Spectroscopic, biological, and molecular modeling studies on the interactions of [Fe(III)-meloxicam] with G-quadruplex DNA and investigation of its release from bovine serum albumin (BSA) nanoparticles

The guanine-rich sequence, specifically in DNA, telomeric DNA, is a potential target of anticancer drugs. In this work, a mononuclear Fe(III) complex containing two meloxicam ligands was synthesized as a G-quadruplex stabilizer. The interaction between the Fe(III) complex and G-quadruplex with sequence of 5′-G3(T2AG3)3-3′ (HTG21) was investigated using spectroscopic methods, molecular modeling, and polymerase chain reaction (PCR) assays. The spectroscopic methods of UV–vis, fluorescence, and circular dichroism showed that the metal complex can effectively induce and stabilize G-quadruplex structure in the G-rich 21-mer sequence. Also, the binding constant between the Fe(III) complex and G-quadruplex was measured by these methods and it was found to be 4.53(±0.30) × 105 M−1). The PCR stop assay indicated that the Fe(III) complex inhibits DNA amplification. The cell viability assay showed that the complex has significant antitumor activities against Hela cells. According to the UV–vis results, the interaction of the Fe(III) complex with duplex DNA is an order of magnitude lower than G-quadruplex. Furthermore, the release of the complex incorporated in bovine serum albumin nanoparticles was also investigated in physiological conditions. The release of the complex followed a bi-phasic release pattern with high and low releasing rates at the first and second phases, respectively. Also, in order to obtain the binding mode of the Fe(III) complex with G-quadruplex, molecular modeling was performed. The molecular docking results showed that the Fe(III) complex was docked to the end-stacked of the G-quadruplex with a π–π interaction, created between the meloxicam ligand and the guanine bases of the G-quadruplex.


Introduction
Guanine-rich DNA sequences at the end region of telomeric DNA(TTAGGG/CCCTAA) n , can fold into structures that consist of stacks of two or more square planar arrays of four Hoogsteen hydrogen-bonded guanines called G-quartets (Gellert, Lipsett, & Davies, 1962;Kumari, Bugaut, & Balasubramanian, 2008;McEachern, Krauskopf, & Blackburn, 2000). The formation of G-quadruplex at the telomeric end can prevent telomere elongation by inhibiting activity of telomerase enzyme, which is activated in 80-85% cancer cells (Paul et al., 2012). Telomerase adds telomere repeats at the end of the chromosome and plays an important role in regulating the length of telomeric DNA. The activity of telomerase is low in the somatic cells of normal tissues and dramatically elevate in the majority of human tumors (Ma et al., 2009). Therefore, telomerase inhibition and G-quadruplex stabilization are the most important factors in designing anticancer drugs (De Cian et al., 2008). A number of small ligands such as berberine derivatives can stabilize the formation of G-quadruplex structure and inhibit telomerase activity (Franceschin et al., 2006;Ma et al., 2009;Zhang et al., 2007). Most of the G-quadruplex binders comprise a planar, aromatic core presumed to create π-π stacking interactions on the terminal tetrads of the G-quadruplex. Nowadays, researchers have shown that metal complexes can strongly and selectively interact with the G-quadruplex DNA (Georgiades, Abd Karim, Suntharalingam, & Vilar, 2010).
Meloxicam (4-hydroxy-2-methyl-N-(5-methyl-2-thiazolyl)-2H-1,2-benzothiazine-3 carboxamide-1,1-dioxide, mel) is an oxicam derivative of the acidic enol-carboxamide class of drugs used to reduce pain, fever, and inflammation (Engelhardt, Homma, Schlegel, Utzmann, & Schnitzler, 1995). The effect of meloxicam on cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) has shown that the meloxicam drug inhibits the growth of COX-2 positive and COX-1 negative colorectal cancer cells and has an anticancer effect (Goldman et al., 1998). Also, recent studies have shown that meloxicam is effective in the treatment of in vitro and in vivo models of urinary bladder cancer (Arantes-Rodrigues et al., 2013). Meloxicam has an aromatic structure and can provide π-π stacking interaction with the G-tetrads. Therefore, based on the importance of Fe(III) in the human body (Lieu, Heiskala, Peterson, & Yang, 2001) as a biological element and also due to the pharmacological properties of meloxicam, we report here the synthesis, structural characterization, and biological properties of a mononuclear Fe(III) complex with meloxicam. We studied the interaction of the Fe(III) complex with G-quadruplex DNA by circular dichroism (CD) spectroscopy, UV-vis absorption spectroscopy, UV-vis, denaturation studies, fluorescence spectroscopy, and polymerase chain reaction (PCR) stop assay.
An efficient approach to increase the efficacy and reduce the side-effects of anticancer drugs is to incorporate drugs with the delivery systems such as nanoparticles (Dreis et al., 2007). One of these nanoparticles is colloidal drug carrier bovine serum albumin (BSA). The major advantages of these nanoparticles are their biocompatibility, low immunogenicity, biodegradability, and the possibility of drug targeting by a modified body distribution (Nitta & Numata, 2013;Weber, Kreuter, & Langer, 2000). In this work, the release pattern of the Fe(III) complex from the BSA nanoparticles (BSA NPs) was also investigated.

Chemicals and reagents
The 5′-G 3 (T 2 AG 3 ) 3 -3′ (HTG21), HTG21rev, 5′-C 3 (TA 2 C 3 ) 3 -3′ ATCGCT 2 CTCGTC 3 TA 2 C 2 oligonucleotide sequences used for this study were purchased from the Bioneer Company (South Korea) and used without any further purification. Tris (hydroxymethyl)-aminomethane (Tris) buffer was an analytical reagent grade and provided by Merck. In order to form intramolecular Gquadruplex, HTG21 was dissolved in 10 mM Tris-HCl, 100 mM KCl, pH 7.4 buffer, heated to 90°C for 5 min, then gradually cooled to room temperature and further incubated at 4°C overnight (Yu et al., 2012). Analytical grade FeCl 3 ·6H 2 O was purchased from Merck. A concentrated stock solution of the Fe(III) complex was prepared by dissolving the complex in DMSO and diluting with Tris-HCl buffer to obtain the required concentration. The DMSO content in the final concentrations did not exceed 0.20% v/v. BSA (purity 98%, 66.5 kDa) and glutaraldehyde 8% solutions were obtained from Sigma (Steinheim, Germany). All chemicals and solvents were high purity and used without any further purification.

Absorption and emission spectra study
Absorption spectra were recorded on a JASCO 7580 UV-vis-NIR spectrophotometer. UV-vis spectra were recorded at room temperature using Tris buffer, which contained 0.2% v/v DMSO. Spectroscopic titrations were performed at room temperature to determine the binding affinity between G-quadruplex DNA and the Fe(III) complex using two-beam spectrometer and sample cuvettes (1 cm path length). During the titration, different concentrations of G-quadruplex DNA solution (concentration of 2-9 μM) were added to the sample cuvette and the solutions were mixed by repeated inversion. The solution of G-quadruplex DNA and the Fe(III) complex were incubated for 7 min before the absorption spectra were recorded. The titration was continued until the spectra were fixed. In order to obtain the exact numbers of the ligand-binding sites, the method of continuous variation analysis (Job plot) was used (Evans et al., 2007;Ingham, 1975;Wei, Jia, Yuan, Feng, & Li, 2006). In this method, the solutions were prepared with a varying mole fraction of the Fe(III) complex and G-quadruplex, while the sum of the Fe(III) complex and G-quadruplex concentration was kept constant at 3 μM. The absorption difference spectra were obtained by subtraction of the absorption spectrum for the Fe(III) complex in the absence of G-quadruplex from that in the presence of it. The difference in the maximum absorbance values at two wavelengths (260 and 363 nm) was plotted vs. the Fe(III) complex mole fraction to generate a Job plot in room temperature.
Fluorescence spectra were measured on a Jasco FP-750 spectrometer. The excitation wavelength was set on 360 nm and the emission spectrum was recorded from 380 to 600 nm. The excitation and emission slits were set at 10 and 5 nm, respectively. The concentration of the Fe(III) complex was 5 μM and the method of titration was similar to the UV-vis titration.

Thermal denaturation experiment
Since nucleic acid structures are sensitive to temperature, UV-denaturation profiles can be used to determine the denaturation temperature (T m ) of the ligand-induced G-quadruplex and the temperature range in which the complex is stable. The denaturation experiment was performed at 3 μM G-quadruplex concentration on a JASCO 7580 UV-vis-NIR equipped with an external thermal bath in the absence and presence of different concentrations of the complex in 10 mM sodium cacodylate buffer, pH 7.4, and 100 mM KCl. The samples were first held at 90°C, then cooled to 15°C with a cooling rate of 1°C min −1 .
2.5. CD spectroscopy CD measurements in the mid-UV region (220-320 nm) were carried out by an Aviv model 215 Spectropolarimeter (Lakewood, NJ, USA) using 1 cm path length, quartz cuvette of 300 μL capacity. A G-quadruplex solution was prepared in 10 mM Tris-HCl buffer, 100 mM KCl at pH 7.4. A G-quadruplex solution (2 μM) was used to obtain the spectra with and without incubation at different concentrations of the Fe(III) complex (0-8 μM) after 7 min. All spectra were collected from 220 to 320 nm and a background correction was performed with the blank solution. The ellipticity values were converted into molar ellipticity using the Equation (1) (Nicoludis, Barrett, Mergny, & Yatsunyk, 2012): where θ is the observed ellipticity (CD signal) in mdeg, C is concentration in mol L −1 , and L is a cuvette path length in cm. All data were smoothed using a Savitzky-Golay smoothing filter with a 13-point quadratic function.

Cell culture
The human cervical carcinoma Hela cells were obtained from the Pasteur Institute of Iran (Tehran, Iran) and cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum, 100 mg/mL penicillin, and 100 mg/mL streptomycin. The cells were maintained at 37°C in a 5% CO 2 incubator, and the media were changed twice weekly.

Cellular uptake
The cytotoxicity of the complex was evaluated by cell viability and determined by counting live cells. Cell viability was calculated by viable cells by dividing the total number of viable cells within the grids on the hemacytometer. Firstly, the harvested Hela cells were incubated in the presence of the Fe(III) complex, then the cells were colored by solution of 0.25% trypan blue. The number of blue staining cells and the number of total cells were counted by microscope.

Preparation of the BSA nanoparticles
The BSA nanoparticles were prepared using desolvation technique (Langer et al., 2003;Weber et al., 2000). For this purpose, 200 mg of BSA were dissolved in 2 mL of purified water and the pH was adjusted to 8.2 with 0.01 M NaOH. Then, 8 mL ethanol was added (1 mL/min) under constant stirring (600 rpm) at room temperature. After protein desolvation, 235 μL of an aqueous glutaraldehyde solution (8%, 1.175 μL/mg BSA) was added to induce particle crosslinking. The crosslinking process was performed under stirring in suspension over 24 h. The purification of the BSA NPs was carried out by three cycles of differential centrifugation (16,100 × g, 20 min) and dispersion of the BSA NPs in purified water was performed by ultrasonication, in order to remove organic solvent and glutaraldehyde. The final nanoparticles suspension were stored at 4°C and protected from light. The prepared nanoparticles were characterized by SEM, XLC Philips instrument. Also, X-ray powder diffraction (XRD) measurement was carried out on an X'pert X-ray diffractometer (Philips) with Cu Kα radiation (λ = 0.15418 nm) in the range of 5°-40°.

Adsorption of the Fe(III) complex onto the BSA nanoparticles
A stock solution of the Fe(III) complex (1.77 × 10 −3 M) was prepared, and volumes of 100-300 μL were added to 50 mg of the BSA NPs. Then, the volume was adjusted with water to 2 mL. The mixture was stirred for 12 h (650 rpm) at room temperature to achieve an adsorption equilibrium of the Fe(III) complex to the particle surface. The nanoparticles were washed as previously indicated. The supernatants of the washing steps were collected and the concentration of the Fe(III) complex was analyzed by UV-vis spectroscopy.

In vitro studies of the release of the Fe(III) complex
The release of the Fe(III) complex was studied in PBS (phosphate buffer pH 7.4, 0.13 M concentration, and 100 mM NaCl) (Faisant, Akiki, Siepmann, Benoit, & Siepmann, 2006;Manoochehri et al., 2013). Each experiment was conducted in triplicate. In order to determine the release of the complex from the nanoparticles, 2 mL of the suspension consisting of the Fe(III) complexloaded BSA nanoparticles was placed within dialysis bags (molecular weight cut-off 12 kDa). The dialysis was performed against 5 mL PBS at 37°C while magnetically stirring at 600 rpm and protecting from light. After suitable time intervals, 500 μL of the release medium was collected and analyzed by UV-vis spectrophotometer (λ = 363 nm).

Molecular modeling
The initial 3D structure of the G-quadruplex DNA in the presence of K + ions at 2.10 Å resolution (PDB ID: 1KF1) was obtained from the Protein Data Bank (PDB). The molecular dynamics (MD) simulation was performed on the structure of G-quadruplex DNA in a water box using the GROMACS 4.5.1 package (Hess, Kutzner, van der Spoel, & Lindahl, 2008). The interaction parameters were computed using the Amber99 force field (Hornak et al., 2006), with the intermolecular (non-bonded) potential represented as a sum of Lennard-Jones (LJ) force and pairwise coulomb interaction and the long-range electrostatic force determined by the particle mesh Ewald (PME) method (Darden, York, & Pedersen, 1993;Essmann et al., 1995). The velocity Verlet algorithm was used for the numerical integrations (Swope, Andersen, Berens, & Wilson, 1982), and the initial atomic velocities were generated with a Maxwellian distribution at the given absolute temperature (Huang, 1987). The Fe(III) complex was docked into G-quadruplex obtained from the MD simulation. The docking studies were performed by the Autodock Vina program (Trott & Olson, 2010). The grid map was set to 20 × 20 × 20 Å 3 along the x, y, and z axes with 1 Å grid spacing. The center of grid map was set to 57.468, 43.374, and 72.401 Å. The grid maps were obtained using AutoGrid. Then, the binding free energy of the Fe(III) complex was calculated. The docking results including the binding site and the inhibitor interactions with G-quadruplex DNA were produced using LIGPLOT+ (Wallace, Laskowski, & Thornton, 1995).

Synthesis and spectroscopic characterization of the Fe(III) complex
The synthesis of the complex in high yield (88%) was achieved by adding a deprotonated alcoholic solution of meloxicam to an alcoholic solution of the iron salt in a 2:1 M ratio. The IR spectrum of the free meloxicam ligand showed four significant stretching vibrations at 3291, 1620, 1347, and 1185 cm −1 which are attributed to the stretching vibrations of ν(N-H) amide , ν(C=O) amide , ν(SO 2 ) asym ., and ν(SO 2 ) sym ., respectively. The IR spectrum of the complex lacks of absorption in the region of 3200-3300 cm −1 . This region was related to the N-H stretching mode because the N-H group of the deprotonated meloxicam ligand is involved in a strong intramolecular hydrogen bond to the enolate oxygen. The stretching vibration of the amide C=O function shifts to 1610 cm −1 in the complex, and the two stretching bands of the SO 2 group also shift slightly to lower frequencies due to hydrogen bonding effect. These data confirm the coordination of the meloxicam ligand to Fe(III). The electronic absorption spectra of the free meloxicam ligand and the Fe(III) complex consist of three wellresolved bands in the range of 200-400 nm that can be attributed to the π → π* and n → π* transitions. The meloxicam shows three absorption bands at 240, 272, and 364 nm. These bands shift to 210, 266, and 363 nm, respectively, which also confirm the coordination of the meloxicam ligand to Fe(III).

CD spectroscopy
CD is a useful technique to study the characterization of the G-quadruplex structures of oligonucleotides (Dai, Carver, & Yang, 2008). CD spectra can be used to distinguish between parallel and antiparallel G-quartet structures (Mergny & Maurizot, 2001). The CD spectrum of the G-quadruplex (HTG21) is presented in Figure 1(A) (violet line). The CD spectrum of the G-quadruplex in buffer containing 10 mM Tris-HCl and 100 mM KCl exhibits a positive peak at 290 nm, a shoulder peak near 260 nm, and a negative band at 238 nm. The equilibrium constant of the G-quadruplex-metal complex formation can be estimated from the change in CD response at a fixed wavelength (290 nm) using the Benesi-Hildebrand equation (Benesi & Hildebrand, 1949;: where ε b and ε f are the extinction coefficients of the bound and the free Fe(III) complex, L T is total concentration of the Fe(III) complex, M is the concentration of the macromolecule, and Δθ is the change of the CD response at 290 nm wavelength. The association constant for the complex formation (K a ) can be calculated from the ratio of the intercept to the slope of obtained line from plot of 1/Δθ vs. 1/L T (Figure 1(B)). According to the above equation, the value of K a was found to be 1.60 × 10 5 M −1 .

UV-vis absorption titration
In order to obtain the binding constant and investigate the binding behaviors of the Fe(III) complex to G-quadruplex, the UV-vis absorption titrations were carried out by adding the different concentrations of G-quadruplex ( Figure 1). The percentage hypochromicity of the maximum absorption wavelength of the Fe(III) complex was calculated using Equation (3) (Wei et al., 2006): (3) The results show a 6 nm red-shift and 22.70% hypochromicity for the Fe(III) complex at the end of titration. The binding constant (K b ), was determined according to Equation (4) where correspond to the extinction coefficients of the fully bound and the free Fe(III) complex at 263 nm, respectively. By plotting [DNA]/(ε b − ε f ) vs.
[DNA] (inset of Figure 1) K b can be obtained from the ratio of the slope to the intercept of the linear plot. The value of the binding constant was found to be 7.50 × 10 5 M −1 . UV-vis titration was used to investigate the Fe(III) complex's selectivity toward G-quadruplex vs. duplex DNA (dsDNA). Since the canonical dsDNA structure in cells has a high excess over other DNA structures (Bhattacharjee et al., 2011), fish sperm DNA was used as main competitor. As shown in Figure S1 (in the supporting information), the binding constant for dsDNA was found to be 4.11 × 10 4 M −1 . Therefore, the Fe(III) complex shows 18-fold selectivity for G-quadruplex over dsDNA.
In order to compare the binding affinity of the Fe(III) ion, the free meloxicam ligand, and the Fe(III) complex to G-quadruplex, their binding constants to HTG21 were measured by absorption spectra according to Equation (4). Figures S2 and S3 (in the supporting information) show the absorption titration spectra of the Fe(III) ion and the free meloxicam ligand with increasing concentration of HTG21, re. The values of the binding constant of Fe(III) and meloxicam to G-quadruplex were found to be 1.14 × 10 4 and 2.00 × 10 4 M −1 , respectively. Therefore, the results indicate that the affinity of the Fe(III) complex to G-quadruplex is an order of magnitude larger than the Fe(III) ion and the free meloxicam to G-quadruplex.
The method of continuous variation analysis (Job plot) was used to determine the number of molecules of Fe(III) complex binding to G-quadruplex. As shown in Figure 2, an inflection point is observed at a Fe(III) complex fraction of 0.5. Based on careful analysis of data obtained from Job plots, it was clear that 1 molecule of Fe(III) complex bind to each molecule of G-quadruplex.

Thermal denaturation profiles
The UV-denaturation experiments were carried out to examine the stability of the Fe(III) complex-induced G-quadruplex (Lane, Chaires, Gray, & Trent, 2008). In order to obtain the optimal wavelength for UV-denaturation profile, the spectra were recorded at high temperature, above T m , where the oligonucleotide is fully dissociated, θ = 0 and at low temperature, below T m , where the oligonucleotide is fully associated, θ = 1. As shown in Figure S4 (in the supporting information), the maximum difference between the absorbance of two spectra occurs at 295 nm, and therefore, this wavelength was selected as an optimal wavelength. In the next step, the absorbance spectra at each temperature were recorded in cacodylate buffer containing KCl at 295 nm. The values of T m in different concentrations of 1, 9, and 120 μM of the Fe(III) complex are 40, 52, and 60°C, respectively, as is illustrated in Figure 3. In order to determine the molecularity of the reaction, concentration of the Fe(III) complex was increased to 120-fold at three different concentrations.
The results indicate that T m is concentration dependent, which is in agreement with a bimolecular process (Liebert, Mergny, & Lacroix, 2003;Mergny & Lacroix, 2009). The analysis of the denaturation profiles allows the determination of the thermodynamic parameters ΔG°, ΔH°, and ΔS°. The Gibbs free energy is related to the affinity constant by the simple relation ΔG°= -RTln(K a ) (Cantor & Schimmel, 1980). In order to calculate the thermodynamic parameters, the absorbance vs. temperature should be converted into a folded fraction (θ) vs. temperature. Folded fraction should be between 0 and 1.  Spectroscopic, biological & molecular modeling studies on interactions of [Fe(III)-meloxicam] with G-quadruplex DNA Therefore, the absorbance (A T ) converts to θ T according to the Equation (5): where A(T), A u (T), and A F (T) correspond to absorbance at each temperature, the baseline values of the unfolded and folded species, respectively. In the case of a bimolecular equilibrium, K a is related to the folded fraction according to the Equation (6): where C 0 is the initial strand concentration. As shown in Figure S5 (in the supporting information), natural logarithm of the affinity constant (ln(K a )) is plotted as a function of the reciprocal of the temperature (1/T K −1 ) (Mills et al., 1999). The analysis is restricted to the temperature range for which 0.15 < θ < 0.85 (Puglisi & Tinoco Jr, 1989). The plot of ln(K a ) vs. 1/T was fitted by a straight line with a slope of -ΔH°/RT and a Y-axis intercept of ΔS°/R according to the Equation (7): The value of ΔH°, ΔS°, and ΔG°were found to be -13.27, -16.87, and -8.66 kcal mol −1 , respectively.

Fluorescence studies
The fluorescence spectroscopy was used to further clarify the nature of the interactions between the Fe(III) complex and G-quadruplex. Figure 4 shows the fluorescence emission spectra of the Fe(III) complex in the absence and presence of G-quadruplex. The fluorescence intensity increases with the addition of G-quadruplex without wavelength shift. The enhancement constant can be obtained from Equation (8)  : where F 0 and F represent the fluorescence intensities in the absence and presence of the enhancer, respectively.
[E] is the concentration of the enhancer. K D is the dynamic enhancement constant and K E is the bimolecular enhancement constant. τ 0 is the average lifetime of the fluorophore in the absence of the enhancer and its value is typically near 10 −8 s (Lakowicz & Weber, 1973;Lu et al., 2010). The values of K D and K E were obtained from plot of F 0 /F vs.
[E] ( Figure S6 in the supporting information) as 1.10 × 10 5 M −1 and 1.10 × 10 13 M −1 s −1 , respectively. The value of K E in this work is 10 3 times higher than the reported maximum scatter collision enhancing constant of 1.0 × 10 10 L mol −1 s −1 for various enhancers with biopolymer . The fluorescence titration data were used to obtain the binding constant (K b ) and the number of binding sites (n) for the complex formation between the Fe(III) complex with the G-quadruplex DNA. The following equation is used for calculation of the binding constant: Figure S7 (in the supporting information) shows a linear plot of log (F − F 0 /F) vs. log [DNA]. The values of K and n were found to be 1.94 × 10 5 M −1 and 1.04, respectively.

PCR stop assay
In this assay, the efficiency of the Fe(III) complex to stabilize G-quadruplex was evaluated. PCR-stop assay showed that Fe(III) complex was bound to HTG21 and stabilized G-quadruplex (Wang, Li, Tan, Ji, & Mao, 2010). In the presence of the Fe(III) complex, the single strand HTG21 was induced into a G-quadruplex structure that blocked hybridization with their complementary strands and the extension of PCR. Different concentrations of the Fe(III) complex were used in this assay and the results show an inhibitory effect as the concentration increases from 0.0 to 65 μM, as shown in Figure 5. The results indicate that the Fe(III) complex can induce and stabilize G-quadruplex in the biological systems to block PCR amplification, and putatively DNA replication and half maximal inhibitory concentration (IC50) was measured to be~20 μM using PCR.

Cellular uptake
The cells were treated with 30 and 60 μM concentrations of the complex in the culture media, and then incubated at 37°C under a 5% CO 2 atmosphere for 48 h. After 10, 24, and 48 h, the cells were assessed for viability by the trypan blue exclusion method. As shown in Figure 6, the percentage of the Hela cells decreases by 36 and 18% after incubation with 30 and 60 μM concentration of the complex.

Molecular modeling
The MD simulation and molecular docking of the Fe(III) complex with G-quadruplex were performed to investigate the binding mode. In order to obtain the conformation of the G-quadruplex DNA, a 100 ns MD simulation was performed on G-quadruplex in a water environment. The topology parameters of the G-quadruplex DNA were created by the Amber99 force field. Then, the system was placed in a cubic box (20 nm × 20 nm × 20 nm) with the TIP4P water model. The solvated system was neutralized by adding eighteen sodium ions in the simulation. Then, the full system was subjected to 100 ns MD. The stability of the system (G-quadruplex DNA, water, and ions) and the structural geometries were examined by means of the root-mean-square deviations (RMSD's) of the G-quadruplex DNA with respect to the G-quadruplex DNA's initial structure. As shown in Figure S8 (in supporting information), after a~60 ns of MD simulation, the structure of the G-quadruplex DNA becomes stable. The average of RMSD value for the G-quadruplex DNA backbone was calculated to be 0.85 ± 0.08 nm. The equilibrated conformation of the G-quadruplex DNA was used for docking. The molecular docking results showed that the Fe(III) complex was end-stacked at the 3′-terminus of the G-quadruplex and π-π interaction created between the aromatic rings of meloxicam and the guanine bases of G-quadruplex. Also, as shown in Figure 7(A), Fe(III) is placed at the center of G-quartet to stabilize the G-quadruplex structure. As illustrated in the 2D schematic interaction model, Figure 7(B), there are three hydrophobic contact points between the Fe(III) complex atoms and the bases of G-quadruplex DNA, viz. (i) between C25, C26, C27 with G15, (ii) between C1 with G9, (iii) between C12, C13, C14, C17, C18, and S3 with G3. The binding free energy of the Fe(III) complex with G-quadruplex was found to be -7.90 kcal mol −1 . In order to validate the reliability of the docking methodology adopted herein, the C 46 H 72 N 12 O 4 (R8G) inhibitor in the X-ray crystallographic structure of 3SC8 was taken as a testing molecule according to our previous work (Ebrahimi & Khayamian, 2013).
3.11. Size, loading, and morphology of the BSA NPs The BSA NPs morphology was evaluated by SEM. In order to obtain the particle size distribution from SEM images, the Digimizer version 4.1.1.0 software was used (Anon, 2014). The histogram of the particle size distribution and average diameter were obtained by measuring about 200 particles in arbitrarily chosen areas in Digimizer. Figure 8(A) and (B) shows a scanning electron microscopy (SEM) image of BSA nanoparticles, as illustrated in this figure, the nanoparticles were spherical in shape, with a mean diameter of 89.82 nm.
To determine the adsorption efficiency, the amount of the unbound Fe(III) complex in the supernatant was determined by UV-vis spectroscopy after separation from the loaded particles by differential centrifugation (16,100 × g; 20 min) and dialysis bag. The amount of the adsorbed Fe(III) complex onto the BSA NPs was calculated by subtracting the concentration of the free Fe (III) complex in the supernatant from the initial Fe(III) complex concentration. For the calculation of the adsorption efficiency (%), the following equation was used: Adsorption efficiency % ¼ m total FðIIIÞ complex À m free FðIIIÞ complex m total FðIIIÞ complex Â 100 (10) Figure 5. Effect of the Fe(III) complex (0-65.8 μM) on the hybridization of HTG21 in the PCR-stop assay. For the optimization of the Fe(III) complex loading on the BSA nanoparticles, the concentration of the Fe(III) complex was varied between 88 and 265 μM. After 15 h, the metal complex-loaded BSA nanoparticles were separated from the free metal complex by centrifugation. As shown in Figure S9 (in supporting information), at low concentrations of the Fe(III) complex, more than 65% of the metal complex is adsorbed onto the surface of the BSA nanoparticles. According to the Equation (10), the loading percent of the Fe(III) complex was found to be 65.42% on the BSA NPs in 88.5 μM of the Fe(III) complex.
In order to investigate morphology of nanoparticles, the SEM image was taken from the Fe(III) complexloaded BSA NPs. As shown in Figure 8(C) and (D), size of the nanoparticles changes, while the morphology of the nanoparticles does not change.
In order to confirm the structure and the phase of the BSA nanoparticles, the XRD analysis was also carried out, as shown in Figure 9. The diffraction peaks confirm that the phase of the nanoparticles does not change before and after the adsorption of the Fe(III) complex.
3.12. In vitro study of the release mechanism of the complex The in vitro study of the release pattern of the Fe(III) complex from the BSA NPs is shown as the percentage of release in phosphate buffer pH 7.4 (0.13 M) at 37°C in Figure S10 (in supporting information). The release of the Fe(III) complex from the BSA NPs followed a bi-phasic release patterns. A continuous release occurs within the initial 9 h when roughly 40% of the Fe(III) complex releases from the BSA NPs. In phase (II), the Fe(III) complex is released at a slower rate within 85 h. The initial release step may be related to the portion of the Fe(III) complexes which are adsorbed onto the nanoparticle surface and controlled by the diffusion of the Fe(III) complex. In the second release phase, the increase in the length of the diffusion pathways leads to a decrease in the release rate or may be related to the entrapped Fe(III) complex inside the BSA NPs (Faisant et al., 2006;Magenheim, Levy, & Benita, 1993;Manoochehri et al., 2013;Siepmann, Faisant, & Benoit, 2002).

Discussion
A mononuclear Fe(III) complex with meloxicam was synthesized and characterized. Interactions of the complex, [Fe(mel) 2 Cl(EtOH)], with the human telomeric G-quadruplex DNA (G 3 (T 2 AG 3 ) 3 ) were investigated using a range of experimental and computational methods.
The CD results showed stabilization of G-quadruplex by the synthesized complex. G-quadruplex was titrated   with Fe(III) complex. The band intensity at 238 nm was gradually decreased by addition of the Fe(III) complex, while the band intensities at 290 and 255 nm were increased. These changes indicate that the guanine-rich DNA is induced to form the G-quadruplex structure upon the binding of the Fe(III) complex.
The hypochromicity in UV-vis spectra shows extent of ligand interaction with G-quadruplex. The observations like red shift of Fe(III) complex bands and hypochromicity indicate that metal complex can interact well with the G-quadruplex DNA. On the basis of absorption titration experiment, the red-shift value of the Fe(III) complex is in range of a groove binding, whereas the hypochromicity percentage is smaller than those of the typical intercalation (>35%). Similar results have been reported with several small molecules that bind to the G-quadruplex DNA (Bhadra & Kumar, 2011;Wei, Han, Jia, Zhou, & Li, 2008;Wei et al., 2009). Therefore, it can be assumed that the Fe(III) complex interacts through the groove and π-π stacking interactions to the G-quadruplex structure at external quartets. UV-vis showed that not only Fe(III) complex interacts strongly with G-quadruplex, but also exhibits a higher selectivity for G-quadruplex vs. dsDNA.
The method of continuous variation analysis revealed that stoichiometry of Fe(III) complex binding to HTG21 was 1:1. UV-melting experiment showed that the denaturation temperature was increased by the Fe(III) complex concentration, indicating stability of G-quadruplex by increasing of the Fe(III) complex concentration.
The increase in the emission intensity could be the result of the stacking of the planar aromatic group of meloxicam and the G-quadruplex base pairs. As a result, the metal complex is protected from solvent water molecules by the hydrophobic environment of the G-quadruplex bases. Therefore, the accessibility of the water molecules to the Fe(III) complex is reduced and this prevents its quenching effect (Li, Yang, Wang, & Qin, 2008). The Fe(III) complex is protected by the hydrophobic bases of G-quadruplex, leading to a decrease in the vibrational modes of relaxation, and a higher emission intensity. Also, PCR-stop assay was used to determine inhibitory effect of Fe(III) complex.
The results of molecular modeling showed that the π-π and hydrophobic interactions are the major interactions between the Fe(III) complex and the G-quadruplex DNA. Also, metal complex placed at the center of G-quadruplex DNA to stabilize structure of quartet.
Finally, BSA nanoparticles with a mean diameter of 89.82 nm were prepared and Fe(III) complex were Scheme 1. Synthesis route to [Fe(mel) 2 Cl(EtOH)]. loaded on BSA NPs. In vitro drug delivery studies indicated controlled slow release profiles of Fe(III) complex.
In summary, the iron(III)-meloxicam complex was synthesized and its interactions with G-quadruplex including the binding constant, type of interactions, bioassays consisting of PCR stop and Cell viability against Hela cells, selectivity over Duplex DNA and its release from BSA nanoparticles were investigated. The results indicated that the complex is a promising anticancer compound.