DNA-binding, cytotoxicity and apoptosis induction of Pt/Fe-based heterometallo-supramolecular polymer for anticancer drug application

Article abstract of DOI:10.1016/j.jorganchem.2019.03.021

We synthesized two Pt/Fe-based metallo-supramolecular polymers with Pt (II) and Fe(II) introduced alternately (polyFePtL1 and polyFePtL2) for anticancer drug application. They have octahedral Fe(II)-centers and square-planar Pt (II)-centers bound with ace

Full text of DOI:10.1016/j.jorganchem.2019.03.021

Accepted Manuscript
DNA-binding, cytotoxicity and apoptosis induction of Pt/Fe-based heterometallo-
supramolecular polymer for anticancer drug application
Utpal Rana, Chanchal Chakraborty, Miki Kanao, Hiromi Morita, Takashi Minowa,
Masayoshi Higuchi
PII:
S0022-328X(19)30123-8
DOI:
https://doi.org/10.1016/j.jorganchem.2019.03.021
JOM 20749
Reference:
To appear in: Journal of Organometallic Chemistry
Received Date: 24 November 2018
Revised Date: 17 March 2019
Accepted Date: 30 March 2019
Please cite this article as: U. Rana, C. Chakraborty, M. Kanao, H. Morita, T. Minowa, M. Higuchi, DNA-
binding, cytotoxicity and apoptosis induction of Pt/Fe-based heterometallo-supramolecular polymer for
anticancer drug application, Journal of Organometallic Chemistry (2019), doi: https://doi.org/10.1016/
j.jorganchem.2019.03.021.
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ACCEPTED MANUSCRIPT
DNA-Binding, Cytotoxicity and Apoptosis Induction of Pt/Fe-Based
Heterometallo-Supramolecular Polymer for Anticancer Drug Application
Utpal Rana,a Chanchal Chakraborty,a,# Miki Kanao,a Hiromi Moritab Takashi Minowa,b
and Masayoshi Higuchi*a
a
Electronic Functional Macromolecules Group, National Institute for Materials Science
(NIMS), Tsukuba 305-0044, Japan
b Nanotechnology Innovation Station, NIMS, 1-2-1 Sengen, Tsukuba 305-0047, Japan
#Present Address: Department of Chemistry, Birla Institute of Technology & Science,
Pilani, Hyderabad Campus, Shameerpet, Jawahar Nagar, Secunderabad, Telangana 500078,
India
*Corresponding author: Masayoshi Higuchi
Tel. & Fax: +81-29-860-4721
E-mail: HIGUCHI.Masayoshi@nims.go.jp
ABSTRACT
We synthesized two Pt/Fe-based metallo-supramolecular polymers with Pt(II) and Fe(II)
introduced alternately (polyFePtL1 and polyFePtL2) for anticancer drug application. They
have octahedral Fe(II)-centers and square-planar Pt(II)-centers bound with acetylide TPY
(4-ethynyl-(2,2′:6′,2″-terpyridine) and ‒PEt3 (polyFePtL1) or ‒PPh3 (polyFePtL2). We
measured their binding affinity to calf-thymus DNA (ct-DNA) by the UV-vis spectral
titration. The binding constant of polyFePtL2 (Kb = 6.0 × 107 M-1) is 15-fold higher than
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that of polyFePtL1 (Kb = 4.0 × 106 M-1). The high binding affinity of polyFePtL2
indicates the intercalative binding of the ‒PPh3 moieties to DNA in addition to the groove
binding of the polymer to DNA. They showed high cytotoxicity against human lung cancer
cell line (A549). PolyFePtL2 has almost twice higher cytotoxicity (41% viable cell) than
polyFePtL1 in 10 µM concentration. We also inspected the cell apoptosis mechanism by
FACS using Annexin-V FITC/PI double staining assay and found that polyFePtL2 induced
apoptosis selectively leading to cancer cell death with ~15% detectable apoptotic cell.
Keywords: Metallosupramolecular polymer; DNA binding; Cytotoxicity; Apoptosis
1. INTRODUCTION
Cancer is one of the major causes of death in the world and the development of anticancer
drugs is still desired. Nearly 50% of present anticancer drugs are based on Pt complexes
such as cisplatin, carboplatin and oxaliplatin.[1-4] Therapeutic activity of the anticancer
drugs crucially depends on the DNA binding affinity. The binding of drugs with DNA
over-stabilizes the DNA duplexes and inhibits the DNA replication, leading to the cellular
apoptosis. There are two binding modes: intercalation and groove binding. In the
intercalation, the planar part of drug stacks with DNA base pairs through the π‒π
interaction. In the groove binding, a drug binds to the minor or major groove of DNA
duplexes based on the ionic interactions etc. The groove binding is normally stronger than
the intercalation.[5] So, we considered anticancer drugs driven by groove bindings are
effective to enhance the binding affinity with DNA and reported anticancer activity of
metallosupramolecular polymers. Metallosupramolecular polymers are composed of metal
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ions and organic ligands. Because metal ions have positive charge(s), the polymers are
regarded as polycation, which can bind phosphate anions of DNA ionically. In our previous
papers, we showed the strong groove binding activity of metallosupramolecular polymers
to DNA and higher cytotoxicity to cancer cells.[6-9] It was also observed that
metallosupramolecular polymer with a helical structure has higher binding affinity to DNA
with the same helicity than the polymer with the opposite helicity.[8] Furthermore, we
revealed that the DNA binding affinity and cytotoxicity of metallo-supramolecular polymer
are controllable by changing the metal-metal distance of polymer.[9]
Recently, heterometallic complexes with two different cytotoxic metals have
received attention as a novel class of anticancer agents.[10, 11] The two metals with
different coordination geometries multiply interact with a biological target and enhance the
anticancer activity. Higher cytotoxicity of heteropolynuclear Ru–Pt anticancer complexes
than monometallic complexes was reported by Reedijk et al.[12] Strong DNA binding and
cytostatic behavior of a Ru–Pt bis(terpyridyl) complex was also reported by Smythe et
al.[13] However, the bimetallic polymeric systems as anticancer drugs are relatively
unexplored and only a few have been studied so far. Inspiring from the above-mentioned
reports, we have designed heterometallo-supramolecular polymers with two metal species
(Fe and Pt) introduced alternately through the polymer chain (Fig. 1) for the anticancer
drug application. We chose Fe(II) due to the wide diversity of coordination, readily
accessible oxidation states and good antitumor activity with fewer side effects.[14] We
chose organometallic Pt(II) as the second metal for toxicity because organometallic Pt-
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compounds are also good anticancer candidates with their large structural variety and
kinetic stability.[15]
Fig. 1 Fe/Pt-based heterometallo-supramolecular polymers (polyFePtL1 and polyFePtL2).
2. EXPERIMENTAL PROCEDURES
2.1 Materials
4-ethynylbenzaldehyde, 2-acetylpyridine, trans-[PtCl2(PEt3)2, CuI and diisopropylamine,
MgSO4 and iron(II) tetrafluoroborate [Fe(BF4)2] were purchased from TCI (Tokyo, Japan)
and Aldrich (St. Louis, MO, USA) and were directly used without further purification.
PolyFePtL2 was prepared according to our previously reported procedure. Dehydrated
DMSO, 28% NH3 solution were purchased from Aldrich. Dry DCM, CHCl3, ethanol and
acetonitrile solvents were purchased from Wako or Kanto Chemical Co. Inc. and used as
received.
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2.2 Instrumentation
UV−vis spectra of polymers and DNA binding titrations were recorded using a Shimadzu
UV-2550 UV−visible spectrophotometer. IR spectra were recorded on a Shimadzu FTIR
8400S Fourier Transform Infrared Spectrophotometer with KBr pellets.1H- and 13C-NMR
spectra were measured at 300 MHz and 75 MHz, respectively, on a JEOL AL 300/BZ
instrument (Tokyo, Japan). Mass spectra (MS) were measured using a Shimadzu/Kratos
TOF Mass spectrometer (Kyoto, Japan). High-resolution mass spectra (HRMS) were
measured using a Shimadzu LCMS-IT-TOF spectrometer. Calf thymus DNA (ct-DNA) (10
mg/mL, 200–500 bp) was purchased from TREVIGEN (Gaithersburg, MD, USA) and the
known molar extinction coefficient of 6600 M-1 at 260 nm was used for ct-DNA
concentration with respect to per nucleotide calculation. The A549 human lung cancer cell
line was purchased from the RIKEN Bio-Resource Centre. A microplate reader (MTP-880,
Corona Electric Co., Ltd., Hitachinaka, Japan) was used for the cytotoxicity assay with
CCK-8. Cell-Counting Kit-8 (CCK-8), and calcein-AM and propidium iodide (PI) double
staining kits were purchased from Dojindo (Kumamoto, Japan). Inverted light and
fluorescence microscopes (DMIL, Leica Microsystems, Wetzlar, Germany) were used to
observe cells stained with the calcein-AM and PI double staining kit. Annexin-V buffer and
Annexin-V (10 µg/mL) were used for FACS measurements and purchased from SONY
(Tokyo, Japan) and SONY SP6800 cell analyzer software was used for FACS analysis.
2.3 Synthesis of 1
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Acetylide terpyridine 1 was prepared as reported previously by refluxing the 4-
ethynylbenzaldehyde (8.92 g, 40.0 mmol), 2-acetylpyridine (9.85 mL, 88.00 mmol) and
KOH (8.98 g, 160.00 mmol) in the mixture of 200 mL of EtOH and 40 mL of 28% NH3
solution for 24 h. The mixture was cooled to room temperature and extracted with DCM.
The combined organic layer was washed with water and brine and dried over MgSO4. After
removal of the solvent, the residue was purified by flash chromatography (1:1 hexane:
DCM) to afford 1 as a white powder (9.4 g, 70 %).
1H NMR (300 MHz, CDCl3, 298 K),
δ (ppm) = 8.73 (s, 2H), 8.69 (d, 2H, J = 7.8 Hz), 7.95
(dd, 4H, J = 8.8 Hz), 7.28 (dd, 2H, J = 7.8 Hz), 7.65 (d, 2H, J = 8.4 Hz), 7.38 (t, 2H, J =4.8,
6.9 Hz), 3.19 (s, 1H).
13C NMR (75 MHz, CDCl3): 156.09, 156.06, 149.28, 149.13, 138.82, 136.88, 132.68,
127.23, 123.90, 122.80, 121.36, 118.71, 83.31, 78.51.
HRMS (m/z): [M + Na] found 356.126; calculated 356.116.
2.4 Synthesis of PtL1
To a solution of 1 (1.0 g, 3.00 mmol) in dry DCM (40 mL), trans-[Pt(PEt3)2Cl2] (0.50 g,
1.00 mmol), CuI (0.03 g, 0.15 mmol) and diisopropylamine (20 mmol) were added. The
mixture was stirred at room temperature under nitrogen for 48 h. The solvent was
evaporated, residue was washed with water, methanol and hot ethanol and finally dried in
vacuum for overnight to afford PtL1 as a light-yellow powder (1.02 g, 93%).
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1H NMR (300 MHz, CDCl3, 298 K),
δ
(ppm)=8.73 (s, 8H), 8.68 (d, 4H, J = 8.1 Hz), 7.90 (t,
4H, J = 7.2 Hz), 7.82 (d, 4H, J = 8.4 Hz), 7.42 (d, 4H, J = 7.8 Hz), 7.37 (t, 4H, J = 5.1, 7.2
Hz), 2.25 (m, 12H), 1.32 (m, 18H).
13C NMR (75 MHz, CDCl3): 155.44, 155.88, 150.01, 149.07, 136.80, 134.70, 131.34,
129.85, 126.86, 123.72, 121.31, 118.40, 110.66, 109.62, 16.69, 8.38.
HRMS (m/z): [M + H] found 1119.293; calculated 1118.384.
2.5 Synthesis of polyFePtL1
The ligand Pt L1, (0.22 g, 0.20 mmol) and Fe(BF4)2,6H2O (0.07 g, 0.20 mmol) were
refluxed at 80oC in argon-saturated dry CHCl3-DMSO (20 mL, 1:1) mixture for 24 h. The
reaction mixture was cooled to room temperature and added in CHCl3. The residue was
filtered and washed with MeOH and CHCl3 several times and dried under vacuum for 24 h
to get polyFePtL1 with >95% yield.
1H NMR (300 MHz, DMSO-d6, 298 K),
δ (ppm)=9.66 (bs, 4H), 9.08 (s, 4H), 8.50 (d, 4H, J
= 5.8 Hz), 8.06 (bs, 4H), 7.58 (bs, 4H), 7.29 (t, 8H), 2.30 (m, 12H), 1.34 (m, 18H).
HRMS (m/z): [M + H] found 576.103; calculated 576.653.
2.6 Cell viability
Cell viability of the cancel cells were measured by Cell Counting Kit-8 (CCK-8) assay.
This assay kit provides an easy-to-use, non-radioactive, and very good method for cell
proliferation, cell viability, and cytotoxicity. CCK-8 assay Kit is a sensitive method for
quantification of viable cells in proliferation and cytotoxicity assay. The method is
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colorimetric and based on the conversion of highly water soluble colorless Dojindo’s
tetrazolium salt to yellow color formazan. WST-8 [2-(2-methoxy-4-nitrophenyl)-3-(4-
nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt] produces a water-
soluble formazan dye upon reduction. Viable cells with active metabolism convert WST-8
into formazan, however, dead cells lose this ability. Thus color formation serves as a useful
and convenient marker of only the viable cells. The measured absorbance (450 nm) is
proportional to the number of viable cells. For cell viability test, the lung cancer cell line,
A549 was grown in DMEM medium (pH 7.4) supplemented with 10% (v/v) FBS, 100
U/mL penicillin, and 100 µg/mL streptomycin (10% FBS-DMEM) and grown in the dark at
37°C in a 5% CO2 humidified environment. Cells were seeded with a density of
approximately 15,000 cells/cm2 into 96-well tissue culture plates. The old medium was
removed from the cell culture medium after 24 h and medium containing different
concentrations polymer was added and incubated for 24 h. For that polymers were
dissolved in 20% acetonitrile aqueous solution and then diluted to 0, 2, 4, 6, 8 and 10 µM in
1% acetonitrile and 99% of 10% FBS-DMEM medium. After 24 h of incubating the cells
with different concentrations polymer in 96-well plates, 10 µL of CCK-8 solution was
added to each well. After an additional 2 h of incubation, the absorbance at 450 nm
(reference 630 nm) was measured using a microplate reader. A higher absorbance value
corresponded to higher cell proliferation, as the water-soluble formazan dye was formed via
cellular respiration.
2.7 Calcein-AM and PI assay
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Calcein-AM and PI double staining assay was performed into 24-well tissue culture plates
at a density of approximately 15,000 cells/cm2. After the cells were incubated for 24 h with
10
µM of polyFePtL1 or polyFePtL2 in 24-well plates, the cells were washed twice with
sterile PBS and 500
µ
L of PBS containing calcein-AM (2 M) and PI (4 M)) were added.
µ
µ
Plates were incubated for 15 min before fluorescence imaging with a microscope.
2.8 Apoptosis analysis
Apoptosis analysis of A549 cell was performed by double staining assay using TACS
Annexin V-FITC Apoptosis Detection Kit and PI. After incubating the cells for 24 h with
10
sterile PBS and then 100
min. The cells were collected in 1.5 mL centrifuge tubes and centrifuged for 3 min (300
g). The medium was removed and 100 L of staining solution was added to the cells and
mixed gently. Next, the cell suspension was kept in the dark at room temperature for 15
min. Finally, 400 L of 1X binding buffer was added and the cells were analyzed by FACS.
µM of polyFePtL1 or polyFePtL2 in 4-well plates, the cells were washed twice with
µ
L of 2% BSA containing trypsin was added and incubated for 5
×
µ
µ
The results were analyzed using SONY SP6800 Software.
3. RESULTS AND DISCUSSION
Pt/Fe-based heterometallo-supramolecular polymers, polyFePtL1 and polyFePtL2 were
synthesized (Scheme 1), where the ditopic ligands of PtL1 and PtL2 with square planar
center and TPY (TPY = 4-ethynyl-(2,2’:6’,2”-terpyridine)) allows an octahedral iron(II)
terpyridyl center. The design and subsequent development of the bimetallic supramolecular
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polymer have been inspired by the cytotoxicity of Fe and Ru-based metallosupramolecular
polymers, which can coordinate to DNA by groove binding.[13,14]
Novel
ligand,
PtL1
was
synthesized
from
trans-Pt[PEt3]2Cl2
(PEt3=triethylphosphine) and 4-ethynyl-phenyl-(2,2’:6’,2”-terpyridine) (1, Fig. S1-S4)
using CuI as a catalyst according to Scheme 1, while the PtL2 was prepared from trans-
[Pt(PPh3)2]Cl2 (-PPh3=triphenylphosphine) treating with 1 according to our previous
1
report.16 The ligand, PtL1 was characterized by H-, 13C-NMR and HRMS analysis (Fig.
S5-S8). The formation of a Pt–C
≡
C bond in PtL1 was confirmed by the 1H-NMR and 13C-
1
NMR spectra. No ethynyl proton signal (at 3.19 ppm) was detected in H-NMR of PtL1
(Fig. S5). The down-field shifting of the ethynyl carbon in 13C-NMR (from 83.31 and 78.51
to 110.66 and 109.62) due to the
π-back-donation from the Pt(II) to each ethynyl center of
ligand indicated the formation of Pt–C
≡
C bond.[17]
The complexation of PtL1 with Fe2+ was monitored by UV–vis spectra. Fig. 2a exhibits the
UV–vis spectral change of ligand by simultaneous addition of Fe2+ in the chloroform-
DMSO solvent mixture. A new metal-to-ligand charge transfer (MLCT) band appeared at
~570 nm, which was increased with metal concentration and became saturated. The
complexation ratio of PtL1 to Fe2+ was determined from the absorbance change at the
MLCT band with respect to the molar ratio of this solution, which indicates 1:1 between
PtL1 and Fe2+ (Fig. 2b). The MLCT band intensity remain unchanged in presence of excess
metal ions, indicated the stability of the polymer in solution.[18,19]
Organometallic metallo-supramolecular polymer polyFePtL1 was prepared in >90% yield
by single-step complexation reaction of PtL1 with Fe2+. Typically, the PtL1 ligand and
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Fe(BF4)2 equimolar were refluxed in an argon-saturated dry DMSO-CHCl3 mixture. The
reaction mixture was cooled to room temperature and added to an excess CHCl3 solution.
The residue was washed with CHCl3 to remove the impurities and dried under vacuum to
1
1
get polyFePtL1. It was characterized by H-NMR and mass analysis. In the H-NMR
spectrum of polyFePtL1 at Fig. S9-10, all the characterized peaks of PtL1 were obtained
with a downfield shifting of terpyridyl protons. The mass spectrometric studies of the
polymer further support for the polymer formation. In the ESI-mass spectrum of FeLPt1, a
peak at m/z 576.10 corresponding to the molecular ion of repeating unit [M+H]+ was
observed. The preparation of polyFePtL2 was reported in our previous paper.[16]
In order to further characterize polyFePtL1 and polyFePtL2, we recorded the
UV−vis spectra in 1% aqueous-acetonitrile solution (5 µM) and representative spectra are
shown in Fig. S11. The polyFePtL1 showed characteristics MLCT absorption at 576 nm
for Fe(II)-terpyridine octahedral complex and at 380 nm for Pt(II)-acetylide square planar
complex. Both the peaks were red-shifted to 580 and 390 nm in polyFePtL2. These
significant red-shifting occurred due to presence of electron withdrawing –PPh3 group (as
lone pair of phosphorous conjugated with phenyl groups) at platinum center of PtL2, which
considerably reduced the lowest unoccupied molecular orbital (LUMO) energy of Pt(II)
containing ligand and facilitated the electron transfer from Fe(dπ) to π*(Pt-acetylide).[17,
20]
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O
O
H
H
+
N
KOH/EtOH
NH4OH, 24 h, Reflux
N
N
N
H
1
(70%)
trans Pt(PEt3)2Cl2
CuI, DCM, 48 h, Reflux
N
N
N
N
P
Pt
P
N
N
PtL1 (93%)
Fe(BF ) ,
CHCl3/DMSO
4 2
24 h, Reflux
-
2BF4
N
N
N
P
Pt
P
Fe2+
N
N
N
n
polyFePtL1
Scheme 1. Synthesis of polyPtL1.
Fig. 2 (a) UV-vis spectral change of PtL1 by addition of Fe(II); (b) Absorption change at
580 nm as a function of the molar ratio of Fe(BF4)2/PtL1.
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The single polymer chain of polyFeLPt1 or polyFeLPt2 contain multiple numbers of
alternative Fe and Pt centers with octahedral and square planar geometry. So, it can interact
strongly with the negative charges of phosphate groups present in the sugar backbone of
DNA. As we know, the DNA is the pharmacological target of many of the useful drugs. So,
interfering DNA replication by targeting is quite easy and conceptually straightforward. [21,
22] Therefore, our prime intention is to stop DNA replication by enhancement of DNA
binding properties of the metallo-supramolecular polymer.
To get insight about DNA binding of these polymers we have employed UV-vis
spectrometric titration method. The binding constant of two polymers to ct-DNA were
determined by UV-vis changes at MLCT band of polymers as a function of DNA
concentration. For that, 5 µM of polymer in 1% aqueous-acetonitrile solution was taken in
a UV-vis cuvette and simultaneously increased the DNA concentration up to
[DNA]/[polymer] ~1.4. As the DNA concentration increased, changes in the MLCT (~580
nm) band of polymers were observed and plateau detected after certain concentration (Fig.
3). The binding constant (Kb) of the polymers were calculated using MvH equation
(equation 1) from the non-linear fitting plot of extinction coefficient change with respect to
[DNA]/[Polymer].[7,8] The Kb(DNA-polyFeLPt2) is 15 times higher than Kb(DNA-
polyFeLPt1). The reason can be explained by van der Waal’s and electrostatic interaction
of the DNA duplex with FePtL2. Presence of extra π-system (planar phenyl ring in ‒PPh3
group) in the PtL2 ligand that extends sufficiently to permit the partial intercalation of the
phenyl group to the DNA bases and enhances binding activity.[23]
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Table 1. DNA binding of polymers.
Hypochromicity
(%)*
bathochromic
shift (nm)*
Kb
×
106 (M-1)
4.07
11.1
14.9
7
4
polyFePtL1
polyFePtL2
60.08
* at 1:1 [DNA]/[Polymer]
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Fig. 3 UV-vis spectral changes of polymers with the addition of ct-DNA and non-
linear fitting of the absorption change for polymer as a function of DNA
concentration, (a) and (b) for polyFePtL1;
(c) and (d) for polyFePtL2, respectively. (e)
Possible conjugate structure of B-DNA with polyFePtL1 and polyFePtL2
.
ꢄ
ꢄ
ꢍ/ꢄ
ꢉ
ꢊ
ꢋ
ꢀꢁ − ꢀꢂ
ꢀꢂ − ꢀꢃ
b − (b − 2ꢅ ꢇꢈ DNA /ꢌ)
ꢆ
=
(1)
2ꢅꢆ ꢇꢈ
Here b = 1+KbCt+Kb[DNA]t/2s, Kb is binding constant between polymer and DNA, the total
polymer concentration in repeat units is Ct, [DNA]t is the DNA concentration per
nucleotide, the size of the binding site per nucleotide is ‘s’ and εa, εf, εb represent the
apparent, free, and bound polymer molar extinction coefficients, respectively. The εb was
determined from the plateau of DNA titration.
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Fig. 4 CCK-8 assay of A549 cells in the presence of polymers (a) polyFePtL1 and (b)
polFePtL2.
In addition, both polymers showed significant hypochromism and red shift of the MLCT
band of Fe(II) upon sequential addition of ct-DNA. The bathochromic shift of polyFePtL1
and polyFePtL2 was 7 and 4 nm with hypochromicity 11.1% and 14.9% respectively at the
plateau. The significant red-shift and maximum hypochromicity of polyFePtL2 indicated
the strong groove binding between the polymer chains and duplex DNA. As the PtL2
contains planar phenyl group, which can intercalate in the DNA duplex and stacked with
DNA base pairs and favors the groove binding of polyFePtL2 and ct-DNA, whereas no
such intercalating groups were present in polyFePtL1. Interestingly, such partial
intercalation was confirmed by the MLCT band change of Pt(II) in presence of DNA. The
Figure 3a unveiled the Pt(II) MLCT of polyFePtL1, which was red shifted from 380 to 387
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nm, whereas for polyFePtL2, blue shifting of Pt(II) MLCT from 390 to 385 nm (Figure 3c)
(at 1:1 DNA−Polymer). The blue shift of the Pt (II) MLCT band is the consequence of
partial intercalation of ligand to the double strand DNA.[24]
As our metallo-supramolecular polymers (polyFePtL1 or polyFePtL2) contain two
cytotoxic metals in the same polymer chain including very strong binding activity to DNA,
it should be a good DNA-replication interfering agent and anticancer drug.[25] The
anticancer activity of polyFePtL1 and polyFePtL2 to human lung cancer cell (A549) were
tested by established CCK-8 assay. For that, the A549 cells were incubated for 24 h and
different concentrations dose of polyFePtL1 or polyFePtL2 was applied for the cell-
viability test. Five different concentrations 2, 4, 6, 8 and 10 µM of polymers were used for
cell viability test. Cell viability decreased with concentration for both polymers and
survival rate reached 71% and 41% for polyFePtL1 and polyFePtL2 at 10 µM
concentrations, respectively showing at Fig. 4. Finally, Calcein-AM and propidium iodide
(PI) double staining colorimetric assay was used for cellular uptake studies and to establish
the cytotoxicity results with cellular levels. This method is fluorescence based for live
(green) and/or dead cells (red) detection with two probes that reflect cellular activities and
plasma membrane integrity.[26] The accumulation of polyFePtL1 and polyFePtL2 to
A549 cell at 10 µM concentration are shown in Fig. 5. An average number of dead/total
cells percentages were calculated from the Fig. 5b and 5d and observed 12% and 58% for
polyFePtL1 and polyFePtL2, respectively. The result also a good agreement with the
DNA-binding and cytotoxicity by CCK-8 assay results. So, from the above results, we
conclude that the polyFePtL2 could effectively enter the cancer cell, and induces the
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apoptosis. Although, the cell death mechanism still not clear to us but we consider that
passive diffusion through the cellular lipid bilayer will be the dominant process involved in
polymer uptake.[27]
Table 2. Live and dead cell assay of the polymers counted from the Figure 5.
Average no. of
live cells (L)
Average no. of
dead cells (D)
D/Total no. of
cells (L+D), %
75
32
10
45
12
58
polyFePtL1
polyFePtL2
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Fig. 5 Calcein-AM and propidium iodide (PI) double staining assay of A549 cells, (a) and
(b) for polyFePtL1; (c) and (d) for polyFePtL2.
Fig. 6 FACS analysis of A549 cells in presence of 10 µM of (a) polyFePtL1 and (b)
polyFePtL2 by Annexin-V FITC/PI double staining.
Considering the essential role of cytotoxicity of polymers are apoptosis induction, the cell
apoptosis mechanism was studied by FACS using Annexin-V FITC/PI double staining
assay. For that, A549 cells were treated with polymers for 24 h with 10 µM concentration
and then cells were collected for staining. As shown in Fig. 6, the populations of apoptotic
cells, including late apoptotic cells and early apoptotic cells for polyFePtL1 were 9.80%
and 3.25% and for polyFePtL2 14.47% and 4.49% respectively. The significant increases
of the apoptotic cells for polyFePtL2 indicates that it effectively inhibits the cell
proliferation and induced apoptosis.
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4. CONCLUSION
In summary, the first organometallic based metallo-supramolecular polymer with
alternative iron and platinum metals have synthesized and its binding to the DNA and
cytotoxicity to cancer cell are presented. The results suggested that the platinum center in
the metallo-supramolecular polymer is competent to interact to the DNA without being
hindered to the iron unit, which simultaneously enhances the electrostatic binding to the
DNA and results the strong cytotoxicity. The polyFePtL2 including planar phenyl group
substituted platinum center showed ~15 times strong binding activity to DNA than
polyFePtL1, which contains ethyl substituted platinum. Strong DNA binding also reflected
in the cytotoxicity properties of the polymers. Approximately double cytotoxicity was
observed for polyFePtL2 than polyFePtL1 for A549 call. Cell death mechanism was
established by FACS and its showed apoptosis is the main reason for cytotoxicity and a
higher percentage of early and late apoptotic cells were observed for polyFePtL2 than
polyFePtL1. The Fe-Pt based hetero-metallo-supramolecular polymer is a unique example
of a potentially antitumor-active polymer which offers stable and improves DNA binding
with anticancer activities.
5. ACKNOWLEDGMENT
The authors would like to acknowledge JST CREST Grant Number JPMJCR1533, Japan
for financial support
6. REFERENCES
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ACCEPTED MANUSCRIPT
[1] E. Wong, C.M. Giandomenico, Current status of platinum-based antitumor drugs,
Chem. Rev. 99 (1999) 2451–2466.
[2] N.P. Farell, Multi-platinum anti-cancer agents. Substitution-inert compounds for
tumor selectivity and new targets, Chem. Soc. Rev. 44 (2015) 8773-8785.
[3] D. Wang, S.J. Lippard, Cellular processing of platinum anticancer drugs, Nat. Rev.
Drug. Discov. 4 (2005) 307-320.
[4] A. Florea, D. Büsselberg, Cisplatin as an anti-tumor drug: cellular mechanisms of
activity, drug resistance and induced side effects, Cancers 3 (2011) 1351–1371.
[5] S. Saha, R. Majumdar, M. Roy, R.R. Dighe, A.R. Chakravarty, An iron complex of
dipyridophenazine as a potent photocytotoxic agent in visible light, Inorg. Chem. 48
(2009) 2652–2663.
[6] J. Li, T. Murakami, M. Higuchi, Metallosupramolecular polymers: versatile DNA
binding and their cytotoxicity, J. Inorg. Organomet. Polym. Mater. 23 (2013)
119−125.
[7] J. Li, Z. Futera, H. Li, Y. Tateyama, M. Higuchi, Conjugation of organic-metallic
hybrid polymers and calf-thymus DNA, Phys. Chem. Chem. Phys. 13 (2011)
4839−4841.
[8] U. Rana, C. Chakraborty, R.K. Pandey, M.D. Hossain, R. Nagano, M. Morita, S.
Hattori, T. Minowa, M. Higuchi, Selective DNA Recognition and cytotoxicity of
water-soluble helical metallosupramolecular polymers, Bioconjugate Chem. 27
(2016) 2307–2314.
21

ACCEPTED MANUSCRIPT
[9] M.D. Hossain, U. Rana, C. Chakraborty, R. Li, R. Nagano, T. Minowa, M. Higuchi,
Effect of metal–metal distance in Ni (ii)-based metallo-supramolecular polymers:
DNA binding and cytotoxicity, RSC Advances 7 (2017) 38008-38013.
[10] J.F. González-Pantoja, M. Stern, A.A. Jarzecki, E. Robles-Escajeda, A. Varela-
Ramirez, R.J. Aguilera, M. Contel, Titanocene–phosphine derivatives as precursors
to cytotoxic heterometallic TiAu2 and TiM (M = Pd, Pt) compounds. Studies of
their interactions with DNA, Inorg. Chem. 50 (2011) 11099−11110.
[11] C.G. Hartinger, A.D. Phillips, A.A. Nazarov. Ruthenium, osmium and gold
complexes. The quest for innovative anticancer chemotherapeutics, Curr. Top.
Med. Chem. 11 (2011) 2688−2702.
[12] K. Schilden, F. Garcia, H. Kooijman, A.L. Spek, J.G. Haasnoot, J. Reedijk, A
Highly flexible dinuclear ruthenium(II)–platinum(II) complex: Crystal structure
and binding to 9-ethylguanine, Angew. Chem. Int. Ed. 43 (2004) 5668 –5670.
[13] V. Ramu, M.R. Gill, P.J. Jarman, D. Turton, J.A. Thomas, A. Das, C. Smythe, A
Cytostatic ruthenium(II)–platinum(II) bis(terpyridyl) anticancer complex that
blocks entry into S Phase by up-regulating p27KIP1, Chem. Eur. J. 21 (2015)
9185–9197.
[14] W.A. Wani, U. Baig, S. Shreaz, R.A. Shiekh, P.F. Iqbal, E. Jameel, A. Ahmad, S.H.
Mohd-Setapar, M. Mushtaqueh, L.T. Hun, Recent advances in iron complexes as
potential anticancer agents, New J. Chem. 40 (2016) 1063-1090.
22

ACCEPTED MANUSCRIPT
[15] C.G. Hartinger, N. Metzler-Nolte, P.J. Dyson, Challenges and opportunities in the
development of organometallic anticancer drugs, Organometallics 31 (2012)
5677−5685.
[16] C. Chakraborty, R.K. Pandey, U. Rana, M. Kanao, S. Moriyama, M. Higuchi,
Geometrically isomeric Pt(II)/Fe(II)-based heterometallo-supramolecular polymers
with organometallic ligands for electrochromism and the electrochemical switching
of Raman scattering, J. Mater. Chem. C 4 (2016) 9428-9437.
[17] M. Shiotsuka, N. Nishiko, K. Keyaki, K. Nozaki, Construction of a photoactive
supramolecular system based on a platinum(II) bis-acetylide building block
incorporated into a ruthenium(II) polypyridyl complex, Dalton Trans. 39 (2010)
1831–1835.
[18] M. Hossain, T. Sato, M. Higuchi, A green copper based metallo-supramolecular
polymer: synthesis, structure, and electrochromic properties, Chem. Asian J. 8
(2013) 76−81.
[19] F.S. Han, M. Higuchi, D.G. Kurth, Metallosupramolecular polyelectrolytes self-
assembled from various pyridine ring-substituted bisterpyridines and metal ions:
photophysical, electrochemical, and electrochromic properties, J. Am. Chem. Soc.
130 (2008) 2073-2081.
[20] S. Zhao, S.M. Arachchige, C. Slebodnick, K.J. Brewer, Synthesis and study of the
spectroscopic and redox properties of RuII,PtII mixed-metal complexes bridged by
2,3,5,6-tetrakis(2-pyridyl)pyrazine, Inorg. Chem. 47 (2008) 6144–6152.
23

ACCEPTED MANUSCRIPT
[21] L.H. Hurley, DNA and its associated processes as targets for cancer therapy. Nat.
Rev. Cancer 2 (2002) 188−200.
[22] C. Troiber, E. Wagner, Nucleic acid carriers based on precise polymer conjugates.
Bioconjugate Chem. 22 (2011) 1737−1752.
[23] P.K.L. Fu, P.M. Bradley, C. Turro, Stabilization of duplex DNA structure and
suppression of transcription in vitro by bis(quinone diimine) complexes of
rhodium(III) and ruthenium(II), Inorg. Chem. 42 (2003) 878-884.
[24] J.M. Veal, R.L. Rill. Noncovalent DNA binding of bis(1,10-phenanthroline)
copper(I) and related compounds. Biochemistry 30 (1991) 1132-1 140.
[25] M. Milkevitch, H. Storrie, E. Brauns, K.J. Brewer, B.W. Shirley, A new class of
supramolecular, mixed-metal DNA-binding agents:ꢀ
The interaction of RuII, PtII
and OsII, PtII bimetallic complexes with DNA, Inorg. Chem. 36 (1997) 4534–4538.
[26] P. Hu, R. Wang, L. Zhou, L. Chen, Q. Wu, M.Y. Han, A.M. El-Toni, D. Zhao, F.
Zhang, Near-infrared-activated upconversion nanoprobes for sensitive endogenous
Zn2+ detection and selective on-demand photodynamic therapy, Anal. Chem. 89
(2017) 3492–3500.
[27] C.A. Puckett, J.K. Barton, Methods to explore cellular uptake of ruthenium
complexes, J. Am. Chem. Soc. 129 (2007) 46−47.
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Highlights:
•
•
•
•
Synthesis of Pt/Fe-based metallo-supramolecular polymers
Pt(II) and Fe(II) ions introduced alternately in the polymers
High binding affinity of the polymers to calf-thymus DNA (ct-DNA)
High cytotoxicity of the polymers against human lung cancer cell line (A549)