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

Article abstract of DOI:10.1039/c5ra11127g

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.

Full text of DOI:10.1039/c5ra11127g

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Imaging of the nuclei of living tumor cells by novel
ruthenium(II) complexes coordinated with 6ꢀchloroꢀ5ꢀ
hydroxylpyrido[3,2ꢀa]phenazine
Yang Ding1, Qiong Wu1, Kangdi Zheng2, Linkun An3*, Xiaoying Hu1, Wenjie Mei1*
1 School of Pharmacy, Guangdong pharmaceutical University, Guangzhou 510006, China.
2 School of Tradition Chinese Medicine, Guangdong Pharmaceutical University, Guangzhou
510006, China.
3 School of Pharmaceutical Sciences, Sun Yatꢀsen University, Guangzhou 510006, China
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Abstract
Two novel ruthenium(II) complexes coordinated with 6ꢀchloroꢀ5ꢀhydroxylpyrido
[3,2ꢀa]phenazine (CQM), [Ru(L)2(CQM)]ClO4 [L = 1,10ꢀphenanthroline, (1) and 2,2'ꢀ
bipyridine, (2)], were investigated as potent fluorescence probes to track the dynamic
changes in the nuclei of living cells. Confocal laser technology was used to observe their coꢀ
location inside the cells. Results showed that both complexes were uptaken by HepG2 cells,
especially for 1, which was localized in the cell nuclei, whereas 2 was distributed in the cell
nuclei and mitochondria. Further studies by realꢀtime fluorescence observation revealed that
1 rapidly entered the living cells, namely, HepG2, HeLa and MCFꢀ7 cells, imagined the
dynamic in the nuclei of living tumor cells, and exhibited low toxicity to cells. Results
demonstrated that 1 may be developed as a novel fluorescence probe for living cell nucleus.
This study facilitates the development of fluorescence chemosensors with metal complexes.
Keywords: Ruthenium(II) complexes; 6ꢀChloroꢀ5ꢀhydroxylpyrido[3,2ꢀa]phenazine (CQM);
Fluorescence probe; Nucleus; Living tumor cell
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1. Introduction
Numerous studies have been performed to develop simple, sensitive, specific, and robust
fluorescence 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 still limited because of their poor water solubility, low photoꢀstability, and
high toxicity.2–5
To overcome the drawbacks of organic dyes, transition metal complexes, especially the
versatile ruthenium(II) complexes, have increasingly attracted attention as potential
fluorescence probes because of their wide spectral range, longꢀterm 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 endosome, in mitochondria, in lysosomes and 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 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=4,7ꢀdiphenylꢀ1,10ꢀ
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phenanthroline) is effectively transported into the cellular interior.19 Furthermore, the
mechanism of cellular entry of a luminescent ruthenium(II) polypyridyl complex
[Ru(DIP)2(DPPZ)]2+ was measured by flow cytometry and supported the passive diffusion of
[Ru(DIP)2(DPPZ)]2+ into the cell.20 Rajendiran et al. also reported that a series of mixed
ligand ruthenium(II) complexes [Ru(5,6ꢀdmp/3,4,7,8ꢀtmp)(diimine)]2+
as fluorescent probes for nuclear and protein components.21 [Ru(phen)2(DPPZ)]2+ has also
been reported to be uptaken by cells and used as an efficient optical probe for staining
nuclear components,22 whereas ruthenium(II) complexes containing 7ꢀFꢀdppz [7ꢀ
fluorodipyrido (3,2ꢀa:2′,3′ꢀc)phenazine] can be uptaken by cells and localize in the nucleus.23
More recently, an alkyl ether chain has been bound into a dppz ligand, which increased the
lipophilicity and membrane permeability of ruthenium(II) complexes.24,25 In addition,
rutheniumꢀoctaarginineꢀconjugated 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 few agents
succeed in direct staining nuclear ascribe to their poor membrane permeability, poor cellular
uptake and high toxicity, and it’s remains a huge challenge to develop novel fluorescence
probe of 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 these complexes
under investigation.
Studies were performed to target metal complexes to the nucleus more effectively. Two
novel ruthenium(II) complexes coordinated with CQM, [Ru(L)2(CQM)]2+, [1,10ꢀ
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phenanthroline, (1) and 2,2'ꢀbipyridine, (2)] (Scheme 1), were synthesized under microwave
irradiation, and their properties on 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 using. Ruthenium chloride
hydrate was obtained from Mitsuwa Chemicals. 8ꢀHydroxyquinoline was purchased from
Aladdin. All the chemicals including solvents were obtained from commercial vendors and
used as received. The TrisꢀKCl 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 Anton Paar Monowave 300 microwave reactor
1
13
(an initiator single mode microwave cavity at 2450 MHz, Biotage). H NMR and C NMR
spectra were recorded on a Bruker DRX 2500 spectrometer in d6ꢀDMSO, and the
electrospray ionisation mass spectrometry (ESIꢀMS) spectra were obtained in acetonitrile on
an Agilent 1100 ESIꢀMS system operating. The electronic absorption spectra were recorded
on a Shimadzu UVꢀ2550 spectrophotometer, the steadyꢀstate emission spectra were recorded
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on a RFꢀ5301 fluorescence spectrophotometer. Fluorescence images of cellular localization
were obtained using EVOS® FL Auto Imaging System. LivingꢀCell Confocal imaging was
performed with an Zeiss LSM 510 META.
2.2 Synthesis and Characterization
6,7ꢀdichloroquinolineꢀ5,8ꢀdione (DCQ) was prepared by using a similar method to that
was reported in the literature.32 In general, a mixture of 8ꢀHydroxyquinoline (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 onꢀstep column
1
chromatography of silicaꢀgel. Yields, 65%. H NMR (500 MHz, DMSOꢀd6): 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). 6ꢀ
Chloroꢀ5ꢀhydroxylpyrido[3,2ꢀa] phenazine (CQM) was synthesized according to the
literature with some improvement.33 In general, a mixture of 6,7ꢀdichloroquinolineꢀ5,8ꢀdione
(1 mmol, 0.23 g) and oꢀPhenylenediamine (1.2 mmol, 0.13 g) in 20 ml of ethanol was heated
at 95 °C for 30 min under microwave irradiation. The yellow precipitate was obtained by
filter while it’s hot and washed with ethanol, then dried in vacuo. Yield: 37%. ESIꢀMS (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 cisꢀ[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 cooled to room temperature, the mixture was stirred for 3 min with the
addition of excess saturated NH4PF6 solution. The orange products were obtained by filter
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and purified by onꢀstep column chromatography of silicaꢀgel (166 mg, 73%), ESIꢀMS (in
1
acetonitrile): m/z 742.2. H NMR (500 MHz, d6ꢀDMSO): δ 9.10 (d, J = 5.2 Hz, 1H), 8.87ꢀ
8.70 (m, 3H), 8.58 (dd, J = 11.6, 8.5 Hz, 2H), 8.48 (d, J = 4.5 Hz, 1H), 8.43ꢀ8.27 (m, 5H),
8.23 (t, J = 9.9 Hz, 1H), 8.15ꢀ8.04 (m, 4H), 8.03ꢀ7.93 (m, 2H), 7.88 (dt, J = 7.4, 5.2 Hz, 2H),
7.82ꢀ7.72 (m, 2H), 7.62 (ddd, J = 8.2, 6.8, 3.9 Hz, 3H). 13C NMR (126 MHz, DMSO),
δ167.46 (s), 154.77 (s), 153.48 (s), 152.35 (s), 152.05ꢀ151.78 (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), 136.59ꢀ136.48 (m), 136.45 ꢀ136.36 (m), 136.16ꢀ135.97
(m), 135.42 (s), 133.53ꢀ133.36 (m), 131.92ꢀ131.78 (m), 131.12 (s), 130.83 (s), 130.62 (s),
129.76ꢀ129.69 (m), 127.56 (s), 127.00ꢀ126.95 (m), 126.92ꢀ126.82 (m), 126.75 (s), 126.51 (s),
126.13 (s), 125.71 (s), 112.67 (s).
Synthesis of [Ru(byp)2(CQM)](ClO4)2 (2)
A mixture of CQM (0.184 mmol, 50 mg) and cisꢀ[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 cooled to room temperature, the mixture was stirred for 3 min with the
addition of excess saturated NH4PF6 solution. The orange products were obtained by filter
and purified by onꢀstep column chromatography of silicaꢀgel (60 mg, 75%), ESIꢀMS (in
1
acetonitrile): m/z 694.2. H NMR (500 MHz, d6ꢀDMSO): 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), 7.93ꢀ7.81 (m, 2H), 7.83ꢀ7.71
(m, 3H), 7.72ꢀ7.64 (m, 1H), 7.53 ꢀ7.34 (m, 3H). 13C NMR (126 MHz, DMSO), δ 167.10 (s),
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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), 150.57ꢀ150.33 (m), 143.54 (s), 142.87 (s), 138.73 (s), 172.86ꢀ109.61 (m), 175.64ꢀ
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 culture
Human cancer cell lines, including the HepG2 (hepatocellular carcinoma cell line),
HeLa (cervical carcinoma cell line), and MCFꢀ7 (breast carcinoma cell line) were purchased
from American Type Culture Collection (ATCC, Manassas,VA). The normal HaCaT
(immortalized human epidermal cell line) 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), and streptomycin (50
U/mL) at 37 °C in a CO2 incubator (95% relative humidity, 5% CO2).
2.4 Cellular localization
The cells were cultured in DMEM medium supplemented with 10% Fetal Bovine Serum
(FBS) at 37 °C and 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 by PBS and then
treated with ruthenium(II) complexes 1 and 2 (200 ꢁM) in DMSO/PBS (pH 7.2, 1:99, v/v)
for 2 h at 37 °C and 5% CO2, respectively. Then cells were stained with Hoechst 33258 and
Mitoꢀtracker green FM for another 20 min and finally luminescence imaging by confocal
microscope.
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2.5 Cytotoxicity assay
The vitro cytotoxicity of the ruthenium(II) complexes 1 and 2 toward HepG2, HeLa,
MCFꢀ7, and HaCaT cells have been studied by the 3ꢀ(4,5ꢀDimethylthiazole)ꢀ2,5ꢀ
diphenyltetraazolium bromide (MTT) assay. Cells were incubated with different
concentrations (0, 25, 50, 100, 150, 200 ꢁM) of complexes for 24 h. 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 96ꢀwell tissue culture
plates for 24 h. After incubation, 20 ꢁL per well of MTT solution (5 mg/mL phosphate
buffered saline) was added and 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 the mean ± the standard deviation. The
following formula was used to calculate the viability of cell growth:
Viability (%) = (mean of Absorbance value of treatment group / mean Absorbance
value of control)·100
2.5 Real-time fluorescence images
The cells were grown in DMEM medium supplemented with 10% Fetal Bovine Serum
(FBS) at 37 °C and 5% CO2. Cells (2×106) were seeded in 75 cm2 culture flasks for 12 h
before imaging. The cells were incubated with the Hoechst 33258 for 10 min at 37 °C under
5% CO2 followed by carefully washing cells with PBS solution and then incubated with the
complexes 1 and 2 (200 ꢁM), respectively. At last, the cells were monitored with
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fluorescence microscopy to obtain realꢀtime fluorescence images every 5 min.
2.6 Cellular uptake of complexes 1 and 2
The cells were cultured DMEM medium supplemented with with 10% Fetal Bovine
Serum (FBS) at 37 °C and 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 to 10 mL of the cell
culture medium solutions containing the complexes 1 and 2 (50 ꢁM) and the flasks were
incubated at 37 °C, 5% CO2 for 6 h. The UVꢀVis spectra of the solutions in HepG2, HeLa
and MCFꢀ7 cells treated with complexes 1 and 2 were tested every one hour, respectively.
2.7 Amphiphilicity measurement
The lipoꢀhydro partition coefficient of the complexes was tested by using octanolꢀ
water twoꢀphase system. In general, equal amounts of octanol and distilled water were
thoroughly mixed in the oscillator for 24 h, then separate twoꢀphase solution. Complexes
were carefully dissolved in water phase and octanol phase for 20 ꢁM solution
with sufficient mixing. After separation, the final concentration of the water phase was
denoted as Cw and the concentration of the octanol phase denoted as Co. Both Co and Cw were
tested by ultravioletꢀvisible (UVꢀvis) spectrophotometer, and the partition coefficient (Po/w)
for the complex was calculated according to the equation: Po/w = Ao/Aw.
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
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chromatography. The chemical structures of the two ruthenium(II) complexes were further
confirmed by ESIꢀMS (Fig. S5). The mass spectra in acetonitrile exhibited a peak at m/z
742.2 (100%) for 1 and 694.2 for 2 (100%) which was ascribed to [MꢀClO4 ꢀH+]+ and agreed
ꢀ
1
with the theoretical value. The chemical shifts (δ) of the H NMR spectra of 1 (Fig. S6),
which were attributed to the protons of each phenanthroline ligand of 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 Ha, Hb, Hc, Hd and He 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 of 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 Ha, Hb, Hc, Hd, and He
appeared at 8.54, 7.76, 7.97, 8.04, and 7.71 ppm, respectively.
To date, 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
lowꢀboiling solvents at high temperature in closed vessels.34–36 Target compounds 1 and 2
were prepared by microwaveꢀassisted synthesis technology.37–42 The temperature of the
reaction system instantly reached 140 °C for 30 min under microwave irradiation, which was
maintained during the whole process (Fig. S2). The corresponding 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 was
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investigated. Complexes 1 and 2 resolved in EtOH, DMEM, and PBS were irradiated under
ultra light (365 nm) to observe their emission fluorescence. Complex 1 exhibited stronger red
fluorescence than 2, and this fluorescence was not quenched by water, contrary to some
compounds with very weak fluorescenceas because their fluorescence can be quenched by
water (Fig. 1A). The electronic absorption spectra of 1 and 2 (100 ꢁM) in TrisꢀKCl buffer
solution (pH 7.2) exhibited characterized MLCT transition absorption at 500 and 450 nm,
respectively. In addition, the characterized intraligand (π→π* charge transition) absorption
for complexes 1 and 2 appeared at 260 and 300 nm, respectively (Fig. 1B). In the emission
fluorescence spectra, when excited at 400 nm, 1 exhibited strong fluorescence in the range of
500–700 nm, with the maximum intensity of 290 at 590 nm. For 2, only a weak fluorescence
with the maximum intensity of 30 appeared at around 580 nm (Fig. 1B). Theses results
suggested that compound 1 coordinated with phen exhibited stronger fluorescence than 2
coodinated with bpy, which confirmed that coplanar molecules have stronger
conjugation effects on enhancing fluorescence emission.
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 Mitoꢀtracker green FM. After treatment
with 1 (100 ꢁM) for 2 h, strong fluorescence ascribed to the 1 was observed in the nucleus,
which was almost completely overlaid with that of Hoechst 33258, and not overlaid with that
of Mitoꢀtracker green FM. For 2, the fluorescence was not only overlaid with Hoechst 33258,
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but also with Mitoꢀtracker green FM (Fig. 1C). Further a cross sectional compositional line
profile of a single cell emission intensity from each probe clearly showed superimposed
emissions of 1 and Hoechst 33258 in a single cell, and confirming that they coꢀlocalized (Fig.
1D). Remarkably, these data indicated that 1 localized mainly in the nuclei, whereas 2
accumulated in the mitochondria and nuclei of HepG2 cells.
Figure 1
Thus, the cellular uptake and distribution by HepG2, HeLa, and MCFꢀ7 cells were
further confirmed. After treatment with either 1 or 2 ([Ru] = 200 ꢁM) for 2 h, both complexes
were confirmed to be uptaken by all cells (Figs. 2A–C). The luminescence intensity of
complexes 1 and 2 in the HepG2, HeLa, and MCFꢀ7 are shown in Figs. 2a–c. The results
showed that the luminescence intensity of 1 was stronger than that of 2 in tumor cell
fluorescence imaging.
Figure 2
3.3 Low cytotoxicity of ruthenium(II) complexes against various cells
Low cytotoxicity against the growth of various cells of both complexes was confirmed
using MTT assay. We examined the various cells in vitro cytotoxicities of these
ruthenium(II) complexes 1 and 2 against HepG2, HeLa, MCFꢀ7 and HaCaT cells by using
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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 cell viability was determined by MTT
assay. After 24 h of treatment with either 1 or 2 at 200 ꢁM, no significant cytotoxic response
was observed for HepG2, HeLa , MCFꢀ7, and HaCaT cells for 1 and 2. The viability for 1 in
the HepG2, HeLa, MCFꢀ7 and HaCaT cells were 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 1 in the
HepG2, HeLa, MCFꢀ7 and HaCaT cells were 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 presented low toxicities for luminescence cell imaging under the applied
conditions.
Figure 3
3.3 Imaging of the nuclei of living tumor cells by ruthenium(II) complexes
Finally, the efficiency of both complexes for imagining living tumor cells was
determined. HepG2, HeLa, and MCFꢀ7 cells were incubated with the two ruthenium(II)
complexes at 37 °C, and the luminescence changes in 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 cell after 20 min and reach to the strongest intensity
was observed at 120 min (Figs. 4A–C). The red fluorescence spots showed that compounds 1
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and 2 were continuously entered the cell during incubation. The transfection rates for 1 in the
HepG2, HeLa, and MCFꢀ7 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
MCFꢀ7 cells were approximately 100%, 52%, and 67%, respectively, after 2 h of incubation
(Figs. 4A–C). The results clearly indicated that 1 and 2 rapidly and selectively highlighted
specific regions of living cells, leading to significant luminescence enhancement. Obviously,
compound 1 entered 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 Po/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, UVꢀvis absorption spectra
showed a decrease in the concentration of both complexes after the time elapsed in the cell
culture medium of cells by UVꢀvis absorption spectra (Fig. S8ꢀ9). The fluorescence intensity
of 1 increased faster and was stronger than that of 2 (Figs. 4D–F), which may be attributed to
the higher lipophilic partition coefficients of 1 (log P = 0.18) than that of 2 (log P = 0.10).
Figure 4
4 Discussion
Ruthenium(II) complexes have been designed and investigated as potential fluorescence
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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 rutnenium(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 to the structure of dppz ligands, were synthesized and demonstrated by
the highly efficient staining of the nuclei of different tumor cells. The cellular uptake of 1 and
2 by HepG2 cells was first confirmed by confocal laser scanning microscopy. Results showed
that 1 localized in the nuclei of cells, whereas 2 accumulated in the nuclei and mitochondria.
This phenomenon was further confirmed on other type of tumor cells, namely, HeLa, and
MCFꢀ7 cells. All cells were stained by either 1 or 2. Thus, the toxicity of both complexes
against various tumor cells was evaluated by MTT methods to determine whether both
complexes can be used to probe living cells. As expected, both complexes exhibited low
toxicity to HepG2, HeLa, MCFꢀ7, and HaCaT cells even at a high concentration 200 ꢁM.
Finally, realꢀtime fluorescence observation was observed to evaluate the efficiency of both
complexes for probing the nuclei of living tumor cells. Thus, the characterized red
fluorescence ascribed to these complexes gradually intensified in the nuclei of the various
cell lines during incubation. The strongest intensity was observed at 90 min, which was
maintained until 120 min. This characteristic was further confirmed by the decrease in the
amounts of 1 and 2 in the cells culture medium. Therefore, both 1 and 2 can be used as novel
fluorescence probes in imaging of the nuclei of living tumor cells. In addition, 1 and 2 were
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also distinguished. 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 uptaken 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 fluorescence probes in targeting imaged nuclei of living tumor
cells using ruthenium(II) complexes coordinated with CQM. The detailed mechanism of
these complexes are under further investigations.
5 Conclusions
In summary, two novel ruthenium(II) complexes coordinated with 6ꢀchloroꢀ5ꢀ
hydroxylpyrido[3,2ꢀa]phenazine (CQM), [Ru(L)2(CQM)]ClO4 (L = phen, 1; and bpy, 2), have
been synthesized. It’s demonstrated that both complexes can be uptaken by tumor cells, with
1 localized mainly in the nuclei, while 2 accumulated in the mitochondria and nuclei of
HepG2 cells. The further studies by real time fluorescence observations show that both 1 and
2, especially 1 can be used to image the nuclei of living tumor cells. In a word, this kind of
complexes may be developed as low toxicity fluorescence probe of tumor cells in the future.
Acknowledgements
This works 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
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(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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Figure Captions
Scheme 1. Molecular structure of ruthenium(II) complexes 1 and 2.
Figure 1. (A) Fluorescence of 1 and 2 in EtOH, DMEM, and PBS buffer solution
(pH 7.2) excited at 365 nm from a portable lamp. [Ru] = 100 ꢀM. (B) Electronic
spectra and emission spectra (λ ex = 400 nm) of 1 and 2 in TrisꢁKCl buffer solution (pH
7.2). [Ru] = 100 ꢀM. (C) Images of 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, Green: Mitoꢁtracker green. [Ru] = 200 ꢀM. (D) Across sectional
compositional line profile of a single cell of 1 and 2 emission intensity under confocal
laser scanning microscopy image.
Figure 2. Cellular uptake of 1 and 2 in HepG2 (A), HeLa, (B) and MCFꢁ7 (C) cells by
fluorescence microscopy. Cells treated with either 1 or 2 ([Ru] = 200 ꢀM) in PBS (1%
DMSO, pH 7.2) for 2 h at 37 °C, followed by 2 ꢀg/mL DAPI for 10 min. Luminescence
intensity of complexes 1 and 2 in the HepG2 (a), HeLa (b), and MCFꢁ7 (c).
Figure 3. In vitro cell viabilities of HepG2 (A), HeLa (B), MCFꢁ7 (C), and HaCaT (D)
cells incubated with 1 and 2 at 37 °C for 24h.

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Figure 4. Realꢁtime fluorescence observation of HepG2 (A), HeLa (B), and MCFꢁ7 (C)
cells after treatment by 1 or 2 (200 ꢀM) 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
MCFꢁ7 (c) cells. Time dependence of changes in concentration of ruthenium(II)
complexes 1 and 2 detected by UVꢁvis absorbance of (D) HepG2, (E) HeLa, (F)
MCFꢁ7 cells; (G) Octanol/water partition coefficients of ruthenium(II) complexes 1
and 2 at room temperature.

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Scheme 1

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Figure 1

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Figure 2

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Figure 3

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Figure 4