7.71 (m, 3H), 7.66 (d, J = 5.5 Hz, 1H), a fluorescent inverted microscope (AM Ex = 495 nm, Em = 520
7
4
C
.63 – 7.58 (m, 3H), 7.36 (dt, J = 17.1, 6.7 Hz, 2H), 5.57 (ddd, J = nm; PI Ex = 530 nm, Em = 620 nm).
8.3, 19.3, 2.2 Hz, 2H), 3.90 (t, J = 2.3 Hz, 1H). TOF-MS for Cell Cycle Analysis
2
+
2+
5
42
H
30
N
8
Ru : Calcl.748.16, found m/z 374.08 (M /2). Anal. U87 MG cells were seeded at a density of 1×10 cells/well in 6-
Ru: calc (%): C, 48.61; H, 2.91; N, 10.80. well plates and left to adhere overnight. The cells were treated
30 12 8 2
data for C42H F N P
found (%): C, 48.69; H, 2.89; N, 10.78.
UV–Vis absorption titration
with PBS, DDP and Ru1 for 24 h, after which cells were
trypsinized, washed, and fixed with 70% alcohol at -4°C for 12
2
000ꢀμL solutions of the blank buffer and Ru (II) complexes h. Next, the fixed cells were stained with PI/RNase A staining
sample (8ꢀμM) were placed in the reference and sample solution (50μL RNase A 450μL PI) for 45 min at 37°C in the dark.
cuvettes (1.0ꢀcm path length), respectively, and then the first Flow cytometry was performed within 1 h.
spectrum was recorded in the range of 200–600ꢀnm. During the In Vivo Safety Evaluation of DDP and Ru1
titration, we added an equal amount of DNA buffer to each To investigate the in vivo toxicity of DDP and Ru1, mice were
cuvette to eliminate the absorbance of the DNA itself, and euthanized and the blood from each group was collected after
mixed the solution by inverting it repeatedly. Before recording treatment on day 10 for blood routine test and biochemistry
the absorption spectrum, complex-DNA solutions were incub- tests. Meanwhile, the heart, liver, spleen, lung and kidney from
ated for 5 min. We repeated the titration processes until there each group were removed and stained by H&E for histological
was no change in the spectrum of at least 4 titrations, analysis.
demonstrating that binding saturation had been reached. At the In Vivo Anti-Tumor Study
end of each titration, Ru (II) complexes concentration change
To establish the tumor subcutaneous model, U87 MG cells
(
due to dilution is negligible.
6
5×10 ) were subcutaneously injected in the right back of male
nude mice (20-22 g). When the volume of these tumors reached
, mice were randomly divided into three
from the Cell Bank of the Chinese Academy of Sciences groups (4 in each) and respectively received the intravenous
Shanghai, China). The cells were incubated in 25 mL cell culture injection of DDP and Ru1 with a dosage of 5 mg /kg or PBS. The
Cell Culture
The Human glioblastoma cell line U87 MG cells were purchased approximately 150mm
3
(
flask with high glucose DMEM with 10% FBS and 1% penicillin- diameter of tumors was measured with a vernier caliper every
streptomycin solution. Cells were cultured in an incubator at 37 2 days along with therapy administration. The volumes of
°
C under a humidified atmosphere containing 5% carbon tumors were calculated via the formula: tumor volume =
dioxide.
Cytotoxicity Assay
(length) × (width)
2
/2. Mice body weight were measured every 2
days after treatment. All mice were sacrificed on day 10 and the
In vitro cytotoxicity of Ru1, Ru2, Ru3, Ru4 and DDP were final tumor weight was monitored directly. The tumors and
measured by the CCK-8 assay. U87 MG cells were seeded into major organs (heart, liver, spleen, lung and kidney) were excised
4
9
6-well plates at a density of 10 cells and 100 μL of DMEM in and stored in 4 % paraformaldehyde for histological
each well. After incubation for 24 h, tumor cells were treated by examination: hematoxylin and eosin (H&E) staining and TUNEL
different concentration of Ru1, Ru2, Ru3, Ru4 and DDP for 24 h. staining.
1
00 μL of DMEM (without FBS) containing 10% CCK-8 was added Histopathological Examination
into each well. After 2 h of culture, the cytotoxicity was Tumors and organs were embedded in paraffin and tissue
determined by the CCK-8 assay.
Cellular Apoptosis
sections of each group were stained with hematoxylin-eosin.
The sections of H&E staining were observed by the optical
U87 MG cells were seeded into 6-well plates at a density of microscope.
5
5
×10 cells and 1 mL of DMEM in each well. After 24 h for Immunofluorescence Histochemical Analysis
incubation, tumor cells were treated by different concentration Tumors and major organs (heart, liver, spleen, lung and kidney)
of Ru1 and DDP for 24 h. The old DMEM was transferred to a were fixed with 4.0% paraformaldehyde, embedded in paraffin,
1
5mL centrifuge tube. The cells were washed with PBS and sectioned and stained with H&E. TUNEL staining was used to
digested with Trypsin Solution without EDTA. After digestion, detect apoptotic cells according to a previously reported
the cells were transferred into the previous 15mL centrifuge method.
tube for centrifugation at 2000 r/min for 5 min. After Statistical Analysis
centrifugation, the supernatant was discarded. The cells were
washed by PBS twice (centrifuged at 2000rpm for 5 min) and
the cells were collected. 500 μL Binding Buffer was added to
Analysis of variance (ANOVA) was performed for all data
analysis and values were presented as means ± standard
6
| J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
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Pleas De ad l to o nn oT tr aa nd sj au cs t ti omn as rgins
Journal Name
ARTICLE
6
7
A.M. Florea and D. Busselberg, Cancers, 2011, 3V, i1ew35Ar1ti-c1le3O7n1lin.e
deviation. Differences were considered statistically significant
at P < 0.05 (*) P < 0.01 (**) and P < 0.001 (***). All the data was
analyzed by SPSS 21.0.
Y. R. Zheng, K. Suntharalingam, T. C. Johnstone, H. Yoo, W. Lin,
DOI: 10.1039/D0DT01877E
J. G. Brooks and S. J. Lippard, J. Am. Chem. Soc., 2014, 136,
8
790-8798.
8
9
K. Suntharalingam, Y. Song and S. J. Lippard, Chem. Commun.,
014, 50, 2465-2468.
D. Yue, S. Xu, Q. Wang, X. Li, Y. Shen, H. Zhao, C. Chen, W.
Mao, W. Liu, J. Liu, L. Zhang, H. Ma, Q. Li, Y. Yang, Y. Liu, H.
Chen and C. Wang, Lancet. Respir. Med., 2018, 6, 863-873.
0 L. Duan, A. Fischer, Y. Xu and L. Sun, J. Am. Chem. Soc., 2009,
131, 10397-10399.
2
Conclusions
In Summary, we have successfully synthesized four new Ru (II)
2
+
2+
complexes, [Ru(phen)
2
podppz] , Ru(bpy)
2
podppz] , [Ru(phe-
1
2
+
2+
n)
1
2
ppip] , [Ru(bpy)
2
ppip] . They were characterized by ESI-MS,
1
1
11 M. R. Gill and J. A. Thomas, Chem. Soc. Rev., 2012, 41, 3179-
192.
H NMR, H- H COSY NMR and elemental analysis. Firstly, the
3
anti-tumor experiments in vitro and vivo confirmed that Ru1
exhibited excellent antitumor efficacy on GBM. Furthermore,
we confirmed the strongest binding ability and the anti-tumor
mechanism of Ru1 via DNA binding experiments and cell cycle
experiments. Moreover, the study in vivo safety and biocom-
patibility of Ru1 was investigated and the results demonstrated
that Ru1 could avoid any detectable side-effects compared with
cisplatin. The results indicate that Ru1 is a very promising drug
candidate and provide a new idea for the follow-up treatment
of GBM.
1
1
1
1
1
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H. H. Harris, J. Biol. Inorg. Chem., 2013, 18, 845-853.
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Nano. Lett., 2017, 17, 2913-2920.
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7 W. Sun, S. Li, B. Häupler, J. Liu, S. Jin, W. Steffen, U. S.
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Conflicts of interest
There are no conflicts to declare.
1
1
8 J.H. Liang, Y. Zheng, X.W. Wu, C.P. Tan, L.N. Ji and Z.W. Mao,
Adv. Sci., 2019, 7, 1901992-1902005.
9 X. Meng, M. L. Leyva, M. Jenny, I. Gross, S. Benosman, B.
Fricker, S. Harlepp, P. Hebraud, A. Boos, P. Wlosik, P. Bischoff,
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Acknowledgements
This work was supported by the National Nature Science
Foundation of China (21671150, 81860547, 21877084,
2
009, 69, 5458-5466.
2
2
0 J. Q. Wang, P. Y. Zhang, L. N. Ji and H. Chao, J. Inorg. Biochem.,
2
015, 146, 89-96.
1 M. Wumaier, J. J. Shi, T. M. Yao, X. C. Hu, R. R. Gao and S. Shi,
8
1573008), Joint projects of Health and Family Planning
J. Inorg. Biochem., 2019, 196, 110681-110691.
Committee of Pudong New Area (PW2017D-10), the 22 M. R. Gill and J. A. Thomas, Chem. Soc. Rev., 2012, 41, 3179-
3
192.
Fundamental Research Funds for the Central Universities
kx0150720173382), Science and Technology Commission of
2
3 W.L. Wang, Z. Guo, Y. Lu, X. C. Shen, T. Chen, R. T. Huang, B.
(
Zhou, C. Wen, H. Liang and B.P. Jiang, ACS. Appl. Mater. Inter.,
Shanghai Municipality Fund (16DZ1930509), Science and
Technology Commission of Shanghai Municipality Fund (No.
2
019, 11, 17294-17305.
2
2
2
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4 H. Huang, P. Zhang, B. Yu, Y. Chen, J. Wang, L. Ji and H. Chao,
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DOI: 10.1039/D0DT01877E
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Dalton Transactions
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DOI: 10.1039/D0DT01877E
Ru1 could most effectively inhibit tumor growth and avoid any detectable side-effects compared
with other ruthenium (II) complexes and cisplatin, demonstrating its potential to be an exciting new
drug candidate for glioblastoma treatment.