DNA Binding and Photocleavage Properties, Cellular Uptake and

Article pubs.acs.org/IC

DNA Binding and Photocleavage Properties, Cellular Uptake and Localization, and in-Vitro Cytotoxicity of Dinuclear Ruthenium(II) Complexes with Varying Lengths in Bridging Alkyl Linkers Ping Liu,† Bao-Yan Wu,† Jin Liu,‡ Yong-Cheng Dai,† You-Jun Wang,‡ and Ke-Zhi Wang*,† †

Beijing Key Laboratory of Energy Conversion and Storage Materials, and Key Laboratory of Radiopharmaceuticals, Ministry of Education, College of Chemistry, and ‡College of Life Sciences, Beijing Normal University, Beijing 100875, P.R. China S Supporting Information *

ABSTRACT: Two new dinuclear Ru(II) polypyridyl complexes containing three and ten methylene chains in their bridging linkers are synthesized and characterized. Their calf thymus DNA-binding and plasmid DNA photocleavage behaviors are comparatively studied with a previously reported, six-methylene-containing analog by absorption and luminescence spectroscopy, steady-state emission quenching by [Fe(CN)6]4−, DNA competitive binding with ethidium bromide, DNA viscosity measurements, DNA thermal denaturation, and agarose gel electrophoresis analyses. Theoretical calculations applying the density functional theory (DFT) method for the three complexes are also performed to understand experimentally observed DNA binding properties. The results show that the two complexes partially intercalate between the base pairs of DNA. Cellular uptake and colocalization studies have demonstrated that the complexes could enter HeLa cells efficiently and localize within lysosomes. The in-vitro antitumor activity against HeLa and MCF-7 tumor cells of the complexes are studied by MTT cytotoxic analysis. A new method, high-content analysis (HCA), is also used to assess cytotoxicity, apoptosis and cell cycle arrest of the three complexes. The results show that the lengths of the alkyl linkers could effectively tune their biological properties and that HCA is suitable for rapidly identifying cytotoxicity and can be substituted for MTT assays to evaluate the cell cytotoxicity of chemotherapeutic agents.

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INTRODUCTION The wide use of Cisplatin and analogous drugs is limited by drug resistance and significant toxic side effects including neurotoxicity, gastrointestinal toxicity, and nepthrotoxicity.1−3 Therefore, it is of great significance to develop other metalbased cancer chemotherapies that are alternatives to platinumbased drugs. A wide variety of ruthenium-based complexes have emerged as leading players among the potential metal-based candidates for cancer treatment because of their promising anticancer activities. Among these complexes, NAMI-A (imidazolium trans-[tetrachloro(dimethyl sulfoxide)(1H-imidazole)-ruthenate(III)]) 4 and KP1019 (indazolium trans[tetrachlorobis(1H-indazole)ruthenate(III)])5 have previously been used in clinical trials.6 A large number of studies have also been performed on the anticancer activities of ruthenium(II) polypyridyl complexes,7,8 but many more studies must be undertaken to obtain insights into the structure−function relationship guiding the rapid developments of Ru-based drugs. DNA has been regarded as an important target of the drug, and adduct formation with the biomolecules was shown to inhibit cell division. The binding of ruthenium(II) polypyridyl complexes with DNA has been studied extensively because the interaction could cause DNA damage in cancer cells, consequently blocking the division of cancer cells and resulting © XXXX American Chemical Society

in cell death. The Ru complexes could also serve as DNA structural probes due to their unique electrochemical and photophysical properties that sensitively response to DNA structures.9a−c The DNA-binding properties of ruthenium(II) polypyridyl complexes have aroused great interest during the past decades in relation to not only anticancer drugs but also to DNA structural probes, cellular imaging, protein monitoring, and sequence-specific DNA cleaving agents.9−11 Subtle modification of the polypyridyl ligands and/or bridging linkers could result in marked differences in the DNA-binding behaviors and anticancer properties of the complexes. Moreover, the dinuclear Ru complex-based threading intercalators, which have displayed greatly enhanced DNA affinities compared to their mononuclear analogs, have received special attention.11 It would be very meaningful to understand the mechanisms controlling DNA threading intercalation, and to this end, we have investigated the effects of the length of the bridging linker of dinuclear ruthenium threading intercalators on DNA binding and anticancer properties. Cell proliferation and cytotoxicity measurement plays a key role in evaluating the anticancer effects of new drugs. The Received: August 25, 2015

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DOI: 10.1021/acs.inorgchem.5b01934 Inorg. Chem. XXXX, XXX, XXX−XXX

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Inorganic Chemistry

cytotoxicity of the Ru complex in cultured HeLa cells. The results demonstrated that the phenomenon and apoptosis rate obtained by HCA and the MTT assay were in close agreement to one another. However, compared to the MTT assay, HCA has been shown to be noninvasive, less time-consuming, highly sensitive and specific to establish subtle changes in cell health and cytotoxicity. The sensitivity of HCA for the detection of cytotoxicity was an order of magnitude greater than that of the MTT assay in assessing cytotoxicity.14 Furthermore, the HCA system acquires the data without damaging the cells, which allows the cells to be used later for the investigation of other end points or kinetic measurements. This new image-based technique is a good alternative to the colorimetric assay to determine the antiproliferative activity of different drugs. Lysosomes are acidic organelles that play essential roles in many cell signaling pathways and physiological processes, including intracellular transportation, protein degradation and recycling, cell membrane recycling, endocytosis, apoptosis, and autophagy.15−17 In addition, tumor metastasis and invasion are mainly associated with changed lysosomal trafficking and increased lysosomal enzyme activity and expression.18a Hence, tracking of lysosomes is essential for drug evaluation, gene delivery, and better understanding of the lysosome-associated events during autophagy.18b,c However, a limited number of fluorescent and bioluminescent lysosome probes have been reported and suffered from the need for biomolecular conjugation, low penetration depth, small Stokes shifts, poor water solubility, poor photostability, and pH sensitivity.19,20 Ru(II) complexes have gained increasing attention in biosensing and bioimaging because of their excellent photophysical properties, e.g., high quantum yields, large Stokes shifts, long-lived phosphorescence, relatively long excitation wavelength, and good photostability.21,22 Among Ru(II) complex-based cellular probes previously reported, most of them targets the nucleus, mitochondria and endosomel vesicle, Lysosome-targeted probes are lacking.9d−k In a recent paper,11d we have reported on the DNA binding properties and HeLa cell cytotoxic and preliminary cellular uptake properties of a six methylene-linked dinuclear Ru(II) complex (see the molecular structure of 2 in Scheme 1). However, the cellular localization of this complex is unclear.11d Herein, we report the synthesis and characterization of two analogous Ru(II) complexes (1 and 3) with ten- and three-methylene in their bridging linkers (see Scheme 1), respectively, and the effects of the linker lengths on their DNA binding and photocleaving, in vitro cytotoxic, and cellular uptake and localization properties. These properties are compared to those of some previously reported representative complexes (see Scheme 1 for their molecular structures).23−27

colorimetric 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay previously described by Mosmann is generally used to measure cytotoxicity and cell proliferation.12 However, HCA is a powerful, multiparameter bioanalytical tool that amalgamates the actions of fluorescence microscopy with automated cell analysis software to understand multiple changes in cellular health. It is capable of combining automated fluorescence microscopy, flow cytometry and advanced image analysis software to achieve quantitative analysis of multiple cellular events in single cells (Figure 1).13 To ensure the agreement between HCA and the MTT assay, a cell proliferation inhibition analysis has been performed to compare the tendency of apoptotic cells determined by the MTT assay and the HCA system, respectively, by assaying the

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EXPERIMENTAL SECTION

Materials. The Ru(II) complexes 1−3 were synthesized based on a route described in Scheme S1. The synthetic and characterization details for 2 have been published,11d and those for 1 and 3 are shown in the Supporting Information. Materials Characterization. The instrumentations and methods for the materials characterization are shown in the Supporting Information. DNA Binding Studies. The instrumentations and methods for the DNA binding studies are shown in the Supporting Information. The intrinsic binding constants, Kb, of the Ru(II) complexes bound to DNA were calculated from eq 1:

Figure 1. Schematic representation of HCA. Instrumentation exploits the combined power of high throughput multiwell automation with epifluorescence microscopy as well as the capability of multiwavelength fluorescent dye analysis. (A) Preparation of plates for HCS and incorporation of fluorescent probes for HCS. (B) Plates loaded into the ImageXpress Micro XLS System HCA platform for analysis. (C) Configure settings for cell cycle. (D) Images acquired at 10× or 20× magnification. (E) Cell Cycle module identifies cell cycle phases: G0/G1 (dark blue), S (light blue), G2 (green), Early M (orange), and Late M (red). (F and G) Preview classification results and interactively adjusted cutoff values. (H) Measurements directly exported to Microsoft Excel.

[DNA]/(εa − εf ) = [DNA]/(εb − εf ) + 1/[Kb(εb − εf )] B

(1)


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Inorganic Chemistry

optimization computations were performed by applying the DFTB3LYP method29,30 and the LanL2DZ basis set31 and assuming the singlet state for the ground state of the complexes.32−34,35a To vividly depict the detail of the frontier molecular orbital interactions, the stereographs of some related frontier molecular orbitals of the complexes were drawn with Gaussview based on the computational results. Cell Culture and in-Vitro Cytotoxicity Assay. HeLa cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with fetal bovine serum (10%), penicillin (100 units/ mL), and streptomycin (50 units/mL) at 37 °C in a CO2 incubator (95% relative humidity, 5% CO2). The cytotoxicity in vitro assay for the complexes was assessed with a standard 3-(4,5-dimethylthiazole)2,5-diphenyltetrazolium bromide (MTT) assay. The cells were placed in 96-well micro assay culture plates (5 × 103 cells per well) and grown overnight at 37 °C in a 5% CO2 incubator. The complexes to be tested were then added to the wells to achieve final concentrations ranging from 5 to 80 μM. The control wells were prepared by the addition of culture medium (100 μL). The culture medium and Cisplatin were used as negative and positive controls, respectively, and the plates were incubated at 37 °C in a 5% CO2 incubator for 48 h. Upon completion of the incubation, stock MTT dye solution (20 μL, 5 mg/mL) was added to each well. After 4 h of incubation, 100 μL of DMSO was added to solubilize the MTT Formosan. The optical density of each well was then measured on a microplate spectrophotometer at a wavelength of 490 nm. The IC50 values were determined by plotting the percentage viability vs concentration on a logarithmic graph and reading the concentration at which 50% of the cells remained viable relative to the control. All of the data were from at least three independent experiments and were expressed as the mean ± standard deviation. Cellular Uptake. To achieve laser confocal imaging, HeLa cells were grown in a laser confocal microscopy 20 mm2 Petri dish at a density of 1.0 × 104 cells per well; the cells were maintained at 37 °C under a 5% CO2 atmosphere for 24 h until they reached 70% confluency, followed by incubation with 20 μM of complexes 1−3 for 24 h at 37 °C. Upon completion of the incubation, the wells were washed three times with phosphate buffer solution (PBS) (pH = 7.4) after removing the culture mediums in the wells. Before imaging, the cell nuclei were stained with Hoechst 33342 (5 μg/mL) for 30 min. Confocal images were analyzed by a ZEISS LSM700 confocal microscope using a EC Plan-Neofluar 40x/1.30 oil objective. The confocal microscope was equipped with an Ar/Kr ion laser, which was used to excite 1−3 at 488 nm. Colocalization Studies. For achieving laser confocal imaging, HeLa cells were cultured on laser confocal microscopy 20 mm2 Petri dishes. The cells were incubated along with three Ru(II) complexes at a 20 μM concentration for 24 h and washed with PBS. Before imaging, the cell lysosomes, mitochondria and nuclei were stained with 10 μM LysoGreen for 2 h, 100 nM Mito-Tracker Green for 30 min and 5 μg/ mL of Hoechst 33342 for 30 min. Confocal images were analyzed by a ZEISS LSM700 confocal microscope using an EC Plan-Neofluar 40x/ 1.30 oil objective. The confocal microscope was equipped with an Ar/ Kr ion laser, which was used to excite 1−3 at 488 nm. High Content Imaging Data Acquisition and Analysis. The ImageXpress Micro (IXM) system (Molecular Devices, Sunnyvale, CA) is an automated field-based high content imaging platform integrated with MetaXpress (MDS Analytical Technologies) Imaging and Analysis software. The IXM system is a fully integrated pointscanning confocal automated field-based high content imaging platform configuration. The details of its optical system and instrumentation can be found at the Web site of Molecular Devices.35b Live-cell imaging was performed using an environmentally controlled ImageXpress Micro system, equipped with a 10× 0.5 NA S-Fluor Dry Objective (Nikon) and a 5% CO2, 37 °C humid-chamber stage incubator. Images were captured using a DAPI filter (387/11 nm Ex, 447/60 nm Em) for propidium iodide (PI) and a GFP filter (472/30 nm Ex, 520/35 nm Em) for Hoechst 33342. HCA Cell Cycle Arrest. HeLa cells (1 × 104 cells/well) were seeded in a 96-well plate. The plate was incubated in a humidified 37

Scheme 1. Molecular Structures of Analogous Ru Complexes

in which [DNA] is the DNA concentration; εa, εf, and εb correspond to the apparent absorption coefficient Aabs/[Ru], the extinction coefficients for the free complex and the complex in the fully bound form, respectively. The Kb values were derived from the ratio of the slop to the intercept of plots of [DNA]/(εa − εf) vs [DNA]. DNA Photocleavage Experiments. The photoinduced DNA cleavage by the complex was examined by agarose gel electrophoresis. Supercoiled pUC18 DNA (0.2 μg) was treated with the ruthenium complex in TAE buffer (40 mM Tris, 20 mM acetic acid, 1 mM EDTA, pH = 8.0), and the solution was then irradiated at room temperature with UV light (360 nm) for 1 h. The samples were analyzed by electrophoresis for 1 h at 80 V on a 0.8% agarose gel in TAE buffer. The gel was stained with 1 μg/mL of ethidium bromide (EB) and photographed under UV light. The percentage of cleavage (C) was calculated according to eq 2:

C=

DII + 2DIII DI + DII + 2DIII

(2)

in which DI, DII, and DIII are the integrated density values of Form I (supercoil form), Form II (nicking form), and Form III (linear form), respectively. Theoretical Calculations. The structural schematic diagram of the title complexes is shown in Scheme S1. The octahedral complex forms from two Ru(II) ions, four bidentate coligands (dipyridyl) and a bridge ligand that consists of different lengths of alkyl chain. The theoretical computations for 1−3 were performed by applying the DFT method. All of the calculations were performed with the Gaussian 98 quantum chemistry program package.28 Full geometry C


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Inorganic Chemistry °C/5% CO2 cell culture incubator. When the cells reached 70% confluence, the media were changed. The cells were treated with 1−3 (50 μM). After incubation for 24 h, the medium was removed, and 100 μL of PI (10 mg/mL) was added to the cell suspensions; they were then incubated at 37 °C for 20 min. For each well, six random field-ofview images were acquired to examine each parameter using a 10× objective magnification. Optimal exposure times were 150 ms for Hoechst 33342 and 500 ms for PI. The images were obtained on the ImageXpressMICRO system using MetaXpress image acquisition software (all Molecular Devices imaging platforms are compatible with the Cell Health Application Module). The images were analyzed with the Cell Cycle Application Module of MetaXpress imaging analysis software. Data for cell number, DNA content, DNA average intensity, and apoptotic classification were acquired on a high content image processing software-MetaXpress Offline. Interactive color-coded graphs allow set classification cutoffs to be easily established. The cell cycle distribution was analyzed using MetaXpress software. The proportions of cells in the G0/G1, S, G2, early M, and late M phases were represented as DNA histograms. Apoptotic cells with hypodiploid DNA contents were measured by quantifying the subG1 peak. HCA Apoptotic Inducing Activity. HeLa cells were seeded into 96-well plates at a density of 1 × 104 cells/well and incubated at 37 °C and 5% CO2 overnight. The medium was removed and replaced with medium containing complexes 1−3. After incubation for 24 h, the medium was removed, and the cells were incubated for 20 min with a mixture of Hoechst 33342 (0.5 mM) and PI (10 mg/mL). The Hoechst 33342 dye was used to measure nuclear morphology (CN, NI and NA). Total cell counts were based on number of Hoechst-stained cell nuclei whereas colocalization with PI staining was scored as necrotic. Filters compatible with Hoechst 33342 and PI include BrightLine Dapi-5060B (Semrock) and Texas Red TXRED-4040B (Semrock), respectively. For each well, six random field-of-view images were acquired to examine each parameter. The images were obtained on the ImageXpressMICRO system using MetaXpress image acquisition software. The images were analyzed with the Cell Cycle Application Module of MetaXpress imaging analysis software. Statistical Analysis. All of the experiments were performed with technical replicates on three independent occasions. Data exported from the MetaXpress High Content Image Processing workstation software were illustrated using GraphPad 5.0 Prism software and were expressed as the mean ± SEM. The statistical analyses were performed in GraphPad using an analysis of variance (ANOVA) with P ≤ 0.05 selected as the criterion of statistical significance.

Figure 2. UV−vis absorption spectra of the complex 1 (1 μM) in the absence and the presence of increasing concentrations of DNA (0−1.6 μM).

DNA, but groove binding and partial intercalation could not be excluded. The values of the intrinsic DNA binding constant Kb were determined by monitoring the changes of absorbance at 287 nm with increasing DNA concentrations with the data over high DNA concentrations ranging from 0.9 to 2.0 μM. The intrinsic DNA binding constants Kb obtained for 1, 2, and 3 were derived to be 2.1 × 106, 2.5 × 106, and 1.2 × 106 M−1, respectively, which were the same order of magnitude as the Kb values of 1.25−4.9 × 106 M that had been previously reported for some proven classical intercalators of 10,25a 11,25b 12,11e and EB,25c and Kb values of 5.0 × 106 M reported for 1311f with mixed binding modes of classic and partial intercalation. However, unlike the threading intercalators of 14−16,11c the Kb values for 1−3 were not significantly enhanced compared to a similar parent Ru(II) complex 17 (Kb = 8.2 × 105 M−1).25d In view of the experimental errors, these values of the binding constant indicate that they are close to one another and are not very sensitive to the linker length; moreover, the two Ru(II) centers may not bind to the same double strands of the DNA simultaneously. Luminescence Spectroscopy Study of the DNA Binding Properties. In contrast to the emission enhancements previously reported for DNA molecular light-switch Ru(II) complexes, such as the dipyrido-[3,2-a:2,3′-c]phenazine (dppz)-based complexes of 1026a and 18,26b dipyrido[2,2d:2′,3′-f ]quinoxaline (dpq)-based complexes of 19,26c 20,26d and 21−2327a and an analogous complexes 24 and 25,27b,c evident emission quenching of 1 (λem = 608 nm) and 3 (λem = 606 nm) by 55% (Figure 3) and 64% (Figure S2), respectively, was observed at saturation {e.g., a saturated concentration ratio of [DNA]/[Ru] = 1.39 were derived from Figure S2, in which the last two points show saturated binding, and were not included for the linear regression} with slight bathochromic shifts less than 5 nm, similar to the behaviors of 2.11d This large emission quenching is unusual compared to previously observed emission quenching for analogous dinuclear Ru(II) complexes of 1211e (by only 16%) with a rigid linker and 14− 1611c with flexible linkers (by less than 10%). The emission quenching followed the Stern−Volmer equation (I0/I = 1 + KSV[DNA]) with Stern−Volmer constant KSV values of (1.08 ± 0.02) × 106 M−1 for 1 and (1.28 ± 0.02) × 106 M−1 (R2 = 0.00904 obtained from Figure S2) for 3, which are similar to

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RESULTS AND DISCUSSION UV−vis Spectroscopy Studies on DNA Binding Properties. The absorption spectra of complexes 1 and 3 in the absence and presence of increasing concentrations of calf thymus DNA (CT-DNA) are shown in Figure 2 and Figure S1, respectively. The spectral behaviors of 1 and 3 are very similar to those of 223 with obvious hypochromism, H% (as defined by H% ≅ 100(Afree − Abound)/Afree), and bathochromism, as indicated by Δλ (Δλ = λbound − λfree). Upon increasing concentrations of CT-DNA to the constant spectra (saturation), the H% (Δλ) values at ∼280 nm were found to be 70 (4 nm), 73 (5 nm), and 63% (3 nm) for 1, 2, and 3, respectively; the H% (Δλ) values at ∼460 nm were found to be 47 (8 nm), 51 (6 nm), and 36% (5 nm) for 1, 2,11d and 3, respectively. It was reported that dinuclear Ru complex-based DNA groove binders of 4,23a 5,23b and 623 and partial intercalators of 7,24a 8,24b and 924c could exhibit obvious bathochromic shifts and hypochromisms in their visible absorption and/or UV bands upon their binding to the DNA, in which 4,23a for example, had H% (Δλ) values of ∼20% (+20 nm). Thus, the evident spectral changes observed for 1−3 suggest a strong interaction between the complexes and the D


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Inorganic Chemistry

Figure 4. Emission spectra of EB bound to DNA ([DNA] = 25 μM, [EB] = 5 μM) in the presence of the complex 1 (0−4.4 μM). The arrows show the intensity changes upon increasing concentrations of the complex. (inset) percentage of EB displaced by 1 from EB bound DNA.

Figure 3. Emission spectra of the complex 1 (1 μM) in the absence and the presence of increasing concentrations of DNA (0−1.6 μM).

the KSV value of (1.30 ± 0.05) × 106 that we previously reported for 2.11d Steady-State Emission Quenching by K4[Fe(CN)6]. Steady-state emission quenching experiments using K4[Fe(CN)6] as a quencher may provide further information about how well the binders could be protected by bound DNA from emission quenching by [Fe(CN)6]4−, although the method could not provide direct evidence for intercalation.36,37 As illustrated in Figures S3 and S4, in the absence of DNA, 1 and 3 were efficiently quenched by [Fe(CN)6]4−, but when bound to DNA, the quenching efficiency of [Fe(CN)6]4− decreased significantly, giving quenching constants of nearly zero. This may be explained by repulsion between the highly negatively charged [Fe(CN)6]4− and the DNA polyanion backbone, which hinders the access of [Fe(CN)6]4− to the DNA-bound complexes38 and indicates that there is a strong interaction between the two complexes and DNA, which is in accordance with the UV−vis absorption and emission observations. R, the ratio of a KSV value in the presence of the DNA to that in the absence of the DNA, was derived to be 0.018 for 1, 0.027 for 2 and 0.034 for 3; these values are smaller than the R value of 0.051 that was previously reported for parent mononuclear complex 24,39 indicating that 1−3 were more effectively protected by the DNA from emission quenching than 24, with 1 being the best protected among 1−3; this appears consistent with the order of their Kb values. Ethidium Bromide Displacement Experiments. The competitive binding experiments with a well-established quenching assay based on the displacement of the intercalating drug EB from CT-DNA may provide strong information for the DNA binding properties of the complexes. If a complex could displace EB from DNA-bound EB, the fluorescence of the solution would be greatly reduced because of the increased free EB molecules that were readily quenched by the surrounding water molecules.40 Figures 4 and S5 show the fluorescence quenching of DNA-bound EB by 1 and 3, respectively. Upon the addition of 1 and 3, sharp decreases in EB emission intensities were observed, indicating that 1 and 3 could displace EB from DNA, similar to the behavior of 2.11d The competitive binding experiment results suggest that 1−3 interact with DNA

through intercalation. The quenching plot (Figures 4 and S5 insets) illustrates that the removal of EB from DNA-bound EB by 1 and 3 are in good agreement with the linear Stern− Volmer equation. We can also determine from the data in Figures 4 and S5 that 50% of the EB molecules were displaced from DNA-bound EB at a concentration ratio of [Ru]/[EB] ≈ 0.47 for 1 and 0.48 for 3. By assuming a DNA binding constant of 2.6 × 105 M−1 for EB,41 the apparent DNA binding constant values were derived to be 3.0 × 106 M−1 for 1 and 2.9 × 106 M−1 (Kb(EB)/([Ru]/[EB]) for 3,41 which are close to a Kb value of 1.25 × 106 M−1 for 2.11d These above-mentioned Kb values are consistent with those derived by UV−vis absorption spectroscopy. Thermal Denaturation of DNA. The thermal behaviors of DNA in the presence of complexes can provide insight into how they can stabilize or destabilize double-stranded DNA and their conformational changes when the temperature increases. It is well-known that as the temperature increases, doublestranded DNA gradually dissociates into single strands and generates a hyperchromic effect on the absorption of DNA base pairs at 260 nm. The melting temperature Tm, which is defined as the temperature at which half of the total base pairs are unbound, is usually examined. The melting curves of CT-DNA in the absence and presence of complexes 1 and 3 are presented in Figures S6 and S7, respectively. The Tm value of free CTDNA was determined as 60.2 ± 0.2 °C, which was increased to 64.4 ± 0.1 and 63.8 ± 0.2 °C in the presence of 1 and 3, respectively, at [Ru]/[DNA] = 1:10. These increased ΔTm values of 4.2 °C for 1 and 3.6 °C for 3 are similar to the ΔTm value of 3.5 °C that was previously reported for 2,11d but are smaller than those previously reported for some dinuclear intercalators, such as 9.3 °C for 1311f and >15 °C for 15 and 16,11c indicating that 1−3 could not effectively stabilize the double strands. In addition, these small ΔTm values could not be warranted to exclude the intercalative binding mode because we have observed that a DNA intercalating and high DNA affinity dinuclear complex 14 (Kb = 5.7 × 107 M−1) only induced a small ΔTm of 1.5 °C.11c Evidence has accumulated to demonstrate that the magnitude of ΔTm values does not invariably parallel the DNA binding affinities. E


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Inorganic Chemistry

DFT Theory Calculations. The different DNA-binding behaviors and some spectral properties of these three complexes can be reasonably explained by the DFT computations and the frontier molecular orbital theory.44−46 The computational selected bond lengths, bond angles, and dihedral angles of 1−3 using the DFT at the B3LYP/LanL2DZ level are shown in Table S1 together with the structural schematic diagrams for 1−3. The dihedral angles of the imidazole and carbazole moieties in 1−3 are all close to 180.00° (174.93°, 174.83°, and 178.16° for 1−3, respectively), suggesting that the alkyl chain length between the two carbazoles does not significantly affect the planarity of the main ligand, but the planarity of 3 appears slightly better than that of 1 and 2. The computed energies of some frontier molecular orbitals (MOs) using the DFT method at the B3LYP/LanL2DZ level are shown in Table S2. The stereocontour graphs of the frontier molecular orbitals (MOs) of the complexes are shown in Figure S10. The electrons of the lowest unoccupied molecular orbitals LUMO + x (x = 4, 5, 6) in the ground state are mainly distributed on the main ligands whereas those of LUMO and LUMO + x (x = 1, 2, 3) are mainly distributed on the ancillary ligands bpy of the complexes. According to frontier molecular orbital theory,47 LUMOs that distributed on the main ligands are closely related to the DNAbinding properties of 1−3. The low energies of these LUMOs of 1−3 are advantageous for accepting the electrons offered from base pairs of DNA and is thus favorable for DNA binding.48 The LUMO + x (x = 4, 5, 6) energies of 1−3 are all negative and rather low, thus showing that these complexes are very excellent electron acceptors in their DNA binding. Moreover, the LUMO + x (x = 4, 5, 6) energies follow decreasing order as 1 > 2 > 3. Obviously, relatively lower LUMO energy and the better planarity of 3 should contribute to greater DNA affinity, which was the opposite of the experimental observation, indicating that the length effect in the alkyl linkers may offset the above-mentioned two factors. In-Vitro Cytotoxic Assay. Cytotoxicity was tested for 1−3 by an MTT assay against HeLa and MCF-7 cell lines as depicted in Figures 6 and S11 respectively, with Cisplatin being

DNA Viscosity Measurements. DNA viscosity measurements are generally viewed as the least ambiguous and the most critical forms of evidence for DNA binding modes in solution in the absence of NMR or crystallographic data.42 A classical intercalation can elongate the DNA double strands due to the increase in distance between adjacent base pairs so as to accommodate the incoming intercalators, leading to the increased DNA viscosities.43 In contrast, a nonclassical or partial intercalation of the binders could bend (or kink) the DNA helix, decreasing the effective length and its viscosity concomitantly.37 To further elucidate the binding modes of 1− 3, DNA viscosity measurements were performed by keeping [DNA] at 0.4 mM and varying the concentrations of 1−3. The changes in the relative DNA viscosities in the presence of 1−3 are compared in Figure 5. Upon increasing the concentrations

Figure 5. Effects of increasing concentrations of the complexes 1−3 on the relative viscosities of the DNA (0.2 mM) in buffered 50 mM NaCl.

of 1−3, the relative viscosities of DNA decreased steadily, suggesting a partial intercalation mode for 1 and 3, which is sharply different from the increased DNA viscosities for 2 with a classic intercalation.42 These facts indicate that the linker lengths in the bridging ligands in 1−3 have profound effects on their DNA binding modes. Photoactivated Cleavage of pUC18 DNA. The results for 1 and 3 to cleave pUC18 DNA studied by gel electrophoresis are shown in Figures S8 and S9, respectively. No DNA cleavage was observed for the control in which complex 1 or 3 was absent (lane 0) or in the incubation of the plasmid with 1 or 3 in the dark (lane 1). Upon increasing concentrations of 1 or 3, the amount of Form I of pUC 18 DNA diminished gradually, and the amount of the nicked circular DNA (Form II) increased. As the concentrations of complexes 1, 2, and 3 were 5 μM, 12.3%, 52.0%,11d and 13.9% of Form I were converted to Form II, indicating that their DNA photocleavage efficiencies follow the order: 2 > 1 ≈ 3. The stronger DNA photocleavage ability of 2 than those of 1 and 3 could be ascribed to the classic intercalative binding mode of 2 with respect to the partial or nonclassical intercalative mode of 1 and 3 as revealed by the viscosity measurements, i.e., the partial or nonclassical intercalative mode may be less favorable for the DNA cleavage than the classical intercalative mode.

Figure 6. In-vitro cytotoxicites of complexes 1, 2, and 3 against HeLa cells measured by MTT assay after 48 h.

as the positive control and 1−3-free cells as the negative control. The results were compared to see the differences that might arise from their linker length changes. A significant difference was also noted for 1−3 in the selected cell lines. From the obtained data, 1 and 3 can effectively inhibit the HeLa cells’ viability in a dose-dependent manner, and it was F


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Inorganic Chemistry

Figure 7. Confocal luminescence microscopy images of HeLa cells without (A, negative control) and with treatments of 20 μM of Ru(II) complexes (red) of 1 (B), 2 (C), 3 (D) for 24 h, stained with Hoechst 33342 (blue) and Mitotracker (green) as well as merged images.

Figure 8. Confocal luminescence microscopy images of HeLa cells without (A, negative control) and with treatments of 20 μM of Ru(II) complexes (red) of 1 (B), 2 (C), 3 (D) for 24 h, stained with Hoechst 33342 (blue) and Lysotracker (green) as well as merged images.

clear that complex 1 was more potent against the selected tumor cell lines than 2 and 3 under identical conditions. However, as compared to Cisplatin with IC50 values being found to be 6.84 μM against HeLa and 21.62 μM against MCF7 cell lines, these complexes all exhibited relatively lower in vitro cytotoxicity: IC50 values were 13.85 μM for 1, > 100 μM for 2, and 46.72 μM for 3 against HeLa cell lines; IC50 values were 70.63 μM for 1, >100 μM for 2, 83.25 μM for 3 against

MCF-7 cell lines. The cytotoxicities of these complexes decrease in the following order: 1 > 3 > 2, which is inconsistent with their DNA-binding affinities, indicating that the cytotoxicities may not originate from the targeting at DNA of 1−3. Cellular Uptake Properties and Colocalization Studies. Similar to the previously reported behavior of 2, the luminescence intensity of the cell population increased G


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Inorganic Chemistry dramatically after the cells were incubated for 24 h with 20 μM of 1 and 3 under a fluorescence microscope compared to the autofluorescence of untreated cells.23 Because of good luminescent stability of 1−3 aqueous solutions, the cellular uptake properties of 1−3 can be conveniently studied using confocal microscopy. To this end, costaining of 1−3 with nucleus and DNA specific dye Hoechst 33342 was conducted in HeLa cells. As shown in Figure S12, the complexes were actually transported into the cellular interior rather than associating solely at the membrane surface as we recently reported for a Ru(II)−Re(I) heteropolynuclear complex.11f The amounts of ruthenium complexes 1−3 accumulated in the cytosol fractions and membrane accounted for approximately 90% of total 1−3 taken up by the cells, and the complexes could interact with cellular components that are present dominantly in the cytosol and which consequently stand for its potential targets. Cellular accumulation in a certain organelle is critical to enable biological activity, and any potential therapeutic and imaging agent must also be able to reach its target within the cell.49 To obtain further information on the exact subcellular localization of complexes, colocalization studies of complexes 1−3 with the lysosomal dye (LysoTracker Green), and the mitochondrial dye (MitoTracker Green) were then assessed by confocal microscopy. First of all, we can see negligible red luminescence from negative control experiments, namely the images in the last column of Figures 7 and 8 that were obtained by 488 nm excitation of Hela cells stained with Hoechst 33342 and the MitoTracker/the LysoTracker in the absence of 1−3. However, the images in the fourth column of Figure 7B−D showed evident red luminescence from 1−3 treated HeLa cells stained with Hoechst 33342 and the MitoTracker; this red coloration does not overlap with the blue coloration from the nucleus stained with Hoechst 33342, and green coloration from mitochondria stained with the MitoTracker, indicative of absence of 1−3 within the mitochondria and once again cell nucleus. In contrast, as shown in Figure 8, the luminescence images of HeLa cells treated with 1−3 and LysoTracker green showed clear regional colocalization. The observed yellow coloration (the merged images in the last column of Figure 8) was caused by the overlapping of the green fluorescence from LysoTracker Green stained lysosomes and the red luminescence from Ru complexes 1−3, which indicated the accumulation of 1−3 into the lysosomes of HeLa cells like LysoTracker Green and once more exclusion of 1−3 in cell nucleus. Image Analysis Using the Cell Cycle Application Module. The effects of 50 μM of 1−3 on HeLa cell cycles were investigated by MetaXpress in PI-stained cells after treatments for 24 h, and the results are shown in Figures S13 and 9. As comparing to the control, although the effects of the complexes on the cell cycles were not very significant, an obvious increase of 10.38 and 7.79% for 1, 13.03 and 4.87% for 2, and 11.20 and 12.44% for 3 of cells in the S phase and the early M phase were observed, respectively, accompanied by a corresponding reduction of 12.73 and 7.94% for 1, 12.21 and 5.93% for 2, and 22.17 and 3.84% for 3 in the percentage of cells in the G0/G1 phase and the G2 phase, respectively (see Figure 9). These results clearly showed that 1 and 3 resulted in more evident changes in the cell cycle than 2, and that the antiproliferative mechanism of 1−3 on HeLa cells is dominantly G0/G1 phase and S phase arrests. HCA Apoptosis Studies. The confocal images were acquired after treating HeLa cells with 50 μM of complexes

Figure 9. Quantitative cell cycle distribution data for HeLa cells after treatment with 50 μM of 1−3 for 24 h, as studied by HCA. Data shown are mean values ± standard deviations of three samples for each treatment.

1−3 and counterstaining with Hoechst 33342. As clearly shown in Figure S12, we observed the morphological differences of nuclei by 1, 2, and 3 with membrane-permeable Hoechst stains. Because complexes 1 and 3 showed toxic effects on HeLa cells, which could induce cell apoptosis, significant morphology changes such as condensed chromatin and fragmented nuclei can be visualized compared to normal cell nuclei with the treatment of 2 under identical conditions. These data confirmed that 1 and 3 could induce apoptosis in HeLa cells. To quantify the apoptosis, we performed Hoechst 33342 and PI staining experiments to examine the percentage of apoptotic cells and changes in the DNA content distribution in the HeLa and MCF-7 cells that were treated with complexes 1−3 with the help of Metaxpress software. PI is membrane-impermeable and generally excluded from viable cells. During late stage apoptosis and necrosis, the membrane increased its permeability, allowing PI to enter the cell and label DNA with red light. Hoechst 33342 can be used as both a marker for all nuclei and for apoptotic nuclei with blue light. HeLa cells were grown, treated with 1−3 for 24 h, and labeled with the two fluorescent markers as indicated in the Experimental Section. As shown in Figure S13, increasing concentrations of 1−3 induced more red fluorescence from PI and less blue fluorescence from Hoechst 33342. The results suggested the presence of apoptotic cells after exposure to 1−3 and occurred in a dose-dependent manner. Based on the HCA experimental results shown in Figure S13, IC50 values were derived to be 29.95 μM for 1, >100 μM for 2, and 58.43 μM for 3 against HeLa cell lines and 60.58 μM for 1, >100 μM for 2, 95.85 μM for 3 against MCF-7 cell lines. These IC50 values are consistent with those derived from MTT experiments in view of experimental errors caused by different techniques. The number of apoptotic cells was estimated by gating on the subdiploid population. Cells with extensive DNA breakdown were not considered apoptotic cells and were gated out. Thus, we could count and differentiate the cells undergoing apoptosis or necrosis. The data were represented as the percentage of apoptotic cells in the entire cell population. The representative results for 80 μM of 1−3 are depicted in Figure 10. For a fixed Ru(II) complex, the apoptotic percentage of Hela cells is lower than that of MCF-7, and the order of the apoptotic percentages for both HeLa and MCF-7 cells are as follows: 1 > 3 > 2, which is consistent with that of MTT studies. Many studies in the literature50 and our study here pointed to a fact that moderate lysosomal H


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Inorganic Chemistry

may contribute to the future development of improved chemotherapeutics against human cancers whereas 2 may contribute to the future development of improved emission lysosomal probes. Currently, this is the first report demonstrating HCA as a new method for understanding cytotoxic and cell cycle arrest properties of ruthenium-based metal complexes. We have given evidence for the facts that inhibition of HeLa cell proliferation by 1−3 could be due to the combination of the induction of apoptosis and cell cycle arrest; moreover, the lysomal uptake of 1−3 may trigger the cell apoptosis. Further mechanistic studies are being planned. As revealed by Table S3, much work is needed to get insights into the structure− property relationship for 1−3 and their analogous complexes.

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Figure 10. Percent of apoptotic HeLa and MCF-7 cells after treatment with 80 μM of 1−3, derived in the experiments as described in the caption of Figure S13. Data shown are mean values ± standard deviations of three samples for each treatment.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.inorgchem.5b01934. Details of synthesis and characterization, instrumentations and methods for characterization and DNA binding properties, synthetic routes to 1−3, figures associated with DNA binding properties of the complex 3 as well as DNA cleavage for complex 1, DNA thermal melting curves for 1, and emission quenching of DNA-free and DNA-bound 1 by K4[Fe(CN)6], stereocontour graphs of the some frontier molecular orbitals, MCF-7 cell viability after treatments with complexes 1−3, confocal luminescence microscopy, brightfield and overlay images of HeLa cells treated with 1−3 and costained by Hoechst 33342, computed selective bond lengths and angles of 1−3, HCA studies on PI and Hoechst 33342 costaining, energies of some related frontier molecular orbitals for complexes 1−3, and comparisons of molecular biological properties of complexes 1−3 and their analogous complexes (PDF)

permeabilization could result in programmed cell death. Further studies are essential to evaluate the involvement of lysosomes in the mechanism of cell death of 1−3.

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CONCLUSIONS In conclusion, two dinuclear ruthenium(II) complexes, 1 and 3, have been synthesized and characterized. The comparative studies of 1 and 3 with 2 by UV−vis absorption and emission spectroscopy revealed that unusually large hypochromism and emission quenching, and effective protection from emission quenching by [Fe(CN)6]2− were elicited because they bound to the DNA with similar DNA affinity on the 106 M−1 order of magnitude. However, 1−3 all stabilize DNA double strands slightly, as indicated by the small increases in DNA melting temperatures (ΔTm = 3.5−4.2 °C). EB displacement and DNA viscosity experiments showed that 1 and 3 bound to the DNA in a partially intercalative mode (the decreases in DNA viscosity), which was in sharp contrast to the classic intercalation exhibited by 2 with an increase in DNA viscosity. The changes in the alkyl chain lengths in 1−3 also brought about profound effects on the following properties: in vitro cytotoxicity against the HeLa cell lines with an order of 1 > 3 > 2, which appears inconsistent with their DNA affinity, indicating that the cytotoxicities may not originate from the targeting at DNA of 1−3; the pUC 18 DNA photocleaving capacity of classic intercalating 2 was stronger than partially intercalating 1 and 3. DFT calculations showed that the length effect in the alkyl linkers may make a dominant contribution to the experimentally observed DNA affinity with the shortest linker-containing 3 being less favorable for DNA binding than other two analogs. The cellular uptake studies revealed that all of the complexes had good uptake properties targeting lysosomes. The cell cycle arrest studies by HCA demonstrated that the antiproliferative effect induced by the Ru complexes on HeLa cells occurred in the G0/G1 phase and in S phase arrest. The apoptosis assay by HCA using Hoechst 33342 staining and PI staining demonstrated that 1 and 3 effectively induced apoptosis of HeLa cells, and the number of apoptotic cells increased with increasing concentrations. This HCA experiment also showed the ease-of-use of HCA and that it is applicable for the rapid screening of a large number of agents for their effects on cell health, replacing the traditional MTT assay; moreover, it is a powerful tool for screening potential toxic therapeutic agents and for analyzing specific pathways of toxicity. Taken together, these findings suggested that 1 and 3

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Fax: (+86-10-58802075). Tel.: (+86-10-58805476). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Financial support by the National Natural Science Foundation of China (21171022 and 21541010), Program for Changjiang Scholars and Innovative Research Team in University, Key Laboratory of Radiopharmaceuticals, Ministry of Education, the Fundamental Research Funds for the Central Universities (2014KJJCB08), and Analytical and Measurements Fund of Beijing Normal University is greatly acknowledged.

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