Chiral Ruthenium(II) Polypyridyl Complexes: Stabilization of G-Quadruplex DNA, Inhibition of Telomerase Activity and Cellular Uptake

Two ruthenium(II) complexes, Λ-[Ru(phen)2(p-HPIP)]2+ and Δ-[Ru(phen)2(p-HPIP)]2+, were synthesized and characterized via proton nuclear magnetic resonance spectroscopy, electrospray ionization-mass spectrometry, and circular dichroism spectroscopy. This study aims to clarify the anticancer effect of metal complexes as novel and potent telomerase inhibitors and cellular nucleus target drug. First, the chiral selectivity of the compounds and their ability to stabilize quadruplex DNA were studied via absorption and emission analyses, circular dichroism spectroscopy, fluorescence-resonance energy transfer melting assay, electrophoretic mobility shift assay, and polymerase chain reaction stop assay. The two chiral compounds selectively induced and stabilized the G-quadruplex of telomeric DNA with or without metal cations. These results provide new insights into the development of chiral anticancer agents for G-quadruplex DNA targeting. Telomerase repeat amplification protocol reveals the higher inhibitory activity of Λ-[Ru(phen)2(p-HPIP)]2+ against telomerase, suggesting that Λ-[Ru(phen)2(p-HPIP)]2+ may be a potential telomerase inhibitor for cancer chemotherapy. MTT assay results show that these chiral complexes have significant antitumor activities in HepG2 cells. More interestingly, cellular uptake and laser-scanning confocal microscopic studies reveal the efficient uptake of Λ-[Ru(phen)2(p-HPIP)]2+ by HepG2 cells. This complex then enters the cytoplasm and tends to accumulate in the nucleus. This nuclear penetration of the ruthenium complexes and their subsequent accumulation are associated with the chirality of the isomers as well as with the subtle environment of the ruthenium complexes. Therefore, the nucleus can be the cellular target of chiral ruthenium complexes for anticancer therapy.


Introduction
Guanine (G)-rich nucleic acid sequences tend to adopt remarkably stable secondary structures known as G-quadruplexes. [1][2][3][4] Human telomeres consist of simple tandem repeats of the Gtract sequence (TTAGGG/CCCTAA) n , which consists of a singlestranded tandem [TTAGGG]-repeated sequence over several hundred bases. [5], Kim et al. [6] reported that telomerase is activated in approximately 85% of cancer cells, whereas it is undetectable in most normal somatic cells. Thus, telomerase inhibition has become an attractive strategy in designing anticancer drugs [7,8]. The folding of telomeric DNA into Gquadruplexes inhibits telomerase by locking the single-stranded RNA component template of the telomerase complex that does not recognize the quadruplex DNA [9]. Therefore, this unique telomerase activity is an ideal probe for tumor diagnosis and a target for cancer chemotherapy, with the potential for selective toxicity to cancer cells.
A number of small-molecule ligands can induce and stabilize the formation of G-quadruplex structure and inhibit telomerase activity, with some showing pronounced effects on cancer cell lines. These ligands include the natural product telomestatin, as well as cationic porphyrins, substituted acridines, polycyclic aceidines, and perylenetetrac arboxylic diimide derivatives [10][11][12][13][14][15]. Metal complexes, particularly those of ruthenium (Ru), have also been shown to interact selectively with G-quadruplexes and to exhibit good antitumor activities [15][16][17]. For example, the [Ru(bpy) 2 (dppz)] 2+ complex has been identified as a distinctive ''light switch.'' This complex can intercalate between duplex DNA base pairs and bind to quadruplex DNA when induced by either Na + or K + over an i-motif, with affinities higher than those obtained for duplex binding [18]. Thomas et al. [19] reported that dinuclear tppz-based systems have high affinities for and thus are bound to quadruplex DNA at high ionic strengths through the 22mer d(AG 3 3 ] human telomeric sequence. However, to the best of our knowledge, only a few studies have reported on the ability of chiral enantiomers to selectively induce and stabilize G-quadruplex formation and to inhibit telomerase. One example is the enantioselective binding of the short linker-containing chiral helicene molecule to telomere repeats and its enantioselective inhibitory activity against telomerase [20]. Meanwhile, Qu et al. [21,22] reported that the metallo supermolecular cylinders [M 2 L 3 ](PF 6 ) 4 and [M 2 L 3 ]Cl 4 (M = Ni or Fe) can selectively stabilize human telomeric G-quadruplex DNA. Only the P enantiomers of these cylinders have a strong preference for Gquadruplex DNA over duplex DNA and can convert the antiparallel G-quadruplex structure to a hybrid structure in the presence of sodium.

[T 2 AG 3 ] 3 )[G
Purified enantiomers generally exhibit very different, and even opposite, biological activities [23,24]. Interestingly, Svensson et al. [25] reported that the D-enantiomer of the [Ru(phen) 2 dppz] 2+ complex has higher DNA binding activity. Our laboratory has also previously examined the interaction of L-[Ru(phen) 2 (p-MO-PIP)] 2+ and D -[Ru(phen) 2 (p-MOPIP)] 2+ with G-quadruplex DNA, as well as their enantioselective inhibitory effect on telomerase activity. Both complexes contain a hydrophobic methoxyl group in their aromatic heterocyclic ligands [26]. The possible correlation between the different biological activities and the isomer chiralities or the DNA complex structure remains to be determined. In addition, the biological activities of the chiral Ru complexes may be related to their ability to bind with the Gquadruplex structure. The ability of these complexes to stabilize G-quadruplex formation may also be related to their telomerase inhibition and anticancer activities. These questions motivated the investigation on the relationships between the anticancer targets of Ru complexes, DNA, and telomerase.
Physical measurements. Elemental analyses (C, H and N) were carried out with a Perkin-Elmer 240C elemental analyzer. 1 H NMR spectra were recorded on a Varian Mercury-plus 300 NMR spectrometer with DMSO-d6 as a solvent and SiMe 4 as an internal standard at 300 MHz at room temperature. An LCQ electrospray mass spectrometer (ESMS, Finnigan) was employed for the investigation of charged metal complex species in CH 3 CN solvent. Emission spectra were measured on a recorded on Perkin-Elmer Lambda-850 spectrophotometer with excitation at 460 nm, and circular dichroism (CD) spectra were measured on a Jasco J-810 spectropolarimeter.  [28]. (p-HPIP) was also prepared according to the literature [29].  O9-dibenzoyl-D-tartrate]?12H 2 O (0.22g, 0.2 mmol), p-HPIP (0.12 g, 0.36 mmol) were added to 20 ml ethylene glycol-water(9:1, v/v). The mixture was refluxed for 6 h under an argon atmosphere. The cooled reaction mixture was diluted with water (40 ml) and filtered to remove solid impurities. Ammonium hexafluorophosphate was added to the filtrate. The precipitated complex was dried, dissolved in a small amount of acetonitrile, and purified by chromatography over alumina, using MeCN-toluene (2:1, v/v) as eluent, yield: 140 mg, 70.01%. 1   [Ru(phen) 2 Cl 2 ] ?2H 2 O (0.12 g, 0.2 mmol) and p-HPIP(0.12 g, 0.36 mmol) were added to 20 ml ethylene glycol-water(9:1, v/v). The mixture was refluxed for 6 h under an argon atmosphere. The cooled reaction mixture was diluted with water (40 ml) and filtered to remove solid impurities. Ammonium hexafluorophosphate was added to the filtrate. The precipitated complex was dried, dissolved in a small amount of acetonitrile, and purified by chromatography over alumina, using MeCN-toluene (2:1, v/v) as eluent, yeild: 1   Absorption spectra studies. Electronic spectra were recorded on a Shimadzu UVPC-3000 spectrophotometer. Spectroscopic titrations were carried out at room temperature to determine the binding capability affinity between DNA and each enantiomer. Initially, 3000 mL solutions of the blank buffer and the ruthenium complex sample (2 mM) were placed in the reference and sample cuvettes (1 cm path length), respectively, and then the first spectrum was recorded in the range 200-600 nm. During the titration, aliquots (1-10 mL) of buffered DNA solution (concentration of 5-10 mM in base pairs) was added to each cuvette to eliminate the absorbance of DNA itself, and the solutions were mixed by repeated inversion. After mixing for 5 min, the absorption spectra were recorded. The titration processes were repeated until there was no change in the spectra for at least four titrations indicating binding saturation had been achieved. The changes in the metal complex concentration due to dilution at the end of each titration were negligible. The UV-Vis is titrations for each sample were repeated at least three times.
Emission measurements. Emission measurements were carried out on a JASCOFP-6500 spectrofluorometer at 20uC. For luminescence titrations a 3000 mL aliquot of the sample solution in a 1 cm path length quartz cuvette was loaded into the fluori-meter sample block, After 5 min to allow the cell to equilibrate, the first spectrum was recorded, and then 1-10 mL of DNA solution (5-10 mM in base pairs) was added to the sample cell, followed by thorough mixing. After 5 min, the spectrum was taken again. Lifetime spectrometer at room temperature with excitation wavelength 460 nm, Exslit 5.00 nm, and emslit 1.50 nm. The titration processes were repeated until there was no change in the spectra for at least four titrations indicating binding saturation had been achieved. The luminescence titrations for each sample were repeated at least three times.
Circular dichroism measurements. All CD experiments were performed at an ambient temperature in aerated buffer solutions in 10 mM Tris-HCl buffer, 100 mM NaCl at pH = 7.4. CD titrations were carried out as follows: concentrated DNA (5-10 mM in base pairs) was added in aliquots to solutions containing Ru(II) complex. All solutions were mixed thoroughly and allowed to equilibrate for 6 min before data collection. The titration process was repeated several times until no change was observed. It showed that binding saturation was achieved. The CD spectra were recorded on a Chirascan (Applied Photophysics) spectrophotometer, using 0.5/1.0 s-per-points from 220 to 350 nm and 1 nm bandwidth at a temperature of 25uC. The CD spectra were obtained by averaging three scans. The instrument was flushed continuously with pure evaporated nitrogen throughout the experiment.
Gel Mobility Shift Assay. The Oligonucleotide at 10 mM was heated to 95uC for 10 min in 10 mM Tris/1 mM EDTA buffer containing 100 mM KCl (pH 7.4). After the DNA was cooled to room temperature, a 2 mL stock solution of the metal complex was added and each sample to produce the specified concentrations. The reaction mixture was incubated for 4 h at room temperature, then loaded onto a native 12% acrylamide vertical gel (1/19 bisacrylamide) in Tris borate EDTA (TBE) buffer, supplemented with 20 mM KCl. After these, each mixture added 8 mL of loading buffer (30% glycerol, 0.1% bromophenol blue, and 0.1% xylene cyanol). Ten microliter solution of each sample were subsequently analyzed by native 12% PAGE (the gel was pre-run for 30 min). Electrophoresis proceeded for 15 h in TBE running buffer containing 20 mM KCl at 4uC. The gels were silver-stained to visualized.
FRET assay. The two double-dye labelled oligonucleotide F21T(59-FAM-G 3 [T 2 AG 3 ] 3 -TAMRA-39) was diluted in Tris-HCl buffer (10 mM, pH 7.4) containing 60 mM KCl and then annealed by heating to 92uC for 5 min, followed by cooling slowly to room temperature overnight. Emission readings were taken at an interval of 1uC over the range 30-95uC, with a constant temperature being maintained for 30 s prior to each reading to ensure a stable value. To test the binding selectivity of the compound to the quadruplex structure, we added various concentrations of competitors: double-stranded DNA (self-complementary ds26 DNA: 59-GTTAGCCTAGCTTAAGCTA GGCTAAC-39). Final analysis of the data was carried out using Origin 7.0(Origin Lab Corp.).
Cell culture. Cells were cultured in RPMI 1640 medium supplemented with 10% heat inactivated fetal bovine serum, 100 mg/ml penicillin, and 100 mg/ml streptomycin. Cells were maintained at 37uC in a 5% CO 2 incubator, and the media were changed twice weekly.
MTT assay. The cytotoxicity of the complexes was evaluated by cell viability and determined by measuring the ability of cells to transform MTT to a purple formazan dye [30]. Cells were incubated at 37uC under a 5% CO 2 atmosphere, and seeded in a 96-well plates (1.0610 3 /well) in growth medium (100 mL) and incubated at 37uC in 5% CO 2 atmosphere for 24 h. Then the cells were treated with various concentrations of complexes in a mixture of growth medium/DMSO (99:1, v/v); The cells was incubated at 37uC under a 5% CO 2 atmosphere for 48 h, MTT (100 ml of 5 mg/ml) was added to each well, and then the plates were further incubated for 4 h, each cell was added in 100 ml cell lysate. After 12 h at 37uC, The absorbance of the solutions at 580 nm was measured with a microplate-reader (the absorbance of the complexes at this wavelength can be neglected [31,32]). The IC 50 values of the complexes were determined by plotting the percentage viability versus concentration on a logarithmic graph and reading off the concentration at which 50% of cells viable relative to the control.
PCR stop assay. Sequences of the tested oligomers were HTG21 (59-G3(T2AG3)3-39) and the corresponding complementary sequence (HTG21rev, ATCGCT 2 CTCGTC 3 TA 2 C 2 ). The reactions were performed in 16PCR buffer, containing 10 mM of each oligonucleotide, 0.16 mM dNTP, 2.5 U Taq polymerase, and different concentrations of complexes. Reaction mixtures were incubated in a thermocycler with the following cycling conditions: 94uC for 3 min, followed by 30 cycles of 94uC for 30 s, 58uC for 30 s, and 72uC for 30 s. PCR products were then analysed on 15% nondenaturing polyacrylamide gels in 16 TBE and silver stained.
Laser Confocal Microscopy Image Analysis. For achieving laser confocal images, HepG2 cells were grown on a laser confocal microscopy 35 mm 2 culture dish at a density of 1.0610 4 cells and maintained culture with at 37uC under a 5% CO 2 atmosphere for 24 h, then was added in the cell layer. Cells were transfected with the complexes L-[Ru(phen) 2 (p-HPIP)] 2+ and D-[Ru(phen) 2 (p-HPIP)] 2+ at a concentration of 20 mM and incubated for different time intervals (24 h and 48 h). After the transfection, the media were removed and the cell layer was washed 3 times with 16PBS. Then, the cell layer was trypsinized and added up to 3 mL PBS. Confocal images were analyzed by a Leica TCS SP5 confocal microscope (Leica Microsystems, Wetzlar, Germany) using a planapochromate 636/NA 1.4 oil immersion objective. The confocal microscope was equipped with an ArKr laser which was used to excite Ru II (488 nm excitation, detection at 560-615 nm (green) and 625-754 nm (red)). Meanwhile, the cell nuclei were stained with Hoechst 33342 solution for 10 min (5 mg mL 21 ).

Results and Discussion
Fluorescence selectivities of Ru complexes to Gquadruplex structures. The binding affinities of these chiral Ru complexes for different DNA structures were investigated via emission spectroscopy. Two different G-quadruplex sequences, HTG21 and G 4 T 2 , were selected for this study [36]. Meanwhile, a complementary oligonucleotide of telomeric DNA (ssDNA) and double-stranded DNA (ds26) were selected as the other DNA structures. All measurements were performed in a Tris buffer containing 10 mM Tris-HCl and 100 mM KCl. The different emission spectra are illustrated in Figure 2. Only a slight increase in emission was observed in the presence of ds26, whereas a decrease in fluorescence was observed in the presence of ssDNA These results are attributed to the inability of ssDNA and ds26 to fold into a quadruplex even in the presence of monovalent cations. However, the emission significantly increased in the presence of the DNA quadruplexes HTG21 and G 4 T 2 . The emission response of L-[Ru(phen) 2 (p-HPIP)] 2+ with G-quadruplexes was approximately four times higher than that with ds26. This can be very obviously enucleated that these chiral complexes exhibited high selectivity for quadruplexes over duplexes, particularly for the human telomeric DNA HTG21.We further examined the interaction between the chiral complexes and HTG21.
Absorption and emission luminescence spectroscopic studies. Electronic absorption spectroscopy is one of the most useful techniques in DNA-binding studies. Hypochromism and bathochroism are usually observed when a complex binds to DNA through intercalation because of the strong stacking interaction between an aromatic chromophore and the DNA base pairs in the intercalation mode. In general, the extent of hypochromism indicates the intercalative binding strength [37].
The absorption spectra of the chiral Ru(II) complexes L-[Ru(phen) 2 (p-HPIP)] 2+ and D-[Ru(phen) 2 (p-HPIP)] 2+ are shown in  Table 1). The spectral characteristics obviously showed that the two Ru(II) complexes interacted with DNA most likely through a mode that involves a stacking interaction between the aromatic chromophore and the DNA base pairs. In addition, the binding constant Circular dichroism spectra. Circular dichroism (CD) spectroscopy was used to investigate the conformational properties of the enantiomeric chiral molecules in relation to the telomeric Gquadruplex. In the absence of salt, the CD spectrum of HTG21 at room temperature exhibited a negative band at 238 nm as well as a major positive band at 257 nm, which probably corresponds to the signal of the HTG21 random coil (characterized by a positive peak at 257 nm). A minor negative band at 280 nm and a positive band near 295 nm were also observed (Figures 4a-4c, black line) [39]. A significant change in the CD spectrum was observed upon addition of L-[Ru(phen) 2 (p-HPIP)] 2+ to the aqueous HTG21 solution (Figure 4a). The bands at 257 nm gradually disappeared with the addition of the complex, eventually leading to the  appearance of a major negative band at 260 nm as well as a significant increase in the band intensity at 295 nm. Meanwhile, a new, strong, positive band gradually appeared near 270 nm. These two changes are consistent with the induction of the G-rich DNA by L-[Ru(phen) 2 (p-HPIP)] 2+ to form the G-quadruplex structure. Thus, all the complexes can convert G-quadruplex from a linear to a hybrid structure. The HTG21 oligonucleotide formed the parallel G-quadruplex structure in the presence of K + (Figures 4d-4f, black line) [40]. The CD spectrum of this structure in the absence of any compound shows a strong positive band at 290 nm, a small positive band at 260 nm, and a minor negative band at 234 nm. The CD spectrum changed upon L-[Ru(phen) 2 (p-HPIP)] 2+ titration to the above solution, showing an enhancement of the maximum band at 290 nm as well as a suppression of the band at 260 nm. A strong, positive, induced CD signal also appeared at 270 nm. The band at 260 nm was gradually suppressed and formed a negative band until the ratio of L-[Ru(phen) 2 (p-HPIP)] 2+ to HTG21 reached 4:1 (Figure 4d). This result indicates the formation of a mixture of anti-parallel and parallel conformations, possibly including hybrid-type forms, as well. This interpretation is further supported by the recent observation of a co-existing equilibrated mixture of antiparallel, hybrid, and parallel topologies of telomeric repeats in native conditions [41]. The results also indicate that L-[Ru(phen) 2 (p-HPIP)] 2+ is more efficient at inducing the formation of G-quadruplexes compared with the other two complexes. The data also suggest that the three complexes, particularly L-[Ru(phen) 2 (p-HPIP)] 2+ , strongly and selectively interacts with G-quadruplex DNA, which is consistent with the experimental results.
We also investigated the interactions in a Na + buffer solution ( Figure S2). The HTG21 oligonucleotide formed the antiparallel G-quadruplex structure in the presence of Na + . However, the CD spectrum remained nearly unchanged upon the addition of the complexes to HTG21 in the Na + buffer solution. These results show that none of the three complexes changed the conformation of the antiparallel G-quadruplex in the Na + solution. Therefore, Na + can stabilize the conformation of the G-quadruplex, and that none of the three Ru complexes can change the conformation of the G-quadruplex at high ionic strengths [42].
The L-[Ru(phen) 2 (p-HPIP)] 2+ and D-[Ru(phen) 2 (p-HPIP)] 2+ complexes induced identical G-quadruplex conformation conversions in the Na + and K + buffer solutions. Nevertheless, we had reported that only the complex L-[Ru(phen) 2 (p-MOPIP)] 2+ could convert the G-quadruplex conformation. Thus, the chiral isomer exhibited enantioselective binding to DNA. This result may be due to the effect of hydrogen bond as L-[Ru(phen) 2 (p-HPIP)] 2+ contains a ligand with a pendant OH functional group. The results also indicate that the interaction between different chiral Ru complexes and DNA were different.
Gel mobility shift assay. The ability of the Ru complexes to promote intermolecular G-quadruplex DNA formation was investigated via electrophoresis. The oligonucleotide HTG21 (59-G 3 (T 2 AG 3 ) 3 -39) contains four repeats of the human telomeric sequence and thus has the potential to form parallel and antiparallel G-quadruplex structures in dimeric (D) and tetrameric (T) forms [43,44]. When the HTG21 oligonucleotide was incubated in Tris buffer (10 mM Tris, 1 mM EDTA, 100 mM KCl, pH = 8.0), gel mobility shift assays show no G-quadruplex structure formation; only the band that correspond to the monomer (M) was observed. The addition of increasing amounts The quantification of the gels is shown in the lower part of Figure 5a. The L-[Ru(phen) 2 (p-HPIP)] 2+ complex efficiently promoted the formation of an intermolecular quadruplex structure. Up to 40% of the HTG21 oligonucleotide adopted a dimeric structure upon the addition of 50 mM L-[Ru(phen) 2 (p-HPIP)] 2+ (Figure 5b). However, the treatment of the HTG21 oligonucleotide with D-[Ru(phen) 2 (p-HPIP)] 2+ resulted in only 29% dimeric formation. These results indicate that the induction of intermolecular G-quadruplex structure formation by D-[Ru(phen) 2 (p-HPIP)] 2+ is clearly less efficient than that of L-[Ru(phen) 2 (p-HPIP)] 2+ . These observations are consistent with the G-quadruplex stabilizing effects shown using other methods.
Studies of telomeric G-quadruplex binding stability and selectivity via fluorescence resonance energy-transfer (FRET) assays. The thermodynamic stabilization activity and selectivity of the complexes to telomeric G-quadruplex DNA were investigated using FRET melting experiments [45]. We used the FRET melting assay to investigate the binding abilities of L-[Ru(phen) 2 (p-HPIP)] 2+ and D-[Ru(phen) 2 (p-HPIP)] 2+ to the Gquadruplex DNA F21T (FAM-G3[T2AG3]3-TAMRA, which mimics the human telomeric repeat) in 100 mM KCl buffer [46]. Figures 6a-6c show that in the absence of any Ru(II) complex, The FRET melting experiments also provide a convenient way of testing the ligand selectivity toward the quadruplex in comparison to the selectivities toward a variety of unlabeled competitors. To determine the selectivity of the two chiral complexes, ds26 was added to quadruplex/ligand mixture as the main competitor during the experiment, given that a duplex is not labeled in the experiment. Although ds26 competes for binding to the ligand, it does not interfere in the emission studies [47]. A major advantage of this technique is that only small amounts of oligonucleotides are used, and that the experiments can be automated using a multiwell plate reader. We used the complex  Figures 6e and 6f show high levels of G-quadruplex stabilization by the chiral complexes; however, the stability was only slightly affected at the 30:1 concentration ratio ( Figure S3). The data also show that the chiral complexes still stabilized the Gquadruplex effectively even with the addition of substantial amounts of ds26. This result may be due to the large planar scaffold of the complexes and is consistent with the emission selectivity results, which demonstrate the high selectivity of the chiral complexes for G-quadruplex DNA over duplex DNA.
Polymerase chain reaction (PCR)-stop. We evaluated the efficiency of L-[Ru(phen) 2 (p-HPIP)] 2+ and D-[Ru(phen) 2 (p-HPIP)] 2+ in stabilizing G-quadruplex DNA. A PCR-stop assay was used to determine whether these complexes were bound to a test oligomer [59-G3(T2AG3)3-39] and therefore stabilized the Gquadruplex structure [48]. In the presence of chiral complexes, the single strand HTG21 was induced into a G-quadruplex structure that blocked hybridization with a complementary strand. A 59-39 extension with Taq polymerase was inhibited, and the final double-stranded DNA PCR product was not detected. Different concentrations of the complexes were used in this assay. L-[Ru(phen) 2 (p-HPIP)] 2+ showed a clearly inhibitory effect as the concentration increased from 0.0 mM to 30.0 mM, with no PCR product detected even at 20.0 mM. However, D-[Ru(phen) 2 (p-HPIP)] 2+ showed a weaker inhibitory effect on the hybridization, eventually inhibiting the hybridization at 20 mM (Figure 7). These results indicate that L-[Ru(phen) 2 (p-HPIP)] 2+ induced the stability of the G-quadruplexes better than D-[Ru(phen) 2 (p-HPIP)] 2+ . The results also indicate that G-quadruplex stabilization  is vital to the inhibition of gene expression, and that all the studied complexes are efficient G-quadruplex binders.
Telomeric repeat amplification protocol (TRAP) assay. The above results encouraged further investigation on the possible inhibitory effects of the two chiral Ru complexes on telomerase activity via a TRAP assay, which has been widely used to provide quantitative estimates of telomerase inhibition [49]. In this experiment, solutions containing different concentrations of L-[Ru(phen) 2 (p-HPIP)] 2+ and D-[Ru(phen) 2 (p-HPIP)] 2+ were added to a telomerase reaction mixture that contains HepG2 cell extracts, which express high levels of telomerase. The IC 50 values were obtained and are shown in vitro cytotoxicity. Figure 8 clearly shows the inhibitory effects of the two chiral Ru complexes on telomerase activity, but at different extents. As the L-[Ru(phen) 2 (p-HPIP)] 2+ concentration increased, the intensity of telomerase activity decreased, particularly at 8 mM (Figure 8), the activity disappeared completely at 32 mM. Meanwhile, the D-[Ru(phen) 2 (p-HPIP)] 2+ complex demonstrated inhibition at 16 mM, but this inhibition was not complete even at 32 mM. Thus, L-[Ru(phen) 2 (p-HPIP)] 2+ has a stronger telomerase inhibitory capability compared with D-[Ru(phen) 2 (p-HPIP)] 2+ , which is consistent with the experimental data from the spectroscopic and PCR-stop analyses.
In vitro cytotoxicity. We investigated the antitumor potential of the Ru complexes using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay to determine the cytotoxicity of the chiral Ru(II) complexes against seven types of cancer cells, namely, human hepatocellular liver carcinoma (HepG2), human cervical cancer (HeLa), human lung carcinoma (A549), human colon colorectal adenocarcinoma (SW480), human melanoma (A375), ishkawa (endometrial adenocarcinoma), human breast cancer(MDA-MB-231) cells and human umbilical vein endothelial cells(HUVEC). All the cells were purchased from Shanghai Sangon Biological Engineering Technology & Services (Shanghai, China). Figure 9 shows the IC 50  These results indicate that the complexes have relatively higher selectivity to cancer cells than to normal cells.
The anticancer activities of the two chiral Ru polypyridyl complexes in vitro demonstrate efficient enantioselection. In addition, the abilities of L-[Ru(phen) 2 (p-HPIP)] 2+ to stabilize quadruplex DNA and inhibit telomerase were stronger than those of D-[Ru(phen) 2 (p-HPIP)] 2+ . These results suggest that the complexes may have anticancer activities, and that the quadruplex DNA and its telomerase may be the anticancer targets.
Cellular uptake. Further investigations of the complexes were conducted based on the previously described results. HepG2 cells loaded with 20 mM complexes were investigated via flow cytometry to obtain the time-dependent uptake profiles [50]. The   results are shown in Figure 10. Upon excitation, the luminescence intensity of the cell population dramatically increased compared with the autofluorescence of untreated HepG2 cells. This result indicates the efficient cellular accumulation of the complexes. The luminescence intensity of HepG2 cells treated with L-[Ru(phen) 2 (p-HPIP)] 2+ is stronger than that of cells treated with D-[Ru(phen) 2 (p-HPIP)] 2+ , which suggest that L-[Ru(phen) 2 (p-HPIP)] 2+ is more effectively interiorized by the cells.
Confocal Microscopy Studies. The intrinsic emission of Ru(II) complexes can be used in the design of Ru(II) complex cellimaging probes that detect the presence of DNA binding via multiple emission peaks [20,51]. Although some Ru(II) complexes can identify cancer cell membrane receptors and can readily accumulate in the cytoplasm of live cells,most are excluded from the nucleus and are mainly localized in the cytoplasm [52,53]. However, a certain amount of Ru(II) complexes can be efficiently transported across the plasma membrane and then accumulate in the nucleus [54,55]. Nuclear accumulation is highly desirable in anticancer agents that target genomic DNA [56]. The intracellular behaviors of L-[Ru(phen) 2 (p-HPIP)] 2+ and D-[Ru(phen) 2 (p-HPIP)] 2+ are observable via confocal microscopy. The confocal microscopic images (Figure 11a) show that the 20 mM L-[Ru(phen) 2 (p-HPIP)] 2+ that were used to incubate the cells for 24 h entered and accumulated inside the cells in the region around the nucleus, subsequently forming very sharp luminescent rings around the nucleus. The nuclear region then exhibited significantly weaker emission, which is indicative of negligible nuclear uptake of the complex. Interestingly, after incubation at 20 mM for 36 h, the green/red signal in the nucleolar region increased. The complex then spread throughout the cell and partly accumulated in the nucleus. These results show that L-[Ru(phen) 2 (p-HPIP)] 2+ can be absorbed by HepG2 cells and can enter the cytoplasm to partly accumulate in the nucleus. However, for D-[Ru(phen) 2 (p-HPIP)] 2+ , the increase in the number of green or red emission dots in the nucleus was limited (Figure 11b). D-[Ru(phen) 2 (p-HPIP)] 2+ accumulated in the cytoplasm and was predominantly excluded from the nucleus after cell incubation at 20 mM for 36 h.
A similar confocal microscopic analysis was also performed using another hydrophilic Ru(II) complex, L-[Ru(phen) 2 (p-DMNP)] 2+ , which contains dimethylamino groups at the same positions on the phenyl ring as L-[Ru(phen) 2 (p-HPIP)] 2+ . After incubation of the HepG2 cells with 20 mM L-[Ru(phen) 2 (p-DMNP)] 2+ for 8 h, green/red emission dots were observed in the cell nuclei (Figure 11c). In addition, L-[Ru(phen) 2 (p-MOPIP)] 2+ completely accumulated in the nuclei after 8 h incubation. This finding suggests that Ru complexes can enter the nucleus and efficiently interact with DNA, which leads to the inhibition of DNA transcription and translation. Therefore, the Ru compounds display promising anticancer activities. The limited capacity of D-Ru in nuclear targeting as well as the selective entry of L-Ru into HepG2 cells is also indicated by the results. The abilities of the complexes to enter the nuclei may be related to their affinities for the constituents of the nucleus as well as to differences in their photophysical properties. Furthermore, the complex containing the appropriate hydrophobic ligand may have the greater ability to enter the cells and accumulate in the nuclei.

Conclusions
One enantiomer of a new chiral Ru(II) complex was synthesized and characterized. This enantiomer showed effective and selective binding to telomeric G-quadruplex DNA and thus inhibited the telomerase activity. The experimental results clearly show that these complexes possess certain binding affinities and significant selectivity for G-quadruplex DNA over duplex DNA. The UV/ Vis, emission spectroscopy, CD spectroscopy, FRET assay, PCRstop assay, GMSA assay, and competition experiment results all demonstrate that L-[Ru(phen) 2 (p-HPIP)] 2+ can selectively stabilize human telomeric G-quadruplex DNA and that it has a strong preference for G-quadruplex over duplex DNA. Although the actual models for the binding of the complexes to the G-  quadruplexes were not identified, our findings imply that the characteristics of the complexes that stabilize the G-quadruplexes can be further rationalized. The TRAP assay results suggest that L-[Ru(phen) 2 (p-HPIP)] 2+ is a potential lead compound for the development of new telomerase inhibitors. These results emphasize the importance of discovering and designing chiral anticancer agents that target G-quadruplex DNA. However, L-[Ru(phen) 2 (p-MOPIP)] 2+ was observed to have more strong ability to interact with quadruplex DNA as it contains a ligand with a methoxy group functional group, which may be involved in H-bonding interaction with the guanine in the external tetrad of Gquadruplex DNA, even the hydroxyl/methoxy group may be changed the electron density of the ligand aromatic ring atom and then the ability of complexes to interact with quadruplex DNA was different. Furthermore, the details of the binding modes of these complexes with G-quadruplex and the structure of Gquadruplex are not clear yet and further studies are needed. The activity of complexes could be adjusted by altering the functional group on the aromatic ring of the ligands. In particular, cellular uptake and confocal microscopic results show that L-[Ru(phen) 2 (p-HPIP)] 2+ can facilitate membrane diffusion into live cells after 24 h and partly reach the cell nucleus at 36 h. However, for D-[Ru(phen) 2 (p-HPIP)] 2+ , only diffusion into the cytoplasm was observed even after 36 h. This difference in cellular localization can be ascribed to the difference in the uptake mechanism of the two chiral complexes. The results also suggest that L-[Ru(phen) 2 (p-HPIP)] 2+ has higher potential as a cellular nucleus-targeting drug. Moreover, although similar to the Lenantiomer, the hydrophobic Ru complex L-[Ru(phen) 2 (p-DMNP)] 2+ can rapidly enter the HepG2 cell nuclei. These studies imply that the accumulation of chiral Ru complexes in the nucleus is associated with the chirality of the isomers as well as with the subtle environment of the complexes (e.g., active ligand and lipophilicity). Therefore, the nucleus is the potential cellular target of chiral Ru complexes for anticancer therapy.