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Synthesis, characterization and cytotoxic activity studies of two ruthenium(II) complexes
Inorganica Chimica Acta xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Inorganica Chimica Acta
journal homepage: www.elsevier.com/locate/ica
Synthesis, characterization and cytotoxic activity studies of two
ruthenium(II) complexes
Wei Li a, Bing-Jie Han a, Ji Wang a, Guang-Bin Jiang a, Yang-Yin Xie a, Gan-Jian Lin a, Hong-Liang Huang b,⇑,
Yun-Jun Liu a,⇑
a
b
School of Pharmacy, Guangdong Pharmaceutical University, Guangzhou 510006, PR China
School of Life Science and Biopharmaceutical, Guangdong Pharmaceutical University, Guangzhou 510006, PR China
a r t i c l e
i n f o
a b s t r a c t
Article history:
Received 25 April 2014
Received in revised form 10 July 2014
Accepted 11 July 2014
Available online xxxx
Two Ru(II) polypyridyl complexes [Ru(phen)2(idpq)](ClO4)2 (1) and [Ru(dmp)2(idpq)](ClO4)2 (2) were
synthesized and characterized. Cytotoxicity, apoptosis, cell cycle arrest, reactive oxygen species and
mitochondrial membrane potential were assayed. The IC50 values of complexes 1 and 2 toward HepG2, A549, MG-63 and HeLa cell lines range from 15.1 ± 2.1 to 24.8 ± 2.4 lM. The complexes can effectively
induce apoptosis and induce cell cycle arrest at G0/G1 phase by an increase of 4.88% for 1 and 6.64% for 2
at G0/G1 phase in HeLa cells. Complexes 1 and 2 can enter the cytoplasm and accumulate in the nuclei.
The complexes can enhance the level of reactive oxygen species. The ratio of the red/green is 0.74 and
0.52 for complexes 1 and 2, which suggests that the complexes induce a decrease of mitochondrial
membrane potential. These complexes induce apoptosis in HeLa through ROS-mediated mitochondrial
dysfunction pathway.
Crown Copyright Ó 2014 Published by Elsevier B.V. All rights reserved.
SI: Antitumor Active Organotin Compounds
Keywords:
Ru(II) complexes
Cytotoxicity in vitro
Apoptosis
ROS
Mitochondrial membrane potential
Cell cycle arrest
1. Introduction
Since the introduction of cisplatin by Rosenberg in 1965, the
area of research on bioinorganic chemistry is a challenging topic
for in vivo and in vitro studies [1–4]. Platinum complexes
represent one of the most successful families of clinically used
anticancer drugs. However, cisplatin, a platinum(II) diamine complex used in 70% of cancer treatment has some drawbacks like
toxic side-effects and lack of activity (drug resistance) against several types of cancer which are problems need to be overcome [5].
These drawbacks have motivated extensive investigations into
alternative metal-based cancer therapies. In recent years, some
other metals have also attracted growing research attention [6].
Among the metal-based compounds, ruthenium complex is one
of the most promising potent drugs. The aqua-complex [(g6-pcymene)Ru(OH2)(j2-N,N-2-pydaT)](BF4)2 (2-pydaT = 2,4-diamino6-(2-pyridyl)-1,3,5-triazine) displays notable pH-dependent
cytotoxic activity in human ovarian carcinoma cells (A2780), its
IC50 values are 11.0 lM at pH 7.4 and 6.58 lM at pH 6.5 [7]. The
monofunctional Ru(II)-arene complex [(g6-arene)Ru(II)(en)Cl]+,
⇑ Corresponding authors. Tel.: +86 20 39352122; fax: +86 20 39352129.
E-mail
(Y.-J. Liu).
addresses:
hhongliang@163.com
(H.-L.
Huang),
lyjche@163.com
where en = 1,2-diaminoethane and the arene is para-terphenyl
exhibits promising cytotoxic effects in human tumor cells
including those resistant to conventional cisplatin, and
investigations have shown that the complex induces apoptosis by
regulating the expression of Bcl-2 family proteins [8]. Many ruthenium(II) complexes have shown interesting properties [9–22].
[Ru(phpy)(bpy)(dppn)]+ (bpy = 2,20 -bipyridine, dppn = benzo[i]
dipyrido[3,2-a:20 ,30 -c]phenazine), is 6 times more active than the
platinum drug in HeLa, and the complex is able to disrupt the mitochondria membrane potential [23]. The [Ru(phen)2(addppn)]2+
induces apoptosis in BEL-7402 cells through ROS-mediated mitochondrial dysfunction pathway [24]. Ru(2,6-bis(2,4,6-trimethylphenyliminomethyl))(2-(phenylazo)-3-methylpyridine) shows very
high cytotoxic activity against EVST-A cells with a low IC50 value
of 0.4 lM [25]. To obtain more insight into cytotoxic activity of
Ru(II) complexes, in this report, two Ru(II) polypyridyl complexes
[Ru(phen)2(idpq)](ClO4)2 (1) (phen = 1,10-phenanthroline, idpq = indeno [1,2-b]dipyrido[3,2-f:20 ,30 -h]-quinoxaline-6-one) [26]
and [Ru(dmp)2(idpq)](ClO4)2 (2) (dmp = 2,9-dimethyl-1,10-phenanthroline, Scheme 1) were synthesized and complex 2 was characterized by elemental analysis, ES-MS and 1H NMR. The
cytotoxicity in vitro was investigated by MTT (MTT = (3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)) assay.
The apoptosis of HeLa cells induced by the Ru(II) complexes was
http://dx.doi.org/10.1016/j.ica.2014.07.017
0020-1693/Crown Copyright Ó 2014 Published by Elsevier B.V. All rights reserved.
Please cite this article in press as: W. Li et al., Inorg. Chim. Acta (2014), http://dx.doi.org/10.1016/j.ica.2014.07.017
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W. Li et al. / Inorganica Chimica Acta xxx (2014) xxx–xxx
Scheme 1. The structures of complexes 1 and 2.
studied with acridine orange (AO) and ethidium bromide (EB)
staining method. The cell cycle arrest was analyzed by flow cytometry. The cellular uptake and co-localization were investigated
with DAPI-stained, and the reactive oxygen species and mitochondrial membrane potential were also studied with fluorescence
microscopy and microplate analyzer.
2. Experimental
2.1. Materials and method
All reagents and solvents were purchased commercially and
used without further purification unless otherwise noted. Ultrapure MilliQ water was used in all experiments. DMSO and RPMI
1640 were purchased from Sigma. Cell lines of HepG-2 (Human
hepatocellular carcinoma cell line), HeLa (Human cervical cancer
cell line), MG-63 (Human osteosarcoma) and A549 (Human breast
cancer) were purchased from the American Type Culture Collection. RuCl33H2O was purchased from the Kunming Institution of
Precious Metals. 1,10-phenanthroline was obtained from the
Guangzhou Chemical Reagent Factory.
Microanalyses (C, H, and N) were obtained with a Perkin–Elmer
240Q elemental analyzer. Electrospray ionization mass spectra (ESMS) were recorded on a LCQ system (Finnigan MAT, USA) using
acetonitrile as mobile phase. The spray voltage, tube lens offset,
capillary voltage and capillary temperature were set at 4.50 kV,
30.00 V, 23.00 V and 200 oC, respectively, and the quoted m/z values are for the major peaks in the isotope distribution. 1H NMR
spectra were recorded on a Varian-500 spectrometer with DMSO
[d6] as solvent and tetramethylsilane (TMS) as an internal standard
at 500 MHz at room temperature.
2.2. The preparation of ligand and complexes
The ligand ipdq and complex [Ru(phen)2(idpq)](ClO4)2 (1) were
synthesized according to the literature [26].
2.2.1. Preparation of [Ru(dmp)2(idpq)](ClO4)2 (2)
A mixture of cis-[Ru(dmp)2Cl2]2H2O [27] (0.312 g, 0.5 mmol)
and idpq (0.167 g, 0.5 mmol) in ethylene glycol (30 mL) was heated
at 150 °C under argon for 8 h to give a clear red solution. Upon
cooling, a red precipitate was obtained by dropwise addition of saturated aqueous NaClO4 solution. The crude product was purified by
column chromatography on neutral alumina with a mixture of
CH3CN–toluene (3:1, v/v) as eluent. The red band was collected.
The solvent was removed under reduced pressure and a red powder was obtained. Yield: 68%. Anal. Calc for C49H34N8Cl2O9Ru: C,
56.01; H, 3.26; N, 10.66. Found: C, 55.89; H, 3.44; N, 10.56%. 1H
NMR (DMSO-d6): d 9.49 (d, 1H, J = 7.0 Hz), 9.33 (d, 1H, J = 7.0 Hz),
8.94 (d, 2H, J = 8.5 Hz), 8.44 (d, 4H, J = 7.0 Hz), 8.27 (dd, 2H,
J = 4.0, J = 4.5 Hz), 8.17 (d, 1H, J = 7.5 Hz), 8.01 (d, 2H, J = 8.5 Hz),
7.97–7.93 (m, 2H), 7.78 (d, 1H, J = 7.0 Hz), 7.69 (d, 1H, J = 6.0 Hz),
7.62 (dd, 2H, J = 5.5, J = 5.5 Hz), 7.58 (d, 1H, J = 5.5 Hz), 7.42 (dd,
2H, J = 8.5, J = 8.0 Hz), 1.95 (s, 6H), 1.79 (s, 6H). ES-MS (CH3CN):
m/z 951.4 ([M-ClO4]+), 425.6 ([M-2ClO4]2+).
Caution: Perchlorate salts of metal compounds with organic
ligands are potentially explosive, and only small amounts of the
material should be prepared and handled with great care.
2.3. Cytotoxicity assay in vitro
Standard
3-(4,5-dimethylthiazole)-2,5-diphenyltetraazolium
bromide (MTT) assay procedures were used [28]. Cells were placed
in 96-well microassay culture plates (8 103 cells per well) and
grown overnight at 37 °C in a 5% CO2 incubator. Complexes tested
were then added to the wells to achieve final concentrations ranging from 106 to 104 M. Control wells were prepared by addition
of culture medium (100 lL). The plates were incubated at 37 °C in
a 5% CO2 incubator for 48 h. Upon completion of the incubation,
stock MTT dye solution (20 lL, 5 mg/mL1) was added to each well.
After 4 h, buffer (100 lL) containing N,N-dimethylformamide (50%)
and sodium dodecyl sulfate (20%) was added to solubilize the MTT
formazan. The culture medium and cisplatin were used as the negative and positive controls, respectively. The optical density of each
well was then measured with a microplate spectrophotometer at a
wavelength of 490 nm. The IC50 values were determined by plotting the percentage viability versus concentration on a logarithmic
graph and reading off the concentration at which 50% of cells
remain viable relative to the control. Each experiment was
repeated at least three times to obtain the mean values. Four different tumor cell lines were the subjects of this study: HepG-2, A549,
MG-63 and HeLa cell lines.
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2.4. Cellular uptake and co-localisation studies
HeLa cells were placed in 24-well microassay culture plates
(4 104 cells per well) and grown overnight at 37 °C in a 5% CO2
incubator. Complexes tested were then added to the wells. The
plates were incubated at 37 °C in a 5% CO2 incubator for 24 h. Upon
completion of the incubation, the wells were washed three times
with phosphate buffered saline (PBS). After removing the culture
medium, the cells were stained with 2-(4-amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI) and visualized by fluorescence microscope.
for 24 h. The cells were cultured in RPMI 1640 supplemented with
FBS (10%) and incubated at 37 °C and 5% CO2. The medium was
removed and replaced with medium (final DMSO concentration,
0.05% v/v) containing complexes 1 and 2 (25 lM). After an incubation of 24 h, the cell layer was trypsinized and washed with cold
PBS and fixed with 70% ethanol. Twenty lL of RNAse (0.2 mg/mL)
and 20 lL of propidium iodide (0.02 mg/mL) were added to the cell
suspensions and the cells were incubated at 37 °C for 30 min. Then
the samples were analyzed with a FACSCalibur flow cytometry. The
number of cells analyzed for each sample was 10 000 [29].
2.9. Statistical analysis
2.5. Apoptosis assay by AO/EB staining method
HeLa cells were seeded onto chamber slides in six-well plates at
a density of 2 105 cells per well and incubated for 24 h. The cells
were cultured in RPMI 1640 supplemented with 10% of fetal bovine
serum (FBS) and incubated at 37 °C and 5% CO2. The medium was
removed and replaced with medium (final DMSO concentration,
0.05% v/v) containing the complexes (25 lM) for 24 h. The medium
was removed again, and the cells were washed with ice-cold PBS,
and fixed with formalin (4%, w/v). Cell nuclei were counterstained
with acridine orange (AO) and ethidium bromide (EB) (AO: 100 lg/mL,
EB: 100 lg/mL) for 10 min. Then the cells were observed and
imaged by a fluorescence microscope (Nikon, Yokohama, Japan)
with excitation at 350 nm and emission at 460 nm.
2.6. Reactive oxygen species (ROS) detection
HeLa cells were seeded into six-well plates (Costar, Corning
Corp, New York) at a density of 2 105 cells per well and incubated
for 24 h. The cells were cultured in RPMI 1640 supplemented with
10% of FBS and incubated at 37 °C and 5% CO2. The medium was
removed and replaced with medium (final DMSO concentration,
0.05% v/v) containing complexes 1 and 2 (12.5 and 25 lM) for
24 h. The medium was removed again. The fluorescent dye 20 ,70 dichlorodihydrofluorescein diacetate (H2DCFDA, 10 lM) was
added to the medium to cover the cells. The treated cells were then
washed with cold PBS–EDTA twice, collected by trypsinization and
centrifugation at 1500 rpm for 5 min, and cell pellets were suspended in PBS–EDTA and then imaged by fluorescence microscope.
The fluorescent intensity was determined by microplate analyzer
(Infinite M200, TECAN, Switzerland) with excitation at 488 nm
and emission at 525 nm. The fluorescent intensity was calculated
by the determined fluorescent intensity minus the fluorescence
intensity of the complexes in the corresponding concentration.
2.7. Mitochondrial membrane potential assay
HeLa cells were treated for 24 h with the complex in 12-well
plates and were then washed three times with cold PBS. The cells
were detached with trypsin–EDTA solution. Collected cells were
incubated for 20 min with 1 lg/mL of JC-1 in culture medium at
37 °C in the dark. Cells were immediately centrifuged to remove
the supernatant. Cell pellets were suspended in PBS and then
imaged by fluorescence microscope. The fluorescent intensity
was determined by microplate analyzer (Infinite M200, TECAN,
Switzerland) with excitation at 488 nm and emission at 525 nm.
The fluorescent intensity was calculated by the determined fluorescent intensity minus the fluorescent intensity of the complexes
in the corresponding concentration.
All of the data were expressed as the mean ± SD. Differences
between two groups were analyzed by a two-tailed Student’s t test.
Differences with P < 0.05 were considered statistically significant.
3. Results and discussion
3.1. Synthesis and characterization
The ligand and complex 1 were synthesized according to the literature [26]. Complex 2 was prepared by the direct reaction of
ligand with [Ru(dmp)2Cl2]2H2O in ethylene glycol. The desired
ruthenium(II) complexes were isolated as the perchlorate and
purified by column chromatography. Each synthetic step involved
here is straightforward and provides a relative high yield of the
desired product in pure form. In the ES-MS spectra of the complex,
all of the expected signals of ([M-ClO4]+) and ([M-2ClO4]2+) were
observed. The measured molecular weights were consistent with
the expected values.
3.2. Cytotoxic activity in vitro assay
Liu reported that complex 1 interacts with CT DNA with a large
DNA-binding affinity (4.0 ± 0.6) 106 M1 [26]. This prompts us to
investigate the anticancer activity of the complex. The cytotoxicity
of complexes 1 and 2 were comparable to that of cisplatin against
HepG-2, HeLa, MG-63 and A549 cells. The IC50 values are listed in
Table 1. Complex 1 shows more sensitive to HepG-2 cells than
complex 2. Treatment of A549 cells with 1 and 2, the two complexes exhibit the same cytotoxic activity. However, treatment of
HeLa and MG-63, complex 2 displays relatively high cytotoxic
activity than complex 1 under identical conditions. Comparing
the IC50 values, the both complexes all show lower cytotoxic effect
than cisplatin and [Ru(phen)2(addppn)]2+ [24] on the selected cell
lines. In addition, these results also suggest that different complexes reveal different cytotoxic activity against different tumor
cell lines.
3.3. Apoptosis assay by AO/EB staining method
To assess whether complexes 1 and 2 cause cell death by apoptosis or necrosis, the AO/EB assays were performed. AO can pass
through cell membrane, but EB cannot. As shown in Fig. 1, in the
Table 1
The IC50 values of complexes 1 and 2 against HepG-2, HeLa, MG-63 and A549 cell
lines.
Complex
2.8. Cell cycle arrest by flow cytometry
HeLa cells were seeded into six-well plates (Costar, Corning
Corp, New York) at a density of 2 105 cells per well and incubated
1
2
Cisplatin
IC50 (lM)
HepG-2
HeLa
MG-63
A549
17.3 ± 1.5
24.8 ± 2.3
11.5 ± 1.2
24.8 ± 2.4
15.8 ± 2.1
7.3 ± 1.4
19.6 ± 2.0
15.1 ± 1.8
6.6 ± 0.5
15.8 ± 1.4
15.8 ± 1.3
6.7 ± 0.8
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Fig. 1. AO/EB staining HeLa cells for 24 h. (a) Control, (b) and (c) treated with 25 lM of complexes 1 and 2. L and A stand for living and apoptotic cells, respectively.
control, the living cells were stained bright green in spots (Fig. 1a)
and exhibit homogeneous nuclei staining. The apoptotic and necrotic cells can be distinguished from one another using fluorescence
microscopy. Treatment of HeLa cells with 25 lM of complexes 1
(Fig. 1b) or 2 (Fig. 1c) for 24 h, green apoptotic cells (typical apoptotic changes, e.g., staining bright, condensed chromatin, and fragmented nuclei) stained by acridine orange were observed. These
results indicate that complexes 1 and 2 can induce apoptosis in
HeLa cells.
3.4. Cellular uptake and co-localization
Most of ruthenium(II) complexes can emit fluorescence at room
temperature [30–33]. Photophysical properties of complexes 1 and
2 were used to evaluate their localization in HeLa cells. Treatment
of HeLa cells with complexes 1 or 2 for 24 h, the cells was stained
with DAPI. As shown in Fig. 2, the blue channel shows DAPI-stained
nuclei, the red channel displays the luminescence of complexes 1
and 2 with an excitation wavelength of 460 nm, and the overlay
represents cellular association of the complexes. These results
suggest that the complexes can be uptaken by HeLa cells and
complexes can accumulate in the cell nuclei.
3.5. Reactive oxygen species (ROS) assay
To determine the effect of complexes 1 and 2 on intracellular
ROS generation, HeLa cells were exposed to 1 and 2 for 24 h. ROS
levels were evaluated with fluorescence microscopy using a
H2DCFDA as fluorescence probe. H2DCFDA is a fluorescent dye that
diffuses through cell membrane and is hydrolyzed by intracellular
esterases to DCFH. DCFH is oxidized to DCF in the presence of ROS,
DCF can emit fluorescence, and its level corresponds to the level of
generated ROS [34]. As shown in Fig. 3, in the control (Fig. 3a), no
fluorescence point was observed. Treatments of HeLa cells with
12.5 lM of complexes 1 (Fig. 3b) and 2 (Fig. 3c), green fluorescence
points were found. In order to investigate the effect of concentration of the complexes on the fluorescent intensity, the levels of
ROS were also studied with microplate analyzer. Comparing with
the control, DCF fluorescent intensities increase (Fig. 4). The
increasing extent of the DCF fluorescent intensity induced by 2 is
higher than that induced by 1 under identical conditions. Furthermore, the levels of ROS induced by 1 and 2 are concentrationdependent. These results suggest that complexes 1 and 2 can
enhance the levels of ROS.
3.6. Mitochondrial membrane potential detection
The changes in mitochondrial membrane potential (DWMMP)
induced by complexes 1 and 2 were assayed using JC-1 as a fluorescent probe. JC-1 with aggregates emits red fluorescence corresponding to high MMP, whereas monomeric JC-1 emits green
fluorescence corresponding to low MMP. As shown in Fig. 5, in
the control (Fig. 5a), the red fluorescent points were observed.
After the HeLa cells were exposed to 12.5 lM of complexes 1
(5b) or 2 (5c), green fluorescent points with little red fluorescence
were found. To quantitatively measure the red and green
Fig. 2. Fluorescence miscroscopy images of HeLa cells exposure to 25 lM of complexes 1, 2 for 24 h. Images show DAPI staining, cellular staining of Ru(II) complexes, and the
overlay.
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Fig. 3. Intracellular ROS was detected in HeLa cells (a) exposure to 12.5 lM of complexes 1 (b) and 2 (c) for 24 h.
Fig. 4. Effects on ROS generation induced by different concentrations of complexes
1 ( ) and 2 ( ) in HeLa cells. Data were calculated from three independent
experiments.
Fig. 6. Assay of HeLa cells mitochondrial membrane potential with JC-1 as
fluorescent probe staining method. HeLa cells exposed to 12.5 and 25 lM of
complexes 1 ( ) and 2 ( ) for 24 h. ⁄p < 0.05 represents significant differences
compared with control.
fluorescent intensity, the ratios of red/green intensity were determined with microplate analyzer. In the control (Fig. 6), the ratio
of red/green is 2.27. Treatment of HeLa cells with 12.5 and
25 lM of complexes 1 and 2, the ratios of red/green are 1.57,
0.74 and 1.54, 0.52, respectively. The changes from red to green
and the decrease of the ratio indicate that the complexes can
induce the decrease of mitochondrial membrane. Moreover, the
DWMMP shows concentration dependent manner.
3.7. Cell cycle arrest studies
The effect of complexes 1 and 2 on the cell cycle arrest of the
HeLa cells was studied using flow cytometry in propidiumiodide-stained method. The status of cell cycle for cells treated
with 25 lM of complexes 1 and 2 for 24 h was shown in Fig. 7,
an increase of 4.88% for 1 and 6.64% for 2 of cells at G0/G1 phase
was observed, accompanied by a corresponding reduction of
7.04% for 1 and 9.92% for 2 in the percentage of cells in the S phase,
Fig. 7. Cell cycle distribution of HeLa exposure to 25 lM complexes 1 and 2 for
24 h. Data were obtained by three independent experiments.
Fig. 5. Assay of HeLa cells mitochondrial membrane potential with JC-1 as fluorescent probe staining method. HeLa cells (control, a) exposed to 12.5 lM of complexes 1 (b)
and 2 (c) for 24 h.
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respectively. In the G2/M phase, a small amount of increase was
found. These data indicate that the anti-proliferative mechanism
induced by complexes 1 and 2 on HeLa cells is G0/G1 phase arrest.
In addition, the apoptotic percentage in the cells increases by
26.03% for 1 and 28.45% for 2. Obviously, complex 2 shows more
effective apoptosis than complex 1 under the same conditions. This
is consistent with the cytotoxic activity (IC50 values) of complexes
1 and 2 against HeLa cells.
4. Conclusion
Two ruthenium(II) complexes were synthesized and characterized. Complex 2 shows higher cytotoxic activity than complex 1
toward HeLa and MG-63 cells. The complexes can effectively
induce apoptosis in HeLa cells. Complexes 1 and 2 can enter into
the cytoplasm and accumulate in the nuclei. These complexes
can enhance the levels of ROS and decrease the mitochondrial
membrane potential. The complexes inhibit the HeLa cell growth
at Go/G1 phase. These results exhibit that complexes 1 and 2
induce HeLa apoptosis through ROS-mediated mitochondrial
dysfunction pathway.
Acknowledgments
This work was supported by the National Nature Science Foundation of China (no. 31070858), High-level Personnel Project of
Guangdong Province in 2013 and the Joint Nature Science fund
of the Department of Science and Technology and the First Affiliated Hospital of Guangdong Pharmaceutical University (No
GYFYLH201315).
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