Synthesis, Crystal Structures, and Anti-cervical Cancer Activity Evaluation of Three Trinuclear Transition Metal(II) Complexes with N,O-Donor Schiff Base Ligand

Article abstract of DOI:10.1002/zaac.201800522

A new bi-nucleating Schiff base ligand, 2-(((3-(dimethylamino)propyl)imino)methyl)-6-methoxyphenol (HL₁) was prepared by a one-pot condensation reaction, which was further used in the construction of three trinuclear Schiff base transition metal(II) complexes [Cu₃(L1)₂(OH)₂(H₂O)₂](NO₃)₂ (1), [Co₃(L1)₂(OH)₂(H₂O)₂](NO₃)₂ (2), and [Cu₃(L1)₂(N₃)₄] (3). Furthermore, a green hand grinding technique was implemented to reduce the particle size of the coordination complexes to generate the nanoscale compounds. The SEM studies reveal the formation of square and spherical particles for nano 1 and 2, and nanorod for nano 3. In addition, the anti-proliferation activity of nano 1–3 was detected on the human cervical cancer Hela cells with CCK-8 assay. The cell viability curves and IC₅₀ values indicated that only nano 1 has anti-proliferation activity on Hela cells. To further investigate the mechanism of nano 1 induced Hela cell death, the Annexin V-FITC/PI double staining assay, western blot assay, and ROS level detection was conducted.

Full text of DOI:10.1002/zaac.201800522

Journal of Inorganic and General Chemistry
DOI: 10.1002/zaac.201800522
ARTICLE
Zeitschrift für anorganische und allgemeine Chemie
Synthesis, Crystal Structures, and Anti-cervical Cancer Activity
Evaluation of Three Trinuclear Transition Metal(II) Complexes
with N,O-Donor Schiff Base Ligand
Teng-Fei Luo,[a] Xiao-Feng Ji,[b] and Cheng-Yi Zhu*[c]
Abstract. A new bi-nucleating Schiff base ligand, 2-(((3-(dimeth- reveal the formation of square and spherical particles for nano 1 and
ylamino)propyl)imino)methyl)-6-methoxyphenol (HL
by a one-pot condensation reaction, which was further used in the con-
struction of three trinuclear Schiff base transition metal(II) complexes CCK-8 assay. The cell viability curves and IC50 values indicated that
1
) was prepared 2, and nanorod for nano 3. In addition, the anti-proliferation activity
of nano 1–3 was detected on the human cervical cancer Hela cells with
[
(
Cu
2), and [Cu
3
(L1)
2
(OH)
2
(H
2
O)
2
](NO
3
)
2
(1), [Co
3
(L1)
2
(OH)
2
(H
2
O)
2
](NO
3
)
2
only nano 1 has anti-proliferation activity on Hela cells. To further
investigate the mechanism of nano 1 induced Hela cell death, the An-
nexin V-FITC/PI double staining assay, western blot assay, and ROS
3
(L1)
2
(N
3 4
) ] (3). Furthermore, a green hand grinding tech-
nique was implemented to reduce the particle size of the coordination
complexes to generate the nanoscale compounds. The SEM studies level detection was conducted.
Introduction
atoms of tetradentate salen type salicylaldimine Schiff bases
prepared by the condensation of several diamines with salicyl-
(
New drugs for the treatment of cancer with better rates of
cure and less severe side effects are needed because cancer has
become one of the most serious diseases that kills millions of
aldehyde derivatives) for the synthesis of various homo- and
hetero-polynuclear complexes of different transition and non-
transition metals.[ Many of these hetero and homo polynu-
clear complexes show anticancer, anti-convulsant, antitumour,
antifungal, antibacterial, anti-tubercular, antioxidant, antima-
larial and anti-inflammatory activities and in corrosion inhibi-
tion. Due to such a wide range of applications, considerable
effort is being directed to the development of new chelating
Schiff base ligands, particularly the poly-nucleating type. In
the research of metal-based anticancer drugs, aside from the
significance of the central metal, the choice of the ligand plays
an important role as well.[
10]
[1]
people every year. In this field coordination chemistry has a
great potential to offer a wide variety of compounds with dif-
ferent arrangement, redox reactivity, and a diversity of mecha-
nisms related to DNA binding, some of them unique to met-
als.[ The usefulness of coordination metal complexes in can-
cer chemotherapy has been demonstrated by Cisplatin and
other platinum coordination compounds, which are amongst
the most successfully used anticancer drugs.[ As a result of
an intense and continued research on coordination complexes
with antitumor activity, compounds of different metals other
than Pt are entering clinical studies. Recent literatures have
2]
3]
11]
To study the metal and ligand effects in the anticancer ac-
tivities of the Schiff base metal complexes, in this study, a
new bi-nucleating Schiff base ligand, 2-(((3-(dimethylamino)
II
II
revealed that the Cu and Co -based coordination complexes
show promising anticancer activities.[4–6]
propyl)imino)methyl)-6-methoxyphenol (HL ) was prepared
1
The design, synthesis, and structural characterization of
Schiff base complexes are still being vividly pursued because
of their diverse spectra of biological and pharmaceutical prop-
by a one-pot condensation reaction, which was further used in
the construction of three trinuclear Schiff base transition
erties.[7–9]
metal(II) complexes, [Cu
3
(L1)
2
(OH)
2
(H
2
O)
2
](NO
3
)
2
(1),
Synthetic inorganic chemists have tremendous fasci-
[
(
Co (L1) (OH) (H O) ](NO ) (2), and [Cu (L1) (N ) ] (3)
Scheme 1). The as-prepared complexes were characterized
3
2
2
2
2
3 2
3
2
3 4
nation to take advantage of the ability of the phenoxo oxygen
*
Dr. C.-Y. Zhu
using various physicochemical techniques, e.g. single-crystal
X-ray diffraction (SCXRD), power X-ray diffraction (PXRD),
elemental analyses (EA) and thermogravimetric analysis
E-Mail: chengyi_zhu666@163.com
[
[
[
a] Department of Gynaecology and Obstetrics
Hangzhou Women’s Hospital
Hangzhou, Zhejiang, P. R. China
(TGA). Furthermore, a green hand grinding technique was im-
b] Department of Gynecology
plemented to reduce the particle size of the coordination com-
plexes to generate nano 1–3. In addition, the anti-proliferation
activity of nano 1–3 on human cervical cancer Hela cells was
studied via the CCK-8 assay, which suggested that only nano
Qingdao Chengyang People’s Hospital
Qingdao, Shandong, P. R. China
c] Department of Gynaecology and Obstetrics
Affliated Dongfeng Hospital
Hubei University of Medicine
Shiyan, Hubei, P. R. China
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/zaac.201800522 or from the au-
thor.
1
exhibits promising anti-cancer activity, and the observed ex-
perimental results were further supported by the Annexin V-
FITC/PI assay and intracellular ROS assay.
Z. Anorg. Allg. Chem. 0000, ᭿,0–0
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X-ray Crystallography: Single crystal X-ray crystal data of 1–3 was
collected with a computer-controlled Oxford Xcalibu E diffractometer
with graphite–monochromated Mo-K radiation (λ = 0.71073 Å) at
α
room temperature. Absorption corrections were applied using SAD-
ABS. The structures were solved by direct methods by the SHELXS-
2
2014 package and refined by full-matrix least-square methods on F
by using the SHELXL-2014/6. All non-hydrogen atoms were refined
anisotropically and all hydrogen atoms were generated in their ideal
locations. Crystallographic data and refinement details are summarized
in Table S1 (Supporting Information).
Scheme 1. Synthetic routes to the title complexes 1–3.
Cell Lines and Cell Culture: The human cervical cancer Hela cell
line and normal human embryonic kidney cell line HEK-293 was pur-
Experimental Section
Materials and Instrumentation: All reagents and solvents employed chase from American Type Culture Collection (ATCC, Rockville,
in this work were commercially available and used without further MD). The Hela cell line was cultured in ATCC-formulated Leibovitz’s
purification. The Schiff base (HL
densation of N,N-dimethyl-1,3-diaminopropane with 3-methoxysali-
cylaldehyde following the literature method. Elemental analyses (C,
H and N), thermogravimetric analysis, infrared spectra, powder X-ray
diffraction (PXRD) analyses, and Scanning Electron Microscopy
1
) ligand was synthesized by the con-
l-15 medium (Gibco, Life Technologies, Carlsbad, CA, USA); the
HEK-293 cell line was cultured in Dulbecco’s modified Eagle’s me-
dium (DMEM; Gibco, Life Technologies, Carlsbad, CA, USA). Both
culture mediums were supplemented with 100 U per mL Penicillin and
Streptomycin solutions (Gibco, Life Technologies), 10% (v/v) heat-
[
13]
(SEM) were determined with a Perkin–Elmer 240 elemental analyzer, inactivated fetal bovine serum (FBS) (HyClone, Logan, UT, USA), and
NETSCHZ STA–449C thermo-analyzer, Nicolet Magna 750 FT-IR 2% l-glutamine. All the cells were cultured at 37 °C in a humidified
spectrometer, Bruker AXS D8 advanced automated diffractometer, and atmosphere of 5% CO with 95% air. The culture medium was re-
2
a JEOL JSM-6700F field-emission microscope, respectively.
placed twice a week according to the cell growth status.
Synthesis of Complex 1:
Cu(NO ·3H O (240 mg, 1 mmol) was added to the methanol solution
of the ligand, HL (222 mg, 1 mmol), and the resulting solution was
refluxed for ca. 30 min. A methanol–water (2:1) solution (10 mL) of
NaNO (168 mg, 2 mmol) was added and the refluxing was continued
for an additional 1 h. Blue block-shaped single crystals of 1 suitable
for X-ray diffraction were obtained after one week on slow evaporation
of the solution in open atmosphere. The crystals were separated by
filtration, washed with MeOH and dried in the air. Yield: ca. 68%
A methanol solution (10 mL) of
Cell Counting Kit-8 (CCK-8) Assay: To assess the viability of the
human cervical cancer Hela cells and human embryonic kidney HEK-
3
)
2
2
1
2
93 cells after treated with nano 1–3. The Cell Counting Kit-8 assay
[20]
was conducted following the manufacturer’s protocols.
In brief, the
Hela and HEK-293 cells were seeded at 5ϫ10 cells per well in 6-
well plates and grown to confluence of 70–80% at 37 °C in a 5% CO
3
5
2
-
humidified atmosphere. Next, the cells were incubated with a series of
concentrations of nano 1–3 (1, 2, 4, 8, 10, 20, 40, 80, 100 μM) for 24
h at 37 °C, 5% CO
2
. After treatment, the cells were harvested with
based on copper(II). C24
H
40Cu
3
N
6
O
14: calcd. C 34.85; H 4.87; N
0.16%; found: C 34.91; H 5.12; N 10.37%. FT-IR: ν˜ = 3348 (m),
026 (m), 2983 (m), 2915 (s), 2827 (s), 1614 (s), 1601 (s), 1526 (s),
1
ϫtrypsin-EDTA (5 min, 37 °C) and centrifuged at 800 rpm for 5
1
3
1
min. The medium was discarded, and the 10% CCK-8 (Dojindo
Laboratories, Kumamoto, Japan) in 100 μL medium without FBS was
added into wells for 2 h incubation at 37 °C in the dark. Afterwards,
Thermo Scientific Microplate Reader was used to measure the ab-
sorbance of each well at 450 nm. The Cell viability curves were calcu-
lated and plotted according to the absorbance values. Three replicate
wells were used to determine each point. The IC50 values were calcu-
lated using SPSS version 22.0.
–
1
446 (s), 1358 (s), 1223 (s), 1062 (w), 853 (w), 774 (w) cm . UV/
Vis: (MeOH): λmax = 321, 410 nm.
Synthesis of Complex 2: The synthesis manipulation for compound 2
was similar to that of compound 1 except that Co(NO
300 mg, 1 mmol) was used to replace the Cu(NO ·3H O in the reac-
tion. Yield: ca. 62% based on cobalt(II). C24 14: calcd. C
4.85; H 4.87; N 10.16%; found: C 35.12; H 4.96; N 10.08%. FT-
IR: ν˜ = 3556 (m), 3459 (m), 3325 (m), 2981 (m), 2912 (m), 1632 (s),
580 (m), 1454 (s), 1428 (s), 1329 (s), 1211 (m), 1075 (m), 1008
m), 916 (s), 743 (m), 721 (s), 678 (s). UV/Vis (MeOH): λmax = 377,
70 nm.
3 2 2
) ·6H O
(
3
)
2
2
3 6
H40Co N O
3
Annexin V-FITC/PI Apoptosis Analysis: To assess the percentage of
apoptotic Hela cells after treated with nano 1–3, the Annexin V-FITC/
PI staining assay (Abcam Apoptosis Detection Kit; ab214663) was
1
(
4
[
21]
conducted according with the manufacturer’s instruction.
In brief,
the Hela cells were seeded in 6-well plates (1ϫ10 cells per well) at
37 °C, 5% CO overnight. After the cells grow into the logarithmic
6
Synthesis of Complex 3: A methanol solution Cu(NO
3
)
2
·3H
2
O
2
(240 mg, 1 mmol) was added to the methanol solution of the ligand,
stage with the confluence reached 70–80%, the Hela cells were ex-
HL
1
(333 mg, 1.5 mmol), and the resulting solution was refluxed for
posed to nano 1–3 at the concentrations as described above for 24 h
ca. 30 min. A methanol–water (2:1) solution (10 mL) of sodium azide at 37 °C, 5% CO
130 mg, 2 mmol) was added and the refluxing was continued for an tive control, and the Cisplatin, known as the apoptosis inducer, was
additional 1 h. Blue block-shaped single crystals of 3 suitable for X- used as the positive control. After that, the cells were collected with
ray diffraction were obtained after one week on slow evaporation of 0.25% w/v trypsin and washed three times with pre-cooled PBS.
2
. The same volume of solvent was used as the nega-
(
the solution in open atmosphere. The crystals were separated by fil- 500 μL Annexin V binding buffer was added to re-suspended the Hela
tration, washed with MeOH and dried in the air. Yield: ca. 54% based cells followed by 5 μL Annexin V-FITC and 5 μL propidium iodide
on copper(II). C27
H
40
N
16
O
4
Cu
3
: calcd. C 38.45; H 4.78; N 26.57%,
(PI) addition for 15 min incubation at 37 °C in the dark. Finally, the
found: C 38.12; H 4.66; N 26.39%. FT-IR: ν˜ = 3564 (br., m), 2982 Hela cells were analyzed for the proportion of intact live cells and
(
1
w), 2725 (w), 1631 (vs), 1545 (m), 1491 (s), 1456 (vs), 1371 (w),
apoptotic cells with flow cytometry (BD Via, New Jersey, USA) at an
625 nm. The results were analyzed using flow cytometry (FACSCali-
325 (w), 129 7(m), 1132 (m), 1081 (s), 951 (m), 852 (w), 746 (m) excitation wavelength of 488 nm and emission wavelengths of 525 and
–1
cm . UV/Vis (MeOH): λmax = 385, 477 nm.
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ARTICLE
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II
III
II
bur, BD Biosciences, USA). Each experiment was performed in tripli- Cd and Co coordination complexes, but the Cu -based co-
cate.
ordination complexes based on the HL1 ligand have not been
reported so far (Table S2, Supporting Information).[
three complexes were prepared by addition of methanol solu-
tion of metal salts to the methanol solution of HL followed
by the addition of NaNO for 1, 2 and NaN for 3. Crystals of
12–14]
The
Intracellular ROS Assay: The DCFH-DA staining method was used
to measure the intracellular ROS generation in Hela cells after treat-
ment with nano 1–3 by flow cytometer following the instruction.[22]
1
The flow fluorescence level reflects the content of intracellular ROS.
3
3
5
The Hela cells were planted in 6-well plates (5ϫ10 cells per well) the complex were obtained after few days on slow evaporation
and incubated at 37 °C, 5% CO
2
before experiment. When the cells of the filtrate.
grow into the logarithmic stage with the confluence reached 70–80%,
nano 1–3 was added at the concentrations described above. After incu-
bation for 24 h, the cells were harvested with 0.25% trypsin and de-
tected with flow cytometer (BD Via, New Jersey, USA) at an excitation
wavelength of 488/530 nm and analyzed using FlowJo7.6 software.
All experiment was performed at least three times.
The complexes were characterized by elemental and spectral
analysis and single-crystal X-ray diffraction studies. The X-
ray crystal structural determination reveals that complexes 1–
3
reveal similar trinuclear cluster-based discrete structures but
with different composing.
The SCXRD analysis study reveals that complex 1 belongs
II
Western Blot Detection: Western blot detection was performed as to the monoclinic space group I2/a featuring a trinuclear Cu -
described in detail.[ In brief, the Hela cells were exposed to different based structure. The asymmetrical unit of 1 contains one and
concentrations of tested nano 1–3 for 24 h, then the cells were har- a half crystallography independent Cu ion, one L1– ligand,
23]
II
vested and lysed with the cell lysis buffer [1% NP-40, 1% sodium
dodecyl sulfate (SDS), 150 mM NaCl, 25 mM Tris-HCl (pH 7.6), 1%
one coordinated water, one μ -OH group, and one disordered
2
NO3– ion (Figure 1a). The Cu1 ion sitting on the twofold axis
deoxycholic acid sodium salt, 1% PMSF] for 30 min on the ice. The
is six-coordinate with four oxygen atoms from two symmetry-
total proteins in Hela cells were isolated using M-PER™ Mammalian
related L1– ligands and two μ -OH groups, resulting in a dis-
2
Protein Extraction Reagent (Thermo Fisher Scientific) and quantified
torted octahedral arrangement. The Cu2 ion locating in the nor-
with BCA Protein Assay Kit (Beyotime Biotechnology) according to
mal site is five-connected by two chelating N atoms from the
the protocol. Afterwards, equal amount of protein was loaded and sep-
arated by commercial IN-GEL (BioRad, Sydney Australia) SDS-
–
–
L1 ligand and three O atoms from the L1 ligand, one μ -OH
2
PAGE, transferred to 0.22 mm polyvinylidene difluoride (PVDF) group, and one coordinated water molecule, shaping a trigonal
II
membranes. The membrane was blocked with PBST containing 5% bipyramidal coordination environment (Figure 1b). The Cu –
nonfat milk for 2 h at room temperature and incubated with primary O bond lengths are in the range of 2.009(2) to 2.378 Å and the
II
antibody at 4 °C overnight, followed by secondary antibody for 2 h.
Cu –N bond lengths are 2.072(3) and 2.219(2) Å, which all
Images were captured and analyzed using the ChemiDoc MP imaging
system (Bio-Rad, Sydney, Australia) and ImageLab software (version
II
II
locate in the normal range of Cu –O and Cu –N bond lengths
II
in other reported Cu -based coordination polymers based on
the similar N,O,O-donor Schiff base ligand.
are held together via phenoxo oxygen atoms of two L ligands
to comprise the trinuclear molecule with 3.263(5) Å. The angle
4.1, Bio-Rad, Sydney, Australia).
[15]
II
Three Cu ions
–
Statistical Analysis: All the experimental data were presented as
meansϮstandard deviation (SD) from three independent experiments.
II
Comparisons between groups were carried out with Prism Software between three Cu ions is 130.097°, indicating the distinctly
version 6.07, GraphPad Software Inc, San Diego, CA). The differ- bent arrangement of the three Cu ions (Figure 1c). There is
II
(
ences were considered significant at *p Ͻ 0.05, **p Ͻ 0.01 and ***p no significant supramolecular interaction (such as hydrogen
Ͻ 0.005.
bond and π–π interaction) observed in the solid-state structure
of complex 1.
Crystallographic data (excluding structure factors) for the structures in
this paper have been deposited with the Cambridge Crystallographic
Data Centre, CCDC, 12 Union Road, Cambridge CB21EZ, UK. Copies
of the data can be obtained free of charge on quoting the depository
numbers CCDC-1884494, CCDC-1884495, and CCDC-1884496.
(Fax: +44-1223-336-033; E-Mail: deposit@ccdc.cam.ac.uk, http://
www.ccdc.cam.ac.uk)
Supporting Information (see footnote on the first page of this article):
1
The supporting information contains the H-NMR spectra of HL
1
li-
gand and the UV-Vis spectra for the complexes 1–3.
Results and Discussion
Crystal Structures of Compounds 1–3
The Schiff base (HL ) ligand could be synthesized by the
1
condensation of N,N-dimethyl-1,3-diaminopropane with 3-
methoxysalicylaldehyde in the MeOH solution via the reflux
method in high yield (Figure S1, Supporting Information). Ac-
Figure 1. (a) View of the molecular unit of 1. (b) The coordination
surroundings for the Cu1 and Cu2 ions. (c) View for the trinuclear
II
cording to the literature, the HL1 ligand was used to produce Cu molecule structure of 1.
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X-ray crystal structural determination reveals that the complex fraction shows that complex 3 reveals a similar structure with
II
2
(
is trinuclear with a central Co lying on a center of inversion complex 1 expect that the coordinated μ -OH group and water
2
Figure 2a). The trinuclear complex is built up of two mononu- are replaced by the coordinated N3– groups (Figure 3a). The
clear CoL moieties linked through phenoxo oxygen atoms to molecular structure is depicted in Figure 3a, which is made up
1
II
the central Co , Co1. The arrangement of the terminal penta- of two asymmetric units (Z = 2). The trinuclear complex is
II
coordinate central Co ion, Co2, is equatorially coordinated by built up of two mononuclear CuL moieties linked through
1
II
one coordinated water molecule, one phenoxo oxygen atom, azide nitrogen and phenoxo oxygen atoms to the central Cu
and one amine nitrogen atom of a deprotonated Schiff base ions. The arrangement of the terminal pentacoordinate Cu
ligand. The coordination environment around the central Co
is distorted octahedral. All the oxidation state of the Co ions 1 –z) is distorted trigonal bipyramidal with Addison parameter
was calculated to be +2 via the bond valence sum (BVS) pro- τ = 0.63. In the distorted trigonal bipyramidal arrangement,
gram.[ The Co –O bond lengths are in the range of 1.914(3) the Cu2 is equatorially coordinated by one nitrogen atom of a
to 2.325(2) Å and the Co –N bond lengths are 2.036(3) and terminal azide, one phenoxo oxygen atom, and an amine nitro-
102(2) Å, which all locate in the normal range of Co –O and gen atom of a deprotonated Schiff base ligand. The axial posi-
II
II
ions (Cu2 and Cu2A, symmetry transformation A = 1/2 –x; y,
II
16]
II
II
II
2
II
II
Co –N bond lengths in other reported Co -based coordination tions are occupied by an imine nitrogen atom of the Schiff
polymers constructed from the similar N,O,O-donor Schiff base ligand and one nitrogen atom of an end-on bridging azide.
base ligand.[ The angle between three central Co atoms The central Cu1 ion is distorted octahedron, which is shaped
17]
II
(
Co2–Co1–Co2A) is 132.195°, which is larger than that of the by four phenoxo bridged oxygen atoms from two deprotonated
II
complex 1, indicating the distinctly bent arrangement of Co
Schiff base ligands and two nitrogen atoms from end-on bridg-
II
ions. There is no significant supramolecular interaction (such ing azides. The angle between three central Cu atoms is
as hydrogen bond and π–π interaction) observed in the solid- 127.435°, which is smaller than that of the complex 1. Further
state structure of the complex 2 as revealed via the PLATON analysis of the structure reveals that there exists two non-clas-
calculation (Figure 2b).
sic hydrogen bond interaction between two adjacent discrete
structures (C8–H8···N6, 2.50 Å and C13–H13A···N4, 2.61 Å),
which extend the structure of 1 into 3D supramolecular net-
work (Figure 3b).
Figure 3. (a) View for the trinuclear CuII molecule structure of 3. (b)
The non-classic hydrogen bond interaction between two adjacent dis-
crete structures.
Figure 2. (a) View for the trinuclear CoII molecule structure of 2. (b) PXRD, Thermogravimetric Analysis, and Nanosizing
The molecular stacking fashion of 2 in the 3D directions.
To characterize the compounds more fully in terms of ther-
Complex 3 was obtained via a slightly modified preparation mal stability, the TGA curves for compounds 1–3 were mea-
method of complex 1 via the replacement of NaNO with sured in a N atmosphere in the temperature range of 25 to
3
2
NaN . The structural analysis of 3 by single-crystal X-ray dif- 800 °C (Figure 4a). For complex 1, a weight loss of 4.1%
3
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Figure 4. The TGA curve for complexes 1–3 (a). The PXRD patterns for 1 (b), 2 (c), and 3 (d).
could be observed from 100 to 186 °C, which could be attrib- 377 nm (for complex 2) and 487 and 385 nm (for complex 3).
uted to the loss of two coordinated water molecules. After a The higher energy transition bands arise from the intra-ligand
long platform till 246 °C, the framework begins to collapse. n–π* and π–π* transitions.
The TGA curve of complex 2 reveals continues weight loss of
Considering the following anticancer activity evaluation, it
4.3% from 62 to 243 °C, which could be ascribed to the re- is necessary to prepare compounds 1–3 in nanoscale, which
moval of two lattice water molecules. Finally, a further heating facilitates the release of drugs to the whole body and absorbed
leads to the framework collapse of the structure. The TGA by the specific tissues by intravenous administration. Accord-
curve of complex 3 reveals no obvious weight loss could be ing to the literature, the mechanical grinding is a simple and
[
18,19]
observed in the temperature range of 25 to 276 °C, which indi- convenient method of preparing nanoscale complexes.
cates that there are no solvents in its crystal lattice, and this is get the crystalline complex 1–3 in nanoscale region, the as-
identical with the observation from the crystal data analysis. prepared crystalline samples were subjected to a mechanical
To
It should be noted that complex 3 shows a higher framework grinding in a mortar and pestle for about 30 min. The crystal-
decomposition temperature than that of complexes 1 and 2, line nature of the nanoscale materials is confirmed by powder
which might be attributed to the formation of the hydrogen X-ray diffraction (PXRD) studies, and it is also noticed that
bond interactions in 3. To check the phase purity of the as- the PXRD pattern of the nanoscale compound matched per-
synthesized crystalline compounds 1–3, power X-ray diffrac- fectly well with the simulated and bulk PXRD patterns. The
tion (PXRD) measurements were carried out at room tempera- formation of the nanostructures was further confirmed by scan-
ture. As shown in Figure 4b–d, the PXRD patterns of the as- ning electron microscopy (SEM) studies that are obtained by
prepared three complexes reveal sharp peaks, indicating their drop-casting a DMSO-dispersed solution of 1–3 on a glass sur-
high crystallinity. Furthermore, the well matched peak posi- face (Figure 5). Nano 1 and 2 adopt almost square and spheri-
tions of the PXRD patterns between the as-prepared samples cal morphologies with average sizes of 240 and 160 nm in
and the simulated patterns confirm that the structure of the order to minimize the interfacial free energy between the par-
three complexes match well with its crystal structure.
ticle and the solvent molecules. Nano 3 exhibits nanorod type
The UV/Vis spectra for the three complexes were also morphology with an average size of 320 nm.
studied in solid state at room temperature (Figure S2, Support-
ing Information). For complex 1, it shows high intensity bands
Anti-proliferation Activity Against Hela Cancer Cells
at 410 and 321 nm assignable to charge transfer transitions
from the pπ orbitals of the phenoxido oxygen and amine nitro-
gen atoms of the Schiff base to the d orbitals of Cu ions. viability was assessed with CCK8 detection kit. Serially di-
The effect of nano 1–3 on human cervical cancer Hela cell
II
Similar charge transfer transitions are observed around 470 and luted nano 1–3 was incubated with Hela cells for 24 h, with
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Figure 5. The SEM micrograph for complexes 1 (a), 2 (b), and 3 (c).
Oxaliplatin used as the positive control drug. The absorbance creased in a concentration-dependent manner when exposed to
values at 570 nm reflect the cancer cells viability after treat- nano 1, whereas nano 2–3 have no inhibitory effect on Hela
ment. As results shown in Figure 6, the Hela cell viability de- proliferation.
The IC50 values calculated according to the CCK8 assays
with SPSS 22.0 are listed in Table 1. The nano 1 has the IC50
value of 1.1Ϯ0.02 μM exhibiting extraordinary anti-prolifera-
tion activity on human cervical cancer cells. This indicated
nano 1 has potential implications for new approaches to human
cervical cancer treatment.
Table 1. IC50 values /μM of nano 1–3 a)
.
Cell / drug
Nano 1
Nano 2
Nano 3
Oxaliplatin
Hela
HEK-293
1.1Ϯ0.02
Ͼ80
Ͼ80
Ͼ80
Ͼ80
Ͼ80
5.2Ϯ0.3
Ͼ80
a) The IC50 values of nano 1–3 against Hela cancer cells and normal
human HEK-293 cells. The IC50 values were calculated according the
cell viability curves presented in Figure 1. Each value showed as
meanϮSD of three independent experiments.
Apoptotic Cell Death in Hela
To further investigate the mechanism of nano 1–3 induced
Hela cell death, the evaluation of live, apoptotic, or dead cells
was determined by Annexin V-FITC/ PI double staining assay.
The apoptosis inducer Cisplatin was used as the positive con-
trol. As the results shown in Figure 7A, after incubated with
Figure 6. Assessment of Hela cell proliferation and cell cycle pro- nano 1, the rate of apoptotic cells significantly increased to
gression after treated with increasing dose of nano 1–3. (A) The Cell 90.46Ϯ3.6%. In accordance with the CCK8 detection, nano 2
viability curves were measured and plotted by CCK8 assay after
treated with serious doses of nanoparticles for 24 h. (B) The cell cycle
Complimentary assessment of apoptosis using the western
progression of Hela cell was analyzed using Muse® Cell Analyzer
and 3 could not induce the apoptotic cell death in Hela.
blot assay under identical treatment conditions showed the key
markers of cell apoptosis cleaved caspase-3 and cleaved cas-
24 h after treatment with various Nano doses. Data represent
meanϮSD. All experiments were performed in triplicate.
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Figure 7. Assessment of Hela cell apoptosis using Annexin V-FITC/ PI double staining method. The apoptotic Hela cells were detected by flow
cytometer following Annexin V-FITC/PI double staining. The cells were exposed to nano 1–3 at the doses as indicated, and the solvent and
Cisplatin was used as the control drug (A). The activation level of caspase 3 and caspase 9 under nano treatment (B).
pase-9 was elevated remarkable after treated with nano 1 (Fig- cer cells with the percentage of 92.72Ϯ4.1% (Figure 8). This
ure 7B), whereas there was no activity changes under the treat- data confirms that nano 1 treatment could cause apoptotic cell
ment of nano 2–3. Together these data indicated that nano 1 death in Hela cancer cells via the ROS related manner.
stimulated a loss in cell viability, which was associated acti-
vation of classical (caspase3, 9 dependent) apoptosis.
Conclusions
We have successfully prepared three trinuclear Schiff base
Intracellular ROS Generation
transition metal(II) complexes based on a newly designed bi-
As reported, the mitochondrial dysfunction is a key factor nucleating Schiff base ligand. The as-prepared three complexes
in apoptotic cell death. Therefore, we speculated the nano 1 were characterized using various physicochemical techniques,
may damage the mitochondrial function and lead to increased e.g. single-crystal X-ray diffraction (SCXRD), power X-ray
ROS and finally induce the Hela cancer cell apoptosis. The diffraction (PXRD), elemental analyses (EA), and thermograv-
ROS production in Hela cancer cells was determined via flow imetric analysis (TGA). Furthermore, a green hand grinding
cytometry. Compared with the nano 2 and 3, nano 1 could technique was implemented to reduce the particle size of the
significantly increase the intracellular ROS level in Hela can- coordination complexes to generate the nanoscale nano 1–3. In
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Figure 8. Nano 1 initiated intracellular ROS generation. (A) The ROS content in Hela cancer cells was quantified by flow cytometry after
treated with nano 1–3 at the doses as indicated. The solvent and Cisplatin was used as the control drug. (B) Statistical results of ROS production
in each group panel A. Each group was repeated three times.
addition, the anti-proliferation effect of nano 1–3 was evaluated References
on human cervical cancer Hela cell line. CCK-8 results indicated
only nano 1 has excellent anticancer activity on Hela cells, and [1] S. Cai, Recent Pat. Anticancer Drug Discov. 2007, 2, 79–101.
this inhibitory effect showed a dose-dependent relationship. The [2] W. I. Sundquist, S. J. Lippard, Coord. Chem. Rev. 1990, 100, 293–
mechanism investigations revealed that nano 1 induces the Hela
cell death via the classical (caspase 3, 9 dependent) pathway. Be-
sides, we also revealed that the caspase pathway is activated by
the increased intracellular ROS production.
322.
[
[
3] B. Lippert, Coord. Chem. Rev. 1999, 182, 263–295.
4] D. S. Raja, N. S. P. Bhuvanesh, K. Natarajan, Dalton Trans. 2012,
41, 4365–4377.
[
5] N. Zhang, Y. Fan, Z. Zhang, J. Zuo, P. Zhang, Q. Wang, S. Liu,
C. Bi, Inorg. Chem. Commun. 2012, 22, 68–72.
[
[
6] Y. Yang, L. Kang, H. Li, Ceram. Int. 2018, 45, 8017–8022.
7] K. Wang, J. Pang, L. Li, S. Zhou, Y. Li, T. Zhang, Front. Chem.
Sci. Eng. 2018, 12, 376–382.
Acknowledgements
The research did not receive any specific funding.
[
8] Y. Feng, H. Fan, Z. Zhong, H. Wang, D. Qiu, Inorg. Chem. 2016,
5
5, 11987–11992.
9] Y. Feng, M. Li, H. Fan, Q. Huang, D. Qiu, H. Shi, Dalton Trans.
015, 44, 894–897.
Keywords: Coordination complex; Schiff base ligand; Nano-
crystals; Cytotoxicity; Apoptosis; ROS
[
2
Z. Anorg. Allg. Chem. 0000, 0–0
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Journal of Inorganic and General Chemistry
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[
10] K. Yin, D. K. Chu, X. R. Dong, C. Wang, J. A. Duan, J. He, [18] M. Kubota, J. Alloys Compd. 2007, 434–435, 294–297.
Nanoscale 2017, 9, 14229–14235.
[19] W. L. Li, N. R. Tao, K. Lu, Scr. Mater. 2008, 59, 546–549.
[
[
11] K. Wu, K. Du, G. Hu, J. Mater. Chem. A 2018, 6, 1057–1066.
12] A. Ray, S. Banerjee, G. M. Rosair, V. Gramlich, S. Mitra, Struct.
Chem. 2008, 19, 459–465.
[20] X. M. Zhang, X. Xiong, C. Tong, Q. Li, S. Huang, Q. S. Li, Y. M.
Liu, H. Y. Li, P. Baker, N. Shan, H. B. Qi, Cell Physiol. Biochem.
2018, 51, 2030–2040.
[
[
[
13] S. Roy, A. Bauzá, A. Frontera, S. Chattopadhyay, Inorg. Chim. [21] H. Gu, J. Chen, Y. Song, H. Shao, Cell Physiol. Biochem. 2018,
Acta 2016, 453, 51–61.
14] S. Roy, K. Harms, S. Chattopadhyay, Polyhedron 2015, 91, 10–
51, 2136–2147.
[22] L. Dai, J. Chen, Y. Cao, L. Del Valle, Z. Qin, J. Cancer. 2018, 9,
4503–4509.
1
7.
˙
[23] W. Sun, W. Ping, Y. Tian, W. Zou, J. Liu, Y. Zu, Cell Physiol.
Biochem. 2018, 51, 2160–2171.
15] I. Gönül, A. Burak, S. Karaca, O. S¸ ahin, S. Serin, Inorg. Chim.
Acta 2018, 477, 75–83.
[
[
16] W. Liu, H. H. Thorp, Inorg. Chem. 1993, 32, 4102–4105.
17] K. Das, A. Datta, P. H. Liu, J. H. Huang, C. L. Hsu, W. T. Chang,
B. Machura, C. Sinha, Polyhedron 2014, 71, 85–90.
Received: December 20, 2018
Published Online: ᭿
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T.-F. Luo, X.-F. Ji, C.-Y. Zhu* ........................................ 1–10
Synthesis, Crystal Structures, and Anti-cervical Cancer Ac-
tivity Evaluation of Three Trinuclear Transition Metal(II)
Complexes with N,O-Donor Schiff Base Ligand
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