Biophysical and biological studies of some polymer grafted metallo-intercalators

Accepted Manuscript Title: BIOPHYSICAL AND BIOLOGICAL STUDIES OF SOME POLYMER GRAFTED METALLO-INTERCALATORS Authors: Yesaiyan Manojkumar, Subramanian Ambika, Rajendran Senthilkumar, Sankaralingam Arunachalam PII: DOI: Reference:

S0927-7765(17)30295-3 http://dx.doi.org/doi:10.1016/j.colsurfb.2017.05.037 COLSUB 8566

To appear in:

Colloids and Surfaces B: Biointerfaces

Received date: Revised date: Accepted date:

8-12-2016 8-5-2017 13-5-2017

Please cite this article as: Yesaiyan Manojkumar, Subramanian Ambika, Rajendran Senthilkumar, Sankaralingam Arunachalam, BIOPHYSICAL AND BIOLOGICAL STUDIES OF SOME POLYMER GRAFTED METALLO-INTERCALATORS, Colloids and Surfaces B: Biointerfaceshttp://dx.doi.org/10.1016/j.colsurfb.2017.05.037 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

BIOPHYSICAL AND BIOLOGICAL STUDIES OF SOME POLYMER GRAFTED METALLO-INTERCALATORS Yesaiyan Manojkumara, Subramanian Ambikaa, Rajendran Senthilkumarb and Sankaralingam Arunachalama*# a

School of Chemistry, Bharathidasan University, Tiruchirappalli-620 024, India. Department of Biosciences, Cell biology, Åbo Akademi University, Turku, Finland. # Plot-46, Nagappa Nagar, Airport (Post), Tiruchirappalli-620007, India. *Corresponding author. Tel.: +91 431 2407053; E-mail address: [email protected] b

Graphical Abstract



HIGHLIGHTS   

BPEI anchored metallo-intercalators display synergic interaction on CT-DNA binding. The geometry of the metal complex on polymeric backbone and degree of coordination play vital role in CT-DNA binding. Polymer-copper(II) complexes are active against metastatic breast cancer cells, MDA-MB-231.

ABSTRACT

Two water-soluble polymer-copper(II) complexes, [Cu(ip)2(BPEI)](ClO4)2.H2O (Complex 1) and [Cu(dppz)2BPEI](ClO4)2.H2O (Complex 2) with different degree of coordination have been synthesized and characterized. The interaction between the prepared complexes and CT-DNA 1

has been assessed by various physico-chemical methods. The spectroscopic and the cyclic voltammetry studies have revealed that both the complexes interact with CT-DNA through intercalation binding mode. Among the two complexes, Complex 2 has higher binding affinity with CT-DNA. The antiproliferative activity of the complexes has been examined on human breast cancer cells, MDA-MB-231, adopting various techniques. The results indicate that both the polymer-copper(II) complexes are effective against the breast cancer cell line and the order of the activity is consistent with the DNA-binding ability.

1.INTRODUCTION Over the years, many metal complexes and organic molecules have been reported as anticancer agents. However, their poor water solubility, metabolic instability and dose-dependant toxicity have restricted them to be used in clinical trails [1-3]. Therefore, many research groups have started to design anticancer drugs without the above mentioned disadvantages. Grafting metal complexes and organic molecules into the polymer make them into good candidate for chemotherapy [4, 5]. The unique features of polymeric drug conjugates mainly depend on the choice of the polymer backbone [6-8]. Especially, the cationic polymer,

branched

polyethyleneimine (BPEI) and its modified forms have been used for gene delivery [9], drug delivery [10], and cell imaging [11]. A recent report indicates that, BPEI increases the cellular uptake of iridium metal complexes in cancer cells [12]. The advantage of buffering capcity of pH of PEI is that it increases the antiproliferative activity in chemotherapeutic drug [9]. Based on the above facts, many reports suggest that BPEI anchored metal complexes have shown promising therapeutic properties than that of ordinary metal complexes [13-15]. Copper is an endogenous metal and essential for angiogenesis, which makes copper based chemotherapeutic agents very popular over the years [16-19]. Further, the redox nature and biocompatibility of copper makes it's complexes as potential drug for a variety of diseases. [2023]. As DNA is the fundamental molecule for cell division, protein synthesis and development of the organism, it serves as a supreme target for many drugs. Moreover, the structure and the composition of DNA offers many target cites for metal complexes. Therefore, studies on the interactions of copper complexes with DNA have been an active field in chemotherapeutic drug

2

desinging. This motivates us to study the importance of polymer anchored copper complexes and their interactions with CT-DNA and proteins [24-26]. In general, the metal complexes containing polypyridyl ligands like ip, dpq and dppz (ip = imidazo[4,5-f]1,10-phenanthroline,

dpq

=

dipyridio[3,2-d;2’,3’-f]quinoxaline,

dppz

=

dipyrido[3,2-a:2′,3′-c]phenazine) are better metallo-intercalators [27-29]. Further, these complexes induce π-stacking interactions with the π- electron clouds of the DNA base pairs which leads to the unwinding of DNA double helix and provide interesting photophysical properties [30, 31]. Our earlier studies show that BPEI anchored copper(II) complexes containing phenanthroline / bipyridine and amino acid mixed ligands displayed electrostatic interaction with

DNA/RNA

[32-34].

However,

BPEI

containing

metallo-intercalator

[Cu(dpq)2BPEI](ClO4)2.2H2O shows dual interactions such as electrostatic and - stacking with DNA/RNA and its binding affinity affects cell viability of the non metastatic MCF-7 cancer cells [35]. Continuing, our efforts in understanding the efficacy of the polymer anchored copper(II) complexes, here, we report the binding ability of BPEI grafted copper complexes containing polypyridyl ligands, (ip and dppz) with DNA. Further, the anticancer activity of these synthesized complexes have been tested on MDA-MB-231 cells.

2. EXPERIMENTAL SECTION 2.1. MATERIAL Calf thymus DNA (CT-DNA), branched polyethyleneimine (BPEI) (Mw ca. 25,000) and copper(II) perchlorate hexahydrate were purchased from Sigma-Aldrich, and ammonium acetate and formaldehyde were obtained from Merck, India. Glacial acetic acid from Qualigens Fine Chemical, India. Ortho phenylenediamine and Tris-HCl were received from Loba Chemie, and sodium chloride (NaCl) from SISCO Research laboratories, India. The ligands (ip and dppz) and the precursor complexes, [Cu(ip)2(H2O)](ClO4)2 and [Cu(dppz)2(H2O)](ClO4)2 were prepared by literature methods [36, 37]. The carbon, hydrogen and nitrogen contents of the samples were determined at SAIF, CERI, Karaikudi, India. Copper analysis was done by a reported procedure [38]. Infra-red spectra were recorded on FT-IR JASCO 460 PLUS spectrophotometer with samples prepared as KBr pellets. EPR spectra were recorded on JEOL-FA200 EPR spectrometer. Human breast cancer cell line, MDA-MB-231 (ATCC, USA.) were cultured as a monolayer in Dulbecco’s Modified Eagles Medium (Biochrom AG, Berlin, Germany), supplemented with 10 3

% fetal bovine serum (Sigma-Aldrich, USA), with 100 μg/mL of streptomycin (Himedia, Mumbai, India) as antibiotics, at 37 oC, in a humidified atmosphere containing 5 % CO2, in a CO2 incubator (Heraeus, Hanau, Germany). 2.2. ABSORPTION SPECTRAL TITRATION The DNA binding experiments were performed at 25.0 ± 0.2 C. A solution of calf thymus DNA in the buffer gave a ratio of UV absorbance at 260 to 280 nm of ~1.8 -1.9:1, indicating that the CT-DNA was sufficiently free of protein [39]. The concentration of CT-DNA in base pairs was determined by UV absorbance at 260 nm by taking the molar extinction coefficient value 13200 M-1cm-1 for CT-DNA at 260 nm [40, 41]. Absorption spectral titration experiments were performed by maintaining constant concentration of the polymer complexes with varying the CT-DNA concentration on a Shimadzu UV-Visible spectrophotometer. An equal amount of CTDNA was added to both the complex and the reference solution to eliminate the absorbance of CT-DNA itself. 2.3. COMPETITIVE BINDING STUDIES Emission spectra were recorded on a JASCO FP 770 spectrofluorimeter. For fluorescence quenching experiments, CT-DNA was pre-treated with ethidium bromide (EB) for 30 min. Solutions of complexes were then added to this mixture and their effect on the emission intensity was measured. The samples were excited at 450 nm and emission was observed between 500 and 700 nm. The spectra were analysed according to the classical Stern-Volmer equation [42], I0/I = 1+ Ksvr, where I0 and I are the fluorescence intensities in the absence and the presence of complex, respectively. Ksv is a linear-Stern-Volmer quenching constant dependent on the ratio of rbE (the ratio of the bound concentration of ethidium bromide to the concentration of DNA). r is the ratio of the total concentration of complexes to that of CT-DNA. 2.4. CYCLIC VOLTAMMETRY All cyclic voltammetry experiments were performed in a single compartment cell with a three electrode configuration on a Princeton EG and G-PARC model potentiostat. Glassy carbon was the working electrode, saturated calomel as the reference electrode and the platinum wire was auxiliary electrode. The supporting electrolyte was 50 mM NaCl / 5 mM Tris- HCl buffer at pH 4

7.1. The electrode surfaces were freshly polished with alumina powder and then sonicated in ethanol and distilled water for 1 min prior to each experiment and the electrode was rinsed with doubly distilled water thoroughly between each polishing step. Before the experiments, solutions were deoxygenated by purging with nitrogen gas for 15 min prior to the measurements. 2.5. SYNTHESIS OF POLYMER GRAFTED COPPER (II) COMPLEXES The precursor complex, [Cu(ip)2(H2O)](ClO4)2 or [Cu(dppz)2(H2O)](ClO4)2 and BPEI, were taken in the 2.5:1 molar ratio in methanol and the mixture was heated between 60-65 0C for 10 h in a water bath. The obtained dark green coloured solution was dialyzed approximately at 15 0C against cold distilled water for 5-6 days, till the absence of colour in the liquid where the dialyzer is placed. Afterwards the solvent was evaporated by a rotary evaporator under reduced pressure. The dark greenish filmy polymer complex obtained was pulverized and dried. Polymer complexes with different degrees of copper(II) complex units grafted onto BPEI were prepared by varying the amount of the precursor copper(II) complex, change the reaction time etc. These complexes are very stable in solution. When we occasionally kept the solutions of the complexes in dialysis bags we never observed the presence of any free quantity of copper complex ion or copper ion in the solution outside the dialysis bag indicating that the polymer-copper(II) complexes are very stable. 2.6. SYNTHESIS OF DIFFERENT DEGREE OF COORDINATION (X) OF POLYMER-COPPER (II) COMPLEXES To a solution of 0.15 g of BPEI in 20 mL of methanol, 0.1 g, 0.2 g and 0.3 g of the precursor complexes, [Cu(ip)2(H2O)](ClO4)2 / [Cu(dppz)2(H2O)](ClO4)2, in 40 mL of methanol was added. This mixture was heated between 60 - 65 C for 6 h, 10 h and 12 h respectively. The ratio of the 1, 2 and 3 amines of the monomeric unit of the BPEI are 1:2:1 [43]. From this ratio, the average monomeric unit of the BPEI consider as (~NH-CH2-CH2~). The ‘x’ represents the degree of coordination, which is the number of moles of copper(II) complex units per mole of the repeating unit (amine groups) of polymeric ligand. If the entire repeating units (amine groups) in the polymer are coordinated to copper, the value of x is 1. It can be calculated either from carbon content [43, 44] or copper content [38]. The degrees of coordination (x) thus obtained for the complex 1 are 0.084, 0.231 and 0.487 for complex 2 are 0.102, 0.119 and 0.144. 2.7. CELL CULTURE

5

MDA-MB-231 cells were cultured in Dulbecco’s Modified Eagles Medium, supplemented with 10 % fetal calf serum (FCS), 100 μg/mL streptomycin, and 100 μg/mL penicillin as antibiotics, at 37 °C, in a humidified 5% CO2 in CO2 incubator. 2.8. WST-1 ASSAY WST-1 is a colorimetric assay for detection of cell cytotoxicity, viability and proliferation. WST1 method was used to determine the cell viability of both the polymer-copper(II) complexes 1 and 2 with MDA-MB-231 cells [45]. The cells were seeded in a 96 well culture plates and incubated for 24 h under 5 % CO2. The cells were then plated at a density of 5000 cells per well into the 96-well plates and incubated overnight. After the time, the culture medium was replaced by complexes with different concentrations (10 - 80 g/mL). The treatment was carried out for 6 and 16 h after that WST-1 was added to each well, incubated for 4h and then measured in a plate reader. The cell viability percentage was calculated from the absorbance value of a Varioskan Microplate Reader at 450 and 620 nm used as a reference. The percentage of cytotoxicity was calculated by (Mean absorbance of untreated cells (control) - Mean absorbance of treated cells × 100) / Mean absorbance of untreated cells (control). Based on the metal analysis, our polymer-copper(II) complexes 1 (x = 0.487) and 2 (x =0.144) sample contains 2.86 ×10-3 and 1.74 ×10-3 M of copper complex units. Therefore, at 16 h the IC 50 value of 19 g/mL for the complex 1 corresponds to 1.7 M of copper complex units and 14 g/mL for the complex 2 corresponds to 1.2 M copper complex units. 2.9. PROPIDIUM IODIDE (PI) ASSAY The chemotherapy induced apoptosis can be determined by PI assay [46]. PI is a fluorescent dye which excluded from viable cells, but it can penetrate cell membranes and strongly bind to DNA of the dead cells. The cells were treated with three different concentrations (10 g/mL, 30 g/mL and 50 g/mL) of the complexes 1 and 2 for 6 h treatment and then stained with PI. When PI bound to DNA; it gave the emission maximum at 615 to 620 nm on excitation at 488 nm laser. The fluorescence emission is proportional to the DNA content of the dead cells and the percentage of apoptosis was measured by flow cytometry. 2.10. CASPASE - 3 ACTIVATION

6

The active form of caspase‐3 was measured by flow cytometry using phycoerythrin (PE) conjugated antibody[47]. Briefly, cells (5 x 105) were treated for 6h, subjected to trypsinization and prepared for flow cytometric analysis using the protocol given in the kit (PE Active Caspase3 Apoptosis Kit; BD Pharmingen, San Diego, CA). The trypsinzed cells were washed once with PBS, and incubated for 20 min in ice with the cytofix/cytoperm solution provided in the kit which fixes and permeabilizes the cells. After fixing the cells were washed twice with perm/wash buffer provided in the kit. The cells were then incubated with caspase-3 in labeled with phyco-erythrinconjugated antibody for 30 min at room temperature and after incubation, cells were washed and re-suspended in the perm/wash buffer and analysed by FACS Calibur flow cytometer (FL-2). 2.11. 4',6 -DIAMIDINO-2-PHENYLINDOLE (DAPI) STAINING ASSAY The nuclear morphology can be investigated by DAPI staining dye. Briefly, the Cancer cells were treated with their corresponding IC50 values of complex 1 and 2. Then they incubated for 6 h. Control and treated cells were rinsed with phosphate buffered saline (PBS). The cells were permeabilized with Triton-X (10% v/v) and stained with 1 µg/mL

4'-6- diamidino-2-

phenylindole (DAPI) for 5-10 min. Cells were washed three times with PBS reduced background and their nuclear morphology was viewed by DAPI filter were examined under fluorescent microscope (confocal laser scanning microscope, LSM 510 META, Carl Zeiss Inc., Thornwood, NY).

3. RESULTS AND DISCUSSION 3.1. SPECTRAL CHARACTERIZATION The IR spectra (Figure S1) provide valuable information on the nature of the functional groups. The precursor complex [Cu(ip)2(H2O)](ClO4)2 shows bands around 1610 and 1483 cm-1 which are assigned as (C=C) and (C=N). In complex 1, these were shifted to 1568 and 1470 cm-1. The bands at 1637 and 1494 cm-1 for [Cu(dppz)2H2O](ClO4)2, were shifted to lower frequency 1598 and 1382 cm-1 in complex 2. The other bands observed around 2926 and 2811 cm-1 for all the complexes assigned to (C-H) of the aliphatic CH2 of the BPEI and the broad bands around 3419 and 3412 cm-1 are assigned to (N-H) of the BPEI [48]. The analytical results for complex 1 with x

=

0.487 is

Calc.:

C-

45.38;

H-3.68

and

N-15.88

and

Found:

C-

45.38;

H-3.58 and N- 15.32. In the case of complex 2, Calc. (%): C -52.32, H-3.62 and N-13.73 and Found (%): C - 52.32, H - 3.56 and N - 12.95 for the degree of coordination x= 0.144. 7

The electronic spectra of the polymer complexes were recorded at room temperature in aqueous solution. In UV-Vis spectra, (Figure S2) the d-d transitions of complexes 1-2 were observed at 642 and 635 nm respectively. The high energy transition due to intra-ligand (IL) (-*) at 252 and 277 nm were observed for complex 1 and 269 nm for complex 2. In most of the case, due to nature, the ligand ip shows two -* transition and dppz shows one -* transition between 250 to 290 nm [49, 50]. Even functionalised ip and dppz ligands also show same number of peaks in the above mentioned region [51, 52]. Similarly our polymer complexes also show -* around that region. EPR spectral studies of paramagnetic transition metal(II) complexes is not only used to understand the distribution of the unpaired electrons present in the complex but also helps to identify coordination mode by which the ligands are bonded with the metal ion. The spectrum of the polymer complexes at room temperature (insert Figure S3) shows a single intense absorption band in the high field and is isotopic due to the tumbling motion of the molecules. The X-band EPR spectra of the complexes, recorded in methanol at liquid nitrogen (Figure S3) and the parameters are given in the Table S1. From the observed values of the complexes, gII  g indicate that the unpaired electron lies predominantly in the dx2-y2 orbital [53] and it is clear that gII  g  2.0023, which suggests that the unpaired electron is localized in the dx2-y2 orbital of square pyramidal geometry [54] in the polymer complexes. The in-plane covalence parameter 2(Cu) was calculated by using the following equation. 2(Cu) = (AII /0.036)+( gII -2.002)+ 3/7 (g -2.002) +0.04 The calculated values suggest that the covalent binding nature [55]. The magnetic moment of the complexes were calculated using the relation 2 = 3/4g2 [56]. It is found to be 2.0259 and 2.0118 B.M. for complexes 1 and 2, respectively. This tells that there is no interaction between Cu-Cu present in these complexes [57]. 3.2. DEGREE OF COORDINATION (x) Polymer-copper(II) complexes 1 and 2 with different degree of coordination were synthesized (Scheme 1) by adding different amount of precursor complexes with constant amount of BPEI.( Table S2). 3.3. DNA BINDING STUDIES 3.3.1. ABSORPTION SPECTRAL STUDIES

8

The binding affinity of polymer complexes with CT-DNA has been studied using various spectroscopic methods. The electronic absorption spectroscopy is one of the most useful techniques to examine the binding mode and the binding ability of metal complexes with DNA [58]. It is important to remember that when a metal complex binds to CT-DNA through a intercalation fashion, it will be reflected in absorption spectra with hypochromic and bathochromic shifts. In the intercalation, a strong stacking interaction between the aromatic chromophore of the complexes and the base pairs of DNA is observed. In general, the greater the extent of hypochromism in the UV-Vis band, stronger the intercalation interaction [59]. Groove binding of the complexes to DNA is resulted in hyperchromism in absorption intensity, indicating the unwinding of the DNA double helix as well as its unstacking and concomitant exposure of the bases [60]. The absorption spectra of the two complexes 1-2 in the absence and the presence of CT-DNA were recorded and given in Figure 1. As the DNA concentration increased, the intensity of intra-ligand (IL) transition band of complexes 1 and 2 show clear hypochromism with red shift. This indicates that both the polymercopper(II) complexes interact with DNA through intercalation mode. The extent of hypochromism and red shift for CT-DNA binding of polymer-copper(II) complex 1 and 2 with various degree of coordination are provided in the Table 1. Furthermore, the intrinsic binding constant (Kb) of complexes 1 and 2 with DNA is determined using eqn. 4. The below constructed equation 4 is used to find the intrinsic binding constants Kb of the copper complex units in the polymer. Kb P

+

Q

PQ

--------- (1)

K b = [P Q ] / [P ] [Q ]

---------- (2)



A b s =  Q [ Q ] +  P Q [P Q ]

------------ (3)

9

where, [P] is the concentration of nucleic acid sites in the base pairs, [Q] is the concentration of copper complex units of the polymer complex and [PQ] is equilibrium concentration of nucleic acid bound copper complex units.

C / A b s-  C = (1 /  Q

Q

Q

PQ-

 Q ) + 1 /(  P Q -  Q ) K b 1 / [P ] --------- (4)

where εQ and εPQ are the molar extinction coefficient of the free copper complex units and apparent molar extinction coefficient of the nucleic acid bound copper complex units respectively. CQ initial concentration of copper complex units and Abs is the experimental absorbance. As per the literature [61] Kb values are calculated (set [Q] equal to CQ and estimate of Kb and (εPQ-εQ) are obtained, then another [Q] is calculated and so on until convergence is achieved). This procedure yields binding constant value (Kb). The Kb values of the polymer-copper(II) complexes 1 and 2 with different degree of coordination are given in the Table 1. As seen from the Table 1, the Kb value increases with degree of coordination i.e., more the number of copper(II) complex units in the polymer, higher the binding constant. Cooperative effect plays a major role in the strong binding nature of these complexes through intercalation mode. It implies that polymer-copper(II) complexes at higher degree of coordination can produce more binding affinity and the extent of hypochromism with CTDNA.

Compared

to

precursor

complexes,

[Cu(ip)2(H2O)](ClO4)2

[36]

and

[Cu(dppz)2(H2O)](ClO4)2 [37] as well as other polymer-copper(II) complexes [26, 32, 33, 62] and surfactant containing metallo-intercalators, [Cu(ip)2DA](ClO4)2 and [Cu(dppz)2DA](ClO4)2 [36] the Kb values for the present complexes are high. Beside the complexes 1 and 2, the Kb value

of

the

[Cu(dppz)2(BPEI)](ClO4)2.H2O

is

much

higher

than

that

of

[Cu(ip)2(BPEI)](ClO4)2.H2O. This is due to the presence of the extended aromatic ring in dppz ligand makes deeper insertion between the base pairs of CT-DNA than the ip ligand.

Interestingly, Complex 2 anchored BPEI has shown more binding affinity towards DNA than its grafted form with graphene oxide [63]. It is important to note that, both the graphene oxide 10

1.2

a

4.59E-007

3.00E-007

1.0 2.50E-007

b

4.08E-007 3.57E-007

1.00E-007

5.00E-008

0.6

[DNA]/ (a-f)

0.8

3.06E-007

0.8

1.50E-007

ance

[DNA]/( a-f)

ance

2.00E-007

2.55E-007 2.04E-007 1.53E-007 1.02E-007

grafted metallo-intercalators and BPEI anchored metallo-intercalators are interacting through intercalation fashion with DNA. In addition to intercalation, the positive charges of BPEI produce electrostatic interaction with negative charge of CT-DNA. This synergic interaction is responsible for enhanced binding affinity of BPEI anchored metallo-intercalators.

Figure 1. (a). Absorption spectra of Complex 1 (x= 0.487): in the absence (dotted line) and in the presence (solid lines) of increasing amounts of DNA. {Inset: Plot of [DNA]/(ε a-εf) vs. [DNA]}. (b). Absorption spectra of Complex 2 (x= 0.144): in the absence (dotted line) and in the presence (solid lines) of increasing amounts of DNA. {Inset: Plot of [DNA]/(ε a-εf) vs. [DNA]} [Complex] = 2×10-4 M, [DNA] = 0 – 1.2× 10-5 M. 3.3.2. COMPETITIVE BINDING STUDIES To further gain insights into the mode of binding between polymer-copper(II) complexes and CT-DNA, competitive binding experiments are carried out. EB is a phenanthiridine fluorescence dye and typical intercalator. It’s fluorescence intensity is very weak, but in the presence of CTDNA, the emission intensity is greatly increased. This is due to the perfect fitting of planar phenanthiridine ring in to the adjacent base pairs of the double helix [64]. As seen in Figure 2, a notable decrease in emission intensities is observed upon increase in the concentration of polymer-copper(II) complexes to the EB-CT-DNA system. It is due to the intercalation of the polymer-copper(II) complexes to DNA base pairs replacing some EB molecules from the EB–CT-DNA system. The fluorescence quenching of EB bound to CT-DNA by the complexes are in good agreement with the linear Stern-Volmer equation which provides further evidence that the complexes bind to CT-DNA. In the linear plot of I0 / I vs. [CPX] / [DNA] (insert Figure 2), the Ksv values are given by the slope to intercept. The Ksv values, thus obtained for the polymer complexes with different degree of coordination are given in the Table 1.

The Ksv value increases with increase in the degree of coordination of copper complex units in the polymer, as observed in the absorption spectral studies. It confirms that the cooperative effect 300

of copper(II) complex units present in the polymer in increasing the overall binding affinity of a 2.1

1.6

1.9

250

I 0 /I

each copper(II) chelate unit to CT-DNA. 225

1.5

b

1.4

I 0 /I

1.7 1.5

200

1.3

1.3

1.2 1.1

Intensity

0.9

150

0

1

2

3

4

5

[CPX]/[DNA]

Intensity

1

1.1

0.9

11

0

150

0.2

0.4

0.6

0.8

[CPX]/[DNA]

100 75

50

0 550

600

650

wavelength, nm

700

750

550

600

650

Wavelength,nm

700

750

Figure 2. Emission spectra of EB bound to DNA: (a). in the absence of the polymer-copper(II) complex 1 (dotted line) and in the presence (solid) of the complex (x=0.487). {insert: Plot of [Complex 1] / [DNA] vs I0 / I} [DNA] = 2×10-4 M ; [complex 1] = 4×10-5 M. (b). in the absence of the polymer-copper(II) complex 2 (dotted line) and in the presence (solid) of the complex(x=0.144). {insert: Plot of [Complex 2] / [DNA] vs I0 / I}. [DNA] = 1×10-4 M ; [complex 2] = 1×10-5 M. 3.3.3. EFFECT OF IONIC STRENGTH ON THE BINDING OF POLYMERCOPPER(II)COMPLEXES WITH CT DNA DNA is an anionic polyelectrolyte. Increasing the concentration of cation can bind to the phosphate group of the DNA by electrostatic force. It leads to the formation of cation atmosphere around DNA. This shields DNA and inhibits the binding of the complexes to the DNA [65]. So decrease in fluorescence intensity or increase in relative fluorescence intensity (I0/I) upon increasing the concentration of NaCl is highly indicative of an electrostatic mode of interaction [66]. Here, I0 represents the fluorescence intensity of the DNA-EB-Complex system in absence of NaCl while I is the fluorescence intensity of the DNA-EB-Complex system in the presence of NaCl. 1.5

Relative Intensity

1.4

Complex 1 Complex 2

1.3

1.2

Figure 3. Titration of1.1DNA (2x10-4 M) - ethidium bromide (1x10-5 M) bound complex with NaCl (from 0 to 4x10-4 M), in the presence of polymer-copper(II) complex 1 (4x 10-4 M) and 1.0 complex 2 (1x 10-5) separately. 0.0000 0.0001 0.0002 out 0.0003 Hence fluorescence experiments were carried to find 0.0004 whether the polymer-copper(II)

[NaCl], M

complexes can show electrostatic interaction with DNA. The competitive binding of Na+ ions to DNA decreases the binding affinity of the polymer-copper(II) complex to DNA. Therefore the relative fluorescence intensity (I0/I) due to ethidium bromide increases on increasing the concentration of NaCl (Figure 3). It clearly indicate the electrostatic interaction between polymer-copper(II) complex and DNA. [67, 68]. 3.3.4. CYCLIC VOLTAMMETRY STUDIES The cyclic voltammetry studies provide useful information on the mode of interactions between DNA and metal complexes [69] which is complement to the UV-Visible spectroscopy and 12

fluorescence spectroscopy. Based on the electrochemical potential shift, i.e., the electrochemical potential

of

the

molecule

will

shift

positively

when

it

intercalates

into

CT-DNA and the potential will shift in the negative direction if the molecule bound to CT-DNA through electrostatic interaction [70, 71]. The typical cyclic voltammograms (CV) of complexes in the absence and presence of CT-DNA in tris-HCl buffer solutions are shown in Figure 4. The complex 1 shows a couple of waves corresponding to Cu(II)/Cu(I) with the cathodic peak and anodic peak potential are -282.5 and 27.5 mV, for complex 2 the cathodic peak and anodic peak potential are -474.5 and -10.5 mV respectively. Separation of anodic and cathodic peak potentials (∆Ep) and formal potentials (E1/2) are found to be at 310 and 127.5 mV for complex 1 and 464 and -242.5 mV for complex 2, respectively. 3.4. IN VITRO CYTOTOXICITY STUDIES OF POLYMER-COPPER(II) COMPLEXES 3.4.1. WST-1 ASSAY The cytotoxicity of the polymer complexes are tested with the MDA-MB-231 cells at two different incubation time intervals (6 and 16 h) and the result for complex 1 and 2 are given in figure 5 as a bar diagram. Our results reveal that both the polymer complexes have shown the pronounced cell death upon increasing the complex concentrations from 10 g/mL – 80 g/mL. The IC50 values of the complexes 1 and 2 at 6 h are 38 g/mL (5.6 M) and 19 g/mL (1.7 M) respectively. At 16 h, the IC50 values of the complexes 1 and 2 are 23g/mL (3.4 M) and 14 g/mL (1.2 M) respectively. The invitro results indicate that both the complexes show better activity than cisplatin. Cisplatin didn’t show any activity against MDA-MB-231 cells up to 24h incubation time [72]. On comparison, it is found that the IC50 value of complex 2 is almost two times lower than that of the complex 1 under the same experimental conditions. The effective cytotoxic potential of complex 2 could be due to many factors viz., generation of reactive oxygen species (ROS) and the consequent lipid peroxidation and/or protein oxidation and/or DNA degradation by the Cu-complexes etc.,[73-76]. Besides, the dppz ligand present in complex 2 might also be attributed

to

the

cytotoxic

effect

as

clearly

seen

in

the

literature

[77, 78] that the dppz ligand produces a strong intercalation with DNA base pairs and leads to the DNA unwinding and finally inhibit the cell division. In the present work, we have presented the results with two systems, one complex with small aromatic ring ligand (ip) another complex with higher aromatic ring ligand (dppz) than the dpq ligand. Both our current complexes of the present study were active against MDA-MB-231cells. So certainly the complex of the previous work, [Cu(dpq)2BPEI][35] which has intermediate aromatic ring size ligand (dpq) is also 13

expected be active against MDA-MB-231 cells. Hence the in vitro cytotoxicity results indicate that BPEI grafted copper(II) complexes are not only active against non metastatic breast cancer cells, MCF-7 [35] but active against metastatic breast cancer cells, MDA-MB-231 also. 3.4.2. PROPIDIUM IODIDE (PI) STAINING

The decrease in cell viability is obtained either from apoptosis or necrosis. It can be evaluated by the propidium iodide assay [79]. Necrotic cells have damaged cell membrane where as apoptotic and normal cells have proper cell membrane. Figure 6. The bar diagram of apoptotic percentage of control and treated cell by PI staining. The cells were treated with Complex 1 and 2 at three different concentrations Therefore PI excludes the apoptotic and normal cells. As PI is a fluorescent intercalating dye it binds with DNA of the necrotic cells. Then uptake of PI by necrotic cells was determined using FACS. The obtained results (Figure 6) indicate that an increase in the concentration of the complexes 1 and 2 have increased the cell death via apoptosis. Among them, complex 2 is an effective one. 3.4.3. CASPASE - 3 ACTIVITY Caspase - 3, Protease, plays a central role in apoptotic programme and the expression of this protease favours the apoptotic cell death [80]. After induction of apoptosis, the quantitative detection of caspase 3 in cellular lysates will be determined by the Caspase - 3 assay. MDA-MB-231 cells are treated with complex 1 and 2 at 50 µM concentration for 6 h The results clearly suggest that there was almost 10 fold induction in caspase-3 activity when compared to control (Figure 7). It demonstrates that both the complexes have the ability to induce cell death by apoptosis in MDA-MB-231 cells. It also indicates that complex 2 induce more apoptotic cell death than complex 1. 3.4.4. DAPI STAINING Further, the cells are stained with DAPI to investigate the apoptotic cell death by the morphological changes of the nucleus. Control cells hardly showed any sort of condensation in comparison to the treated cells. The treated cells are irregular, nuclear shrinkage, anomalies and show nuclear blebbings (Figure 8).

14

In the case of both the complexes, complex 2 produces more apoptotic morphology which is another important hallmark of apoptosis. It should be noted that the initial study of the above in vitro cytotoxicity experiment is to explore the anticancer activity of the present polymer copper(II) complexes. whilst more pharmacological studies are still needed to fully understand the molecular mechanism of cytotoxicity.

4. CONCLUSIONS Two new water soluble and modified phenanthroline ligand containing polymer-copper(II) complexes with different degree of coordination have been synthesized and characterized by various physicochemical methods. The interactions of these complexes with CT-DNA have been studied by using various spectroscopic methods. All these studies have revealed that both the complexes bind to CT-DNA through intercalation mode and show π-π stacking interactions. Interestingly, the extended planar dppz containing complex 2 has shown more binding affinity with CT-DNA. These square pyramidal polymer-copper(II) complexes are found to have ability of showing greater - stacking with CT-DNA than our previously reported cobalt based polymer complexes. The investigation of cytotoxicity against metastatic breast cancer cells, MDA-MB- 231 shows that complex 2 is more effective than complex 1. The better IC50 value of complex 2 is due to the hydrophobic ligand (dppz). The BPEI increase the cellular uptake of the copper(II) complexes and dppz act as an effective DNA intercalator. The decrease in the cell viability via apoptosis has been confirmed by various assays. The morphological change of the cell clearly indicate the apoptotic cell death which has been determined by DAPI staining. The degree of coordination of polymer-copper(II) complexes directly influences the CT-DNA binding affinity. This tells that more number of metallo-intercalators unit in the polymer backbone

will

lead

to

more

binding

affinity

with

CT-DNA. Based on these results, we have concluded that the geometry of the metal complex on the polymeric back bone and the degree of coordination play vital role in CT-DNA binding. Therefore, considerable interest has to be devoted to the geometry and choice of ligand of the metal complexes while designing the polymer metal complexes for anticancer drug development. Hence, these encouraging results made us to investigate the complex containing dppz ligand for further development of DNA molecular probes and new therapeutic drugs. ACKNOWLEDGEMENTS

15

Y.M acknowledges the UGC-BSR-RFSMS for financial support through Senior Research Fellowship and S.A acknowledges the UGC -RFSMS for the financial support in the form of Research Fellowship.The authors are grateful to the UGC-SAP & COSIST and DST-FIST programmes of the Department of Chemistry, Bharathidasan University. Corresponding author thanks the sanction of research schemes from funding agencies; CSIR [Grant no. 01(2461)/11/EMR-II] and UGC [Grant no. 41-223/2012(SR)]. We sincerely thank Prof. John E. Eriksson, Department of Biosciences, Cell biology, Åbo Akademi University, Turku, Finland for anticancer studies. AUTHOR INFORMATION Yesaiyan Manojkumar and Subramanian Ambika are contributed equally. Sankaralingam Arunachalam is the corresponding author. REFERENCE [1] C. Lipinski, Am Pharm Rev, 5 (2002) 82-85. [2] L.R. Kelland, Expert Opinion on Investigational Drugs, 9 (2000) 1373-1382. [3] N. Larson, H. Ghandehari, Chemistry of Materials, 24 (2012) 840-853. [4] M. Callari, J.R. Aldrich-Wright, P.L. de Souza, M.H. Stenzel, Prog. Polym. Sci., 39 (2014) 1614-1643. [5] F.M. Veronese, O. Schiavon, G. Pasut, R. Mendichi, L. Andersson, A. Tsirk, J. Ford, G. Wu, S. Kneller, J. Davies, Bioconjugate chemistry, 16 (2005) 775-784. [6] S.K. Samal, M. Dash, S. Van Vlierberghe, D.L. Kaplan, E. Chiellini, C. Van Blitterswijk, L. Moroni, P. Dubruel, Chem. Soc. Rev., 41 (2012) 7147-7194. [7] X. Pang, H.-L. Du, H.-Q. Zhang, Y.-J. Zhai, G.-X. Zhai, Drug Discov.Today, 18 (2013) 1316-1322. [8] J. Yang, J. Kopeček, J. Controlled Release, 190 (2014) 288-303. [9] U. Lungwitz, M. Breunig, T. Blunk, A. Göpferich, Eur. J. Pharm. Biopharm., 60 (2005) 247-266. [10] D.W. Dong, S.W. Tong, X.R. Qi, J.Biomed.Mater.Res. A, 101 (2013) 1336-1344. [11] M.A. Mintzer, E.E. Simanek, Chem. Rev., 109 (2008) 259-302. [12] S.P.Y. Li, T.S.M. Tang, K.S.M. Yiu, K.K.W. Lo, Chem. Eur. J., 18 (2012) 13342-13354. [13] R.S. Kumar, V. Periasamy, C.P. Paul, A. Riyasdeen, S. Arunachalam, M. Akbarsha, Med. Chem. Res., 20 (2011) 726-731. [14] J. Lakshmipraba, S. Arunachalam, D.A. Gandi, T. Thirunalasundari, Eur. J. Med. Chem., 46 (2011) 3013-3021. [15] S. Nehru, S. Arunachalam, R. Arun, K. Premkumar, J. Biomol. Struct. Dyn., 32 (2014) 1876-1888. [16] C. Duncan, A.R. White, Metallomics, 4 (2012) 127-138. [17] D. Denoyer, S. Masaldan, S. La Fontaine, M.A. Cater, Metallomics, (2015). [18] R.A. Festa, D.J. Thiele, Curr. Biol., 21 (2011) R877-R883. [19] F. Bacher, O. Dömötör, A. Chugunova, N.V. Nagy, L. Filipović, S. Radulović, É.A. Enyedy, V.B. Arion, Dalton Trans., 44 (2015) 9071-9090. [20] A.K. Boal, A.C. Rosenzweig, Chem. Rev., 109 (2009) 4760-4779. [21] G. Crisponi, V.M. Nurchi, D. Fanni, C. Gerosa, S. Nemolato, G. Faa, Coord. Chem. Rev., 254 (2010) 876-889. [22] J.L. Burkhead, L.W. Gray, S. Lutsenko, Biometals, 24 (2011) 455-466. 16

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Scheme 1. Synthesis of polymer-copper(II) complexes, x is the degree of coordination [average monomer unit is considered as (~NH–CH2–CH2~)].

25

20

20



b

a

15

=

15

10

I,A

I, A

10 5

Figure 4. (a). CV spectra of complex 1 (x=0.487) in the absence (black dotted line) and in the Complex 2 presence (red line) of DNA. (b). CV Complex spectra of 1complex 2 (x=0.144) in the absence (black dotted line) and in the presence (red line) of DNA. [complex] = 1×10-3 M; [DNA] = 0-3×10-4 M. 5

0

0

-5

-10

-5

-15

-10

400

200

0

-200

-400

-600

-800

600

E, mV

400

200

0

-200

E, mV

18

-400

-600

-800

Figure 5. The cell viability of MDA-MB-231 cells. Cells were cultured in 96 well plate for 24h and then treated with polymer-copper(II) complexes 1 and 2 for 6h and 16h.

Control

CPX 1

Figure 7. Effect of polymer-copper(II) complexes, [CPX 1] and [CPX 2] = 50 µM on caspase - 3 activation. (A) Representative dot plot and (B) Bar diagram depicting % of apoptotic A A cell death. CPX 2

Figure 8. Apoptotic morphological changes of MDA-MB-231 cells with polymer complexes 1 A

B

B C A and 2. (A) cells treated with complex 1, (B) cells treated with complex 2 and (C) untreated control

cells.

Table 1. The binding constant (Kb), percentage of hypochromism (H%) and stern-volmer constant (Ksv) of the polymer-copper(II) complexes 1 and 2 with CT-DNA.

Table 2. Electrochemical parameters for the complex 1 and 2 with CT-DNA.

Complexes

Complex 1

Complex 2

Degree of coordination (x) 0.084 0.231 0.487

∆max (nm)

Hypochromism (H%)

Kb (M-1)

Ksv

4 6 10

15.75 21.75 24.56

1.22×105 8.76×105 1.04×106

0.1778 0.2228 0.2674

0.102 0.119 0.144

20 23 25

19.81 24.40 28.36

1.52×106 2.24×106 5.04×106

0.7644 0.9765 1.1834

19

Complex

Epa

∆Ep

(mV)

(mV)

-282.5

27.5

310

-127.5

-252.5

30.5

222

-111.0

-474.5

-10.5

464

-242.5

-458.5

-4.5

454

-231.5

Epc (mV)

Complex 1 Complex 1+DNA Complex 2 Complex 2+DNA

E1/2 (mV)

It suggests that both the complexes show quasi reversible one electron process. After the addition of DNA, no new peaks appeared in the cyclic voltammograms. However, the current intensity decreased in the cathodic peak in both the complexes. In presence of CT-DNA, the cathodic and anodic peak potentials show positive shifts which indicate that both the complexes bind to CTDNA through intercalation (Table 2).

20