Synthesis, characterization and DNA binding investigations of a new binuclear Ag(I) complex and evaluation of its anticancer property

Article abstract of DOI:10.2147/IJN.S225038

Aim: A new Ag(I) complex (A₃) was synthesized and evaluated for its anticancer activity against human cancer cell lines. Materials and Methods: The complex A₃ was characterized by¹ H,¹³ C, and³¹P nuclear magnetic resonance (NMR), infrared (IR) spectra, elemental analysis, and X-ray crystallography. The interaction of the complex with CT-DNA was studied by electronic absorption spectra, fluorescence spectroscopy, and cyclic voltammetry; cell viability (%) was assessed by absorbance measurement of the samples. Results: The interaction mode of the complex A₃ with DNA is electrostatic, and this complex shows good potential in anticancer properties against HCT 116 (human colorectal cancer cells) and MDA-MB-231 (MD Anderson-metastatic breast) cell lines with 0.5 micromolar concentrations. Conclusion: The Ag(I) complex could interact with DNA noncovalently and has anticancer properties.

Full text of DOI:10.2147/IJN.S225038

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O R I G I N A L R E S E A R C H
Synthesis, Characterization and DNA Binding
Investigations of a New Binuclear Ag(I) Complex
and Evaluation of Its Anticancer Property
This article was published in the following Dove Press journal:
International Journal of Nanomedicine
Mian-Hong Zheng1,*
Aim: A new Ag(I) complex (A3) was synthesized and evaluated for its anticancer activity
Fahime Bigdeli2,*
against human cancer cell lines.
1
Materials and Methods: The complex A3 was characterized by H, 13C, and 31P nuclear
Lan-Xing Gao1,*
Deng-Ze Wu1
magnetic resonance (NMR), infrared (IR) spectra, elemental analysis, and X-ray crystal-
lography. The interaction of the complex with CT-DNA was studied by electronic absorption
Xiao-Wei Yan3
spectra, fluorescence spectroscopy, and cyclic voltammetry; cell viability (%) was assessed
Mao-Lin Hu3
Ali Morsali
by absorbance measurement of the samples.
2
Results: The interaction mode of the complex A3 with DNA is electrostatic, and this
1College of Chemistry and Materials
Engineering, Wenzhou University,
Wenzhou 325035, People’s Republic of
complex shows good potential in anticancer properties against HCT 116 (human colorectal
cancer cells) and MDA-MB-231 (MD Anderson-metastatic breast) cell lines with 0.5
China; 2Department of Chemistry,
Faculty of Sciences, Tarbiat Modares
University, Tehran, Iran; 3College of
Materials and Chemical Engineering, and
Guangxi Key Laboratory of Calcium
Carbonate Resources Comprehensive
Utilization, Hezhou University, Hezhou,
Guangxi 542800, People’s Republic of
China
micromolar concentrations.
Conclusion: The Ag(I) complex could interact with DNA noncovalently and has anticancer
properties.
Keywords: binuclear Ag(I) complex, X-ray crystallography, DNA, electrostatic binding,
anticancer
*These authors contributed equally to
this work
Introduction
Cancer is a challenging disease and global deaths owing to it are increasing.1
Important methods for the treatment of cancer patients include surgery, chemical
medication, radiotherapy, biological immunization, and chemotherapy. In che-
motherapy, 5-fluorouracil (5-Fu) is the main anticancer drug, possessing an inhibi-
tory capability against many cancers, particularly for gastrointestinal tumors.2,3
However, critical side effects have been observed associated with the clinical use
of 5-Fu.4 In addition, 5-Fu possesses a short half-life and is swiftly removed after
operation. Many studies have been carried out to combat these problems and
increase the antitumor activity of 5-FU; some procedures include modulations of
5-FU in combination with other compounds and advanced delivery systems.2,5,6
Choi et al indicated that chloroquine can augment the antitumor effect of 5-FU by
cell cycle blockage in human colon cancer cells.7 More research is needed to find
new types of anticancer drugs. Metal complexes display good potential in cancer
therapy due to their unique features, for example, various coordination routes,
reactivity, and redox ability against organic materials. Therefore, based on these
characteristics, it is possible to design metal complexes with the ability to interact
Correspondence: Deng-Ze Wu; Ali
Morsali
Email wudengze@wzu.edu.cn;
morsali_a@modares.ac.ir
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with target biomolecules, and, as a result, to modify the interaction can unwind and break the DNA double helix.19
cellular cycle.8 In some cases, metal complexes show Metal ions may play the role of a counter ion as a result of
better antitumor properties than the ligands. The com- interaction with DNA noncovalently.20 Also, the groove
plexes may act via their impact on electron transport, binding of some octahedral complexes with DNA with
inhibition of DNA replication by the metal ion center, base pairs has been reported.18,21–23
perturbation in the proteins and enzyme actions as well
as redox balance in cells, reactive oxygen species (ROS) tion of DNA. This is important in the development of
production and alteration of the cell membrane.9,10
biotechnological areas and also the ligands of the com-
Silver compounds can interact with DNA via metaliza-
Cisplatin is a compound that is currently used in cancer plexes can intercalate between bases of DNA. DNA may
treatment. Despite some favorable results, the function of be metalized by the coordination of silver to bases of
cisplatin is restricted to only some types of tumor, and its DNA15 or with the aid of electrostatic binding.24,25
usage leads to toxicity and resistance in clinical applica-
This report describes the synthesis and characterization of
tion. Hence, it is essential to develop new types of antic- a new binuclear Ag complex, [(PPh3)3Ag(L1)].[(PPh3)2Ag
ancer compounds. Many researchers have focused on (L1)] (A3), where PPh3=triphenylphosphine, L1=5-fluorour-
coinage metals (Au, Cu, and Ag) because the complexes acil-1-yl acetic acid, and its interaction with calf thymus
of these metals exhibit a wide range of anticancer activity DNA (CT-DNA). Also, we investigated the anticancer activ-
with less toxicity to non-cancerous cells.11 Among coinage ity of the complex A3 toward human cancer cell lines.
metals, silver is of special importance since it is compa-
tible with biological systems, and silver complexes display
Materials and Methods
Apparatus and Reagents
low toxicity to human cells, are selective against cancer
cells, and act as antimicrobial agents.8,12 A few active
anticancer complexes of Ag(I) such as carboxylate, phos-
phane, and bipyridine complexes have been reported.12–14
The presence of the ligands in the silver complexes
leads to the improvement of the silver function.15
Moreover, the high number of Ag(I) center ions in
a complex can improve the biological activity of silver
complexes,16 and the cytotoxic effect of binuclear silver(I)
complexes is significantly more in cancer cells in compar-
ison with the normal cells.14 Haque et al showed that silver
ions have a fundamental importance in the destruction of
tumor cells.3,12
The ligand was prepared according to the method men-
tioned in this report, and all other chemicals and solvents
were purchased from Aldrich Chemical Company or other
1
commercial vendors. H, 13C, and 31P nuclear magnetic
resonance (NMR) spectra were recorded on an AVANCE
III AV 500 spectrometer using tetramethylsilane (TMS) as
an internal standard and DMSO-d6 as the solvent at room
temperature. IR spectra were recorded on a Nicolet IS10
spectrophotometer. Elemental analysis was carried on
a EURO EA 3000 Elemental Analyzer. UV–vis absorption
spectra were provided by a UV-6100 double beam spectro-
photometer. Fluorescence measurements were made on an
F-2700 FL spectrophotometer at room temperature. The
electrochemical experiments were performed with a CHI
1030b electrochemical workstation (Shanghai Chenhua
Co.). The absorbance (A) of wells in biological investiga-
Anticancer agents mostly act through the DNA
molecule.15 Molecules may interact with DNA by chan-
ging or preventing its operation, disordering gene sequen-
cesand introde in transcription.17
The main binding types in DNA complexes are cova-
lent and non-covalent. The mechanism of action of many
anticancer drugs is by covalently binding to DNA.
Covalent binding is observed at several binding sites
including the nitrogen centers of the bases, and the phos-
phates. The antitumor activity of the covalent binding
leads to blockage of DNA, irreversibly and completely.
tions was measured using
a multiplate reader on
a Multiskan Mk3, Thermo Scientific, Waltham, USA, at
450 nm. All cell lines were purchased commercially from
the Institute of Biochemistry and Cell Biology, Chinese
Academy of Sciences (Shanghai, China).
The non-covalent interactions with DNA consist of elec- X-Ray Crystallography
trostatic bindings with the negatively charged phosphate, The specifications of the single crystal of the A3 class,
intercalation between adjoining base pairs, and groove orientation matrix, and cell dimensions were determined
interaction.18 Non-covalent interaction has advantages by the founded methods and Lorentz polarization and
including reversibility and less cytotoxicity. This type of absorption corrections were performed. Empiric absorption
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corrections were provided by the SADABS program. Most the suspension was sealed and heated to 70°C for 20 h.
of the non-hydrogen atoms were determined according to After cooling to room temperature, the solution was fil-
direct procedures, and the rest were located by subsequent tered off. After slow evaporation of the colorless and
Fourier synthesis. All non-hydrogen atoms were refined transparent solution, colorless crystals were obtained.
anisotropically, and all hydrogen atoms were maintained
Yield: 85.23%. M.p:171.1ºC-173.7ºC. 1H NMR, 13C
changeless and took in the final stage of full-matrix least NMR, and 31P NMR are given in Supporting Information,
squares refinement based on F2 by the SHELXS-97, Figure 1S–3S.
SHELXL-97 and SHELXTL programs.
DNA Binding and Anti-Cancer Activity
Experiments
Syntheses
The binding of A3 with CT-DNA was investigated by elec-
tronic absorption spectra, fluorescence spectroscopy, and
cyclic voltammetry studies. Biological activity tests of com-
plex A3 as a drug, with a molecular size of 14.46×20.05Å2 as
measured using Diamond Software, have been performed in
the solution and on a molecular scale. The size of complex A3
is suitable for delivery and penetration as a drug in the
body.27 To determine the anticancer activity of the complex,
the cell viability (%) was investigated by absorbance mea-
surement of the samples.
Synthesis of 5 FUAA Ligand 5-Fluorouracil-1-yl
Acetic Acid (L1)
First, 31.2 g (240 mmol) 5-fluorouracil was added to 160 mL
of aqueous solution of 51.2 g (92 mmol) potassium hydro-
xide and heated up to 40°C; then, 50 g (360 mmol) 80 mL
bromoacetic acid solution was slowly added in the mixture
for 120 min. Under this temperature mixing reaction was
stirred overnight, cooled to room temperature, adjusted pH
to 5.5 by concentrated hydrochloric acid and put in the
refrigerator frozen for 2 h. After removing precipitate, sedi-
ment was filtered off and a solution of strong hydrochloric
acid was added to it to bring the pH value to 2 then it was put
in the refrigerator frozen for 6 h. After filtering sediment and
washing in cold water three times, the compound was dried
(Figure 1).26 Yield: 89%. M. p: 241ºC, Anal. Calc. for C6H5
N2FO4 (%): C, 38.45; H, 2.48; N, 13.85. Found (%): C,
Preparation of DNA-Modified Electrode
For preparation of the electrode-modified DNA, gold disk
electrodes were glossed by the alumina powder (1.0, 0.5,
and 0.05 μm). Then, the Au electrode was refined and left
in fresh piranha solution (30% H2O2 and 70% H2SO4) to
remove adsorbed impurities, then it was placed in an
ultrasound bath for 5 min.
1
38.31; H, 2.68; N, 14.89. H NMR (500 MHz, DMSO):
δ/ppm, 4.36 (2H, s), 8.07 (1H, d, J = 8.5 Hz), 11.91 (1H, d,
J = 8.5 Hz), 13.22 (1H, s), 13C NMR (126 MHz, DMSO)
δ130.70, 139.52, 149.85, 157.65, 169.45, IR: 3272.34;
3414.80; 1699.31; 1377.31; 1242.58 cm−1.
In order to prepare a neat Au electrode, the surface of the
electrode was activated electrochemically through sweeping
from −0.3 to +1.5 V in 0.1 M H2SO4 until a stable cyclic
voltammogram was created. After washing with water, the
Au electrode was modified by pouring a droplet of 10 μL of
1.0 μg μL−1 CT-DNA solution onto its surface, then drying in
the air overnight. Then, the DNA-modified electrode (named
CT-DNA/Au all over) was immersed in water for about 4 h to
eliminate any unadsorbed CT-DNA.
Synthesis of Complex [(PPh3)3Ag(L1)].[(PPh3)2Ag
(L1)] (A3)
AgBF4 (0.039 g, 0.3 mmol) was dissolved in (5 mL) CH3
CN solution of PPh3 (0.052 g, 0.3 mmol) under ultrasoni-
cation; then, 42 μL Et3N (0.3 mmol) was added, and
a white cloudy solution was obtained. Then, 5-FUAA
(0.038 g, 0.2 mmol) was added to the resulting solution,
The Preparation of the Samples for
Cytotoxic Measurements
The cell viability of A3 was measured in cell lines of HCT
116 (human colorectal cancer cells) and MDA-MB-231
(MD Anderson-metastatic breast) using the cell counting
kit-8 (CCK-8) assay. Cancer cells were seeded in 96-well
plate with a cell density of 10,000 cells/well and incubated
in a humidified 37°C incubator with an atmosphere of 5%
CO2/95% air. After 24 h, A3 with or without NIR irradia-
tion (808 nm laser, 0.2 W/cm2 for 1 h) was diluted to 0.5,
Figure 1 Synthesis of ligand 5-fluorouracil-1-yl acetic acid (L1).
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Table 1 Data Collection and Refinement Parameters for A3
1, 2, 4, and 6 μM, respectively, and added into the plate.
For comparison, free 5-Fu with the same concentrations
was also added into the plate. 24 h post-treatment, the
medium in each well was refreshed with the culture med-
ium (200 μL with 10 μL CCK-8 reagent). After 2 h, the
absorbance (A) of each well was measured at 450 nm.
Compound
A3
Identification code
Empirical formula
Formula weight
p
C
102H83Ag2F2N4O8P5
1901.31
296(2) K
0.71073 Å
Triclinic, P-1
Temperature
Results
Structural Studies
Wavelength
Crystal system, space group
Unit cell dimensions
The complex [(PPh3)3Ag(L1)].[(PPh3)2Ag(L1)] (A3) was
formed by reaction of AgBF4 with CH3CN solution of
PPh3, Et3N and 5-FUAA. Slow evaporation of the result-
ing solution led to the formation of colorless and transpar-
ent crystals. After preparation of A3 it was characterized
by Fourier-transform infrared spectroscopy (FT-IR) spec-
a = 13.806(3) Å alpha = 75.622(4)
deg.
b = 14.652(3) Å beta = 79.104(4)
deg.
c = 27.796(5) Å gamma = 62.666(3)
deg.
1
troscopy, H NMR, 13C NMR, 31P NMR, and X-ray crys-
tallography. The structural data and description of the
Volume
4820.1(17) Å3
diffraction experiment are listed in Table
1 and
Z, Calculated density
Absorption coefficient
F(000)
2, 1.310 Mg/m3
0.549 mm−1
a schematic representation of the complex is given in
Figure 2. The structural details are discussed in the
Discussion section.
1948
Anal.Calc.for C102H83O4N4F2P5Ag2 (%): C, 64.38; H,
4.37; N, 2.95. Found (%): C, 64.45; H, 4.32; N, 2.95.
1H NMR (500 MHz, DMSO) δ 7.46 (t, J = 7.1 Hz,
15H), 7.40–7.29 (m, 60H), 4.10 (s, 4H).
Crystal size
0.310 x 0.230 x 0.120 mm
0.759 to 28.440 deg.
Theta range for data
collection
13C NMR (126 MHz, DMSO) δ 133.45 (d, J = 16.7
Hz), 132.05 (s), 131.85 (s), 130.45 (s), 128.98 (d, J = 9.5
Hz). 31P NMR (202 MHz, DMSO) δ 7.89 (s). IR: 1763,
1377.95, 1057, 444.05, 428.14, 408.85 cm−1.
Limiting indices
−17≤h≤18, −10≤k≤19, −36≤l≤37
42,714/23,856 [R(int) = 0.0412]
98.4%
Reflections collected/unique
Completeness to theta =
25.242
Absorption correction
Refinement method
None
Electronic Absorption Spectra Studies
UV-vis absorption spectroscopy is one of the prevalent
methods for studying the interactions between DNA and
complexes. Absorption titration experiments were car-
ried out at a constant concentration of complex A3
(10 μM) with changing concentrations of (0–0.045 mg
mL−1) from CT-DNA in buffer containing KH2PO4
/NaOH (at pH = 6.2, 7.2, and 8.2). Absorption spectra
were measured at each increase in the CT-DNA solution
(Figure 3).
Full-matrix least-squares on F2
23,856/120/1108
0.953
Data/restraints/parameters
Goodness-of-fit on F2
Final R indices [I>2sigma(I)]
R indices (all data)
R1 = 0.0723, wR2 = 0.2139
R1 = 0.1345, wR2 = 0.2737
n/a
Extinction coefficient
Largest diff. peak and hole
1.147 and −1.396 e. Å3
Fluorescence Studies
A simple method to check the interaction characteristics
between the complex and DNA is fluorescence spectro-
fluorescence when binding to DNA.28 Emission spectra
were carried out with EtBr (0.05 mM) connected to CT-
scopy. Ethidium bromide, 3,8-diamino-5-ethyl-6-phenyl DNA (0.12 mg mL−1) in the absence and presence of A3 in
phenanthridium bromide (EtBr), is widely used to study buffer KH2PO4/NaOH (pH = 7.2) with a range of concen-
small molecule–DNA interactions because of its high trations of 0–0.1 mM (Figure 4).
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Figure 2 Molecular diagram of [(PPh3)3Ag(L1)].[(PPh3)2Ag(L1)] (A3).
5 mM Fe(CN)63-/4-. From Figure 6, it can be seen that the
anodic peak currents increased with increasing the scan rate,
and there is a good linear relation between peak current and
scan rate in the range of 0.01–0.13 V s −1. These results
show that the electrochemical kinetic process is a typical
surface adsorption-regulated electrochemical process.
Electrochemical Characterization
Cyclic Voltammetry
Voltammetric measurements were performed in
a prevalent three-electrode cell contains a bare gold or CT-
DNA/Au as the working electrode, a saturated calomel
electrode (SCE), and a platinum wire auxiliary electrode.
The CV experiments were performed using a 5 mM solu-
tion of K3[Fe(CN)6]/K4[Fe(CN)6] (1:1) in 0.05 M KH2PO4
/NaOH buffer at pH 7.2 with 0.1 M KCl at room tempera-
ture (25ºC). All solutions were deaerated with pure nitro-
gen, and the electrochemical experiments were carried out
at a scan rate of 0.1 V s−1.
Since complex A3 is a non-electroactive organic small
3-/4-
molecule, Fe(CN)6
was employed as a redox probe to
study the interactions of non-electroactive of the complex
with CT-DNA. Cyclic voltammogram changes of
3-/4-
Fe(CN)6
at different concentrations of A3 at CT-DNA
/Au are observed in Figure 7.
As observed in Figure 7, both the reduction and oxida-
tion peak currents gradually decrease with seven increas-
ing concentrations from 0.26 mM to 2.50 mM of A3 (some
concentrations have been omitted in the Figures). This
A typical cyclic voltammogram of the bare Au
electrode (curve a) and CT-DNA/Au (curve b) in 5.0 mM
3－/4－
−1
Fe(CN)6
solution at a scanning rate of 0.1 V s is
observed in Figure 5. As seen from Figure 5, two redox peaks
were obtained in both cases. In addition, the peak current at
CT-DNA/Au was lower than that in the bare Au electrode.
This is because DNA acts as an electron and mass transfer
process can be ascribed to the fact that DNA films cause
3/4-
the redox activation of Fe(CN)6
to be harder at the Au
electrode because of the physical blockage and possible
electrostatic repulsion. After adding the prodrugs to the
solution, they interact with DNA, resulting in the forma-
3－/4－
blocker layer, which prevents the motion of Fe(CN)6
toward the electrode surface. The results also demonstrate the
good modification of the DNA on the surface of Au elec-
trode. The CVof the DNA-modified Au electrode remained
tion of a denser DNA film; therefore, the migration of
3-/4-
Fe(CN)6
ions via the film becomes harder and the
3-/4-
stable after 20 scans in the KH2PO4/NaOH buffer solution, redox peak current of Fe(CN)6
is decreased.
As shown in Figure 8, both the peak currents of the
cyclic voltammograms decreased with increasing the con-
representing electrochemical stability of the DNA films.
3-/4-
The electrochemical behaviors of Fe(CN)6
at CT-
DNA/Au were further investigated by a change in scan centrations of drugs and reached a saturation value accord-
rate in KH2PO4/NaOH buffer solution (pH 7.14) containing ing to Langmuir adsorption behavior.
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Figure 3 Electronic absorption spectra of the A3 (10 μM) in the absence and presence of amounts of CT-DNA (0, 0.005, 0.01, 0.015, 0.020, 0.025, 0.030, 0.035, 0.040,
0.045 mg mL−1) in KH2PO4/NaOH buffer ((A) pH = 6.2, (B) pH = 7.2, (C) pH = 8.2). Inset: Plot of A0/A-A0 vs.1/[DNA] for the titration of the A3 with CT-DNA. The
binding constants (Kb) of A3 are 36.03 mg mL−1 (A), 50.40 mg mL−1 (B) and 41.1 mg mL−1 (C), respectively.
the drugs, respectively; ΔIP, max is the maximum difference
Biological Activity
of the oxidation peak current; and [DRUG] shows the
concentration of the drug. Therefore, the binding constant
In order to perform a quantitative comparison of the bind-
ing strength of A3 with CT-DNA, the binding constant (K)
was calculated according to the Langmuir formula in
Equation (A.1).29 Based on the procedure of Qu et al30 it
is considered that DNA and DRUG only create a single
complex DNA.DRUGm.
3
−1
(K) is 3.13 ×10 L mol
.
Anti-Cancer Activity
Anticancer potential of A3 on both HCT 116 (human
colorectal cancer cells) and MDA-MB-231 cancer cell
lines was evaluated by cell counting kit-8 (CCK-8) assay.
The results were reported as percentage inhibition of cell
proliferation ( SD). The relative cell viability was calcu-
lated by the following equation:
DNA þ mDRUG , DNA ꢀ DRUGm
(A)
Using Equations 1S-8S in Supporting Information,
Equation (A.1) is obtained.
1
1
1
1
Cell viability ð%Þ ¼ ðAtreatmentꢃAblankÞ=ðAcontrolꢃAblank
ꢀ 100%
Þ
¼
þ
ꢁ
(A:1)
ΔIP ΔIP;max ΔIP;max ꢁ K ½DRUGꢂ
where ΔIP =IP0-IP, IP and IP0 show the oxidation peak
Ablank indicates the absorbance of the well without adding
in the presence and absence of CCK-8 reagent and Acontrol indicates the absorbance of the
3-/4-
current of Fe(CN)6
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Figure 4 Emission spectra of EtBr (0.05 mM) connected to CT-DNA (0.12 mg mL−1) in the absence and presence of A3 (0.00, 0.02, 0.04, 0.06, 0.08, 0.10 mM) in KH2PO4
/NaOH buffer (pH = 7.2). Inset: Stern–Volmer quenching curve. The KSV value is 2.789 × 103 M−1
.
in pH 7.2 KH2PO4/NaOH buffer solution at a bare Au electrode (a), CT-DNA/Au (b). The scan rate is 0.1 V s −1 and the
3
－/4－
Figure 5 Cyclic voltammograms of Fe(CN)6
－/4－
concentrations of Fe(CN)63
and KCl are 5 mM.
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Figure 6 The relationship between anodic peak current and the scanning rate (0.01–-0.13 V s −1) for CT-DNA/Au.
Figure 7 Cyclic voltammograms of Fe(CN)63-/4- in KH2PO4/NaOH buffer solution (pH 7.2) containing different concentrations of drugs. The scan rate is 0.1 V s −1 and the
3-/4-
concentrations of Fe(CN)6
and KCl are 5 mM.
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Figure 8 Adsorption isotherm of drug A3 on CT-DNA/Au. The solid line corresponds to the Langmuir model. Inset: The relationship between 1/[DRUG] and
1/ΔIP.
well with the addition of saline buffer instead of any drugs. The
Discussion
Structural Characterization
compounds A3+NIR and A3 in counteranions (0.5–6µM) were
found to be well active (Figures 9 and 10). At the various
Characteristic absorption bands of the ligands PPh3 and L1 are
concentrations, significant differences in the activity of com-
pounds 5-A3+NIR and A3 were observed. Increasing the con-
centrations led to an enhancement of compounds' activities.
observed in the FT-IR spectra for complex [(PPh3)3
Ag(L1)].[(PPh3)2Ag(L1)] (A3) with the some extent shift
except of absorption band of OH (3414 cm−1) that is related
Figure 9 Cell viability of A3+NIR, A3 and 5-Fu in HCT 116 cell line. Significant differences are indicated as ***P<0.001, **P<0.01, *P<0.05, n=3.
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Figure 10 Cell viability of A3+NIR, A3 and 5-Fu in MDA-`MB-231 cell line. Significant differences are illustrated as **P<0.01, *P<0.05, n=3.
to the carboxylic functional group due to coordination bonding EtBr. As given in Figure 4, with an increase in the con-
of it as an O-donor moiety in A3. Single X-ray crystal analysis
shows that there are two Ag(I) ions with different coordination
environments consisting of AgOP3 and AgO2P2. Ag1 is coor-
dinated by one O atom of carboxylate: Ag1-O1: 2.3787(64) Å
and three P atoms of PPh3 molecules: Ag1-P1: 2.5316(22) Å,
Ag1-P2: 2.5817(15) Å, Ag1-P3: 2.5855(15) Å. The coordina-
tion sphere of Ag2 includes two O atom belonging to
a carboxylate group: Ag2-O5: 2.4990(55) Å, Ag2-O6:
2.4507(69) Å and two P atoms of PPh3 molecules, Ag1-P4:
2.4209(21) Å, Ag1-P5: 2.4550(17) Å; therefore, four coordi-
nated and binuclear complexes are formed in which the Ag
−Ag distance of the two silver atoms Ag1 and Ag2 is
8.5318(13) Å. The structure of A3 crystallizes in the triclinic
space group Pī with lattice constants a = 13.806(3) Å,
b =14.652(3), c= 27.796(5), Z = 2 (Figure 2).
centration of the complex, the intensity of the emission
band in the EtBr-DNA system decreased as indicated by
the replacement of the EtBr with the complex at DNA
binding sites. The fluorescence quenching efficiency is
evaluated by the Stern–Volmer constant (KSV) based on
the classical Stern–Volmer equation: F0/F = 1 + KSV[Q], in
which F0 and F are related to the fluorescence intensities
of EtBr-DNA in the absence and presence of the complex,
respectively, and [Q] is the concentration of the complex.
KSV is a linear Stern–Volmer quenching constant achieved
from the linear regression of F0/F with [Q].
Voltammetric measurements display good electroche-
mical stability of the DNA on the Au electrode and the
correlation between the peak currents of the cyclic voltam-
mograms and concentrations of drugs shows a Langmuir
adsorption behavior.
The Interaction of the Complex with the
DNA Molecule
Anti-Cancer Activity
According to the literature, silver complexes exhibit
anticancer activity by various mechanisms.3,16 Herein
silver(I) ions interfere with the function of cancer cells
by interacting with the phosphate group of DNA
molecules.
The binding mode between DNA and complexes can be deter-
mined by absorption spectroscopy based on the shifts and
absorption strength. According to Figure 3 the absorption
spectra of the complex incubated with increasing concentra-
tions of CT-DNA. The absorption bands of A3 undergo hyper-
chromism in molar absorptivity, which is ascribed to the
electrostatic interaction between the metal ions of the complex
and phosphate group of DNA.31 The higher Kb value for b in
comparison to a (or c) indicates that neutral environments
provide more favorable conditions for this interaction.
In the other investigation, the study of complex binding
The cell viability of A3 with or without NIR irradiation
was evaluated in both HCT 116 (human colorectal cancer
cells) and MDA-MB-231 (MD Anderson-metastatic
breast) cell lines by cell counting kit-8 (CCK-8) assay.
According to Table 2 and the IC50s (half-maximal inhibi-
tory concentrations), the comparison of the anticancer
to DNA was carried out using the fluorescence feature of activities of compounds is as follows: A3+NIR˃A3˃5-Fu.
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Zheng et al
Table 2 IC50s of A3+NIR, A3 and 5-Fu (µm). IC50s Were Calculated
Based on CCK-8 Results by IBM SPSS Statistics Software
4. Bounous G, Pageau R, Regoli D. Enhanced 5-FU mortality in rats
eating definded formula diets. Int J Clin Pharmacol Biopharm.
1978;16:265–267.
Group
HCT116
MDA-MB-231
5. Shen L, Hu J, Wang H, Wang A, Lai Y, Kang Y. Synthesis and
biological evaluation of novel uracil and 5-fluorouracil-1-yl acetic
acid-colchicine conjugate. Chem Res Chin Univ. 2015;31:367–371.
doi:10.1007/s40242-015-4445-3
6. Zhou W-M, He -R-R, Ye J-T, Zhang N, Liu D-Y. Synthesis and biologi-
cal evaluation of new 5-fluorouracil-substituted ampelopsin derivatives.
Molecules. 2010;15:2114–2123. doi:10.3390/molecules15042114
7. Choi JH, Yoon JS, Won YW, Park BB, Lee YY. Chloroquine
enhances the chemotherapeutic activity of 5-fluorouracil in a colon
cancer cell line via cell cycle alteration. Apmis. 2012;120:597–604.
doi:10.1111/j.1600-0463.2012.02876.x
A3+NIR
A3
4.51 0.25
4.93 0.22
9.61 1.06
3.34 0.51
4.15 0.21
9.91 0.81
5-FUAA
The results demonstrate a significant influence of the silver
complex on the anticancer activity.
8. Ndagi U, Mhlongo N, Soliman ME. Metal complexes in cancer
therapy–an update from drug design perspective. Drug Des Devel
Ther. 2017;11:599. doi:10.2147/DDDT.S119488
9. Cuin A, Massabni AC, Pereira GA, et al. 6-Mercaptopurine com-
plexes with silver and gold ions: anti-tuberculosis and anti-cancer
activities. Biomed Pharmacother. 2011;65:334–338. doi:10.1016/j.
biopha.2011.04.012
10. Zhang P, Sadler PJ. Advances in the design of organometallic antic-
ancer complexes. J Organomet Chem. 2017;839:5–14. doi:10.1016/j.
jorganchem.2017.03.038
11. Tan SJ, Yan YK, Lee PPF, Lim KH. Copper, gold and silver com-
pounds as potential new anti-tumor metallodrugs. Future Med Chem.
2010;2:1591–1608. doi:10.4155/fmc.10.234
Conclusion
In summary, a new Ag(I) complex, [(PPh3)3Ag(L1)].
[(PPh3)2Ag(L1)] (A3), L1=5-fluorouracil-1-yl acetic acid,
was prepared and the crystal structure of it was studied by
single crystal X-ray analysis. The binding of the complex to
CT-DNA was studied by electronic absorption spectra, fluor-
escence emission, and cyclic voltammetry, and the results
suggested an electrostatic interaction with DNA. The prepared
binuclear complex, because it includes ion metal centers (Ag+)
and ligand L1 (5-fluorouracil-1-yl acetic acid) with a molecu-
lar size of 14.46×20.05 Å2, can be regarded as a new agent for
evaluation of anticancer properties. The results of the biologi-
cal study indicated that complex A3 possesses a good potential
in antiproliferative activity against human cancer cell lines,
with the 0.5 micromolar concentrations.
12. Haque RA, Nasri SF, Iqbal MA.
A new dinuclear Ag (I)–
N-heterocyclic carbene complex derived from para-xylyl linked bis-
imidazolium salt: synthesis, crystal structure, and in vitro anticancer
studies. J Coord Chem. 2013;66:2679–2692. doi:10.1080/00958972.
2013.813492
13. Eloy L, Jarrousse AS, Teyssot ML, et al. Anticancer activity of
silver–N-heterocyclic carbene complexes: caspase-independent
induction of apoptosis via mitochondrial Apoptosis-Inducing Factor
(AIF). ChemMedChem. 2012;7:805–814. doi:10.1002/cmdc.201200
055
14. Iqbal MA, Umar MI, Haque RA, Ahamed MBK, Asmawi MZB,
Majid AMSA. Macrophage and colon tumor cells as targets for
Acknowledgments
Support of this investigation by the National Natural
Science Foundation of China (nos. 21371137 and
21571143) and Tarbiat Modares University is gratefully
acknowledged. Also, the authors thank Huacheng He
(Hezhou University) for his valuable guidance on testing
activities and providing high-quality figures.
a
binuclear silver(I) N-heterocyclic carbene complex, an
anti-inflammatory and apoptosis mediator. Inorg Biochem.
2015;146:1–13. doi:10.1016/j.jinorgbio.2015.02.001
J
15. Kasyanenko N, Qiushi Z, Bakulev V, Osolodkov M, Sokolov P,
Demidov V. DNA binding with acetate bis (1, 10-phenanthroline)
silver(I) monohydrate in a solution and metallization of formed
structures. Polymers. 2017;9:211. doi:10.3390/polym9060211
16. Asif M, Iqbal MA, Hussein MA, et al. Human colon cancer targeted
pro-apoptotic, anti-metastatic and cytostatic effects of binuclear
Silver(I)–N-Heterocyclic carbene (NHC) complexes. Eur J Med
Chem. 2016;108:177–187. doi:10.1016/j.ejmech.2015.11.034
17. García-Giménez JL, Hernández-Gil J, Martínez-Ruíz A, et al. DNA
binding, nuclease activity, DNA photocleavage and cytotoxic proper-
ties of Cu (II) complexes of N-substituted sulfonamides. J Inorg
Biochem. 2013;121:167–178. doi:10.1016/j.jinorgbio.2013.01.003
18. Topala T, Bodoki A, Oprean L. R. Oprean, experimental techniques
employed in the study of metal complexes-DNA–interactions. Exp
Tech. 2014;62:6.
19. LePecq J-B, Paoletti C. A fluorescent complex between ethidium
bromide and nucleic acids: physical—chemical characterization.
J Mol Biol. 1967;27:87–106. doi:10.1016/0022-2836(67)90353-1
20. Hossain Z, Huq F. Studies on the interaction between Ag+ and DNA.
J Inorg Biochem. 2002;91:398–404. doi:10.1016/S0162-0134(02)
00454-3
21. Benjamin Garbutcheon-Singh K, Grant MP, Harper BW, et al.
Transition metal based anticancer drugs. Curr Top Med Chem.
2011;11:521–542. doi:10.2174/156802611794785226
Disclosure
The authors report no conflicts of interest in this work.
References
1. Dewangan J, Srivastava S, Mishra S, Divakar A, Kumar S, Rath SK.
Salinomycin inhibits breast cancer progression via targeting HIF-1α/
VEGF mediated tumor angiogenesis in vitro and in vivo. Biochem
Pharmacol. 2019;164:326–335. doi:10.1016/j.bcp.2019.04.026
2. Li M, Liang Z, Sun X, Gong T, Zhang Z. A polymeric prodrug of
5-fluorouracil-1-acetic acid using a multi-hydroxyl polyethylene glycol
derivative as the drug carrier. PLoS One. 2014;9:e112888.
3. Iqbal MA, Haque RA, Nasri SF, et al. Potential of silver against human
colon cancer:(synthesis, characterization and crystal structures of xylyl
(Ortho, meta, & Para) linked bis-benzimidazolium salts and Ag(I)-
NHC complexes: in vitroanticancer studies). Chem Cent J. 2013;7:27.
doi:10.1186/1752-153X-7-27
submit your manuscript | www.dovepress.com
International Journal of Nanomedicine 2020:15
963
DovePress

Zheng et al
Dovepress
22. Łęczkowska A, Vilar R. Interaction of metal complexes with nucleic
acids. Ann Rep Section” A”(Inorganic Chemistry). 2013;109:299–316.
doi:10.1039/c3ic90029k
28. Olmsted J III, Kearns DR. Mechanism of ethidium bromide fluores-
cence enhancement on binding to nucleic acids. Biochemistry.
1977;16:3647–3654. doi:10.1021/bi00635a022
23. Gago F. Stacking interactions and intercalative DNA binding.
Methods. 1998;14:277–292. doi:10.1006/meth.1998.0584
24. Espinosa Leal LAS, Karpenko A, Swasey S, et al. The role of
hydrogen bonds in the stabilization of silver-mediated cytosine
tetramers. J Phys Chem Lett. 2015;6:4061–4066. doi:10.1021/acs.
jpclett.5b01864
25. Swasey SM, Gwinn EG. Silver-mediated base pairings: towards
dynamic DNA nanostructures with enhanced chemical and thermal
stability. New J Phys. 2016;18:045008. doi:10.1088/1367-2630/18/4/
045008
26. Qu J-Q, Qu L, Jia X-F. 5-Fluoro-uracil-1-acetic acid. Acta Crystallogr
Sect E. 2006;62:o1504–o1506. doi:10.1107/S1600536806009676
27. Enriquez GG, Rizvi SA, D’Souza MJ, Do DP. Formulation and
evaluation of drug-loaded targeted magnetic microspheres for cancer
therapy. Int J Nanomedicine. 2013;8:1393. doi:10.2147/IJN.S37465
29. Chakravarty S, Dureja V, Bhattacharyya G, Maity S, Bhattacharjee S.
Removal of arsenic from groundwater using low cost ferruginous
manganese ore. Water Res. 2002;36(3):625–632. doi:10.1016/
S0043-1354(01)00234-2
30. Qu X, Trent JO, Fokt I, Priebe W, Chaires JB. Allosteric,
chiral-selective drug binding to DNA. Proc Natl Acad Sci. 2000;97
(22):12032–12037. doi:10.1073/pnas.200221397
31. Kyros L, Banti C, Kourkoumelis N, Kubicki M, Sainis I, Hadjikakou S.
Synthesis, characterization, and binding properties towards CT-DNA
and lipoxygenase of mixed-ligand silver(I) complexes with
2-mercaptothiazole and its derivatives and triphenylphosphine. J Biol
Inorganic Chem. 2014;19:449–464. doi:10.1007/s00775-014-1089-6
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