Imaging of the nuclei of living tumor cells by novel ruthenium(II) complexes coordinated with 6-chloro-5-hydroxylpyrido[3,2-a]phenazine

Abstract

Two novel ruthenium(II) complexes coordinated with 6-chloro-5-hydroxylpyrido[3,2-a]phenazine (CQM), [Ru(L)₂(CQM)]ClO₄ [L = 1,10-phenanthroline, (1) and 2,2′-bipyridine, (2)], were investigated as potential fluorescent probes to track dynamic changes in the nuclei of living cells. Confocal laser technology was used to observe their co-location inside the cells. The results showed that both complexes were taken up by HepG2 cells, especially 1, which was localized in the cell nuclei, whereas 2 was distributed in the cell nuclei and mitochondria. Further studies by real-time fluorescence observations revealed that 1 rapidly entered the living cells, namely, HepG2, HeLa and MCF-7 cells, imaged the dynamic changes of the nuclei of living tumor cells, and exhibited low toxicity toward cells. The results demonstrated that 1 may be developed into a novel fluorescent probe for living cell nuclei. This study facilitates the development of fluorescent chemosensors with metal complexes.

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1. Introduction

Numerous studies have been performed to develop simple, sensitive, specific, and robust fluorescent probes/sensors for biochemistry, molecular biology, and clinical diagnostics.¹ Although many organic dyes have been designed and investigated, the utilization of these organic dyes is limited because of their poor water solubilities, low photo-stabilities, and high toxicities.^2–5

To overcome the drawbacks of organic dyes, transition metal complexes, especially versatile ruthenium(II) complexes, have increasingly attracted attention as potential fluorescent probes because of their wide spectral range, long-term luminescence and large Stokes shifts.^6–10 Several recent notable reviews have shown that luminescent ruthenium(II) complexes have emerged as promising candidates for wide application in chemosensors, biolabeling, in vivo tumor imaging, and live cell compartmentalization staining, such as in the nucleus, in the cytoplasm, in the endosome, in mitochondria, in lysosomes and in the endoplasmic reticulum.^11–16

Ruthenium complexes with dipyridophenazine (dppz) ligands have been frequently investigated because of their strong DNA binding and their extraordinary photophysical properties.^17,18 In particular, these compounds have been paid much attention because of their “light switch effect”. These compounds are highly luminescent when intercalated into DNA and are virtually nonemissive in aqueous solution, which is advantageous for fluorescence microscopy. For example, Barton et al. explored the cellular uptake of ruthenium(II) complexes and found that the complex cation [Ru(dip)₂(dppz)]²⁺ (dip = 4,7-diphenyl-1,10-phenanthroline) can be effectively transported into the cellular interior.¹⁹ Furthermore, the mechanism of cellular entry of the luminescent ruthenium(II) polypyridyl complex [Ru(dip)₂(dppz)]²⁺ was investigated by flow cytometry, which supported the passive diffusion of [Ru(dip)₂(dppz)]²⁺ into the cell.²⁰ Rajendiran et al. also reported a series of mixed ligand ruthenium(II) complexes [Ru(5,6-dmp/3,4,7,8-tmp)(diimine)]²⁺ as fluorescent probes for nuclear and protein components.²¹ [Ru(phen)₂(dppz)]²⁺ has also been reported to be taken up by cells and can be used as an efficient optical probe for staining nuclear components,²² while ruthenium(II) complexes containing 7-F-dppz [7-fluorodipyrido(3,2-a:2′,3′-c)phenazine] can be taken up by cells and localized in the nucleus.²³ More recently, an alkyl ether chain was bound to a dppz ligand, which increased the lipophilicity and membrane permeability of the corresponding ruthenium(II) complexes.^24,25 In addition, ruthenium–octaarginine-conjugated polypyridyl peptides have been investigated.^26,27 Ruthenium(II) estradiol polypyridine complexes and dinuclear ruthenium(II) complexes have also been considered as probes for cellular imaging.^28–31 However, there are still relatively few agents which have succeeded in direct nuclear staining, which is ascribed to their poor membrane permeability, poor cellular uptake and high toxicity, and it remains a huge challenge to develop novel fluorescent probes for living cells. Thus, imaging of living cells is still challenging because of the high toxicity, limited uptake in living cells, and limited nuclear accumulation of the complexes under investigation.

In this work, studies have been performed to target metal complexes more effectively toward the nucleus. Two novel ruthenium(II) complexes coordinated with CQM, [Ru(L)₂(CQM)]²⁺, [1,10-phenanthroline, (1) and 2,2′-bipyridine, (2)] (Scheme 1), were synthesized under microwave irradiation, and their properties for imaging of the nuclei of living tumor cells were investigated.

Scheme 1 Molecular structure of ruthenium(II) complexes 1 and 2.

2. Experimental section

2.1 Materials and methods

All reagents were purchased from commercial suppliers without further purification. Solvents were dried and purified by conventional methods prior to use. Ruthenium chloride hydrate was obtained from Mitsuwa Chemicals. 8-Hydroxyquinoline was purchased from Aladdin. All chemicals, including solvents, were obtained from commercial vendors and used as received. The Tris–KCl buffer consisted of Tris (10 mM) and NaCl (100 mM), and the pH value was adjusted to 7.2 with HCl (0.1 mol).

The complexes were synthesized using an Anton Paar Monowave 300 microwave reactor (initiator single mode microwave cavity at 2450 MHz, Biotage). ¹H NMR and ¹³C NMR spectra were recorded on a Bruker DRX 2500 spectrometer in d⁶-DMSO, and electrospray ionisation mass spectrometry (ESI-MS) spectra were obtained in acetonitrile on an Agilent 1100 ESI-MS system. Electronic absorption spectra were recorded on a Shimadzu UV-2550 spectrophotometer, and steady-state emission spectra were recorded on a RF-5301 fluorescence spectrophotometer. Fluorescence images of cellular localization were obtained using an EVOS® FL Auto Imaging System. Living-cell confocal imaging was performed using a Zeiss LSM 510 META.

2.2 Synthesis and characterization

6,7-Dichloroquinoline-5,8-dione (DCQ) was prepared by using a similar method to that reported in the literature.³² In general, a mixture of 8-hydroxyquinoline (45 mmol, 6.5 g), NaOH (25 mmol, 10 g), NaClO₃ (28 mmol, 3 g) and high concentration hydrochloric acid (150 mL) was heated at 80 °C for 3 h; the products were obtained by one-step column chromatography on silica-gel. Yields, 65%. ¹H NMR (500 MHz, DMSO-d⁶): δ 9.05 (dd, J = 4.6, 1.6 Hz, 1H), 8.46 (dd, J = 7.9, 1.6 Hz, 1H), 7.90 (dd, J = 7.9, 4.7 Hz, 1H) (Fig. S3†). 6-Chloro-5-hydroxylpyrido[3,2-a]phenazine (CQM) was synthesized according to the literature with some improvement.³³ In general, a mixture of 6,7-dichloroquinoline-5,8-dione (1 mmol, 0.23 g) and o-phenylenediamine (1.2 mmol, 0.13 g) in 20 mL of ethanol was heated at 95 °C for 30 min under microwave irradiation. A yellow precipitate was obtained by filtration while it was hot and washed with ethanol, then dried in vacuo. Yield: 37%. ESI-MS (in chloroform): m/z 282.5 (Fig. S4†).

Synthesis of [Ru(phen)₂(CQM)](ClO₄)₂ (1). A mixture of CQM (0.6 mmol, 165 mg) and cis-[Ru(phen)₂Cl₂]·H₂O (0.4 mmol, 228 mg) in ethylene glycol (20 mL) was heated at 140 °C for 30 min under microwave irradiation. After cooling to room temperature, the mixture was stirred for 3 min with the addition of excess saturated NH₄PF₆ solution. Orange products were obtained by filtration and purified by one-step column chromatography on silica-gel (166 mg, 73%), ESI-MS (in acetonitrile): m/z 742.2. ¹H NMR (500 MHz, d⁶-DMSO): δ 9.10 (d, J = 5.2 Hz, 1H), 8.87–8.70 (m, 3H), 8.58 (dd, J = 11.6, 8.5 Hz, 2H), 8.48 (d, J = 4.5 Hz, 1H), 8.43–8.27 (m, 5H), 8.23 (t, J = 9.9 Hz, 1H), 8.15–8.04 (m, 4H), 8.03–7.93 (m, 2H), 7.88 (dt, J = 7.4, 5.2 Hz, 2H), 7.82–7.72 (m, 2H), 7.62 (ddd, J = 8.2, 6.8, 3.9 Hz, 3H). ¹³C NMR (126 MHz, DMSO), δ 167.46 (s), 154.77 (s), 153.48 (s), 152.35 (s), 152.05–151.78 (m), 149.88 (s), 149.88 (s), 148.76 (s), 148.69–148.59 (m), 148.40 (s), 147.91 (s), 147.91 (s), 143.52 (s), 142.91 (s), 138.72 (s), 137.49 (s), 136.82 (s), 136.59–136.48 (m), 136.45–136.36 (m), 136.16–135.97 (m), 135.42 (s), 133.53–133.36 (m), 131.92–131.78 (m), 131.12 (s), 130.83 (s), 130.62 (s), 129.76–129.69 (m), 127.56 (s), 127.00–126.95 (m), 126.92–126.82 (m), 126.75 (s), 126.51 (s), 126.13 (s), 125.71 (s), 112.67 (s).

Synthesis of [Ru(bpy)₂(CQM)](ClO₄)₂ (2). A mixture of CQM (0.184 mmol, 50 mg) and cis-[Ru(bpy)₂Cl₂]·H₂O (0.115 mmol, 80 mg) in ethylene glycol (20 mL) was heated at 140 °C for 30 min under microwave irradiation. After cooling to room temperature, the mixture was stirred for 3 min with the addition of excess saturated NH₄PF₆ solution. Orange products were obtained by filtration and purified by one-step column chromatography on silica-gel (60 mg, 75%), ESI-MS (in acetonitrile): m/z 694.2. ¹H NMR (500 MHz, d⁶-DMSO): δ 9.40 (dd, J = 8.2, 1.3 Hz, 1H), 8.76 (ddd, J = 56.4, 35.8, 6.5 Hz, 5H), 8.22 (dd, J = 8.5, 0.9 Hz, 1H), 8.11 (t, J = 8.0 Hz, 3H), 8.04 (dtd, J = 19.0, 8.0, 1.4 Hz, 2H), 7.97 (t, J = 7.5 Hz, 1H), 7.93–7.81 (m, 2H), 7.83–7.71 (m, 3H), 7.72–7.64 (m, 1H), 7.53–7.34 (m, 3H). ¹³C NMR (126 MHz, DMSO), δ 167.10 (s), 159.31 (s), 158.22 (s), 157.94 (s), 157.69 (s), 153.49 (s), 152.64 (s), 152.29 (s), 152.05 (s), 151.54 (s), 150.57–150.33 (m), 143.54 (s), 142.87 (s), 138.73 (s), 172.86–109.61 (m), 175.64–100.01 (m), 137.57 (s), 137.43 (s), 137.22 (s), 136.71 (s), 136.47 (s), 133.45 (s), 131.89 (s), 129.72 (s), 128.82 (s), 128.77 (s), 128.08 (s), 128.00 (s), 127.79 (s), 127.56 (s), 127.47 (s), 125.83 (s), 124.91 (s), 124.66 (s), 124.54 (s), 124.41 (s), 112.74 (s).

2.3 Cell lines and cell cultures

Human cancer cell lines, including HepG2 (hepatocellular carcinoma cell line), HeLa (cervical carcinoma cell line), and MCF-7 (breast carcinoma cell line) were purchased from the American Type Culture Collection (ATCC, Manassas, VA). Normal HaCaT (immortalized human epidermal cell line) was also obtained from ATCC. All cell lines were cultured in 25 cm² culture flasks in DMEM medium (Gibco, Gaithersburg, MD, USA) supplemented with fetal bovine serum (10%), penicillin (100 U mL⁻¹), and streptomycin (50 U mL⁻¹) at 37 °C in a CO₂ incubator (95% relative humidity, 5% CO₂).

2.4 Cellular localization

Cells were cultured in DMEM medium supplemented with 10% fetal bovine serum (FBS) at 37 °C under 5% CO₂. Cells in complete growth medium at 2 × 10⁶ cells per mL were incubated for 24 h at 37 °C, unless otherwise stated. Cells were washed with PBS and then treated with ruthenium(II) complexes 1 and 2 (200 μM) in DMSO/PBS (pH 7.2, 1 : 99, v/v) for 2 h at 37 °C under 5% CO₂. Then, cells were stained with Hoechst 33258 and Mito-tracker green FM for another 20 min and finally, luminescence imaging was carried out by confocal microscopy.

2.5 Cytotoxicity assay

The in vitro cytotoxicity of ruthenium(II) complexes 1 and 2 toward HepG2, HeLa, MCF-7, and HaCaT cells was studied using a 3-(4,5-dimethylthiazole)-2,5-diphenyltetraazolium bromide (MTT) assay. Cells were incubated with different concentrations (0, 25, 50, 100, 150, 200 μM) of complexes for 24 h. The cell viability was determined by measuring the ability of cells to transform MTT into a purple formazan dye, which was carried out as described previously. Cells were seeded in 96-well tissue culture plates for 24 h. After incubation, 20 μL per well of MTT solution (5 mg mL⁻¹ phosphate buffered saline) was added and the plates were incubated for 4 h. The color intensity of the formazan solution, which reflects the cell growth conditions, was measured at 490 nm using a microplate spectrophotometer (SpectroAmaxt 250). All data were from at least three independent experiments and are expressed as means ± the standard deviations. The following formula was used to calculate the viability of cell growth:

Viability (%) = (mean absorbance value of treatment group/mean absorbance value of control) × 100

2.6 Real-time fluorescence images

Cells were grown in DMEM medium supplemented with 10% fetal bovine serum (FBS) at 37 °C under 5% CO₂. Cells (2 × 10⁶) were seeded in 75 cm² culture flasks for 12 h before imaging. The cells were incubated with Hoechst 33258 for 10 min at 37 °C under 5% CO₂ followed by carefully washing the cells with PBS solution and then incubating with complexes 1 and 2 (200 μM). The cells were then monitored by fluorescence microscopy to obtain real-time fluorescence images every 5 min.

2.7 Cellular uptake of complexes 1 and 2

Cells were cultured in DMEM medium supplemented with 10% fetal bovine serum (FBS) at 37 °C under 5% CO₂. After 24 h incubation, the cells were washed with PBS and then the cell culture medium of the cell culture flasks was replaced with 10 mL of the cell culture medium solutions containing complexes 1 and 2 (50 μM). The flasks were then incubated at 37 °C, 5% CO₂ for 6 h. UV-vis spectra of the solutions of HepG2, HeLa and MCF-7 cells treated with complexes 1 and 2 were measured every hour.

2.8 Amphiphilicity measurements

The lipo–hydro partition coefficient of the complexes was tested by using an octanol–water two-phase system. In general, equal amounts of octanol and distilled water were thoroughly mixed in an oscillator for 24 h, then separated into a two-phase solution. Complexes were carefully dissolved in the water phase and the octanol phase to give a 20 μM solution with sufficient mixing. After separation, the final concentration of the water phase was denoted as C_w and the concentration of the octanol phase was denoted as C_o. Both C_o and C_w were tested by ultraviolet-visible (UV-vis) spectrophotometry, and the partition coefficient (P_o/w) for the complex was calculated according to the equation: P_o/w = A_o/A_w.

3. Results

3.1 Synthesis and characterization

Targeted complexes 1 and 2 were synthesized under microwave irradiation at 140 °C for 30 min with corresponding yields of 73% and 75%. The complexes were purified by chromatography. The chemical structures of the two ruthenium(II) complexes were confirmed by ESI-MS (Fig. S5†). The mass spectra in acetonitrile exhibited a peak at m/z 742.2 (100%) for 1 and at 694.2 for 2 (100%) which was ascribed to [M − ClO₄⁻ − H⁺]⁺ and agreed with the theoretical value. The chemical shifts (δ) of the ¹H NMR spectra of 1 (Fig. S6†), which were attributed to the protons of each phenanthroline ligand, H₁, H₂, H₃, H₄ and H₅, appeared at 9.10, 8.79, 8.55, 8.45, and 8.35 ppm, respectively. The chemical shifts attributed to the phenazine ring H_a, H_b, H_c, H_d and H_e appeared at 8.35, 7.76, 7.97, 7.83, and 7.62 ppm, respectively. The chemical shifts (δ) of the ¹H NMR spectra of 2, which were attributed to the protons for each bipyridyl ligand, H₁, H₂, H₃, and H₄, appeared at 8.22, 7.44, 7.97 and 8.76 ppm, respectively. The chemical shifts attributed to the phenazine ring H_a, H_b, H_c, H_d, and H_e appeared at 8.54, 7.76, 7.97, 8.04, and 7.71 ppm, respectively.

At present, microwave irradiation, as an alternative heat source, is becoming increasingly popular in chemistry because this preparation method is simple and facilitates rapid heating and cooling, concurrent heating and cooling, and energy efficient “green” synthesis with low-boiling point solvents at high temperature in closed vessels.^34–36 Target compounds 1 and 2 were prepared using microwave-assisted synthesis technology.^37–42 The temperature of the reaction system instantly reached 140 °C after 30 min under microwave irradiation, and was maintained at this temperature during the whole process (Fig. S2†). The yields for 1 and 2 under microwave irradiation were approximately 73% and 75%.

3.2 Photophysical properties of complexes 1 and 2

The fluorescence properties of the two ruthenium complexes in different solvents were investigated. Complexes 1 and 2 dissolved in EtOH, DMEM, and PBS were irradiated under ultraviolet light (365 nm) to observe their fluorescence emission. Complex 1 exhibited stronger red fluorescence than 2, and this fluorescence was not quenched by water, contrary to some compounds with very weak fluorescence whose fluorescence can be quenched by water (Fig. 1A). The electronic absorption spectra of 1 and 2 (100 μM) in Tris–KCl buffer solution (pH 7.2) exhibited characteristic MLCT transition absorption at 500 and 450 nm, respectively. In addition, characteristic intraligand (π → π* charge transition) absorption for complexes 1 and 2 appeared at 260 and 300 nm, respectively (Fig. 1B). In the fluorescence emission spectra, when excited at 400 nm, 1 exhibited strong fluorescence in the range of 500–700 nm, with a maximum intensity of 290 at 590 nm. For 2, only weak fluorescence with a maximum intensity of 30 appeared at around 580 nm (Fig. 1B). These results suggested that compound 1 coordinated with phen exhibited stronger fluorescence than 2 coordinated with bpy, which confirmed that coplanar molecules have stronger conjugation effects in enhancing fluorescence emission.

Fig. 1 (A) Fluorescence of 1 and 2 in EtOH, DMEM, and PBS buffer solution (pH 7.2) excited at 365 nm using a portable lamp. [Ru] = 100 μM. (B) Electronic spectra and emission spectra (λ_ex = 400 nm) of 1 and 2 in Tris–KCl buffer solution (pH 7.2). [Ru] = 100 μM. (C) Images from confocal laser scanning microscopy of HepG2 cells incubated with 1 and 2. Cells were treated with ruthenium(II) complexes for 2 h at 37 °C. Blue: Hoechst 33258, green: Mito-tracker green. [Ru] = 200 μM. (D) A cross sectional compositional line profile of a single cell of 1 and 2 showing the emission intensity under confocal laser scanning microscopy.

3.3 Cellular uptake and distribution

The cellular distributions of complexes 1 and 2 were confirmed using confocal laser scanning microscopy. The nuclei of HepG2 cells were stained blue using Hoechst 33258, whereas the mitochondria were stained green using Mito-tracker green FM. After treatment with 1 (100 μM) for 2 h, strong fluorescence ascribed to 1 was observed in the nucleus, which was almost completely overlaid with the fluorescence of Hoechst 33258, and not overlaid with that of Mito-tracker green FM. For 2, the fluorescence was not only overlaid with the Hoechst 33258 fluorescence, but also with that of Mito-tracker green FM (Fig. 1C). Furthermore, a cross sectional compositional line profile of the single cell emission intensity from each probe clearly showed superimposed emissions of 1 and Hoechst 33258 in a single cell, confirming that they co-localized (Fig. 1D). Remarkably, these data indicated that 1 localized mainly in the nuclei, whereas 2 accumulated in the mitochondria and nuclei of HepG2 cells.

Thus, the cellular uptake and distribution in HepG2, HeLa, and MCF-7 cells were further confirmed. After treatment with either 1 or 2 ([Ru] = 200 μM) for 2 h, both complexes were confirmed to be taken up by all cells (Fig. 2A–C). The luminescence intensities of complexes 1 and 2 in HepG2, HeLa, and MCF-7 are shown in Fig. 2a–c. The results showed that the luminescence intensity of 1 was stronger than that of 2 in tumor cell fluorescence imaging.

Fig. 2 Cellular uptake of 1 and 2 in HepG2 (A), HeLa (B), and MCF-7 (C) cells observed by fluorescence microscopy. Cells were treated with either 1 or 2 ([Ru] = 200 μM) in PBS (1% DMSO, pH 7.2) for 2 h at 37 °C, followed by 2 μg mL⁻¹ DAPI for 10 min. Luminescence intensity of complexes 1 and 2 in HepG2 (a), HeLa (b), and MCF-7 (c).

3.4 Low cytotoxicity of ruthenium(II) complexes toward various cells

Low cytotoxicity against the growth of various cells for both complexes was confirmed using an MTT assay. We examined the in vitro cytotoxicities of these ruthenium(II) complexes 1 and 2 toward HepG2, HeLa, MCF-7 and HaCaT cells by using an MTT assay. All cells were treated with varying concentrations of ruthenium(II) complexes at 37 °C for 24 h to explore their antitumor potential, and the cell viability was determined by MTT assay. After 24 h of treatment with either 1 or 2 at 200 μM, no significant cytotoxic response was observed for HepG2, HeLa, MCF-7, and HaCaT cells toward 1 and 2. The viability for 1 in the HepG2, HeLa, MCF-7 and HaCaT cells was 87.2%, 78.5%, 68.1% and 66.9% respectively, after 24 h of incubation at 37 °C for 24 h. By contrast, the viability for 2 in the HepG2, HeLa, MCF-7 and HaCaT cells was 89.0%, 72.2%, 68.5% and 65.3%, respectively, after 24 h of incubation at 37 °C for 24 h (Fig. 3). These results demonstrated that complexes 1 and 2 generally present low toxicities for luminescence cell imaging under the applied conditions.

Fig. 3 In vitro cell viabilities of HepG2 (A), HeLa (B), MCF-7 (C), and HaCaT (D) cells incubated with 1 and 2 at 37 °C for 24 h.

3.5 Imaging of the nuclei of living tumor cells by ruthenium(II) complexes

Finally, the efficiency of both complexes for imaging living tumor cells was determined. HepG2, HeLa, and MCF-7 cells were incubated with the two ruthenium(II) complexes at 37 °C, and the luminescence changes of both complexes were recorded every 5 min. Prior to incubation with the ruthenium(II) complexes, very weak luminescence was observed in the cells. After the cells were incubated with either 1 or 2, a continuous bright luminescence gradually appeared in the cells after 20 min and the strongest intensity was observed after 120 min (Fig. 4A–C). The red fluorescent spots showed that compounds 1 and 2 continuously entered the cells during incubation. The transfection rates for 1 in the HepG2, HeLa, and MCF-7 cells were approximately 95%, 100%, and 100%, respectively, after 2 h of incubation. By contrast, the transfection rates for 2 in the HepG2, HeLa, and MCF-7 cells were approximately 100%, 52%, and 67%, respectively, after 2 h of incubation (Fig. 4A–C). These results clearly indicated that 1 and 2 rapidly and selectively highlighted specific regions of the living cells, leading to significant luminescence enhancement. Obviously, compound 1 entered the cells faster than 2 and the transfection rate of 1 also exceeded that of 2. Furthermore, the possible effectors of the cellular internalization process were investigated by evaluating the lipophilicities of ruthenium(II) complexes 1 and 2 using the octanol/water partition coefficient (log P_o/w). The measured partition coefficients of the ruthenium(II) complexes were 0.10 for 2 (lipophilic) to 0.18 for 1 (lipophilic) (Fig. 4G). Obviously, compound 1 is comparatively more lipophilic than 2. In addition, UV-vis absorption spectra showed a decrease in the concentration of both complexes with prolonged time in the cell culture medium (Fig. S8 and S9†). The fluorescence intensity of 1 increased faster and was stronger than that of 2 (Fig. 4D–F), which may be attributed to the higher lipophilic partition coefficient of 1 (log P = 0.18) than that of 2 (log P = 0.10).

Fig. 4 Real-time fluorescence observations of HepG2 (A), HeLa (B), and MCF-7 (C) cells after treatment with 1 or 2 (200 μM) in DMSO and PBS (pH 7.2, 1 : 99, v/v) for 2 h at 37 °C. Time course of the transfection rates of 1 and 2 in HepG2 (a), HeLa (b), and MCF-7 (c) cells. Time dependence of changes in concentration of ruthenium(II) complexes 1 and 2 detected by UV-vis absorbance of (D) HepG2, (E) HeLa, and (F) MCF-7 cells; (G) octanol/water partition coefficients of ruthenium(II) complexes 1 and 2 at room temperature.

4. Discussion

Ruthenium(II) complexes have been designed and investigated as potential fluorescent probes for cells, but they have not been successfully used in imaging of the nuclei of living cells.⁴³ The most promising complexes reported are polypyridine ruthenium(II) complexes coordinated with dppz, which is an enlarged aromatic ring intercalating ligand.^{17,18,44–46} However, the utilization of these complexes is limited because of their high toxicity and low efficacy.^{27,28,45–49} In the present study, two novel ruthenium(II) complexes coordinated with CQM, which mimics the structure of dppz ligands, were synthesized and their use as fluorescent probes was demonstrated by highly efficient staining of the nuclei of different tumor cells. The cellular uptake of 1 and 2 by HepG2 cells was confirmed by confocal laser scanning microscopy. The results showed that 1 localized in the nuclei of cells, whereas 2 accumulated in the nuclei and mitochondria. This phenomenon was further confirmed for other types of tumor cell, namely, HeLa, and MCF-7 cells. All cells were stained with either 1 or 2. The toxicity of both complexes toward various tumor cells was evaluated using MTT methods to determine whether both complexes can be used to probe living cells. As expected, both complexes exhibited low toxicity toward HepG2, HeLa, MCF-7, and HaCaT cells, even at a high concentration of 200 μM. Finally, real-time fluorescence observations were made to evaluate the efficiency of both complexes for probing the nuclei of living tumor cells. The characteristic red fluorescence ascribed to these complexes gradually intensified in the nuclei of the various cell lines during incubation. The strongest intensity was observed after 90 min, and was maintained until 120 min. This characteristic was further confirmed by the decrease in the amounts of 1 and 2 in the cell culture medium. Therefore, both 1 and 2 can be used as novel fluorescent probes in the imaging of the nuclei of living tumor cells. In addition, 1 and 2 were also distinguishable. The hydrophobicity of ancillary ligands plays a key role in promoting the penetration of a complex into the membrane. Complexes with high hydrophobicity are generally easily taken up by cells.^{19,21,24–27} In our studies, the octanol/water partition coefficients (log P) obtained for 1 and 2 were approximately 0.18 and 0.10, respectively. These results were consistent with the reported values.⁵⁰ In summary, a method has been constructed to develop novel fluorescent probes for targeted imaging of the nuclei of living tumor cells using ruthenium(II) complexes coordinated with CQM. The detailed fluorescence mechanism of these complexes is under further investigation.

5. Conclusions

In summary, two novel ruthenium(II) complexes coordinated with 6-chloro-5-hydroxylpyrido[3,2-a]phenazine (CQM), [Ru(L)₂(CQM)]ClO₄ (L = phen, 1; and bpy, 2), have been synthesized. It was demonstrated that both complexes can be taken up by tumor cells, with 1 localized mainly in the nuclei, while 2 accumulated in the mitochondria and nuclei of HepG2 cells. Further studies by real time fluorescence observations showed that both 1 and 2, especially 1, can be used to image the nuclei of living tumor cells. This kind of complex may be developed as a low toxicity fluorescent probe for tumor cells in future.

Acknowledgements

This work was supported by the National Nature Science Foundation of China (81373257), the Provincial Major Scientific Research Projects in Universities of Guangdong Province (2014KZDXM053), the Science and Technology Item Foundation of Guangzhou (2013J4100072) and the Joint Natural Sciences Fund of the Department of Sciences and Technology & the First Affiliated Hospital of Guangdong Pharmaceutical University (GYFYLH201309).

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Footnotes

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c5ra11127g

‡ These authors contributed equally to the work.

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