Hypoxia-activated NIR photosensitizer anchoring in the mitochondria for photodynamic therapy

Article abstract of DOI:10.1039/c9sc03355f

Photodynamic therapy is considered as a promising treatment for cancer, but still faces several challenges. The hypoxic environment in solid tumors, imprecise tumor recognition and the lack of selectivity between normal and cancer cells extremely hinder t

Full text of DOI:10.1039/c9sc03355f

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Hypoxia-activated NIR photosensitizer anchoring
in the mitochondria for photodynamic therapy†
Cite this: Chem. Sci., 2019, 10, 10586
a
a
a
a
a
a
All publication charges for this article Feng Xu, Haidong Li, Qichao Yao, Haoying Ge, Jiangli Fan,
Wen Sun,
have been paid for by the Royal Society
of Chemistry
b
ac
Jingyun Wang
and Xiaojun Peng
*
Photodynamic therapy is considered as a promising treatment for cancer, but still faces several challenges.
The hypoxic environment in solid tumors, imprecise tumor recognition and the lack of selectivity between
normal and cancer cells extremely hinder the applications of photodynamic therapy in clinics. Moreover,
the “always on” property of photosensitizers also increases the toxicity to normal tissues when exposed
to light irradiation. In this study, a hypoxia-activated NIR photosensitizer ICy-N was synthesized and
successfully applied for in vivo cancer treatment. ICy-N is in the inactivated state with low fluorescence
whereas its NIR emission (lem ¼ 716 nm) was induced via reduction caused by nitroreductase at the
tumor site. In addition, the reduced product ICy-OH was specially located in the mitochondria and
demonstrated a high singlet oxygen production under 660 nm light irradiation, which efficiently induced
cell apoptosis (IC50 ¼ 0.63 mM). The in vivo studies carried out in Balb/c mice indicated that ICy-N was
suitable for precise tumor hypoxia imaging and can work as an efficient photosensitizer for restraining
tumor growth through the PDT process.
Received 8th July 2019
Accepted 1st October 2019
DOI: 10.1039/c9sc03355f
rsc.li/chemical-science
enhancing the specicity of photosensitizers for tumour cells.
Because the photosensitizer is always “on”, ROS can be
Introduction
In recent decades, cancer has gradually become the major cause produced once it is exposed to light irradiation even in healthy
23
of death in both developed and less developed countries due to cells, which consequently leads to undesired side effects.
the growth and aging of the population and the incidence rate is That's why patients should be kept out of light before being
1
,2
increasing every year.
Therefore, multiple treatments exposed to certain light irradiation. Hence, “stimuli-activatable”
3
,4
5,6
7,8
including radiotherapy, chemotherapy, immunotherapy,
photosensitizers are urgently needed for precise photodynamic
photodynamic therapy therapy and minimizing side effects.
and so on have been developed for treating cancer.
The hypoxic environment is an important feature of
9,10
photothermal therapy (PTT),
11–14
(PDT)
Among them, photodynamic therapy has attracted great tumours due to the insufficient supply of oxygen. Several
interest due to its minimal invasiveness, negligible drug resis- methods have been reported for hypoxic tumour therapy, such
2
4
tance, controllable treatment area selection and excellent ther- as those based on thermoresponsive nanoplatforms,
15,16
25,26
apeutic effects.
with a photosensitizer and certain light irradiation on the transfer mediated upconversion nanoparticles (UCNPs),
A typical PDT process involves pretreatment decomposition of the endogenous H
2
O
2
system,
electron
2
7
2
8
tumour region which can activate the photosensitizer to light-controlled drug release complex conjugates and so on.
17,18
produce reactive oxygen species (ROS) to damage cells.
Thus, Nitroreductase (NTR) is a well-known specic enzyme over-
the capacity of photosensitizers to produce ROS and tumour expressed in solid tumours due to the hypoxic microenvi-
recognition remain the most important factors for maximum ronment which can effectively reduce the nitroaromatic to the
therapeutic effects and minimum side effects. Up to now, corresponding arylamine by using reduced nicotinamide
2
9,30
numerous efficient photosensitizers have been developed based adenine dinucleotide (NADH) as an electron donor.
on various uorophores.
Based
19–22
But little effort has been devoted to on this reduction reaction, many nitroaromatic decorated
3
1–35
probes have been developed for hypoxia imaging.
Li's
group proved that nitroreductase was over-expressed when
the O2 concentration was below 10% and the para-nitro
benzoate group was an ideal substrate for NTR with a fast
response, high sensitivity and excellent selectivity because of
a
State Key Laboratory of Fine Chemicals, Dalian University of Technology, 2 Linggong
Road, Dalian 116024, P. R. China. E-mail: pengxj@dlut.edu.cn
b
School of Life Science and Biotechnology, Dalian University of Technology, 2 Linggong
Road, Dalian 116024, P. R. China
c
3
1
Shenzhen Research Institute, Dalian University of Technology, Nanshan District, the strong hydrogen-bonding interaction and spatial match
Shenzhen 518057, P. R. China
Herein, a hypoxia-activated photosensitizer ICy-N was
designed, not only for hypoxic imaging, but also for PDT of
†
Electronic supplementary information (ESI) available. See DOI:
0.1039/c9sc03355f
1
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cancer cells under hypoxic conditions, by decorating hemi-
cyanine dye Cy-OH with 4-nitrobenzyl bromide as the recog-
Results and discussion
nition site and the introduction of iodine into the indole ring Due to its unique properties including excellent photostability,
38
was used to improve the intersystem crossing (ISC) process so controllable NIR emission and good water solubility, Cy-OH
3
6
as to increase singlet oxygen production. As depicted in with iodine and 4-nitrobenzyl bromide substituents was
Scheme 1, in normal tissue, ICy-N was in a uorescence-off selected for both precise tumour imaging and photodynamic
state because the Intramolecular Charge Transfer (ICT) therapy in this work. The water solubility and stability were
process was restrained and the energy of the excited state was characterized by transmission electron microscopy (TEM) and
mainly released through the vibration relaxation pathway due UV-vis spectroscopy. As shown in Fig. S1,† ICy-N showed slight
3
7
to the vibration of the nitro group and the ISC process was aggregation in water and exhibited spherical morphology with
blocked. As a result, the ability to produce singlet oxygen was a diameter of about 5 nm. Meanwhile, the absorbance
restricted and the toxicity was minimized in normal cells. But measured during 6 h changed scarcely which indicated that ICy-
in the tumour region, ICy-N will be reduced by NTR and N was stable and dispersed well in water solution. The recog-
consequently ICy-OH will be released, serving as an efficient nition mechanism of the ICy-N response to nitroreductase is
type II PS for photodynamic therapy. When irradiated with shown in Scheme 2. ICy-N was non-uorescent in PBS buffer
6
60 nm light, ICy-OH can be excited and reach the singlet while its reduced product ICy-OH emitted strong uorescence
state (S ), which can be used for emitting uorescence at 710 at 710 nm, which was extremely important for tumour hypoxia
1
nm. Meanwhile, some of the excited molecules will reach the imaging (Fig. 1a). Along with the change of uorescence
triplet state (T ) by the ISC process because of the “heavy atom spectra, the maximum absorption peak of ICy-N red shied
1
effect” of the introduced iodine atom. Finally, the energy from 575 nm to 655 nm (Fig. 1b). This is a favorable result for
transfer from triplet states to the ground state of molecular PDT because longer wavelengths have deeper penetration in
1
oxygen results in the formation of singlet oxygen ( O
2
) which tissues when exposed to light irradiation during a therapeutic
serves as a strong oxidant to induce cell apoptosis. Therefore, process. In addition, the quenching of the excited state by the
tumour recognition and precise PDT were simultaneously nitro group of ICy-N may also lead to lower triplet state
achieved.
production which consequently resulted in a lower singlet
oxygen quantum yield (F ) in contrast to ICy-OH. To conrm
D
this hypothesis, the ability of ICy-N and ICy-OH to produce
1
singlet oxygen ( O
2
) was tested and compared by using 1,3-
1
2
diphenylisobenzofuran (DPBF) as the O trapping agent and
methylene blue (MB) as a reference. As seen in Fig. 1d, the
absorbance of DPBF at 415 nm decreased dramatically in the
presence of ICy-OH under 660 nm light irradiation, while ICy-
N only resulted in slight bleaching of DPBF under identical
conditions (Fig. 1c). The singlet oxygen quantum yield (FD)
was calculated to be 0.72% and 1.80% for ICy-N and ICy-OH
respectively, suggesting that the reduced product ICy-OH
displayed stronger ROS production capacity and thus may
result in higher toxicity to cancer cells (Table S1†). What's
1
more, the O
2
production of ICy-OH was also proved by Singlet
Oxygen Sensor Green (SOSG) and electron paramagnetic
Scheme 1 Proposed strategy of ICy-N for hypoxia imaging and cancer resonance (EPR) measurements (Fig. S5†), showing that ICy-
therapy.
Scheme 2 Recognition mechanism of ICy-N with NTR.
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Fig. 1 (a) Fluorescence and (b) UV-vis spectra of ICy-N and ICy-OH in
PBS buffer (pH ¼ 7.4, 10 mM). (c) Photodegradation curves of DPBF
with ICy-N and (d) ICy-OH under 660 nm light irradiation for different
times (0–2100 s).
OH can serve as an efficient photosensitizer under 660 nm
light irradiation.
To conrm the feasibility of this protocol, 4T1 cells were Fig. 2 (a) Confocal microscopy imaging of 2.5 mM ICy-N in 4T1 cells
concentrations. (b) Subcellular colocalization
preincubated under hypoxia (10%, 2% and <1% O
2
) conditions under different O
2
assays of ICy-N in 4T1 cells. The green channel of MitoTracker Green
and LysoTracker Green was excited at 488 nm and collected at 500–
for 8 h respectively to induce the overexpression of nitro-
reductase. Cells that were incubated under normoxic conditions
were also taken as the control group. Then the cells were stained
with ICy-N and incubated for another 60 min before imaging
under a laser scanning confocal microscope. As expected, no
obvious uorescence was observed under normoxic conditions
550 nm. The blue channel of Hoechst 33342 was excited at 405 nm
and collected at 440–480 nm.
39–41
molecules with positive charge.
It is quite important for
(Fig. 2a) due to the low expression of nitroreductase, whereas
improving the PDT efficiency of photosensitizers because
mitochondrial damage and the following release of cytochrome
C to trigger apoptosis was considered to be the main pathway
the hypoxia (10%, 2% or <1% O ) induced overexpression of
2
nitroreductase gave rise to a signicant red uorescence signal.
The result demonstrated that ICy-N was reduced by nitro-
reductase, followed by releasing ICy-OH with red uorescence
when excited with a 635 nm laser (as illustrated in Scheme 2). To
further investigate the subcellular organelle localization of the
reduced product, various commercial trackers and ICy-N (2.5
mM) were applied to the cells that were pretreated under hypoxic
42
during the PDT process.
Then, the PDT properties of the reduced product were
studied with a 660 nm LED lamp. 4T1 cells were preincubated
under hypoxic conditions (10% O , 5% CO ) for 8 h and treated
2 2
with 2.5 mM ICy-N for 60 min, followed by exposure to 660 nm
ꢀ2
light (12 mW cm ) for 5 min. Aer that, annexin V-FITC and PI
2
conditions (2% O ) for 60 min respectively (Fig. 2b). Apparently,
(Propidium Iodide) were utilized for assessing cell apoptosis
a poor colocalization region uorescence signal was observed
between the reduced product and LysoTracker Green (a lyso-
somal tracker) or Hoechst 33342 (a nuclear tracker). In contrast,
the red uorescence overlapped well with the MitoTracker
Green (a mitochondrial tracker), indicating that ICy-N can be
triggered by NTR efficiently and nally gathered in the mito-
chondria. When MCF-7 cells were treated with carbonyl cyanide
induced aer the treatment. When apoptosis occurs, phos-
phatidylserine inside the cytomembrane will turn over to the
outside which can be recognized by annexin V with high affinity
and specicity. With the development of apoptosis, the
permeability of the cytomembrane changes so that PI can enter
the cells and combine with DNA to emit red uorescence,
whereas it is inaccessible in living cells. As shown in Fig. 3a, the
3-chlorophenylhydrazone (CCCP,
a
membrane potential

uorescence signal of FITC was detected in the cytomembrane
uncoupler) to induce the loss of mitochondria membrane
potential, the uorescence signal of ICy-N decreased dramati-
cally, whereas, the uorescence signal of MitoTracker Green FM
in the ICy-N + Light group, indicating that apoptosis was
induced. Strong red uorescence in the cell nucleus in the PI
channel can also be observed, which indicated cell death aer
PDT. This phenomenon was in accordance with the one treated
with ICy-OH under the same conditions (Fig. S8†). In contrast,
no uorescence signal was observed from FITC and PI channels
in Light and ICy-N groups, because the cells were still surviving
(a mitochondrial probe whose uptake is independent of
membrane potential) remained unchanged (Fig. S6†). This
result can be attributed to the negative membrane potential
(ꢀ180 mV) of the mitochondria which facilitates the uptake of
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concentration of ICy-N increased under 10% O
only 20% cells survived at a concentration of 1.5 mM, whereas,
0% cells were still alive in the 21% O treated group at the
2
conditions and
8
2
same concentration. This result was consistent with the
hypothesis we proposed that a higher singlet oxygen quantum
yield of ICy-OH can lead to stronger cytotoxicity so that the side
effects in normal tissues can be minimized during the PDT
process.
To have a better understanding of the PDT efficiency of ICy-
N, a clinical photosensitizer Ce6 was used as a contrast. Similar
cell viability was observed between ICy-N and Ce6 under dark
conditions which further veried the good biocompatibility of
ICy-N. However, quite different PDT efficiency was found in the
light treated groups. Ce6 seems nontoxic at low concentrations
(0–1.5 mM) while strong cytotoxicity was found in the group
treated with ICy-N under 10% O conditions. When the
2
concentration increased to 3 mM, comparative cell viability was
observed and the IC50 values of ICy-N under 10% O conditions
2
and Ce6 were determined to be 0.63 mM and 2.79 mM respec-
tively, indicating that ICy-N demonstrates a better PDT effi-
ciency than Ce6 under identical conditions.
To get more proof of the photo-induced cytotoxicity of the
Fig. 3 (a) Confocal microscopy imaging of annexin V – FITC and PI reduced product ICy-OH, DCFH-DA was used for tracking the
stained 4T1 cells after different treatments. The green channel of
annexin V – FITC was excited at 488 nm and collected at 500–550
found that strong uorescence of DCF was observed aer irra-
nm; the red channel of PI was excited at 561 nm and collected at 590–
ROS production during the PDT process. Striking contrast was
diation with 660 nm light, whereas the signal was low in the
6
40 nm. Cytotoxicity of photosensitizers under dark conditions (b) and
irradiation with 660 nm light (c) at various concentrations.
dark indicating that ICy-OH can work as an efficient photo-
sensitizer and produce ROS under irradiation (Fig. S7†). In
addition, the ROS induced apoptosis was visible even in the
in these control groups. To get a better understanding of the bright image (Fig. S10†). Aer being irradiated with a 635 nm
cytotoxicity mechanism, the JC-1 assay was carried out in MCF-7 laser for 1 min, obvious vesicles were observed with the exten-
cells. As shown in Fig. S9,† when MCF-7 cells were treated with sion of incubation time indicating the development of
2.5 mM ICy-N and irradiated with 660 nm light, the uorescence apoptosis. In contrast, when the cells without treatment with
signal of the red channel which belongs to the aggregation of ICy-OH were exposed to the same laser irradiation, no
JC-1 in the mitochondria decreased dramatically while the red morphological change was found. Then, the cell death was

uorescence in the dark group was obvious, indicating mito- further proved by ow cytometry statistically. As seen in
chondrial damage. This result demonstrated that cell apoptosis Fig. S11,† the PI signal in the ICy-OH treated group was nega-
was induced by mitochondrial dysfunction during the PDT tive. Aer irradiation with 660 nm light, a positive signal was
process. All these results proved that the reduced product can observed, showing the death of MCF-7 cells.
work as an efficient photosensitizer and induce cell apoptosis
All these results indicated that ICy-N was in a uorescence-
under 660 nm light irradiation.
off state and had negligible cytotoxicity under dark conditions
To further investigate the cytotoxicity of ICy-N and the and low PDT efficiency without reduction by NTR (in normal
reduced product ICy-OH, the MTT assay was carried out (Fig. 3b tissues). But, it can be turned on under hypoxic conditions by
and c). Negligible cytotoxicity was observed in the ICy-N treated NTR (in tumour tissues) and the reduced product ICy-OH
group under 21% O and the viability of MCF-7 cells was still showed higher singlet oxygen production. Thus, ICy-OH is an
2
above 98% at a concentration of 4 mM under dark conditions. efficient photosensitizer and is promising for PDT under 660
However, considerable damage was found aer 12 h incubation nm light irradiation.
in the 10% O
2
group which was caused by the reduced product
Inspired by the excellent performance of ICy-N during the
ICy-OH. The higher cytotoxicity of ICy-OH was benecial for PDT process in vitro, an in vivo study was carried out in 4T1
tumour therapy because only in tumour tissues can ICy-N be tumour bearing Balb/c mice. Firstly, Balb/c mice were implan-
reduced to ICy-OH. Next, the PDT effect was studied under 660 ted with a 4T1 tumour subcutaneously at the le armpit and
ꢀ
2
nm light irradiation (12 mW cm ) aer incubation with ICy-N then divided into four groups (Control, Light, ICy-N, and ICy-N
3
for 60 min under 21% and 10% O conditions, respectively. As + Light) when the tumour volume reached 200 mm . The
2
shown in Fig. 3c, the photo-induced cytotoxicity of ICy-N was feasibility of ICy-N as an NIR uorescent probe for hypoxia
improved with increasing ICy-N concentration. But the PDT was imaging was tested by intratumoral injection of ICy-N in PBS
still inefficient in the 21% O
2
group in contrast to that treated buffer in 4T1 tumour bearing Balb/c mice. As depicted in Fig. 4,
with 10% O . The cell viability decreased rapidly when the an obvious uorescence signal (680–720 nm) was observed in
2
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The tumour volume remained almost unchanged for 14 days
while other groups exhibited above 5-fold increase. The anti-
cancer capability in vivo was as good as Ce6 (Fig. S12†). More
importantly, scars were observed at the tumour surface in the
PDT group, demonstrating that severe damage was caused in
tumours through the PDT treatment (Fig. S13†).
The body weight of mice changed scarcely both in PDT and
control groups indicating that no obvious injury was induced in
mice during the treatment. To further prove this, the mice were
euthanized aer 14 days and dissected to get the organs of the
four groups, which were made into paraffin slices and stained
with hematoxylin and eosin (H&E). As seen in Fig. 4e and S9,†
the organs in the PDT group didn't show any difference
compared with the PBS treated one. Additionally, no physio-
logical morphology abnormalities or histopathological lesions
were observed, indicating low toxicity of ICy-N to normal
tissues. All these results conrmed that ICy-N was suitable for
precise tumour imaging and served as an efficient photosensi-
tizer to restrain tumour growth under light irradiation.
Conclusions
In summary, an iodine substituted NIR hemicyanine dye ICy-N
was synthesized and successfully applied for tumour hypoxia
imaging and photodynamic therapy in this study. ICy-N is in
a uorescence-off state and has poor singlet oxygen quantum
yield due to the vibration of the nitro group in normal tissues,
Fig. 4 In vivo fluorescence imaging of nitroreductase in Balb/c mice.
(
a1–a6) PBS (100 mL) was injected subcutaneously and (b1–b6) ICy-N
(
100 mL, 50 mM in PBS) was injected intratumorally and observed with whereas it can be reduced by the over-expressed nitroreductase
excitation at 660 nm and emission at 680–720 nm. The red circle in
in the tumour region. The reduced product ICy-OH displayed
a strong uorescence emission at 710 nm which is suitable for
in vivo bioimaging. More importantly, the improved singlet
oxygen production of ICy-OH makes it an efficient photosensi-
tizer to induce cell apoptosis under 660 nm light irradiation.
The in vivo antitumor experiment further conrms that ICy-N
can serve as a promising NIR photosensitizer both for precise
tumour hypoxia imaging and photodynamic therapy aer the
activation of nitroreductase. Overall, this work provides a novel
strategy for establishment of an “activatable” photosensitizer
for precise imaging and therapy.
(
(
a2–a6) represents the injected region. (c) Relative tumor volume and
d) body weight of mice after different treatments. (e) H&E staining of
organs from the PBS group and PDT group after 14 days of treatment.
Scale bar: 50 mm.
the tumour region (b1–b6) due to the overexpression of NTR
under hypoxic conditions of tumours. As time went by, the
intensity gradually increased and remained unchanged aer
180 min, suggesting that the reduction of ICy-N by NTR was

nished aer 3 h, which was selected as the preincubation time
before exposure to 660 nm light during the PDT process. As
a comparison, Balb/c mice without tumour were injected with
PBS subcutaneously and imaged at the same time period. No
Experimental section

uorescence signal was captured in spite of the extension of Materials and instruments
incubation time indicating that ICy-N was in a uorescence-off
state in normal tissues and can only be turned on by NTR
specically and the NIR uorescence emission was suitable for
bioimaging in vivo. Further, the PDT effect was assessed
through the tumour volume growth and body weight of mice
which were collected every two days (Fig. 4c and d). Distinctly,
the tumour volume increased quickly in the PBS treated group
whether the mice were irradiated with 660 nm light or not.
Besides, the group treated with ICy-N under dark conditions
showed a similar tumour growth rate to the control group
indicating the low cytotoxicity of ICy-N under dark conditions.
In contrast, the PDT group that was irradiated with a 660 nm
All reagents and solvents used were of reagent grade and were
obtained from commercial suppliers. Flash column chroma-
tography was performed with silica gel (200–300 mesh) and
dichloromethane/methanol or petroleum ether/ethyl acetate
was used as eluent. H NMR and C NMR spectra were ob-
tained using a BrukerAvance II 400 MHz spectrometer. Chem-
1
13
3 6
ical shi (d) was reported as ppm in CDCl or DMSO-d with
TMS as the internal standard. The mass spectrum was recorded
on LTQ Orbitrap XL MS instruments. ICy-OH and ICy-N were
dissolved in DMSO at a concentration of 1 mM respectively as
ꢁ
the stock solution and stored at ꢀ20 C for further use. UV-vis
spectra and uorescent spectra were achieved from an Agilent
Technologies CARY 60 UV-vis spectrophotometer (serial no.
MY1523004) and Varian CARY Eclipse uorescence
ꢀ2
lamp (100 mW cm ) for 20 min aer pretreatment with ICy-N
for 3 h demonstrated evident suppression of tumour growth.
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ꢁ
spectrophotometer (serial no. MY15210003) respectively. The the 4T1 cells were incubated with 2.5 mM ICy-N at 37 C for 60
excitation and emission slit widths were adjusted to obtain the min and washed with PBS 3 times before scanning under the

uorescence intensity in the appropriate range. Cell imaging confocal microscope. Finally, photosensitizers were excited with
was performed on Olympus FV1000 and FV3000 confocal a 635 nm laser and the signal of the uorescence channel at
microscopes (Olympus, Japan). Mitotracker Green, Lysotracker 700–750 nm was gathered with a 60ꢂ objective lens (N ¼ 3).
Green and Hoechst 33342 were purchased from Life Technol-
ogies Co. (USA). Reactive oxygen species detection kit DCFH-DA
Apoptosis assessment with annexin V-FITC & PI under
a confocal microscope
(2,7-dichlorodihydrouorescein diacetate) and annexin V-FITC
and PI apoptosis kits were purchased from Nanjing KeyGen
Biotech. Co. Ltd. (China).
4T1 cells were pre-incubated under hypoxic conditions as
mentioned before and incubated with 2.5 mM ICy-N for 60 min.
Then, the cells were washed with DMEM (containing 10% FBS)
Determination of singlet oxygen quantum yield (FD)
3
times and irradiated with 660 nm light for 10 min and incu-
ꢁ
The singlet oxygen quantum yield was determined by the re- bated for another 30 min at 37 C to induce apoptosis. Aer
43
ported method. Briey, Methylene Blue (MB) was used as the that, DMEM culture medium was replaced with 1ꢂ binding
reference and 1,3-diphenylisobenzofuran (DPBF) was used as buffer and 5 mL annexin V and 5 mL PI were added and mixed
ꢁ
the singlet oxygen trapper. Firstly, the concentration of DPBF in slightly. Aer 30 min incubation at 37 C, the cells were imaged
mL DCM was modied to adjust the absorbance at 415 nm under an Olympus FV-1000. The green channel (annexin V) was
3
close to 1.0. Then, appropriate concentrations of MB and excited at 488 nm and the uorescence signal at 500–550 nm
samples were added respectively to make sure that the absor- was collected. The red channel (PI) was excited at 561 nm and
bance of the mixture at 660 nm was between 0.2 and 0.4. Aer the uorescence signal at 590–640 nm was collected (N ¼ 3).
that, the mixture solutions were exposed to 660 nm light irra-
diation and the absorption spectrum was recorded with a UV-vis
spectrophotometer. At last, the singlet oxygen quantum yield
was calculated with the following equation:
Detection of cellular ROS generation within DCFH-DA
MCF-7 cells were seeded at confocal culture dishes at a density
4
of 5 ꢂ 10 and pre-incubated under hypoxic conditions. Then,
F
D
¼ FMB ꢂ (k(ps) ꢂ F(MB))/(k(MB) ꢂ F(PS)
)
various concentrations of ICy-N and 10 mM DCFH-DA were
added and incubated for 60 min. Then, the cells were washed
FD represents the singlet oxygen quantum yield of the tested with PBS 3 times and irradiated with a 635 nm laser for 2 min
photosensitizer; FMB represents the singlet oxygen quantum and scanned under a confocal microscope at 488 nm excitation,
yield of MB and the value is 0.57 in DCM; PS represents the whereas, the uorescence channel at 500–550 nm was collected
tested photosensitizer ICy-N or ICy-OH; k represents the slope of (N ¼ 3).
the decrease of the absorbance at 415 nm of DPBF with the
addition of irradiation time; F is the correction factor which is
calculated by the following equation:
Cytotoxicity assay
The cytotoxicity in the presence or absence of light was evalu-
F ¼ 1 ꢀ 10ꢀ
OD
ated by reducing MTT (3-(4,5)-dimethylthiazol-2-yl)-3,5-diphe-
nyltetrazolium bromide) to formazan crystals with
mitochondrial dehydrogenases. MCF-7 cells were seeded in
OD represents the absorbance of the mixture at 660 nm.
5
a 96-well plate with a concentration of 1 ꢂ 10 cells per mL in
Cell incubation
100 mL DMEM medium (containing 10% FBS). Aer 24 h
attachment in a cell incubator, the cells were then treated with
various concentrations of ICy-N, ICy-OH and Ce6 in 100 mL
medium for 60 min (N ¼ 6). Then the plate was irradiated with
Breast cancer cells (MCF-7 cells and 4T1 cells) were purchased
from the Institute of Basic Medical Sciences (IBMS) of the
Chinese Academy of Medical Sciences and cultured with Dul-
becco's modied Eagle's medium (DMEM, Invitrogen) con-
taining 10% fetal bovine serum (Invitrogen) and 1% antibiotics
ꢀ
2
6
60 nm light at an average optical density of 12 mW cm for 10
min and then incubated in the cell incubator. As for the dark
group, the cells were kept out of light with the other treatments
unchanged. Cells cultured with free medium were used as
control and six replicates were set for each group to eliminate
contingency. Aer 12 h incubation, the medium was replaced
ꢀ
1
(
penicillin/streptomycin, 100 U mL ) in an atmosphere of CO
2
/
ꢁ
air ¼ 5%/95% at 37 C.
Endogenous nitroreductase imaging in 4T1 cells
ꢀ1
with 100 mL MTT (5 mg mL in DMEM) solution and cultured
ꢁ
For hypoxia incubation, 4T1 cells were seeded in confocal for another 4 h at 37 C in the cell incubator. Finally, the MTT
4
culture dishes at an approximate concentration of 2 ꢂ 10 cells solution was removed carefully and the formed crystals were
per mL and incubated in a cell incubator under normal dissolved in 100 mL dimethyl sulfoxide (DMSO). The plate was
conditions for 12 h and then the dishes were moved to a sealed then carefully shaken for 10 min and the absorbance was
box lled with 2% (or 10%) O and 5% CO for 8 h to induce the determined on a microplate reader (Thermo Fisher Scientic) at
2
2
overexpression of NTR. As for lower O
2
concentration, Anaero- 570 nm and 630 nm. The viability was expressed as a percent of
2
Pouch was placed with cell dishes to remove residual O . Then, the controlled one using the following equation:
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Cell viability (%) ¼ (ODdye ꢀ ODdye630)/(ODcontrol570
acid and heated to reux for 4 h under continuous stirring.
Then, the reaction mixture was poured into 200 mL water and
extracted with ethyl acetate (50 mL ꢂ 3). The organic layer was
washed with saline and dried with anhydrous Na SO . Finally,
5
70
ꢀ
ODcontrol ) ꢂ 100
6
30
2
4
the organic layer was evaporated to dryness and the crude
Visualization of nitroreductase and photodynamic therapy in
the 4T1 tumor bearing mice model
product was puried by silica gel column chromatography to
obtain 2 as a deep red gummy liquid (4.4 g, 72.4%). H NMR
1
(
400 MHz, CDCl
3
) d 7.62 (dd, J ¼ 8.0, 1.7 Hz, 1H), 7.59 (d, J ¼ 1.3
All procedures were carried out in compliance with the Guide
for the Care and Use of Laboratory Animal Resources and the
National Research Council, and were approved by the Institu-
tional Animal Care and Use Committee of the NIH. 4–5 week old
Hz, 1H), 7.29 (d, J ¼ 7.6 Hz, 1H), 2.25 (s, 3H), 1.28 (s, 6H). ESI-
+
+
MS: m/z calcd for C H IN [M + H] : 286.01, found: 286.05.
1
1
13
Balb/c mice were chosen for transplanting 4T1 cells at a density Synthesis of compound 3 (1-ethyl-5-iodo-2,3,3-trimethyl-3H-
6
of 5 ꢂ 10 under the armpit. When the tumour volume reached indol-1-ium iodide)
3
2
(
00 mm , the xenogra tumour mice were divided into 4 groups
4
.4 g 2 (15.4 mmol) was dissolved in 20 mL toluene and heated
Control, Light, ICy-N, and ICy-N + Light, N ¼ 5). The tumour
to reux. Then 7.2 g iodoethane (46.2 mmol) in 5 mL toluene
was added through a syringe in batches under continuous
stirring. A pink precipitate was obtained aer stirring for 6
hours and ltered under reduced pressure. The precipitate was
washed with ethyl acetate 3 times and dried over a vacuum oven
to afford 3 (4.7 g, 69.1%). H NMR (400 MHz, DMSO-d
volume was measured using a vernier caliper and calculated
using the following equation:
2
V ¼ 1/2 ꢂ a ꢂ b
1
6
) d 8.31 (s,
V represents the tumour volume of mice, a represents the
longest diameter of the tumour region, and b represents the
diameter in the vertical direction according to a of the tumour
region.
1
H), 8.00 (d, J ¼ 8.3 Hz, 1H), 7.78 (d, J ¼ 8.2 Hz, 1H), 4.46 (q, J ¼
6
.9 Hz, 2H), 2.80 (s, 3H), 2.50 (s, 6H), 1.41 (t, J ¼ 7.1 Hz, 3H). ESI-
+
+
MS: m/z calcd for C H IN [M + H] : 314.04, found: 314.07.
1
3
17
As for nitroreductase imaging, the mice injected with 50 mM
ICy-N were narcotized and imaged with a NightOWL II LB983
small animal in vivo imaging system with 660 nm excitation and
Synthesis of compound 4 (2-bromocyclohex-1-
enecarbaldehyde)
7
1
00 ꢃ 20 nm emission lters. The light power for mice PDT was 12.4 mL PBr was dissolved in 20 mL CHCl and slowly added to
3
3
ꢀ
2
00 mW cm
.
a mixture of 11.2 mL anhydrous DMF and 30 mL CHCl during
3
ꢁ
a period of 1 hour at 0 C. Then 5 mL cyclohexanone (48.4
Synthesis of ICy-N
mmol) was added dropwise at that temperature. Aer stirring
for 1 hour, the mixture was heated to room temperature and
stirred for another 12 hours. The mixture was poured into a 200
The synthetic route to ICy-N is displayed in Scheme S1.†
3
mL ice-water mixture and neutralized with a NaHCO solid until
the pH is approximately 7. The resulting solution was extracted
Synthesis of compound 1((4-iodophenyl) hydrazine)
10 g 4-iodoaniline (45.7 mmol) and 2 mL concentrated hydro-
with CH Cl (50 mL ꢂ 3) and dried over anhydrous Na SO .
2
2
2
4
chloric acid were dissolved in 40 mL water in a round-bottom
Finally, the organic layer was concentrated in vacuo to give an
orange oil (6.8 g) which was used without further purication.
ꢁ

ask and cooled to 0 C. Then, 3.5 g (50.7 mmol) NaNO
2
in 10
mL water was added to the turbid solution dropwise and stirred
for 1 hour at that temperature. The reaction mixture was then
heated to room temperature and 17.3 g SnCl (91.4 mmol) dis-
2
Synthesis of compound 5
2
00 mg 2-hydroxy-4-methoxybenzaldehyde (1.3 mmol) in 6 mL
solved in 16 mL water and 4 mL concentrated hydrochloric acid
was slowly added to the reaction mixture under continuous
stirring. A light brown precipitate was obtained aer 2 hour
stirring and ltered under reduce pressure. Aer washing with
water 3 times, the precipitate was dissolved in 200 mL saturated
NaOH solution and extracted with ethyl acetate (50 mL ꢂ 3).
anhydrous DMF was added to 295 mg 4 (1.56 mmol) and 847 mg
ꢁ
Cs
hours in a N
ethyl acetate (20 mL ꢂ 3). Aer being dried over anhydrous
Na SO , the organic layer was evaporated and the crude product
was puried by silica gel column chromatography to obtain 5 as
a yellow solid (125 mg, 39.7%). H NMR (400 MHz, CDCl3)
2
CO
3
(2.6 mmol). The mixture was stirred at 25 C for 12
2
atmosphere and extracted with water (60 mL) and
2
4
The organic layer was then washed with Na
dried with anhydrous Na SO . Finally, the organic layer was
evaporated to dryness to give compound 1 as a brown solid (8.4
2 2 3
S O and saline and
1
2
4
d 10.31 (s, 1H), 7.08 (d, J ¼ 9.1 Hz, 1H), 6.69–6.63 (m, 3H), 3.84
1
(
(
s, 3H), 2.57 (t, J ¼ 6.0 Hz, 2H), 2.44 (t, J ¼ 6.0 Hz, 2H), 1.74–1.68
g, 78.5%). H NMR (400 MHz, DMSO-d
6
) d 7.35 (d, J ¼ 8.8 Hz,
m, 3H).
2
H), 6.89 (s, 1H), 6.62 (d, J ¼ 8.8 Hz, 2H), 4.05 (s, 2H). ESI-MS: m/
+
+
z calcd for C H IN [M + H] : 234.97, found: 234.97.
6
8
2
Synthesis of compound 6
Synthesis of compound 2 (5-iodo-2,3,3-trimethyl-3H-indole)
2
42 mg 5 (1 mmol), 441 mg 3 (1 mmol) and 0.2 mL piperidine
ꢁ
5g (4-iodophenyl) hydrazine (21.3 mmol) and 3.7 g 3-methyl-2- were dissolved in 10 mL acetic anhydride and heated to 60 C
butanone (42.6 mmol) were dissolved in 50 mL glacial acetic for 2 hours. The resulting blue mixture was poured into 100 mL
10592 | Chem. Sci., 2019, 10, 10586–10594
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water and extracted with CH
was dried over anhydrous Na SO and evaporated under
2
Cl
2
(20 mL ꢂ 3). The organic layer
Acknowledgements
2
4
reduced pressure. The crude product was further puried by This work was supported by the National Science Foundation of
silica gel column chromatography to obtain 6 as a blue solid China (project 21421005, 21576037 and U1608222).
1
(
1
1
432 mg, 64.9%). H NMR (400 MHz, DMSO-d
6
) d 8.58 (d, J ¼
4.8 Hz, 1H), 8.15 (d, J ¼ 1.3 Hz, 1H), 7.88 (dd, J ¼ 8.3, 1.4 Hz,
Notes and references
H), 7.62–7.56 (m, 2H), 7.48 (d, J ¼ 8.4 Hz, 1H), 7.12 (d, J ¼ 2.2
Hz, 1H), 7.04 (dd, J ¼ 8.7, 2.3 Hz, 1H), 6.50 (d, J ¼ 14.9 Hz, 1H),
.38 (q, J ¼ 7.1 Hz, 2H), 3.93 (s, 3H), 2.74 (t, J ¼ 5.8 Hz 2H), 2.69
t, J ¼ 5.8 Hz, 2H), 1.87–1.79 (m, 2H), 1.75 (s, 6H), 1.34 (t, J ¼ 7.1
1 L. A. Torre, F. Bray, R. L. Siegel, J. Ferlay, J. Lortet-Tieulent
and A. Jemal, Ca-Cancer J. Clin., 2015, 65, 87.
4
(
2 W. Chen, R. Zheng, P. D. Baade, S. Zhang, H. Zeng, F. Bray,
A. Jemal, X. Q. Yu and J. He, Ca-Cancer J. Clin., 2016, 66, 115.
3 J. Du, Z. Gu, L. Yan, Y. Yong, X. Yi, X. Zhang, J. Liu, R. Wu,
C. Ge, C. Chen and Y. Zhao, Adv. Mater., 2017, 29, 1701268.
+
+
Hz, 3H). ESI-MS: m/z calcd for C H INO [M] : 538.12, found:
2
8
29
2
538.28.
4
N. Ma, F.-G. Wu, X. Zhang, Y.-W. Jiang, H.-R. Jia, H.-Y. Wang,
Y.-H. Li, P. Liu, N. Gu and Z. Chen, ACS Appl. Mater.
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Synthesis of compound ICy-OH
g BBr (4.0 mmol) in 10 mL anhydrous CH
dropwise to 20 mL anhydrous CH Cl solution containing 200
mg 6 (0.3 mmol) at 0 C with continuous stirring. Aer 1 hour
stirring at that temperature, the solution was heated to room
temperature and stirred for another 12 hours. Then the solution
1
3
2 2
Cl was added
2
2
5
6
ꢁ
was poured into 100 mL water and extracted with CH
2
Cl
2
(20 mL
and
ꢂ 3). The organic layer was dried over anhydrous Na
2
SO
4
7
8
evaporated to give a blue solid. The crude product was then
puried by silica gel column chromatography to obtain ICy-OH
1
as a blue solid (180 mg, 92.1%). H NMR (400 MHz, DMSO-d )
6
d 8.42 (d, J ¼ 14.4 Hz, 1H), 8.08 (d, J ¼ 1.3 Hz, 1H), 7.81 (dd, J ¼
8
8
.3, 1.4 Hz, 1H), 7.62 (s, 1H), 7.48 (d, J ¼ 9.2 Hz, 1H), 7.34 (d, J ¼
.4 Hz, 1H), 6.84–6.78 (m, 2H), 6.31 (d, J ¼ 14.5 Hz, 1H), 4.27 (q,
J ¼ 7.0 Hz, 2H), 2.71 (t, J ¼ 5.8 Hz, 2H), 2.67 (t, J ¼ 5.8 Hz, 2H),
1
.85–1.78 (m, 2H), 1.71 (s, 6H), 1.30 (t, J ¼ 7.1 Hz, 3H). ESI-MS:
+ +
2
m/z calcd for C27H27INO [M] : 524.11, found: 524.23.
2
443.
Synthesis of ICy-N
50 mg ICy-OH (0.23 mmol), 60 mg 1-(bromomethyl)-4-nitro-
9
Z. Zhou, Y. Yan, L. Wang, Q. Zhang and Y. Cheng,
Biomaterials, 2019, 203, 63.
1
benzene and 95 mg K
DMF. The mixture was heated to 60 C under a N
and monitored by TLC. When the reaction was completed, the
2
CO
3
(0.69 mmol) were dissolved in 10 mL 10 J. Gao, M. Sanchez-Purra, H. Huang, S. Wang, Y. Chen, X. Yu,
ꢁ
2
atmosphere
Q. Luo, K. Hamad-Schifferli and S. Liu, Sci. China: Chem.,
2017, 60, 1219.
mixture was washed with 60 mL water and extracted with 11 M. Li, T. Xiong, J. Du, R. Tian, M. Xiao, L. Guo, S. Long,
CH Cl (20 mL ꢂ 3). The organic layer was dried over dried over
J. Fan, W. Sun, K. Shao, X. Song, J. W. Foley and X. Peng, J.
Am. Chem. Soc., 2019, 141, 2695.
2
2
anhydrous Na SO and evaporated in vacuo. The crude product
2
4
was then puried by silica gel column chromatography to 12 L. Huang, Z. Li, Y. Zhao, Y. Zhang, S. Wu, J. Zhao and G. Han,
1
obtain ICy-N as a blue solid (86 mg, 47.6%). H NMR (400 MHz,
DMSO-d
8
J. Am. Chem. Soc., 2016, 138, 14586.
6
) d 8.55 (d, J ¼ 14.8 Hz, 1H), 8.32 (d, J ¼ 8.5 Hz, 2H), 13 J. P. Celli, B. Q. Spring, I. Rizvi, C. L. Evans, K. S. Samkoe,
.18 (s, 1H), 7.89 (d, J ¼ 8.4 Hz, 1H), 7.80 (d, J ¼ 8.6 Hz, 2H), 7.60
S. Verma, B. W. Pogue and T. Hasan, Chem. Rev., 2010,
110, 2795.
(
d, J ¼ 8.6 Hz, 1H), 7.56 (s, 1H), 7.51 (d, J ¼ 8.5 Hz, 1H), 7.21 (s,
1
2
2
H), 7.13 (d, J ¼ 8.8 Hz, 1H), 6.52 (d, J ¼ 15.0 Hz, 1H), 5.49 (s, 14 W. Chen, Y. Wang, M. Qin, X. Zhang, Z. Zhang, X. Sun and
H), 4.40 (q, J ¼ 7.2 Hz, 2H), 2.75–2.65 (m, 4H), 1.87–1.77 (m,
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1
3
H), 1.74 (s, 6H), 1.33 (t, J ¼ 7.0 Hz, 3H). C NMR (100 MHz, 15 D. E. J. G. J. Dolmans, D. Fukumura and R. K. Jain, Nat. Rev.
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6
) d 176.68, 161.50, 161.30, 154.22, 147.65, 145.52,
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+
+
MS: m/z calcd for C34
H
32IN
2
O
4
[M] : 659.1407, found: 659.1405.
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06, 1098.
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1
8 M. Li, S. Long, Y. Kang, L. Guo, J. Wang, J. Fan, J. Du and
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This journal is © The Royal Society of Chemistry 2019