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Cyclometalated Ir(III) Complexes as Lysosome-Targeted Photodynamic Anticancer Agents.
We have designed and synthesized two Ir(III) complexes
( Ir1 and Ir2 ) coordinated with an 8-sulfonamidoquinoline
derivative ligand as photosensitizers, which exhibit strong red phosphorescence
emission and a long phosphorescence lifetime. The Ir(III) complexes
exhibit a high population of triplet states, which enable red phosphorescence
and efficient singlet oxygen generation. Ir1 and Ir2 rapidly enter the cancer cells and accumulate in lysosomes,
producing large amounts of intracellular singlet oxygen when exposed
to light irradiation, eventually leading to cancer cell death, and
the phototoxic indexes of complexes Ir1 and Ir2 against cancer cells are in the range of 76–228. Overall,
our studies indicate that the synthesized Ir(III) complexes with quinoline
ligands exhibit photosensitizing properties, effectively inducing
cancer cell death when exposed to light. These promising results suggest
their potential application in photodynamic therapy.
## Introduction
Introduction Cancer is a major human health threat
that urgently needs to be
overcome. 1 Noninvasive treatment strategies
activated by external stimuli have attracted promising attention for
their controllable treatment methods, minimal damage to normal tissues,
and significant therapeutic effects. 2 , 3 Light has long
been used as an external stimulus in many therapeutic processes. 4 , 5 As a clinical noninvasive therapeutic method, photodynamic therapy
(PDT) has attracted widespread attention and achieved good therapeutic
effects. 6 , 7 PDT is a technique in which photosensitizers
are activated by the light of an appropriate wavelength in the presence
of oxygen, producing reactive oxygen species (ROS) by transferring
electrons from photosensitizers to oxygen and resulting in oxidative
cellular damage and destruction. 8 , 9 Various organic and
inorganic materials, including organic dyes, semiconductor materials,
and metal complexes, can be used as photosensitizers to produce ROS
under light stimulation. 7 , 10 In particular, metal
complexes exhibit rich triplet excited-state properties based on the
heavy atom effect, revealing the promising potential for PDT. 8 , 11 Cyclometalated Ir(III) complexes exhibit high fluorescence
quantum
efficiency and triplet excited-state lifetimes, 12 , 13 which facilitate electron transfer with oxygen, resulting in a high
singlet oxygen ( 1 O 2 ) generation quantum yield. 14 , 15 In the past few years, several Ir(III) complexes functioning as
photosensitizers for PDT treatment have been reported. 16 – 18 In particular, the red-emitting cyclometalated Ir(III) complexes
are widely used in PDT. 19 – 22 Furthermore, cyclometalated Ir(III) complexes containing
different carbon–nitrogen (ĈN) and diamine (N̂N)
ligands exhibited different phototoxicity indexes (PI, the ratio of
the toxic effects in dark and upon light irradiation) for cancer cells,
indicating the key role of ligands in the PDT of cyclometalated Ir(III)
complex. 23 – 25 Quinoline derivatives exhibit low cytotoxicity,
high cell permeability,
and relatively convenient synthetic and functionalization methods
and can be used as a versatile ligand for preparation of metal complexes. 26 , 27 8-Quinolinol is a potential radioprotective agent, as a ligand and
fluorophore in the Zn(II) complex, which can reduce the acute side
effects of radiotherapy in cancer treatment. The synthesis and photophysical
properties of quinolinolate-Ir(III) complexes have been reported,
but their phosphorescence properties are weak. 28 The effect of the quinoline ligand on the singlet oxygen
production capacity and PDT efficacy is still unknown. Based
on the above background, we choose 4-methyl- N -quinoline-8-ylbenzenesulfonamide
ligand as an ancillary ligand to
synthesize two phosphorescent cyclometalated Ir(III) complexes ( Figure 1 , Ir1 and Ir2 ) as photosensitizers. We use two different
N ligands (N = 1-phenylisoquinoline (1pq) and 2-(2-pyridyl)-benzothiophene
(pbt)) to modulate the luminescence properties of two cyclometalated
Ir(III) complexes. Ir1 and Ir2 feature high
triplet state population enabling red phosphorescence, long phosphorescence
lifetime, and efficient singlet oxygen generation. Ir1 and Ir2 can rapidly enter the cancer cells and accumulate
in lysosomes, producing large amounts of singlet oxygen intracellularly
when exposed to the light irradiation and eventually leading to cancer
cell death. In these assays, quinoline-functionalized cyclometalated Ir(III) complexes
with a high phototoxic potential against cancer cells were identified,
suggesting that quinoline functionalization was favorable for the
application of cyclometalated Ir(III) complexes in PDT. Figure 1 Chemical structures
of the cyclometalated Ir(III) complexes ( Ir1 and Ir2 ).
## Results and Discussion
Results and Discussion Synthesis and Characterization Two novel cyclometalated
Ir(III) complexes ( Ir1 and Ir2 ) containing
4-methyl- N -quinoline-8-ylbenzenesulfonamide ligand
as the ancillary ligand were synthesized according to the methods
in the Experimental Section ( Figure S1 ). Briefly, the iridium dimer ([(L) 2 Ir(m-Cl)] 2 ), (L = 1pq or pbt) was reacted with the 4-methyl- N -quinoline-8-ylbenzenesulfonamide ligand in dichloromethane
and ethanol (v/v = 4:1) overnight. Ir1 ang Ir2 were purified by Al 2 O 3 column chromatography
and obtained in relatively high yield, characterized by NMR and electron
spray ionization mass spectrometry (ESI-MS) ( Figures S2–S9 ). 29 Photophysical Properties The photophysical properties
of the two cyclometalated Ir(III) complexes in different solvents
and the effects of the solvent polarity and viscosity on their properties
were investigated. The UV–visible absorption and emission spectra
of Ir1 and Ir2 were measured in different
solvents at 25 °C; all of the photophysical data of Ir1 and Ir2 are tabulated in Table 1 . The complexes showed similar absorption
bands ( Figure 2 ); both
complexes displayed strong absorption at 250–300 nm, which
was attributed to spin-allowed ligand center transition ( 1 LC), and a weaker absorption at 400–500 nm due to a combination
of spin-allowed metal-to-ligand charge transfer ( 1 MLCT)
and ligand-to-ligand charge transfer ( 1 LLCT) characters. Ir1 and Ir2 showed a weak absorption between
400 and 500 nm assigned to the spin-forbidden of 3 MLCT
and 3 LLCT transitions, which were compared with similar
reported ionic iridium(III) complexes. 30 , 31 Ir1 and Ir2 exhibited strong red phosphorescence between
580 and 630 nm, and their emission quantum yields are comparable to
the standard Ru(bpy) 3 Cl 2 ( Figure 2 b, Table 1 ), at room temperature upon excitation at 455 nm. Notably, Ir1 and Ir2 exhibited polarity-sensitive UV–vis
absorption and phosphorescence, higher UV–vis absorption, and
fluorescence intensity in low-polarity solvents, such as dichloromethane
solution. 32 Table 1 Photophysical Data of Complexes at
298 K complexes λ abs /nm (H 2 O) λ em (max)/nm (H 2 O) quantum yield
(Φ em ) a (CH 3 CN) quantum yield
Φ (1O 2 ) b (H 2 O) lifetime (τ)/ns (CH 3 CN) (Air/N 2 ) Ir1 350, 450 610 0.035 0.65 352.6/568.8 Ir2 340,
460 600 0.028 0.68 353.9/383.2 a Φ em , the luminescence
quantum yields. b Φ
( 1 O 2 ), 1 O 2 quantum yield.
[Ru(bpy) 3 ] 2+ was used as a standard compound
(Φ em =
0.028, Φ ( 1 O 2 ) = 0.2, 465 nm light irradiation). Figure 2 UV–vis (a) and emission spectra (b) of 10 μM Ir1 or Ir2 in various solvents (CH 2 Cl 2 , CH 3 CN, CH 3 OH, and H 2 O), l ex = 455 nm. Since the complexes exhibited higher UV–vis
absorption and
fluorescence intensity in nonpolar solvents, we investigated their
phosphorescence intensity in different polarities of solvents composed
of water-1,4-dioxane. As shown in Figure S10 , as the proportion of 1,4-dioxane increases, the phosphorescence
intensity of the complexes was greatly enhanced. Furthermore, oxygen
severely affected the phosphorescence intensity ( Figure S11 ) and lifetime ( Figure S12 ) of Ir1 and Ir2 , which was longer in methylene
chloride than in ultrapure water. Aqueous Stability A good photosensitizer should have
high stability for the anticancer application. 33 Therefore, we monitored the stability of Ir1 and Ir2 by UV–vis absorption spectroscopy. As
shown in Figure S13 , no obvious changes
in the absorption spectra of Ir1 and Ir2 can be observed in phosphate-buffered saline (PBS) at 25 °C
for 24 h. Additionally, Ir1 and Ir2 also
showed high photostability under 465 nm light (6.5 mW/cm 2 ) irradiation for 1 h and were also stable at different pH values
( Figure S14 ) and temperatures ( Figure S15 ). Cellular Localization Since Ir1 and Ir2 showed excellent phosphorescence in extracellular experiments, Ir1 and Ir2 were selected to be used in subsequent
experiments for intracellular experiments. The intracellular localization
of Ir1 and Ir2 was monitored by a confocal
microscope. After Hep-G2 cells adhered to the confocal dish, the complexes
were incubated with Hep-G2 cells for 1 h. Both complexes emitted red
phosphorescence in the cytoplasm of the Hep-G2 cells. In order to
study their subcellular localization more accurately, we used commercial
organelles dyes (Mito-Tracker Red, ER-Tracker Red, and Lyso-Tracker
Red) for coincubation with Hep-G2 cells for 0.5 h to stain the mitochondria,
endoplasmic reticulum, and lysosomes. As shown in Figures 3 and 4 , the bright phosphorescence points of these complexes mainly have
high overlap coefficients with the organelles of lysosomes, which
shows that the complexes have high targeting efficiency and can be
used for lysosome-targeted photosensitizers. Figure 3 Colocalization images
of the Hep-G2 cells costained with Ir1 (10 μM,
1 h, l ex = 458
nm, l em = 600 ± 30 nm) and commercial
organelles dyes: ER-Tracker Red (333 nM, 30 min, l ex = 543 nm, l em = 615 ±
30 nm); Mito-Tracker Red (100 nM, 30 min, l ex = 543 nm, l em = 599 ± 30 nm); and
Lyso-Tracker Red (75 nM, l ex = 543 nm, l em = 590 ± 30 nm). Scale bar: 20 mm. Figure 4 Colocalization images of the Hep-G2 cells costained with Ir2 (10 μM, 1 h, l ex = 458
nm, l em = 600 ± 30 nm) and commercial
organelles dyes: ER-Tracker Red (333 nM, 30 min, l ex = 543 nm, l em = 615 ±
30 nm); Mito-Tracker Red (100 nM, 30 min, l ex = 543 nm, l em = 599 ± 30 nm); and
Lyso-Tracker Red (75 nM, 30 min, l ex =
543 nm, l em = 590 ± 30 nm). Scale
bar: 20 mm. Cytotoxicity Test Through 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) toxicity test, the dark- and photocytotoxicity of Ir1 and Ir2 was investigated against a variety
of cells , including liver cancer cell line (Hep-G2), lung cancer
cell line (A549), cisplatin-resistant lung cancer cell line (A549R),
cervical cancer cell line (Hela), and normal liver cell line (L02). Table 2 summarizes the toxicity
of iridium complexes and cisplatin (used as controls) to various cancer
cell lines for 48 h under dark and 465 nm light (6.5 mW/cm 2 ) for 0.5 h. IC 50 is called the half-inhibition rate,
which represents the concentration required to inhibit half the cell
proliferation and is negatively related to toxicity. The experimental
results show that these complexes have very low cytotoxicity in the
dark for all cancer cells and normal cells after 48 h of drug exposure
and incubation ( Figure S16 ). However, for
photocytotoxicity screening, the IC 50 of both iridium complexes
with four different types of cancer cells is lower than that of cisplatin,
and Ir1 and Ir2 exhibited high photocytotoxicity
(IC 50 less than 5 μM) to Hela and A549 with a high
photocytotoxicity index [PI, ratio of IC 50 (dark)/IC 50 (light)] of more than 110, which indicates that the complexes
are highly effective on cancer cells under light. Importantly, Ir1 and Ir2 showed very strong photocytotoxicity
against cisplatin-resistant lung cancer cell, A549R, with IC 50 values of 13.2 and 12.7 ± 0.2, respectively ( Figure 5 ). While the IC 50 value of cisplatin was above 50 μM ( Figure S17 ), the above results suggest that cyclometalated Ir(III)
complexes have great potential in cancer PDT. In addition, both Ir1 and Ir2 were virtually noncytotoxic to normal
liver L02 cells in the absence of light irradiation (the IC 50 value was above 100 mM, Figure S18 ).
The excellent photodynamic anticancer properties of the complexes
prompted us to investigate their in vivo safety. Zebrafish with vascular
expression of green fluorescent protein (GFP) were used to investigate
the biosafety of the complexes at the in vivo level. No damage was
observed in the vessels of zebrafish treated with Ir1 or Ir2 for 96 h ( Figure S19 ). The above results indicated that Ir1 and Ir2 were promising photosensitizer drugs for efficient PDT. Table 2 Cytotoxicity (IC 50 , mM)
of the Complexes toward Cancer Cell Lines a Hep-G2 Hela A549 A549R complex light (dark) PI b light (dark) PI b light (dark) PI b light (dark) PI b Ir1 4.3 ± 0.2 (>100) >23.2 0.5 ± 0.3 (>100) >200 0.8 ± 0.2 (>100) >133 13.2 ± 0.2 (>100) >7.8 Ir2 0.4 ± 0.2 (>100) >228 0.7 ± 0.1 (>100) >153 0.9 ± 0.1 (>100) >113 12.7 ± 0.2 (>100) >7.9 Cisplatin 21.8 ± 0.2 (24.8 ± 0.2) 1.14 43.2 ± 2.3 (48.2 ± 2.7) 1.11 57.5 ± 1.7 (60.7 ± 1.7) 1.06 a Data are presented as the means
± standard deviations. b PI = IC 50 (dark)/IC 50 (light). Figure 5 Cytotoxic effects of Ir1 (a) or Ir2 (b)
on different types of cells with 465 nm light irradiation. ROS Generation The 1 O 2 generation
quantum yields of the complex were measured by a commonly method. 1 O 2 oxidized the imidazole derivative to form a trans -annular peroxide adduct, which quenched the absorbance
of p -nitrosodimethyl aniline (RNO) to quantify 1 O 2 , and the results are presented in Figure S20 and Table 1 . 34 As can be
seen from the picture, the singlet oxygen yield of the complexes in
acetonitrile was higher than that of the standard compound. Furthermore, as a site of oxidative metabolism in eukaryotes, mitochondria
are the main source of ROS, and the PDT process has been reported
to cause lysosomal damage by ROS. Confocal microscopy was used to
detect the increase of ROS in liver cancer cells induced by Ir1 or Ir2 . Hep-G2 cells were incubated with Ir1 or Ir2 for 1 h, and then, Hep-G2 was treated
with 2′,7′-dichlorodihydrofluorescein diacetate (H 2 DCFDA) staining for 0.5 h and finally irradiated under 465
nm light (6.5 mW/cm 2 ) for 10 min. Confocal images clearly
show that accompanied by the generation of ROS, the green fluorescence
intensity of DCF increased in a concentration-dependent manner ( Figure 6 ). Compared with
the control cells without light treatment, the fluorescence intensity
of DCF increased significantly in cells treated with complexes and
irradiation. In addition, the cells pretreated with sodium azide (NaN 3 , an effective 1 O 2 scavenger) significantly
inhibited the fluorescence signal of the DCF. Figure 6 Analysis of ROS generation
by Ir1 and Ir2 mediated by PDT using fluorescence
microscopy. Hep-G2 cells were
incubated with Ir1 or Ir2 for 1 h and irradiated
with blue light (465 nm, 6.5 mW/cm 2 ) for 10 min. Scale
bar: 200 mm.
## Synthesis and Characterization
Synthesis and Characterization Two novel cyclometalated
Ir(III) complexes ( Ir1 and Ir2 ) containing
4-methyl- N -quinoline-8-ylbenzenesulfonamide ligand
as the ancillary ligand were synthesized according to the methods
in the Experimental Section ( Figure S1 ). Briefly, the iridium dimer ([(L) 2 Ir(m-Cl)] 2 ), (L = 1pq or pbt) was reacted with the 4-methyl- N -quinoline-8-ylbenzenesulfonamide ligand in dichloromethane
and ethanol (v/v = 4:1) overnight. Ir1 ang Ir2 were purified by Al 2 O 3 column chromatography
and obtained in relatively high yield, characterized by NMR and electron
spray ionization mass spectrometry (ESI-MS) ( Figures S2–S9 ). 29
## Photophysical Properties
Photophysical Properties The photophysical properties
of the two cyclometalated Ir(III) complexes in different solvents
and the effects of the solvent polarity and viscosity on their properties
were investigated. The UV–visible absorption and emission spectra
of Ir1 and Ir2 were measured in different
solvents at 25 °C; all of the photophysical data of Ir1 and Ir2 are tabulated in Table 1 . The complexes showed similar absorption
bands ( Figure 2 ); both
complexes displayed strong absorption at 250–300 nm, which
was attributed to spin-allowed ligand center transition ( 1 LC), and a weaker absorption at 400–500 nm due to a combination
of spin-allowed metal-to-ligand charge transfer ( 1 MLCT)
and ligand-to-ligand charge transfer ( 1 LLCT) characters. Ir1 and Ir2 showed a weak absorption between
400 and 500 nm assigned to the spin-forbidden of 3 MLCT
and 3 LLCT transitions, which were compared with similar
reported ionic iridium(III) complexes. 30 , 31 Ir1 and Ir2 exhibited strong red phosphorescence between
580 and 630 nm, and their emission quantum yields are comparable to
the standard Ru(bpy) 3 Cl 2 ( Figure 2 b, Table 1 ), at room temperature upon excitation at 455 nm. Notably, Ir1 and Ir2 exhibited polarity-sensitive UV–vis
absorption and phosphorescence, higher UV–vis absorption, and
fluorescence intensity in low-polarity solvents, such as dichloromethane
solution. 32 Table 1 Photophysical Data of Complexes at
298 K complexes λ abs /nm (H 2 O) λ em (max)/nm (H 2 O) quantum yield
(Φ em ) a (CH 3 CN) quantum yield
Φ (1O 2 ) b (H 2 O) lifetime (τ)/ns (CH 3 CN) (Air/N 2 ) Ir1 350, 450 610 0.035 0.65 352.6/568.8 Ir2 340,
460 600 0.028 0.68 353.9/383.2 a Φ em , the luminescence
quantum yields. b Φ
( 1 O 2 ), 1 O 2 quantum yield.
[Ru(bpy) 3 ] 2+ was used as a standard compound
(Φ em =
0.028, Φ ( 1 O 2 ) = 0.2, 465 nm light irradiation). Figure 2 UV–vis (a) and emission spectra (b) of 10 μM Ir1 or Ir2 in various solvents (CH 2 Cl 2 , CH 3 CN, CH 3 OH, and H 2 O), l ex = 455 nm. Since the complexes exhibited higher UV–vis
absorption and
fluorescence intensity in nonpolar solvents, we investigated their
phosphorescence intensity in different polarities of solvents composed
of water-1,4-dioxane. As shown in Figure S10 , as the proportion of 1,4-dioxane increases, the phosphorescence
intensity of the complexes was greatly enhanced. Furthermore, oxygen
severely affected the phosphorescence intensity ( Figure S11 ) and lifetime ( Figure S12 ) of Ir1 and Ir2 , which was longer in methylene
chloride than in ultrapure water.
## Aqueous Stability
Aqueous Stability A good photosensitizer should have
high stability for the anticancer application. 33 Therefore, we monitored the stability of Ir1 and Ir2 by UV–vis absorption spectroscopy. As
shown in Figure S13 , no obvious changes
in the absorption spectra of Ir1 and Ir2 can be observed in phosphate-buffered saline (PBS) at 25 °C
for 24 h. Additionally, Ir1 and Ir2 also
showed high photostability under 465 nm light (6.5 mW/cm 2 ) irradiation for 1 h and were also stable at different pH values
( Figure S14 ) and temperatures ( Figure S15 ).
## Cellular Localization
Cellular Localization Since Ir1 and Ir2 showed excellent phosphorescence in extracellular experiments, Ir1 and Ir2 were selected to be used in subsequent
experiments for intracellular experiments. The intracellular localization
of Ir1 and Ir2 was monitored by a confocal
microscope. After Hep-G2 cells adhered to the confocal dish, the complexes
were incubated with Hep-G2 cells for 1 h. Both complexes emitted red
phosphorescence in the cytoplasm of the Hep-G2 cells. In order to
study their subcellular localization more accurately, we used commercial
organelles dyes (Mito-Tracker Red, ER-Tracker Red, and Lyso-Tracker
Red) for coincubation with Hep-G2 cells for 0.5 h to stain the mitochondria,
endoplasmic reticulum, and lysosomes. As shown in Figures 3 and 4 , the bright phosphorescence points of these complexes mainly have
high overlap coefficients with the organelles of lysosomes, which
shows that the complexes have high targeting efficiency and can be
used for lysosome-targeted photosensitizers. Figure 3 Colocalization images
of the Hep-G2 cells costained with Ir1 (10 μM,
1 h, l ex = 458
nm, l em = 600 ± 30 nm) and commercial
organelles dyes: ER-Tracker Red (333 nM, 30 min, l ex = 543 nm, l em = 615 ±
30 nm); Mito-Tracker Red (100 nM, 30 min, l ex = 543 nm, l em = 599 ± 30 nm); and
Lyso-Tracker Red (75 nM, l ex = 543 nm, l em = 590 ± 30 nm). Scale bar: 20 mm. Figure 4 Colocalization images of the Hep-G2 cells costained with Ir2 (10 μM, 1 h, l ex = 458
nm, l em = 600 ± 30 nm) and commercial
organelles dyes: ER-Tracker Red (333 nM, 30 min, l ex = 543 nm, l em = 615 ±
30 nm); Mito-Tracker Red (100 nM, 30 min, l ex = 543 nm, l em = 599 ± 30 nm); and
Lyso-Tracker Red (75 nM, 30 min, l ex =
543 nm, l em = 590 ± 30 nm). Scale
bar: 20 mm.
## Cytotoxicity Test
Cytotoxicity Test Through 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) toxicity test, the dark- and photocytotoxicity of Ir1 and Ir2 was investigated against a variety
of cells , including liver cancer cell line (Hep-G2), lung cancer
cell line (A549), cisplatin-resistant lung cancer cell line (A549R),
cervical cancer cell line (Hela), and normal liver cell line (L02). Table 2 summarizes the toxicity
of iridium complexes and cisplatin (used as controls) to various cancer
cell lines for 48 h under dark and 465 nm light (6.5 mW/cm 2 ) for 0.5 h. IC 50 is called the half-inhibition rate,
which represents the concentration required to inhibit half the cell
proliferation and is negatively related to toxicity. The experimental
results show that these complexes have very low cytotoxicity in the
dark for all cancer cells and normal cells after 48 h of drug exposure
and incubation ( Figure S16 ). However, for
photocytotoxicity screening, the IC 50 of both iridium complexes
with four different types of cancer cells is lower than that of cisplatin,
and Ir1 and Ir2 exhibited high photocytotoxicity
(IC 50 less than 5 μM) to Hela and A549 with a high
photocytotoxicity index [PI, ratio of IC 50 (dark)/IC 50 (light)] of more than 110, which indicates that the complexes
are highly effective on cancer cells under light. Importantly, Ir1 and Ir2 showed very strong photocytotoxicity
against cisplatin-resistant lung cancer cell, A549R, with IC 50 values of 13.2 and 12.7 ± 0.2, respectively ( Figure 5 ). While the IC 50 value of cisplatin was above 50 μM ( Figure S17 ), the above results suggest that cyclometalated Ir(III)
complexes have great potential in cancer PDT. In addition, both Ir1 and Ir2 were virtually noncytotoxic to normal
liver L02 cells in the absence of light irradiation (the IC 50 value was above 100 mM, Figure S18 ).
The excellent photodynamic anticancer properties of the complexes
prompted us to investigate their in vivo safety. Zebrafish with vascular
expression of green fluorescent protein (GFP) were used to investigate
the biosafety of the complexes at the in vivo level. No damage was
observed in the vessels of zebrafish treated with Ir1 or Ir2 for 96 h ( Figure S19 ). The above results indicated that Ir1 and Ir2 were promising photosensitizer drugs for efficient PDT. Table 2 Cytotoxicity (IC 50 , mM)
of the Complexes toward Cancer Cell Lines a Hep-G2 Hela A549 A549R complex light (dark) PI b light (dark) PI b light (dark) PI b light (dark) PI b Ir1 4.3 ± 0.2 (>100) >23.2 0.5 ± 0.3 (>100) >200 0.8 ± 0.2 (>100) >133 13.2 ± 0.2 (>100) >7.8 Ir2 0.4 ± 0.2 (>100) >228 0.7 ± 0.1 (>100) >153 0.9 ± 0.1 (>100) >113 12.7 ± 0.2 (>100) >7.9 Cisplatin 21.8 ± 0.2 (24.8 ± 0.2) 1.14 43.2 ± 2.3 (48.2 ± 2.7) 1.11 57.5 ± 1.7 (60.7 ± 1.7) 1.06 a Data are presented as the means
± standard deviations. b PI = IC 50 (dark)/IC 50 (light). Figure 5 Cytotoxic effects of Ir1 (a) or Ir2 (b)
on different types of cells with 465 nm light irradiation.
## ROS Generation
ROS Generation The 1 O 2 generation
quantum yields of the complex were measured by a commonly method. 1 O 2 oxidized the imidazole derivative to form a trans -annular peroxide adduct, which quenched the absorbance
of p -nitrosodimethyl aniline (RNO) to quantify 1 O 2 , and the results are presented in Figure S20 and Table 1 . 34 As can be
seen from the picture, the singlet oxygen yield of the complexes in
acetonitrile was higher than that of the standard compound. Furthermore, as a site of oxidative metabolism in eukaryotes, mitochondria
are the main source of ROS, and the PDT process has been reported
to cause lysosomal damage by ROS. Confocal microscopy was used to
detect the increase of ROS in liver cancer cells induced by Ir1 or Ir2 . Hep-G2 cells were incubated with Ir1 or Ir2 for 1 h, and then, Hep-G2 was treated
with 2′,7′-dichlorodihydrofluorescein diacetate (H 2 DCFDA) staining for 0.5 h and finally irradiated under 465
nm light (6.5 mW/cm 2 ) for 10 min. Confocal images clearly
show that accompanied by the generation of ROS, the green fluorescence
intensity of DCF increased in a concentration-dependent manner ( Figure 6 ). Compared with
the control cells without light treatment, the fluorescence intensity
of DCF increased significantly in cells treated with complexes and
irradiation. In addition, the cells pretreated with sodium azide (NaN 3 , an effective 1 O 2 scavenger) significantly
inhibited the fluorescence signal of the DCF. Figure 6 Analysis of ROS generation
by Ir1 and Ir2 mediated by PDT using fluorescence
microscopy. Hep-G2 cells were
incubated with Ir1 or Ir2 for 1 h and irradiated
with blue light (465 nm, 6.5 mW/cm 2 ) for 10 min. Scale
bar: 200 mm.
## Conclusions
Conclusions In conclusion, we have reported two red
luminescent 8-sulfonamidoquinoline
ligand-functionalized cyclometalated Ir(III) complexes that accumulated
in the lysosomes of cancer cells. These complexes display high 1 O 2 quantum yields upon 465 nm light irradiation.
The phototoxic indexes of complexes Ir1 and Ir2 against cancer cells are in the range 76–228. Therefore,
it can be used as a highly effective photodynamic therapeutics agent.
## Experimental Section
Experimental Section Materials and Instruments IrCl 3 ·H 2 O, 1-phenylisoquinoline(1pq), 2-(2-pyridyl)-benzothiophene
(pbt), MTT, DMSO- d 6 , and PBS were obtained
from Macklin. Cervical cancer cell line (Hela), human lung carcinoma
cell line(A549), cisplatin-resistant A549 (A549R), human liver cancer
cell line (Hep-G2), and human normal liver cell line (L02) were obtained
from the ATCC cell bank. Dulbecco’s modified Eagle’s
medium (DMEM) and fetal calf serum were purchased from Sigma-Aldrich.
Mito-Tracker Red, ER-Tracker Red, and Lyso-Tracker Red were obtained
from Beyotime Biote chnology. 1 H NMR and 13 C NMR spectra data were obtained utilizing
a BrukerAV-500 spectrometer. ESI-MS data were recorded using Agilent
6130B. UV–vis absorption spectra were measured using a UV-2550
spectrophotometer. The fluorescence spectra were recorded with a Hitachi
F-7000 Fluorimeter. Preparation of the Iridium Complex The specific synthesis
steps are as follows: The precursors were prepared from iridium chloride
(IrCl 3 ) and 1-phenylisoquinoline (1 pq) or 2-(2-pyridyl)-benzothiophene
(pbt). Specifically, 0.05 mmol of solid iridium chloride and 0.1 mmol
of 1 pq or pbt powder were added to 40 mL of solution containing ethylene
glycol ether and water (v/v = 3:1) in a two-neck round-bottom flask;
the mixture was stirred at 120 °C and refluxed overnight in a
nitrogen atmosphere. After the reaction solution was cooled to ambient
temperature, it was filtered under vacuum. The precipitate was washed
3 times with diethyl ether. Next, a mixture of dichloro-bridged
dimeric (0.1 mmol) complexes ([(L) 2 Ir(m-Cl)] 2 ), (L = 1 pq or pbt), 8-(tosylamino)quinoline (tlq) (0.22 mmol),
and triethylamine (4.5 mmol) was completely dissolved in dichloromethane
and ethanol (v/v = 4:1) in a sealed reaction container according to
the measured molar ratio. The mixture was then refluxed for 20 h in
a nitrogen atmosphere and heated at 55 °C. After the solvent
was removed under vacuum filtration, the precipitate was washed with
ethanol to remove unreacted ligands and dried under vacuum to obtain
solid products Ir1 or Ir2 . [Ir(1pq) 2 (tlq)]Cl ( Ir1 ) Yield
= 51.6% (92.68 mg). Red color solid. 1 H NMR (500 MHz, DMSO- d 6 ): δ 8.94–8.89 (t, J = 7.5 Hz, 2H), 8.86 (d, J = 6.5 Hz, 1H), 8.29 (d, J = 8.3 Hz, 1H), 8.24 (d, J = 7.9 Hz, 1H),
8.14 (d, J = 8.0 Hz, 1H), 8.06–8.01 (m, 2H),
7.97 (d, J = 8.0 Hz, 1H), 7.84 (dd, J = 6.6, 3.2 Hz, 4H), 7.62 (d, J = 6.6 Hz, 1H), 7.49
(d, J = 7.6 Hz, 2H), 7.47–7.43 (t, J = 7.5 Hz, 1H), 7.38–7.33 (t, J = 8.0 Hz, 2H), 7.28 (d, J = 7.9 Hz, 1H), 7.03 (t, J = 7.0 Hz, 1H), 6.88–6.78 (m, 4H), 6.62 (d, J = 7.3 Hz, 1H), 6.57 (d, J = 8.1 Hz, 2H),
6.21 (d, J = 7.6 Hz, 1H), 6.12 (d, J = 7.8 Hz, 1H), 1.96 (s, 3H) ppm. ESI-MS ( m / z ): Calcd for C 46 H 33 N 4 O 2 S( 193 Ir) (M + ), 899.07; found, 899.20
[M + ]. Elemental Anal. Calcd for Ir1 C 46 H 33 N 4 O 2 SIr: C, 61.39; H,
3.67; N, 6.23; S, 3.56. Found: C, 61.26; H, 3.64; N, 6.32; S, 3.52. [Ir(pbt) 2 (tlq)]Cl ( Ir2 ) Yield
= 77.0% (144 mg). Yellow color solid. 1 H NMR (500 MHz,
DMSO- d 6 ): δ 9.10 (d, J = 5.7 Hz, 1H), 8.37 (d, J = 8.3 Hz, 1H), 7.94–7.84
(m, 4H), 7.80 (d, J = 7.9 Hz, 1H), 7.71 (d, J = 8.0 Hz, 1H), 7.61 (t, J = 5.8 Hz, 2H),
7.51 (t, J = 8.1 Hz, 1H), 7.42 (dd, J = 8.4, 5.0 Hz, 1H), 7.32 (d, J = 8.0 Hz, 1H), 7.24
(dd, J = 15.4, 6.3 Hz, 2H), 7.17 (t, J = 7.6 Hz, 1H), 7.09 (t, J = 7.5 Hz, 1H), 6.94–6.89
(m, 5H), 6.82 (dt, J = 18.9, 7.7 Hz, 2H), 6.05 (d, J = 8.1 Hz, 1H), 5.97 (d, J = 8.1 Hz, 1H),
2.23 (s, 3H). ESI-MS ( m / z ): Calcd
for C 42 H 29 N 4 O 2 S 3 ( 193 Ir) (M + ), 910.12; found, 933.00 [M + +Na] + . Elemental Anal. Calcd For Ir2 C 42 H 29 N 4 O 2 S 3 Ir: C, 55.38; H, 3.19; N, 6.15; S, 10.55. Found: C, 55.36; H, 3.14;
N, 6.18; S, 10.52. HPLC Investigation The prepared 50 μM Ir1 or Ir2 solutions contained 1% DMSO in PBS/acetonitrile
(CH 3 CN) (V/V, 3:7). Analytical high-performance liquid
chromatography (HPLC) was used for analysis with an injection volume
of 20 μL. The mobile phase was a linear gradient of 0.1% HCOOH
in H 2 O and 0.1% HCOOH in CH 3 CN. The absorbance
wavelength was set to 280 nm. Each sample was analyzed by a high-performance
liquid chromatograph (Thermo Fisher) equipped with a C18–H
HPLC column at a flow rate of 1.0 mL/min and a column temperature
of 25 °C. Retention time for Ir1 is 3.108 min and
that for Ir2 is 2.867 min. Cellular Localization Hep-G2 cells were placed in 35
mm confocal dishes for about 12 h and then incubated with 10 mM Ir1 or Ir2 for 1 h before staining with Lyso-Tracker,
Mito-Tracker, or ER-Tracker for 30 min. Cells were washed with PBS
and imaged with confocal microscopy. Dark Cytotoxicity and Phototoxicity Hela, A549, A549R,
Hep-G2, and L02 cells were cultured in mixed DMEM supplemented with
10% v/v FBS and 1% v/v penicillin–streptomycin and maintained
in a humidified incubator at 37 °C under an atmosphere of 5%
CO 2 . 29 The cytotoxicity
of complexes Ir1 , Ir2 , and cisplatin on
A549, A549R, Hela, and Hep-G2 cells for 48 h was evaluated by a colorimetric
MTT assay. First of all, about 5000 of each kind of cells were plated
and seeded per well in 96-well plates; then, the cells were incubated
with the complexes in different concentrations at 37 °C. In the
dark toxicity, the complexes’ exposure period was 48 h. As
for phototoxic operations, after the cells were incubated with complexes Ir1 or Ir2 for 1 h, the complexes were removed,
and the 96-well plate was rinsed twice with PBS, and then, the nondrug
medium was placed. After that, the 96-well plate was irradiated with
465 nm light for 30 min and then cultured in the incubator for another
47.5 h. Subsequently, MTT (25 μL, 5 mg/mL) was added and incubation
continued for 4 h to form purple formazan. Finally, 100 μL of
DMSO was added to each well to dissolve purple formazan, and the absorbance
at 490 nm was recorded by a microplate reader. Cells in the blank
control group were treated with DMSO (1%, v/v). Determination of Singlet Oxygen Quantum Yield An air
saturation of acetonitrile solution, containing the complexes under
test ( A = 0.1 at 405 nm), p -nitrosodimethyl
aniline (RNO, 24 μM), and imidazole (12 mM) in a quartz fluorescent
cuvette, was irradiated at 405 nm, and the absorbance was recorded
at various time intervals. Plots of A 0 – A at 420 nm ( A 0 is the absorbance before irradiation) versus the irradiation time
were prepared, and the linear regression slope was calculated ( S complex ). Phenalenone was used as the reference
compound (Φ ref ( 1 O 2 ) = 95%)
to obtain S ref and then to calculate the
singlet oxygen quantum yields (Φ complex ) for each
complex 1 2 where I (absorbance correction
factor) was calculated from eq 2 , I 0 is the light intensity of
the irradiated source, and A is the absorbance of
the complex. Determination of Intracellular ROS ROS accumulation
in Hep-G2 cells induced by Ir1 or Ir2 under
light irradiation was evaluated by a fluorescence microscope and 2′,7′-dichlorofluorescein
diacetate (H 2 DCFDA). DCFH-DA is an ROS fluorescent probe
that diffuses into the cell and is rapidly oxidized by intracellular
ROS to form the highly fluorescent 2′,7′-dichlorofluorescein
(DCF, l ex = 488 nm, l em = 520 ± 20 nm). The fluorescence intensity of
DCF is believed to be positively correlated with the amount of ROS
formed in cells. Biosafety in Zebrafish The GFP-transfected Singapore
zebrafish (<5 bpf) were obtained from the Center of Experiment
Animals at Sun Yat-Sen University. Zebrafish were housed in E3 medium
in 12-well plate dishes (five zebrafish per well) at 28 °C. Zebrafish were incubated with 50 μM Ir1 or Ir2 for 96 h. And then, all zebrafish were exposed to E3 medium
with anesthetic tricaine (0.2 mg/mL). Zebrafish mortality and the
fluorescence of GFP were visualized with an Olympus XI51 microscope.
GFP: λ ex = 460 ± 20 nm, λ em = 530 ± 20 nm. 30
## Materials and Instruments
Materials and Instruments IrCl 3 ·H 2 O, 1-phenylisoquinoline(1pq), 2-(2-pyridyl)-benzothiophene
(pbt), MTT, DMSO- d 6 , and PBS were obtained
from Macklin. Cervical cancer cell line (Hela), human lung carcinoma
cell line(A549), cisplatin-resistant A549 (A549R), human liver cancer
cell line (Hep-G2), and human normal liver cell line (L02) were obtained
from the ATCC cell bank. Dulbecco’s modified Eagle’s
medium (DMEM) and fetal calf serum were purchased from Sigma-Aldrich.
Mito-Tracker Red, ER-Tracker Red, and Lyso-Tracker Red were obtained
from Beyotime Biote chnology. 1 H NMR and 13 C NMR spectra data were obtained utilizing
a BrukerAV-500 spectrometer. ESI-MS data were recorded using Agilent
6130B. UV–vis absorption spectra were measured using a UV-2550
spectrophotometer. The fluorescence spectra were recorded with a Hitachi
F-7000 Fluorimeter.
## Preparation of the Iridium Complex
Preparation of the Iridium Complex The specific synthesis
steps are as follows: The precursors were prepared from iridium chloride
(IrCl 3 ) and 1-phenylisoquinoline (1 pq) or 2-(2-pyridyl)-benzothiophene
(pbt). Specifically, 0.05 mmol of solid iridium chloride and 0.1 mmol
of 1 pq or pbt powder were added to 40 mL of solution containing ethylene
glycol ether and water (v/v = 3:1) in a two-neck round-bottom flask;
the mixture was stirred at 120 °C and refluxed overnight in a
nitrogen atmosphere. After the reaction solution was cooled to ambient
temperature, it was filtered under vacuum. The precipitate was washed
3 times with diethyl ether. Next, a mixture of dichloro-bridged
dimeric (0.1 mmol) complexes ([(L) 2 Ir(m-Cl)] 2 ), (L = 1 pq or pbt), 8-(tosylamino)quinoline (tlq) (0.22 mmol),
and triethylamine (4.5 mmol) was completely dissolved in dichloromethane
and ethanol (v/v = 4:1) in a sealed reaction container according to
the measured molar ratio. The mixture was then refluxed for 20 h in
a nitrogen atmosphere and heated at 55 °C. After the solvent
was removed under vacuum filtration, the precipitate was washed with
ethanol to remove unreacted ligands and dried under vacuum to obtain
solid products Ir1 or Ir2 . [Ir(1pq) 2 (tlq)]Cl ( Ir1 ) Yield
= 51.6% (92.68 mg). Red color solid. 1 H NMR (500 MHz, DMSO- d 6 ): δ 8.94–8.89 (t, J = 7.5 Hz, 2H), 8.86 (d, J = 6.5 Hz, 1H), 8.29 (d, J = 8.3 Hz, 1H), 8.24 (d, J = 7.9 Hz, 1H),
8.14 (d, J = 8.0 Hz, 1H), 8.06–8.01 (m, 2H),
7.97 (d, J = 8.0 Hz, 1H), 7.84 (dd, J = 6.6, 3.2 Hz, 4H), 7.62 (d, J = 6.6 Hz, 1H), 7.49
(d, J = 7.6 Hz, 2H), 7.47–7.43 (t, J = 7.5 Hz, 1H), 7.38–7.33 (t, J = 8.0 Hz, 2H), 7.28 (d, J = 7.9 Hz, 1H), 7.03 (t, J = 7.0 Hz, 1H), 6.88–6.78 (m, 4H), 6.62 (d, J = 7.3 Hz, 1H), 6.57 (d, J = 8.1 Hz, 2H),
6.21 (d, J = 7.6 Hz, 1H), 6.12 (d, J = 7.8 Hz, 1H), 1.96 (s, 3H) ppm. ESI-MS ( m / z ): Calcd for C 46 H 33 N 4 O 2 S( 193 Ir) (M + ), 899.07; found, 899.20
[M + ]. Elemental Anal. Calcd for Ir1 C 46 H 33 N 4 O 2 SIr: C, 61.39; H,
3.67; N, 6.23; S, 3.56. Found: C, 61.26; H, 3.64; N, 6.32; S, 3.52. [Ir(pbt) 2 (tlq)]Cl ( Ir2 ) Yield
= 77.0% (144 mg). Yellow color solid. 1 H NMR (500 MHz,
DMSO- d 6 ): δ 9.10 (d, J = 5.7 Hz, 1H), 8.37 (d, J = 8.3 Hz, 1H), 7.94–7.84
(m, 4H), 7.80 (d, J = 7.9 Hz, 1H), 7.71 (d, J = 8.0 Hz, 1H), 7.61 (t, J = 5.8 Hz, 2H),
7.51 (t, J = 8.1 Hz, 1H), 7.42 (dd, J = 8.4, 5.0 Hz, 1H), 7.32 (d, J = 8.0 Hz, 1H), 7.24
(dd, J = 15.4, 6.3 Hz, 2H), 7.17 (t, J = 7.6 Hz, 1H), 7.09 (t, J = 7.5 Hz, 1H), 6.94–6.89
(m, 5H), 6.82 (dt, J = 18.9, 7.7 Hz, 2H), 6.05 (d, J = 8.1 Hz, 1H), 5.97 (d, J = 8.1 Hz, 1H),
2.23 (s, 3H). ESI-MS ( m / z ): Calcd
for C 42 H 29 N 4 O 2 S 3 ( 193 Ir) (M + ), 910.12; found, 933.00 [M + +Na] + . Elemental Anal. Calcd For Ir2 C 42 H 29 N 4 O 2 S 3 Ir: C, 55.38; H, 3.19; N, 6.15; S, 10.55. Found: C, 55.36; H, 3.14;
N, 6.18; S, 10.52.
## [Ir(1pq)
[Ir(1pq) 2 (tlq)]Cl ( Ir1 ) Yield
= 51.6% (92.68 mg). Red color solid. 1 H NMR (500 MHz, DMSO- d 6 ): δ 8.94–8.89 (t, J = 7.5 Hz, 2H), 8.86 (d, J = 6.5 Hz, 1H), 8.29 (d, J = 8.3 Hz, 1H), 8.24 (d, J = 7.9 Hz, 1H),
8.14 (d, J = 8.0 Hz, 1H), 8.06–8.01 (m, 2H),
7.97 (d, J = 8.0 Hz, 1H), 7.84 (dd, J = 6.6, 3.2 Hz, 4H), 7.62 (d, J = 6.6 Hz, 1H), 7.49
(d, J = 7.6 Hz, 2H), 7.47–7.43 (t, J = 7.5 Hz, 1H), 7.38–7.33 (t, J = 8.0 Hz, 2H), 7.28 (d, J = 7.9 Hz, 1H), 7.03 (t, J = 7.0 Hz, 1H), 6.88–6.78 (m, 4H), 6.62 (d, J = 7.3 Hz, 1H), 6.57 (d, J = 8.1 Hz, 2H),
6.21 (d, J = 7.6 Hz, 1H), 6.12 (d, J = 7.8 Hz, 1H), 1.96 (s, 3H) ppm. ESI-MS ( m / z ): Calcd for C 46 H 33 N 4 O 2 S( 193 Ir) (M + ), 899.07; found, 899.20
[M + ]. Elemental Anal. Calcd for Ir1 C 46 H 33 N 4 O 2 SIr: C, 61.39; H,
3.67; N, 6.23; S, 3.56. Found: C, 61.26; H, 3.64; N, 6.32; S, 3.52.
## [Ir(pbt)
[Ir(pbt) 2 (tlq)]Cl ( Ir2 ) Yield
= 77.0% (144 mg). Yellow color solid. 1 H NMR (500 MHz,
DMSO- d 6 ): δ 9.10 (d, J = 5.7 Hz, 1H), 8.37 (d, J = 8.3 Hz, 1H), 7.94–7.84
(m, 4H), 7.80 (d, J = 7.9 Hz, 1H), 7.71 (d, J = 8.0 Hz, 1H), 7.61 (t, J = 5.8 Hz, 2H),
7.51 (t, J = 8.1 Hz, 1H), 7.42 (dd, J = 8.4, 5.0 Hz, 1H), 7.32 (d, J = 8.0 Hz, 1H), 7.24
(dd, J = 15.4, 6.3 Hz, 2H), 7.17 (t, J = 7.6 Hz, 1H), 7.09 (t, J = 7.5 Hz, 1H), 6.94–6.89
(m, 5H), 6.82 (dt, J = 18.9, 7.7 Hz, 2H), 6.05 (d, J = 8.1 Hz, 1H), 5.97 (d, J = 8.1 Hz, 1H),
2.23 (s, 3H). ESI-MS ( m / z ): Calcd
for C 42 H 29 N 4 O 2 S 3 ( 193 Ir) (M + ), 910.12; found, 933.00 [M + +Na] + . Elemental Anal. Calcd For Ir2 C 42 H 29 N 4 O 2 S 3 Ir: C, 55.38; H, 3.19; N, 6.15; S, 10.55. Found: C, 55.36; H, 3.14;
N, 6.18; S, 10.52.
## HPLC Investigation
HPLC Investigation The prepared 50 μM Ir1 or Ir2 solutions contained 1% DMSO in PBS/acetonitrile
(CH 3 CN) (V/V, 3:7). Analytical high-performance liquid
chromatography (HPLC) was used for analysis with an injection volume
of 20 μL. The mobile phase was a linear gradient of 0.1% HCOOH
in H 2 O and 0.1% HCOOH in CH 3 CN. The absorbance
wavelength was set to 280 nm. Each sample was analyzed by a high-performance
liquid chromatograph (Thermo Fisher) equipped with a C18–H
HPLC column at a flow rate of 1.0 mL/min and a column temperature
of 25 °C. Retention time for Ir1 is 3.108 min and
that for Ir2 is 2.867 min.
## Cellular Localization
Cellular Localization Hep-G2 cells were placed in 35
mm confocal dishes for about 12 h and then incubated with 10 mM Ir1 or Ir2 for 1 h before staining with Lyso-Tracker,
Mito-Tracker, or ER-Tracker for 30 min. Cells were washed with PBS
and imaged with confocal microscopy.
## Dark Cytotoxicity and Phototoxicity
Dark Cytotoxicity and Phototoxicity Hela, A549, A549R,
Hep-G2, and L02 cells were cultured in mixed DMEM supplemented with
10% v/v FBS and 1% v/v penicillin–streptomycin and maintained
in a humidified incubator at 37 °C under an atmosphere of 5%
CO 2 . 29 The cytotoxicity
of complexes Ir1 , Ir2 , and cisplatin on
A549, A549R, Hela, and Hep-G2 cells for 48 h was evaluated by a colorimetric
MTT assay. First of all, about 5000 of each kind of cells were plated
and seeded per well in 96-well plates; then, the cells were incubated
with the complexes in different concentrations at 37 °C. In the
dark toxicity, the complexes’ exposure period was 48 h. As
for phototoxic operations, after the cells were incubated with complexes Ir1 or Ir2 for 1 h, the complexes were removed,
and the 96-well plate was rinsed twice with PBS, and then, the nondrug
medium was placed. After that, the 96-well plate was irradiated with
465 nm light for 30 min and then cultured in the incubator for another
47.5 h. Subsequently, MTT (25 μL, 5 mg/mL) was added and incubation
continued for 4 h to form purple formazan. Finally, 100 μL of
DMSO was added to each well to dissolve purple formazan, and the absorbance
at 490 nm was recorded by a microplate reader. Cells in the blank
control group were treated with DMSO (1%, v/v).
## Determination of Singlet Oxygen Quantum Yield
Determination of Singlet Oxygen Quantum Yield An air
saturation of acetonitrile solution, containing the complexes under
test ( A = 0.1 at 405 nm), p -nitrosodimethyl
aniline (RNO, 24 μM), and imidazole (12 mM) in a quartz fluorescent
cuvette, was irradiated at 405 nm, and the absorbance was recorded
at various time intervals. Plots of A 0 – A at 420 nm ( A 0 is the absorbance before irradiation) versus the irradiation time
were prepared, and the linear regression slope was calculated ( S complex ). Phenalenone was used as the reference
compound (Φ ref ( 1 O 2 ) = 95%)
to obtain S ref and then to calculate the
singlet oxygen quantum yields (Φ complex ) for each
complex 1 2 where I (absorbance correction
factor) was calculated from eq 2 , I 0 is the light intensity of
the irradiated source, and A is the absorbance of
the complex.
## Determination of Intracellular ROS
Determination of Intracellular ROS ROS accumulation
in Hep-G2 cells induced by Ir1 or Ir2 under
light irradiation was evaluated by a fluorescence microscope and 2′,7′-dichlorofluorescein
diacetate (H 2 DCFDA). DCFH-DA is an ROS fluorescent probe
that diffuses into the cell and is rapidly oxidized by intracellular
ROS to form the highly fluorescent 2′,7′-dichlorofluorescein
(DCF, l ex = 488 nm, l em = 520 ± 20 nm). The fluorescence intensity of
DCF is believed to be positively correlated with the amount of ROS
formed in cells.
## Biosafety in Zebrafish
Biosafety in Zebrafish The GFP-transfected Singapore
zebrafish (<5 bpf) were obtained from the Center of Experiment
Animals at Sun Yat-Sen University. Zebrafish were housed in E3 medium
in 12-well plate dishes (five zebrafish per well) at 28 °C. Zebrafish were incubated with 50 μM Ir1 or Ir2 for 96 h. And then, all zebrafish were exposed to E3 medium
with anesthetic tricaine (0.2 mg/mL). Zebrafish mortality and the
fluorescence of GFP were visualized with an Olympus XI51 microscope.
GFP: λ ex = 460 ± 20 nm, λ em = 530 ± 20 nm. 30