Derivatization of chlorin e6 with maleimide enhances its photodynamic efficacy in HepG2 cells

Article abstract of DOI:10.1142/S1088424620500248

Three derivatives of chlorin e6 (1-3) were synthesized by introduction of maleimide, cysteine and glutathione at C-13 carboxyl of the chlorin scaffold. The evaluation of their PDT effects showed that compound 1, the derivative with a maleimide group, exhibited more potent photocytotoxicity against HepG2 cells (IC50 3.2 μM) than 2 (IC50 6.7 μM) and 3 (IC50 10.2 μM), although the cellular uptake of 1 was slightly lower than that of 2 and 3. The high PDT effect of 1 was found to be in agreement with the high level of intracellular singlet oxygen. Further investigation of the mechanism revealed that 1 can significantly lower the GSH level in HepG2 cells due to the addiction reaction of maleimide and GSH, thus resulting in the reduction of ROS scavenging and the enhancement of cellular oxidative stress. This approach to improve PDT effects of photosensitizers by means of interfering with the cellular redox system and enhancing cellular oxidative stress offers a new strategy for development of photosensitizers in cancer therapy.

Full text of DOI:10.1142/S1088424620500248

This is a JAM - Just Accepted Manuscript
Journal of Porphyrins and Phthalocyanines
Article Title:
Derivatization of chlorin e6 with maleimide enhances its photodynamic
efficacy in HepG2 cells
Author(s):
DOI:
Xiuhan Guo, Shisheng Wang, Fan Zhang, Guangzhe Li，Yueqing Li, Weijie
Zhao
10.1142/S1088424620500248
To be cited as:
Xiuhan Guo, Shisheng Wang, Fan Zhang, Guangzhe Li，Yueqing Li, Weijie Zhao, Derivatization
of chlorin e6 with maleimide enhances its photodynamic efficacy in HepG2 cells, Journal of
Porphyrins and Phthalocyanines, doi: 10.1142/S1088424620500248
Received: 19 January 2020
Accepted: 2 May 2020
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Derivatization of chlorin e6 with maleimide enhances its
photodynamic efficacy in HepG2 cells
Xiuhan Guoa,b, Shisheng Wang*a,b , Fan Zhangc, Guangzhe Lia，Yueqing Lia, Weijie
Zhaoa,b
aSchool of Chemical Engineering, Dalian University of Technology, Dalian 116024, P. R. China.
bState Key Laboratory of Fine Chemicals, Dalian University of Technology, Dalian 116024, P. R. China.
cThe Second Hospital of Dalian Medical Universty,Dalian,116024, P. R. China.
Received date (to be automatically inserted after your manuscript is submitted)
Accepted date (to be automatically inserted after your manuscript is accepted)
ABSTRACT: Three derivatives of chlorin e6 (1~3) were synthesized by introduction of maleimide, cysteine
and glutathione at C-13 carboxyl of chlorin scaffold. The evaluation of their PDT effects showed that
compound 1, the derivative with maleimide group, exhibited more potent photocytotoxicity against HepG2
cells (IC50 3.2 M) than 2 (IC50 6.7 M) and 3 (IC50 10.2 M), although the cellular uptake of 1 was slightly
lower than that of 2 and 3. The high PDT effect of 1 was found to be agree with the high level of intracellular
singlet oxygen. Further investigation of mechanism revealed that 1 can significantly lower the GSH level in
HepG2 cells due to the addiction reaction of maleimide and GSH, thus resulting in the reduction of ROS
scavenging and the enhancement of cellular oxidative stress. This approach to improve PDT effects of
photosensitizers by means of interfering cellular redox system and enhancing cellular oxidative stress offered
a new strategy for the development of photosensitizers in cancer therapy.
KEYWORDS: Chlorin e6, Glutathione, Maleimide, Singlet oxygen, Oxidative stress, Photodynamic therapy

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*Correspondence to: wangss@dlut.edu.cn, +86 411 84986200, School of Chemical Engineering, State Key Laboratory of Fine
Chemicals, Dalian University of Technology, Dalian 116024, P. R. China.

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INTRODUCTION
Photodynamic therapy (PDT) is a promising and minimally invasive approach for the treatment of cancer. In the presence
of a photosensitive drug, light of a specific wavelength and molecular oxygen, a series of photochemical reactions are
initiated to produce reactive oxygen species (ROS) [1-4], in particular singlet oxygen (1O2). ROS are the major cytotoxic
agents and readily react with electron-rich biomolecules in surrounding unsaturated lipids, amino acids and DNA, resulting
in tumor cell death by necrotic and apoptotic mechanisms [5-8]. Biological systems have evolved an effective and
complicated network of defense mechanisms which enable cells to cope with lethal oxidative environments [9-11].
Glutathione (GSH) is one of the most abundant intracellular antioxidants, and is readily oxidized to glutathione disulfide
(GSSG) by ROS under the catalysis of glutathione peroxidase (GPx) , which may subsequently cause the reduction of
intracellular GSH level [12-14].
Currently the inhibitory effects of photosensitizers against tumor cell growth have been improved through enhancement
of targeting ability, cellular uptake or ROS productivity [1,15-24]. Considering that the intracellular redox system can
eliminate the ROS produced by photosensitizers, we proposed a new strategy that lowering the intracellular GSH level
would enhance the PDT effect through interfering redox circle and reducing ROS scavenging capability which results in the
increase of singlet oxygen concentration [10,25-27].
In our previous study on the PDT effects of chlorin derivatives, we fulfilled the synthesis of three compounds 1~3 (Fig.1)
via the modification of chlorin e6, which was prepared from Pheophytin a through successively ring-opening and hydrolysis
[6,28-31]. The interesting result of their PDT evaluation was that the photocytotoxicity of compound 1 was three times
more potent than that of compound 3, although the cellular uptake of 1 was slightly lower than that of 3. In the present study,
we proved that the higher PDT effect of 1 was largely attributed to the existence of maleimide moiety, which reacted with
thiol of cysteine or GSH via addiction reaction under neutral conditions [32-34], consequently increasing the concentration
of intracellular singlet oxygen and promoting the oxidative stress of tumor cells.
<Fig. 1.>
RESULTS AND DISCUSSION

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PDT effects, intracellular singlet oxygen and cellular uptake of compounds 1~3
The phototoxicity of compounds 1~3 against HepG2 cells was evaluated and their IC50 values were determined to be
3.2, 6.7 and 10.2 uM, respectively (Fig. 2).
<Fig.2. >
We further measured the concentration of intracellular 1O2 produced by compounds 1~3 with ATTA-Eu3+ as a selective
1
1O2 probe (Fig. 3) [35]. Compared with 2 and 3, compound 1 exhibited higher ability to produce cellular O2, which
corresponded to their phototoxicity in HepG2 cells. Nevertheless, the cellular uptake experiments indicated that the cellular
uptake of 1 was slightly lower than that of 2 and 3 (Fig. 4), which might be related to the better biocompatibility of amino
acid and peptide residues in 2 and 3. In normal cells, the uptake rates of compounds1~3 were similar, but were slightly
lower than those in tumor cells. (see supporting information) Combining these experimental results, we can conclude that
1
the PDT effect of these photosensitizers 1~3 is closely related to their ability of O2 production rather than the cellular
uptake.
<Fig.3. >
<Fig.4.>
Influence of 1 and 3 on GSH level in cell or cell-free system
Since GSH is the principal endogenous antioxidant in cells, the level of intracellular GSH can markedly influence the
1
ability of cells to scavenge ROS. In order to explore the mechanism involved in different production capacity of O2 by 1
and 3, we investigated the influence of 1 and 3 on GSH level in PBS solution and in cells.

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We firstly determined the change of GSH level in PBS buffer (10 mM, pH 7.4) caused by 1 and 3 in the dark or with
LED irradiation (660 nm) using monochlorobimane (mBCl) as a reagent for GSH detection. mBCl reacts with GSH to
produce a fluorescent substance which can be detected by luminoscope (Ex：395 nm，Em: 460 nm)[36]. As a result, the
GSH level was decreased by 1 in the dark with a concentration-dependent manner, while 3 didn’t influence the GSH level in
the same condition (Fig. 5). The decrease of GSH level in the solution of 1 was attributed to be the addition reaction
between GSH and 1 because of the maleimide group in 1, and the reaction product was testified by HPLC-MS/MS analysis
(m/z 1054.50, [M+H]+, see Fig. S4).
<Fig. 5.>
Under the irradiation of light (660 nm) for 10 min, the GSH levels in solutions of 1 and 3 were both much lower than
those in the dark condition (Fig. 5), which was inferred that 1 and 3 produced ROS under the light irradiation and thus
consumed the GSH in the solutions. The synergistic effect of maleimide addition reaction with GSH and ROS oxidation of
GSH in solution of 1 resulted in the significant decrease of GSH level under the light irradiation.
We further determined the GSH levels in HepG2 cells treated with 1 and 3 in the dark or with light irradiation. With
the same results as in PBS solutions, the GSH levels in compound 1-treated HepG2 cells were lower than that in compound
3-treated HepG2 cells with or without light irradiation (Fig. 6). The intracellular GSH level was markedly decreased by
compound 1 under the light irradiation due to its high reactivity with GSH and good productivity of ROS in HepG2 cells,
which was consistent with its potent photocytotoxicity. The addition reaction between 1 and GSH in HepG2 cells was also
confirmed by HPLC-MS/MS analysis of the product after cell lysis and extraction (m/z 1054.50, [M+H]+, Fig. S5).
<Fig.6. >
CONCLUSIONS

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We synthesized three photosensitizers 1~3 with chlorin e6 as the starting material and evaluated their PDT effects in
HepG2 cells. Compound 1, the derivative of chlorin e6 with a maleimide group, exhibited three times more potent
photocytotoxicity than compound 3. The increased PDT effect of 1 was proved to be the contribution of maleimide group,
which enabled 1 to react with GSH in the cells, and thus resulted in the increase of intracellular ROS level and enhancement
of cellular oxidative stress. It is expected that the introduction of other groups with the similar reactivity with maleimide
group would enhance the PDT effects of photosensitizers, which will be confirmed in the future study. This approach to
improve PDT effects of photosensitizers by means of consuming intracellular GSH and enhancing cellular oxidative stress
offered a new strategy for the development of photosensitizers.
EXPERIMENTAL SECTION
Synthesis of photosensitizers
Compounds 1~3 were prepared as previously reported, and their spectroscopic data agreed with the published data[31].
Determination of intracellular singlet oxygen
The HepG2 cells and photosensitizers were incubated for 24 h. After addition of ATTA-Eu3+ and incubation for 4 h, the
medium was replaced with Tris-HCl buffer (10 mM, pH 7.4). The cells were exposed to a LED light at different times. A
fluorescence microscope that can be used for normal fluorescence imaging and time-gated fluorescence imaging was
equipped with a time-gated digital CCD camera system, xenon flash lamp, and UV-2A fluorescence filters (excitation filter,
330-380 nm; dichroicmirror, 400 nm; emission filters, >590 nm). The time-gated luminescence imaging measurements were
carried out with the following conditions: delay time, 33 μs; gate time, 1.0 ms; lamp pulse width, 80 µs; and exposure time
for normal fluorescence, 0.218 s; exposure time for time-gated fluorescence, 1.11 s.
Cellular uptake of photosensitizers
The HepG2 and L-O2 cells were plated at 5000 cells per well in a 96-well plate and allowed to grow for 24 h at 37℃
and 5 % CO2, then the cells were incubated with photosensitizers (50 M) for 0, 2, 4, 6, 8, 10 h at 37℃ and 5 % CO2. Cells

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were washed three times with PBS, and then 100 L methanol was added. Cellular uptake was measured with ELIASA by
determining the fluorescence emission at 660 nm. All experiments were carried out in triplicate independent experiments.
Measurements of the reduced glutathione in PBS buffer
Reduced glutathione (GSH) were measured as described previously. The GSH in PBS (10 mM) was added to the
photosensitizer solutions of different concentrations at a final GSH concentration of 200 μM. After standing for 24 h, the
solutions were irradiated with LED light (18 W, 660 nm) for 10 min (1.7 J / cm2 light dose). Monochlorobimane (mBCI,
200 μM) was added, and fluorescence at 460 nm in response to excitation at 395 nm was measured after standing for 1h in
the dark. The measurements of GSH in the dark were performed with the method as above described except for light
irradiation. All experiments were carried out in triplicate independent experiments.
Measurements of the reduced glutathione in cells
The HepG2 cells were plated at 5000 cells per well in a 96-well plate and allowed to grow for 24 h at 37℃ and 5 %
CO2. Then the cells were incubated with photosensitizer for overnight at 37℃and 5 % CO2, washed three times with PBS
solution, and replaced with 100 μL of fresh culture medium. The cells were irradiated with LED light (18 W, 660 nm) for
10 min (1.7 J / cm2 light dose). The medium was aspirated off and the cells were washed with PBS. Monochlorobimane
(mBCI, 200μM) was added, and the fluorescence at 460 nm in response to excitation at 395 nm was measured after standing
for 1h in the dark. The measurements of GSH in the dark were performed with the method as above described except for
light irradiation. All experiments were carried out in triplicate independent experiments.
HPLC-MS/MS analysis of cell lysis solution
The HepG2 cells were plated at 5000 cells per well in a 96-well plate, allowed to grow for 24 h at 37℃and 5 % CO2, and
incubated with photosensitizer overnight at 37℃and 5 % CO2. After the cells were washed three times with PBS and
replaced with 100 μL of fresh culture medium, they were irradiated with LED light (18 W, 660 nm) for 10 min (1.7 J / cm2

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light dose). The medium was aspirated off and the cells were washed with PBS. 200 μL of methanol was added into the
cells and incubated for 2h. The suspension liquid was filtrated by 0.45 μm micropore filter to yield methanol solution for
HPLC-MS/MS analysis. The HPLC conditions are as follows: Hypersil Gold C18 column (150 mm×2.1 mm, 5μm); solvent
system: acetonitrile /water (containing 0.3% acetic acid), from 50% acetonitrile to 100% acetonitrile for 20 min; flow rate:
1.0 mL/min, detection wavelength: 254 nm.
Acknowledgements
We thank professor Jingli Yuan (Dalian University of Technology, China) for providing the compound ATTA-Eu3+. This
work was financially supported by the Natural Science Foundation of China (Grant No. 21506024), Natural Science
Foundation of Liaoning Province(2019-MS-060)，Scientific Research Foundation for the Returned Overseas Chinese
Scholars of State Education Ministry of China (Grant No. 49)，Fundamental Research Funds for the Central Universities
(DUT20LK08)
Supporting Information
A full list of UV–vis, HPLC, MS/MS and 1H NMR spectra for compounds 1and 3 are given in the supplementary
material. This material is available at http://www.u-bourgogne.fr/jpp/.
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FIGURE CAPTIONS
Fig. 1. Chemical structures of compounds 1~3
Fig.2. Phototoxicity of 1~3 against HepG2 cells. The cell viability was determined by MTT method after 12 h treated with 1~3 at the
concentration ranges of 0~100 μM under irradiation of 660 nm light for 10 min. Values represent as the mean ± standard deviation (SD)
of three independent experiments.
Fig.3. Measurement of 1O2 produced by 1~3 in HepG2 cells with different irradiation times (LED light, 660 nm) using ATTA-Eu3+ as a
fluorescent probe
Fig.4. Cellular uptake of photosensitizers 1~3 in HepG2 cells with different incubation times after irradiation (LED light, 660 nm)
Fig. 5. Measurements of GSH level in PBS buffer (10 mM, pH 7.4) by using mBCI. (a) The comparison of GSH level in PBS buffer
containing GSH (200 μM) and different concentrations of 1 and 3 with or without light irradiation (18 W, 660 nm, 10 min) .(b) The
comparison of GSH level in PBS buffer with different concentrations of 1 and 3 in the dark. (c) The comparison of GSH level in PBS
buffer with different concentrations of 1 and 3 with light irradiation .
Fig.6. Measurements of GSH level in HepG2 cells treated with different concentrations of 1 and 3. (a) With light irradiation. (b) In the
dark.

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Fig. 1.

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Fig.2.

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Fig.3.

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Fig.4.

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Fig. 5.

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Fig.6.

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Derivatization of chlorin e6 with maleimide enhances its photodynamic efficacy in HepG2 cells
Xiuhan Guo, Shisheng Wang*, Fan Zhang, Guangzhe Li，Yueqing Li, Weijie Zhao
Chlorin derivatives containing maleimide functional group are photocytotoxic in cancer cells in visible light. Chlorin derivatives
containing maleimide functional group singlet oxygen generation level was evaluated in HepG2 cells. A rareearth ion ATTA-Eu3+ as the
probe for detection of singlet oxygen. Maleimide group may lead to GSH consumption through reacting with GSH in cells. Highly
photocytotoxicity related to GSH consumption in cells.

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Journal of Porphyrins and Phthalocyanines
Supporting Information
Derivatization of chlorin e6 with maleimide enhances its
photodynamic efficacy in HepG2 cells
Xiuhan Guoa,b, Shisheng Wang*a,b , Fan ZhangC, Guangzhe Lia，Yueqing Lia, Weijie
Zhaoa,b
aSchool of Chemical Engineering, Dalian University of Technology, Dalian 116024, P. R. China.
bState Key Laboratory of Fine Chemicals, Dalian University of Technology, Dalian 116024, P. R. China.
cThe Second Hospital of Dalian Medical Universty,Dalian,116024,P. R. China.

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Table of Contents
General information…………………………………………………………………...3
UV–vis spectra data of compounds 1~3……………………………………………….3
Fig. S1. HPLC chromatogram of compound 1(100 μM) and GSH (200 μM) PBS solution without light
treatment with UV detector…………………………………….4
Fig. S2. HPLC chromatogram of compound 1(100 μM) and GSH (200 μM) PBS solution without light
treatment with UV detector with MS detector…………………4
Fig. S3. HPLC chromatogram of compound 1(100 μM) and GSH (200 μM) PBS solution after 10 min
irradiation (LED light, 660 nm) with UV detector……………..5
Fig. S4. HPLC chromatogram of compound 1(100 μM) and GSH (200 μM) PBS solution after 10 min
irradiation (LED light, 660 nm) with MS detector……………..5
Fig. S5. HPLC chromatogram (A and B) of cell extracts after added compound 1 and irradiated 10 min
(LED light, 660 nm) with MS detector…………………………….6
Fig. S6. MS/MS spectrum of compound 1…………………………………………....7
Fig. S7. MS/MS of the product of compound 1 in cells…………………………...….7

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Fig. S8. MS spectrum of compound 1…………………………………………… ..…8
Fig. S9. 1H NMR spectrum of compound 1…………………………………….…….8
Fig. S10. MS spectrum of compound 3……………………………………………….9
Fig. S11. 1H NMR spectrum of compound 3…………………………………………9
Fig. S12. Detection of 1O2 in HepG2 cells……………………………………..……10
Fig.S13.Cellular uptake of photosensitizers 1~3 in L-O2 cells ………………..……10
1. General information
All reactions conducted under standard conditions were monitored by thin-layer chromatography (T
LC) on gel F254 plates. The silica gel (200～300 mesh, Qingdao Haiyang Chemical Co., Ltd, China) w
as used for column chromatography. All the reactions were performed under N2 atmosphere. After wor
kup, the organic layer was driedoveranhydrous Na2SO4.1H-NMRspectra were recorded on Bruker Av-a
nce 400 MHz instruments, and spectra data were reported in ppm relative to tetramethylsilane (TMS) a
s internal standard. ESI-MS spectra were recorded on a Thermo Electron LTQ-Orbitrap XL hybrid mas
s spectrometer (ThermoFinnigan, Bremen, Germany) equipped with an electrospray ionization interfac
e. Electronic absorption spectra were measured on a JASCO V-550 UV/VIS spectrophotometer.Compo

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und stock solutions were prepared at 32 mM in DMSO. All compound working solutions were prepare
d by diluting the 32 mM stock solution with culture medium or PBS buffer.
2. UV–vis spectra data of compounds 1~3 (in DMSO)
Photo
Comp.
λmax (ε/M-1cm-1)
IC50/μM
3.2±0.1
6.7±1.8
10.2±0.1
41.2±3.3
1
2
3
664(48340) 609(4200) 502(13910) 401(152130)
664(57530) 609(4800) 502(15210) 401(172030)
663(54340) 609(4700) 502(14110) 401(152190)
Chlorin e6 663(49980) 609(4290) 502(13910) 401(146170)
Chlorin e6
trimethyl
ester
664(50540) 609(5300) 502(16910) 403(150710)
26.5±3.6
NL
2.43E
Channel
2.
2400
U
2200
2000
1800
1600
1400
1200
1000
800
15.6
600
400
1.8
200
2.1
13.9
8.3
10.5
0.2
7.4
10.8
6.5
0
-
-
-
0
2
4
6
8
1
1
1
1
1
2
2
2
2
2
Time

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Fig. S1. HPLC chromatogram of compound 1(100 μM) and GSH (200 μM) PBS solution without light
treatment with UV detector.
Fig. S2. HPLC chromatogram of compound 1(100 μM) and GSH (200 μM) PBS solution without light
treatment with UV detector with MS detector.

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Fig. S3. HPLC chromatogram of compound 1(100 μM) and GSH (200 μM) PBS solution after 10 min
irradiation (LED light, 660 nm) with UV detector.

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Fig. S4. HPLC chromatogram of compound 1(100 μM) and GSH (200 μM) PBS solution after 10 min
irradiation (LED light, 660 nm) with MS detector.
A
B

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Fig. S5. HPLC chromatogram(A and B) of cell extracts after added compound 1 and irradiated 10 min
(LED light, 660 nm) with MS detector.
21061101-GXH-P-LCMS3 #1431-1513 RT: 15.02-15.84 AV: 27 SB: 37 14.52-15.07 , 16.17-16.81 NL: 6.15E5
F: FTMS + p ESIFull ms2 747.35@cid35.00 [205.00-800.00]
581.3093
100
95
90
85
80
75
70
65
60
55
50
45
40
35
30
25
20
673.3104
665.3862
15
10
687.3260
607.2887
5
0
715.3210
549.2837
550
507.2729
283.9561
229.5381
250
321.9742 363.0168
415.0353 449.7189
400 450
639.9473
650
758.9215 797.0890
750 800
300 350
500
m/z
600
700
Fig. S6. MS/MS spectrum of compound 1 (RT 15.54 min).

Accepted manuscript to appear in JPP
21061101-GXH-P-LCMS3 #670-709 RT: 7.00-7.37 AV: 13 SB: 36 6.20-6.75 , 8.01-8.56 NL: 3.48E5
F: FTMS + p ESIFull ms2 1054.43@cid35.00 [290.00-1100.00]
579.2940
100
95
90
85
80
75
70
65
60
55
50
45
40
35
30
25
20
15
1036.4189
925.3875
781.3348
607.2889
961.3875
10
5
547.2680
533.2528
912.6152
868.3666
851.3513
813.7075
994.4091
493.2574
747.3469
750
454.1643
450
707.2986
359.5170
350
645.6511
650
1072.9877
1050 1100
0
300
400
500
550
600
700
m/z
800
850
900
950
1000
Fig. S7. MS/MS of the productof compound 1 in cells (RT = 7.13 min).
ZF-M746 #18-28 RT: 0.05-0.08 AV: 11 SB: 17 0.66-0.70 NL: 3.17E4
T: ITMS + c ESIFull ms [200.00-1500.00]
747.45
100
95
90
85
80
75
70
65
60
55
50
45
40
35
310.27
30
25
351.40
20
769.45
15
10
5
518.98
301.12
437.28
210.85
360.37
1493.86
1500
455.57
578.92
600
1110.12
1100
811.61
667.93
700
1182.94
1200
1317.08
1300
866.62
1044.76
1000
1399.13
1400
939.36
0
200
300
400
500
800
900
m/z
Fig. S8. MS spectrum of compound 1

Accepted manuscript to appear in JPP
Fig. S9. 1HNMR spectrum of compound 1
D:\data\...\20131025_ZhangF_MS_1
10/25/2013 3:18:14 PM
20131025_ZhangF_MS_1 #8-20 RT: 0.13-0.30 AV: 6 SB: 5 0.05-0.09 , 0.64-0.77 NL: 2.90E5
F: + c ESI Q1MS [200.000-1100.000]
527.82
100
95
90
85
80
75
70
65
60
55
50
45
40
35
536.01
1054.36
30
25
20
15
10
5
432.16
212.91
278.88
437.03
414.91
586.21
268.82
230.67
304.07
546.83
560.61
345.05
350
610.12
613.60
460.12
391.10
1070.43
328.71
499.59
500
738.29
770.35
685.23
837.01
805.84
800
893.84
900
955.62
950
990.61
1020.63
0
200
250
300
400
450
550
600
650
m/z
700
750
850
1000
1050
1100
Fig. S10. MS spectrum of compound 3

Accepted manuscript to appear in JPP
Fig. S11. 1H NMR spectrum of compound 3
c
a
b
Fig. S12. Detection of 1O2 in HepG2 cells .The HepG2 cells and photosensitizer were incubated for 24
h. Then ATTA-Eu3+ was added, and incubated for 4 h, and the medium was replaced with Tris-
HClbuffer (10 mM, pH 7.4). The cells were exposed to a LED light 2 min.(a) The bright field, (b)
fluorescence of compound 1(red, localization of compound 1); (c) fluorescence of the stable complex
generated by ATTA-Eu3+ and 1O2(white, for localization of 1O2).