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Synthesis, visible light photocleavage, antiproliferative and cellular uptake properties of ruthenium complex [Ru(phen)2(mitatp)]2+

By Anthony Foster,2014-09-10 21:07
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Synthesis, visible light photocleavage, antiproliferative and cellular uptake properties of ruthenium complex [Ru(phen)2(mitatp)]2+

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    Synthesis, visible light photocleavage, antiproliferative and

    cellular uptake properties of ruthenium complex

    2+[Ru(phen)(mitatp)] 2

    5 Yu Huijuan, Yu Lin, Hao Zhifeng, Zhou Lihua, Xie Zhenming

    (Faculty of Chemical Engineering and Light Industry, Guangdong University of Technology,

     GuangZhou 510006)2+ Abstract: A new ruthenium complex [Ru(phen)(mitatp)]( phen = 1,10-phenanthroline, mitatp = 2

    5-methoxy-isatino[1,2-b]-1,4,8,9-tetraazatriphenylene) has been synthesized and characterized. The

    10 interaction of the complex with DNA has been studied and the results indicate that 2+ [Ru(phen)(mitatp)]could efficiently photocleave pBR322 DNA under irradiation at visible light and 21the singlet oxygen Owas proved to be reactive species in the photocleavage process. The cytotoxicity 2 2+ has also been evaluated by MTT method, and [Ru(phen)(mitatp)]shows prominent anticancer 2activity against various cancer cells. Live cell imaging study and flow cytometric analysis demonstrate

    15 that the complex could cross cell membrane accumulating in the nucleus and inducing cell death by induction of G0/G1 cells cycle arrest and apoptosis.

    Keywords: Ruthenium complex; DNA photocleavage; Cytotoxicity; Apoptosis

    0 Introduction

    20 Although cis-platinum and other platinum complexes as anticancer drugs has acquired remarkable success in treatment of various cancers, such as testicular, ovarian, cervical, bladder, head and neck, and lung cancers, their clinical drawbacks are also apparent, including the limited applicability, the acquired resistance, and the serious side effects [1-4]. The limitations of

    cis-platinum have motivated extensive investigations into alternative metal-based anticancer

    25 agents. Among the non-platinum metal anticancer agents, ruthenium complexes have drawn much attention due to their favorable properties, such as high cytotoxicity against cancer cells, lower toxicity toward healthy tissues, various oxidation states under physiological conditions, light-activated reactions with DNA and rich synthetic chemistry [4-8]. Many ruthenium

    complexes have been synthesized and some of them have shown potential application in

    30 chemotherapy and photodynamic therapy (PDT) as evidenced by in vitro and in vivo studies, such

    as NAMI-A and KP1019, have successfully entered clinical trials [9,10].

    Indolo[2,3-b]quinoxaline and its derivatives have been reported to be a kind of important DNA intercalating agents endowed with antiviral, cytotoxic and DNA cleaving activities [11,12]. So numerous indolo[2,3-b]quinoxaline analogues have been synthesized and studied as antiviral

    35 and antitumor drugs [13-15], while the research of metal complex based on

    indolo[2,3-b]quinoxaling or its derivative has been rarely reported. In previous study, we have

    2+ 2+ synthesized two Ru(II) complexes [Ru(bpy)(mitatp)]2 (Fig. 1) and [Ru(bpy)(nitatp)](bpy = 22

    2,2bipyridine, nitatp = 5-nitro-isatino[1,2-b]-1,4,8,9-tetraazatriphenylene) by modification dppz with indolo[2,3-b]quinoxaline [16]. The introduction of indolo[2,3-b]quinoxaline has effectively

    12+40 prolonged the excited lifetime and enhanced Oquantum yields of [Ru(bpy)(dppz)], thus 2 2

    improving its photocleavage activity and giving rise to a potential PDT agent. The success of modifying dppz encouraged us to develop other Ru(II) complexes based on

     indolo[2,3-b]quinoxaline and explore their biological properties. Some reports have suggested that

    Foundations: This work was supported by the Specialized Research Fund for the Doctoral Program of Higher Education, China (No. 20104420120006), the Foundation for Distinguished Young Talents in Higher Education of Guangdong, China (No. LYM10068), the 211 Key Program of Guangdong, China.

    Brief author introduction:Yu Huijuan, (1979-), Female, Dorctor,Bioinorganic Chemistry. E-mail:

    xiaoheiyu79@163.com

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    the ancillary ligands play an important role in biomolecular interactions and recognition processes, 45 thus variation of ancillary ligands of the Ru(II) complexes may create some difference in the

    biological activities [5,17-19]. In this paper, we reported the synthesis and characterization of the

    2+ complex [Ru(phen)(mitatp)]1 (Fig. 1). The DNA binding and photocleavage ability, antitumor 2

    activity, cellular uptake, as well as cell-cycle arrest and apoptosis were studied. For comparison

    2+ purpose, the biological properties of the analogue complex [Ru(bpy)(mitatp)]2 were also 2

    50 explored.

     2+ 2+ Fig. 1. Chemical structures of [Ru(phen)(mitatp)]1 and [Ru(bpy)(mitatp)]2. 22

    1 Experimental

55 1.1 Physical measurements

    Microanalysis (C, H, and N) was carried out with a Perkin-Elmer 240Q elemental analyzer.

    1H NMR spectra were recorded on a Varian-500 spectrometer and all chemical shifts are given

    relative to tetramethylsilane (TMS). Electrospray mass spectra (ES-MS) were recorded on a LCQ

    system (Finnigan MAT, USA). The spray voltage, tube lens offset, capillary voltage and capillary 60 temperature were set at 4.50 KV, 30.00 V, 23.00 V and 200 oC, respectively, and the quoted m/z

    values are for the major peaks in the isotope distribution. UV-Vis spectra were recorded on a

    Perkin-Elmer Lambda 850 spectrophotometer. Emission spectra were recorded on a Perkin-Elmer

    LS 55 spectrofluorophotometer at room temperature. Time-resolved emission measurements were

    conducted on an FLS 920 combined fluorescence lifetime and steady state spectrometer. Confocal 65 microscopy was performed using a Leica TCS SP5 (Germany) confocal laser scanning

    microscope.

    1.2 Materials

    Calf Thymus DNA (CT-DNA), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium

    bromide (MTT), propidium iodide (PI) and 4,6-diamidino-2-phenyindole (DAPI) were purchased

    70 from the Sigma Company. Cisplatin was purchased from Qilu Pharmaceutical Co., Ltd.

    RPMI-1640 medium was purchased from Invitrogen Co. (USA) and DMEM medium could be got

    from Life Technologies. Tris-HCl buffer (5 mM Tris-HCl, 50 mM NaCl, pH = 7.2) solution was

    prepared using doubly distilled water. A solution of Calf Thymus DNA in the buffer gave a ratio

    of UV absorbance at 260 and 280 nm of about 1.8-1.9:1, indicating that the DNA was sufficiently 75 free of protein [20]. The DNA concentration per nucleotide was determined by absorption

    -1-1spectroscopy using the molar absorption coefficient (6600 M.cm) at 260 nm [21].

    1.3 Preparation

    The compounds [Ru(phen)Cl].2HO [22] and [Ru(phen)(1,10-phenanthroline-5, 2222

    6-diamine)](ClO)[23] were synthesized according to literature methods, and other materials 42

    80 were commercially available and of reagent grade.

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     (mitatp)](ClO)1.3.1 Synthesis of [Ru(phen) 242 A mixture of [Ru(phen)(5,6-diamino-1,10-phenanthroline)](ClO)(0.35 g, 0.4 mmol) and 242 5-methoxy-isatin (0.071 g, 0.4 mmol) in acetic acid (50 mL) was refluxed for 6 h. Upon cooling, a

    red precipitate was obtained by addition of 200 mL HO, then filtered, washed with ether and 2

    dried in vacuum. The crude product was purified by column chromatography on alumina with 85

     acetonitrile-toluene (1:1, v/v) as eluent. The solvent was removed under reduced pressure and a red powder was obtained, Yield: 0.34 g, 85%. Anal. Calc for CHClNORu: C, 53.42; H, 2.89; 45292991 N, 12.46. Found: C, 53.28; H, 2.81; N, 12.60%. H NMR (500 MHz, d-DMSO): δ 12.69 (s 6 (singlet),1H, -NH), 9.71 (d (doublet), 1H, H, J = 7.5 Hz), 9.54 (d, 1H, H, J = 7.5 Hz), 8.78 (d, cc

    4H, H, H, J = 8.5 Hz), 8.40 (s, 4H, H, H), 8.24 (t (triplet), 2H, H, J= J= 5 Hz), 8.17 (d, 1H, 90 445521 2 H, J = 5 Hz), 8.14 (d, 1H, H, J = 6 Hz), 8.09 (t, 2H, H, J= J= 4.5 Hz), 7.99 (d, 1H, H, J = 2.5 aa21 2 g 7.92-7.88 (m (multiplet), 2H, H, H), 7.81-7.77 (m, 4H, H, H), 7.69 (d, 1H, H, J = 9Hz), Hz), bb33f2+ 7.46 (d, 1H, H, J= 6Hz), 3.96 (s, 3H, -CH). ES-MS (CHCN): m/z 406 ([M-2ClO]). e334 1.4 DNA-binding experiments

    Viscosity measurements were carried out using an Ubbelodhe viscometer maintained at a 95

     constant temperature at 30.0 ? 0.1 ºC in a thermostatic bath. DNA samples approximately 200 base pairs in average length were prepared by sonicating in order to minimize complexities arising

     from DNA flexibility [24]. Flow time was measured with a digital stopwatch, and each sample was measured three times, and an average flow time was calculated. Data were presented as 01/3 (η/η)versus binding ratio [25], where η is the viscosity of DNA in the presence of complex and 100 0 ηis the viscosity of DNA alone. Absorption spectra titrations were carried out at room temperature to determine the binding

     affinity between DNA and complex. 3 mL solutions of the blank buffer and the Ru(II) complex sample ([Ru] = 20 μM) were placed into two 1 cm path cuvettes respectively, then an aliquots (~4

    µL) of buffered DNA solution (concentration of 4-5 mM in base pairs) was added to each cuvette 105

     to eliminate the absorbance of DNA itself. The Ru(II)-DNA solutions were allowed to incubate for 5 min before the absorption spectra were recorded. The intrinsic binding constant K and the binding size n of complexes to DNA were calculated using the following equation from Eq. (1) [26] 221/2110(ε-ε)/(ε-ε) = (b-(b-2KC[DNA]/n ))/2KC(1a) afbftt b = 1+KC+K[DNA]/2n (1b) t Where [DNA] is the concentration of CT-DNA in base pairs, the apparent absorption

     coefficients ε, ε, and εcorrespond to A/[Ru], the absorbance for the free ruthenium complex, afb obsd and the absorbance for the ruthenium complex in the fully bound form, respectively. K is the ?1equilibrium binding constant in M, Cis the total metal complex concentration, and n is the115 t binding size. Thermal denaturation studies were carried out with a Perkin-Elmer Lambda 35 spectrophotometer equipped with a Peltier temperature-controlling programmer (? 0.1 ºC). The absorbance at 260 nm was continuously monitored for solutions of CT-DNA (50 μM) in the

    absence and presence of the Ru(II) complex in the buffer (1.5 mM NaHPO, 0.5 mM NaHPO, 120 2424

    0.25 mM NaHEDTA, pH = 7.2). The temperature of the solution was increased by 1 ºC. 22

    1.5 DNA photocleavage experiments

    The photo-induced DNA cleavage by Ru(II) complex was examined by gel electrophoresis

    experiment. Supercoiled pBR322 DNA (0.1 µg) was treated with the Ru(II) complex in the buffer

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125 (50 mM Tris-HCl, 18 mM NaCl, pH = 7.2), and the solution was then irradiated at room

     temperature with a UV lamp (365 nm, 10 W, 45 min) or Xe lamp (450 nm, 150 W, 60 min). The samples were analyzed by electrophoresis for 1.5 h at 80 V on a 1% agarose gel in TBE buffer (89

     HEDTA, pH = 8.3). The gel was stained with 1 µg/mL ethidium mM Tris-borate acid, 2mM Na22 bromide and photographed on an Alpha Innotech IS-5500 fluorescence chemiluminescence &

    visible imaging system. 130

     In order to identify the actual reactive oxygen species responsible for DNA damage, a number of control experiments were carried out using various types of quenchers. Histidine (15 1 mM) and NaN(15 mM) were used as Oquenchers, superoxide ditmutase (SOD 100 unit) was 3 2 used as a superoxide anion radical quencher, mannitol (15 mM) and ethanol (15 mM) were used as

    OH scavengers. 135

     1.6 Cell lines and cell culture The cell lines, including hepatocellular carcinoma HepG2, melanoma A375, cervical cancer

     Hela, colorectal adenocarcinoma SW620 and lung adenocarcinoma cell A549 were supplied by Center of Experimental Animal Sun Yat-sen University (Guangzhou, China). Cells were routinely

    kept in DMEM or RPMI-1640 supplemented with 10% fetal bovine serum (Hyclone), penicillin G 140 -1? (Sigma-Aldrich, 100 U/mL) and streptomycin (Sigma-Aldrich, 100 mg mL) at 37 C in a humidified atmosphere containing 5% COAfter growth to confluence, the cells were detached 2. with a 0.25% trypsin for passage, and the cells were ready for the study until the cell growth was in a stable state and the logarithmic growth phase unless otherwise specified.

    1.7 Cytotoxicity assays 145 Cell viability was measured by its ability to transform tetrazolium to a purple formazan dye

     of MTT, according to previously reported procedures [27]. Firstly, cells were plated in 96-well 3 culture clusters (Costar) to grow overnight at a density of 2.5×10cells/well. When growing at

     30% confluence, the cells were treated with the test compounds in different concentrations for 48 h. After adding 20 μL/well of MTT solution (MTT working solution, 5 mg/mL phosphate buffered 150 saline) and being incubated for 5 h, the medium was aspirated and replaced with 150 μL/well

     DMSO to dissolve the formazan. The color intensity of the formazan solution, which reflects the cell growth condition, was quantified at 570 nm using a microplate spectrophotometer

     (SpectroAmaxTM 250). All datas were from at least three independent experiments and expressed

    as mean ? standard deviation. 155 1.8 Cellular uptake properties The complexes were co-incubated in the Hela cell lines with DAPI (a cell permeable agent that can enter cells and accumulate in nucleus). Cells were incubated with 10 μM complex and

     DAPI for 15 min at 37 ?C, 5% COin the dark, then washed with fresh growth medium for 1h to 2 remove loosely bound drug, and gently rinsed with PBS immediately prior to microscopy. The 160

     cells were illuminated through a filter cube containing a 450 nm excitation filter, a 590 nm long-pass emission filter for detection of the complexes. DAPI was detected using a 358nm

     excitation filter and a 460 nm long pass emission filter. Images of the complex and DAPI localization were taken in rapid succession.

    1.9 Flow cytometric analysis 165

    The cell cycle distribution was analyzed by flow cytometry as previously described [6]. Cells

    exposed to the complexes were trypsinized and washed with PBS. After adding with 70% ethanol

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     oC, the trypsinized cells were stained with propidium iodide (PI) for and overnight fixation at ?20

    4 h in darkness. The DNA content was measured by Epics XL-MCL flow cytometer (Beckman

    Coulter, Miami, FL) and cell cycle distribution was analyzed by MultiCycle software (Phoenix 170

     Flow Systems, San Diego, CA). The proportions of cells in G0/G1, S, and G2/M phases were represented as DNA histograms. Apoptotic cells with subdiploid DNA content were measured by

     quantifying the sub-G1 peak in the cell cycle pattern. Each experiment per sample was determined by recording 10,000 events.

     175 2 Results and discussion

     2.1 Synthesis and characterization

     2+ Complex [Ru(phen)(mitatp)]1 was prepared by direct reaction of 2 2+ [Ru(phen)(1,10-phenanthroline-5,6-diamine)]with the appropriate mole ratios of isatin 2 derivatives in acetic acid. The desired Ru(II) complex was isolated as perchlorate salts and was 1180 purified by column chromatography and characterized by H NMR, ESI-MS and elemental

    analyses. In the electrospray ionization mass spectrometry of the complex, only the signals of

    ?2+?+[M?ClO ] and [M?2ClO ] were observed, the measured molecular weight was consistent 4 4 11 H-H COSY (COSY = correlated spectroscopy) with expected values. With the aid of 1experiments and comparison with those of similar compounds [16, 28, 29], fully assigned H

    NMR spectra was obtained in DMSO. 185 2.2 Electronic absorption spectra 2+ The absorption spectra of [Ru(phen)(mitatp)]1 is characterized by intense π-π* ligand 2 transitions in the UV and metal-to-ligand charge transfer (MLCT) transition in the visible region. The broad MLCT absorption band appears at 457 nm and the peak below 200 nm is assigned to * the internal π-πtransition of the ligands. For metallointercalators, DNA binding is associated 190

     with hypochromism and a red shift in the MLCT and ligand bands [30]. The absorption spectra of 2+ [Ru(phen)(mitatp)]1 in the absence and presence of CT-DNA (at a constant concentration of 2

     complex, [Ru] = 20μM) are given in Fig. 2. Upon increasing concentration of DNA, the complex exhibits pronounced hypochromism and the hypochromism in the MLCT band at 457 nm reached as high as 16.7% at a ratio of [DNA]/[Ru] of 10:1. Similar hypochromicity was reported for 195 2+ 2+ [Ru(phen)(dppz)]and [Ru(bpy)(dppz)], which are believed to bind to DNA through 22 intercalation of the dppz ligand. In order to compare quantitatively the binging strength of 2+ [Ru(phen)(mitatp)]1, the intrinsic binding constant K of Ru(II) complex to DNA was 2 determined by monitoring the changes in absorbance at 457 nm (shown in the insets of Fig. 2) using Eq. (1) [26]. The intrinsic binding constants K and binding size n derived were (2.77 ? 0.40) 200 6 -1 2+ × 10M(n = 2.34 ? 0.05 bp). The K value is much larger than that of Δ-[Ru(phen)(dppz)]2 5 -12+ 5 -1 (3.2×10M), Λ-[Ru(phen)(dppz)](1.7×10M) [31], and is comparable with 2 2+ 6 -16 -1[Ru(phen)(phehat)](2.5 × 10M) [32] and the known DNA intercalator EB (1.4 × 10M) 2 2+ [33]. This result suggests that [Ru(phen)(mitatp)]1 intercalatively binds to DNA, involving a 2 strong stacking interaction between the aromatic chromophore and the base pairs of the DNA. 205

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     1.00 1.6 0.75 ε ?ε ) 0.50 )/( 1.2 ε ?a f 0 f ε 0.25 ( 0.00 0.8 0 5 10 15 20 5[DN A]*10 Absorbance

    0.4

     0.0

    300 400 500 600 700

     Wavelength / nm2+ Fig. 2. Absorption spectra of [Ru(phen)(mitatp)]1 in the presence increasing amount of CT-DNA. [Ru] = 20 2µM. Inset: plot (ε- ε)/(ε- ε) vs [DNA], and the nonlinear fit curve. a fb f

210 2.3 Viscosity Measurements A useful technique to prove intercalation is viscosity measurements, which are sensitive to length change of DNA and regarded as the least ambiguous and the most critical tests of binding mode in solution in the absence of crystallographic structural data or NMR spectra [34]. Under appropriate conditions, intercalation of drugs like ethidium bromide (EB) causes a significant

    215 increase in viscosity of DNA solution due to the increase in separation of base pairs of

     intercalation sites and hence results an increase in overall DNA contour length. On the other hand, drug molecules binding exclusively in the DNA grooves cause less pronounced or no changes in 2+ DNA solution viscosity [35]. The effects of [Ru(phen)(mitatp)]1 together with 2 2+ 2+ [Ru(phen)(dppz)]and [Ru(bpy)]on the viscosity of rod-like DNA are shown in Fig. 3. 23 2+ 220 [Ru(phen)(dppz)]increases the relative specific viscosity for the lengthening of the DNA 22+ double helix through the intercalation mode. While for complex [Ru(bpy)], which has been 3 known to bind with DNA in electrostatic mode, it exerts essentially no effect on DNA viscosity.

     On increasing the amounts of the complex, the relative viscosity of DNA increasing steadily, 2+which is similar to the behavior of the [Ru(phen)(dppz)]. The result suggests that 2 2+ [Ru(phen)(mitatp)]1 shows an intercalative binding mode to DNA. 225 2

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     1.26

     1.20 1/3) 0 η / 1.14η(

     1.08

     1.02

     0.00 0.04 0.08 0.12 0.16

    [Ru]/[DNA]

    2+ 2+ 2+ Fig. 3. Effect of increasing amounts of [Ru(phen)(mitatp)]1 (?), [Ru(phen)(dppz)](?) and [Ru(bpy)](?) 223on the relative viscosity of CT-DNA at 30 (? 0.1) ?C. The total concentration of DNA is 0.25 mM.

    230 2.4 DNA Thermal Denaturation Studies Thermal behaviors of DNA in the presence of complexes can give insight into DNA conformational changes when temperature is raised, and offer information about the interaction

     strength of complexes with DNA. According to the literature [36, 37], the intercalation of natural or synthesized organic- and metallo-intercalators generally results in a considerable increase in

    melting temperature (T). The melting curves of CT-DNA in the absence and presence of the 235 m complex are presented in Fig. 4. Here, the thermal denaturation experiment carried out for DNA in

     othe absence of Ru(II) complexes revealed a Tof 53.2?0.2 C under our experimental conditions. m

     The observed melting temperature in the presence of complex successively increased upon increasing its concentration. The melting point increased by 14?C at a concentration ratio of

    [Ru]/[DNA] = 0.10. The large increase in Tof DNA with the Ru(II) complex is comparable to 240 m

    2+ 2+that observed for classical intercalators Δ-[Ru(phen)(dppz)](16?C), Λ-[Ru(phen)(dppz)] 222+ (5?C) and EB (13?C) [31]. Thus, it could be rationally concluded that [Ru(phen)(mitatp)]1 is a 2

    typical DNA intercalator, which is consistent with the absorption spectra and viscosity results

    above.

     1.0

     0.8

     0.6

     0 f 0 DNA R=0.05 0.4A-A )\(A -A ) (R=0.10 0.2

     0.0 40 50 60 70 80 90o Tempreature / C 2452+ Fig. 4. Melting curves of CT-DNA (50 μM) at different concentration of [Ru(phen)(mitatp)]1: DNA alone (?); 2R ([Ru]/[DNA]) = 0.05 (?); 0.10 (?).

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     2.5 Luminescence spectroscopic studies 2+ [Ru(phen)(mitatp)]1 luminesces intensively in CHCN (φ = 0.121), but the emission 23

    intensities significantly decreased in Tris-NaCl buffer at ambient temperature(φ = 0.049). The 250

     results likely suggest that the more polar the solvent, the smaller relative intensities are observed. Similar phenomenon has also been found with the Ru(II) dppz complexes and its analogue 2+ [Ru(bpy)(mitatp)]2 [38]. The luminescence titration experiment was carried out in aqueous 2 solution. As shown in Fig. 5, upon the addition of CT-DNA, an obvious increase in emission

    intensity was observed. The emission intensity (λ= 594 nm) showed about 14 times 255 max enhancement and saturated at a [DNA]/[Ru] ratio of 11:1. This implies that the complex has a strong interaction with DNA, since the hydrophobic environment inside the DNA helix reduces

     the accessibility of water molecules to the complex and the complex mobility is restricted at the binding site, leading to decrease of the vibrational modes of relaxation. 2+ Excited state lifetime of [Ru(phen)(mitatp)]1 was also measured at a ratio of [DNA]/[Ru] 260 2

     of 11:1. The decay profile fits mono-exponential decay curves and the lifetime was determined to be 535ns in the absence of DNA. Upon binding to DNA, the complex exhibits biexponential decay

     curve with the lifetime increasing to microsecond scale 605, 1838ns (shown in the insets of Fig. 5). 2+ These values are much larger than that of [Ru(phen)(dppz)](120, 750 ns) [39] and its analogue 2 2+ 2+ [Ru(bpy)(mitatp)]2 (385, 1137 ns) [16] and comparable with [Ru(phen)(dppp2)](400, 1400 265 222+ 2+ ns) [39], [Ru(phen)(dppp3)](350, 1300 ns) [39], [Ru(phen)(dppn)](370, 1250 ns) [39]. 22

     800 1 1+DNA 600Intensity(a.u)

     400 0 2 4 6 8 10 Time / μs

     200Intensity (a.u.)

     0 500 550 600 650 700 750 800

    Wavelength / nm

     .2+ Fig. 5. Emission spectra of [Ru(phen)(mitatp)]1 in Tris-HCl buffer in the absence and presence of CT-DNA. 2 Arrow shows the intensity change upon increasing DNA concentrations. [Ru] = 2.5 µM. Inset: typical decay 2+ 270 profile for [Ru(phen)(mitatp)]1 in the absence and presence of CT-DNA. ([Ru] = 10 µM, [DNA]/[Ru] = 11) 2

     2.6 Photoactivated cleavage of pBR 322 DNA by Ru (II) complex 2+ As shown in Fig. 6 (a), [Ru(phen)(mitatp)]1 is able to photocleave pBR322 DNA under 2 irradiation at UV light (λ= 365 nm). No DNA cleavage was observed for the control in which

    metal complex was absent (lane 0). As the concentration of the Ru(II) complex increased (lanes 275

    1-4 ), the amount of Form I of pBR322 DNA diminished gradually, and the amount of the nicked

    circular DNA (form II) increased remarkably. When the concentration reached just 6 μM, DNA

    was completely converted from Form I to Form II, showing the complex to be an efficient

    2+ photocleaver of DNA. More interestingly, [Ru(phen)(mitatp)]1 can also efficiently damage 2

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280 λ= DNA under irradiation at visible light. As shown in Fig. 6 (b), after irradiation at visible light ( 450 nm) for 60 min, the complex can also completely cleavage DNA at 10 μM. Futhermore, the 2+ 2+photocleavage ability of [Ru(phen)(mitatp)]1 is stonger than its analogous [Ru(bpy)(mitatp)] 22

     2, who cleaves DNA at 10 μM concentration (λ= 365 nm) [16]. This may be attributed to its 32+ longer MLCT excited state lifetim (605, 1838ns) than [Ru(bpy)(mitatp)]2 (385, 1137 ns). The 2 2+ 285 longer excited lifetime makes [Ru(phen)(mitatp)]1 have longer time to exchange energy with 2 molecular oxygen of the air, thus generating more reactive oxygen species (ROS). In order to establish the reactive species responsible for the photoactivated cleavage of the

     plasmid, we further investigated the influence of different potentially inhibiting agents. Fig. 7 shows the typical results, the cleavage of the plasmid was not inhibited in the presence of hydroxyl radical (OH) scavengers such as mannitol (lane 4) and ethanol (lane 5), indicating that hydroxyl 290 radical (OH) was not likely to be the cleaving agent. In the presence of the superoxide anion -) scavenger superoxide dismutase (SOD) (lane 6), no inhibition was observed, which radical (O 2 -may also not be the reactive species. However, the addition of the single oxygen indicated that O 2 1O) quencher Histidine (lane 2) and NaN(lane 3) significantly inhibited the photocleavage (23 1295 activity of the Ru(II) complex, suggesting that Ois likely to be the reactive species responsible 2 2+ 1for the cleavage reaction. Complex [Ru(phen)]has also been reported to involve an O-based 32 mechanism [40]. 2+ Fig. 6. Photoactivated cleavage of pBR 322 DNA in the presence of [Ru(phen)(mitatp)]1, (a) after irradiation at 2 365 nm for 45 min. Lane 0, DNA alone; Lanes 1-4, in the different concentrations of complex: (1) 2; (2) 4; (3) 6; 300

    (4) 8 μM. (b) after irradiation at 450 nm for 60 min. Lane 0, DNA alone; Lanes 1-5, in the different concentrations of complex: (1) 2; (2) 4; (3) 6; (4) 8; (5) 10 μM. 2+ Fig. 7. Photocleavage cleavage of pBR322 DNA in the presence of [Ru(phen)(mitatp)]1 and different inhibitors 2 after irradiation at 365 nm for 45 min, lane 0, DNA control; lane 1, no inhibitor ( [Ru] = 8 μM); lanes 2-7: (2) Ru + 305 Histidine (15 mM), (3) Ru + NaN(15 mM), (4) Ru + Mannitol (15 mM), (5) Ru + ethanol (15 mM), (6) Ru + 3 SOD (100 unit).

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     2.7 In vitro cytotoxicity 2+310 Experiments were designed to investigate the in vitro cytotoxicity of [Ru(phen)(mitatp)] 2 1 against several human cancer cell lines, including hepatocellular carcinoma HepG2, melanoma A375, cervical cancer Hela, colorectal adenocarcinoma SW620 and lung adenocarcinoma cell

     2+ A549. For comparison purpose, the antiproliferative effect of the analogue [Ru(bpy)(mitatp)]2 2 has also been examined. Both complexes exhibit dose-dependent growth inhibitory effect against

    the tested cell lines. Table 1 shows the ICvalues of the Ru(II) complexes by MTT assay after a 315 50 48h treatment. Both complexes exhibited a broad spectrum of inhibition on human cancer cells, with ICvalues ranging from 20 to 130 μM, which were comparable with those of cis-platinum, 50 indicating great cytotoxic effects of the complexes on cancer cells. The SW620 cell was especially 2+ susceptible to [Ru(phen)(mitatp)]1, with a lower IC(21μM) than that of cis-platinum (30μM), 250 2+ displaying the higher antiproliferative activity of [Ru(phen)(mitatp)]1 than cis-platinum. 320 22+ 2+Notably, the ICvalues of [Ru(phen)(mitatp)]1 were lower than those of [Ru(bpy)(mitatp)] 50 222+ 2, manifesting its higher antiproliferatvie activity than [Ru(bpy)(mitatp)]2. 2

     2+ 2+ Table 1. ICvalues (μM) of [Ru(phen)(mitatp)]1 and [Ru(bpy)(mitatp)]2 against the selected cell lines. 50 22Complex HepG2 A375 Hela SW620 A549 2+1 [Ru(phen)(mitatp)] 58 37 27 21 20 22+123 52 36 34 52 2 [Ru(bpy)(mitatp)] 2cis-platium 15 17 15 30 19

    325 2.8 Cellular Uptake property The cellular uptake characteristics of transition metal-based PDT agents are important factors influencing their cytotoxicity and PDT efficacy [5]. It has been reported that for both ruthenium and platinum complexes, the rate of cellular uptake is significantly correlated with the cytotoxic 2+ activity [41-44]. To explore the cellular uptake property of [Ru(phen)(mitatp)]1 and 2 2+ 330 [Ru(bpy)(mitatp)]2, live cell imaging has been carried out using HeLa cells and the inherent 2 high fluorescence quantum yield of the two complexes facilitating visualization of fluorescence microscopy. As determined by fluorescence microscopy (Fig. 8), just after incubation with HeLa

     cells for 15min at 37 ?C, both complexes can cross membrane entering into cells, exhibiting prominent cellular uptake efficacy. But there is slight difference in the distribution for the two

    335 Ru(II) complexes, complex 1 appears predominantly localized in the nucleus, with lesser

    accumulation in the cytoplasm, while for complex 2, it could be observed both in nucleus and

    cytoplasm. The possible reason causing this difference is that the larger π-system of ancillary

    ligand phen might confers a lipophilic character to complex 1, thereby enhancing its cellular

    uptake efficacy [5, 41, 43].

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