Enhanced singlet oxygen generation of a soft salt through efficient energy transfer between two ionic metal complexes

Article abstract of DOI:10.1039/c8dt00720a

In this study, a soft salt complex based photosensitizer has been developed for photodynamic therapy (PDT) of cancer cells. The iridium(iii) complex [Ir(L)(L′)]³⁺(PF₆^-)₃ (C1) with L and L′ being terpyridine ligands (L = 4′-phenyl-2,2′:6′,2′′-terpyridine, L′ = 3-([2,2′:6′,2′′-terpyridin]-4′-yl)-9-hexyl-9H-carbazole) was chosen as the cationic component, and the iridium(iii) complex [Ir(dfppy)₂CN₂]^-Bu₄N⁺ (A1) was selected as the anionic component. Complexes C1 and A1 are directly connected through electrostatic interaction to form a soft salt based photosensitizer (S1), which exhibited an enhanced singlet oxygen generation rate because of efficient energy transfer between two ionic metal complexes. Furthermore, this novel photosensitizer was successfully applied in photodynamic therapy (PDT) of cancer cells for the first time.

Full text of DOI:10.1039/c8dt00720a

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Efficient extraction of sulfate from water using
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DOI: 10.1039/C8DT00720A
Table of Content
Enhanced singlet oxygen generation of a soft salt through
efficient energy transfer between two ionic metal complexes
Yun Ma, Shujun Zhang, Huanjie Wei, Yafang Dong, Liang Shen, Shujuan Liu, Qiang Zhao*
Li Liu* and Wai-Yeung Wong*
A novel soft salt based photosensitizer was successfully developed for the application in
photodynamic therapy of cancer cells for the first time.

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DOI: 10.1039/C8DT00720A
Journal Name
ARTICLE
Enhanced singlet oxygen generation of a soft salt through efficient
energy transfer between two ionic metal complexes
Received 00th January 20xx,
Accepted 00th January 20xx
Yun Ma,ab Shujun Zhang,b Huanjie Wei,b Yafang Dong,b Liang Shen,b Shujuan Liu,b Qiang Zhao,b* Li
Liu,c* and Wai-Yeung Wongad*
DOI: 10.1039/x0xx00000x
In this study, a soft salt complex based photosensitizer has been developed for photodynamic therapy (PDT) of cancer
cells. The iridium(III) complex [Ir(L)(L')]3+(PF6 )3 (C1) with L and L' being terpyridine ligands (L = 4'-phenyl-2,2':6',2''-
www.rsc.org/
-
terpyridine, L' = 3-([2,2':6',2''-terpyridin]-4'-yl)-9-hexyl-9H-carbazole) was chosen as the cationic component, and the
iridium(III) complex [Ir(dfppy)2CN2]-Bu4N+ (A1) was selected as the anionic component. Complexes C1 and A1 are directly
connected through electrostatic interaction to form a soft salt based photosensitizer (S1), which exhibited enhanced
singlet oxygen generation rate because of efficient energy transfer between two ionic metal complexes. Furthermore, this
novel photosensitizer was successfully applied in photodynamic therapy (PDT) of cancer cells for the first time.
challenge because of their complicated excited-state
properties.9 Soft salt is composed of an anionic component
and a cationic component linked through electrostatic and van
Introduction
Photodynamic therapy (PDT) is based on the concept that
cancer cells are killed by cytotoxic reactive oxygen species
(ROS), which is generated from photosensitizers upon light
irradiation.1 As one of the most common toxic ROS, singlet
oxygen (1O2) is generated by energy transfer from the triplet
excited state of photosensitizers to the ground state of O2.2 Up
to now, numerous photosensitizers have been developed for
the generation of 1O2, such as organic dyes,3 conjugated
polymers,4 nanoparticles5 and transition metal complexes.6
Among them, transition-metal complexes are most widely
der Waals interactions.10 Recently, soft salts have been
successfully applied in organic light-emitting diodes by
Thompson and his co-workers.10a We have also applied them
as phosphorescent ratiometric and lifetime probes for
intracellular pH detection.10b Nevertheless, much less
attention has been paid to the research work of soft salts. The
cyclometalated ligands of the two ionic complexes can be
easily modified, which are helpful in tuning the absorption and
emission peaks of the cationic and anionic components of a
soft salt. Therefore, soft salt could be a promising platform for
1
used as the photosensitizers due to their high O2 generation
rate and photostability.7
1
the design of new photosensitizer to enhance O2 generation
by energy transfer. Until now, however, the use of soft salts in
PDT is still an unexplored area.
In recent years, a design strategy has been provided for the
improvement of the 1O2 generation rate through Förster
resonance energy transfer (FRET).8 However, complex
synthesis or modification procedures are required for the
preparation of these systems. Especially, the design of
transition-metal complex based energy transfer system is still a
In the present study, the first example of a soft salt based
photosensitizer for the enhancement of 1O2 generation by
energy transfer is reported. The iridium(III) complex
-
[Ir(L)(L')]3+(PF6 )3 (C1) with L and L' being terpyridine ligands
F
3
F
N
N
N
N
a.Institute of Molecular Functional Materials and Department of Chemistry, Hong
Kong Baptist University, Waterloo Road, Kowloon Tong, Hong Kong, P. R. China.
E-mail: rwywong@hkbu.edu.hk
CN
CN
-
N
N
3PF6
Ir
F
N
N
Ir
N
N
b.Key Laboratory for Organic Electronics & Information Displays (KLOEID) and
Institute of Advanced Materials (IAM), Nanjing University of Posts &
Telecommunications (NJUPT), 9 Wenyuan Road, Nanjing 210023, Jiangsu, P. R.
China. E-mail: iamqzhao@njupt.edu.cn
F
C1
A1
F
c. Hubei Collaborative Innovation Center for Advanced Organic Chemical Materials,
Ministry of Education Key Laboratory for the Synthesis and Application of Organic
Functional Molecules, School of Chemistry and Chemical Engineering, Hubei
University, Wuhan 430062, Hubei, P. R. China. E-mail: liulihubei@hubu.edu.cn
d.Department of Applied Biology and Chemical Technology, The Hong Kong
Polytechnic University, Hung Hom, Hong Kong, P. R. China. E-mail: wai-
yeung.wong@polyu.edu.hk
3
F
N
N
N
CN
N
N
Ir
N
Ir
N
N
CN
N
3
F
F
S1
† Electronic Supplementary Information (ESI) available: [Synthetic details, UV-
visible spectra and cell imaging data]. See DOI: 10.1039/x0xx00000x
Scheme 1 Chemical structures of complexes A1
,
C1 and S1.
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tetrabutylammonium cyanide (5 equiv) in dichloromethane for 5 h.
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The terpyridine ligands and their corresponDdOinIg: 1c0a.1t0io3n9i/cC8irDidTi0u0m72(I0IIA)
complex (C1) were prepared according to the previous methods.11
Then, the complex S1 was obtained by stirring A1 and C1 (3 : 1
molar ratio) in the mixture of acetone-water at room temperature.
The desired complexes were characterized by NMR spectroscopy,
MALDI-TOF spectrometry and elemental analysis.
Photophysical properties
The photoluminescence (PL) spectrum of A1 and absorption
spectrum of C1 were investigated in CH3CN solution (10 μM). From
Fig. 1a, it can be seen that A1 exhibited emission peaks at 451 and
475 nm, and the absorption band of C1 was located at around 454
nm. The emission peak of A1 and the absorption band of C1 are
well overlapped, indicating that an efficient energy transfer might
occur from A1 to C1. The quenching study was carried out based on
the equation of I0/I = 1 + kq[Q], where I0 and I refer to the PL
intensity of A1 with and without the quencher C1, kq represents the
experimental quenching rate constant and [Q] indicates the molar
concentration of the quencher. Upon addition of an increasing
amount of C1 into A1 a CH3CN solution (3 × 10-5 M), the blue
emission was gradually decreased in intensity, demonstrating the
Fig. 1 (a) Normalized absorption and photoluminescence spectra of A1 and C1 in CH3CN
solution. (b) Stern-Volmer plot of the quenching study between A1 and C1 ([Q] is the
concentration of quencher). The final concentrations for A1 and C1 are 1 × 10-5 M and
3 × 10-5 M.
(L
=
4'-phenyl-2,2':6',2''-terpyridine, L'
=
3-([2,2':6',2''-
terpyridin]-4'-yl)-9-hexyl-9H-carbazole) was selected as the
cationic component. This type of iridium(III) complexes
possesses a high cationic character (charge +3), which displays
the following advantages: (i) three anionic iridium(III)
complexes are required for the formation of a soft salt
complex, and (ii) the electrostatic interactions with the anionic
iridium(III) complex would be strong. These features ensure
the efficient energy transfer between the two ionic complexes.
Besides, the iridium(III) complex [Ir(dfppy)2CN2]-Bu4N+ (A1) was
chosen as the anionic component because its emission peak
overlaps with the absorption band of C1. Soft salt complex S1
was formed by these two components through electrostatic
interaction. Complex S1 exhibited enhanced 1O2 generation
rate and it has been successfully applied as a photosensitizer
for PDT.
Results and discussion
Synthetic procedures
Fig. 2 (a) The decrease of DPBF absorption at 412 nm as a function of irradiation
time in the presence of complexes A1 (30 μM), C1 (10 μM), S1 (10 μM) and
[Ru(bpy)3]2+(Cl-)2. The control plot was with only light for S1 without DPBF. (b)
The soft salt S1 complex was synthesized in two steps. Firstly, the
respective anionic A1 and cationic C1 iridium(III) complexes were
prepared. Complex A1 was synthesized through refluxing
The bleaching of DPBF for C1/A1 versus irradiation time at a fixed equivalent of
C1 with varying A1 equivalents. [C1] = 10 µM.
[Ir(dfppy)2Cl]2 (dfppy
=
2-(2,4-difluorophenyl)pyridine) and
2 | J. Name., 2012, 00, 1-3
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1
efficient energy transfer/quenching process between the two ionic transfer from A1 to C1 was dependent on their equivalent, the O2
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metal complexes. The calculation of the Stern–Volmer plot of the generation as a function of an equivalent raDtiOoIw: 1a0s.1a0l3s9o/Cd8eDteTr0m07in2e0dA
two components yielded a kq value of 0.94 × 106 M-1, which by the DPBF indicator. Fig. 2b illustrates the time curves of the
suggested the superquenching behavior of A1 by C1. From Fig. 1b, it decrease of absorbance of DPBF at 1.0 equivalent of C1 with A1
indicates that the emission of A1 and the absorption of C1 coincide equivalent varying from 1.0 to 4.0. The bleaching rate of DPBF was
with each other and the FRET is likely to be responsible for the observed to improve as A1 equivalent increased, reached a peak
energy transfer in this system.
after 3.0 equivalents (Fig. 2b). The observations mentioned above
further demonstrated that the capability of 1O2 generation was
increased by the energy transfer from A1 to C1. A possible reason is
that the energy transfer between the two complexes influenced the
triplet excited state generation, singlet state generation, and
intersystem crossing in the whole system. In addition, the stability
of S1 in acetonitrile/buffer (1 : 9, v : v) and cell culture medium at
37 °C was investigated. Fig. S5 shows that the PL intensity at 451 nm
barely changed at 37 °C even after 3 h, indicating the good stability
of S1.
Singlet oxygen generation rate
1,3-Diphenylisobenzofuran (DPBF) was employed to detect the
generation of 1O2 of A1, C1 and S1. In the presence of 1O2, the
absorption band of DPBF at 412 nm will decrease.2e As illustrated in
Fig. 2a, the capability of A1, C1 and S1 to generate 1O2 was
measured by irradiation with a white light source (12 mW cm−2).
The decrease of DPBF absorption at 412 nm as a function of the
irradiation time indicated that a small DPBF absorption intensity
change was observed for A1 or C1. In sharp contrast, the DPBF
absorbance for S1 was remarkably decreased. The DPBF bleaching
PDT of cancer cells
rate constants (kobs) in the presence of A1, C1 and S1 were To study the dark cytotoxicity and phototoxicity of A1, C1 and
measured to be 1.49 × 10-2 s-1, 8.71 × 10-3 s-1, and 3.68 × 10-2 s-1, S1, the standard methyl thiazolyl tetrazolium (MTT) assay was
respectively. Compared with the bleaching rate for S1, that for C1 conducted to measure the relative viabilities of HeLa cells. Fig.
was 4.23 times slower and that for A1 was 2.46 times slower. 3a shows that there is no obvious cellular death after different
1
Therefore, the generation of O2 benefited greatly from the energy concentrations of A1
,
C1 and S1 were treated to cells for 24
transfer between the two components. In addition, the singlet hours under the dark environment. The cellular viabilities were
oxygen generation ability of [Ru(bpy)3]2+(Cl-)2 has been measured as assessed to be approximately 90% even at
a high
the reference (Fig. 2a). Compared to the [Ru(bpy)3]2+(Cl-)2, S1 concentration of 50 μM, which suggested the low cytotoxicity
showed lower singlet oxygen generation ability. As the energy of these complexes in the dark. However, the cellular viabilities
drastically decreased to 61%, 69% and 37% for A1, C1 and S1
(10 μM) when the cells were exposed to the white light (12
mW cm−2) for 30 min. These results are consistent with the
amount of 1O2 generation measured in the solution.
Afterwards, a systematic study was performed in order to
better demonstrate the cytotoxic effect of S1 on cancer cell
viabilities.
The generation of 1O2 was detected in living cells using the
indicator of DCFH-DA (2’,7’-dichlorofluoresceindiacetate), which
can be altered to DCF (2,7-dichlorofluorescein, λem = 529 nm) with a
1
green fluorescence in the presence of O2. Fig. S6 shows that HeLa
cells treated with DCFH-DA followed by a white light irradiation
displayed very weak green fluorescence. However, intense green
fluorescence was observed when S1 was used to pre-process the
1
cells, demonstrating the generation of intracellular O2 as displayed
in Fig. S7. In addition, the dual fluorescence of Annexin V–
FITC/propidium iodide (PI) was applied to monitor the cell death
caused by S1-mediated PDT. It can be seen from Fig. 4 that an
increasing number of apoptotic cells was observed when the
irradiation time was prolonged, showing that the PDT effect was
enhanced by increasing the irradiation time and cytotoxicity of S1
was low in the dark. Furthermore, flow cytometry was employed to
study the PDT effect of S1. In flow cytometry experiments, the cell
population was measured by Annexin V–FITC+/PI– of viable cells,
early apoptotic cells and necroticor late-stage apoptotic cells at
various phases of cell death. In the control groups of S1 under the
dark environment and blank exposure to white light, less than 5.0%
cellular death was detected (Fig. 5). In addition, the 25.8% and 18.9%
cellular deaths were measured after the cells were treated by A1
(30 μM) and C1 (10 μM) for 24 hours and then irradiated by white
Fig. 3 In vitro cell viability of HeLa cells. (a) Cell viability values (%) assessed using
an MTT test versus incubation concentrations of A1 C1 and S1 in dark. (b) The
cells were incubated with A1 (30 μM), C1 (10 μM) and S1 (10 μM) and then
irradiated by a white light (12 mW cm−2) with a xenon lamp for 10, 20 and 30 min,
respectively.
,
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Fig. 4 Time-lapse confocal microscopy images of Annexin V–FITC/PI stained S1 loaded Hela cells after 30 min white light irradiation (12 mW cm−2).
generation of transition-metal complexes through an efficient
energy transfer from C1 to A1. Our future work will focus on
the development of soft salt complexes with long-wavelength
absorption in order to perform in vivo PDT.
light for 30 minutes. On the contrary, 28.3% cellular death was
monitored when the cells were treated by S1 (10 μM) for 24 hours
and then irradiated by white light for 10 minutes. Prolonged
irradiation time resulted in more dead cells, and 50.4% cellular
death was observed when the irradiation time was 30 minutes (Fig.
5). These results indicated that S1 could be an effective Experimental
phototherapeutic candidate to cancer treatment.
Materials and methods
Commercially available chemical reagents were used without
Conclusions
further purification. All solvents were purified before use. The
solvents were carefully dried and distilled from appropriate drying
In summary, a novel soft salt based photosensitizer, which
consists of two oppositely charged ionic complexes, has been
developed. The soft salt complex S1 can generate singlet
oxygen to effectively kill the cancer cells, demonstrating its
potential application in PDT. Moreover, it has demonstrated
1
agents prior to use. H and 13C NMR spectra were recorded with a
Bruker Ultrashield 400 MHz FT-NMR spectrometer. Mass spectra
were obtained with
a Bruker Autoflex matrix assisted laser
desorption/ionization time of flight (MALDI-TOF) mass
spectrometer. UV-visible absorption spectra were recorded with a
HP UV-8453 spectrophotometer. Photoluminescent spectra were
measured with an Edinburgh Instrument FLS920. The irradiation
during the PDT process was conducted with a xenon lamp (CEL-HXF
1
that the O2 generation rate for S1 was 4.23-fold faster than
that for C1 and 2.46-fold faster than that for A1. Therefore,
our design principle provides a new strategy for enhancing O2
1
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Fig. 5 Flow cytometry quantification of Annexin V–FITC/PI labeled HeLa cells under different conditions: (a) blank, (b) blank was exposed to white light (12 mW cm−2) for 30 min),
(c) S1 (10 μM) under the dark and (c-f) S1 with white light irradiation (12 mW cm−2) for 10, 20 and 30 min, respectively. (g, h) C1 and A1 with white light irradiation (12 mW cm−2
for 30 min
)
300, P = 300 W). Confocal luminescence imaging was carried out on Hz, 1H), 8.01 (dd, J = 8.5, 1.5 Hz, 1H), 7.51 (m, 3H), 7.33 (t, J = 7.1
an Olympus FV1000 laser scanning confocal microscope equipped Hz, 1H), 4.34 (t, J = 7.3 Hz, 2H), 1.96–1.80 (m, 2H), 1.46–1.20 (m,
with a 40 immersion objective lens. Flow cytometry experiments 6H), 0.86 (t, J = 7.0 Hz, 3H).
were conducted on FlowSight.
Synthesis and characterization
Synthesis of C1. Firstly, the trichloroiridium(III) terpyridine
4'-Phenyl-2,2':6',2''-terpyridine. To
a
stirred mixture of complexes [Ir(ptpy)Cl3] was synthesized from the reaction of
benzaldehyde (1 mmol) and 2-acetylpyridine (4 mmol) in EtOH (10 IrCl3·3H2O and the ptpy ligand in degassed ethylene glycol at 160 °C
mL), NaOH powder (4.2 mmol) and ammonia (3.0 mL) were added. for 15 min. Then, a mixture of [Ir(ptpy)Cl3] (1 mmol) and tpyc ligand
After the dark pink solution had been stirred at 25 °C for 12 h, the (1 mmol) in degassed ethylene glycol was heated at 180 °C for 20
precipitate was isolated by filtration and washed with EtOH. min under an inert atmosphere of nitrogen in the dark. The mixture
Purification was accomplished readily by recrystallization from was then cooled to room temperature and a saturated aqueous
1
ethanol. Yield 69%. H NMR (400 MHz, CDCl3) δ 8.76–8.71 (m, 4H), solution of KPF6 (2 mmol) was added to precipitate an orange-red
8.67 (t, J = 6.4 Hz, 2H), 7.94–7.85 (m, 4H), 7.49 (dt, J = 20.0, 4.9 Hz, solid. The solid was washed with cold water and then a mixture of
3H), 7.35 (dt, J = 10.8, 5.2 Hz, 2H).
methanol and ether, and then dried in vacuo. Subsequent
recrystallisation of the complex from acetone–diethyl ether
afforded C1 as air-stable orange-red crystals. Yield 46%. 1H NMR
(400 MHz, DMSO-d6): δ 9.68 (s, 2H), 9.61 (s, 2H), 9.30 (s, 1H), 9.27–
9.18 (m, 4H), 8.59 (d, J = 8.5 Hz, 1H), 8.44 (d, J = 7.6 Hz, 2H), 8.37
(dt, J = 14.5, 7.4 Hz, 5H), 8.05 (d, J = 8.9 Hz, 1H), 7.99 (d, J = 5.1 Hz,
2H), 7.95 (d, J = 5.3 Hz, 2H), 7.84 (t, J = 7.7 Hz, 2H), 7.76 (dd, J =
12.8, 7.9 Hz, 2H), 7.59 (dt, J = 12.6, 7.0 Hz, 5H), 7.40 (t, J = 7.6 Hz,
1H), 4.58 (t, J = 7.0 Hz, 2H), 1.94–1.74 (m, 2H), 1.50–1.17 (m, 6H),
0.82 (t, J = 7.1 Hz, 3H). 13C NMR (400 MHz, DMSO-d6): δ 158.9, 158.8,
3-([2,2':6',2''-Terpyridin]-4'-yl)-9-hexyl-9H-carbazole. To a stirred
mixture of 9-hexyl-9H-carbazole-3-carbaldehyde (1 mmol) and 2-
acetylpyridine (4 mmol) in EtOH (10 mL), NaOH powder (4.2 mmol)
and ammonia (3.0 mL) were added. After the dark pink solution had
been stirred at 25 °C for 12 h, the precipitate was isolated by
filtration and washed with EtOH. Purification was accomplished
readily by recrystallization from ethanol. Yield 76%. 1H NMR (400
MHz, CDCl3) δ 10.10 (s, 1H), 8.62 (d, J = 1.3 Hz, 1H), 8.16 (d, J = 7.8
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155.5, 155.0, 154.5, 154.5, 153.7, 143.9, 142.9, 142.8, 141.4, 135.3, and were cultured at 37 °C with 5% CO2 for 24 h. Different
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132.4, 130.1, 130.0, 129.9, 128.8, 127.7, 127.6, 127.4, 126.5, 125.6, concentrations of A1, C1 and S1 (5, 10, 20D, O50I: 1a0n.1d03190/0C8μDMT0)0w72e0rAe
124.0, 123.5, 123.1, 122.8, 121.5, 120.9, 120.5, 118.6, 111.0, 110.8, then added into the wells. The cells were subsequently incubated
31.5, 29.1, 26.6, 22.5, 14.3, 1.6. MS (MALDI-TOF): m/z 1274.3 (M+). for 24 h at 37 °C under 5% CO2. Then, MTT (10 μL/well, 5 mg/mL)
Elemental analysis (calcd, found for C54H45F18IrN7P3): C (45.70, was added to each well and the plate was incubated for an
45.58), H (3.20, 3.36), N (6.91, 7.14).
additional 4 h at 37 °C under 5% CO2. The medium was then
replaced with 150 μL dimethyl sulfoxide (DMSO) per well, and
OD570 was monitored by an enzyme-linked immunesorbent assay
(ELISA) reader. The following formula was used to calculate the
inhibition of cell growth:
Synthesis
of
A1.
0.1
mmol
Iridium(III)
bis(2-(2,4-
difluorophenyl)pyridine) dichloro-bridged dimer was combined with
0.1 mmol tetrabutylammonium cyanide in dichloromethane at 50
°C for 4 h. After removing dichloromethane under reduced
pressure, the product was purified by column chromatography
aluminum oxide with dichloromethane and methanol (10 : 1, v : v)
as the eluent. Yield 73%. 1H NMR (400 MHz, DMSO-d6): δ (ppm)
9.53 (d, J = 8 Hz, 2H), 8.20 (d, J = 8 Hz, 2H), 8.02 (t, J = 16 Hz, 2H),
7.44 (t, J = 12 Hz, 2H), 6.64–6.59 (m, 12 H), 5.52 (d, J = 8 Hz, 2H),
3.33–3.17 (m, 8H), 1.61–1.53 (m, 8H), 1.34–1.25 (m, 8H), 0.92 (t, J =
16 Hz, 12H). 13C NMR (100 MHz, DMSO-d6): δ (ppm) 170.2, 163.4,
155.0, 145.8, 139.8, 137.4, 132.2, 129.6, 124.7, 123.4, 121.6, 120.1,
59.5, 31.2, 23.5, 19.7, 14.0. MS (MALDI-TOF): m/z 625.2 [M-].
Elemental analysis (calcd, found for C40H48F4IrN5): C (55.41, 55.78),
H (5.58, 5.89), N (8.08, 8.27).
Cell viability (%) = (mean of Abs. value of treatment group/mean
Abs. value of control) × 100%
Cytotoxicity assay of PDT
Cells were seeded into a 96-well cell culture plate at 104 /well and
allowed to adhere for 24 h. After that, the cells were incubated with
A1, C1 or S1 for 22 h at 37 °C with 5% CO2. Next, the cells were
irradiated by white light (12 mW cm-2) with a xenon lamp for 10, 20
and 30 min, respectively. The cell viability was measured through
MTT assays.
Synthesis of S1. C1 (0.1 mmol) and A1 (0.3 mmol) were added to Annexin V/ Propidium iodide (PI) assay
acetone (10 mL). The reaction mixture was stirred for 2 h at room
Hela cells were planted and allowed to adhere for 24 h. Then the
temperature and then extracted with CHCl3. Next, the solution was
washed by water for several times for removing the counterions
and then concentrated by rotary evaporation. The resulting solid
was washed by diethyl ether to afford S1 as a red solid. Yield 65%.
1H NMR (400 MHz, DMSO-d6) δ 9.54 (m, 4H), 9.35 (s, 2H), 9.25 (d, J
= 4.0 Hz, 1H), 9.14 (s, 2H), 8.97 (d, J = 7.6 Hz, 2H), 8.78 (d, J = 8.0 Hz,
2H), 8.44 (d, J = 7.6 Hz, 1H), 8.16–8.30 (m, 14H), 7.76-7.82 (m, 17H),
7.66 (d, J = 7.6, 2H), 7.60 (t, J = 7.6, 2H), 7.47 (m, 5H), 7.28 (t, J = 7.2
Hz, 1H), 7.08 (t, J = 7.2 Hz, 4H), 6.34-6.42 (m, 6H), 5.64 (dd, J = 8 Hz,
3H), 4.58 (t, J = 7.0 Hz, 2H), 1.26–1.45 (m, 8H), 0.82 (t, J = 7.1 Hz,
3H). 13C NMR (100 MHz, DMSO-d6): δ 168.4, 168.3, 167.2, 158.9,
158.8, 155.5, 155.1, 154.5, 154.1, 153.8, 152.6, 152.4, 150.3, 145.3,
143.0, 142.9, 142.8, 142.6, 141.4, 139.7, 138.5, 135.2, 132.0, 130.1,
128.8, 127.8, 127.6, 127.4, 127.4, 126.5, 125.6, 124.0, 123.5, 123.2,
127.1, 122.8, 122.5, 121.6, 121.2, 121.0, 120.5, 120.3, 111.0, 110.8,
31.4, 29.0, 26.5, 22.6, 14.3, 1.6. MS (MALDI-TOF): m/z 1274.3 [M+],
625.3 [M-]. Elemental analysis (calcd, found for C126H81F12Ir4N19): C
(52.95, 52.78), H (2.86, 3.13), N (9.31, 9.15).
cells were incubated with S1 for 12 h at 37 °C with 5% CO2. Then the
cells were irradiated by white light (12 mW cm-2) with a xenon
lamp. The cell was stained with annexin V-FITC (5 μL) and PI (10 μL)
at room temperature for 10 min in the dark. The fluorescence
intensity of the cells was measured by confocal microscopy and
flow cytometry (FlowSight) with excitation at 405 nm. Cells were
viewed in green channel for annexin V (λem = 500-560 nm) and red
channel for PI (λem = 600-680 nm), respectively.
Conflicts of interest
The authors declare no competing financial interest.
Acknowledgements
We are grateful for the financial support from the Hong Kong
Research Grants Council (HKBU 12304715), the Hong Kong
Polytechnic University (1-ZE1C), Ms Clarea Au for the Endowed
Professorship (847S), the National Program for Support of Top-
Notch Young Professionals, Natural Science Foundation of
Jiangsu Province of China (BK20160885), the National Natural
Science Foundation of China (21701087 and 21671061),
Nanjing University of Posts and Telecommunications
(NY216026), and Postgraduate Education Reform Project of
Jiangsu Province (SJLX16-0335).
Cell culture
The cell lines Hela (human cervical cancer) were provided by the
Institute of Biochemistry and Cell Biology, SIBS, CAS (China). The
cells were grown in DMEM (Dulbecco's modified Eagle’s medium)
supplemented with 10% FBS (fetal bovine serum), 100 mg/mL
streptomycin and 100 U/mL penicillin at 37 °C with 5% CO2.
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