A glutathione-responsive photosensitizer with fluorescence resonance energy transfer characteristics for imaging-guided targeting photodynamic therapy

Article abstract of DOI:10.1016/j.ejmech.2020.112203

Here, we have synthesized and characterized a novel activatable photosensitizer (PS) 8a in which two well-designed boron dipyrromethene (BODIPY) derivatives are utilized as the photosensitizing fluorophore and quencher respectively, which are connected by a disulfide linker via two successive Cu (?) catalyzed click reactions. The fluorescence emission and singlet oxygen production of 8a are suppressed via intramolecular fluorescence resonance energy transfer (FRET) from the excited BODIPY-based PS part to quencher unit, but both of them can be simultaneously switched on by cancer-related biothiol glutathione (GSH) in phosphate buffered saline (PBS) solution with 0.05% Tween 80 as a result of cleavage of disulfide. Also, 8a exhibits a bright fluorescence image and a substantial ROS production in A549 human lung adenocarcinoma, HeLa human cervical carcinoma and H22 mouse hepatoma cells having a relatively high concentration of GSH, thereby leading to a significant photocytotoxicity, with IC₅₀ values as low as 0.44 μM, 0.67 μM and 0.48 μM, respectively. In addition, the photosensitizer can be effectively activated and imaged in H22 transplanted hepatoma tumors of mice and shows a strong inhibition on tumor growth. All these results suggest that such a GSH-responsive photosensitizer based on FRET mechanism may provide a new strategy for tumor-targeted and fluorescence imaging-guided cancer therapy.

Full text of DOI:10.1016/j.ejmech.2020.112203

Journal Pre-proof
A glutathione-responsive photosensitizer with fluorescence resonance energy transfer
characteristics for imaging-guided targeting photodynamic therapy
Jing-Jing Cao, Ming-Shan Zhang, Xiao-Qiang Li, De-Chao Yang, Gan Xu, Jian-Yong
Liu
PII:
S0223-5234(20)30170-7
DOI:
https://doi.org/10.1016/j.ejmech.2020.112203
EJMECH 112203
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 12 December 2019
Revised Date: 1 March 2020
Accepted Date: 1 March 2020
Please cite this article as: J.-J. Cao, M.-S. Zhang, X.-Q. Li, D.-C. Yang, G. Xu, J.-Y. Liu, A glutathione-
responsive photosensitizer with fluorescence resonance energy transfer characteristics for imaging-
guided targeting photodynamic therapy, European Journal of Medicinal Chemistry (2020), doi: https://
doi.org/10.1016/j.ejmech.2020.112203.
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2020 Published by Elsevier Masson SAS.

A glutathione-responsive photosensitizer with fluorescence
resonance energy transfer characteristics for imaging-guided
targeting photodynamic therapy
a
a
a
a
a
Jing-Jing Cao , Ming-Shan Zhang , Xiao-Qiang Li , De-Chao Yang , Gan Xu ,
a, b, c, *
Jian-Yong Liu
a
National & Local Joint Biomedical Engineering Research Center on Photodynamic Technologies,
College of Chemistry, Fuzhou University, Fuzhou 350108, China
b
Key Laboratory of Molecule Synthesis and Function Discovery, Fujian Province University,
College of Chemistry, Fuzhou University, Fuzhou 350108, China
c
State Key Laboratory of Photocatalysis on Energy and Environment, College of Chemistry,
Fuzhou University, Fuzhou 350108, China
*
Correspondence: lkw82@fzu.edu.cn; Tel.: +86-591-22867105
Abstract
Here, we have synthesized and characterized a novel activatable photosensitizer
(PS) 8a in which two well-designed boron dipyrromethene (BODIPY) derivatives are
utilized as the photosensitizing fluorophore and quencher respectively, which are
connected by a disulfide linker via two successive Cu (І) catalyzed click reactions. The
fluorescence emission and singlet oxygen production of 8a are suppressed via
intramolecular fluorescence resonance energy transfer (FRET) from the excited
BODIPY-based PS part to quencher unit, but both of them can be simultaneously
switched on by cancer-related biothiol glutathione (GSH) in phosphate buffered saline
(PBS) solution with 0.05% Tween 80 as a result of cleavage of disulfide. Also, 8a
exhibits a bright fluorescence image and a substantial ROS production in A549 human
lung adenocarcinoma, HeLa human cervical carcinoma and H22 mouse hepatoma
cells having a relatively high concentration of GSH, thereby leading to a significant
photocytotoxicity, with IC50 values as low as 0.44 µM, 0.67 µM and 0.48 µM,
respectively. In addition, the photosensitizer can be effectively activated and imaged in
H22 transplanted hepatoma tumors of mice and shows a strong inhibition on tumor
growth. All these results suggest that such a GSH-responsive photosensitizer based on
FRET mechanism may provide a new strategy for tumor-targeted and fluorescence
imaging-guided cancer therapy.
1

Keywords
Photodynamic therapy; activatable photosensitizers; GSH-responsive;
fluorescence resonance energy transfer (FRET)
1
. Introduction
Photodynamic therapy (PDT) utilizes light as the energy source to excite the
photosensitizer (PS), then the excited PS reacts with oxygen in tissues inducing the
production of reactive oxygen species (ROS), which results in localized cell apoptosis
and necrosis. PDT as a new modality, has several distinct advantages over traditional
treatments (including chemotherapy, radiotherapy, and surgery) in solid tumors, such
as low side effects, minimally invasiveness and negligible multidrug resistance (MDR)
[
1-3]. However, the clinical use of this treatment is limited by the low cancer
selectivity of currently available photosensitizers leading to phototoxicity against
normal tissues [4, 5]. To address these problems, there are several commonly used
methods. One is conjugation of PS to a targeting ligand, such as biotin [6, 7], folic
acid [8, 9], saccharide [10], peptides [11, 12], antibody [13] and tamoxifen [14] etc.,
which can greatly improve the specificity of PS to tumor tissues. Another is
encapsulating the photosensitizers with nanomaterials [15, 16], in which utilizes the
enhanced permeability and retention (EPR) effect to achieve passive transport of the
photosensitizers to the tumor tissues. Alternatively, activatable photosensitizers are
novel but effective agents for selective PDT which have drawn widespread attention
in recent years for their unique characteristics [5, 17].
Activatable photosensitizers that mainly based on fluorescence resonance energy
transfer (FRET) [18-20], intramolecular charge transfer (ICT) [21, 22], photo-induced
electron transfer (PET) [23, 24] and self-quenched mechanisms [25, 26] are in a
passive state in normal tissues. However, when going into tumor tissues, with regard
to the special microenvironments like hypoxia [27, 28], specific physiological
enzymes [29, 30], acidic environment [31, 32] and so on, the photo-physical and
photo-chemical properties of which can be restored, including fluorescence emission
and photodynamic effect. Considering the significant differences in the concentration
2

of glutathione (GSH) between the intracellular (~ 10 mM) and the extracellular (~
µM) [33, 34], and the much higher levels in cancer cells than in normal cells [35],
2
GSH-responsive photosensitizers has attracted much attention [36-39].
As a class of versatile fluorescent molecules, boron dipyrromethenes (BODIPYs)
have many excellent photo-physical and photo-chemical properties, such as large
molar extinction coefficients, high photo-stability and low sensitivity to physiological
environment [40, 41]. More importantly, their spectral properties can be finely tuned
by modifying the BODIPY core [42], which is beneficial to the design of activatable
photosensitizer. In this study, we choose two carefully designed BODIPY derivatives as
the photosensitizing fluorophore and quencher respectively, both are linked each other
through a disulfide bridge to construct a glutathione-responsive photosensitizer 8a for
near infrared (NIR) fluorescence imaging-guided targeting photodynamic therapy
(Scheme 1). Due to intramolecular FRET process between PS and quencher moieties,
the activatable PS remains a silent state, as a result, the fluorescence emission, ROS and
photodynamic activity can be obviously quenched. However, they will be
simultaneously restored when treated with glutathione (Scheme 1). The
GSH-responsive properties of this photosensitizer has been validated in vitro as well as
in vivo.
Scheme 1. Schematic illustration of proposed activation mechanism.
3

2
. Results and discussion
2
.1. Molecular design and synthesis
The rationally designed GSH-responsive PS includes three modules, a
BODIPY-based photosensitizing chromophore, a BODIPY-based quencher and a
bio-reducible disulfide linker. Here, BODIPY derivatives are selected as the PS and
quencher, which are mainly due to their excellent photo-physical and photo-chemical
properties, such as strong absorptions in the visible and near-infrared region (NIR),
and high photo-stability. It is noteworthy that their spectral properties can be finely
tuned by reasonable chemical modification of the BODIPY core. Fig. 1 shows the
detailed synthetic route for the activatable PS 8a. According to the previously
reported procedures, we first synthesized BODIPY 4 [9] being as the photosensitizing
agent, the disulfide linker 5a [43] and BODIPY-based quencher 7 [44]. Both PS 4 and
quencher 7 have an alkyne group, they were then linked by diazide 5a via two
successive Cu (І) catalyzed azide-alkyne cycloaddition reactions to afford 8a with the
overall yield of 48%. Two iodine atoms were anchored to the pyrrole rings of
1
BODIPY core, boosting intersystem crossing and singlet oxygen ( O ) production,
2
made it be an ideal PS. Owing to intramolecular charge transfer (ICT), quencher 7
bearing two N,N-dimethylamino moieties had a relatively weak fluorescence emission
and a red-shift absorption at ca. 705 nm, which well overlapped with the emission of
BODIPY 4. Therefore, upon PS unit was excited, there would be a highly efficient
energy transfer in 8a between the PS and quencher moieties. As a result, the
1
fluorescence emission and O production were inhibited. When GSH was added, both
2
of them were restored. By contrast, the control group with the non-cleavable C-C
linker, 8b, was also synthesized by employing a similar protocol. Briefly, BODIPY 4
was treated with 5b, followed by 7 to give 8b in a moderate yield of 50% (Fig. 1). All
the new compounds were purified readily by column chromatography on silica gel
and characterized with various spectroscopic methods.
4

Fig. 1. Synthesis of the GSH-responsive photosensitizer 8a and the control compound 8b.
2
.2. Spectroscopic and photosensitizing properties
The photo-physical and photo-chemical properties of the target compound 8a
together with the references 8b, 4 and 7 were measured in both
N,N-dimethylformamide (DMF) and phosphate buffered saline (PBS) solution (pH=7.4,
with 0.05% Tween 80), respectively. The results are summarized in Table 1. As shown
in Fig. 2, PS 4 had an absorption maximum of 662 nm and fluorescence emission at
6
20-850 nm in DMF. In particular, this emission was fully covered by the absorption
spectra of BODIPY-based quencher 7 (Fig. 2b). Thus, when compound 8a (or 8b) was
selectively excited at 610 nm, the emission from the quencher moiety around 768 nm
(769 nm) was observed while the emission of PS unit at 698 nm (697 nm) was
negligible (Fig. 2c), which indicated a highly efficient FRET process was occurred in
8a (or 8b) from PS to quencher unit. The fluorescence quantum yield of BODIPY 4 was
0.24 in DMF, while that of the BODIPY donor part of 8a was significantly reduced to
0.003. So, the quenching efficiency of the aniline-substituted BODIPY in 8a was
5

estimated to be 0.99. The singlet oxygen quantum yields of 8a, 8b and 4 were also
determined by employing 1,3-diphenylisobenzofuran (DPBF) as the indicator and
unsubstituted zinc(II) phthalocyanine (ZnPc) as the reference [45]. It could be seen
from Table 1 that BODIPY 4 was a highly efficient singlet oxygen generator in DMF
and PBS containing 0.05% Tween 80 (Φ = 0.38 and 0.30). As expected, after
∆
conjugation with the aniline-substituted BODIPY, the products 8a and 8b exhibited
extremely low Φ in both DMF and PBS. The pH-responsive properties of 7 and 8a
∆
were then investigated by electronic absorption and fluorescence emission spectra. As
shown in Fig. S2 and Fig. S3, both compounds are insensitive to neutral and weakly
acidic environment with the pH value higher than 3. So we believe that 8a would not
be protonated whether it is in vitro and in vivo experiments.
Table 1. Photo-physical/photo-chemical data of compounds in different solvents.
ꢂꢄꢅ
ꢇꢁ
a
b
c
Solvent
Compounds
ꢀ
(nm)
ꢆꢀ (nm)
ꢁꢂꢃ
ΦF
Φ∆
ꢁ
ꢂꢃ
4
7
662
705
698
773
768
769
697
744
742
744
0.24
0.043
0.052
0.049
0.11
0.38
0.007
0.018
0.020
0.30
DMF
8
8
a
664, 704
664, 705
662
b
4
PBS
7
708
0.014
0.021
0.019
0.006
0.015
0.016
(
0.05% Tween 80)
8a
668, 709
669, 708
8
b
a
b
Excited at 610 nm; Relative to unsubstituted zinc(II) phthalocyanine (ZnPc) in DMF as the reference (Φ =
F
c
0
.28). Relative to unsubstituted zinc(II) phthalocyanine (ZnPc) in DMF as the reference (Φ = 0.56).
∆
Fig. 2. (a) UV-Vis absorption spectra of 4 (−⋅−), 7 (− −), 8a (⋅ ⋅ ⋅) and 8b (—) (all at 10 µM) in DMF; (b) Overlaps
between the fluorescence spectrum (λex = 610 nm) of PS 4 (−⋅−) and the absorption spectrum of quencher 7 (− −) in
DMF and (c) fluorescence emission spectra (λex = 610 nm) of 4 (−⋅−), 7 (− −), 8a (⋅ ⋅ ⋅) and 8b (—) (all at 10 µM) in
DMF.
6

The activation of 8a by GSH was examined by monitoring its spectral changes in
PBS solution containing 0.05% Tween 80. Here, we set the GSH concentrations to 2
µM and 10 mM, which simulates the extracellular and intracellular bio-distribution of
glutathione respectively. As shown in Fig. 3a and Fig. S4, when 2 µM GSH was
added, even if incubated for 720 min in PBS with 0.05% Tween 80, the changes in
fluorescence of 8a at 698 nm were negligible. However, when 10 mM GSH was
added, the fluorescence of 8a at 698 nm was steadily enhanced with the culture time.
After co-culture for 720 min, the fluorescence quantum yield of 8a from BODIPY
photosensitizer was recovered to be 0.085. Given that the fluorescence quantum yield
of compound 4 in PBS was 0.11, 77% of 8a was estimated to be cleaved by 10 mM
GSH for 720 min. This process followed pseudo-first-order kinetics with kobs = 5.8 ×
−
3
−1
1
0
min for 8a (Fig. S5). These results suggested that the disulfide bond of 8a
could be efficiently cleaved resulting in fluorescence restoration of BODIPY-based PS
by a millimolar concentration of GSH, but not a micromolar concentration of GSH. It
means that compound 8a can keep stable in a quenched state during blood circulation,
but upon it enters into cancer cells, will be activated by intracellular GSH. In addition,
we also investigated the control group and found that the fluorescence intensity of 8b
remained almost unchanged regardless of the amount of GSH added.
1
The ability of 8a and 8b to produce O was also explored in PBS solution with
2
1
0
.05% Tween 80 by using 9,10-dimethylanthracence (DMA) as the O scavenger.
2
1
DMA can be decomposed by O from PS under light illumination. As a result, the
2
fluorescence emission of DMAat 432 nm was vanished. As depicted in Fig. 3b and Fig.
S6, there was not obvious changes for fluorescence of DMA at 432 nm in the presence
of 8a and 8b under irradiation at 670 nm, which indicated both 8a and 8b couldn’t
effectively generate singlet oxygen. However, treated 8a with millimolar concentration
of glutathione, the fluorescence of DMA at 432 nm decreased dramatically (Fig. 3b and
1
Fig. S6). This was because that 8a was activated by GSH to generate O inducing
2
1
degradation of DMA. And 8a treated with GSH (10 mM) exhibited a slight lower O
2
generation efficiency than the reference BODIPY 4. Besides, there was no chance to
7

activate the reference compound 8b with the non-cleavable linker by using any
concentration of GSH. In brief, the intramolecular FRET process in 8a quenches most
of their singlet excited state, reducing the fluorescence emission and singlet oxygen
generation efficiency, and the glutathione with millimolar concentration can activate
them.
Fig. 3. (a) Changes in the fluorescence intensity of 8a (10 µM) and 8b (10 µM) at 698 nm in PBS with 0.05%
Tween 80 in the presence of GSH (2 µM and 10 mM, respectively); (b) Comparison of the degradation rates of
DMA in PBS with 0.05% Tween 80 as monitored by the decrease in fluorescence intensity at 432 nm with
irradiation time both using 8a and 8b as the photosensitizers (both at 10 µM) in the presence of GSH (2 µM and 10
mM, respectively).
2
.3. In vitro studies
To study the activation of 8a at cellular level, we first investigated the fluorescence
imaging by incubating this dye with HeLa, A549 and H22 cancer cells as well as HELF
human embryonic lung fibroblast cells. As demonstrated in Fig. 4, when treatment of
HeLa, A549 and H22 cells with 8a (10 µM) for 8 h, a bright intracellular fluorescence
was observed. The results indicated that 8a could be well uptaken by the three cancer
cells, and then efficiently activated by intracellular biothiols. By contrast, the
fluorescence in HELF normal cells with a lower concentration of GSH was much
weaker than that in the three cancer cells. On the other hand, these four cells pretreated
with exogenous GSH (5 mM) for 2 h and followed by 8a for another 8 h, the
intracellular fluorescence intensity dramatically increased. These results suggested that
8
a remained in a quenched state in normal cells, but could be activated by biothiols of
higher concentration in cancer cells, thereby leading to an enhanced fluorescence
8

emission. Besides, for the non-cleavable analogue 8b, no matter whether pre-treated
with GSH or not, there was almost negligible fluorescence in all the four cell lines. And
the same conclusion can be reached from flow cytometry analysis (Fig. S7).
To account for the different activated behaviors of 8a between the cancer and
normal cell lines, we investigated the intracellular GSH concentrations in HeLa, A549,
H22 and HELF cells with Total Glutathione Assay Kit (Fig. S8). It could be seen that
the GSH levels in HeLa, A549 and H22 cancer cells were much higher than that in
HELF normal cells, which well explained the higher activation of 8a in the three
cancer cells.
Fig. 4. (a) Confocal laser scanning microscopy (CLSM) images of HeLa, A549, H22 and HELF cells after
incubation with 8a (or 8b) (10 µM) for 8 h, or cells pre-treated with GSH (5 mM) for 2 h, followed by treatment of
8
a (or 8b) (10 µM) for another 8 h; (b) quantitative results of (a) (** p < 0.01, *** p < 0.001).
Reactive oxygen species (ROS) is an important factor in PDT. Next, we examined
intracellular ROS levels of 8a and 8b in HeLa, A549, H22 and HELF cells under light
irradiation by using 2’,7’-dichlorofluorescein diacetate (DCFH-DA) as the ROS probe.
DCFH-DA itself does not emit fluorescence, but its oxidized product DCF by ROS is
highly fluorescent. Therefore, the ROS level in living cells can be determined by
monitoring intracellular DCF fluorescence. As shown in Fig. 5, a substantial green
fluorescence of DCF in the green channel and a strong red fluorescence of PS in the red
9

channel were observed when the A549 cells were illuminated by NIR light after
treatment with 8a for 12 h. It was believed that the intracellular thiols could cleaved 8a,
which led to a turn-on fluorescence emission accompanying by a substantial ROS.
Moreover, the intracellular fluorescence in both green and red channels was found to be
stronger when the A549 cells were pretreated with exogenous GSH (5 mM), followed
by 8a, which indicated exogenous GSH promoted the release of PS, thereby elevating
the intracellular ROS level. When Vitamin C (VC) (100 µM), a ROS inhibitor, was
added, the green fluorescence of DCF substantially decreased, but there was not
obvious change in fluorescence intensity of 8a. This is because the ROS produced by
activated 8a was heavy eliminated by VC, resulting in a reduced DCF fluorescence
emission. The same experiment results were demonstrated in HeLa and H22 cells (Fig.
S9 and Fig. S10). In contrast, negligible fluorescence in both channels was observed in
HELF cells and the exogenous GSH can promote the intracellular fluorescence
emission and ROS production. For the control 8b, the fluorescence in both channels
was few captured for all the four cell lines in any cases.
1
0

Fig. 5. CLSM images of (a) A549 and (b) HELF cells incubated with 8a (or 8b) (10 µM) for 12 h, pretreated with
GSH (5 mM) for 2 h and then incubated with 8a (or 8b) (10 µM) for another 12 h or pretreated with VC (100 µM)
for 1 h and then incubated with 8a (or 8b) (10 µM) for another 12 h. The green fluorescence is DCF signals, the
red fluorescence is PS signals; (c) quantitative results of DCF green fluorescence in (a) and (b) (* p < 0.05, ** p <
0
.01, *** p < 0.001).
11

In addition, the cytotoxicity of 8a and 8b was evaluated by MTT assay in HeLa,
A549, H22 and HELF cells. The results were described in Fig. 6. Compound 8a was
basically noncytotoxic against all the four cell lines up to 10 µM without light.
−2
−2
However, once exposed to light (λ = 670 nm, 20 mW·cm , 2.4 J· cm ), it was found
that there was a high cytotoxicity to HeLa, A549 and H22 cancer cells, but not HELF
normal cells. The IC values, defined as the drug concentrations required to kill 50% of
5
0
the cells, are as low as 0.67 µM (for HeLa cells), 0.44 µM (for A549 cells) and 0.48
µM (for H22 cells), respectively. While, the reference 8b showed few toxicity to any
cells in the absence or in the presence of light. It was concluded that 8a could
selectively be activated in cancer cells with relatively high biological thiols leading to a
specific attack to cancer cells.
Fig. 6. Cytotoxic effects of 8a (circles) and 8b (triangles) against (a) HeLa, (b) A549, (c) H22 and (d) HELF cells
−2
−2
in the absence (closed symbols) and presence (open symbols) of light (λ = 670 nm, 20 mW·cm , 2.4 J· cm ). Data
are expressed as the mean value ± standard error of the means of three independent experiments, each performed
in sextuplicate.
To ascertain the distribution of PS in cellular organisms, the subcellular
localization of 8a was also studied in HeLa, A549 and H22 cells (Fig. 7, Fig. S11, Fig.
1
2

S12). The cells were first cultured with 8a, then stained with DAPI, Mito-Tracker
Green and Lyso-Tracker Red which are specific fluorescence probes for nucleus,
mitochondria and lysosomes, respectively. Finally, the intracellular fluorescence
images of PS and probes were captured via two different channels by confocal laser
scanning microscopy (CLSM). As demonstrated in Fig. 7a, the fluorescence of
Mito-Tracker and 8a were well superimposed. And the line traces in the last column
also illustrated this conclusion. In addition, the experimental results in lysosomes were
similar to those of mitochondria (Fig. 7b). By contrast, the fluorescence images of 8a
and the DAPI could not be superimposed well (Fig. 7c). All above suggested that
GSH-mediated cleavage of 8a could occur in mitochondria and lysosomes.
Fig. 7. Visualization of the intracellular fluorescence of A549 cells for Mito-Tracker (in green, a), Lyso-Tracker (in
green, b) and DAPI (in blue, c). The last column is fluorescence intensity profiles of Mito-Tracker, Lyso-Tracker,
DAPI and 8a (10 µM) traced along the white line in (a, b, c).
2
.4. In vivo study
To further confirm the activation of 8a in solid tumors, the in vivo fluorescence
imaging was performed on H22 tumor-bearing mice by fluorescence molecular
TM
tomography (FMT) using the FMT 2500 system (PerkinElmer Inc.). As shown in
1
3

Fig. 8a, the apparent fluorescence signals in tumor were observed for mouse after
administration with 8a (2 mg/kg, 200 µL) via tail-intravenous injection for 2 h and the
signal intensity increased along with time, implying that 8a could be efficiently
accumulated and activated in tumor tissues. While, the signals in the surrounding
normal tissues were insignificant, which might be due to low accumulation or poor
activation of 8a in these area. In comparison, for the control 8b, significantly weak
fluorescence signals were captured in the whole body including tumor tissues as a
result of non-cleavable linker. The same experimental phenomenon was observed in
the ex vivo fluorescence imaging for the harvested tumor and major organs (heart,
liver, spleen, lung and kidney) (Fig. 8b).
Encouraged by the above results, next, we assessed the in vivo photodynamic
effects of 8a by using H22 tumor-bearing mice. They were randomly divided into five
groups treated with saline, 8a, 8a+light, 8b and 8b+light, respectively. It was found
that for the saline control group after 15 days administration, there was a fast tumor
growth which could be reflected by the huge changes in tumor sizes (Fig. 9b). Similar
results were obtained for the groups treated with only 8a or 8b without light
suggesting negligible dark toxicity. After laser irradiation, 8a exhibited a significant
inhibition for tumor growth. Comparatively, only a slight tumor growth inhibition was
achieved for the mice treated with 8b+light. The excellent photodynamic antitumor
performance of 8a was also verified by the average tumor weights (Fig. 9c) and tumor
photographs (Fig. 9d). In order to further confirm in vivo anti-tumor effect, tumors
were excised and subjected to histological analysis via hematoxylin and eosin (H&E)
staining assay. A high level of cell necrosis and apoptosis was observed in the 8a+light
treated tumor tissues, which was indiscernible for the mice receiving the other four
treatments (Fig. 10).
Considering biosafety, we simultaneously monitored the body weights of all the
mice every day during the treatment (Fig. 9a), as illustrated there were no abnormal
changes among all the groups after injection during 15 days, which demonstrated good
1
4

biocompatibility and low bio-toxicity of 8a and 8b. Moreover, the H&E staining
images (Fig. S13) for the major organs (heart, liver, spleen, lung, and kidney) in the
mice treated with 8a+light and 8b+light further manifested no systemic toxicity of 8a
and 8b. All the results indicate that 8a can be served as a promising fluorescence
imaging-guided targeting antitumor PS.
Fig. 8. (a) In vivo fluorescence images of mice bearing H22 hepatocellular tumor taken at different time after
intravenous injection of 8a, 8b and saline; (b) Ex vivo images of the different organs of mice, including kidney,
lung, spleen, liver, heart, and tumor, which were sacrificed at 24 h after injection (He: heart; Li: liver; Sp: spleen;
Lu: lung; Ki: kidney; Tu: tumor).
1
5

Fig. 9. (a) Changes in body weight; (b) Changes in tumor volume; (c) Mean tumor mass; (d) Photograph of the
excised tumors, after treatment with 8a, 8b, 8a+light, 8b+light and saline. Data are expressed as the mean value ±
standard error of the means of three independent experiments, (* p < 0.05, ** p < 0.01, *** p < 0.001).
Fig.10. H&E staining images of tumors for different groups after 15 days treatment. Red arrows
represent areas of apoptosis and black arrows are areas of necrosis.
3
. Conclusions
In summary, a novel GSH-responsive NIR PS based on FRET mechanism has been
synthesized and characterized. This PS can be activated by exogenously added GSH
with a concentration of millimolar in PBS solution with 0.05% Tween 80, and
endogenous biothiol in the A549, HeLa and H22 cancer cells companying with
significant fluorescence emission, high ROS production, and photodynamic effect. The
in vivo studies for H22 tumor-bearing mice demonstrate a strong tumor-imaging and a
substantial tumor growth inhibition ability of 8a with good biosafety. All results
strongly indicate that this kind of GSH-responsive photosensitizer has a great potential
for tumor-targeted and fluorescence imaging-guided cancer therapy.
1
6

4
. Experimental
4
.1. Genernal
Experimental details regarding the purification of solvents, instrumentation and
synthesis of compounds 1-5 and 7 presented in electronic supplementary information.
4
.1.1. Synthesis of 6a
A mixture of 4 (50 mg, 44 µmol) and 5a (41 mg, 200 µmol) in dichloromethane (6
mL) was added to a solution of CuSO ·5H O (10 mg, 40 µmol) and sodium ascorbate
4
2
(30 mg, 150 µmol) in a 1:1 mixture of water and ethanol (1 mL). The mixture was
stirred at room temperature overnight, and then was poured into water (20 mL),
extracted with dichloromethane three times (3 × 20 mL). The crude product was further
purified by column chromatography on silica gel using CH Cl /CH OH (30:1, v/v) as
2
2
3
1
the eluent to give 6a as a green solid (36 mg, 67%). H NMR (400 MHz, CDCl ): δ =
3
8
.13 (d, J = 16.4 Hz, 2 H, CH=CH), 7.75 (s, 1 H, triazole-H), 7.60 (d, J = 8.4 Hz, 4 H,
ArH), 7.58 (d, J = 16.4 Hz, 2 H, CH=CH), 7.19 (d, J = 8.4 Hz, 2 H, ArH), 7.14 (d, J =
.4 Hz, 2 H, ArH), 6.96 (d, J = 8.4 Hz, 4 H, ArH), 5.29 (s, 2 H, OCH ), 4.74 (t, J = 6.4
8
2
Hz, 2 H, NCH ), 4.19 (t, J = 4.8 Hz, 4 H, OCH ), 3.89 (t, J = 4.8 Hz, 4 H, OCH ),
2
2
2
3
.78-3.74 (m, 4 H, OCH ), 3.72-3.65 (m, 8 H, OCH ), 3.61 (t, J = 6.4 Hz, 2 H, CH N ),
2 2 2 3
3
.58-3.54 (m, 4 H, OCH ), 3.38 (s, 6 H, OCH ), 3.21 (t, J = 6.4 Hz, 2 H, SCH ), 2.90 (t,
2
3
2
13
J = 6.4 Hz, 2 H, SCH ), 1.49 (s, 6 H, CH ); C NMR (150.7 MHz, CDCl ) δ: 159.95,
2
3
3
1
1
6
59.16, 150.42, 145.66, 143.52, 139.08, 138.30, 133.20, 129.74, 129.69, 129.24,
27.93, 123.69, 116.74, 115.69, 114.98, 82.70, 71.93, 70.87, 70.65, 70.57, 69.67, 67.54,
2.02, 59.05, 49.84, 48.81, 37.70, 37.52, 17.71 ppm; HRMS (ESI): m/z calcd for
+
C H BF I N NaO S : 1357.2202 [M+Na] ; found 1357.2205.
54
63
2 2
8
9 2
4
.1.2. Synthesis of 6b
According to the above procedure, 4 (50 mg, 44 µmol) was treated with 5b (40 mg,
10 µmol) in the presence of CuSO ·5H O (10 mg, 40 µmol) and sodium ascorbate (30
2
4
2
1
mg, 150 µmol) to give 6b as a green solid (34 mg, 73%). H NMR (400 MHz, CDCl ):
3
δ = 8.13 (d, J = 16.8 Hz, 2 H, CH=CH), 7.66 (s, 1 H, triazole-H), 7.60 (d, J = 7.6 Hz, 4
1
7

H, ArH), 7.58 (d, J = 16.8 Hz, 2 H, CH=CH), 7.19 (d, J = 7.6 Hz, 2 H, ArH), 7.14 (d, J
7.6 Hz, 2 H, ArH), 6.96 (d, J = 7.6 Hz, 4 H, ArH), 5.28 (s, 2 H, OCH ), 4.40 (t, J = 6.8
=
2
Hz, 2 H, NCH ), 4.22-4.16 (m, 4 H, OCH ), 3.92-3.86 (m, 4 H, OCH ), 3.78-3.73 (m, 4
2
2
2
Hz, OCH ), 3.72-3.64 (m, 8 H, OCH ), 3.59-3.53 (m, 4 H, OCH ), 3.39 (s, 6 H, OCH ),
2
2
2
3
3
1
1
1
6
.28 (t, J = 6.8 Hz, 2 H, CH N ), 2.01-1.92 (m, 2 H, CH ), 1.65-1.56 (m, 2 H, CH ),
2 3 2 2
13
.49 (s, 6 H, CH ), 1.46-1.36 (m, 4 H, CH ); C NMR (150.7 MHz, CDCl ) δ: 160.09,
3
2
3
59.33, 150.58, 145.78, 143.67, 139.24, 138.44, 133.34, 129.89, 129.86, 129.39,
28.06, 122.82, 116.90, 115.84, 115.13, 82.84, 72.07, 71.01, 70.80, 70.71, 69.82, 67.69,
2.26, 59.20, 51.37, 50.47, 30.32, 28.78, 26.30, 26.21, 17.83 ppm; HRMS (ESI): m/z
+
calcd for C H BF I N NaO : 1321.3074 [M+Na] ; found 1321.3076.
5
6
67
2 2
8
9
4
.1.3. Synthesis of GSH-responsive PS 8a
According to the procedure for 6a, treatment of 7 (26 mg, 41 µmol) with 6a (50 mg,
8 µmol), CuSO ·5H O (10 mg, 40 µmol) and sodium ascorbate (30 mg, 150 µmol)
3
4
2
1
afforded 8a as a green solid (55 mg, 71%). H NMR (400 MHz, pyridine-d ): δ = 8.54
5
(
d, J = 16.4 Hz, 2 H, CH=CH), 8.40 (s, 1 H, triazole-H), 8.38 (s, 1 H, triazole-H), 8.28
d, J = 16.0 Hz, 2 H, CH=CH), 8.19 (d, J = 16.4 Hz, 2 H, CH=CH), 7.74 (d, J = 8.4 Hz,
(
4
H, ArH), 7.68 (d, J = 8.8 Hz, 4 H, ArH), 7.64 (d, J = 16.4 Hz, 2 H, CH=CH), 7.42 (d,
J = 8.4 Hz, 2 H, ArH), 7.35 (d, J = 8.4 Hz, 2 H, ArH), 7.33 (d, J = 8.4 Hz, 2 H, ArH),
7
6
4
.21 (d, J = 8.4 Hz, 2 H, ArH), 6.99 (d, J = 8.8 Hz, 4 H, ArH), 6.85 (s, 2 H, pyrrole-H),
.63 (d, J = 8.8 Hz, 4 H, ArH), 5.54 (s, 2 H, OCH ), 5.50 (s, 2 H, OCH ), 4.90-4.84 (m,
2
2
H, NCH ), 4.16 (t, J = 4.4 Hz, 4 H, OCH ), 3.83 (t, J = 4.8 Hz, 4 H, OCH ), 3.74-3.70
2
2
2
(
m, 4 H, OCH ), 3.70-3.64 (m, 8 H, OCH ), 3.54 (t, J = 4.8 Hz, 4 H, OCH ), 3.39 (t, J =
2 2 2
5
.6 Hz, 4 H, SCH ), 3.37 (t, J = 5.6 Hz, 4 H, SCH ), 3.29 (s, 6 H, OCH ), 2.77 (s, 12 H,
2
2
3
1
3
NCH ), 1.55 (s, 6 H, CH ), 1.54 (s, 6 H, CH ); C NMR (150.7 MHz, CDCl ): δ =
3
3
3
3
160.08, 159.27, 158.69, 152.84, 160.00, 150.49, 145.78, 143.90, 143.62, 140.98,
139.18, 138.47, 136.56, 133.30, 130.18, 130.03, 130.00, 129.84, 129.80, 129.34,
129.18, 128.38, 127.92, 125.10, 123.93, 123.78, 117.38, 116.86, 115.83, 115.34,
115.11, 114.92, 112.23, 82.82, 72.05, 70.98, 70.77, 70.69, 69.79, 67.67, 61.99, 59.16,
48.83, 40.36, 37.92, 37.80, 17.82, 14.95; HRMS (ESI): m/z calcd for
1
8

+
C H B F I N O S [M+H] , 1975.5568, found, 1975.5582.
94
103 2 4 2 12 10 2
4
.1.4. Synthesis of reference 8b
According to the procedure for 6a, treatment of 7 (26 mg, 41 µmol ) with 6b (52
mg, 39 µmol), CuSO ·5H O (10 mg, 40 µmol) and sodium ascorbate (30 mg, 150 µmol)
4
2
1
afforded 8b as a green solid (52 mg, 68%). H NMR (400 MHz, CDCl ): δ = 8.13 (d, J
3
=
16.4 Hz, 2 H, CH=CH), 7.63-7.57 (m, 8 H, CH=CH and triazole-H and ArH), 7.55 (d,
J = 16.4 Hz, 2 H, CH=CH), 7.51 (d, J = 8.4 Hz, 4 H, ArH), 7.21-7.14 (m, 4 H, CH=CH
and ArH), 7.13-7.09 (m, 4 H, ArH), 7.06 (d, J = 7.6 Hz, 2 H, ArH), 6.95 (d, J = 8.0 Hz,
4
H, ArH), 6.69 (d, J = 8.0 Hz, 4 H, ArH), 6.58 (s, 2 H, pyrrole-H), 5.22 (s, 4 H, OCH2),
.31 (t, J = 6.8 Hz, 4 H, NCH ), 4.17 (t, J = 4.0 Hz, 4 H, OCH ), 3.87 (t, J = 4.4 Hz, 4 H,
4
2
2
OCH ), 3.77-3.73 (m, 4 H, OCH ), 3.72-3.64 (m, 8 H, OCH ), 3.55 (t, J = 4.0 Hz, 4 H,
2
2
2
OCH ), 3.38 (s, 6 H, OCH ), 3.00 (s, 12 H, NCH ), 1.92-1.84 (m, 4 H, CH ), 1.46 (s, 6
2
3
3
2
13
H, CH ), 1.43 (s, 6 H, CH ), 1.37-1.31 (m, 4 H, CH ); C NMR (150.7 MHz, CDCl ): δ
3
3
2
3
=
160.09, 159.29, 158.70, 152.88, 150.99, 150.52, 145.76, 143.92, 143.64, 140.93,
1
1
7
2
39.21, 138.45, 136.53, 133.31, 130.18, 129.83, 129.36, 129.20, 128.48, 127.96,
25.17, 122.94, 122.84, 117.30, 116.87, 115.84, 115.37, 115.10, 115.00, 112.23, 82.81,
2.06, 70.99, 70.79, 70.71, 69.81, 67.67, 62.19, 62.14, 59.19, 50.26, 40.40, 30.15,
9.83, 25.95, 17.81, 14.94; HRMS (ESI): m/z calcd for C H B F I N NaO
10
9
6
106
2
4 2 12
+
[
M+Na] , 1961.6259, found, 1961.6380.
4
4
.2. Photo-physical and photo-chemical studies
.2.1. GSH-responsive fluorescence emission
8
a and 8b were dissolved in DMF to obtain solutions with concentration of 1 mM,
which were diluted to 10 µM with PBS containing 0.05% Tween 80. And then GSH
was dissolved in PBS to obtain a 0.5 M GSH stock solution. To investigate the effect
of GSH on the disulfide bond, we prepared a mixture of 8a (10 µM) with GSH (2 µM
and 10 mM) and 8b (10 µM) with GSH (2 µM and 10 mM). All of these solutions
were kept at room temperature during the test. And the fluorescence spectra (ꢈ = 610
ꢉꢊ
nm) of these solutions were recorded at different time intervals.
1
9

4
.2.2. GSH-responsive singlet oxygen generation
In order to study the singlet oxygen generation efficiency, Firstly, 1 mM stock
solution of 9,10-dimethylanthracence (DMA), 1 mM stock solution of compounds and
the stock solution of GSH (0.5 M and 1 mM) were prepared in DMF. Then the mixture
of 8a (or 8b) (10 µM) and DMA with GSH (2 µM and 10 mM) were prepared in PBS
−
2
with 0.05% Tween 80, followed by illumination with light (670 nm, 20 mW·cm ).
The value were recorded by monitoring the decay of fluorescence intensity of DMA at
4
32 nm (λ =370 nm).
ꢋꢌ
4
.3. In vitro studies
4
.3.1. Intracellular fluorescence imaging, flow cytometry analysis and intracellular
ROS measurements
5
HeLa, A549, H22 and HELF cells with a density of 10 cells in 1000 µL of DMEM
were seeded in a confocal dish and incubated overnight at 37°C with 5% CO2.
Removing the medium and rinsing thrice with PBS. After that, replacing the medium
with fresh DMEM containing GSH (5 mM, 1 mL). In addition, we set up a control
group without GSH. Specifically, the cell culture medium (1 mL) was added under the
same conditions, followed by incubation at 37 °C, 5% CO for 2 h, and then the old
2
medium was removed. After rinsing twice with PBS, 8a (10 µM) or 8b (10 µM) were
added and cultured in a cell culture incubator for 8 h, and intracellular fluorescence was
detected by laser confocal (excitation wavelength was 633 nm). And under the same
conditions, the intracellular fluorescence intensity was measured by flow cytometry.
As for intracellular ROS measurements, after adding the compound and culturing
for 12 h, we added DCFH-DA with DMEM and co-cultured for 1 h, rinsing with PBS
for 5 times and then changing to serum-free, 1 mL without phenol red DMEM medium,
−2
the cells were irradiated with light at 670 nm (20 mW·cm ) for 3 minutes, and
intracellular fluorescence was detected by laser confocal scanning. (λꢋꢌ = 488 nm for
DCF green fluorescence and λꢋꢌ = 633 nm for 8a, 8b red fluorescence, respectively).
At the same time, in order to prove that the green fluorescence is caused by the ROS
2
0

generated by the photosensitizer, we added a set of pre-treated cells with VC (100 µM,
mL) (ROS scavenger) for 1 h and then added 8a, 8b stock solution. Intracellular
fluorescence was detected by laser confocal microscopy under the same method.
1
4
.3.2. Determination of intracellular GSH concentrations
HeLa, A549, H22 and HELF cells with a density of 2×10 cells in 10 mL of
6
DMEM were seeded in a 96-well plates and incubated overnight at 37°C with 5% CO2.
After washed with phosphate buffer saline (PBS), lysed with 200 µL of cell lysates on
ice and then were centrifuged (10000 × g) for 10 min at 4°C and the supernatant (10 µL)
was mixed with 150 µL of the Glutathione Assay Kit. Then added 50 µL of 0.5 mg/mL
NADPH solution and mixed well. The level of intracellular GSH was determined by
recording the absorbance at 412 nm via a microplate reader 25 minutes later. And the
protein concentration was then detected using a microplate reader with the BCAProtein
Assay Kit. The final intracellular GSH concentration is expressed in nmol/mg-protein.
4
.3.3. Photocytotoxicity studies
To study the cytotoxicity and photocytotoxicity, MTT assays against HeLa, A549,
H22 and HELF cells were conducted. Approximately 6000 cells (100 µL) each well
were seeded in 96-well plates and inoculated under 5% CO overnight at 37°C. Then
2
replacing the medium with fresh DMEM containing 8a or 8b (1% DMSO, 0.05%
Tween 80) with different concentrations, respectively. After incubating for 24 h under
dark conditions, treating the 96-well plates with light or not after replacing with fresh
DMEM. For the irradiation group, the 96-well plates were irradiated with a 670 nm
−
2
LED lamp for 2 minutes with a power of 20 mW·cm . Then the cells were inoculated
at 37°C overnight under 5% CO . After that, the MTT (10 mL, 5 mg/mL) was added to
2
per well and incubated for 4 h. Next, the DMEM was removed and added DMSO (100
µL) to each well. Finally, the OD values at 490 nm of each well were measured.
4
.3.4. Subcellular localization
HeLa, A549, H22 and HELF cells with a density of 50000 cells in 1 mL of DMEM
were seeded in a confocal dish and incubated overnight at 37°C with 5% CO . Then 8a
2
2
1

(10 µM) or 8b (10 µM) were added and cultured in a cell culture incubator for 12 h.
DAPI, Lyso-Tracker Red and Mito-Tracker Green in the DMEM were added into the
dish and incubated for minutes (DAPI: 20 min, Lyso-Tracker Red and Mito-Tracker
Green are both 60 min). Subsequently, washed by PBS for 5 times and imaged. And the
DAPI, Lyso-Tracker Red and Mito-Tracker Green were excited at 405 nm, 543 nm and
4
88 nm, and detected at 425-475 nm, 550-620 nm and 510-570 nm, respectively. And
the compounds were excited at 633 nm and detected at 650-750 nm.
4
.4. In vivo studies
Female KunMing mice (20-25 g) were purchased from the Laboratory Animal
Services Centre of Wushi, Fuzhou, China. All animal experiments had been approved
by the Animal Experimentation Ethics Committee of Fuzhou University. The mice
were kept under pathogen-free conditions with free access to water and food. Selecting
7
mice in good health and then H22 cells (10 cells/mL, 100 µL) were inoculated into the
subcutaneous tissue on the back of the mice and when the average size of the tumor
3
reached 50 mm , a series of experiments were carried out. For in vivo fluorescence
imaging study, nine mice were randomly divided into three groups and treated with a
single intravenous injection of saline, 8a and 8b (2 mg/kg, 200 µL), respectively. And
collecting the whole body images at 0 h, 2 h, 6 h, 12 h and 24 h post-injection. And the
mice were sacrificed at 24 h after injection to harvest the tumor and normal tissues,
including heart, liver, spleen, lung, kidney for the ex vivo fluorescence images.
On the other hand, another 25 mice were randomly divided into five groups of five
each and the mice were injected with saline, 8a and 8b (2 mg/kg, 100 µL) for three
times on the first, fourth and seventh day respectively and then subjected to laser
irradiation or not after 12 h of each administration: group 1 for saline (blank control),
2
group 2 for 8a, group 3 for 8a with laser (670 nm, 100 mW/cm ), group 4 for 8b, group
2
5
for 8b with laser (670 nm, 100 mW/cm ). Monitoring the tumor volume and body
weight every day for 15 days. Then the mice were sacrificed, then tumors were excised
and weighed. For histological examination, tumors from all five groups and normal
tissues (heart, liver, spleen, lung, and kidney) from group 4 and group 5 were harvested,
2
2

fixed overnight in 4% polyformaldehyde solution, embedded into paraffin, sectioned
and stained with hematoxylin and eosin (H&E), and detected by digital microscopy.
Acknowledgments
This work was supported by the National Natural Science Foundation of China
(project Nos. 21471033), the Key Project of Science and Technology Development of
Fujian Province (project No. 2015Y0086), the Independent Research Project of State
Key Laboratory of Photocatalysis on Energy and Environment (project Nos. 2014C04).
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.org/xxx.
Conflicts of interest
The authors declare no conflict of interest.
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Highlights:
1. A novel glutathione-responsive photosensitizer has been synthesized.
2. The photosensitizer can be activated by glutathione in solution and cancer cells.
3. The photosensitizer exhibits a high photodynamic activity to cancer cells.
4. The photosensitizer can be efficiently accumulated and activated in tumor.
5. The photosensitizer shows a strong inhibition on tumor growth.

Declaration of interests
☒
The authors declare that they have no known competing financial interests or personal relationships
that could have appeared to influence the work reported in this paper.
☐
The authors declare the following financial interests/personal relationships which may be considered
as potential competing interests: