Nucleic acid binding study of surfactant copper(II) complex containing dipyrido[3,2-a:2′-3′-c]phenazine ligand as an intercalator: in vitro antitumor activity of complex in human liver carcinoma (HepG2) cancer cells

Abstract

A new surfactant copper(II) complex, [Cu(dppz)₂DA](ClO₄)₂, where dppz = dipyrido[3,2-a:2′-3′-c]phenazine and DA-dodecylamine, has been synthesized and characterized by physico-chemical and spectroscopic methods. The critical micelle concentration (CMC) value of this surfactant copper(II) complex in aqueous solution was determined from conductance measurements. Specific conductivity data at different temperatures was obtained for the evaluation of the temperature-dependent CMC and the thermodynamics of micellization (ΔG⁰_m, ΔH⁰_m and ΔS⁰_m). The binding interaction of this complex with nucleic acids (calf thymus DNA and yeast t-RNA) was investigated using electronic absorption, fluorescence spectroscopy, viscometry, cyclic voltammetry (CV) and thermal denaturation studies. In the presence of the nucleic acids, the UV-Vis spectrum of our complex exhibited a redshift of the absorption band at 268 nm along with significant hypochromicity, indicating intercalation of our complex with the nucleic acids. The intrinsic binding constant values are K_b = 1.1 × 10⁶ M⁻¹ for DNA and 1.6 × 10⁶ M⁻¹ for RNA. The viscosity measurements confirmed that the complex–nucleic acid interaction occurs through intercalation. A competitive binding study with ethidium bromide (EB) showed that the complex exhibits the ability to displace the nucleic acid-bound EB, indicating that the complex binds to nucleic acids in strong competition with EB for the intercalative binding site. CV results also confirmed this mode of binding. Some significant thermodynamic parameters of the binding of the titled complex to DNA were also determined. The antimicrobial and antifungal screening tests of this complex have shown good results. The copper(II) complex exhibited pronounced activity against a human liver carcinoma (HepG2) cancer cell line.

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1. Introduction

In the past two decades, extensive studies on DNA and DNA site-specific cleavage have been conducted.¹ Several biological studies have proven that DNA is the major intracellular target of certain anticancer drugs. Small molecules interact with DNA and may damage the DNA in cancer cells, which halts cell division and finally results in cell death.² Because of this, there are efforts that stem from the search for an understanding of drug–nucleic acid interactions on a molecular level, and based on them, to develop novel chemotherapeutics and diagnostic agents.³ There are several types of sites in the DNA molecule where binding of small molecules can occur among base pairs,⁴ groove, and helix.⁵ Among the small molecules studied, transition metal complexes are very important for detecting possible next generation drugs. A number of useful applications of transition metal complexes require that the complex binds to DNA via an intercalative mode with the ligand intercalating into the DNA base pairs. Because of the uncommon binding properties and general photoactivity, these coordination compounds are probably used as DNA secondary structure probes,⁵ photocleavers, and antitumor drugs.⁶ In contrast to DNA, very little attention has been paid to the binding properties of transition metal complexes with RNA. These metal complexes have been used as catalysts of RNA hydrolysis cleavage,⁷ shape-selective probes of RNA tertiary structure, and agents of RNA oxidation cleavage and mismatch approval.⁸ But only a few reports have investigated the interaction between transition metal complexes and RNA.⁹

The ligand (dppz = dipyrido[3,2-a:2′-3′-c]phenazine) (dppz) is a familiar heterocyclic aromatic compound, which has been principally used in synthetic inorganic chemistry in the arrangement of supramolecular motifs.¹⁰ This ligand can be synthetically modified to produce different derivatives that have also been reported in detail in the literature.¹¹ This class of ligands containing multiple pyridine rings is commonly known as ‘polypyridines’ and has been used in coordination chemistry on a large scale. These transition metal-dppz compounds have been under extensive investigation due to their differential electrochemical, optical, and photophysical properties.¹² Transition metal-dppz interacts with different biological substrates either by intercalation or by coordinative binding.¹³ Multidirectional applications include use as antitumor agents, protein probing agents, and radiotherapeutics.¹⁴

Copper is found in all living organisms and is a resolvable trace element in redox chemistry, growth, and development.¹⁵ It is a biologically required element, and many enzymes that depend on copper for their activity have been identified. Because of its biological relevance, a large number of copper(II) complexes have been synthesized and researched to determine their biological activities.¹⁶ Among the copper complexes, attention has been mainly focused on the copper(II) complexes with modified phenanthroline ligands due to their high nucleolytic efficiency, antitumor, and antimicrobial activities.¹⁷ The number of publications based on phenanthroline and modified phenanthroline-containing coordination compounds has been extensively growing in the last decade. Among a detailed study of articles, only a very limited number of studies deal with the biological aspects of these groups of ligands or compounds. Some derivatives of modified phenanthroline and their respective coordination compounds with copper have been synthesized, but DNA interaction studies or cell viability assays have not yet been performed.

In our laboratory, we have been focusing on the design, development, and interaction of surfactant metal complexes with nucleic acids.^18,19 Surfactant metal complexes with chelating ligands are attractive for metallobiomolecules that can be used with the appropriate systems for binding and activating simple molecules, catalysis, and magnetic interactions.²⁰ In these surfactants, the metal complex containing the central metal ion with its primary coordination sphere acts as the head group, and the hydrophobic part of one or more ligands acts as the tail. Like any other surfactant, these surfactant metal complexes form micelles. The critical micelle concentration (CMC) is the concentration of the surfactant above which a surfactant aggregates into micelles. Thus, the CMC represents a phase separation between single molecules of surfactant and surfactant aggregates in dynamic equilibrium.²¹ Understanding micelle behavior in biological systems is important because the state of aggregation or micelle phase of naturally occurring molecules, drug molecules, or added surfactant may influence the biological effects.^22,23 We have recently established that complexes of [Co(diimine) (DA)₂](ClO₄)₃ [diimine = bipyridine (bpy), 1,10-phenanthroline (phen), etc.] interact with DNA through their long chains. In the present investigation, the complex [Cu(dppz)₂(DA)]²⁺ containing ligand with extended aromaticity and a long aliphatic chain has been prepared, and its interaction with calf thymus (CT) DNA and yeast tRNA was studied by UV-visible absorption, emission spectroscopy, viscosity, thermal denaturation, and cyclic voltammetric methods. The antimicrobial and antibacterial characteristics of this surfactant copper(II) complex were also tested against Gram +ve and Gram −ve bacteria and fungus. In addition, the anticancer activity of this complex has been tested against human liver cancer cells (HepG2).

2. Experimental

2.1. Materials and methods

All of the reagents used in the preparation of ligand and its metal complex were of reagent grade (Sigma). The solvents used in the synthesis of ligand and metal complex were distilled before use. Calf thymus DNA (CT-DNA), dodecylamine (DA), and ethidium bromide (EB) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). All of the experiments involving interaction of the surfactant complex with nucleic acids were carried out in buffer containing 5 mM Tris and 50 mM NaCl and adjusted to pH 7.2 with hydrochloric acid. A solution of CT-DNA gave a ratio of UV absorbance at 260 and 280 nm of approximately 1.8–1.9, indicating that the CT-DNA was sufficiently free of protein.²⁴ Ligands were prepared by the Schiff-base condensation of 1,10-phenanthroline-5,6-dione with the appropriate diamino compound in ethanol under reflux.²⁵ The dione was prepared by oxidation of 1,10-phenanthroline following the method of Gillard et al.²⁶

The elemental analyses (C, H, and N) of the samples were determined at SAIF, Cochin University, Cochin, Kerala. The electronic spectra were recorded on a Shimadzu UV-3101PC spectrophotometer using cuvettes of 1 cm path length, and emission spectra were recorded on a JASCO FP 770 spectrofluorimeter. Conductivity measurements were carried out in aqueous solutions of the complex with an Elico conductivity bridge type CM 82 and a dip-type cell with a cell constant of 1.0. FTIR spectra were recorded on a FTIR JASCO 460 PLUS spectrophotometer with samples prepared as KBr pellets. EPR spectra were recorded on a JEOL-FA200 EPR spectrometer at room temperature and at liquid nitrogen temperature (LNT) in methanol solution.

2.2. Preparation of [Cu(dppz)₂(Cl)]Cl

The precursor copper(II) complex, [Cu(dppz)₂(Cl)]Cl, was prepared using a similar method to that described in the literature.²⁷ The complex was prepared by reacting CuCl₂·2H₂O (0.17 g; 1.0 mmol) with dppz (0.74 g, 2.5 mmol) in methanol (50 cm³). The solution was stirred at 45 °C for 2 h followed by cooling to ambient temperature. The solid product thus obtained was isolated, washed with ethanol and diethyl ether, and finally dried in a vacuum desiccator.

2.3. Synthesis of surfactant copper(II) complex

To a solution of [Cu(dppz)₂(Cl)]Cl in 15 cm³ of water, slightly more than the calculated amount of dodecyl amine in 3 cm³ of ethanol was added dropwise over a period of 30 min. The green solution gradually became red, and the mixture was set aside at 40 °C for 2 days until no further change in colour was observed. Afterwards, a saturated solution of sodium perchlorate in very dilute perchloric acid was added. Slowly, the complex [Cu(dppz)₂(DA)](ClO₄)₂ was separated as a pasty solid mass and was filtered off, washed with small amounts of alcohol and acetone, and then dried over air. The semidried solid was further dried in a drying pistol over fused calcium chloride and stored in a vacuum desiccator. The structure of [Cu(dppz)₂(DA)](ClO₄)₂ is shown in Scheme 1.

Scheme 1 Structure of the surfactant copper(II) complex.

2.4. CMC determination

The conductivity measurements of the appropriate concentration range of the surfactant copper(II) complex were obtained by using a continuous dilution of a concentrated solution into water. The conductivity of these solutions was measured at 303, 308, 313, 318, and 323 K. The conductivity was recorded when its fluctuation was less than 1% within 2 min. After each addition, the solution was mixed carefully without the formation of foam. The breakpoint in the plot of specific conductance versus surfactant concentration was taken as the CMC.

2.5. Nucleic acid binding experiments

The DNA/RNA binding experiments were performed at 25.0 ± 0.2 °C. The nucleic acid concentration per nucleotide was determined by UV-visible spectroscopy using the known molar extinction coefficient value of 6600 M⁻¹ cm⁻¹ and 9250 M⁻¹ cm⁻¹ for DNA and RNA at 260 nm, respectively.²⁸ The absorption titration was performed with a fixed concentration of the copper(II) surfactant complex to which an increasing concentration of the DNA/RNA stock solution was added to both the complex solution and reference solution to eliminate the absorbance of nucleic acids.

Thermal denaturation studies were carried out on a JASCO V530 spectrophotometer using cuvettes of 1 cm path length that contained buffer with a JULABO F32H Peltier system (±0.1 °C). With the use of the thermal melting program, the temperature of the cell containing the cuvette was ramped from 40 to 110 °C. The absorbance at 260 nm was monitored every 1 °C for solutions of CT-DNA (80 μM) in the absence and presence of the title complex at different concentrations. The melting temperature T_m, which is defined as the temperature where half of the total base pairs is unbounded, was determined from the midpoint of the melting curves. ΔT_m values were calculated by subtracting the T_m of the DNA alone from that of DNA–complex adduct.

Cyclic voltammetry measurements were made on a Princeton EG&G-PARC model potentiostat. The supporting electrolyte consisted of 50 mM NaCl/10 mM Tris HCl buffer (pH = 7.2). A standard three-electrode system was used containing a glassy carbon working electrode, a platinum wire auxiliary electrode, and a saturated calomel reference electrode (SCE). Solutions were deoxygenated by purging with N₂ prior to measurements.

Ethidium bromide emits intense fluorescence in the presence of DNA/RNA due to its strong intercalation between adjacent DNA/RNA base pairs. It has been previously reported that this fluorescence can be quenched by the addition of a second molecule.²⁹ The extent of fluorescence quenching of EB bound to nucleic acids can be used to determine the extent of binding between the second molecule and nucleic acids. These competitive binding experiments were used to find out the extent of binding of the surfactant copper(II) complex with nucleic acids. The fluorescence spectra of EB were measured using an excitation wavelength of 520 nm, and the emission range was set between 550 and 750 nm.

Viscosity experiments were carried out on a Ubbelohde viscometer that was immersed in a thermostatic water bath maintained at 30 ± 0.1 °C. The flow time was recorded three times for each sample, and an average flow time was calculated. The data are presented as (η/η₀)^1/3 versus the ratio of the concentration of the complex to nucleic acid, where η is the viscosity of nucleic acid in the presence of complex, and η₀ is the viscosity of nucleic acid alone. Viscosity values were calculated from the observed flow time of nucleic acid-containing solutions (t > 100 s) corrected for the flow time of the buffer alone (t₀), η = (t – t₀)/t₀.³⁰

2.6. Cytotoxicity assay

The cytotoxicity of the surfactant copper(II) complex was measured using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) dye assay as previously described.³¹ The complex was first dissolved quantitatively in dimethyl sulfoxide (DMSO, Sigma) to make the stock solution. Briefly, HepG2 liver cancer cells were seeded at a density of 5 × 10⁴ cells per well into 96-well plates. After 24 h, the cells were treated with surfactant copper(II) complex at various concentrations (10, 30, 60, 90 μg mL⁻¹) and incubated for 24 and 48 hours as indicated. At the end of the incubation, 10 μl of MTT (5 mg mL⁻¹) per well was added and the plates were incubated in the dark at 37 °C for 4 hours. The formazan crystals formed after 4 hours were solubilized in 100 μl of DMSO after aspirating the medium. The absorbance was monitored at 570 nm (measurement) and 630 nm using a 96-well plate reader (Bio-Rad, Hercules, CA, USA). The IC₅₀ value was defined as the concentration of compound that produced a 50% reduction of cell viability.

2.6.1. Evaluation of apoptosis using acridine orange and ethidium bromide staining. Acridine orange (AO) and ethidium bromide staining was performed as described by Spector et al.³² Twenty-five microliters of cell suspension of each sample (both attached, released by trypsinization, and floating), containing 5 × 10⁵ cells, was treated with AO and EB solution (one part of 100 mg mL⁻¹ AO and one part of 100 mg mL⁻¹ EO in PBS) and examined under a fluorescent microscope (Carl Zeiss, Germany) using a UV filter (450–490 nm). Three hundred cells per sample were counted in tetraplicates for each dose point. Cells were scored as viable, apoptotic, or necrotic as judged by the staining, nuclear morphology, and membrane integrity, and percentages of apoptotic and necrotic cells were then calculated. Morphological changes were also observed and photographed.

2.6.2. Dye preparation and drug preparation. For cell staining, 200 μL of dye mixture (100 μL mg⁻¹ AO and 100 μL mg⁻¹ EB⁻¹ in distilled water) was mixed with 2 mL of cell suspension (30 000 cells per mL) in a 6-well plate. The suspension was immediately examined and viewed under an Olympus inverted fluorescence microscope (Ti-Eclipse) at 200× and 400× magnification. We observed untreated cells as controls and cells treated with testing material IC₅₀ concentrations for 24 h of exposure.

2.6.3. Drug treatments. HepG2 cells were seeded in a 24-well plate at 50 000 cells per well. After 24 h of incubation, the medium was replaced with 100 μL of medium containing an IC₅₀ dose of the testing material. Untreated cells served as the control. After 24 h, the medium was aspirated, and the cells were treated with the prepared dye and observed under a fluorescent microscope.

2.6.4. Hoechst 33342 staining. Staining with Hoechst 33342 dye was optimized with cells at 1–2 × 10⁶ mL, in buffered media, pH 7.2; 2% fetal calf serum was added to maintain the cells. The copper(II) complex was added and the cells were incubated for 24 and 48 hours. Spent medium was removed by aspiration, and 1 mL of saline was added; then the cells were centrifuged at 1500 rpm for 10 min. After removal of the saline supernatant, the cells were stained with 0.5 mL of dye solution (3.5 μg mL⁻¹ in PBS) and incubated for 30 min in a 37 °C incubator. After 30 min, the Hoechst 33342 solution was discarded and the cells were observed for apoptosis at 490–520 nm under a fluorescent microscope. Time is a critical factor due to the transport of the dye, with samples being observed 30 minutes after staining because the signal begins to degrade after approximately 120 minutes. The staining kinetics were empirically defined.

3. Results and discussion

3.1. Characterization

The surfactant copper(II) complex synthesized in the present study was characterized by UV-visible, IR, and EPR spectral techniques. The purity of the complex was checked by microanalyses (C, H, and N), and the results were found to be in good agreement with the calculated values (found: C, 56.59%; H, 4.57%; N, 12.27% C₄₈H₄₇Cl₂CuN₉O₈; calcd for: C, 56.95%; H, 4.68%; N, 12.45%). The electronic absorption spectra are often very helpful in the evaluation of results furnished by other methods of structural investigation. The electronic spectral measurements were used to assign the stereochemistries of the metal ions in the complexes based on the positions and number of d-d transition peaks. The electronic absorption spectrum of the complex was recorded at room temperature. The spectrum shows multiple transitions in the ultraviolet region due to intraligand transitions at 362 nm and 382 nm caused by metal to ligand charge transfer transitions (MLCT). This type of MLCT band at approximately 350 nm has been reported for copper complexes of phenanthroline.³³ A broad band was observed at the lower frequency of approximately 648 nm in the spectrum, corresponding to the d–d transitions of copper(II) and indicating square pyramidal geometry around copper(II).^34,35 Similar observations were observed by Pradeep et al.³⁵

In the IR region, our surfactant copper(II) complex showed bands at approximately 1586 cm⁻¹, 1351 cm⁻¹, and 3392 cm⁻¹, which can be attributed to the ring stretching frequencies [γ(C=C), γ(C=N) and γ(N–H)] of the dipyrido[3,2-a:2′-3′-c]phenazine ligand whose values in the free state are 1594 cm⁻¹, 1383 cm⁻¹, and 3483 cm⁻¹, respectively. These shifts can be explained by the fact that each of the two nitrogen atoms of dipyrido[3,2-a:2′-3′-c]phenazine ligands donates a pair of electrons to the central copper metal, thus forming a coordinate covalent bond. The other bands observed for this complex at approximately 2920 cm⁻¹ and 2850 cm⁻¹ can be assigned to C–H asymmetric and symmetric stretching vibrations of aliphatic CH₂ of dodecylamine. The perchlorate bands that appear at approximately 1100 and 620 belong to an ionic species, which indicates that the counter-ion is not involved in the copper–ligand coordination.^36,37 The solid state EPR spectra of our surfactant copper(II) complex were recorded in X-band frequencies at room temperature (ESI Fig. 1†) as well as in frozen solution (77 K). The spectral features at both temperatures are quite similar. The complex exhibits well-defined single isotropic features near g = 2.09 in the solid state at RT as well as at LNT (ESI Fig. 2†), revealing that such isotropic lines are usually the results of intermolecular spin exchange, which broaden the lines, suggesting that dx² − dy² is the ground state with the d⁹ (Cu²⁺) configuration and square pyramidal geometry.

3.2. Critical micelle concentration (CMC)

The CMC value of our surfactant copper(II) complex was determined at five different temperatures (303, 308, 313, 318, and 323 K) using a conductivity method that was previously reported.³⁸ Determination of the CMC value from the conductivity measurements was carried out through a change in the slope when the specific conductivity versus surfactant concentration for surfactant solutions was plotted. These conductivity measurements (ESI Fig. 3†) were repeated three times, and the accuracy of the CMC values (ESI Table 1†) was found to be within ± 2%. Similar to our previous reports,^18,38 the CMC value for the copper(II) surfactant complex of the present study is also very low compared to that of the simple organic surfactant, dodecylammonium chloride (CMC = 1.59 × 10⁻² mol dm⁻³), indicating that our surfactant metal complex has a greater capacity to associate into micellar aggregates. In addition, the CMC value of our complex containing modified phenanthroline ligand is lower than that of the corresponding phenanthroline coordinated complex (9.75 × 10⁻⁵).³⁸ This is expected, as the dppz ligand is more hydrophobic than phenanthroline ligand, which facilitates higher micellization.

3.3. Thermodynamics of micelle formation

Various thermodynamic quantities such as the free energy (ΔS⁰_m), the enthalpy (ΔH⁰_m), and the entropy (ΔS⁰_m) of micellization were obtained using the following relationships and the temperature dependence of CMC fitted equations:

ΔG⁰_m = RT ln CMCΔH⁰_m = −RTdln CMC/dTΔS⁰_m = (ΔH⁰_m − ΔG⁰_m)/T

The standard free energy of micelle formation per mole of monomer, ΔG⁰_m, is given by

ΔG⁰_m = RT(2 − α_ave)ln CMC, (1)

where R, T, and α_ave are gas constants, absolute temperature, and average degree of micellar ionization, respectively.

The enthalpy and entropy of micelle formation can be obtained by applying the Gibbs–Helmholtz equation to eqn (1)

ΔH⁰_m = −RT²(2 − α_ave)dln CMC/dT (2)

ΔS⁰_m = (ΔH⁰_m − ΔG⁰_m)/T (3)

The thermodynamic parameters thus obtained for the surfactant copper(II) complex are shown in ESI Table 1.† As seen from the table, the Gibbs free energy of micellization is found to be negative, which indicates that the micellization was spontaneous. A linear correlation between the enthalpy and entropy of micellization was observed for this surfactant copper(II) complex, as shown in ESI Fig. 4.† As the temperature is increased, the enthalpy contribution to the free energy increased, whereas the entropic contribution decreased. Further ΔH⁰_m of micellization is negative, and ΔS⁰_m of micellization is positive. Nusselder and Engberts³⁹ have suggested that negative ΔH⁰_m values will indicate London dispersion forces (LDFs) as a major force in the micelle formation. Positive values for ΔS⁰_m indicate that the micellization of the surfactant complex in aqueous solution is governed mainly by hydrophobic interactions between the surfactant cations, resulting in the breakdown of the structured water surrounding the hydrophobic groups.

3.4. Binding studies with nucleic acids

3.4.1. Absorption studies. The binding of intercalated complexes to the DNA helix can be characterized through absorption spectral titration by following the changes in absorbance and wavelength. Due to the intercalative mode involving a strong stacking interaction between the aromatic chromophore and the DNA base pairs, hypochromism along with a redshift can be observed. The extent of the hypochromism is commonly consistent with the strength of the intercalative interaction.^40,41 Thus, in order to provide evidence for the intercalative binding of surfactant copper(II) complex to DNA, the binding process was monitored using absorption spectroscopy by following the changes in the absorption band intensity and its position. The absorption spectra of the complex in the absence and presence of nucleic acids are shown in Fig. 1 and 2, which indicates that with the increase in the concentration of CT-DNA or yeast tRNA, the absorption spectrum of the surfactant copper(II) complex showed strong hypochromism (H% = 100% (A_free − A_bound)/A_free) in the absorbance bands, denoting a strong stacking interaction (intercalation) between the aromatic chromophore and the base pairs of the nucleic acid. With DNA binding, the hypochromism reaches as high as 29.16% with a slight redshift, and with yeast tRNA binding, the hypochromism reached as high as 38.77% with a slight redshift. It has been previously suggested that hypochromism is due to a strong interaction between the extended aromaticity of the ligand and that of the DNA bases.^42,43 This affinity is basically consistent with data found for phenanthroline-based complexes of copper.¹⁹

Fig. 1 Absorption spectra of the surfactant complex in the absence (dotted lines) and in the presence of increasing amounts of CT-DNA (solid lines), [Complex] = 3.0 × 10⁻⁵ M, [DNA] = 0–5.5 × 10⁻⁶ M. The arrow shows the absorbance changes upon increasing DNA concentrations. Inset: plot of [DNA]/(ε_a − ε_f) versus [DNA].

Fig. 2 Absorption spectra of complex in the absence (dotted lines) and in the presence of increasing amounts of RNA (solid lines), [Complex] = 3.0 × 10⁻⁵ M, [RNA] = 0–7.4 × 10⁻⁶ M. The arrow shows the absorbance changes upon increasing RNA concentrations. Inset: plot of [RNA]/(ε_a − ε_f) versus [RNA].

Our results may suggest that the ligand, dppz, structurally provides one aromatic moiety extending from the metal center to overlap with the DNA base pairs by intercalation. The long aliphatic chain present in the surfactant copper(II) complex enhances this intercalation with the base pairs of DNA through a strong hydrophobic effect. The observed spectroscopic changes are thus consistent with the intercalation of complex into the DNA base stacks. From the absorption titration data, the binding constant (K_b) was determined using the following equation (eqn (4)),²⁸

[NA]/(ε_a − ε_f) = [NA]/(ε_b − ε_f) + 1/K_b(ε_b − ε_f) (4)

where [NA] is the concentration of nucleic acid expressed in base pairs, and ε_a, ε_f, and ε_b are the apparent, free, and fully bound copper(II) complex extinction coefficients, respectively. In plots of [NA]/(ε_a − ε_f) versus [NA], K_b is given by the ratio of the slope to intercept. The K_b values thus obtained for the surfactant copper(II) complex with CT-DNA and yeast tRNA are given in Table 1. The large hypochromism and K_b values observed in the electronic absorption titration experiments of the surfactant copper(II) complex with yeast tRNA binding compared to CT-DNA indicate a strong stacking interaction between the aromatic chromophore (dppz) and the base pairs of RNA. It should be noted that the binding constant of the surfactant complex of the present study is higher than that of the surfactant complexes containing bipyridine, phenanthroline, and dpq ligands reported by us earlier.^18,19 Compared to these ligands, the dppz ligand can provide more aromaticity by extending from the metal center to overlap with the DNA base pairs via intercalation. Additionally, the K_b of our surfactant copper(II) complex is much higher than that for the ordinary copper(II)/cobalt(III) complexes, such as [Cu(dppz)₂(Cl)]Cl,²⁷ [Co(bpy)₃]³⁺, (K_b, 9.3 × 10³ M⁻¹),⁴⁴ [Co(bpy)₂(imp)]³⁺, and (K_b, 1.1 × 10⁴ M⁻¹).⁴⁵ This indicates that the long aliphatic chain amine ligands present in the surfactant copper(II) complex play a definite role in the intercalative binding between the surfactant copper(II) complex and nucleic acids through their hydrophobic effect.

Table 1 The binding constant (K_b) and K_SV of [Cu(dppz)₂DA](ClO₄)₂ with nucleic acid using Tris buffer

Complex	Kb (M^−1)		% Hypochromism		Ksv M^−1
	DNA	RNA	DNA	RNA	DNA	RNA
[Cu(dppz)2DA] (ClO4)2	1.1 × 10^6 ± 0.17	1.6 × 10^6 ± 0.26	29.16	38.77	7.88 × 10^3	1.13 × 10^4

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3.4.2. Thermal denaturation studies. DNA melting experiments are useful for establishing the extent of intercalation because the intercalation of the complex into DNA base pairs causes stabilization of base stacking and therefore raises the melting temperature of the double-stranded DNA.⁴⁶ It is well-accepted that when the temperature of the solution increases, double-stranded DNA gradually dissociates to single strands, generating a hyperchromic effect in the absorption spectra of the DNA bases (λ_max = 260 nm). Therefore, the transition temperature of double strands to single strands can be determined by monitoring the absorbance of the DNA bases at 260 nm as a function of temperature.⁴⁷ Previous research⁴⁸ demonstrates that the intercalation of complexes into DNA generally results in a considerable increase of T_m. The melting curves of CT-DNA in the absence and presence of [Cu(dppz)₂DA](ClO₄)₂ are presented in ESI Fig. 4.† A melting temperature (T⁰_m) of CT-DNA in buffer was determined as 74.0 ± 0.2 °C under our experimental conditions. The DNA intrinsic binding constant of the title complex at T_m was calculated using McGhee's equation (eqn (5)),^49,2 where T⁰_m is the melting temperature of CT-DNA alone, T_m is the melting temperature in the presence of the Cu(II) complex, ΔH_m is the enthalpy of DNA (per base pair), R is the gas constant, K is the DNA binding constant at T_m, L is the free complex concentration (approximated by the total complex concentration) at T_m, and n is the size of the binding site. For the CT-DNA used in these studies, under identical solution conditions, a melting enthalpy of 6.9 kcal mol⁻¹ was determined by differential scanning calorimetry.² On the basis of the neighbor-exclusion principle, the value of n for the title complex was assumed to be 2.0 bp.⁵⁰

1/T⁰_m − 1/T_m = (R/ΔH_m)[ln(1 + KL)]^1/n (5)

By substitution of the required parameters into eqn (5), K was determined to be 6.68 × 10⁵ M⁻¹ for the title complex at 85 °C.

3.4.3. Thermodynamic parameters. Only a few thermodynamic parameters such as free energy, enthalpy, and entropy changes upon binding of metal complexes to DNA have been measured, although there have been many reports on the interaction of metal complexes with DNA. It is essential to obtain the thermodynamic parameters of DNA-complex formation for a thorough understanding of the driving forces behind the binding of metal complexes to DNA.⁵¹ The change in standard enthalpy was determined according to the van't Hoff's equation (eqn (6)). The changes in standard free energy and standard entropy of the binding of the title complex to DNA were determined according to eqn (7) and (8), where K₁ and K₂ are the DNA binding constants of the complex at temperatures T₁ and T₂, respectively; ΔG⁰_T, ΔH⁰, and ΔS⁰ are the changes in standard free energy, standard enthalpy, and standard entropy of the binding of the title complex to CT-DNA, respectively.

ln(K₁/K₂) = ΔH⁰/R(T₁ − T₂/T₁T₂) (6)

ΔG⁰_T = −RT ln K (7)

ΔG⁰_T = ΔH⁰ − TΔS⁰ (8)

The value of ΔH⁰ is derived to be −82.8 kJ mol⁻¹ by substituting K₁ = 1.17 × 10⁶ M⁻¹ (T₁ = 298 K) and K₂ = 6.68 × 10⁵ M⁻¹ (T₂ = 358 K) into eqn (6). By substituting K₁ = 1.17 × 10⁶ M⁻¹ (T₁ = 298 K) and ΔH⁰ = −82.8 kJ mol⁻¹ into eqn (7) and (8), ΔG⁰_{298 K} = −34.6 k J mol⁻¹ and ΔS⁰ = −16.2 J mol⁻¹ K⁻¹ at 25 °C were derived.

3.4.4. Binding mode between surfactant copper(II) complex and CT-DNA. The experimental results clearly demonstrate that the complex formation for all the cases was spontaneous with negative ΔG⁰ values. The negative binding free energy suggests that the energy of the complex–DNA adduct is lower than the sum of the energies of the free complex and DNA, and the binding of the title complex to CT-DNA is favorable at room temperature. The negative enthalpy denotes that the binding at 25 °C is exothermic and thus is driven by enthalpy. The negative entropy change implies that the degree of freedom of the title complex and DNA conformation is reduced upon complex–DNA binding. According to the thermodynamic data, interpreted as follows, the model of interaction between a drug and biomolecule can be:⁵² (1) ΔH < 0 and ΔS < 0, hydrophobic forces; (2) ΔH > 0 and ΔS > 0, van der Waals interactions and hydrogen bonds; and (3) ΔH > 0 and ΔS < 0, electrostatic interactions.⁵³ In order to elucidate the interaction of our complex with DNA, the thermodynamic parameters were calculated. When we apply this analysis to the binding of the complex with CT-DNA, we find that ΔH < 0 and ΔS < 0. Therefore, intercalations via hydrophobic interactions are most likely the main forces in the binding of the investigated complex to CT-DNA. These results indicate that the complex [Cu(dppz)₂DA](ClO₄)₂ undoubtedly interacts with CT-DNA in an intercalation mode.

3.4.5. Competitive binding studies. No luminescence was observed for the surfactant copper(II) complex in any solvent or even in the presence of nucleic acid. Therefore, competitive binding experiments using a surfactant complex as a quencher may afford further information for studying the binding of complex to nucleic acids. Ethidium bromide (EB) is known to emit intense fluorescence in the presence of nucleic acids due to its strong intercalation between the base pairs of nucleic acids. It has been reported that the enhanced fluorescence can be quenched, at least partially, by the addition of a second molecule.⁵⁴ The extent to which the fluorescence of nucleic acids-bound EB can be quenched can be used to determine the extent of binding between the second molecule and nucleic acids. The emission spectra of nucleic acids-bound EB in the absence and the presence of complex are shown in Fig. 3 and 4. The binding was analyzed through the Stern–Volmer equation, I₀/I = 1 + K_sv[Q], where I₀ and I are the fluorescence intensities in the absence and presence of the complex, respectively, K_sv is the linear Stern–Volmer constant, and Q is the concentration of surfactant copper(II) complex.⁵⁵ A plot of I₀/I vs. [Q] was drawn, and K_sv was obtained from the slope (Table 1). As seen from this table, the K_sv values of our surfactant copper(II) complex are 7.88 × 10³ with DNA and 1.13 × 10⁴ with RNA, suggesting that our complex binds with RNA more strongly than with DNA, which was observed through absorption obtained by the spectral method.

Fig. 3 Emission spectra of EB bound to CT-DNA in the absence ([dash dot, graph caption]) and in the presence (—) of complex [EB] = 2 × 10^–5 M, [DNA] = 1 × 10^–4 M, [Complex] = 0–7.04 × 10^–5. The arrow shows intensity changes upon increasing the concentration of the complex.

Fig. 4 Emission spectra of EB bound to RNA in the absence ([dash dot, graph caption]) and in the presence (—) of complex [EB] = 2 × 10^–5 M, [RNA] = 1 × 10^–4 M, [Complex] = 0–7.43 × 10^–5. The arrow shows intensity changes upon increasing the concentration of the complex.

3.4.6. Electrochemical studies. The change in the electrochemical properties upon binding of surfactant copper(II) complex with nucleic acids was studied by cyclic voltammetry in Tris buffer solution that contained 5 mM Tris-HCl/50 mm NaCl at pH 7.0. Based on the shift of the formal potentials in the cyclic voltammograms, the relative binding modes of the metal complexes with DNA can be determined.⁴⁶ The cyclic voltammogram (CV) of our complex exhibits one redox couple in the potential range of +1 V to −1.5 V. The typical CVs of surfactant copper(II) complex in the absence and presence of nucleic acids are shown in ESI Fig. 5 and 6,† and the peak potentials are provided in Tables 2 and 3. When DNA was added to a solution of complex, both the anodic and cathodic peak current heights of the complex increased in the same manner as that which was observed after increasing additions of RNA. During the addition of nucleic acids, the anodic peak potential (E_pa), cathodic peak potential (E_pc), and E_1/2 (calculated as the average of E_pc and E_pa) all showed positive shifts. These positive shifts are considered to be evidence for intercalation of the complex into the nucleic acids because this kind of interaction is due to hydrophobic forces. Therefore, the positive shift in the CV peak potentials of complex is indicative of an intercalative binding mode of the complex with nucleic acids.⁴⁷

Table 2 Electrochemical parameters for the interaction of RNA with [Cu(dppz)₂DA](ClO₄)₂

Surfactant copper(II) complex	Epc	Epa	ΔEp	E1/2	Ipa/Ipc
[Cu(dppz)2DA](ClO4)2	−0.78	0.13	4.88	−0.31	0.22
[Cu(dppz)2DA](ClO4)2 + CT-DNA	−0.64	−0.04	0.91	−0.32	0.41

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Table 3 Electrochemical parameters for the interaction of DNA with [Cu(dppz)₂DA](ClO₄)₂

Surfactant copper(II) complex	Epc	Epa	ΔEp	E1/2	Ipa/Ipc
[Cu(dppz)2DA](ClO4)2	−0.69	0.09	0.78	−0.49	0.20
[Cu(dppz)2DA](ClO4)2 + RNA	−0.57	−0.06	0.63	−0.25	0.28

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3.4.7. Viscosity studies. Viscosity measurements were carried out for further clarifying the CT-DNA binding nature of the complex. According to the classical intercalation concept put forward by Lerman,⁴⁸ the presence of the intercalation bond between a drug and the base pairs of DNA forces these base pairs away from each other, and therefore, unwinds the double helix and lengthens a given amount of DNA, which in turn, increases the viscosity of the DNA solution. In contrast, groove binding or electrostatic interactions typically cause less pronounced (positive or negative) or no change in the DNA solution viscosity.^45,2 In the absence of crystallographic structural data, these hydrodynamic methods are the most suitable methods to support an intercalative binding model. The effects of surfactant copper(II) complex on the viscosity of nucleic acid solution is given in ESI Fig. 7.† The results show that the presence of the surfactant copper(II) complex increased the relative viscosity of nucleic acid solutions, indicating that there was an intercalative interaction between the complex and the nucleic acid, which is in good agreement with the above findings obtained by electronic absorption, thermal denaturation, fluorescence binding, and cyclic voltammetry methods.

3.5. Cytotoxicity studies

3.5.1. MTT assay. The cytotoxicity of the surfactant copper(II) complex was tested by combining cultured HepG2 liver cancer cells with the complex at 90 μg mL⁻¹ in medium for 24 and 48 h. The in vitro antitumor activity of this complex was determined according to the percentage of nonviable cells (%NVC), which was calculated by the following equation:

NVC% = [number of NVC/total number of cells] × 100

The increasing concentration of surfactant copper(II) complex was accompanied by a progressive decrease in the viable cell (VC) %. This was due to the fact that by increasing the concentration of cationic surfactant complex, the adsorption of ions on cell membranes increases, leading to an increase in penetration and antitumor activity.

The inhibition of cell viability showed that the surfactant copper(II) complex is the most active one at a concentration of 90 μg mL⁻¹, with the VCs increasing up to 7.3 (±0.11)%. This indicates that the drug at this concentration causes the death of most of the tumor cells. For the 24 h treatment period, higher concentrations of the complex were required to kill the cells, whereas for the 48 h treatment, cells were killed at lower concentrations of complex. The cytotoxic activity on human tumor cell lines was determined according to the dose values of drug exposure required to reduce survival in the cell lines to 50% (IC₅₀). The IC₅₀ value of the complex was slightly higher for the 24 h treatment groups, i.e., in the range of 7.3 (±0.11) – 90 μg mL⁻¹, whereas for the 48 h treatment groups, the IC₅₀ value fell to 5.9 (±0.19) – 90 μg mL⁻¹. It should be noted that the action of the complex as an antitumor agent was found to be dependent on the type of tumor cell line tested. However, as shown from the results, the surfactant copper(II) complex showed good cytotoxic activity against all tumor cell lines, and at very low concentrations, reduced the survival to 50%. This is due to the fact that the copper(II) complex has the capacity to reduce the energy status in tumors as well as to enhance tumor hypoxia. It is known that phenanthroline-containing metal complexes have a wide range of biological activities such as antitumor, antifungal, apoptotic;⁵⁶ they also interact with DNA so that replication, transcription, and other nuclear functions are inhibited. In general, the high selectivity of action by the copper(II) complex upon tumors is due to its specific reactivity.⁵⁷ From these results, the surfactant copper(II) complex seems to offer promise due to the high electron affinity of the metal, which increases its ability to bind DNA, and the ready reducibility of the compounds.⁵⁸

3.5.2. Apoptosis studies (AO and EB staining). AO/EB staining also revealed apoptosis from the perspective of fluorescence. After HepG2 liver cancer cells were exposed to different concentrations of the surfactant copper(II) complex for 24 h, acridine orange and ethidium bromide (AO/EB) were used in a double staining assay.^59–61 Acridine orange is taken up by both viable and nonviable cells and emits green fluorescence if intercalated into double stranded nucleic acid (DNA) or red fluorescence if bound to single stranded nucleic acid (RNA). Ethidium bromide is taken up only by nonviable cells and emits red fluorescence by intercalation into DNA. We distinguished four types of cells according to the fluorescence emission and the morphological aspect of chromatin condensation in the stained nuclei: (1) viable cells showing light green fluorescing nuclei with a highly organized structure; (2) early apoptotic cells showing bright green fluorescing nuclei with chromatin condensation and nuclear fragments; (3) late apoptotic cells with orange to red fluorescing nuclei and condensed or fragmented chromatin; and (4) necrotic cells that fluoresced red without chromatin fragmentation. Viable cells have uniform bright green nuclei with organized structure. Apoptotic cells have orange to red nuclei with condensed or fragmented chromatin. Necrotic cells have uniform orange to red nuclei with condensed structure (Fig. 5). Our results indicate that surfactant copper(II) complex induced apoptosis at the concentrations evaluated, in agreement with the cytotoxic results. The AO/EB staining results suggest that the complex treatment caused increased cell apoptosis in HepG2 liver cancer cells.

Fig. 5 Photomicrographs of control and AO/EB-stained HepG2 liver cancer cells incubated for 24 hours with the surfactant copper(II) complex. (A) Untreated control cells. (B) Surfactant copper(II) complex-treated control cells (90 μg mL⁻¹); viable (light green), early apoptotic (bright green fluorescing), late apoptotic (red to orange fluorescing), and necrotic (red fluorescing) cells were observed. Magnification = 200×.

3.5.3. Apoptosis detection using Hoechst 33342 DNA staining. It is possible to perform an apoptosis detection assay with Hoechst 33342 (H342) dye (Sigma B-2262), but the increase in fluorescence seen in the apoptotic cells may be less dramatic. H342 is a “vital” DNA stain that binds preferentially to A-T base-pairs. The cells require no permeabilization for labeling, but do require physiologic conditions because the dye internalization is an active transport process. The physiologic conditions typically vary among cell types (Stander et al., 2009). To investigate if HepG2 liver cancer cells will undergo apoptosis due to the exposure of the surfactant copper(II) complex, cells were treated with IC₅₀ concentrations of surfactant copper(II) complex (90 μg mL⁻¹) for 24 and 48 h, and then stained with Hoechst 33342. The cells were observed under a fluorescent microscope for morphological changes that indicated that apoptosis had occurred. Cytological changes such as chromatin fragmentation and condensation, binucleation, cytoplasmic vacuolation, nuclear swelling, and cytoplasmic blebbing indicating late apoptosis (Fig. 6) were observed, whereas untreated cells did not show such changes. Cells with normal and abnormal nuclear features were manually counted. Both apoptotic and necrotic cells increased in a dose-dependent manner. These data clearly indicated that higher doses of surfactant copper(II) complex resulted in remarkable chromatin condensation and nuclear fragmentation in HepG2 liver cancer cells.

Fig. 6 The surfactant copper(II) complex induces apoptosis in HepG2 liver cancer cells. Representative fluorescent micrographs of HepG2 liver cancer cells stained with Hoechst 33342 fluorescent dye after complex exposure for 24 and 48 hours. (A and C) Untreated control cells; (B and D) surfactant copper(II) complex-treated control cells (90 μg mL⁻¹). (Aand B) 24 hours; (Cand D) 48 hours. Magnification = 200×.

4. Conclusions

In this work, we have reported the synthesis, characterization, and CMC value of a new surfactant copper(II) complex containing a substituted phenanthroline ligand with extended aromaticity and its binding with DNA and RNA. The critical micelle concentration value of this surfactant copper(II) complex is very low, and the complex has increased capacity to associate and form aggregates when compared to those of ordinary synthetic organic surfactants. Electronic spectroscopy revealed the ability of this complex to bind to nucleic acid. A competitive binding study with EB was also performed using fluorescence spectroscopy. All of these experiments show that the interaction between the nucleic acids and the surfactant copper(II) complex took place via an intercalative mode. Viscosity measurements of nucleic acid solutions in the presence of our complex have confirmed that intercalation was the most likely binding mode. Cyclic voltammetric studies have also established the intercalating binding ability of our surfactant copper(II) complex and its affinity to nucleic acids. This intercalation is due to the presence of the more extended aromaticity of the dppz ligand, and the presence of long aliphatic chains in the complex enhanced this intercalation. The complex also exhibited anticancer properties on human liver carcinoma cell lines.

Acknowledgements

We are grateful to the UGC-SAP and DST-FIST programmes of the Department of Chemistry, Bharathidasan University, and UGC-RFSMS fellowship to one of the authors, K. Nagaraj, by Bharathidasan University. Financial assistance from the CSIR (Grant no. 01(2461)/11/EMR-II), DST (Grant no. SR/S1/IC-13/2009) and UGC (Grant no. 41-223/2012(SR) sanctioned to S. Arunachalam are also gratefully acknowledged.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c4ra08049a

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