Synthesis of new ultrasonic-assisted palladium oxide nanoparticles: an in vitro evaluation on cytotoxicity and DNA/BSA binding properties

Article abstract of DOI:10.1080/07391102.2018.1546619

Better solubility and improved toxicity of palladium complexes compared with cisplatin were major reasons for synthesis of novel Pd(II) complex, [Pd(8Q)(bpy)]NO₃ (8Q=8-hydroxyquinolinate, bpy=2,2′-bipyridine). Interaction between the [Pd(8Q)(bp

Full text of DOI:10.1080/07391102.2018.1546619

Journal of Biomolecular Structure and Dynamics
ISSN: 0739-1102 (Print) 1538-0254 (Online) Journal homepage: http://www.tandfonline.com/loi/tbsd20
Synthesis of new ultrasonic-assisted palladium
oxide nanoparticles: an in vitro evaluation on
cytotoxicity and DNA/BSA binding properties
Ziba Sorinezami, Hassan Mansouri-Torshizi, Mohammad Aminzadeh, Arezou
Ghahghaei, Nasimeh Jamgohari & Mostafa Heidari Majd
To cite this article: Ziba Sorinezami, Hassan Mansouri-Torshizi, Mohammad Aminzadeh,
Arezou Ghahghaei, Nasimeh Jamgohari & Mostafa Heidari Majd (2019): Synthesis of new
ultrasonic-assisted palladium oxide nanoparticles: an in vitro evaluation on cytotoxicity
and DNA/BSA binding properties, Journal of Biomolecular Structure and Dynamics, DOI:
10.1080/07391102.2018.1546619
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JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS
https://doi.org/10.1080/07391102.2018.1546619
Synthesis of new ultrasonic-assisted palladium oxide nanoparticles: an in vitro
evaluation on cytotoxicity and DNA/BSA binding properties
a
b
b
c
Ziba Sorinezami , Hassan Mansouri-Torshizi , Mohammad Aminzadeh , Arezou Ghahghaei , Nasimeh
b
d
Jamgohari and Mostafa Heidari Majd
a
b
Department of Chemistry, University of Zabol, Zabol, Iran; Department of Chemistry, University of Sistan and Baluchestan, Zahedan, Iran;
c
d
Department of Biology, University of Sistan and Baluchestan, Zahedan, Iran; Faculty of Pharmacy, Zabol University of Medicinal Sciences,
Zabol, Iran
Communicated by Ramaswamy H. Sarma
ABSTRACT
ARTICLE HISTORY
Better solubility and improved toxicity of palladium complexes compared with cisplatin were
Received 17 July 2018
Accepted 23 October 2018
major reasons for synthesis of novel Pd(II) complex, [Pd(8Q)(bpy)]NO
3
(8Q¼8-hydroxyquinolinate,
0
bpy¼2,2 -bipyridine). Interaction between the [Pd(8Q)(bpy)]NO complex and calf thymus DNA in
3
KEYWORDS
Pd(II) complex; cytotoxicity;
DNA/BSA binding;
aqueous solution has been investigated by circular dichroism (CD), UV-Visible absorption and
fluorescence spectroscopic techniques. These experiments showed that prepared Pd(II) complex
can effectively intercalate into CT-DNA and weakly bind to BSA in which the bovine serum albu-
min molecule was unfolded slightly. The cytotoxicity of the prepared complex has been eval-
uated on the MCF-7 and DU145 cell lines by MTT and TUNEL assay. The MTT results were
8
-hydroxyquinoline;
nanoparticle
showed that in DU145, the CC50 values of [Pd(8Q)(bpy)]NO
10.4 and 8.3 lM, respectively), unlike MCF-7. Accordingly, TUNEL assay was performed on DU145
and apoptosis was clearly obvious by 43% DNA fragmentation in the treated cell lines. So, we
can suggest the [Pd(8Q)(bpy)]NO as alternative drug for cisplatin in the future which has great
3
and cisplatin are very close together
(
3
potential in DNA denaturation and apoptosis specially on prostate cancer. PdO nanoparticles
were successfully prepared without supported any surfactants via sonochemical approach. The
synthesized PdONPs were characterized using UV-Vis and FTIR spectroscopy, X-ray diffraction
(
XRD), dynamic light scattering (DLS), energy-dispersive X-ray spectroscopy (EDX), scanning elec-
tron microscopy (SEM) and transmission electron microscopy (TEM).
Abbreviations: CT-DNA: Calf thymus deoxyribonucleic acid; BSA: Bovine Serum Albumin; CD: Circular
Dichroism; Kapp: Apparent binding constant; Ksv: Stern-Volmer constant; K : Quenching constant; EtBr:
q
Ethidium Bromide; DLS: dynamic light scattering; SEM: Scanning Electron Microscopy; TEM: transmis-
sion electron microscopy; EDX: energy-dispersive X-ray spectroscopy; XRD: X-ray diffraction; MTT: 3-
(4,5-Dimethylthiazol-2-Yl)-2,5-Diphenyltetrazolium Bromide
1
. Introduction
(Dahm, Bailly, Karmazin, & Bellemin-Laponnaz, 2015; Liang
et al., 2017).
For several decades, identification of new cytotoxic com-
pounds led to development of therapeutic agents for treat-
ment of cancer. It is noticeable that less than 40 agents were
routinely used in the clinical stage over 600,000 compounds
in the testing phase, due to limitation of their application as
antitumor drugs (Schwartsmann, Winograd, & Pinedo, 1988).
In addition, application of some common anticancer drugs
widely used in cancer therapy is limited because of their
severe side effects. For example, cisplatin have several
Many palladium complexes were synthesized and character-
ized because of their structural similarity with platinum based
drugs and anticancer properties, such as less kidney toxicity
(Hadian Rasanani et al., 2018; Kelland, 2007; Mansouri-Torshizi,
Moghaddam, Divsalar, & Saboury, 2009; Saeidifar et al., 2017;
Ulukaya et al., 2011). Chelating ligands with N, O, S donor
groups, such as phenanthroline derivatives, N-heterocyclic car-
benes and Schiff bases enhanced the activity of Pd(II) com-
plexes (Carvalho et al., 2015; Ray et al., 2007). This activity is
undesirable effects for patients including its toxicity to the also increased using lipophilic ligands which less by improved
brain, drug resistance, low stability and low solubility in transferring through cell membranes (Hadjikakou & Hadjiliadis,
0
physiological media. These limitations caused improvement 2009). 2,2 -bipyridine (bpy) as N,N-donor chelating ligand can
in synthesis of new derivatives with anticancer activity form a planar complex with transition metal complexes and act
CONTACT Ziba Sorinezami
soori@uoz.ac.ir
Department of Chemistry, University of Zabol, Zabol, Iran; Hassan Mansouri-Torshizi
hmtorshizi@
hamoon.usb.ac.ir Department of Chemistry, University of Sistan and Baluchestan, Zahedan, Iran
Supplemental data for this article can be accessed online at https://doi.org/10.1080/07391102.2018.1546619
ß 2019 Informa UK Limited, trading as Taylor & Francis Group

2
Z. SORINEZAMI ET AL.
1
3
Scheme 1. Preparation route of [Pd(8Q)(bpy)]NO complex with H NMR numbering.
as DNA intercalator (Aminzadeh, Mansouri-Torshizi, & Modarresi-
During recent years, the interest for transition metal com-
Alam, 2017; Guney, Yilmaz, & Kazak, 2010; Shahraki et al., 2014; plexes bearing planar extended aromatic ligands has tremen-
Tom ꢀa s-Mendivil, D ꢀı ez, & Cadierno, 2013). Metal complexes bear- dously increased due to their application as probes capable to
ing lipophilic ligands can bind to DNA which occurred by non- utilize the nucleic acid structures (Sun et al., 2016; Tarushi et al.,
covalent interactions, such as both van der Waals and hydropho- 2017). This article reports the synthesis and characterization of
0
bic forces. During recent years, considerable attention has been a new water-soluble Pd (II) antitumor complex containing 2,2 -
directed toward 8-hydroxyquinoline (8Q) and derivatives because bipyridine (bpy) and 8Q as planar and aromatic chelating
ligands. PdO nanoparticles were also prepared without using
any surfactants via sonochemical approach which is an efficient
method for production of desirable nano-materials with differ-
ent morphologies and sufficient nanoparticles (Abbasian &
Ghanbari, 2015; Ghanbari, Salavati-Niasari, & Ghasemi-Kooch,
of diverse types of biological activities, such as antibacterial, anti-
biotic and antifungal efficiency (Mahamoud, Chevalier, Davin-
Regli, Barbe, & Pages, 2006; Musiol et al., 2007; Palit et al., 2009).
Recently, transition metal complexes containing 8Q derivatives
have been reported and used with good anticancer activity
2014). The synthesized PdONPs were characterized using UV-Vis
(Mansouri-Torshizi, Rezaei, Kamranfar, & Majd, 2016; Mansouri-
spectroscopy, Fourier transform-infrared (FTIR) spectroscopy, X-
ray diffraction (XRD), dynamic light scattering (DLS), energy-dis-
persive X-ray spectroscopy (EDX), scanning electron microscopy
Torshizi, Saeidifar, Rezaei-Behbehani, Divsalar, & Saboury, 2010;
Saeidifar et al., 2014; Sorinezami & Mansouri-Torshizi, 2016;
Sorinezami, Mansouri-Torshizi, & Ghanbari, 2017).
Interaction studies of small molecules with DNA are important
for further investigating of the structure and function of DNA
and designing new drugs. Small molecules can bind to DNA by
three kinds of non-covalent interactions, such as electrostatic
binding, groove binding and intercalation binding modes
(SEM) and transmission electron microscopy (TEM).
Since design of new drugs that can intercalate in DNA is
very important, we have designed a new Pd(II) complex which
can intercalate from both sides (bpy or 8Q) between base pairs
of DNA as both of the ligands are planar and aromatic,
(Scheme 1). In addition, this complex is water soluble which is
(Chitrapriya, Jang, Kim, & Lee, 2011; Shi, Lou, Zhou, & Pan, 2018).
an important first step for possible applications in biological
systems. In vitro cytotoxic activity of [Pd(8Q)(bpy)]NO3 com-
pared to cisplatin was investigated against both prostate cancer
DU145 cells and breast cancer MCF-7 cells. The apoptotic effect
of complex in DU145 cell lines was examined by TUNEL assay.
Furthermore, the binding affinity toward the BSA as well as
binding properties to CT-DNA have been evaluated by circular
dichroism (CD), UV-Vis absorption spectroscopy and fluores-
cence quenching measurements.
The drug–protein interaction in the blood stream, serum
or plasma may cause to formation of a protein–drug complex
that greatly influences on the distribution, metabolism and
toxicity of drugs in the body. Albumin is one of the most
numerous plasma protein in mammalians with diverse func-
tions in transportation of substances and in the metabolism
and distribution of endogenous and exogenous molecules
(
Luo, Shi, Hu, Zhao, & Huang, 2012; Nicholson, Wolmarans, &
Park, 2000; Shahraki, Shiri, Majd, & Razmara, 2017). Bovine
serum albumin (BSA) is mainly selected as an adequate pro-
tein model for drug–protein interactions, due to its availabil- 2. Experimental
ity, relatively low cost, structural similarity with human serum
albumin and high affinity binding sites to metal complexes
2
.1. Materials
(
Eslami Moghadam et al., 2016; Milutinovi ꢀc et al., 2017; Palladium(II) chloride, sodium chloride, highly polymerized
Shahraki, Shiri, & Saeidifar, 2018; Shiri, Shahraki, & Shahriari, calf thymus DNA sodium salt (CT-DNA), 8Q, 2,2 -bipyridine,
0
2
018; Wang, Herting, Wallinder, & Blomberg, 2015).
AgNO , Tris-HCl buffer and (3-(4,5-dimethylthiazol-2-yl)-2, 5-
3

JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS
3
diphenyltetrazolium bromide (MTT) were purchased from 50 ml of water-ethanol mixture (1:1 v/v) and then added
Merck (Germany). Ethidium bromide (EtBr) and sephadex dropwise to the above solution while stirring vigorously.
G-25 were obtained from Fluka (Switzerland). BSA was pur- Stirring was continued for another 2 h at room temperature.
chased from Aldrich Chemical Co (USA) and used as received. The yellowish precipitate so obtained was filtered and
MCF-7 and DU145 cell lines were obtained from Pasture washed several times with water, ethyl alcohol, and acetone
ꢁ
Institute (Tehran, Iran). T-Octylphenoxypolyethoxyethanol and was dried at 40 C. Yield was 1.53 g (92%). In the second
Triton X-100), Phosphate buffered saline powder, sodium cit-
(
step, for preparation of [Pd(8Q)(bpy)]NO3 (Scheme 1),
Pd(bpy)Cl ] (0.333 g, 1.0 mmol) was dispersed in 20 ml water,
rate and paraformaldehyde were obtained from sigma (USA).
TdT-mediated dUTP Nick-End Labeling (TUNEL) kit was
bought from Roche Applied Science (Germany). All media
and cell culture components were obtained from Life
Technologies (USA). Solvents used were of reagent grade
and purified before being used by the standard methods.
The double-distilled water was used for preparation of
all solutions.
[
2
then NaHCO3 (0.084 g, 1 mmol) and 8Q (0.145 g, 1.0 mmol),
dissolved in 10 ml water, was added to this solution. The
ꢁ
mixture was stirred at 45–50 C for 2 h. Volume of the solu-
tion was increased to 60 ml with distilled water and then
AgNO3 (0.34 g, 2.0 mmol) was added and the solution was
ꢁ
stirred at 55 C in the dark for 7 h. Stirring was continued at
room temperature in the dark for 10 h. The AgCl precipitate
was filtered and the clear reddish filtrate was evaporated
ꢁ
under vacuum to dryness at 35–40 C. The orange powder so
2
.2. Measurement instruments
obtained was dissolved in 55 ml of acetonitrile-methanol (1:1
v/v) and then was filtered. The ether diffusion into the filtrate
was resulted the dark orange microcrystals which formed
after three days. The crystals were isolated by filtration,
Infrared spectrum of the metal complex was determined
with KBr pellet on a JASCO-460 plus FT-IR spectrometer in
the range of 4000–400 cm . H NMR spectrum of the com-
plex was recorded with a Brucker AC-80 Avance spectrom-
eter at 500 MHz in DMSO-d6 using tetramethylsilane as
internal reference. Electronic absorption spectra of
ꢀ
1 1
ꢁ
washed with 15 ml acetone, and dried at 40 C (Yield: 0.31 g,
6
6.2%). All our attempts to grow single crystals were unsuc-
cessful. Anal. Calcd. for C18 H14 N4 O4 Pd (468 g/mol): C,
8.72; H, 2.99; N, 11.96%. Found: C, 48.70; H, 3.03; N, 12.01%.
[
Pd(8Q)(bpy)]NO complex were measured on a JASCO UV-
3
4
Vis 7850 recording spectrophotometer and the fluorescence
spectra were recorded on a Cary Eclipse spectrofluorimeter
FT-IR spectrum of the Pd(II) complex shows five characteristic
ꢀ1
bands at 1108, 1319, 3059, 1608 and 1384 cm assigned to
(
Varian, USA). Circular dichroism (CD) spectra were measured
m (CAO), m (CAN), m (CAH aromatic), m (C ¼ C aromatic) and
on an Aviv Spectropolarimeter model 215 (USA) in a 1 mm
path length cylindrical quartz cell. XRD analysis of the sam-
ples were carried out on a Bruker D8 ADVANCE X-ray diffract-
ometer equipped with a Cu Ka source (k ¼ 0.154 nm) at
ꢀ
NO (uncoordinated counter ion) stretching modes, respect-
3
1
ively (Nakamoto, 2008). H NMR (500 MHz, DMSO-d , ppm):
6
Protons of quinoline moiety resonate at 6.42 (d, 1H, H-f),
6
1
.86 (d, 1H, H-d), 7.13 (t, 1H, H-e), 7.55 (q, 1H, H-b), 8.15 (d,
4
0 kV and 30 mA. Morphologies of samples were examined
0
H, H-c), 8.24 (d, 1H, H-a) and the protons of 2,2 -bipyridine
by HITACHI S-570 scanning electron microscopy (SEM). The
elemental analysis of the prepared particles was also imple-
mented by a SEM equipped with an energy-dispersive X-ray
spectrum (EDX). The morphology of the prepared samples
was characterized using Philips EM208 TEM with an acceler-
ating voltage of 100 kV. The size distribution and zeta poten-
tial were carried out by DLS measurements using Zetasizer
Nano S90 (Malvern Instruments Ltd., U.K.). Conductivity
measurement of the Pd(II) complex was carried out on a
Systronics conductivity bridge 305. Microchemical analysis of
carbon, hydrogen and nitrogen for the complex was done
using Herause CHNO-RAPID elemental analyzer. Treated cells
were analyzed using Olympus IX81 compound fluorescence
microscope equipped with XM10 monochrome camera
0
0
moiety resonate at 7.53 (t, 2H, H-2,2 ), 8.12 (d, 2H, H-4,4 ),
0
0
1
8
.25 (t, 2H, H-3,3 ), 8.31 (t, 2H, H-1,1 ). H NMR spectrum of
the Pd(II) complex is shown in supplementary data,
Figure 1s.
Electronic absorption spectrum of the Pd(II) complex
shows four bands at 204 (log Ɛ ¼ 3.60) (I), 238 (log Ɛ ¼ 3.25)
(
II), 312 (log Ɛ ¼ 2.74) (III) and 396 nm (log Ɛ ¼ 2.42) (IV).
Bands (I), (II) and (III) are assigned to the first and higher
ꢂ
0
p ! p transitions of 2,2 -bipyridine and 8-hydroxyquinoli-
nate ligands (Mansouri-Torshizi et al., 2010). However band
(IV) show blue shift of 10 nm from less polar ethanol to more
polar water. This band may be assigned to charge transfer
0
from Pd(II) to 2,2 -bipyridine and 8-hydroxyquinolinate
ꢁ
ligands. The compound was decomposed at 256 C. The
(
Olympus, Hamburg, Germany).
preparation route is shown in Scheme (1).
2
.3 Synthesis of [Pd(8Q)(bpy)]NO3
2
.4 Synthesis of PdONPs
0
For the first step, 2,2 -bipyridinedichloropalladium(II),
[
(
Pd(bpy)Cl2], was synthesized as described previously The sonochemical synthesis of PdO nanoparticles were
Mansouri-Torshizi et al., 2016; Saeidifar, Mansouri-Torshizi, obtained by ultrasonic irradiation of [Pd(8Q)(bpy)]NO com-
3
Divsalar, & Saboury, 2013). PdCl2 (0.885 g, 5.0 mmol) and pound. The complex was dissolved in 50 ml of water under
NaCl (2.922 g, 50 mmol) was dissolved in a 100 ml flask con- ultrasonic waves (150 W, 30 min) followed by addition of
taining 100 ml distilled water and was stirred for 30 min at sodium hydroxide as precipitating agent. The obtained prod-
ꢁ 0
ꢁ
0 C. 2,2 -bipyridine (0.780 g, 5.0 mmol) was dissolved in uct was characterized after calcination at 400 C for 2 h.
6

4
Z. SORINEZAMI ET AL.
Figure 1. XRD patterns of PdO nanoparticles synthesized by sonochemical method.
2
.5 Interaction with DNA and BSA
cultivation and during experiments. Cytotoxicity assay was
conducted in both DU145 and MCF-7 cells. Cells (1 ꢃ 10
4
Earlier, we described the procedures of interaction studies
between metal complexes with CT-DNA (Islami-Moghaddam,
Mansouri-Torshizi, Divsalar, & Saboury, 2009). For interaction
studies of our new complex, different UV absorption and
fluorescence methods were used and stock solutions of Pd(II)
complex (1.4 mM), CT-DNA (4 mg/mL) and BSA (10 mM) were
prepared in Tris-HCl buffer (20 mM, pH 7.0) containing 20 mM
sodium chloride. The absorption titration measurements were
studied at kmax¼312 (nm) and complex-DNA/BSA systems
were incubated for 1 h both at 300 and 310K, respectively,
before spectral measurements. In the fluorescence measure-
ments, the excitation wavelength were set at 471 and 295nm
and emission spectra were recorded in the range of
cells/well) were cultivated onto 96-well plates. At 40–50%
confluency (24 h post-seeding), the cultured cells were
treated with different concentrations (i.e. ranging from 12.5
to 200 lM) of Pd(II) complex and cisplatin as an anti-cancer
drug. The treated cells were incubated for different time
frames (i.e. 24, 48, and 72 h), and then subjected to 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT).
The media was removed and 150 mL of fresh media plus
5
0 mL MTT solutions (prepared as 2 mg/mL in FBS) were
ꢁ
added to each well and incubated for 4 h at 37 C in a CO
2
incubator. The media was removed and the cells were
washed (3ꢃ), and then the formed formazan crystals were
dissolved by adding DMSO (200 mL) and Sorenson’s buffer
5
50–700nm and 300–500nm for CT-DNA and BSA systems,
(25 mL) to each well. The absorbance was read at 570 nm
respectively. DNA concentrations were determined by UV
spectrophotometry by absorption intensity at 260nm using
using a spectrophotometer (BioTek Instruments, Inc., Bad
Friedrichshall, Germany).
ꢀ
1
the molar absorption coefficient of DNA (Ɛ ¼6600 M
2
60
ꢀ1
cm ) (Eslami Moghadam et al., 2016). CT-DNA solution gave
a ratio of UV absorbance at 260 and 280nm (A260/A280) of 2.7 Apoptotic study by TUNEL assay
approximately 1.8–1.9 indicating that the DNA was sufficiently
DU145 cells were treated with 10.5 lM of prepared palladium
free of protein (Mansouri-Torshizi et al., 2010). The concentra-
tion of BSA solution was determined spectrophotometrically
complex for 72 h. Cells were washed with phosphate buf-
fered saline (PBS) and then control and treated cells were
fixed in 4% paraformaldehyde in PBS for 1 h and rinsed (3ꢃ)
with PBS. A solution containing 0.1 g Triton X-100 and 0.1 g
ꢀ
1
ꢀ1
using Ɛ 280¼44,300 M cm (Alarcon et al., 2010). For CD
measurements, each samples were scanned in the range of
2
00–350nm in Tris–HCl buffer solution at ambient tempera-
sodium citrate in 100 mL of molecular grade H O was added
2
ture. The spectral data were collected with [Complex]/[macro-
molecule] ratio (1/R)¼1, [DNA] ¼ 50 mM and [BSA] ¼ 3 mM.
for 15 min at RT to permeabilize the cells. After washing with
PBS (3ꢃ), cells were incubated with TUNEL reaction mixture
ꢁ
for 1 h at 37 C in a humidified atmosphere in the dark. Cells
2
.6 Cell culture and in vitro cytotoxicity analysis:
were rinsed with PBS (3ꢃ) and the cells were photographed
using fluorescent microscope. For quantification of apoptotic
cells, a total of 200–400 cells were counted.
MTT assay
Prostate cancer DU145 cells and breast cancer MCF-7 cells
4
2
were cultured at a seeding density of 4.0 ꢃ 10 cells/cm into
the T-50 flasks using RPMI 1640 media supplemented with 3. Results and discussion
0% FBS, 100 units/mL penicillin G, and 100 mg/mL strepto-
mycin (Heidari Majd, Akbarzadeh, & Sargazi, 2017; Sargazi,
Kuhestani, Nosrat Nahoki, & Heidari Majd, 2015). The cultured The new Pd(II) complex of formula [Pd(8Q)(bpy)]NO (where
1
3
.1 Characterization of the complex
3
ꢁ
0
cells were kept at 37 C in a humidified CO incubator during bpy ¼ 2,2 -bipyridine and 8Q ¼ 8-hydroxyquinolinate has
2

JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS
5
0
diaquo-2,2
been
synthesized
by
interaction
of
bipyridinepalladium(II) nitrate with sodium salt of 8Q. The
molar conductance value of this complex in water is 108
ꢀ
1
ꢀ1
2
(X
mol cm ). This value is unchanged after 2 days and
thus suggests that it is 1:1 electrolyte (Geary, 1971).
In spite of the presence of four aromatic rings in the
structure of the complex, it is soluble enough in water being
the first important step for possible applications in biological
systems. This may be due to presence of a positive charge
ꢀ
on complex ion being balanced by an ionizable NO3 acting
as counter ion. Satisfactory results of spectral and elemental
analysis revealed that the complex is of good purity. The
analytical data of the complex are given in Section 2.3. Non-
spectral (elemental analysis and conductivity measurement)
1
and spectral (UV-Vis, FT-IR and H NMR) characterization
data, support the proposed structure of the metal complex
shown in Scheme 1, where a bidentate coordination of both
ligands are proposed. The crystalline phases and morpholo-
gies of the samples were characterized by XRD and scanning
electron microscope (SEM). A detailed account of cytotoxic
activity (CC ), binding ([L]1/2, m) and refolding/unfolding
5
0
ability of the complex to CT-DNA and BSA are discussed in
the following sections.
3.2 FT-IR spectra and XRD analysis
The FT-IR spectra of the prepared products without calcina-
tions and after calcinations are shown in supplementary
data, Figure 2s. FTIR spectrum of PdO nanoparticles exhibited
ꢀ
1
peak at near 669 cm , corresponding to the Metal–Oxygen
stretching mode which is assigned to Pd–O bond in PdO
(
Kliche, 1985). The crystalline nature of the PdONPs was clari-
fied with XRD analysis. The XRD pattern of PdO reveals the
typical diffraction patterns of pure substance with character-
istic reflection peaks, corresponding to the face-centered
cubic structure of the crystalline Pd (JCPDS NO.: 046-121).
Using Scherrer equation, D ¼Kk/bCosh, where b is the width
c
of the observed diffraction peak at its half maximum inten-
sity, K is about 0.9, as the shape factor and k is the X-ray
wavelength (CuKa radiation, equals to 0.154 nm) was about
1
6.7 nm (Bayat, Ghanbari, & Salavati-Niasari, 2016). XRD pat-
tern for sample including PdO nanoparticles are shown in
Figure 1.
3.3 DLS measurements, SEM/TEM images and energy-
dispersive X-ray spectrum (EDX)
The DLS measurement presented the size distribution of the
PdO nanoparticles with an average size of about 126 nm. The
zeta potential analysis showed that PdONPs had negative
value (ꢀ64.4mV) indicating that PdO nanoparticles possess
negatively charged surface. The size obtained by the DLS is
different because it gives the average size of the particles as
Figure 2. SEM images of PdO nanoparticles (a and b). TEM images of PdO
nanoparticles (c and d). DLS diagram (b, inserted).
with lower particle size were synthesized. For better estima-
well as non-homogeneous dispersion of the samples. SEM tion of the particle size of nanoparticles, transmission electron
images of nanostructures synthesized by sonochemical microscopy (TEM) was used. TEM image of PdONPs is shown
approach are shown in Figure 2a, b, which implies nanostruc- in Figure 2c, d that confirms synthesis of nanoparticles with
tures with average diameter of 56–119 nm were obtained. By average particle size of about 60nm. The EDX analysis of
applying ultrasonic waves at 180 W and 60min, nano-products nanoparticles prepared by ultrasonic-assisted process is also

6
Z. SORINEZAMI ET AL.
appeared in the Figure 3. The EDX quantitative analysis con- both cells was continued until no further changes were
firms the nanostructure which consists of about 70.30 wt% Pd, observed in the absorption readings. The experiment was
8
.60wt% sodium, and 21.00 wt% oxygen.
examined at 300 and 310 K, separately. The conformational
changes of native CT-DNA and BSA interaction by
[
Pd(8Q)(bpy)]NO3 as a function of spectral changes at two
3
.4 DNA refolding and BSA unfolding
temperatures (300 and 310 K) are illustrated in Figure 4, that
shows an increase (left) and a decrease (right) in the absorp-
tion of fixed amount of CT-DNA and BSA respectively upon
The interaction of complexes with macromolecules (DNA or
BSA) may cause electronic perturbations which can be
observed through spectroscopic titrations (Arjmand & Aziz,
addition of increasing amount of [Pd(8Q)(bpy)]NO complex.
3
This behavior mirrored to refolding of CT-DNA and unfolding
of BSA molecules. Refolding and unfolding causes hidden of
purine and pyrimidine bases of CT-DNA and exposure of
tryptophan residues of BSA to water molecules present in
the medium leading to decrease in absorbance of CT-DNA
and increase in absorbance of BSA.
2
009; Chaveerach, Meenongwa, Trongpanich, Soikum, &
Chaveerach, 2010). For this experiment, the sample cell and
the reference cell were filled with 1.8 ml of Tris-HCl buffer
solution of CT-DNA (0.136 mM) and BSA (0.011 mM), respect-
ively that the absorption of CT-DNA in this concentration
was ꢄ0.8. Then, 25 ml of [Pd(8Q)(bpy)]NO (1.4 mM) was
3
The concentrations of Pd(II) complex in the midpoint of
transition from native form to refolded (CT-DNA) or unfolded
added to both sample and reference cuvette and after 3 min,
the absorption spectra were recorded at 260 and 280 nm for
CT-DNA and BSA, respectively. Addition of metal complex to
(BSA) by the Pd(II) complex, [L]1/2 value, are gathered in
Table 1. The low values of [L]1/2 clearly confirmed the high
ability of the [Pd(8Q)(bpy)]NO3 complex, as a good anti-
cancer drug, in modification of the native CT-DNA and
BSA molecule.
3
.5 Absorption titration measurements
This study provides a depth insight for interaction of physio-
logical macromolecules with an antitumor metal complex.
The calculated parameters may be useful for structural evalu-
ation of the denatured macromolecules which are induced
by the Pd(II) complex. In the first experiments a fixed
amount of this complex (0.1 mM) was titrated with increasing
Figure 3. EDX of nanoparticles prepared by ultrasonic-assisted process.
3 t
Figure 4. The changes in UV absorbance for CT-DNA (right) and BSA (left) due to increasing the total concentration of [Pd(8Q)(bpy)]NO , [L] at 300 and 310 K.
3
Table 1. Binding parameters for interaction between CT-DNA and BSA (data in bracket) with the [Pd(8Q)(bpy)]NO complex in
2
0 mM Tris-HCl buffer and pH¼ 7.0.
a
b
c
d
4
ꢀ1
e
Compound
Temp (K)
[L]1/2 (mM)
DAmax
g
K ꢃ 10 (M)
n
[
Pd(8Q)(bpy)]NO
3
300 310 56 (66) 47 (60) 0.14 (0.48) 0.09 (0.37) 4 (4) 4 (4) 6.02 (4.56) 7.12 (7.93) 5.27 (5.25) 5.41 (4.36)
a
The concentrations of the complex in the midpoint of transition.
Change in the absorbance when all the binding sites on DNA were occupied by metal complex.
b
c
The number of binding sites per 1000 nucleotides.
d
The apparent binding constant.
The Hill coefficient (as a criterion of cooperativity).
e

JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS
7
Figure 5. The changes in the absorbance of fixed amount of metal complex in the interaction with varying amount of macromolecules at 300 K and 310 K. The
linear plot of the reciprocal of DA vs the reciprocal of [DNA] (right) and [BSA] (left) for the complex [Pd(8Q)(bpy)]NO
3
.
3
Figure 6. Scatchard plots for binding of the complex [Pd(8Q)(bpy)]NO to CT-DNA (right) and BSA (left) at constant temperatures of 300 K and 310 K.
concentration of DNA or BSA at two temperatures, 300 and 2016; Shiri, Shahraki, Shahriyar, & Majd, 2017). The calculated
3
10 K, and spectral changes are monitored based on UV–Vis apparent binding constant are comparable with those of
technique. The values of DAmax, in which all binding sites on 2,2 -bipyridine-palladium complexes reported earlier, acting
0
DNA and or BSA are bound by the complex, are determined as DNA intercalators (Aminzadeh et al., 2017; Saeidifar
which are shown in Figure 5 and Table 1.
et al., 2014).
In the next experiments a fixed amount of each macro-
molecule was titrated with varying amounts of the Pd(II)
complex at 300 and 310 K, separately. As shown in Figure 6,
3
.6 Fluorescence spectroscopic studies
the Scatchard plots for the interaction of the Pd(II) complex Fluorescence spectroscopy is the most important technique
with DNA or BSA are given using the values of m/[L] versus to study the interaction of probes with macromolecules
f
m, where [L] is the concentration of free metal complex, and because of high sensitivity. The DNA binding of the
f
m is the ratio of the concentration of bound metal complex [Pd(8Q)(bpy)]NO3 complex was explored using competitive
to total DNA or BSA concentration (Scatchard, 1949). At both ethidium bromide (EtBr) binding experiments in Tris-HCl buf-
temperatures, the positive slope is observed in the plots, fer solution at pH ¼7.0. It is well known that upon binding
suggesting cooperative binding properties during interaction to DNA, the fluorescence intensity of EtBr is significantly
(
Saeidifar et al., 2013).
To obtain the binding parameters, the above experimental base pairs (Kumar & Arunachalam, 2007; Olmsted III &
data (m and [L] ) were substitute in the following equation Kearns, 1977). Addition of Pd(II) complex to the EtBr-DNA
increased due to its strong intercalation between the DNA
f
(
Hill equation; Hill, 1910).
system, resulted in gradually decrease in fluorescence inten-
sity due to replacement of the EtBr molecule by the complex
À
Á
À
Á
n
n
m ¼ g K½Lꢅ = 1 þ K½Lꢅ
(1-3)
f
f
(Liu, Yuan, Zhang, Yan, & Yang, 2008). The emission spectra
Using Eureka software to resolve the above equation, the of EtBr bound to CT-DNA in the absence and the presence
theoretical values of unknown parameters (g, n, K) can be of the compound are given in Figure 7. In this figure, a weak
extracted, as shown in Table 1.
intensity spectrum due to free EtBr near the base line is pre-
The values of n were calculated as 5.27 and 5.41 at 300 sent. This implies that, in the competition between the two
and 310 K, respectively indicating cooperative DNA binding probes, the EtBr and Pd(II) complex, intercalation affinity of
with identical binding site (Shahraki, Shiri, Majd, & Razmara, the Pd(II) complex is more than that of EtBr which cause
2
017; Shiri, Shahraki, Baneshi, Nejati-Yazdinejad, & Majd, releasing of EtBr molecules from CT-DNA.

8
Z. SORINEZAMI ET AL.
Table 2. Binding parameters for the palladium(II) complex on the fluores-
cence of EtBr in the presence of CT-DNA.
a
bK ꢃ 10ꢀ5 (M)ꢀ1
c
Compound
Pd(8Q)(bpy)]NO
r
f
n
[
3
0.000
0.574
0.351
0.004
0
.017 0.034 0.050
0
0
.264
.206
a
Formal ratio of metal complex to nucleotide concentration.
b
c
Association constant.
Number of binding sites per nucleotide.
Figure 7. Florescence emission spectra of interacted EtBr-DNA in the absence
(
1) and presence of different concentration of the palladium(II) complex: 5 mM
(
2), 10 mM (3), 15 mM (4), 20 mM (5), 25 mM (6), EtBr alone (7).
Figure 9. Fluorescence emission spectra of BSA (8 mM) in the absence and
presence of different concentrations of the [Pd(8Q)(bpy)]NO complex in a
3
Tris–HCl buffer (kmax¼295 nm). The arrow shows the changes in emission
intensity upon increasing the concentrations of the Pd(II) complex.
The characteristic fluorescence emission behavior of BSA is
originated from Trp-134 and Trp-212 residues that provide the
study of protein-small molecules interaction with fluorescence
spectroscopic method (Messori, Orioli, Vullo, Alessio, & Iengo,
Figure 8. Competition between the complex [Pd(8Q)(bpy)]NO
the binding sites of DNA (Scatchard plot). In curve no. 1 (D), Scatchard’s plot
3
with EtBr for
was obtained with calf thymus alone, [DNA] ¼ 60 mM. In curves nos. 2 (᭹), 3 2000; Wang et al., 2011). Figure 9 shows the emission spectra
(
᭿) and 4 (᭜) respectively, 30, 80 and 120 mM of [Pd(8Q)(bpy)]NO , were
3
of the BSA in the absence and presence of the
Pd(8Q)(bpy)]NO3 complex at ambient temperature. A fixed
added, corresponding to molar ratio [complex]/[DNA] of 0.017, 0.034 and 0.05.
Solution were made in 20 mM NaCl, 20 mM Tris-HCl (pH¼ 7.0).
[
concentration of BSA (8 mM), was titrated by various amounts
of the Pd(II) complex (0.0–3.2 mM). Upon BSA binding of the
Pd(II) complex, the emission intensity maximum at 345 nm is
remarkably decreased without any significant change in the
position of the maximum emission wavelength. The binding of
the complex to BSA also quenches the intrinsic fluorescence
attributed to the tryptophan residues in BSA (Hu, Liu, Pi, & Qu,
In addition, to characterize the mode of binding of
[
Pd(8Q)(bpy)]NO3 to DNA, Scatchard analysis were carried
out in which the number of EtBr molecules intercalated to
DNA in the presence of different concentrations of the Pd(II)
complex was calculated (Scatchard, 1949). For this purpose,
appropriate amount of Pd(II) complex was added to the solu-
tion of CT-DNA pretreated with EtBr. The samples were incu-
bated for 2 h and finally processed for fluorescence spectral
measurements. The fluorescence scatchard plots were con-
structed for EtBr-DNA solution at different rf values (as
defined [Pd(II) complex]/[DNA]) as shown in Figure 8 and the
results are summarized in Table 2. These values are compar-
able with those observed for the analogous complexes
2
005). The linear Stern–Volmer quenching constant for serum
albumins has been measured from the following equation:
F0
¼
1 þ kq s0 ½Qꢅ ¼ 1 þ Ksv ½Qꢅ
(2-3)
F
where F0 and F are the fluorescence intensities in the
absence and presence of quencher respectively, Ksv is the
Stern-Volmer quenching constant and [Q] is concentration of
quencher, k is the bimolecular quenching rate constant and
q
(
Mansouri-Torshizi et al., 2010).
equal to k ¼K /s , and s is the average lifetime of the BSA
q
sv
0
0
ꢀ
8
Figure 8 shows increasing the concentration of Pd(II) com- in the absence of quencher. The value of s is equal to 10
0
plex, decreases the value of Kapp (i.e. apparent association s for tryptophan fluorescence in proteins (Zhang, Ma, Wang,
constant) while the number of binding sites, n, is remain con- Zhang, & Zhou, 2012). The plot of F /F versus [Q] should
0
stant. This mode of DNA binding (type-A behavior) implies gave a straight line with a slope equal to Stern-Volmer
that the complex inhibits the EtBr binding to CT-DNA, com- quenching constant. The value of Ksv was obtained
5
ꢀ1
petitively (Howe-Grant, Wu, Bauer, & Lippard, 1976).
6.29 ꢃ 10 M for Pd(II) complex (Figure 10).

JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS
9
Table 3. Quenching constant (Ksv), binding constant (Kbin) and number of
binding sites (n) for BSA interaction of the [Pd(8Q)(bpy)]NO
3
complex.
ꢀ
1
ꢀ1 ꢀ1
ꢀ1
Complex
Pd(8Q)(bpy)]NO
K
sv (M
)
K
q
(M
S
)
K
bin (M
)
n
5
13
5
[
3
6.29 ꢃ 10
6.29 ꢃ 10
6.87 ꢃ 10
1.23
Figure 10. The Stern–Volmer curve of the fluorescence quenching of BSA by
Pd(8Q)(bpy)]NO complex in Tris-HCl buffer at 300 K.
[
3
Figure 11. Plot of log[(F
plex-BSA binding constant and the number of binding sites on BSA.
0
ꢀF)/F] vs. log[Q] for determination of the Pd(II) com-
Moreover, fluorescence quenching phenomena is classi-
fied into two important quenching mechanisms, the dynamic
Figure 12. (a) CD spectra of CT-DNA (50 mM) in the absence (dashed line) and
and static quenching. The value of k was calculated as presence (solid line) of the Pd(II) complex. (b) CD spectra of free BSA (dashed
q
1
3
ꢀ1 ꢀ1
line) and Pd-BSA system (solid line); [BSA] ¼ 3 mM.
6
.29 ꢃ 10
M
s , which is greater than the maximum dif-
1
0
ꢀ1 ꢀ1
fusion collision rate constant (2.0 ꢃ 10
M
s ) for a bio-
molecules, suggesting the existence of static quenching
process during BSA binding of the Pd(II) complex
quenching is the possible mechanism in the binding process
between BSA and complex.
(
Ramachandran, Raja, Bhuvanesh, & Natarajan, 2013). To
evaluate the binding constant (K ) and the number of the
A
Pd(II) complex bound per protein (n), the following equation
was used for static quenching mechanism (Cui et al., 2003):
3
.7 CD spectral studies
ðF0ꢀFÞ
CD spectroscopy is a quite sensitive technique in biological
chemistry and structural biology to investigate the changes
in macromolecule morphology during interaction with small
molecules. The CT-DNA in right-handed B form is character-
ized in CD spectrum by a positive band at 275 nm due to
base stacking and also a negative band at around 247 nm
correlated to helicity (Loganathan et al., 2012). By addition of
the Pd(II) complex to the DNA solution the intensities of
Log
¼ Log Kbin þ n Log ½Qꢅ
(3-3)
F
where Kbin is the apparent binding constant for the forma-
tion of BSA–drug complex, n is the number of the binding
sites on the BSA molecule and [Q] and [P] are total concen-
tration of quencher and protein, respectively. By plotting of
versus Log [Q] the values of Kbin and n are
obtained from the intercept and slope, respectively (Figure
t
ðF0ꢀFÞ
Log
F
1
1). The calculated Kbin and n values for the BSA–complex both positive and negative CD bands are clearly decreased,
system are presented in Table 3.
which supporting the intercalation mode of interaction
The results indicate that the value of apparent association between the complex and DNA (Behnamfar et al., 2015). The
constant for Pd(II) complex is higher than that of the Stern- CD spectra of CT-DNA in the absence and presence of Pd(II)
Volmer quenching constant. This implies that static complex are displayed in Figure 12a.

1
0
Z. SORINEZAMI ET AL.
3
Figure 13. The impact of [Pd(8Q)(bpy)]NO in the breast cancer MCF-7 cells (panel A–C) and prostate cancer DU145 cells (panel D–F).
In order to obtain an insight into the structure of the BSA, is the cell path length (1 mm). The results showed that with
CD spectra are also monitored to examine the conform- addition of the Pd(II) complex decreasing tendency of the
ational aspects in BSA molecule upon environmental pertur- a-helix content from 55.21 to 48.82% is observed.
bations due to the interaction with Pd(II) complex. The CD
spectrum of BSA exhibited two negative peaks at 208 and
2
22 nm, which is characteristic of the a-helix structure of the
3
.8 In vitro effects of Pd(II) complex and cisplatin on
MCF-7 and DU145 cell lines
protein. Upon BSA interaction of the complex, the intensity
of both negative peaks is decreased without any obvious
shift of the bands indicating the unfolding of the peptide Forasmuch as the cytotoxicity of palladium complexes
strand in protein secondary structure. The CD spectra were against human breast cancer cell lines and prostate cancer
recorded in the absence and presence of the Pd(II) complex cell lines has been confirmed (Khan et al., 2011; Ulukaya
that are shown in Figure 12b which clearly showed that the et al., 2011), the MCF-7 and DU145 cell lines were selected
a-helical content in the BSA protein is decreased upon inter- for cytotoxicity evaluation of [Pd(8Q)(bpy)]NO in comparison
3
action with metallodrug. The a-helix content of BSA before to cisplatin. Figure 13 represents the impact of
and after the addition of the Pd(II) complex is evaluated [Pd(8Q)(bpy)]NO3 in the breast cancer MCF-7 cells (panel
from MRE value at 208 nm according to the following equa- A–C) and prostate cancer DU145 cells (panel D–F). In DU145
tion (Chen, Yang, & Martinez, 1972):
cells, Pd(II) complex resulted in prolonged inhibitory up to
72 h in sustained manner compared to cisplatin (about 80%
toxicity for Pd(II) complex). In MCF-7 cells, the cytotoxic
effects of [Pd(8Q)(bpy)]NO3 were partly low after 72 h in
compared to DU145 (i.e. ꢄ60–65% toxicity). In DU145 cells,
ꢀ
MRE208ꢀ4000
a ꢀ helixð%Þ ¼
ꢃ 100
(4-3)
3
3000 ꢀ 4000
where MRE208 is the experimental MRE values at 208 nm. The
MRE value for pure is 33,000 and its value for b-form and
random coil conformation is 4000 at 208 nm. The MRE208
used to express variations in the secondary structure of BSA
molecule are calculated by the following equation:
the CC50 values of [Pd(8Q)(bpy)]NO and cisplatin were eval-
3
,
uated 10.4 and 8.3 lM, respectively. But In MCF-7 cells, the
CC50 value of the Pd(II) complex was very different with the
cisplatin: 70 and 12.5 lM, respectively. The cytotoxic effect of
observedCDðmdegÞ2
08
MRE208 ¼
(5-3) [Pd(8Q)(bpy)]NO
on breast cancer cells seems to be time
3
Cp ꢃ n ꢃ l ꢃ 10
dependent, while it display a trend of time and concentra-
where n is the number of amino acid residues (for BSA mol- tion dependent inhibitory effects on prostate cancer cells.
ecule is equal to 583), C is the molar concentration of BSA, l Similar to our results, Khan et al. (2011) have reported that
p

JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS
11
3
Figure 14. Assessment of apoptosis by TUNEL assay in DU145 cells. (a) Untreated cells. (b) The cells were exposed to 10.5 lM of [Pd(8Q)(bpy)]NO for 72 h.
their new synthesized Pd(II) complexes are highly effective strands of the BSA. On the other hand, according to cytotox-
against DU145 cells.
icity studies, the apoptosis ability of the [Pd(8Q)(bpy)]NO on
3
prostate cancer cells was confirmed. All these studies
prompted us to propose this novel palladium complex as a
potential therapeutic compound for the treatment of the
cancer cells.
3
.9 Apoptosis assay
According to previous evidence, [Pd(8Q)(bpy)]NO3 showed
good cytotoxic effects on DU145 prostate cancer cells. To
determine whether the cell death was attributable to
Acknowledgments
[
Pd(8Q)(bpy)]NO -induced apoptosis, TUNEL assays were per-
3
The authors are grateful for all support provided by University of Sistan
and Baluchestan, University of Zabol and Zabol University of
Medical science.
formed (Yang et al., 2015) for the course of 72 h treatment
with 10.5 lM [Pd(8Q)(bpy)]NO (CC value of Pd(II) complex).
3
50
As shown in Figure 14, a detectable level of apoptosis was
evident in DU145 cells after treatment with prepared Pd(II)
complex in compared to untreated cells. After counting the References
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