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Cite this: RSC Adv., 2017, 7, 38008

Eﬀect of metal–metal distance in Ni(II)-based
metallo-supramolecular polymers: DNA binding
and cytotoxicity†
Md. Delwar Hossain,‡a Utpal Rana,a Chanchal Chakraborty,a Jinghua Li,§a
Reiko Nagano,b Takashi Minowab and Masayoshi Higuchi *a
Two Ni(II)-based metallo-supramolecular polymers (polyNiL1 and polyNiL2) with diﬀerent metal–metal

Received 19th May 2017
Accepted 25th July 2017

distances were synthesized via (1 : 1) complexation between the Ni salts with bis(1,10-phenanthroline)sand their DNA binding properties and cytotoxicity were revealed. Short metal–metal distances (1.35 nm)
in polyNiL2 showed 7 times stronger DNA binding properties and remarkably higher cytotoxicity to

DOI: 10.1039/c7ra05644c

Hela-Fucci cells than long metal–metal distances (1.95 nm) in polyNiL1. Enhancement of Ni polymer-

rsc.li/rsc-advances

induced cell killing has been observed in a cell cycle study.

Introduction
The construction of small molecules as anticancer drugs has
been an intensive subject in the past few decades. Transition
metal complexes as potential therapeutic agents have received
more attention because the central metal atom can bound toa surrounding array of molecules or anions and the modiedligands can specically recognize the sequence of a nucleic
acid.1 It is potentially useful to understand the binding of metal
complexes to DNA, for designing the structure and conformation of metal complexes. The mode of recognition is a kind ofreversible non-covalent binding primarily based upon intercalation, major or minor groove-binding, hydrogen bonding andelectrostatic interactions, which are dependent on the spatialconguration and specic modication of small molecules.2 As
such, the development of new metal complex structures having
high binding aﬃnity with DNA is of increasing importance indrug design.2b In addition to the studies on the interaction
between short strand planar complex and DNA, metal nanoparticles and polymer complexes have recently received more
attention for application in antitumor targeted drug delivery.3
Previously, we reported high binding aﬃnity of terpyridinebased metallo-supramolecular polymers to DNA.4a,b These
results indicate that the metallo-supramolecular polymersaElectronic Functional Macromolecules Group, National Institute for Materials Science(NIMS), 1-1 Namiki, Tsukuba 305-0044, Japan. E-mail: HIGUCHI.Masayoshi@nims.
go.jp; Fax: +81-29-860-4721; Tel: +81-29-860-4721b

Nanotechnology Innovation Station, NIMS, 1-2-1 Sengen, Tsukuba 305-0047, Japan† Electronic supplementary
10.1039/c7ra05644c

information(ESI)

available.

See

DOI:

‡ Present Address: Department of Chemistry, Faculty of Science, Jagannath
University, Dhaka-1100, Bangladesh.
§ Present Address: Laboratory of So Matter and Biological Physics, Institute ofPhysics, Chinese Academy of Sciences, Beijing 100190, China.

38008 | RSC Adv., 2017, 7, 38008–38013

become a new candidate of anticancer drugs, because suchstrong binding to DNAs should prevent multiplication of thecancer cells. Recently, we reported that, the right handed helicalmetallo-supramolecular polymer exhibited strong DNA binding
properties to B-DNA and high cytotoxicity to cancer cell.4c
Metallo-supramolecular polymer can bind to the DNA by strong
electrostatic interaction and groove binding. Electrostaticinteraction occurred between the positively charge metal centerof polymer and negatively charge phosphate backbone of DNA.
The phosphate–phosphate distance of B-DNA is 0.7 nm is wellknown.4d So, the metal–metal distance 0.7 nm in metallosupramolecular polymer should be a potential route for
improving DNA binding properties, as a result of cytotoxicity. In
order to enhance the possibility of metallo-supramolecularpolymers as anticancer drugs, we planned to investigate the
DNA binding activity of tetrahedral or square planar coordination geometry type metallo-supramolecular polymers withvariation of the metal–metal distances. However, variety ofN-hetero ligands such as terpyridines, bipyridines and porphyrins are well known for synthesis of metallo-supramolecularpolymers but very few papers were reported with 1,10-phenanthroline based ligands.5 Phenanthroline analogues formed
coordinate metal complexes with variety of metal ions including
Ni2+, Cu2+, Pt4+ and have been shown numerous biologicalactivities such as antitumour,6a anti-candida,6b antimycobacterial,6c antimicrobial,6d activities etc. Moreover,considerable attention has been focused on the use of phenanthroline complexes as intercalating agents of DNA1a and asarticial nucleases.7 With the aim to generate biological activities of phenanthroline-based metallo-supramolecular polymers,herein we report the binding properties of Ni(II)-based polymers
with ct-DNA and their cytotoxicity to cancer cells (Hela-Fucci). In
addition, the variation of the metal–metal distances and theirThis journal is © The Royal Society of Chemistry 2017

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relationship to the binding aﬃnity to DNA as well as anticancer
activity to cancer cells also investigated.

Experiment
Materials
Calf-thymus (ct) DNA (TREVIGEN, 10 mg mL1, 200–500 bp),
was highly puried and qualied. Human cervical cancer (Hela)cells expressing the Fucci probes (Hela-Fucci) were purchasedfrom RIKEN BRC, Japan. Cell Counting Kit-8 (CCK-8) was obtained from Dojindo Laboratories (Kumamoto, Japan). Fetal
bovine serum (FBS) was obtained from Bioserum (Hiroshima,
Japan). Dulbecco's Modied Eagle Medium (DMEM), Dulbecco's phosphate buﬀered saline (PBS), and trypsin–EDTA (0.05%
trypsin, 0.53 mM EDTA–4Na) were obtained from Invitrogen(Carlsbad, CA, USA). Cell culture dishes were from BD Bioscience (San Jose, CA, USA). All reagents were the highest purity
commercially available and, unless otherwise noted, were used
as obtained without further purication.

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37  C in a humidied incubator at 5% CO2. The adherent
cultures were grown as monolayer and were passaged once in 3–
4 days by exposure to 0.05% trypsin–EDTA. For cell viability
assay, the Hela-Fucci cells were seeded in 96-well plates andcultured for 24 h. The medium was removed, and Ni polymers
were added, which were prepared by medium including 5%acetonitrile and reached the nal concentration 0, 5, 10, 15, 20,
25 mM respectively. The 5% acetonitrile was used as a control.
Aer 24 h of incubation, 10 mL of CCK-8 solution was added toeach well and check the cell viability. Further 24 h was incubated and check the cell viability. Cell viabilities were normalized to (OD450–OD620) for the untreated cells. Assays were
performed in quadruplicate.
Fluorescence imaging
Fluorescence images were taken using a uorescence microscope (DMIL, Leica Microsystems, Germany). For time-lapse
imaging, cells were held in an incubation chamber at 37  C ina humidied atmosphere containing 5% CO2 (Tokai Hit, Fujinomiya, Japan).

Instrumentation
UV-vis spectra were recorded at 30  C using a Shimadzu UV2550 UV-vis spectrophotometer. A Model 550 Microplate
Reader (Bio-Rad Laboratories, Inc.) was used for cell viability
test. Total Internal Reection Fluorescence (TIRF), Leica
microsystems were used for cell cycle study.
Binding constant
The concentration of ct-DNA per nucleotide was calculated fromits known extinction coeﬃcient at 260 nm (6600 M1 cm1,
200–500 base pairs) and the concentration of polymers wereexpressed in repeat units.8 The binding constants of the polymers to ct-DNA were determined by absorption titration at 30  C
in 5% acetonitrile-aqueous solution. Dilution of the polymers at
the end of each titration was negligible. The DNA binding
constant, Kb, of the polymers were determined from tting thechanges in the absorption of the polymer as a function of ct
DNA concentration using eqn (1).10 1=2b  b2  2Kb 2 Ct ½DNAt s
3a  3f¼(1)
3b  3f
2Kb CtResults and discussion
Bis(1,10-phenanthroline)s with diﬀerent substituent L1–L2
were synthesized according to our previous report.9a The polyNiL1 and polyNiL2 (Scheme 1) were prepared by 1 : 1complexation equimolar L1 or L2 (in CH2Cl2), and Ni(ClO4)2$6H2O(CH3CN) under inert atmosphere.9b The reaction mixture
was stirred 1 h at room temperature. The desired polymers wereobtained as a pink solid (PolyNiL1: 92%; polyNiL2: 90%) and ishighly soluble in acetonitrile. The molecular weight (Mw) of
polyNiL1 and polyNiL2 in acetonitrile were determined byhere b ¼ 1 + KbCt + Kb[DNA]t/2s, Kb is binding constant between
polymer and DNA, Ct represents the total polymer concentration in repeat units, [DNA]t is the DNA concentration per
nucleotide, ‘s’ is the size of the binding site per nucleotide and3a, 3f, 3b represent the apparent, free, and bound polymer molar
extinction coeﬃcients, respectively. The value of 3b was determined from the plateau of DNA titration, at which point addition of DNA did not result in any further changes to theabsorption spectrum.
Cell viability
Human cervical cancer cell lines (Hela-Fucci) were maintainedin DMEM medium supplemented with 5% fetal bovine serum(FBS), 100 IU mL1 of penicillin, 100 mg mL1 of streptomycin at
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Scheme 1

Structures of the polyNiL1 and polyNiL2.

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SEC-viscometry-RALLS
method(size
exclusion
chromatography-viscometry-right-angle light scattering) using
polyethylene oxide as a standard and to be 1.24  105 Da and1.28  105 Da respectively.9
The coordination geometry of Ni(II) in polymers were justied by UV-vis spectrophotometric titration of Ni(II) ions andligand L1 (Fig. S1, ESI†) or L2.9b Interestingly, the spectrum
changed during the addition of 0.0–1.0 equivalents of the Ni(II)salt to the ligand and then remained unaﬀected by the additionof more than 1.1 equivalents of the Ni(II) salt. The titration plots
between the absorbance and the molar ratio of [the Ni salt]/[ligand] clearly showed the 1 : 1 complexation. These results
indicated that Ni(II) bind 1 : 1 way and formed tetrahedral
geometry during the formation of polyNiL1 and polyNiL2.9a,b
The UV-vis spectra of polyNiL1 and polNiL2 were recorded in
5% acetonitrile aqueous solution (concentration ¼ 5 mM). The
polyNiL1 showed three absorption peaks at 230, 280 and
324 nm whereas polyNiL2 showed two absorption peaks at 232
and 277 nm. The peaks at 230 and 280 nm for n / p* and p/ p* transition of phenanthroline unit and 324 nm for p / p*transition of uorene unit (Fig. S2, ESI†).9
The interaction between the polymers (polyNiL1 and polyNiL2) and DNA was investigated by UV-vis spectral measurement (Fig. 1). Metallo-supramolecular polymers contains Ni(II)ions with bis(phenanthroline)s containing aromatic heterocycles, can be immensely powerful tools for probing nucleic acids.
ct-DNA belongs to B-form DNA having wide major groove withmoderate depths easily accessible to molecules. In the UV-vis
titration with increasing concentrations of ct-DNA, all of Ni(II)polymers were found to exhibit noticeable hypochromism andbathochromic shi with several isosbestic points (Table 1).
During the titration of ct-DNA to Ni(II) polymers, a hypochromicshi at 280–350 nm was observed due to electrostatic interaction between the polymer complexes and DNA (Fig. 1a and b). In
UV-vis spectra, the hypochromicity along with bathochromicshis indicate an electronic interaction between the polymers
and ct-DNA. The spectral change was saturated at 1 : 1 molar
ratio of [nucleotide of DNA]/[unit of polymer] (Fig. 1a andb insets). According to the ts of titration data (eqn (1)), thebinding constants (Kb) of polyNiL1, and polyNiL2 were determined to be 2.77, and 19.76  106 M1 respectively by using thenon-linear curve tting (Table 1).10 PolyNiL1 with uorene
spacer has approximately seven times smaller binding constant
than without spacer polyNiL2. The reason can be explained as
the metal–metal distance in the polymer chains. The metal–
metal distance for polyNiL1 and polyNiL2 was 1.95 and 1.33 nm
respectively, which was calculated by simple Chem3D soware.
In contrast, the phosphate–phosphate distance of B-DNA are
known to be approximately 0.7 nm. The charge of the Ni(II) atomin the polymer is +2, whereas the charge of the phosphate in
DNA is 1. So, one Ni(II) center of polymer can interact with two
phosphate groups of DNA. The metal–metal distance of polymers to phosphate–phosphate distance of DNA of polyNiL1 andpolyNiL2 is approximately 2 and 3 respectively. So, strong
electrostatic bonding between ct-DNA and polyNiL2 occurred
due to the close matching of the metal–metal distance of thispolymer with phosphate–phosphate distance of B-DNA.

38010 | RSC Adv., 2017, 7, 38008–38013

Paper

Fig. 1 UV-vis titration spectra of (a) polyNiL1 (7 mM: based on repeatunit), and (b) polyNiL2 (7 mM: based on repeat unit), in the absence andpresence of increasing concentrations of ct-DNA (up to 7 mM: pernucleotide of DNA) in aqueous solution including 5% CH3CN. Red
color for only polymers. The insets show titration plots at maximum
absorbance for [nucleotide of DNA]/[unit of polymer] used to obtain
the binding constant. (c) Estimation of conjugate structures of B-DNA
and polyNiL1 and polyNiL2.

Table 1

Calf thymus DNA binding of Ni(II) polymers

Polymer

Hypochromicity (%)

Kb (106 M1)s

polyNiL1
polyNiL2

13
14

2.77
19.76

0.17
0.49

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Compared with the mono-metal complexes, the binding
constants of the polymer complexes are larger than the monometal complexes.11 Such a strong binding properties of thepolymer complexes to ct-DNA will be caused by the collaborativeeﬀect on stabilization between the neighboring binding sites in
the polymer complex. Therefore, very strong electrostaticinteraction can be revealed between the Ni polymers and ctDNA.
The structural changes of DNA in the presence of polymers
were studied by circular dichroism (CD) titration and the results
are shown in Fig. 2. The B-form of ct-DNA exhibited two positive
bands at 277 and 220 nm and a negative band at 248 nm (ablack spectrum in Fig. 2a and b), implying the right-handed
helical structure of DNA. When the polymer was added to the
DNA solution, the spectrum was dramatically changed. Thechanges caused by polyNiL2 were more signicant than thoseby polyNiL1. Interestingly, with increasing the concentration of
polyNiL2, the band at 277 nm in Fig. 2b was blue-shied to
267 nm and almost vanished when the mass ratio of DNA/polyNiL2 reached 1.0 : 0.2 (a green spectrum in Fig. 2b), indicating
the original helical structure of DNA was collapsed. With thefurther addition of polyNiL2 to the solution, a new band at
283 nm appeared in the negative region. This wavelength (283nm) corresponds with that of an intense absorption (280 nm) in
the UV-vis spectrum of polyNiL2 (a black spectrum of Fig. S2,
ESI†). This result indicates the helicity in polyNiL2 was induced
by DNA in the polyNiL2-DNA conjugates, probably because of
the strong and favorable electrostatic interaction between the
cationic polyNiL2 and anionic DNA. It is considered that thepolymer chains of polyNiL2 take on a helical conformation
along the DNA helicity.12a As for polyNiL1, with increasing thepolymer concentration, the band at 277 nm in Fig. 2a became
small but no new peaks appeared in the negative region. It
suggests the electrostatic interaction with DNA is weaker thanthat of polyNiL1.
It is commonly considered that DNA is the major target ofmany anticancer agents.12b The aim of many studies is toestablish new anticancer agents with greater selectivity tocancer cells. The cytotoxicity of the polymers (polyNiL1, polyNiL2) were studied using cell counting kit, CCK-8 (WST-8) assay
with Hela-Fucci cell. The cytotoxicity of the polymers were
measured as the percentage ratio of the absorbance of thetreated Hela-Fucci cells to the untreated controls. Cell viability
experiment showed 59.5% and 1% of viable cells with using poly
NiL1 (25 mM) and polyNiL2 (25 mM) respectively (Fig. 3). The
polyNiL2 polymer without spacer largely decreased the cellFig. 2

CD spectra of (a) polyNiL1 and (b) polyNiL2.

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Cytotoxicity of polyNiL1 (0, 5, 10, 15, 20, 25 mM) and polyNiL2(0, 5, 10, 15, 20, 25 mM) in cell culture medium against Hela-Fucci cells.

Fig. 3

viability in a dose-dependent manner than polyNiL1 with
uorene spacer because the binding aﬃnities of polyNiL2(Kb ¼ 19.76  106 M1) with ct-DNA is higher than polyNiL1(Kb ¼ 2.77  106 M1). It seems that the cytotoxicity of thepolymers is largely dependent on the binding constant.
Cellstain-double staining kit is utilized for simultaneousuorescence staining of viable and dead cells. This kit contains
calcein-AM and propidium iodide (PI) solutions, which stain
viable (green) and dead cells (red), respectively. Aer the cells
were incubated for 24 h with 25 mM of PolyNiL1 or polyNiL2 in
24 well plates, the cells were washed twice with sterile PBS, and
500 mL of PBS containing calcein-AM (2 mM) and PI (4 mM) wereadded. Plates were incubated for 15 min before uorescenceimaging with a microscope. PolyNiL2 (Fig. 4b) showed morenumber of dead cells (red uorescence) than polyNiL1 (Fig. 4a).
Fluorescence microscopic analysis suggested that PolyNiL2 hasa greater potential to induce the death of Hela-Fucci cancer cells(Fig. 4) and indicates the higher cytotoxicity of polyNIL2.
Cell cycle study is the most important process for understanding the mechanism of DNA replication within a denedtime period. Cell cycle occurs in four phases, G1(gap1)/
S(synthesis)/G2(gap2)/M(mitosis).13 A uorescent probe thatlabels diﬀerent phases of cell cycle like G1 phase nuclei in red
and S/G2/M phase nuclei in green. Sakaue-Sawano et al. developed a cell-cycle visualization system called uorescentubiquitination-based cell-cycle indicator (Fucci), which takes
advantage of the cell cycle-dependent ubiquitination of Cdt1
protein and geminin protein.13 Fucci is a sophisticated technology for easy determination of G1 and S/G2/M phases of thecell cycle. Fucci-G1 red is a fusion protein of a fragment ofFig. 4 Fluorescence images of Hela-Fucci cells treated with polyNiL1(a) and polyNiL2 (b).

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Fig. 5 Eﬀects of Ni polymers in Hela-Fucci cells using time-lapse imaging. (a) Representative images taken 0 to 72 h after treatment with
polyNiL1. (b) 0 to 16 h after treatment with polyNiL2; red and green phases were changed with time by the treatment of (c) polyNiL1 and (d)polyNiL2.

human Cdt1 with the orange uorescent, mKO2 (monomeric
Kusabira-Orange2) that indicates the G1 phase, whereas FucciS/G2/M green is a fusion protein of a fragment of humangeminin with the green uorescent protein, mAG1 (monomericAzami-Green1) that visualize S/G2/M phase.13c In this system, G1
and S/G2/M cells emit red and green uorescence by TIRF
microscope, respectively. In our experiment, we added Ni
polymer solution in Hela-Fucci cells in DMEM then held in TIRF
microscope. In Fig. 5a and c, we have shown that G1 (red phase)and S/G2/M (green phase) Hela-Fucci cells gradually decreased
during 0 h to 72 h treatment with polyNiL1 (25 mM). In Fig. 5band d, G1 and S/G2/M cells drastically decreased during 0 h to
16 h with polyNiL2 (25 mM) and nally only G1 cells remained.
This result indicated that G1 phase is arrested with the treatment of polyNiL2 and predicted to enhance cell killing. Thus,
we concluded that the polyNiL2 showed very strong cytotoxicity
than polyNiL1.

Conclusions
In an eﬀort to select the new candidates for anti-cancer drugs,
we used our synthesized polymers (polyNiL1, and polyNiL2) todo the DNA binding experiments. The very strong electrostatic

38012 | RSC Adv., 2017, 7, 38008–38013

interaction was conrmed between the polymers and ct-DNA by
UV-vis experiment. The polyNiL2 exhibited higher binding
constants than polyNiL1 to ct-DNA. Cell viability experimentshowed 59.5% and 1% of viable cell for polyNiL1 and polyNiL2
to cancer cells (Hela-Fucci) respectively. Cell cycle studies
showed living cancer cell dead rapidly with the treatment with
polyNiL2.

Acknowledgements
The authors would like to acknowledge JST CREST Grant
Number JPMJCR1533, Japan for nancial support.

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