A near-infrared fluorescent probe that can image endogenous hydrogen polysulfidesin vivoin tumour-bearing mice

Article abstract of DOI:10.1039/d0ob02253e

Hydrogen polysulfides (H₂S_n,n> 1), which are important reactive sulfur species, play crucial roles in H₂S-related bioactivities, including antioxidation, cytoprotection, activation of ion channels, transcription factor functions and tumour suppression. Monitoring H₂S_nin vivois of significant interest for exploring the physiological roles of H₂S_nand the exact mechanisms of H₂S_n-related diseases. Herein, we conceive a novel near-infrared fluorescent probe, NIR-CPS, that is used to detect H₂S_nin living cells andin vivo. With the advantages of high sensitivity, good selectivity and a remarkably large Stokes shift (100 nm), NIR-CPS was successfully applied in visualizing H₂S_nin living cells and mice. More importantly, NIR-CPS monitored H₂S_nstimulated by lipopolysaccharide in tumour-bearing mice. These results demonstrate that the NIR-CPS probe is a potentially powerful tool for the detection of H₂S_nin vivo, thus providing a valuable approach in H₂S_n-related medical research.

Full text of DOI:10.1039/d0ob02253e

Organic &
Biomolecular Chemistry
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PAPER
A near-infrared fluorescent probe that can image
endogenous hydrogen polysulfides in vivo in
tumour-bearing mice†
Cite this: Org. Biomol. Chem., 2021,
9, 911
1
a,b
a
a
a
a
Ling Zhang,
Yunsheng Xue*
*
Huizhen Liu, Chunli Wu, Youguang Zheng, Xiaoning Kai and
a
Hydrogen polysulfides (H
related bioactivities, including antioxidation, cytoprotection, activation of ion channels, transcription
factor functions and tumour suppression. Monitoring H in vivo is of significant interest for exploring
the physiological roles of H and the exact mechanisms of H -related diseases. Herein, we conceive
a novel near-infrared fluorescent probe, NIR-CPS, that is used to detect H in living cells and in vivo.
2 n 2
S , n > 1), which are important reactive sulfur species, play crucial roles in H S-
2 n
S
2
S
n
2 n
S
2 n
S
With the advantages of high sensitivity, good selectivity and a remarkably large Stokes shift (100 nm),
NIR-CPS was successfully applied in visualizing H2Sn in living cells and mice. More importantly, NIR-CPS
Received 12th November 2020,
Accepted 23rd December 2020
monitored H
2
S
n
stimulated by lipopolysaccharide in tumour-bearing mice. These results demonstrate that
in vivo, thus providing a valu-
DOI: 10.1039/d0ob02253e
the NIR-CPS probe is a potentially powerful tool for the detection of H
2 n
S
rsc.li/obc
able approach in H
2
S
n
-related medical research.
1
0–15
bioactivities,
although this remains controversial.
Introduction
S-Sulfhydration of cysteine residues (SH → SSH) was originally
Reactive sulfur species (RSS), which are a family of sulfur-con- attributed to H S. Recent results revealed that H S is more
2
2 n
9
,11–14,16–18
taining molecules, play essential roles in cell transduction, effective than H
2
S in protein S-sulfhydration.
has been implicated in a number of physiological and patho-
Representative RSSs include thiols, hydrogen sulfide (H S), logical processes. H S was found to activate tumour suppres-
2 n
H S
1
–3
redox
homeostasis,
and
metabolic
regulation.
2
2 n
persulfides (RSSH), polysulfides (RSS
n
SR), S-nitrosothiols sor protein, lipid phosphatase and the tensin homologue
1
4
(
SNO) and sulfenic acids (SOH), as well as inorganic sulfur (PTEN) by S-sulfhydration.
Additionally, H
2
S
n
exhibited
4
2+
derivatives. Among them, H S is the most attractive, as it has higher potency than H S in inducing Ca influx in astrocytes
2
2
been demonstrated to be the third gaseous signalling mole- by activating transient receptor potential ankyrin 1 (TRAP1)
5
18–20
cule. H
2
S has diverse biological functions, including vasodila- channels.
2 n
H S can effectively relax vascular smooth
2
1
tion, neurotransmission, insulin secretion, inflammation regu- muscle by activating protein kinase G1a (PKG1α). H S also
2
n
6
–8
lation, apoptosis and ischaemia/reperfusion-induced injury.
(hydrogen polysulfides) and H S always coexist in bio- milieu. This is due to H
logical systems. H S can be derived from H S in the presence signalling, subsequently causing the elevation of intracellular
exhibited crucial antioxidant activities in the cellular redox
H
2
S
n
2
2 n
S
mediating the activation of Nrf2
2
n
2
9
of reactive oxygen species (ROS), and H
into a precursor of H S. Recent research suggests that H
2 n
S can also degrade GSH levels and the expression of HO-1 (a Nrf2-regulatory
2
2
2
2
S
n
2 n
gene). These results demonstrate that H S possesses diverse
may be the actual signal transduction molecule in H S-related biological functions, including antioxidation, cytoprotection,
2
activation of ion channels, transcription factor functions and
2 n
tumour suppression. In addition, H S appears to be involved
in several diseases, including cancers, ethylmalonic encepha-
a
Jiangsu Key Laboratory of New Drug Research and Clinical Pharmacy, Jiangsu
Center for the Collaboration and Innovation of Cancer Biotherapy, School of
Pharmacy, Xuzhou Medical University, Xuzhou, 221002, P. R. China.
E-mail: zhamgling1999@163.com, xzmcysxue@sina.com
lopathy, Parkinson’s disease (PD), and Huntington’s disease
2
3
(HD). However, compared with other biothiols (RSH), the
fundamental chemical biology of H2Sn has yet to be further
elucidated.
b
NHC Key Laboratory of Nuclear Medicine, Jiangsu Key Laboratory of Molecular
Nuclear Medicine, Jiangsu Institute of Nuclear Medicine, Wuxi, 214063, P. R. China
†
Electronic supplementary information (ESI) available: Additional materials,
To better explore the physiological and pathologic roles of
methods and figures, including fluorescence spectra, cell imaging, live mouse
imaging, NMR and HRMS spectra, and a detailed protocol for synthesis. See
DOI: 10.1039/d0ob02253e
H S , a sensitive and effective detection method for H S is
2
n
2 n
urgently needed. The traditional methods for detecting poly-
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Organic & Biomolecular Chemistry
sulfides, such as UV-Vis spectroscopy and mono-bromobimane nitrofluorobenzoate group. The fluorobenzoate group would
(
MBB)-based detection, are limited by poor sensitivity and trap H2Sn by replacing the F atom, forming the persulfide
1
4,18,24
destructiveness.
Fluorescence assays are powerful tools intermediate with the –SSH group; then, –SH, as a nucleophile,
2 n
S , owing to their high sensitivity, excellent continues to react with the carbonyl group, which further pro-
for monitoring H
spatiotemporal resolution ability, and nondestructiveness.
The first H fluorescent probe was developed by the Xian fluorescence emission (Scheme S1†). The NIR-CPS probe was
group, and several H probes have been subsequently prepared by the reaction of compound 1 with intermediate 2
reported. To date, various types of probes have been reported in the presence of piperidine (Scheme S2†). The structure of
for detecting H , including ratiometric probes, two-photon the NIR-CPS probe was characterized by NMR spectroscopy
2
5,26
motes cyclization to release the fluorophore NIR-OH with NIR
2 n
S
2
7
2 n
S
2 n
S
probes, near-infrared probes, dual response probes, mitochon- and mass spectrometry (Fig. S14–S22†).
dria-targetable probes, lysosome-targetable probes, and endo-
4
,28–46
Response mechanism of NIR-CPS
-selective fluo- To assess the sensing performance of NIR-CPS, the product of
rescent probes by taking advantage of the strong nucleophili- the NIR-CPS reaction with H2Sn was purified and analysed.
city and reduction ability of H , including H -mediated The fluorescence and UV-vis analyses of the mixture solution
aromatic substitution cyclization, the H -mediated ring- show the same emission spectra and absorption spectra as the
opening reaction of aziridine and H S -mediated reduction of compound NIR-OH (Fig. S1†). The results of NMR spec-
plasmic reticulum-targetable probes.
strategies have been used in the design of H
Several sensing
S
2 n
2
S
n
2 n
S
2 n
S
2
n
4
the nitro group. Nevertheless, the selective detection of H
2
S
n
troscopy and mass spectrometry confirmed the mechanism of
is still challenging due to the interference of other biothiols, this reaction as illustrated in Scheme 1.
such as H S, GSH, Cys, Hcy, and thiol-containing proteins,
2
Fluorescence response of NIR-CPS to H
that have similar chemical properties to H
2 n
physiological levels of endogenous H S are low in cells/ The absorption and fluorescence sensing of NIR-CPS towards
2 n
S in vitro
2 n
S . Moreover, as the
plasma/tissues, the sensitivity of the current H2Sn probes H2Sn were both tested under simulated physiological con-
could still be improved. Additionally, probes for the detection ditions. With the addition of Na S (0–300 μM) to a solution
2
4
of endogenous H
2 n
S in vivo, especially in pathological con- containing NIR-CPS, the maximum absorption changed from
ditions, such as cancers, are still sparse. Therefore, it is impor- 552 nm to 570 nm (Fig. S1†). In the absence of Na S , a negli-
2
4
tant to develop an NIR fluorescent probe for imaging H
2
S
n
gible fluorescence signal was observed (Fig. 1). After treatment
with desirable characteristics, especially high sensitivity and of NIR-CPS with different concentrations of Na S (0–300 μM),
2
4
the ability to monitor endogenous H2Sn in vivo in tumour- a significant fluorescence emission peak at 670 nm gradually
bearing mice.
increased (20-fold) and reached a plateau at 100 μM Na2S4
Compared with H
2
S
n
probes with emission in the visible (Fig. 1). Moreover, there was a linear relationship between the
region, near-infrared (NIR) probes are more desirable for fluorescence intensity and the concentrations of Na2S4 from
detection in vivo due to deep tissue penetration, minimal 0–20 μM (Fig. 1). The detection limit was determined to be as
optical damage to biological samples, and minimal inter- low as 18 nM in PBS buffer, which was lower than most pre-
4
7,48
ference from background autofluorescence.
Herein, we viously reported H S probes (Table S1†). It should be noted
2 n
conceived a novel NIR fluorescent probe, NIR-CPS, for H
2
S
n
that the NIR-CPS probe has a remarkably large Stokes shift
detection in vivo. The NIR-CPS probe could provide good (100 nm) (Fig. S1†), which can eliminate the measurement
selectivity, high sensitivity (a detection limit of 18 nM) and a interference and self-quenching induced by the excitation and
remarkably large Stokes shift (100 nm). NIR-CPS was utilized scattered light. These results indicate the feasibility of
to image endogenous
Significantly, the application of NIR-CPS in tumour-bearing under complex biological conditions.
mice suggested that the probe could image intratumoural
The absorption and emission spectra of both NIR-CPS and
in vivo, highlighting its great potential for exploring the NIR-CHO in different solvents (PBS/DMSO (v/v 99 : 1), DMSO
H
2
S
n
in living cells and mice. NIR-CPS for the quantitative and qualitative detection of H2Sn
2 n
H S
anticancer mechanism of polysulfide.
and acetonitrile) are also obtained. As anticipated, NIR-COH
showed very faint fluorescence emission, and NIR-COH
Results and discussion
Design and synthesis of NIR-CPS
In the design of an NIR probe for H S , the Changsha ana-
2
n
logue dye icyanoisophorone was selected as a fluorophore due
4
9–51
to its NIR emission and large Stokes shift.
p-Nitrofluorobenzoate served as the H S recognition group as
2
n
2
7
well as a fluorescence quencher.
both an effective nucleophile and electrophile. We anticipated
that the fluorescence of a fluorophore can be quenched by the Scheme 1 Proposed response mechanism of NIR-CPS for H
2 n
H S has been proven to be
4
2 n
S .
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rescence was almost quenched at pH 9.0 (Fig. S5†). The
observed fluorescence profile of NIR-CPS towards Na S under
2
4
different pH conditions is mainly due to the pH dependence
5
1
of the emission profiles of NIR-COH. There is an equilibrium
50
between the phenolic and phenolate form in NIR-COH.
As shown in Fig. S6,† the free probe showed negligible fluo-
rescent signal. Within 60 min, the very faint emission intensity
of the free probe did not change, showing good stability of
NIR-CPS.
2 n
Selectivity of NIR-CPS for H S in vitro
To evaluate the selectivity of NIR-CPS towards H
2 n
S , fluo-
rescence responses were measured in the presence of various
biological substances. As shown in Fig. 2, only Na2S2 and
Na
2
S
4
exhibited
a
dramatic fluorescence enhancement.
S, Cys, GSH, CysSSCys, Hcy,
However, other RSS, such as Na
2
2
−
2−
2−
GSSG, S , S2O3
,
SO3
, did not elicit any fluorescence enhancement. The
NIR-CPS probe was not responsive to reactive oxygen species
,
SO4
,
Cys-polysulfide, and
8
3 3
CH SSSCH
−
•
1
2−
(
H O , OCl , OH, tBuOOH, O , O ) or reactive nitrogen
2 2 2
−
−
−
2 3
species (NO , ONOO , NO, NO ) (Fig. S7†). Various ions,
−
+
2+
2+
2+
3+
2+
2−
−
such as Na , K , Cu , Mg , Zn , Fe , Fe , CO
3
, HCO
3
,
−
−
−
2−
−
Cl , Br , I , HPO , and H PO , could not trigger inter-
4
2
4
ference (Fig. S8†). In addition, when Na
2
S
2
(20 μM) and
various other species coexisted, there was no obvious inter-
ference with fluorescence responses (Fig. 2, S7 and S8†). These
results clearly indicate that NIR-CPS showed high selectivity to
2 n
recognize H S over other biological species.
Fluorescence imaging of H S in living cells
2
n
2 n
Fig. 1 Fluorescence response of NIR-CPS to H S . (A) Fluorescence Next, we exploited the capability of NIR-CPS to detect H
2 n
S in
spectra of NIR-CPS (10 μM) with Na
0, 35, 40, 60, 80, 100, 200 and 300 μM) in PBS buffer (20 mM, pH =
.4, 1% DMSO, containing 1 mM CTAB) at 37 °C for 30 min. (B) The fluor-
escence intensity changes of NIR-CPS (10 μM) at 670 nm after incu-
bation with different concentrations of Na (0–300 μM). Inset: The
2 4
S (0, 0.5, 1, 2, 4, 6, 8, 10, 15, 20, 25,
living cells. Prior to cell imaging, standard MTT assays were
performed to investigate the cytotoxicity of the probe. MCF-7
cells were selected as the test model for cell fluorescence
imaging. When incubated with different concentrations of
3
7
2 4
S
linear relationship between the fluorescence intensity and the concen- NIR-CPS (5 μM, 10 μM, 15 μM, 20 μM), the cell viability was
trations of Na
mean ± SD (n = 3).
2 4
S (0–20 μM) in PBS buffer. Data are presented as the
higher than 90% (Fig. S9†). As shown in Fig. S10,† the cell via-
bility showed almost no significant change over different incu-
bation times (6 h, 12 h, 18 h, 24 h, 48 h). These results indicate
that NIR-CPS has low cytotoxicity to MCF-7 cells at a concen-
showed maximum emission peaks around 670 nm–675 nm in tration of 10 μM.
different solvents (Fig. S2†). The fluorescence quantum yield
The untreated MCF-7 cells showed almost no fluorescence
of NIR-CHO is approximately 12.5% (in DMSO) and 12.8% (in (Fig. 3). Cells treated with the free NIR-CPS probe (10 μM)
acetonitrile); ICG in DMSO (Φ = 0.13) was used as a fluo- exhibited relatively weak fluorescence emission (Fig. 3). To
f
rescence standard. As shown in Fig. S3,† NIR-CPS has almost confirm the source of the weak fluorescence, the cells were pre-
4
0
the same absorption spectra in different solvents. Additionally, treated with NMM (an efficient eliminator of H S , 1 mM) for
2
n
NIR-COH also has the same absorption spectra in different 1 h to remove the physiological levels of H S . Compared with
2
n
solvents.
The reaction time of the probe with Na
the untreated cells, a significant decrease in the fluorescence
was further intensity was observed in the NMM-treated cells, indicating
2
S
4
explored. As illustrated in Fig. S4,† the fluorescence response that the NIR-CPS fluorescence change in the cells arises from
to Na reached saturation within approximately 20 min, and the physiological levels of H2Sn (Fig. 3). Then, treatment of
the fluorescence intensity did not change over 60 min, probe-pretreated cells with different concentrations of Na2S4
showing good stability of NIR-CPS.
(10 and 20 μM) led to a remarkable increase in the fluo-
The pH effects on the response of NIR-CPS and its response rescence emission (Fig. 3). Notably, the fluorescence intensi-
towards Na were also investigated. From pH 4.0 to 8.0, the ties in these cells were further quantified, and the data indi-
fluorescence intensities gradually decreased, and the fluo- cate that the fluorescence induced by exogenous H S was con-
2 4
S
2 4
S
2
n
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2 n
Fig. 3 Confocal fluorescence imaging of exogenous H S in living
MCF-7 cells using NIR-CPS. Cells were untreated (A) or incubated (B)
with NIR-CPS (10 μM) for 20 min at 37 °C. Cells were pretreated with
N-ethylmaleimide (NMM, 1 mM) for 1 h, and then incubated with
NIR-CPS (10 μM) for 20 min (C). Cells were incubated with NIR-CPS
(
2
10 μM) for 20 min and then further incubated with Na
0 min (D). Cells were incubated with NIR-CPS (10 μM) for 20 min and
then further incubated with Na (10 μM) for 20 min (E). The average
2 4
S (20 μM) for
2 4
S
fluorescence intensity of the above images (F). Scale bars = 10 μm. Data
#
are presented as the mean ± SD (n = 3). p < 0.001 vs. (A) column.
2 n
induce the production of endogenous H S in cells. After the
addition of NIR-CPS to the culture of the LPS-loaded cells, the
fluorescence signal dramatically increased (2.8-fold, Fig. 4).
However, the cells were pretreated with PAG (CSE inhibitor, DL-
propargylglycine) and subsequently incubated with LPS and
the NIR-CPS probe. A remarkable decrease in fluorescence
intensity was observed, suggesting that the fluorescence signal
Fig. 2 The selectivity of NIR-CPS for H
2
S
n
. (A) Fluorescence spectra of
NIR-CPS (10 μM) towards Na
reactive sulfur species (20 μM Na
CysSSCys; 100 μM Hcy; 10 mM GSH; 1 mM GSSG; 500 μM S
Na ; 500 μM Na SO ; 500 μM NaHSO ; 500 μM Na SO ; 1 mM Cys-
polysulfide; 100 μM CH SSSCH ; 1 mM S-nitroso glutathione) in PBS
buffer (20 mM, pH = 7.4, 1% DMSO, containing 1 mM CTAB) at 37 °C for
0 min. (B) Fluorescence responses of NIR-CPS (10 μM) towards Na
100 μM), Na (100 μM) and various reactive sulfur species at 37 °C for
0 min. In each group, the red bars represent the relative responses at
70 nm of NIR-CPS to RSS and the mixture of RSS with 100 μM Na . 1.
Na (100 μM); 2. Na (100 μM); 3. Na S (20 μM); 4. Cys (1 mM); 5.
GSH (1 mM); 6. CysSSCys (1 mM); 7. Hcy (100 μM); 8. GSH (10 mM); 9.
GSSG (1 mM); 10. S (500 μM); 11. Na (500 μM); 12. Na SO
500 μM); 13. Na SO (500 μM); 14. Cys-poly sulfide (1 mM); 15.
CH SSSCH (100 μM); 16. NaHSO (500 μM); 17. S-nitrosoglutathione
1 mM). Data are presented as the mean ± SD (n = 3).
2 4 2 2
S (100 μM), Na S (100 μM) and various
S; 1 mM Cys; 1 mM GSH; 1 mM in Fig. 4 was caused by H S through LPS stimulation. These
2 n
2
8
; 500 μM
results revealed the capability of NIR-CPS to image endogen-
ously produced H in living cells.
2
S
2
O
3
2
3
3
2
4
2 n
S
3
3
Fluorescence imaging of H S in vivo
3
2
S
4
2 n
(
2 2
S
The excellent features of NIR-CPS, including NIR emission,
high sensitivity and good selectivity, make the probe more
favourable for fluorescence imaging of H S in vivo. Inspired
2 n
3
6
2 4
S
2
S
4
2
S
2
2
8
2
S
2
O
3
2
3
(
2
4
3
3
3
(
centration dependent (4-fold, 5.6-fold). These results imply
that NIR-CPS is capable of monitoring varying concentrations
of exogenous and physiological H
Having demonstrated the capability of NIR-CPS to monitor
exogenous H in cells, we further validated the capability of MCF-7 cells using NIR-CPS. The cells were induced by LPS (1 μg mL
NIR-CPS to visualize endogenously produced H
cells. Cystathionine γ-lyase (CSE) is regarded as the major
2 n
S inside living cells.
2 n
Fig. 4 Confocal fluorescence imaging of endogenous H S in living
−1
2
S
n
)
for 16 h, and then incubated with NIR-CPS (10 μM) for 20 min (A). Cells
were preincubated with DL-propargylglycine (PAG, 200 μM) for 30 min
and further treated with LPS (1 μg mL− ) for 16 h at 37 °C. Then the cells
were incubated with NIR-CPS (10 μM) for 20 min (B). The average fluor-
2 n
S in MCF-7
1
5
2
enzyme for H
2
S
n
generation. Lipopolysaccharide (LPS, 1 μg
−
1
mL ) can upregulate CSE expression, resulting in an increase
in endogenous H2Sn levels in cells. Therefore, LPS can presented as the mean ± SD (n = 3). p < 0.001 vs. (B) column.
escence intensity of the above images (C). Scale bars = 10 μm. Data are
5
3
#
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by the advantages of NIR-CPS, we further investigated the showed increased fluorescence emission (5.5-fold) (Fig. 5).
capability of the probe to visualize H S in living mice. Mice With the i.p. injection of LPS into PAG-pretreated mice, the
2
n
were divided into several groups. One group was i.p. injected fluorescence of the mice was remarkably weakened (Fig. 5),
with the free probe as the control group. The other three indicating that a strong fluorescence was triggered by endogen-
groups were i.p. injected with the NIR-CPS probe, followed by ous H S induced by LPS.
2
n
i.p. injection with various amounts of Na
2
S
4
(0.2, 2.0 and 4.0
Next, the fluorescence intensities of the whole mice were
equiv.). The other group was injected with LPS and then detected in real time within 40 min after i.p. injection of the
injected with the free probe after 24 h. The last group was pre- NIR-CPS probe. As illustrated in Fig. S13,† obvious fluo-
treated with DL-propargylglycine for 30 min and then i.p. rescence was observed within 1 min, and the maximum fluo-
injected with LPS for 24 h followed by a subsequent i.p. injec- rescence intensity of the mice was obtained at approximately
tion of NIR-CPS. Fluorescence signals from the whole-body 25 min, and the intensity was maintained for 40 min. The data
area of the mice were imaged and quantified using a Night show that NIR-CPS had a fast response to H S , and the fluo-
2 n
OWL IILB 983 small animal in vivo imaging system. The mice rescence intensities in vivo were stable. All these data establish
in the control group exhibited almost no fluorescence (Fig. 5). that NIR-CPS is sensitive enough to monitor various levels of
However, compared with the control group, the mice treated
with both Na (0.2, 2.0 and 4.0 equiv.) and the probe dis- animals.
played a marked elevation in the fluorescence intensities (3.2-,
.5-, and 10.2-fold), indicating that our probe shows the desir-
able penetration for imaging exogenous H in vivo (Fig. 5). The imbalance between ROS generation and cellular anti-
Moreover, the mice injected with LPS and the probe also oxidant capacity is an important pathogenic factor in several
2 n 2 n
H S and is suitable for long-term monitoring of H S in living
2 4
S
Bioluminescence imaging of H S in tumour-bearing mice
2
n
6
2 n
S
5
3
diseases, such as tumours. Reactive sulfur species (RSS) have
2
,54
a crucial function in regulating the intracellular redox state.
Specifically, H2Sn displays strong reducing and nucleophilic
abilities and is associated with scavenging oxidants and intra-
1
6,55–58
cellular electrophiles.
The levels of H
2
S
n
4
in vivo might
1
be involved in the pathogenesis of tumours. In addition,
2 2 2 4
several inorganic polysulfide donors (Na S and Na S ) and
garlic-derived organic polysulfide also exhibited anticancer
5
9–61
activities.
anticancer effects of H
can monitor level changes of H
further elucidating the physiological and pathological role of
in tumours.
We next sought to investigate the feasibility of the NIR-CPS
probe for imaging H S in tumour-bearing mice. MCF-7 cells
Nevertheless, the underlying mechanisms of the
are not clear. Therefore, probes that
in tumours are critical for
2 n
S
2 n
S
2 n
H S
2
n
were grafted into nude mice to produce murine xenograft
tumour models. The nude mice were divided into several
groups. The mice were intravenously injected with NIR-CPS
alone as the control group. One group was intratumourally
injected with LPS for 24 h and then intravenously injected
Fig. 5 Representative fluorescence images visualizing exogenous and with the NIR-CPS probe. The other mice were intratumourally
2 n
endogenous H S in living mice using NIR-CPS. The mice were i.p.
injected with DL-propargylglycine for 30 min and then intratu-
mourally injected with LPS for 24 h, followed by a subsequent
intravenous injection of NIR-CPS. The fluorescence signals
from the tumours in the nude mice were imaged and quanti-
injected with the NIR-CPS probe (2 mM, 100 μL DMSO) as the control
group (A). The mice were i.p. injected with the NIR-CPS probe (2 mM,
1
00 μL DMSO) for 15 min, followed by i.p. injection of 0.2 equiv. of
2 4
Na S (0.4 mM, 100 μL saline) (B). The mice were i.p. injected with the
NIR-CPS probe (2 mM, 100 μL DMSO) for 15 min, followed by i.p. injec- fied. Fig. 6 shows almost no fluorescence emission in the
tion of 2 equiv. of Na
injected with the NIR-CPS probe (2 mM, 100 μL DMSO) for 15 min, fol-
lowed by i.p. injection of 4 equiv. of Na (8 mM, 100 μL saline) (D). The
mice were i.p. injected with LPS (10 μg mL , 100 μL in 1 : 9 DMSO/saline
v/v) for 24 h, followed by i.p. injection of NIR-CPS (2 mM, 100 μL DMSO)
2 4
S (4 mM, 100 μL saline) (C). The mice were i.p.
probe-loaded group. After the intratumoural injection of LPS
into the nude mice, the fluorescence intensities in the
tumours remarkably increased (27.5-fold, Fig. 6). However, the
fluorescence in the nude mice pretreated with DL-propargylgly-
2 4
S
−1
(
(
E). The mice were pretreated (i.p. injection) with DL-propargylglycine cine decreased dramatically, indicating that the fluorescence
2 mM, 100 μL in saline) for 30 min, then i.p. injected with LPS (10 μg
2 n
in the tumour was triggered by LPS-induced endogenous H S
−1
mL , 100 μL in 1 : 9 DMSO/saline v/v) for 24 h, and a subsequent i.p.
injection of NIR-CPS (2 mM, 100 μL DMSO) (F). Quantification of the
relative fluorescence intensities from the abdominal area of the mice in
the above groups (G). Images were taken after incubation for 25 min.
(
Fig. 6). Subsequently, the fluorescence intensities were quanti-
fied in real time within 50 min after intravenous injection of
the NIR-CPS probe. A significant fluorescence signal could be
detected within the first 5 min, indicating the fast response of
#
Data are presented as the mean ± SD (n = 3). P < 0.001 vs. Group A.
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Organic & Biomolecular Chemistry
NIR-CPS towards H
2
S
n
in the tumour. Moreover, the fluo-
rescence intensities in the tumour region reached their peak
value at 15 min (28-fold) and gradually decreased after 40 min
(
2 n
Fig. 7). These results suggest that our probe could detect H S
level changes in tumours.
Conclusions
To conclude, we developed a novel fluorescence probe that
showed a remarkably large Stokes shift, high sensitivity, and
ideal selectivity towards H S . This probe was successfully
2
n
applied for the selective imaging of endogenous H S induced
2
n
by LPS in living cells and mice. Notably, fluorescence imaging
of H S in nude mice bearing MCF-7 tumours indicated that
2
n
the fluctuations of H S in cancerous tissues could be selec-
2
n
tively monitored by our probe. We anticipate that this new
probe will show great potential as an effective tool in the
exploration of H S -related roles in tumours.
2
n
Experimental
Synthesis of NIR-CPS
4
9–51
Fig. 6 Representative fluorescence images of NIR-CPS in tumour- Compound 1 (0.2 g, 0.53 mmol),
compound 2 (0.23 g,
bearing mice. (A) Mice were intravenously injected with NIR-CPS (2 mM, 0.80 mmol) and piperidine (5.2 μL, 0.05 mmol) were dissolved
1
00 μL saline, 3% DMSO, 1% Tween 80) as the control group. (B) Mice
1
in dry ethanol (15 mL). The mixture was refluxed for 6 h. The
solvent was removed under reduced pressure, and then, the
crude product was dissolved in 100 mL of dichloromethane and
−
were intratumourally injected with LPS (10 μg mL , 100 μL in saline) for
2
3
4 h, followed by intravenous injection of NIR-CPS (2 mM, 100 μL saline,
% DMSO, 1% Tween 80). (C) The mice were pretreated (intratumoural
injection) with DL-propargylglycine (2 mM, 100 μL in saline) for 30 min washed with 30 mL of water. The organic layers were dehydrated
and then intratumourally injected with LPS (10 μg mL− , 100 μL in 1 : 9 with Na
1
2
4
SO , and then the solvent was evaporated. The crude
DMSO/saline v/v) for 24 h, followed by intravenous injection of NIR-CPS
2 mM, 100 μL DMSO). (D) Quantification of the fluorescence intensity
from the tumour area of mice from all groups. Data are presented as the
product was purified using a silica gel column to produce com-
pound NIR-CPS as a purple solid (50 mg, 14.5%). TLC (silica,
(
1
CH Cl
2 2
: CH
3
OH, 10 : 1 v/v): R
f
= 0.40; H NMR (400 MHz,
mean ± SD (n = 3).
CDCl ): δ 9.03 (dd, J = 5.6, 2.8 Hz, 1H), 8.48–8.52 (m, 1H), 7.96
3
(
d, J = 7.6 Hz, 1H), 7.65 (t, J = 7.2 Hz, 1H), 7.56 (t, J = 7.6 Hz,
1
6
H), 7.40–7.48 (m, 4H), 7.23–7.25 (m, 1H), 7.28 (s, 1H),
.35–6.51 (m, 3H), 3.36 (q, J = 6.8 Hz, J = 14.0 Hz, 4H), 2.64–2.83
(
m, 2H), 2.05–2.09 (m, 2H), 1.60–1.70 (m, 2H), 1.18 (t, J = 7.2
13
Hz, 6H); C NMR (100 MHz, CDCl
3
): δ 170.14, 168.86, 164.15,
1
1
1
1
60.72, 152.59, 152.29, 149.41, 148.96, 146.83, 144.05, 135.80,
34.60, 131.30, 130.76, 130.35, 130.25, 129.36, 128.66, 127.64,
25.06, 124.10, 123.58, 121.22, 118.964, 118.72, 108.84, 108.41,
+
04.80, 97.31, 44.52, 27.24, 23.11, 22.48, 12.65. HRMS (ESI ):
+
(
M) calcd for C38
2 7
H32FN O , 647.2118; found, 647.2190.
Fluorometric analysis in vitro
All fluorescence measurements were accomplished on a Hitachi
2 4 2 2
F4600 Fluorescence Spectrophotometer. Na S (or Na S ) was
2 n
Fig. 7 Representative fluorescence images of visualizing the H S levels
used as the H S source in all experiments. The NIR-CPS probe
at different times in tumour-bearing mice using NIR-CPS. Mice were
2 n
intratumourally injected with LPS (10 μg mL− , 100 μL in saline) for 24 h, solution (DMSO) was placed in a quartz cuvette. With the probe
1
followed by intravenous injection of NIR-CPS (2 mM, 100 μL saline, 3%
DMSO, 1% Tween 80). Images were taken after incubation with LPS for
diluted to 10 μM with 20 mM PBS buffer, various amounts of
Na S were added. The resulting solution was then incubated
2
4
1
min, 5 min, 10 min, 15 min, 20 min, 25 min, 30 min, 35 min, 40 min,
for 30 min, and fluorescence spectra were obtained with an exci-
tation wavelength of 570 nm. The emission spectra were col-
and 50 min. Quantification of the fluorescence intensity from the
tumour area of mice from all groups. Data are presented as the mean ±
SD (n = 3).
−1
lected from 600 nm to 850 nm at a velocity of 1200 nm min
.
916 | Org. Biomol. Chem., 2021, 19, 911–919
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Paper
A photomultiplier voltage of 1000 V was used. Data are pre- saline) for 30 min, then i.p. injected with LPS (10 μg mL−1
,
sented as the mean ± SD (n = 3).
100 μL in 1 : 9 DMSO/saline v/v) for 24 h and given a sub-
sequent i.p. injection of NIR-CPS (2 mM, 100 μL DMSO). For
Fluorescence imaging of living cells
the time-dependent experiment, the mice were i.p. injected
The MCF-7 cells were treated with 10% (v/v) FBS (foetal bovine with LPS (10 μg mL− , 100 μL in saline) for 24 h, followed by i.
1
−1
serum) and penicillin/streptomycin (100 μg mL ) at 37 °C in a p. injection of NIR-CPS (2 mM, 100 μL DMSO). The fluo-
% CO incubator. Cells were permitted to grow to 80% conflu- rescence intensities were then recorded after incubation of
5
2
ence before harvesting and were seeded in a glass-bottom plate. NIR-CPS at different times (0 min; 1 min; 5 min; 10 min;
NIR-CPS solution (the final concentration of 10 μM, 1.0 mM 15 min; 20 min; 25 min; 30 min; 35 min; 40 min). The mice
stock solution in DMSO) was added to the cell media and incu- were imaged using a Night OWL IILB 983 small animal in vivo
bated under the previous conditions for 20 min. For exogenous imaging system, with an excitation filter of 485 nm and an
H
2
S
n
imaging, the probe-treated cells were then further incu- emission filter of 680 nm. The fluorescence signals were quan-
bated with Na (1.0 mM stock solution in DI H O, the final tified with Olympus software (FV10-ASW). All data are
concentration of 10 μM or 20 μM) for 20 min. For endogenous expressed as the mean ± SD (n = 3).
2
S
4
2
imaging, the cells were pretreated with LPS (1 μg mL− ) for
1
2 n
H S
Fluorescence imaging in tumour-bearing mice
1
6 h and then incubated with NIR-CPS (the final concentration
of 10 μM) for 20 min. All cells were rinsed thrice with PBS
buffer prior to imaging. Confocal fluorescence imaging was per-
formed on an Olympus FV1000 confocal laser scanning micro-
scope with 60× oil objectives. The excitation wavelength was
Female BALB/c nude mice received a subcutaneous injection
7
of 10 MCF-7 tumour cells. Tumours with a volume of approxi-
3
mately 200 mm were formed over a period of 4 weeks. Mice
bearing implanted tumours were anaesthetized by i.p. injec-
tion of 10% chloral hydrate (0.04 mL per 10 g) and then ran-
domly divided into several groups. Mice were intravenously
injected with NIR-CPS (2 mM, 100 μL saline, 3% DMSO, 1%
Tween 80) as the control group. The experimental group was
intratumourally injected with LPS (10 μg mL− , 100 μL in
saline) for 24 h, followed by intravenous injection of NIR-CPS
5
1
70 nm. The fluorescence images (670 nm) were obtained at
024 × 1024 pixels and were analysed with Olympus software
(FV10-ASW). All data are expressed as the mean ± SD (n = 3).
Animals and administration
1
All animal experiments were carried out in compliance with
the Chinese legislation on the use and care of laboratory
animals and were approved by the Institutional Animal Care
and Use Committee of Xuzhou Medical University. Adult male
Kunming mice weighing 20–25 g were provided by the
Experimental Animal Centre of Xuzhou Medical College.
Female nude mice weighing 20–25 g (six to eight weeks old)
were purchased from Shanghai Sippr-BK Laboratory Animal
Co., Ltd. All mice were housed in a room with regulated temp-
erature (22 ± 2 °C) and humidity (50 ± 10%) on a 12 h light/
dark cycle. The animals had ad libitum access to standard com-
mercial animal feed and pure water, and were maintained
under specific pathogen-free conditions. Mice were acclimat-
ized for 1 week prior to experiments.
(
2 mM, 100 μL saline, 3% DMSO, 1% Tween 80). The other
mice were pretreated (intratumoural injection) with DL-propar-
gylglycine (2 mM, 100 μL in saline) for 30 min, then intratu-
mourally injected with LPS (10 μg mL− , 100 μL in saline) for
1
24 h and given a subsequent intravenous injection of NIR-CPS
(
2 mM, 100 μL saline, 3% DMSO, 1% Tween 80). After injection
of the NIR-CPS probe, fluorescence images were obtained at a
series of time points within 40 min (0 min; 1 min; 5 min;
10 min; 15 min; 20 min; 25 min; 30 min; 35 min; 40 min)
using a Night OWL IILB 983 small animal in vivo imaging
system with an excitation filter of 485 nm and an emission
filter of 680 nm. All data are expressed as the mean ± SD (n = 3).
Statistical analyses
Fluorescence imaging of living mice
All statistical analyses were performed using SPSS software,
version 16.0 (SPSS Inc., Chicago, IL, USA). Values are reported
as the mean ± SD (standard deviation of the mean). The data
were analysed with one-way analysis of variance (ANOVA).
Statistical significance was set at p < 0.05.
Prior to in vivo imaging, the mice were anaesthetized by i.p.
injection of 10% chloral hydrate (0.04 mL per 10 g), and their
abdominal fur was removed. The mice were randomly selected
and divided into several groups. The mice were given i.p. injec-
tion of the free NIR-CPS probe (2 mM, 100 μL DMSO) as the
control group. For exogenous H2Sn imaging, the other three
groups were i.p. injected with the NIR-CPS probe (2 mM,
Conflicts of interest
1
00 μL DMSO) for 15 min, followed by i.p. injection of 0.2
e
q
u
i
v
.
o
f
N
a
S
(
0
.
4
m
M
,
1
0
0
μ
L
s
a
l
i
n
e
)
,
2
e
q
u
i
v
.
o
f
N
a
S
2
4
2
4
There are no conflicts to declare.
(
4 mM, 100 μL saline), or 4 equiv. of Na
saline). For endogenous H imaging stimulated by LPS, the
other groups were i.p. injected with LPS (10 μg mL , 100 μL in
2 4
S (8 mM, 100 μL
2 n
S
−
1
Acknowledgements
1
: 9 DMSO/saline v/v) for 24 h, followed by i.p. injection of
NIR-CPS (2 mM, 100 μL DMSO). The last group was pretreated This project was financed by the Open Program of NHC Key
i.p. injection) with DL-propargylglycine (2 mM, 100 μL in Laboratory of Nuclear Medicine and Jiangsu Key Laboratory of
(
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